Glandular-Affectors: The Driving Operations in the Physiology of Man Chapter 23 James T. Fulton–October, 2018 [xxx material removed from Chapter 16 in order to form a new chapter 23. Needs very heavy reformation and editing ]

Abstract: The “glandular-affectors” consist of - - - [xxx rewrite Abstract copied FROM CHAPTER 16 ] the stage 7 neurons stimulating the glandular modality, the muscular modality and the skeletal modality of the physiological system of animals. Both inorganic molecules and biologicals are employed effectively to provide adequate neuroaffector flexibility within the physiological system. The neuroaffectors are founded on the Electrolytic Theory of the Neuron and employ the same coordinate chemistry principles as all neurons. Specifically, the stage 8 neuroaffectors employ the same dual antiparallel coordinate bond, DACB, found through out the chemical sensory neurons of the animal physiology. In the course of developing the theoretical framework of the neuroaffectors, a hybrid situation was uncovered defining a stage 8 hybrid gland/neuron configuration. The hybrid output hormones, as in the conventional stage 7 neuroaffectors, secrete peptides of variable complexity. Simultaneously, these hybrids accept chemical stimulants at their input structure(s) like a stage 1 chemical sensory neuron. Internally, the stage 8 neuron incorporated the electrolytic processes and mechanisms (including an Activa (electrolytic liquid- crystalline semiconductor amplifying device) common to all neurons. The stage 8 neural hybrids form the principle functional structures within the hypophysis (pituitary gland). The hypothalamus, pituitary, thyroid axis, H-P-T axis, is described in detail as an exemplar of the other axes of the glandular system controlled by the neuroaffectors. The exemplar includes more detailed flow diagrams, including details of the feedback signal employed, than previously published.

Key Words: Hormonal affector, glandular-affector, , Acetylcholine, hypothalamus, hypophysis, endocrine, exocrine, paracrine, tropic hormones, TSH 2 Neurons & the Nervous System

The NEURONS and NEURAL SYSTEM: a 21st CENTURY PARADIGM

This material is excerpted from the full β-version of the text. The final printed version will be more concise due to further editing and economical constraints. A Table of Contents and an index are located at the end of this paper. A few citations have yet to be defined and are indicated by “xxx.”

James T. Fulton Neural Concepts [email protected]

January 5, 2019 Copyright 2011-18 James T. Fulton 23 Glandular–affectors & the hormonal system1

In neurobiology, a central aim is to determine the components and circuitry that are responsible for a given brain function. Wilson et al. (2002) The above quotation can be equally applied to the Crine modality, with endocrinology a subordinate system, as defined in this work. 23.1 Introduction

This chapter will expand on the role of glandular–affectors first defined in Chapter 1, Section 1.1.2.3. It will focus particularly on distinct definitions for many of the biological agents affecting the glandular and other tissue systems of the body. This will draw attention specifically to the current concepts of the NANC, the non-adrenergic and non-cholinergic neuro-affectors. One of the most important members of NANC is the unusual inorganic chemical nitric oxide. Nitric oxide is commonly described as a short lived free radical. During the 21st Century, the importance of nitric oxide has become more important in biochemistry. Because of its importance, it has led to the description of a new type of neuro-affector labeled nitrergic or nitroergic (for nitroxide-ergic).

When discussing the interface of the neural system and the glandular and muscular systems, another important situation occurs. The stage 7 neurons (block diagram shown below and in Section 1.1.5) terminate in a unique axon pedicle that releases various chemicals in order to stimulate muscle tissue or additional elements of the glandular system. These stage 7 neurons are consistent with detailed definitions of glandular cells. As a result these stage 7 neurons can be defines as ending with a –a morphologically and electrolytically defined connection between a stage 7 neuron and either one or more sarcomere (muscle) cells or one or more glandular cells involving the transfer of chemical agents.

The stage 7 chemical synapse (with a cleft) is the original type of synapse studied by researchers, such as Cajol, at the beginning of the 1900's and giving impetus to the concept defining all synapses as chemically based during the 20th Century. All other synapses of stages 1 through 6 involved electrical (tight junction) synapses.

The tight junction synapses of stage 1 through stage 6 have been shown to be electrically reversible. This capability is easily demonstrated using multiple probe techniques 2 This capability places a serious impediment in the interpretation of the generic synapse as a chemically dependent functional structure. It is easy to cause the reversal of the diode characteristic exhibited by the tight junction synapse by simply interchanging the three potentials at its terminals. The diode characteristic can be reversed within microseconds. Under the chemical theory of the neuron, this reversal requires the interchange of positions between the vesicles of the axon and the sites of the neurites as well as their attaining functional normality within microseconds. The time requirement is needed to correctly exhibit the diode reversal property. It is proposed this interchange is impossible via the chemical theory of the neuron and the chemical theory of the interneuron synapse

1Released: 5 January 2019

2Eliasof, S. et. al. (1998) Localization and function of five glutamate transporters cloned from the salamander retina. Vision Res. vol. 38, pp 1443-1454 2 Neurons & the Nervous System

is invalid. The agents transferred between stage 7 neurons and muscle tissue is most generally acetylcholine (at striated muscle) or nitric oxide (at ) (Section 5.1.2.4). - - - - 2nd draft of opening material – October 14, 2018 Because of the need to understand the potential roles of stage 7 neurons relative to the muscular and glandular modalities, it has been necessary to investigate these modalities more than initially expected. As a result, the Chapter is more properly labeled, Neuroaffectors, and the hormonal and muscular modalities. The outline of the chapter reflects this labeling. [xxx re-outline to focus on the glandular affectors and receptors ] Section 23.1 focuses on a broad range of background information needed to properly interpret later sections. Section 23.2 focuses on the architecture of the stage 6 & 7 neurons. Section 23.3 focuses on the anatomy of the neuro-glandular interface. It also defines more precisely the multiple aspects of the Crine (hormonal) modality. Section 23.4 focuses on the secretions of the endocrine submodality. Section 23.5 focuses on the neuro-muscular interface and paracrine submodality. Section 23.6 addresses the interplay between the neural-glandular-muscular modalities in animal physiology. [xxx move to after 23.10 ?? ] Section 23.9 focuses on the secretions of the exocrine submodality. Section 23.10 addresses the cataloging of hormones by their chemical structure. Section 23.11 explores the potential receptors of the Crine (hormonal) modality. Section 23.10 Some remarks on nominal Crine (hormonal) system operations

[xxx rewrite with a focus on this new chapter ] The investigation into the glandular and particularly the muscular modalities will be brief and leave many aspects of these modalities un-investigated as they diverge from the main theme of this work. This is also true with regard to the muscular modality because of the limited data on how muscle tissue functions at the detailed level (what is the actual chemical or electronic mechanism involved in contraction?).

- - - -

The analogy between the processing of L-glutamate into GABA electrostenolytically at the surface of the neural lemma is addressed in Section 3.2. The processing of L-arginine into L- citrulline will be developed in detail (Section 23.5.2.4).

The interaction between the neural system and the release of various hormones has not been studied in great detail as part of this work. Early secretion based experiments have been based primarily on the concentration of various chemicals near the axons of neurosecretory neurons. Glutamate and GABA have been identified routinely at these locations and therefore claimed to be examples of neuro affectors, when in fact they are neuro-facilitators and neuro-inhibitors respectively through their participation in the adjacent electrostenolytic process powering the neurons. Acetylcholine (Ach) was one of the earliest potential neuro-affectors based on its ease of chemical identification. While its choline component, a nitrogenous alcohol, is a source of nitrogen and may contribute to the production of nitric oxide, NO, its precise functional role remains a subject of discussion. The study of type 4 conexi in the early 20th Century (by Cajal, Golgi & others) gave rise to the belief that all interfaces between tissue of the body involved the transfer of chemical agents. However, it is now clear that within the neural system, signal transfer between neurons is entirely by electrons and involves type 1, type 2 and type 3 conexi (Section 5.1.2.4). Only stimulation of stage 1 sensory neurons and the exudate of stage 7 axon pedicles involve species other than electrons. Hormonal-affectors 23- 3

The description of the neural system in an electrolytic context provides an abundance of new concepts to be explored. In this context, the major glands can be considered stage 7 neural interfaces with the exocrine and endocrine systems. In addition, the neuron containing endothelial tissue interfacing with the smooth muscle of the cardiac and arterial system becomes a complex of considerable interest. The interface between the neural system and the glandular system appears to involve a variety of stereo-chemical processes. Many of the molecules involved as precursors to the hormones exhibit considerable stereo-specificity. The neural/glandular interface appears to be a new and very active area of study. This work will show the intimate connection between stage 7 of the neural system and the initial elements of the hormonal system. Schmidt & Walter describe NO as “partly a and partly a hormone3.” This work will consider NO as a “localized ” affecting only tissue within a limited distance due to the short lifetime of this free radical. Describing the operation of stage 7 neuro-affectors involves very complex reactions described in a previous time as stereochemical. However, this term is now reserved for the description of individual molecules. The stereo-specific interactions between molecules are now considered only a feature of more complex processes that frequently involve catalytic (an inorganic term) or enzymatic (an organic term) reactions. These catalytic processes are known to involve not only stereo-specific interactions but also significant changes in the energy states associated with the components. The complexity of these reactions and resultant molecules appears to be separating into two distinct fields, the development of supramolecules in both the materials engineering and the enzymatic contexts and the design and development of bioorganic molecules in the biological and pharmacological domains. Supramolecules are restricted to multiple molecule complexes that do not employ covalent bonds.

The term bioorganics has become necessary to delineate between the broad field of physical organic chemistry outside of the biological context and the equally or broader field of physical organic chemistry within the biological sphere.

The receptor concept was introduced by pharmacologists in the 19th Century to explain the selective toxicity of plant alkaloids and certain synthetic chemotherapeutic agents4. The receptor-substrate context has expanded immensely from the earlier lock-and-key concept of Fischer promulgated in the early 20th Century.

Section 23.9 is included here to discuss the important enteric neural system and its role in the overall enteric system. The enteric system, the complete digestive system, operates largely autonomously and employs several mechanisms that are unique to it and serve to isolate it from the other physiological systems of the organism.

This Chapter will also include some material on the striate and smooth muscle systems, major targets of the neuro-affector neurons and neuro-affector discharges. This discussion will highlight how specific neuro-affectors only affect certain types of muscle tissue and thereby provide a degree of isolation between the neuro-affector chemicals, as well as the operation of the individual muscle types. It will also highlight why the smooth muscles do not require the presence of end-plates as used in striate muscle. - - - - 23.1.1 xxx Organization of the stage 6 & 7 neuro-affector neurons

The study of the neuro-affectors associated with the glands is complicated by a number of

3Schmidt, H. & Walter, U. (1994) NO at work vol 78, pp 919-925

4Robison, G. Butcher, R. & Sutherland, E. (1971) Cyclic AMP. NY: Academic Press page 22 4 Neurons & the Nervous System factors. C Much of the work has been performed in the clinical setting where avoiding invasive techniques on humans has been stressed. C Much of the work has focused on reflex activity that is easily observed in the clinical setting. C Unlike the external sensory modalities, the somatosensory modality involves a much more entwined arrangement of sensory receptors with multiple types of highly modified end organs. C It is very difficult to stimulate only one internal sensory receptor neuron without employing invasive techniques. C It is difficult to excite only a single muscle via the efferent neural pathway. Redundant stage 7 neurons and stage 7 neuron axon pedicles are typical in the excitation of muscle tissue. C Stage 6 commands to the muscular system typically address multiple muscles simultaneously. C As observed in the second figure below, some shortcuts have been employed when studying reflex activity. It is common to omit the location of the soma of neurons and employ an unusual symbol related to the synapse. The study of the neuro-affectors associated with the glandular modality is complicated primarily by the diversity of the interface and limited data on the operation of the neural/glandular synapses. The above conditions make it difficult to isolate the individual glandular elements and determine their precise individual roles.

23.1.1.1 Xxx Role of neuro-affectors in overall system

Neuro-affectors are one of the terminal types of neurons in the neural system. The neuro- affectors are found at multiple locations within the system. The locations addressed in this work are primarily at the muscular modality and the glandular modality interfaces. They act to control the muscles and to control the operation of other glandular tissue types primarily through hormonal action. Figure 23.1.1-1 places the stage 7 neuro-affector neurons in context within the overall system. Hormonal-affectors 23- 5

Figure 23.1.1-1 The stage 7 neuro-affector neurons within the overall neural system shown in context. They are fundamentally stage 3 signal projection neurons where the last axon segment has been modified to act as a neuro-glandular interface. They are the major controllers of the muscles and the primary participants in the glandular system. They are believed to produce, and release under neural control, a wide range of organics, non-biologicals and biologicals.

The neuro-affectors are major terminal elements in the neural system and major points of origination in the endocrine and exocrine systems (as well as the expanded crine modality described below. The recognized types of stage 7 terminal elements are shown in the lower left quadrant.

23.1.1.2 Xxx Role of glandular-affectors in overall system EMPTY [xxx see 23.3.1.3 use a single path version of that figure ] 23.1.2 Common inter-modality functional blocks

After completing the analyses in support of Chapter 16 and Chapter 23, a common functional block appeared that could be used for neuroaffectors, glandular-affectors and muscular tissue. The generic functional blocks associated with each of these interfaces is shown in Figure 23.1.2- 1. 6 Neurons & the Nervous System

Figure 23.1.2-1 Common inter-modality functional blocks ADD.

Frame A shows the neuron functional block used throughout this work (except for the more complex stage 1 sensory receptor neurons). It includes a differential electrical input structure that makes the labels of adrenergic and cholinergic obsolete with respect to orthodromic signaling. It also accounts for the sensitivity of neurons to chemical modulators. It also provides for the generation of the negative voltage of the internal chambers of the neuron via the electrolytic energy supplies. The output of the neuron is typically a voltage except in the case of the neuroaffectors where it is typically acetylcholine or nitric oxide.

Frame A illustrates a neuron as displayed previously but with a defined modulator input. The receptor at this location is primarily to accommodate glandular inputs, such as epinephrine or norepinephrine. The concept is that there can be separate receptors for one or more glandular inputs to a neuron besides the required electrolytic receptor, receptors of neural (electrical) signals and means to accept chemical supplies if required (to support stage 7 neurons). These defined receptor locations are distinct from those required for the cell to maintain homeostasis. Frame B shows a typical muscle functional block. It exhibits the same generic input structure as the neuron and the same inputs for chemical modulators and electrolytic supplies. In addition, it exhibits a chemical supplies terminal where additional sources of energy can be introduced. It appears that striate muscle is only susceptible to stimulation by acetylcholine at the input accompanied by a positive polarity sign. Conversely, the tension inherent in smooth muscle is repressed by stimulation by nitric oxide at the input terminal accompanied by a negative polarity sign. Hormonal-affectors 23- 7 Frame C shows a typical gland functional block. It exhibits the same generic input structure as the neuron and the same inputs for chemical modulators and electrolytic supplies. In addition, it exhibits a chemical supplies terminal where additional sources of energy can be introduced. Based on the modeling of the hypophysis (pituitary) gland by Labrie et al5,, at least the glands within the hypophysis also exhibit a differential input structure. The modulator and energy supply terminals in A, and probably in B and C, do not employ protein receptors of the GPCR family. See Section 3.2.4. The receptors at these locations are typically amino-. The receptor for the electrolytic supplies is known to be phosphatidylserine molecules in a liquid crystalline array forming the outer bilayer of a region of type 2 lemma of the cell involved. This is the proposed common functional block used in these major modalities in the architecture of the physiology of animals (including humans). 23.2 Background in literature

During the 1960–1980 period, many significant volumes were published that provided a great deal of data on the anatomy and histology of the hypothalamus. These included Haymaker et al6 and Reichlin et al7 . These volumes largely ignore the cytology of the hypothalamus and its functional aspects. They do provide some low resolution mapping of specific hormones within the hypothalamus and hypophysis. They do provide global functional aspects associated with several medical conditions. However, they are basically archaic, except for histological data mining. In 1979-80, a large scale handbook was published in four volumes8,9. Volume one introduces the electrophysiology of the hypothalamus with the primitive protocols of that time. On page 865, Renaud notes, “The hypothalamus, being a relatively small and poorly defined area of brain, has tended to escape the detailed electrophysiological observation that has provided us with some understanding of neural mechanisms operative in more accessible and structured brain regions. . .” Chapter 2 of volume 2 attributes most of the hormones of the hypothalamus to their origins within “–containing neurons and their projections.” Page 77 is particularly important. The chapter does not address the cytology of these neurons. All of these volumes lack significant intellectual continuity because of the number of authors in each of these volumes.

The molecules released by stage 7B neurons interfacing with the glandular modality appear to be poorly documented and not widely discussed within the neuroscience community at this time. Chapter 2 of volume 2 of Morgane & Panksepp contains some work in this area. There has been very little academic literature on this subject since 1980. As late as 2005, Harbuz & Lightman10 (writing in Melmed & Conn) provide only a conceptual cartoon addressing the neural/glandular interface. They suggest both the brainstem and hippocampus provide stimulation to the paraventricular nucleus, PVN, of the hypothalamus. In the same text, Bichet identified two molecules understood to be produced by stage 7 neurons with pedicels located

5Labrie,F. Drouin, J. De Lean, A. et al. (1976) In Levey, G. ed. Hormone-receptor interaction: molecular aspects. Volume 9 of Modern Pharmacology-Toxicology NY: Marcel Dekker

6Haymaker, W. Anderson, E. & Nauta, W. eds (1969) The Hypothalamus. Sprinfield, Il: Charles C. Thomas

7Reichlin, S. Bailessarini, R. & Martin, J. eds. (1978) The Hypothalamus, Volume 56 in a series. NY: Raven Press

8Morgane, P. & Panksepp, J. eds. (1979) Anatomy of the hypothalamus, vol 1 of Handbook of the Hypothalamus. NY: Marcel Dekker

9Morgane, P. & Panksepp, J. eds (1980) Physiology of the Hypothalamus, vol 2 of Handbook of the Hypothalamus. NY: Marcel Dekker

10Harbuz, M. & Lightman, S. (2005) The Neuroendocrine-Immune Interface In Melmed, S. & Conn, P. eds Endocrinology, 2nd Edition Totowa, NJ: Humana Press Chapter 8 8 Neurons & the Nervous System in the posterior hypophysis11. “OT and AVP are synthesized in separate populations of magnocellular neurons (cell body diameter of 20-40 microns of the supraoptic and paraventricular nuclei” of the hypothalamus. OT is oxytocin and AVP is arginine vasopressin. The two peptides differ by only two amino acids. Section 23.3.3.3 will address these molecules in greater detail. Knigge et al. in Chapter 2 of volume 2 of Morgane & Panksepp identified closer to a dozen originating in the hypothalamus in 1980. They noted at that time, “During the last 10 years it has become obvious that hormones other than oxytocin and vasopressin are synthesized and release by neurons of the central nervous system.” The concepts in the literature related to the hormonal (Crine) modality prior to the revolution caused by the emergence of DNA sequencing are highly conflicting. As a result, this literature must be looked upon as a source of factual information but not for a dependable concept or framework of hormonal modality!! 23.2.1 Developing a modern definition of a hormone

Quoting Guillemin in 1978(page 189 in Reichlin et al.), “Perhaps time has come to redefine the hormone. The hypothalamic hypophysiotropic peptides, TRF, LRF and , are really not hormones according to the current definition, which is still that proposed by Starling in 1905. The definition implies a single source of secretion and, fundamentally, the direct ingress of the secretory product into blood vessels for distribution to a distant receptor tissue, itself thus triggered to respond by its own secretion, change in metabolism, etc.”

The definition quoted by Guillemin is totally inadequate at this time (2018). On page 213, he offered a new definition of a hormone(apparently for purposes of discussion), “as a substance made by certain (secretory) cells known to affect the function of other cells. Such a simple statement would not be a contradiction to the definition given by Starling in 1905; it would be somewhat more encompassing.”

Current dictionaries provide a variety of definitions for a hormone (apparently depending on their perspective). The definitions in Merriam-Webster, at Dictionary.com and in Wikipedia exhibit much too narrow perspectives. They generally only address the , not the broader Crine modality.

The 2ndEdition of Endocrinology (2005) presents a focus on endocrinology that is too narrow.

As Guillemin noted in 1978, “it is time to think seriously about questions of nomenclature before they become problems of nomenclature.” Now, it is even more important to address this task.

A useful definition must address several fundamental conditions; 1. Recognize the presence of hormones within all systems of a broader Crine modality than just the endocrine system of that modality. 2. Address whether hormones are limited to only direct signaling paths or whether they are also involved in modulating the direct signaling paths, whether neural or glandular. 3. Recognizing the three major types of hormones; the neuro-hormones emanating from the hypothalamus, the neuro-hormones emanating from the posterior hypophysis,, the hormones emanating from the adeno- and central hypophysis and the hormones emanating from peripheral glands of the Crine modality. 4. Address whether those chemicals supporting the electrostenolytic process powering the neurons are hormones or more a matter of fuels for the neurons with no modulatory action. 5. Address whether the digestive juices of the traditionally labeled pancreas gland should be considered ducted hormones.

11Bichet, D. (2005) Posterior pituitary hormones In Melmed, S. & Conn, P. eds Endocrinology, 2nd Edition Totowa, NJ: Humana Press Chapter 14 Hormonal-affectors 23- 9 6. Address the fact that many hormones are closely coupled to carrier proteins.

23.2.1.1 Expanding on the 6 aspects converging on the definition of a hormone EMPTY The following text uses numbered paragraphs that correlate with those of the previous listing. 1. The character of the “hormones” secreted by the different systems, paracrine, pericrine endocrine and exocrine, under the general heading of the Crine modality vary considerably. While in the past, different investigators have adopted a variety of local perspectives on the definition of a hormone, the need is for a comprehensive definition. The comprehensive definition may then be narrowed with prefixes for the purpose of a given investigator or class of investigators. 2. There are a vast number of hormones (and derivative hormones found in other species) among the animal world. In this context, the human hormones will be considered the parent types. They can be divided into two distinct groups; those exerting a direct impact on orthodromic internal or external receptor organs, and those exerting an indirect impact on any physiological element of the organism. The former can be considered signaling hormones leading to a definable action and the latter can be considered parametric modulating (or mediating) hormones.

3. At least five major types of hormones can be readily described; (1) those emanating from the hypothalamus and transported via the portal to the hypophysis (pituitary), (2) those emanating from the Posterior portion of the hypophysis, (3) those emanating from the anterior portion of the hypophysis, and (4)those emanating from specific peripheral glands and targeting a broad variety of target tissues within the animal or target tissue of other animals of the same species. (5) There are also the hormones of the neuroaffectors targeting individual muscular element(s).

4. The pairs of glutamate & GABA and aspartate & glycine have frequently been described as hormones when their actual role is in fueling the neurons and other cells of the body by providing the negative potential(s) found within the cells. While these molecules have frequently been shown to affect a cell transiently when applied topically in vitro, this is not useful in interpreting their in vivo role. In their in vivo role, these materials are present through out the organism, particularly concentrated near the neurons. Regardless of their location, they are normally present in concentrations that remain constant over time. Failure to maintain these concentrations are associated with disease in the animal.

5. The pancreas and other glands associated with the digestive tract secrete a broad range of molecules that are designed to digest food. These molecules are not designed to participate in orthodromic signaling to, or parametric modulation of, other cells of the organism. These materials are generally characterized as enzymes (catalysts) and not hormones.

6. Many generally recognized hormones are not sufficiently soluble in the vascular circulation and they are generally conjugated with, frequently larger, carrier proteins. It is important to identify the molecular weight of the hormone separately from the molecular weight of the carrier or the conjugate structure. This has not always been done by different investigators across the academic literature. 23.2.1.2 Proposed working definition of a hormone

A propose definition of a hormone should respect the situations discussed in the previous sections in order to be useful to the entire bioscience community. In order to be both as explicit as possible and simultaneously as comprehensive as possible, a format will be used here that follows the Unites States Patent Office format for “claims.” They begin with a primary claim that is limited to one sentence (with an initial capital letter and ends with a period) that cannot include any disjunctive expression such as the word “or.” It cannot include any abbreviation, dash, parentheses or quotation marks. One or more secondary claims can refer to a primary claim and expand the particulars of that claim. The claims are numbered sequentially beginning with the primary claim. 10 Neurons & the Nervous System

Therefore, the following definition is offered in 2018;

A hormone is: 1. One or more molecules of a single stereographic configuration created within an organism capable of causing dynamic changes in the performance of a target cell for finite intervals in vivo. A. The molecules of article 1 wherein the hormones are distributed throughout the organism via the vascular circulation. B. The molecules of article 1 wherein the hormones are distributed via the portal between the hypothalamus and the hypophysis. C. The molecules of article 1 wherein the hormones are excreted into the exterior environment of the organism.

D. The molecules of article 1 wherein the hormones are distributed via a dedicated duct between the source of the hormones and it’s target tissue.

E. The molecules of article D wherein the dedicated duct refers to a synaptic cleft between a neuroaffector and a cell of a muscle.

F. The molecules of article 1 that are complexed with a carrier protein to increase their solubility in the vascular circulation.

The definition is compatible with molecules consisting of small organic molecules, e.g. @N=O, catecholines, peptides and steroids. This definition excludes the digestive juices (enzymes). It does not include glutamate & aspartate and GABA & glycine as hormones since their primary role is as fuels and residues in electrostenolytics. They can be considered part of the homeostatic system of the organism rather than as a signaling or modulating function. Overall the proposed definition is considerably broader than that provided by Starling in 1905 or suggested by Guilleimin in 2005 (Section 23.2.1).

This definition is based on specific molecular structures and not defined in terms of one or more aspects of biological activity where the potential failure to observe a crucial aspect of the total activity is ever-present. The individual definitions provided apply to various portions of the Crine modality.

Definition Application

1. The primary definition applicable to all hormones of the Crine modality. A. Applies to hormones of the endocrine system. B. Applies to hormones of the pericrine system. C. Applies to hormones of the exocrine system. D. Applies primarily to hormones of the peripheral glands. E. Applies to hormones of the paracrine system. F. Applies to some hydrophobic and large hormones, primarily of the endocrine system. Section 23.3 provides maps of the Crine modality and its neural/glandular interface in support of this definition of a hormone.

23.3 Anatomy of the neuro-glandular interface Hormonal-affectors 23- 11 The medical–physiology community makes note of the interface between the neural system and the glandular system. [xxx edit the following ] The hypophysis is neural material, originating within the CNS, that secretes significant hormonal material outside of the blood-brain barrier. The hypophysis is more commonly known as the pituitary gland. Ganong has noted, using different terminology than in this work12, “The adrenal medulla is in effect a sympathetic ganglion in which the postganglionic neurons have lost their axons and become secretory cells.. The cells secret when stimulated by the preganglionic nerve fibers. . . .” The above trend leading to a new field of neuroendocrinology has been summarized in a 50th Year editorial by Henderson & Dores13. “The composite nature of the perfusion of hypothalamo-hypophysial system in most vertebrates, the widespread identification of the phenomenon of neurosecretion and associated neurohaemal organs, the characterisation of steroidal, amine, tyrosine-derived, and protein hormones, including the universal , are all key events in endocrinology’s more recent history (at best a documentation of information). A new term also arose – neuroendocrinology – as an all embracing description uniting the older, outdated divisions between neurophysiology and endocrinology. Molecular and cellular processes gradually accrued within the endocrine vocabulary. Hormones as excitatory substances would now be modified and see them as inhibitors or moderators of other hormonal actions. The circulation of blood was no longer the sole route of access; local synthesis and delivery of hormones, to a neighbouring target became apparent. More neologisms appeared – paracrine, autocrine and are part of the latest endocrine vocabulary.”

Henderson14 has simultaneously issued a new chapter available on line reflecting the most recent perspective from an “endocrinologist.” Interestingly, he uses the expression “neurotransmitter/neuromodulator” for substances delivered by “diffusion/vascular transport” in his first table addressing the multifarious nature of hormones (and cited as a modification of Bern15). This description meets the definition of a neuromodulator of this work but not the definition of a neurotransmitter. Hopefully, this indicates he is moving toward separating the term neurotransmitter from a diffusion-based phenomenon. Bern did not address and Henderson does not expand on his understanding of the neuron-to-neuron signaling function, or his description of a neurotransmitter.

His insights appear very worthy of study;

“This chapter does not take fully into account the fact that the descriptions of classical endocrine glands, as noted above, are becoming increasingly blurred and often indistinct. There are unequivocal demonstrations that many hormones are produced outside their "normal" sources and that the paracrine nature of many substances, viewed originally as emanating from the gut, brain, or kidney, are produced for local use within the liver, heart, pancreas, or blood vessels.”

Henderson also provides an interesting role for the diencephalon in the hormonal system;

“The diencephalon is the most relevant portion of the "endocrine brain." This posterior portion of the forebrain (proencephalon) contains in its lateral walls, abutting onto the lumen of the third cerebral ventricle, several important endocrine structures that are present in all vertebrates: the epithalamus, the dorsal thalamus, the ventral thalamus, and the hypothalamus, which is connected to the pituitary gland.”

12Ganong, W. (1975) Review of Medical Physiology, 7th Ed. Los Altos, CA: Lange Medical Publications pg 267

13Henderson, I. & Dores, R. (2011) Editorial: Animals to atoms Gen Comp Endocrin vol 170, pp 1–2 & 115-116

14Henderson, I. (2011) Endocrinology of the vertebrates In Handbook of Physiology-comparative Physiology Chapter 10.

15Bern, H. (1990). The "new" endocrinology: its scope and its impact. Am Zool vol 30, pp 877-885 12 Neurons & the Nervous System

“Turning to perhaps the most major endocrine unit of the brain, an interface is encountered which represents a highly intricate association between neural and nonneural structures, the hypothalamo-hypophyseal neuroendocrine complex. Before an examination of the various relationships of this extraordinarily intricate union of tissues, some broad generalities give an insight into the pertinence of the subtle permutations described later. In all vertebrates, at the base of the brain rests the hypothalamus, which consists of a series of aggregations of neurons (nuclei) to form distinct hypothalamic nuclei.” All of the above occurs within the first seven pages of 127. Later (pg 698), he accepts nitric oxide into the family of hormones; “There are several chemical categories of hormone: the steroid/sterol variety, the peptide/protein types, those derived from tyrosine/phenylalanine, and those derived from arachidonic acid (, thromboxanes, prostacyclins, and leukotrienes). To this chemical classification may now be added nitric oxide (NO). Originally defined as endothelium derived relaxing factor (EDRF) by Furchgott and Zawadsi, it was essentially regarded as a local regulator of vascular tone. However, the widespread occurrence of NO in the brain, gut, and elsewhere renders it likely that it is a ubiquitous signaling molecule, and what is now known as the L-arginine-NO-citrulline pathway participates in diverse endocrine processes, from parturition to the regulation of sodium excretion.”

His figure 10.63 only addresses the enzymatic generation of NO.

While addressing where, Henderson does not address the how or why hormones are released from their point of fabrication, as illustrated by a brief quotation,

“Once produced, a hormone of whatever type may encounter its specific receptor, but there are extracellular influences that, in a sense, authorize this interaction.”

As appropriate for a handbook, the chapter provides well over 900 citations.

This work will focus on a broader terminology in discussing the hormonal system than many works in order to maintain consistency in a complex area. Figure 23.3.1-1 describes that terminology from the perspective of the neural system. The frame on the right describes the non-hormonal signaling between two neurons (based on the transfer of electrical charges within a tight junction. The other three frames all address the classification of hormones operating at a distance, and described here as neuroaffectors. Paracrine neuroaffectors operate over a very short distance within a confined space. They are typically nitric oxide and acetylcholine and released within the immediate vicinity of their targets, typically on a one neuron to one target cell basis. The target cells are typically muscle or gland cells. (see GnRH in Section 23.1.3.1.4 for a potential 3rd molecule to be secreted by stage 7 neurons). The endocrine neuroaffectors are a major system that releases hormones within the tissue of the organism in order to affect targets at longer distances greater than involved in a (up to several meters away). The term synapse loses its meaning in such situations. The exocrine neuroaffectors release hormones into the environment outside the organism itself (both the external environment and the internal environment of the enteric system (or digestive tract). These neuroaffectors are shown moving down a duct to escape the physiological environment of the organism. The endocrine and exocrine systems broadcast their secreted molecules and do not operate on a one neuron to one cell basis. A special case involves those hormones released within the brain-blood-barrier of the CNS. These hormones will be described as pericrine here. They act in the manner of the hormones secreted by the endocrine system but are released and broadcast only within the CNS. Hormonal-affectors 23- 13

Figure 23.3.1-1 The classifications of the hormonal system used in this work ADD. Signaling within the electrolytic synapse is by electrons. Signaling within the paracrine system appears to employ electrostenolysis on the surface of the neuron rather than vesicles. The complexity of the hormones of the endocrine, paracrine and exocrine system appears to require mitochondria involvement and vesicles for hormone release. The tight junction of the electrolytic synapse is 20–30 Angstrom wide. The gap junctions of the crine modality are 200 Angstrom or more wide. Based on Pappas, 1975.

Figure 23.3.1-2 provides a broader context for these materials in the context of the Electrolytic Theory. This figure separates the CNS from the rest of the peripheral system, both neural and hormonal. The blood-brain-barrier effectively isolates the CNS from the hormonal system designed to control the rest of the body. Only a portion of the paracrine system, represented by the hypothalamus, is found within the BBB. While the hypophysis (the pituitary gland) is generally associated with the CNS, it is effectively outside of the BBB and serves the non-CNS portions of the organism (Section 23.3.2.1). The hypothalamus and hypophysis act as the major neuron to non-neuron interface. They are stage 7 neuroaffectors in the framework of this work. All of the other endocrine and exocrine glands are also outside of the BBB.

The figure can be compared with the older works of Ganong (1975) pg 168, 449-450 and the BBB on page 450, fig 22-2 in Eyzaguirre (1975). 14 Neurons & the Nervous System

Figure 23.3.1-2 A caricature of the neural-glandular system UPDATE xxx. The blood-brain-barrier is semipermeable with respect to both direction and species but highly selective. The hypothalamus is the dominant hormonal source within the CNS. The hypophysis (pituitary) is the master gland of the multi-unit endocrine system outside of the CNS. It releases ACTH, adrenocorticotropic hormone; FSH, follicle-stimulating hormone; GH, growth hormone; LH, luteinizing hormone; PRL, prolactin; and TSH, thyroid-stimulating hormone. The hypophysis also influences the pancreas in the release of many exocrine secretions.

The hypothalamus is believed to control secretion of dopamine by the substantia nigra within the CNS as a method of overall control of the CNS16. The hypothalamus is known to be the major source of neural control over the hypophysis (pituitary gland). Are emanations of the hypothalamus stage 6 or stage 7 neurons?

It is generally assumed that dopamine is produced by the hypothalamus from the simple tyrosine as follows: tyrosine (tyrosine hydroxylase) L-Dopa (L-Dopa decarboxylase) dopamine. (eyzaguirre pg 306) If dopamine was produced by the hypothalamus, it would clearly consist of, at least some, stage 7 neurons. If however, it controlled the release of dopamine produced by the hypophysis, the hypothalamus would be a clear example of a stage 6 neuron. This area of physiology may require more careful study. Similarly, the boundary between the ducted and ductless portions of the glandular system may require more careful definition. It is also becoming clear that the emanations of the stage 6 and stage 7 neurons may also participate in elements at the neural/glandular interface that are portions of a servoloop

16Kalat, J. (1998) Biological Psychology, 6th Ed. Ca: Pacific Grove Brooks/Cole page 439 Hormonal-affectors 23- 15 involving chemically based feedback elements (Section 23.4.3.1). The figure shows the signaling paths involved in the neural system, including those controlling the stage 7 neural and neuro-glandular elements. The direct neural affectors connect to the muscles while the indirect neuro-affectors and neuro-modulators impact a wide range of body tissues. The indirect elements include: C the walls of the vascular system that are impacted by paracrine glands, C the conventional endocrine gland elements (six major glands) and C the sweat glands, gastric glands of the exocrine system. The latter appear to be under a combination of neural control and hormonal control (originating in the hypophysis and pancreas). Toward the end of the 20th Century, the hormone associated with the endothelium-derived relaxation factor (EDRF) was identified. To the surprise of many, it was identified as the simple molecule nitric oxide. Currently, hormones are described as either a protein or peptide assembled from amino acids, a steroid or the molecule nitric oxide. Some investigators also define the prostaglandins (C20 fatty acids containing a five-member ring) as hormones. See the previous material from Henderson. The significance of the value 20 was not discussed. Why not 16 carbons, as in the case of protirelin (a.k.a. Thyroid-releasing hormone, TRH), which also incorporates three five-member rings but does not include a fatty-acid component. 23.3.1 The differentiation between neuro-affectors and hormones

The biological literature is inconsistent in the use of the concepts of communications to describe cellular interaction. Dugas suggests cellular communications follows three primary pathways17. The first involves the transmission of electrical impulses via the nervous system. The second involves chemical messengers or hormonal secretions. The third involves de novo protein synthesis. While this work will generally follow this concept, it is not clear why de novo protein synthesis constitutes communications. His first pathway is the path of true supported by the Electrolytic Theory of the Neuron. His second pathway will be expanded to include all neuro-affectors (signals) emanating from the terminal (stage 7) neurons of the neural system. These stage 7 signals may be neuro-muscular or neuro-glandular interactions. The stage 7 neurons typically secrete the paracrine hormones, @N=O(nitric oxide) and acetylcholine. More chemically complex hormones are released by glandular tissue of the crine modality. 23.3.1.1 The definition of a neuro-hormone

Beyond this point in this chapter, and generally in this work, a neuro-hormone is the secreted molecule at the pedicle of a terminal (stage 7) neuron of the neural system. It may be a molecule affecting a muscle, a gland, or it may be a chemical material (signal) affecting any tissue of the body (see GnRH in Section 23.1.3.1.4) or the external environment. While the term signal is frequently associated with hormones, it should be clear the term is used in a narrow sense.

A distinction is made here between the actual interneural signal transmitter, the electron (or hole), and the many chemical pseudo-neurotransmitters of the literature. These pseudo- neurotransmitters were actually the result of two activities; C laboratory experiments on stage 7 (terminal) neurons contacting muscle tissue during the early part of the 20th Century that are actually neuro-hormones, and C cytological documentation of every molecule found at the location of a synapse whether the molecule participated in signaling or not, followed by considerable prognostication by the investigators as to how these molecules might be employed in signaling. This latter approach resulted in the excessively complex and poorly supported figure of Wilcox & Gonzales (1995) reproduced in Fuster in 2008, page 61. The neuro-hormones of this work include the “hormone releasing hormones” of the

17Dugas, H. (1996) Op. Cit. pg 122 16 Neurons & the Nervous System hypothalamus but not the “stimulating hormones” of the adenohypophysis, a.k.a., the anterior hypophysis. The hormones of the neurohypophysis (a.k.a. posterior hypophysis) are necessarily included by definition. They are the peptides, oxytocin and vasopressin. 23.3.1.2 The dual character of the efferent neural system

The efferent neural system is conventionally divided into two major divisions, the autonomous system and the volition (sometimes somatic) system. The volition system will not be addressed in this section (See Section 18.1.1.5). The autonomous system is further divided into two branches providing a push/pull or agonist/antagonist operating capability. Such opposing actions allow a fine degree of process control to be exercised by the system. The two branches are labeled the (ortho)sympathetic and parasympathetic neural systems. However, these definitions do not segregate “on” actions into one branch and “off” actions into the other. 23.3.1.3 Introductory schematics from the stage 7neurons to their targets

The glandular modality can be described as operating in stages, not unlike the neural system. Figure 23.3.1-3 provides a block diagram expanding and extending stage 7B of the neural block diagram of Section 23.1.1. Only the major hormone of endocrinology are shown. Because of the extent and complexity of the glandular modality, the next figure will extend the description of the modality from a different perspective. Both figures refrain from illustrating feedback paths. Such feedback paths are abundant and complex. What is known about these paths will be introduced in Section 23.4.2.1.

Figure 23.3.1-3 Top Block diagram of the glandular modality, without feedback, expanding and extending the similar Top Block of the Neural modality. An unknown number, n, of stage 7B neuroaffectors stimulate the hypothalamus. The hormones passing through the portal are considered “tropins,” hormones that stimulate creation of other hormones within the hypophysis but that do not circulate in the blood stream. See text. Compare to Labrie et al., 1976. Hormonal-affectors 23- 17

The neuro-hormones produced in the hypothalamus are believed to be all peptide-based according to Knigge et al. (Section 23.4). OT & AVP are identified as monoamines in some previous articles. but are now believed to be oligopeptides. There is considerable conflict in the literature concerning the hormones produced by the anterior hypophysis. A significant portion of the hormones associated with the anterior hypophysis (pituitary) are steroid-based hormones; FSH, LH, LtH & ACTH, according to Henley et al. Following the explosion in knowledge related to DNA in the 1990's, FSH has been described as a glycoprotein by Rose et al. in 200018. They assert FSH consists of two portions of 111 residues and 92 residues respectively, and which are products of different genes. Each subunit has two N-linked glycosylation sites, which are on Asn 52 and Asn 78 for the a-subunit and Asn 7 and Asn 24 for the b-subunit and which are essential for expression of FSH bioactivity. They have provided citations to the relevant research. They also assert that “(i.e., FSH, TSH, LH, and CG) are all members of the glycoprotein family. In their conclusion, they assert, “The molecular structure of FSH has been largely determined but variation in key structures leads to heterogeneity and renders impossible a definition of a single molecular entity as FSH. Biologically, FSH exerts multiple effects at its target tissues so that there is no single action that defines FSH.” In many cases within both the hypothalamus and the hypophysis, large proto-hormones (a.k.a. pre-hormones) are present that are cleaved into multiple smaller hormones represented by the abbreviated names above.

- - - -

The recent text of Melmed & Conn, 2005, does not include any comprehensive graphic similar to that above. Their chapter 1 by Conn, “Introduction to Endocrinology,” is only 2.5 pages long and includes no figures.

The figure shows that the glandular modality can be delineated into three distinct stages; the generation of hypothalamic “tropin-releasing” hormones, stage G1, used to control the generation of other “tropin hormones” within the hypophysis (pituitary), stage G2, that are ultimately distributed to lower level glands and other tissue), stage G3, by the vascular circulation. The hormones released by the hypophysis are generally labeled trophic (growth) hormones or tropin–stimulating hormones.

The hormones released by the hypophysis are deposited directly into the capillary bed of the hypophysis and ultimately into the general vascular circulation.

The hormones output by the hypothalamus are not released into the vascular circulation, but are transported to the hypophysis by a dedicated “portal” extending between the two morphological elements.

The names of individual hormones have arisen over many decades of investigations and, unfortunately, there is little structure to the current names. The abbreviations shown in this figure will be associated with the full hormone name in the following paragraph. The last two letters of more recently identified hormones may indicate their function; –RH indicates a releasing harmone, –IH indicates an inhibiting hormone, and –SH indicates a stimulating hormone. Labrie et al. provided the following caption for their version of this figure. “TSH, thyrotropin; LH, luteinizing hormone; ACTH, adrenocortcotropin; FSH, follicle-stimulating hormone; GH, growth hormone; PRL, prolactin; TRH, thyrotropin-releasing hormone; GH-RH, growth hormone releasing hormone; CRH, corticotropin-releasing hormone; PR–IH, prolactin release-inhibiting hormone; LH–RH, luteinizing hormone-releasing hormone; GH–RIH, growth hormone-releasing inhibiting- hormone (somatostatin); PRH, prolactin–releasing hormone.” In some cases, they omitted the

18Rose, M. Gaines Das, R. & Balen, A. (2000) Definition and Measurement of Follicle Stimulating Hormone Endocrine Reviews 21(1): pp 5–22 18 Neurons & the Nervous System word hormone from the full name. The hormones of the hypothalamus, while labeled tropin-releasing in character, are processed by the glands of the hypophysis as either stimulating or inhibiting. The inhibiting signal paths are shown by dashed paths passing through the portal. How this “differencing” is achieved at the entrance to the individual glands of the hypophysis without a differential input structure similar to that of neurons appears to be unknown. It may be the inhibiting hormone may be occupying the receptor site(s) targeted by the stimulating hormone. In this case, inhibition would only be effective in the presence of a stimulating hormone. The glandular portion of the hypothalamus receives a currently unknown number of stage 7B neuroaffectors from the CNS. Some of these stage 7B neuroaffectors appear to originate in the non-glandular portion of the hypothalamus They were labeled parvi-neurons and magno- neurons because of their size, by Swanson in a later figure. It is not known whether the bulk of the stage 7B neuroaffectors are individually targeted to glandular tissue of the hypothalamus or whether a matrix approach is employed to reduce the number of neuroaffectors required.

There is one stage 7B neuroaffector that apparently operates differently from the others (note dash-dot path). It circumvents the glandular portion of the hypothalamus and may circumvent the hypophysis as well, hereby secreting one or more “hormones” directly into the vascular circulation. The molecules named oxytocin, OT, and arginine vasopressin, AVP, are unusual. They are described as arising at many locations within the organism, and these locations do not relate to the neural system. There remains a controversy as to whether OT and AVP might originate in the posterior hypophysis, as indicated by the dashed box. The posterior pituitary gland is also known as the neurohypophysis. When using this nomenclature, the anterior lobe is known as the adenohypophysis.

Figure 23.3.1-4 shows a potential expanded flow diagram related to the stage 7B neuroaffector neurons, hypothalamus-hypophysis and the target tissue. The figure can be described as introductory for two reasons,

1. It is difficult to achieve completeness in a figure of this complexity and 2. The relationships illustrated will be more clearly described in the following discussion. Hormonal-affectors 23- 19

Figure 23.3.1-4 Schematic of stage 7 neuroaffectors and their targets UPDATE xxx. The stage 7 neurons operate differently when supporting the paracrine, endocrine and exocrine systems, or sub-modalities. Within the paracrine regime, they actually secrete the hormone stimulating the target tissue. When supporting the endocrine system, they provide secretions used to create the growth hormones of the hypothalamus, that in turn stimulate the creation of a large number of hormones within the hypothalamus that are distributed to a variety of tissue types via the blood stream. The operation of the exocrine submodality involves more situations. See text.

This figure justifies providing a new name for the overall modality, the Crine modality.

There are a myriad of morphological, histological and cytological figures attempting to characterize the functions and operations occurring between the stage 7B neuroaffectors and their ultimate target tissues. There is little consistency in labeling individual functional elements among this myriad of figures. Noback provides a clear set of figures (11.1 & 11.2 in the 1967 edition) that is detailed but does not define the “magnocellular neurons found in [Figure 23.3.3- 2] from Swanson (2003). Noback does show multiple “neurosecretory granules” along the length of axons terminating in the posterior (neuro–) hypophysis. Globally, the above figure defines three major functional paths, the paracrine, the endocrine and the exocrine with all of the stage 7B neuroaffector neurons receiving their stimulation from other neurons within engines within the CNS. The paracrine path illustrates two distinct types of stage 7 neuroaffectors; those secreting nitric oxide to stimulate cytosol receptors that in turn control the tension of smooth muscle, and those secreting acetylcholine to stimulate end plates on the surface of, and subsequently control the tension of striate muscle. The endocrine path illustrates two sub-paths. The anterior hypophysis path supports multiple stage 7 neuroaffectors stimulating multiple glandular elements within the hypothalamus that in 20 Neurons & the Nervous System turn secrete multiple hormones generally labeled growth hormones or growth factors in older literature. These hormones travel via the portal to stimulate additional glandular elements within the anterior (or adeno-) hypophysis to secrete more targeted hormones that are released into the blood stream (for ductless distribution throughout the anatomy). There remains a question as to whether the hormones travel through the portal in separate “labeled” channels or in a common channel.

The posterior hypophysis path supports stage 7 neuroaffectors that are reported to have their neuritic structures and their soma in the hypothalamus but their axons traversing the portal to support their pedicles secreting at least two hormones directly, oxytocin and vasopressin. These endocrine neurons operate similarly to the paracrine neurons except their secretions are more broadly targeted and are released into the blood stream.

The portal extending from the hypothalamus to the hypophysis is structurally very complex. It forms one afferent signal path from within the blood-brain-barrier to outside of it. It primarily carries hormonal signals but is reported to carry some neural signals via long axons extending from the hypothalamus to the posterior hypophysis. Some authors indicate an arterial supply entering the portal and exiting through an anterior venous drainage, and a posterior venous drainage.

In many cases, the hypophysis appears to develop a set of pre-hormones consisting of long proteins. These proteins are cleaved at more than one point to generate shorter snippets that constitute a large number of individually identified hormones. An example of one of this set of proteins is the pre-hormone, thyroglobulin, Tg. It is a large glycoprotein (Section 23.4.3.4).

The exocrine path illustrates three distinct signal sub-paths. The first subpath is focused and ultimately deposits into the kidney or bladder for excretion along with urine. The second sub-path is much broader with individual stage 7 neuroaffector terminations located adjacent to nearly every hair follicle of the external skin. When the subject is perspiring the pheromones secreted by the neuroaffectors are mixed with the sweat. Some of the mixture is sufficiently volatile for the pheromones to travel significant distances. The third subpath is in support of the digestive system of the alimentary canal. Morphologically, the digestive system operates outside of the subjects body and is therefore an element of the exocrine modality. For purposes of this figure, the stage 7 neurons terminate in the pancreas, secreting distinctive hormones into various ducts leading to pores emptying into the digestive tract of the alimentary canal.

Alternately, the neurons shown may be of stage 6 and interfacing with a “minibrain” associated with the pancreas. This minibrain would control a wide variety of stage 7 neuroaffectors within the pancreas secreting their hormones into dedicated ducts. The pancreas also responds to one or more hormones originating in the hypophysis. The ducts of the pancreas are subject to various disease conditions that may be temporary or permanent. The author encountered what was thought to be a permanent problem with digesting long chain fats, butterfat, avocado fat and banana fat. The problem arose when in his late 20's and caused serious diarrhea when ingesting these fats. The condition abruptly disappeared 50 years later at age 82, suggesting some form of duct blockage within the pancreas as the source of the disease. The author can now eat ice cream without concern. - - - - Hormonal-affectors 23- 21 23.3.2 The anatomy and physiology of the paracrine system EDIT

Section 23.1.2 presents background material relating to the muscles involved in the paracrine interface with the neural system. The heyday of muscle research appears to have been in the 1960's, even though the capability of the available instrumentation was limited. The emphasis at that time appears to have been on fish and frog muscles. Freygang et al19. have provided some electrical and response data on frog muscles. As noted in Section 23.1.1 and Section 23.5.3, Pierrot-Deseilligny & Burke have provided a more recent summary of human striate muscle responses by recording the responses to external stimulus. Albuquerque et al20. have provided an extensive review of what has come to be known as the “nicotinic Acetylcholine receptors.” nAChR. After reviewing the 20th Century very briefly in their introduction, they assert, “Despite these numerous developments, our understanding of the contributions of specific neuronal nAChR subtypes to the many facets of physiology throughout the body remains in its infancy.” Their introduction also notes, “ Subsequent studies aided by the Torpedo electric organ, a rich source of muscle-type nicotinic receptors (nAChRs), and the discovery of α-bungarotoxin, a snake toxin that binds pseudo-irreversibly to the muscle nAChR, resulted in the muscle nAChR being the best characterized -gated hitherto.” The last assertion is far from direct evidence of the role of nAChR in the neural-striate muscle interface of mammals!

Nicotinic and muscaronic are, like ionotropic and metabatropic, are adjectives that are not precisely defined. They do not relate to any molecular entity or mechanism.

On page 74, Albuquerque et al. assert, “The fundamental functional studies of Sir Bernard Katz, Sir John C. Eccles, and Stephen Kuffler laid the groundwork for much of our current knowledge of cholinergic synaptic transmission at the neuromuscular junction” These gentlemen were all active more than 50 years ago. To say there has been little progress since that time should be embarrassing to the entire bioscience community. The review does not address striate or smooth muscle!

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The 1955 paper of Castillo & Katz21 is very carefully edited, contains considerable experimental data and includes very important information. As an example, they note the ACh is only effective when applied to the external side of the sarcolemma. “These transient changes furnish strong additional evidence for the view that a release of ACh is effective only when it occurs on the outer surface of the end-plate.” And, “The experiments have shown that very small quantities of ACh can produce a powerful depolarization of the muscle fibre if they are applied externally to the surface of the end-plate, but the same doses applied intracellularly have no effect.” These findings strongly suggest that the lemma is not porous to ACh and ACh is not effective on the inside of the lemma!! Therefore, the ACh receptor is on the exterior of the lemma, a surface receptor as expected for striate muscle. The paper did not differentiate between striate and smooth muscle! - - - -

19Freygang Jr., W. Rapoport, S. & Peachey, L. (1967) Some Relations between Changes in the Linear Electrical Properties of Striated Muscle Fibers and Changes in Ultrastructure J Gen Physiol vol 50, pp 2437–2458

20Albuquerque, E. Pereira, E. Alkondon, M. & Rogers, S. (2009) Mammalian Nicotinic Acetylcholine Receptors: From Structure to Function Physiol Rev vol 89, pp 73–120 doi:10.1152/physrev.00015.2008.

21del Castillo, J. & Katz, B. (1955) On the Localization of Acetylcholine Receptors J Physiol vol 128, pp 157-181 22 Neurons & the Nervous System

In 1972, Vogel et al22 reported they had grown ACh receptors of muscle in vitro. These receptors were from chicks and rat muscles. They did not determine the molecular chemistry of their receptors. They did note the receptors tended to cover the lemma of neonatal and embryonic muscle until neural synapses were established. They also suggested their receptors were similar to those of mammalian striate muscle. In 1979, Burden et al. performed some interesting regeneration experiments23. “Acetylcholine receptors (AChRs) on skeletal muscle fibers are selectively concentrated at the neuromuscular junction. The density of receptors in the portion of myofiber plasma membrane beneath the nerve terminal is 1,000-5,000 times greater than in extra–junctional regions.” “This paper concerns the formation of AChR accumulations at synaptic sites in regenerating adult muscle. After damage to nerve and muscle, both axons and myofibers degenerate and are phagocytized, but the Schwann cells that capped the nerve terminals and the basal lamina sheaths of the myofibers survive.” “New myofibers develop within the basal lamina sheaths of the original myofibers, axons grow to the original synaptic sites on the basal lamina, and functional synapses are formed.” In 1999, Richmond & Jorgensen24 reported “One GABA and two acetylcholine receptors function at the C. elegans neuromuscular junction.” The fact that a GABA receptor is a mislabeling of the electrostenolytic receptor where GABA is produced as a waste product is noted (Section 3.2.2). The two acetylcholine receptors were not explicitly identified at the molecular level. They did assert their receptors were ligand-gated ion channels without providing any evidence. Their studies were based primarily on mutants. They did show the potential errors in the genetic code of their mutants. “A defect in [neuroaffectation] was confirmed by directly assaying postsynaptic currents in response to nerve stimulation. We stimulated the ventral nerve cord while recording from muscles by whole-cell patch clamp.” They did show that their mutations did arise in the presynaptic axon or pedicle. “Thus, the uncoordinated phenotype of unc-13 mutants is not caused by failure of motor neurons to extend processes to muscles. Alternatively, formation of either pre- or postsynaptic elements of the neuromuscular junction might be disrupted. We determined whether neuromuscular junctions were present by visualizing clusters of synaptic vesicles tagged with green fluorescent protein, GFP.

Aamodt25 contributed a microscope picture of the C. elegans of Richmond & Jorgensen

The above papers suggest the appropriately equipped laboratory should be able to assay the AChR surface receptor of striate muscle at the molecular level.

Linstrom et al. may have avoided identifying the AChR26. They chose to identify it by its affinity for snake venom. “AChR is routinely identified and quantitated by binding of radioactively labeled krait or cobra venom toxins, which are competitive inhibitors of acetylcholine binding.” They did note the wide presence of AchR on the exterior surface of muscle cells, with a greater accumulation at the neuronal system interface, the end plate.

22Vogel, Z. Sytkowski, A. & Nirenberg, M. (1972) Acetylcholine Receptors of Muscle Grown In Vitro Proc Nat Acad Sci USA vol 69(11), pp 3180-3184

23Burden, S. Sargent, P. & McMahan, U. (1979) Acetylcholine Receptors in Regenerating Muscle Accumulate at Original Synaptic Sites in the Absence of the Nerve J Cell Biol vol 82, pp 412-425

24Richmond, J. & Jorgensen, E. (1999) One GABA and two acetylcholine receptors function at the C. elegans neuromuscular junction Nature Neurosci vol 2, pages 791–797

25Aamodt, S. (1999) Synaptic physiology in C. elegans Nature Neurosci vol 2, pages 782–790

26Lindstrom, J. Einarson, B. & Tzartos, S. (1981) Production and assay of antibodies to acetylcholine receptors Methods Enzymol vol 74, pages 432-460 Hormonal-affectors 23- 23 23.3.2.1 The physiology of the neuroaffector-smooth muscle interface EMPTY

With the recent recognition (1980–1990) of the role of nitric oxide as a hormone affecting smooth muscle, there has been little in depth laboratory investigations through 2018. It is clear that nitric oxide passes directly through the sarcolemma of smooth muscle, There is no need for a surface receptor on the sarcolemma. It appears that the nitric oxide receptor is associated with an element outside of the nucleus of the myocyte and it is therefore labeled here as a cytosol receptor. It is likely that the cytosol element is mitochondria based on the very few remarks in the literature. Role and Berg, 1996 questioned any role for nitric oxide within the mammalian CNS. 23.3.2.2 The physiology of the neuroaffector-striate muscle interface EMPTY

[Figure 23.1.2-2] shows in caricature the operation of the neural system-striate muscle interface. However, the details related to frames B & C are sorely lacking. Even the character of the acetycholine receptor remains unidentified. [Figure 23.1.2-3] illustrates the typical end plate of a striate muscle. [Figure 23.1.2-4] provides data on the tension of a striate muscle as a function of stimulation. What will be developed here is the mechanism by which the individual myofibrils within the myocyte are caused to contract.

Cully has described the operations of the striated muscle recently27,28,29. In Cully et al. (2016) the Abstract noted, “Little is known of the Ca2+-handling properties of the t-system as the small Ca2+ fluxes conducted are difficult to resolve with conventional methods. To advance knowledge in this area we calibrated t-system-trapped rhod-5N inside skinned fibres from rat.”

Launikonis, Cully et al. have also published recently30. They have provided considerable laboratory data on fast-twitch mammalian fibers and “In conclusion, this study shows that the t-system of fast-twitch mammalian fibers has (a) a relatively large proton permeability coefficient that contributes to the relatively fast change in intracellular pH in the fibers and (b) an NHE system whose activity differs markedly between individual fibers and that plays a major role in the regulation of pH under resting conditions.”

Hobson specifically defines acetylcholine, dopamine, norepinephrine and as neuromodulators rather than neurotransmitters (page 36). He notes, “Although they do not themselves convey signals about content, the neuromodulators alter the form– or mode–of processing of the content.” He provides the chemical structures of these chemicals along with the location of their natural sources within the brain. ADD

Hobson provides a series of EEG waveforms attempting to define the different states of human sleep stages by associating them with different characteristic waveforms (page 51).

Hobson defines an “emotional salience” (page 59) but does not develop it sufficiently to relate it to the overall saliency map of stage 4. His four principles of the conscious and unconscious (potentially nonconscious in this work)

27Cully, T. (2017) Milestones and frontiers in muscular physiology: Ca2+ handling in T-tubules Iatreia vol 31(1-S)

28Cully T, Edwards J, Murphy R, Launikonis B. (2016) A quantitative description of tubular system Ca(2+) handling in fast- and slow-twitch muscle fibres. J Physiol vol ;594, pp 2795-2810

29Cully T, Murphy R, Roberts L, et al. (2017) Human skeletal muscle plasmalemma alters its structure to change its Ca2+-handling following heavy-load resistance exercise. Nat Commun vol 8, pg 14266.

30Launikonis, B. Cully, T. Csernoch, L. & Stephenson, G. 20187) NHE- and diffusion-dependent proton fluxes across the tubular system membranes of fast-twitch muscle fibers of the rat J Gen Physiol vol 150(1), pp 95–110 https://doi.org/10.1085/jgp.201711891 24 Neurons & the Nervous System associated with a bottom-up approach to neurodynamic psychology are interesting. “1. Most of the information in the brain-mind is unconscious (nonconscious) and had better remain so. 2. Consciousness contains a very small part of the information in the brain-mind, but it is critical because it is the only channel by which the unconscious (nonconscious) can be noticed and voluntarily influenced, and the only portal by which the more complex communications of language can leave or enter the system. 3. The dynamic relationship of the conscious mind to the unconscious (nonconscious) is the nub of the contras between the psychoanalytic model and the neurodynamic model. 4. Neurodynamics recognized and respects the power of the unconscious (nonconscious) but is rather more grateful than fearful that so many biologically critical bookkeeping task are being taken care of automatically.” He notes, “I fully realize that all of these points are highly debatable and very much in deen of more detailed elaboration.”

Hobson also introduces a “state-space concept” or graphic but only semi-quantitative presentation to define the input, mode and activation associated with pharmaceutical intervention. He uses the acronym, AIM, for his presentation.

Fuster has discussed the neuroaffectors of the paracrine portion of the CNS (page 66) using a graphic from Arnsten &Robbins. He also defines a broader, but not exclusive, range neuroaffectors than Figure [23.3.1-1]. 23.3.3 The anatomy and physiology of the endocrine system

The endocrine subsystem consists of six major glands: 1. The hypophysis (pituitary) gland. 2. The thyroid gland. (T3 ~ 40 micrograms/day, T4 ~ 80 micorgrams/day) 3. The parathyroid glands. 4. The adrenal glands. 5. The gonads. 6. The islets of Langerhans in the pancreas.

The kidneys are frequently considered a gland of the semi-endocrine type. The interior of the kidneys is extensively ducted. However, its input and output are directly with the vascular circulation.

As discussed below, all of these glands are found outside the BBB of the CNS. This is not to say there are not other glandular material providing hormonal support within the CNS.

The thyroid and parathyroid glands generally stimulate biological activity in a wide variety of tissues. resulting in increase O2 consumption. The adrenal glands, adrenal cortex and adrenal medulla, located above the kidneys are complex. They appear to secrete both neuromodulators (affecting other neural tissue) and neuro-affectors (affecting non-neural tissue). The adrenal medulla can be surgically removed and the subject can survive (suggesting its role is largely that of a neuromodulator (producing epinephrine and norepinephrine (Ganong pg 269). The adrenal cortex is critical to life. It appears to produce a variety of steroids critical to the metabolism of carbohydrates and proteins. The hypophysis is frequently characterized as the master gland. It produces at least 6-10 hormones (Ganong, page 167-168 & 298). Many of these hormones are considered hormone–generating– hormones as they appear to cause the release of other hormones by the peripheral glands. These specialized hormones were previously labeled factors, but are generally labeled stimulating hormones, SH (such as thyroid stimulating hormone, TSH). If the digestive juices produced by the pancreas are considered hormones due to their Hormonal-affectors 23- 25 enzymatic structures, the pancreas produces a long list of hormones (Section 23.10). Section 3.5.6 also addresses the pharmacological aspects of hormones. 23.3.3.1 The unique physical position of the hypophysis

The transition between the neural system and the endocrine system is formed by the morphologically defined combination of the hypothalamus and the hypophysis (the pituitary). The hypophysis is the classic example of a neurosecretory structure. It receives instructions from the hypothalamus and releases vasopressin and oxytocin along with a variety of other hormones.

Discussions of the hypothalamus are frequently unclear as to whether this structure releases any hormones. Some discussions consider the output of the hypothalamus entirely neural while others suggest it generates “releasing hormones controlling the stimulating hormones” (particularly directed toward the hypophysis). There are indications that the hypothalamus does act as a pericrine gland within the brain environment and releases dopamine, a major mood controlling hormone (Ganong, pg 159). The hypothalamus will be shown to be a pericrine gland in that its hormonal outputs travels to the hypophysis (pituitary) gland via a “portal” rather than via the vascular circulation. The portal is a form of duct conveying both hypothalamus generated hormones and stage 7 axons. Hobson has provided useful information on the hormonal system of the CNS (within the blood- brain-barrier). His Figure 23.3.3-1 focuses on the brainstem and glands associated anatomically with the thalamus but then focuses on the CNS31. While the title might suggest a popular book, it contains much material of an academic character.

The heavy black line has been added to emphasize the hypophysis is outside of the BBB. The precise location of the barrier was not completely resolved during the 1970's. As Lal & Rastogi note, “Part of the hypothalamus, the median eminence, lies outside the blood-brain-barrier, BBB, where it is in close proximity to capillaries of the pituitary portal vasculature. No barrier exists between the portal blood vessels and the substance of the median eminence.” See Section 23.3.3.1.1.

31Hobson, J. (2001) The Dream Drugstore. Cambridge, MA: MIT Ptess 26 Neurons & the Nervous System

Figure 23.3.3-1 Sagittal view of the left human hemisphere focused on the brainstem. Note the dorsal raphe, the locus coeruleus, the brainstem cholinergic nuclei, the pituitary gland and the hypothalamus. A heavy line has been added to represent the BBB and show the pituitary is outside of that barrier. See text. From Hobson, 2001.

Hormonal-affectors 23- 27 Figure 23.3.3-2 provides a clear division between the dual endocrine roles of the system32. The magno-neuron path of the hypothalamus directs the release of vasopressin (VAS) and Oxytocin (OXY) in to the artero-vascular circulation. It may consist of stage 7 neurons with their soma within the hypothalamus but their axons and secreting pedicles extending into the posterior hypophysis (pituitary). See Section 23. 4.2.4. The parvi-neuron path directs the release of a wide variety of hormones in to the circulation. These hormones in turn control the release of other hormones by subsidiary glands. Note the feedback loop formed by the estrogen, testosterone and cortisone hormones.

Figure 23.3.3-2 The two divisions of the neuro-endocrine system in this parasagittal view of the hypothalamus (left of the isthmus) and the anterior lobe (A) of the hypophysis (pituitary gland) of the rat. I; intermediate lobe. P; posterior lobe of hypophysis. A; adrenal cortex. ACTH; adrenocorticotropic hormone. CORT; corticosterone/cortisol. E; estrogen. FSH; follicle- stimulating hormone. G; gonads. GH; growth hormone. LH; luteinizing hormone. PRL; prolactin. T; testosterone. TH; thyroid gland. TSH; thyroid-stimulating hormone. T3/T4; thyroid hormones. A blood-brain-barrier (BBB) has been added to emphasize the hypophysis is outside of this barrier. See text. From Swanson, 2003.

The prefixes magno- and parvi- refer to the size of the neurosecretory cells within the hypothalamus/hypophysis. Swanson has noted the large size and high metabolism of the magno-neurons due to their high production of the neuro-affectors, VAS and OXY (page 127). While the Swanson figure suggests that most signals between the hypothalamus and the hypophysis are neural in character, most author suggest the hypothalamus is a source of a type of chemical hormones, known as tropic hormones or “tropins.”

32Swanson, L. (2003) Brain Architecture: Understanding the Basic Plan. NY: Oxford Univ Press page 127 28 Neurons & the Nervous System

An interesting feature of the hormonal system is the interface between the hypothalamus and the hypophysis (pituitary gland). Contrary to the view suggested by the dashed lines in the Swanson figure that most signals between the hypothalamus and the hypophysis are neural in character, most author suggest the hypothalamus is a source of a type of chemical hormones, known as tropic hormones or “tropins.”, The hypothalamus secretes a wide variety of hormones destined for the pituitary gland via a portal (not the general cardiovascular pathway). In this sense, the portal qualifies as a “duct “while the hormones secreted by the pituitary are distributed via the “ductless” cardiovascular system. The tropic hormones of the hypothalamus are designed to control the secretion of other hormones by an intermediate gland rather than being directed to an end user. This portal appears to contain a series of parallel channels acting like labeled glandular pathways (in analogy to the labeled neural pathways of the neural system). The posterior lobe of the hypophysis (also known as the neurohypophysis) is functionally different from the anterior lobe (also known as the adenohypophysis). Both the prefixes, neuro- and adeno-, are poorly supported from a functional perspective.. Among other elements, the posterior lobe contains the pedicles of stage 7 neurons while their soma remain in the hypothalamus. Some of the literature suggests these pedicles secrete either oxytocin (OXY, or OT) or vasopressin (VAS, also known as arginine vasopressin, AVP). These are very complex organics. There may be an intermediary gland between the secretions from the pedicles and these hormones. In the absence of such an intermediate, the list of secretions from stage 7 neurons will need to be expanded. Arginine is a known chemical in the pedicles of stage 7 neurons. Bichet (page 212) has offered a method of creating and delivering these two hormones.

[xxx may want to use my version here to avoid duplication ] Figure 23.3.3-3 from Labrie et al. provides a graphic showing this alternative33.

33Labrie,F. Drouin, J. De Lean, A. et al. (1976) In Levey, G. ed. Hormone-receptor interaction: molecular aspects. Volume 9 of Modern Pharmacology-Toxicology NY: Marcel Dekker Hormonal-affectors 23- 29

Figure 23.3.3-3 Schematic of the hypothalamohypophysis complex. The letter abbreviations follow the above figure, with some exceptions. The upper row of labels represent tropins. The arrows and polarities are not reviewed here. Refer to the original work. The hypophysis appears to produce one or more protohormones that are used to produce the labeled hormones shown. From Labrie et al., 1976

The Labrie et al. graphic shows that the pituitary gland both receives (via the portal) and secretes (into the vascular circulation) a wide variety of hormones. See Section 23.10 for identification of these hormones.

At least three of the hormones released by the hypophysis (pituitary gland) affect other glands directly, causing the release of additional hormones. ACTH (adrenocorticotropic hormone) affects the adrenal cortex, contributing to the release of CORT (corticosterone/cortisol). TSH (thyroid-stimulating hormone) affects the thyroid, contributing to the release of T3 and T4 (both thyroid hormones). FSH (follicle-stimulating hormone) and LH (luteinizing hormone) both appear to affect the gonads, contributing to the release of E (estrogen) and T (testosterone).

E, T and CORT are known to affect the CNS, thus closing a feedback loop that began with the CNS causing the release of the initial hormones by the hypophysis.

The central and posterior portions of the hypophysis are known to generate an entirely separate family of hormones. Many of the hormones labeled as -stimulating hormones, such as ACTH, a-MSH, ß-MSH, and γ-MSH are derived by proteolysis (cleavage) of a single precursor peptide, proopiomelanocortin, POMC. https://en.wikipedia.org/wiki/Melanocyte-stimulating_hormone includes a table showing these points of cleavage. The text on that page, as of 2018, is inconsistent as to whether some of these hormones are created in the hypothalamus or hypophysis (pituitary). These inconsistencies frequently relate to the rapid pace of laboratory investigations and changing terminology between the 1960's and now.

MSH, melanocyte-stimulating hormone, affects the pigmentation of the skin. It will not be 30 Neurons & the Nervous System addressed further in this work. The different forms of MSH belong to a group called the melanocortins. This group includes ACTH, a-MSH, ß-MSH, and ?-MSH; these peptides are all cleavage products of a large precursor peptide called (POMC). a-MSH is the most important melanocortin for pigmentation.

23.3.3.1.1 Hypophysis & part of the hypothalamus are outside the BBB

The generalization in the literature that places the hypophysis within the brain and therefore on the brain-side of the blood-brain-barrier (BBB) requires discussion. The descriptions in the previous paragraph provide the first functional reason why the hypophysis is located on a stalk clearly separated from the hypothalamus and the rest of the brain. As shown in Noback (page 169), and also stated clearly in Ganong, the hypophysis is located on the underside of the brain on a stalk that is physically outside of the blood-brain-barrier formed by the arachnoid arterial system. The hypophysis is serviced by the anterior hypophysial artery from the carotid artery and the circle of Willis. This arrangement bypasses the question of how hormones pass through the BBB from the hypophysis. The more recent text by Seeley, Stephens & Tate shows similar, but less detailed, graphics in color34. For a more detailed graphic and discussion, see the Ciba Collection of Medical Illustrations, Vol 4, Endocrine System, Section 1, Plate 9. The graphic found there is reproduced in the 3rd Edition of Werner & Ingbar as figure 12-1.

Lal & Rastogi, writing chapter 8 in Hrdina & Singhal35 have discussed the straddling of the BBB by the hypothalamus with the result that most of the hormonal activity of the hypothalamus and all the activity of the hypophysis, occurs outside of the BBB. 23.3.3.2 The configuration of the neuroendocrine signal paths

Martin & Reichlin have provided a caricature of the stage 7 circuits of the neuroendocrine system36. They did not develop the physiology of the neurons in these circuits. They also used a figure from Krieger to illustrate the differences between autocrine, paracrine and endocrine cells.

[xxx expand and provide more physiology here. No need for stage 3 going to hypophysis ] Renaud37 (page 630) has described the “tuberoinfundibular” neurons as parvicellular hypothalamic neurons whose fine unmyelinated axons terminated on median eminence capillaries of the pituitary portal circulation. The axons of these neurons are known to divide into short branches near termination sites, pedicles, on capillaries.

The row of termination (pedicles) sites along the external surfaces of the median eminence are frequently described as varicosities associated with the overall neuroaffector neuron. 23.3.3.3 Background on oxytocin & arginine vasopressin

Bichet (2005), writing chapter 14 in Melmed & Conn, presents a complex role and flow diagram for these two hormones. His text is sometimes difficult to interpret, and his graphics (fig.2. frames A and B) are difficult to compare. The frames were apparently generated by using different

34Seeley, R. Stephens, T. & Tate, P. (2006) Anatomy & Physiology, 7th Ed. NY: McGraw-Hill pg 449

35Lal, S. & Rastogi, R. (1981) Effect of neuroleptics on pituitary function in man In Hrdina, P. & Singhal, R. eds. Neuroendocrine regulation and altered behavior. London: Croom Helm

36Martin, J. & Reichlin, S. (1987) Clinical Neuroendocrinology, 2nd Ed. Philadelphia, PA: Davis

37Renaud, L. (1978) Neurophysiology and neuropharmacology of medial hypothalamic neurons and their extrahypothalamic connections In Morgane, P. & Panksepp, J. eds. (1979) Anatomy of the hypothalamus, vol 1 of Handbook of the Hypothalamus. NY: Marcel Dekker Chapter 9 Hormonal-affectors 23- 31 visualizer software packages employing molecular stereographic description files from different sources. In fact, the state of the art in these two types of software packages is quite primitive. It is virtually impossible to compare molecules of this complexity using black and white printing. A better rendition of Fig. 2 appears in color in chapter 8 by Bichet in “The Pituitary38.” Figure 23.3.3-4 provides a better rendition of these two frames. F3 (phenylalanine is replaced by I3 (isoleucine and R8 ( is replaced by L8 (Leucine).

Figure 23.3.3-4 Comparison of OT and AVP by Bichet . “The peptides differ only by two amino acids (F3–>I3 and R8–>L8 in AVP and OT.” See text. From Bichet, 2017 and replacing Bichet, 2005.

Bichet labels the two molecules as both hormones and polypeptides in the same sentence. Referring to [Figure 23.3.3-1], “oxytocin, OT, and arginine vasopressin, AVP are synthesized in separate populations of magnocellular neurons (cell body diameter of 20–40 microns).” This pathway leads to the posterior pituitary. “Another pathway from parvocellular neuron (cell body diameter of 10-15 micron) to the hypophysial portal system transports high concentration of AVP to the anterior pituitary gland. AVP produced by this pathway and the corticotrpoin-releasing hormone , CRH, are two major hypothalamic secretagogues regulation the secretion of adrenocorticopropic hormone, ACTH, by the anterior pituitary.”

“OT is most recognized for its key role in parturition and milk letdown in mammals.” “Disorders of water balance are a common feature of clinical practice. An understanding of the physiology and pathophysiology of vasopressin(AVP), perception of thirst, vasopressin receptors, and aquaporin water channels is key to the diagnosis and management of these disorders. “

38Melmed, S. (2017)The Pituitary, 4th ED. NY: Elsevier https://doi.org/10.1016/B978-0-12-804169-7.00008-8 32 Neurons & the Nervous System

Witter has described the sequence of amino acids for these two peptides in Figure 23.3.3-5 . The sequence differences noted by Bichet are easier to identify in this figure. LVP and AVT are other variants of this family. LVP appears only in the family Suidae and the hippopotamus. AVT appears in all major vertebrate groups in addition to AVP.

23.3.3.3.1 Potential formation of AVP–by Bichet

On page 212, Bichet describes a unique role for the production of AVP. “AVP and its corresponding carrier protein, neurophysin II, are synthesized as a composite precursor by the magnocellular and parvocellular neurons described previously. In contrast to conventional neurotransmitters (e.g., acetylcholine, excitatory and inhibitory amino acids, monoamines,), which are synthesized in nerve terminals and can be packaged locally in recycled small vesicular membranes, the biosynthesis and secretion of neuropeptides such as OT and AVP in the Figure 23.3.3-5 Family of OT & AVP hormones. LVP hypothalamo-neurohypophysial system and AVT are only found in a very few families and requires continual de novo transcription and species of animals. See text. From Witter & de translation of peptide precursor proteins.” Wied. 2000. This quotation has an impact on how stage 7 neuroaffectors are categorized in this work (Section 23.1.1 and Section 23.2). It leaves unsaid what material is secreted by the pedicle of the neurons. It is possible these neurons projecting to the anterior hypophysis should be classified as stage 6 neurons termination in a separate stage 7 neuron within the hypophysis and secreting AVP. Bichet addresses this situation more clearly in his 2017 variant that follows.

Bichet (2017) provides a clearer and more advanced text in a different volume by Melmed39. “The hypothalamus, a small, one cubic centimeter structure in humans, is located at the anterior and ventral part of the thalamus and embodies a group of nuclei that form the floor and ventrolateral walls of the triangular-shaped third ventricle. The neurohypophysis consists of:

(1) a set of hypothalamic nuclei, namely the supraoptic nuclei (SONs) and paraventricular nuclei (PVNs), which house the perikarya of the magnocellular neurons; (2) axonal processes of the magnocellular neurons form the supraoptical hypophyseal tract; and (3) the neurosecretory material of these neurons which is transported to the posterior pituitary gland. His figure 8.4 provides detailed information about the osmoregulatory circuits in the mammalian brain. “Oxytocin and AVP (his Fig. 8.6) are synthesized in different populations of magnocellular neurons from the supraoptic, paraventricular, and accessory nuclei. During evolution, secretion of these peptides in the cerebrospinal fluid (CSF) has been replaced by a vascular secretion. In advanced vertebrates, the axonal projections of these neurons are toward the posterior pituitary and also to forebrain, telencephalon, and diencephalon regions, probably explaining the behavioral effects of oxytocin and vasopressin.”

39Melmed, S. (2017) The Pituitary. NY: Elsevier chapter 8. Hormonal-affectors 23- 33 His new Fig 8.6 replaces figure 2 in his (2005) chapter and has been adopted above. It is in color and of better quality for comparing AVP with OT. “AVP and its corresponding carrier protein, neurophysin II, are synthesized as a composite precursor by the magnocellular and parvocellular neurons described previously. The precursor is packaged into neurosecretory granules and transported axonally in the stalk of the posterior pituitary (Fig. 8.7) [25]. On route to the neurohypophysis, the precursor is processed into the active hormone (his Fig. 8.7). Preprovasopressin has 164 amino acids and is encoded by the 2.5-kb AVP gene located in chromosome region 20p13 [26]. The AVP gene (coding for AVP and neurophysin II) and the OXT gene (coding for oxytocin and neurophysin I) are located in the same chromosome region, at a very short distance from each other (12 kb in humans) in a head-to-head orientation.” 34 Neurons & the Nervous System

Figure 23.3.3-6 reproduces Bichet’s figure 3 of 2005 with some detail added based on his 2017 text. A problem quickly arises with Bichet basing his analysis on a 2-terminal neuron based on the chemical theory of the neuron. This basis is emphasized in his figure 8.7 of 2017. His baseline assumes “A dendrite with release of dense core vesicles containing vasopressin is represented in the left upper part of the figure. In the central part, a microtubule is tracting a dense core vesicle along the very long axon.”

Figure 23.3.3-6 Potential mechanism of vasopressin production. The 2017 variant shows only one “dendrite” at upper left. It displays a group of vesicles containing vasopressin. The portal region, between the hypothalamus on the left and hypophysis on the right, has been extended. The inset in the original is shown for completeness. See text. The reader is referred to Bichet (2005) for a discussion of the inset. The details are not supported in this work. Modified from Bichet, 2005.

“Neurophysins were first thought to be carrier proteins for vasopressin and oxytocin. It is now recognized that NPI (for oxytocin) and NPII (for vasopressin) belong to the precursor of the respective hormone (see above section on the vasopressin and the oxytocin gene). After synthesis in the hypothalamic neurons, the vasopressin precursor migrates along the neuronal axons, many of which terminate in the posterior pituitary. The time from synthesis to release of the hormone into the systemic circulation is about 1.5 hours.” This assertion is clearer than in the 2005 version. However, it remains unclear where all of the steps in precursor hormone secretion Hormonal-affectors 23- 35 and actual hormone secretion takes place. It may be the stage 7 affector neuron axon need not be extended into the posterior hypophysis. “Both cleaved and uncleaved precursors [63] are present in neurosecretory granules of the posterior pituitary. Only a small percentage of synthesized peptide is released; some vasopressin-containing neurosecretory granules move away from nerve endings and are unavailable for release. Once secreted into the circulation, vasopressin is accompanied, but not bound, by its specific neurophysin. Neurophysins themselves do not appear to have biological activity.” “The plasma half-life of vasopressin is short, being about 5- 15 minutes. Clearance is independent of plasma vasopressin concentration as it involves a liver and kidney-dependent process.” Bichet describes the receptors associated with AVP and OT in the context of coupling to G- protein coupled receptors (GPCR) without explaining in detail the operation of this circuitry. Their description is not supported in this work. See Section 3.2.4. Bichet proceeds to discuss the medical and clinical aspects related to AVP and OT that are not of interest in this work. Oxytocin

Molecular FormulaC43H66N12O12S2 Average mass1007.187 Da Monoisotopic mass1006.436462 Da ChemSpider ID388434

Vasopressin

Molecular FormulaC46H65N15O12S2 Average mass1084.232 Da Monoisotopic mass1083.437866 Da ChemSpider ID559126

23.3.3.3.2 Critique of Bichet mechanism for AVP production

Based on The Electrolytic Theory of the Neuron,

1. Any neuron is a 3-terminal active device (an Activa), except for stage 1 neurons that exhibit more than three terminals due to their containing more than one Activa, 2. The Activa is formed by a tight junction between the dendrolemma and the axolemma with the podaplasm having electrical access to the contents of the tight junction (liquid crystalline water). 3. The tight junction is formed between the outer bilayers of two regions of type 2 lemma adjacent to each other. 4. The transport of any molecules, or vesicles, through the type 2 lemma of the dendrite and/or axon is highly unlikely and such transport must be justified by Bichet. xxx ADD ] Figure 23.3.3-7 shows an alternate mechanism leading to the secretion of AVP into the hypophysis space.

23.3.3.4 Endocrinolgy of aging

Lamberts40 has addressed ageing in the context of the endocrine system in 2005. The Introduction focuses on the medical observations of aging. It then addresses the changes in

40Lamberts, S. (2005) Endocrinology of aging in Melmed, S. & Conn, P. eds Endocrinology, 2nd Edition Totowa, NJ: Humana Press chapter 28 36 Neurons & the Nervous System hormone activity with aging. He notes, “the two clinically most prevalent and important changes in endocrine activity during aging that represent pathology (i.e., diseases) are in glucose tolerance and thyroid function.” He also notes when discussing physical capacity, “‘use it or lose it;’ activity is an extremely important treatment for aging.” The first few pages are important for senior citizens to read but are primarily qualitative.

23.3.4 The anatomy and physiology of the exocrine subsystem MAY DUPLICATE

The physical configuration of the exocrine system is more complex than the paracrine and endocrine subsystems. One major portion of the subsystem is shared between at least one external animal source and a set of internal sensory receptors forming the stage 1 signal generators of the oskonatory sensory modallity of the subject (8.6.11). At the same time, the subject animal may be secreting a set of hormones with the purpose of communicating with the stage 1 sensory neurons of the oskonatory modality of the other animal. These hormones are generally secreted through the pores of the skin (hair follicles) or in the urine. A second portion of the subsystem is intimately involved with the digestive system as presented in this work. It involves hormones secreted from the pancreas and delivered through a duct system to the digestive tract. Chemically, these secreted pepsins and lipases quallify as hormones even though they do not target chemical receptors within the animal subject.

Section 23.9 provides more details. 23.3.5 Terminology EDIT

Phosphatidyl amino acids– The primary receptors, both neural and glandular, within the mammalian physiology under the Electrolytic Theory of the Neuron. The following receptors are related to the glandular modality. The phosphatidyl amino acid fundamental to the electrostenolytic process powering all biological cells, including the neurons will be found in Section 3.2. Those specific to the gustatory modality will be found in Section 8.5. Those specific to the gustatory modality will be found in Section 8.6.

Phosphatidylcysteine, PtdCys– Proposed glandular receptor for triiodothyronin, T3 under The Electrolytic Theory of the Neuron. d-value = 3.219 Angstrom based on L-cysteine_5653..

Phosphatidylproline, PtdPro– Proposed glandular receptor for triiodothyronin, T3, and quadiodothyronine, T4, both under The Electrolytic Theory of the Neuron. d-value = 2.749 Angstrom based on L-proline_128566. Found principally within the hypothalamus and hypophysis (pituitary) but also anywhere the concentration of both T3 and T4 is sensed.

Sinusoid– (s-n(y)-sid) A minute endothelium-lined space or passage for blood in the tissues of an organ (as the liver). thyrotropin-releasing hormone, TRH, under The Electrolytic Theory of the Neuron, a hormone produced by the hypothalamus and passed to the hypophysis along the portal between them. Endothelium– (en-d-th-l-m) An epithelium of mesoblastic origin composed of a single layer of thin flattened cells that lines internal body cavities (as the serous cavities or the interior of the heart) 23.4 Neuro-affectors releasing endocrine hormones–Global perspective

This section is an extension and expansion of the discussion in Section 23.3.2 and Section 23.3.3. The stage 7 neuroaffectors providing efferent signals to the glandular system are grouped into the hypothalamus. The output signals from the hypothalamus are chemical in character and largely directed at the hypophysis, a.k.a. the pituitary gland. The hypothalamus secretes substances known as neurohormones that start and stop the secretion of pituitary hormones. The hypothalamus may also provide some neural (electrical) signals to the pituitary gland. In a sense, the hypothalamus is the primary or most important gland of the glandular system. Hormonal-affectors 23- 37 However, in a sense, the hypothalamus is only the primary command center of the glandular system and the pituitary is the most functionally important gland because of the range of hormones it releases and thereby the number of body functions it controls, or at least influences. There are three commonly recognized sources of hormones of the Crine modality, those originating in, 1. The hypothalamus, 2. The hypophysis (pituitary and 3. The glands of the peripheral Crine system. The hypothalamus is the principle neural/glandular interface, although some of the neural soma of the hypothalamus support hormone producing pedicles in the hypophysis (pituitary). The pituitary gland is divided into two major parts, the anterior and posterior parts, based on their distinctive roles. These parts support the following glandular foci as presented by Stacy & Santolucito using the language of their day41. A. Posterior pituitary gland 1. Antidiuretic hormone (ADH) 2. Corticotropin releasing hormone (CRH) 2. Vasopressin 3. Oxytocin

B. Anterior pituitary gland 1. Growth hormone (somatotrophic) 2. Diabeogenic hormone 3. Thyrotrophic hormone (TSH) 4. Adrenocorticotrophic hormone (ACTH) 5. Gonadotrophic hormones (FSH, LH, LtH) 6 Prolactin (PRL)

Stacy & Santolucito have noted that the antidiuretic hormone, ADH, may actually originate in the stage 7 neuro-affectors of the hypothalamus. When supporting the neural/musulature interface, stage 7 neurons typically secrete NO and acetylcholine the classic NANC molecules,, Section 23.5.3.2) but it is clear the stage 7 neurons support the formation of many other hormones within the hypothalamus/hypophysis. The NANC designation (nonadrenergic/noncholinergic) is archaic; the noncholinergic designation does not refer to acetylcholine but to the catacholamines ]

The prolactin inhibiting hormone (PIH) is also known as dopamine(DA)

Melmed has provided a 2017 edition of “The Pituitary” that appears to be quite up to date42. Chapter 1, by Drouin, describes the morphology and general operation of the pituitary in considerable detail. Drouin discusses each of the trophs in detail, see Section 23.4. Chapter 8 by Bichet is discussed in Section23.4.2.4. There are 24 chapters spanning both academic and clinical aspects of the pituitary. A significant portion of the hormones associated with the anterior pituitary are steroid-based hormones; FSH, LH, LtH & ACTH, according to Henley et al. They are not peptide-based; whereas the neuro-hormones. produced in the hypothalamus are believed to be all peptide-based according to Knigge et al. Figure 23.4.1-1 shows the major hormones released by the pituitary gland, as noted above, the

41Stacy,R. & Santolucito J. (1966) Modern college physiology, St Louis, MO: C. V. Mosby

42Melmed, S. ed. (2017) The Pituitary, 4th Ed. Online NY: Elsevier 38 Neurons & the Nervous System primary functional neurosecretory structure, and their targets43. As noted in Section 23.3.2 and Section 23.3.3, the morphological label hypophysis is frequently used interchangeably with the physiological label pituitary. An important fact to note is that most hormones are not protein- based. Many or cholesterol-based and NO and acetylcholine are clearly not protein-based. This major section will focus on the hypothalamus-pituitary-epinephrine axis, H-P-E axis (Section 23.4.2), and the hypothalamus-pituitary-thyroxine axis, H-P-T axis (Section 23.4.3), of the glandular system. Henley et al. (page 58) have addressed the hypothalamus-pituitary-gonad, H-P-G, axis. To economize on space, the H-P-T axis associated with the thyroid system will be treated as an exemplar of the glandular system interfacing with the hypothalamus.

43Carpenter, M. & Sutin, J. (1983) Human Neuroanatomy, 8th Ed. London: Williams & Wilkins pg 570 Hormonal-affectors 23- 39

Figure 23.4.1-1 Schematic diagram of the target organs of the pituitary hormones. Note the “portal” capillaries between the hypothalamus and the pituitary. Hormones secreted by cells in the anterior and intermediate lobes of the pituitary are regulated by hypothalamic hypophysiotrophic peptides conveyed by the hypophysial portal system. The posterior lobe of the pituitary contains vasopressin and oxytocin secreted by cells of the supraoptic and paraventricular nuclei of the hypothalamus. The figure should be expanded to distinguish between the hormones built from short peptides, proteins and steroids. See text. Modified from Carpenter & Sutin, 1983 who cited Schally of the late 1970's.

23.4.1 Background relative to the hormonal system

Our understanding of the operations of the hormonal system, from the hypothalamus to the tissues of the body as consumers of the hormones, have changed dramatically with the end of the 20th Century. The change is so significant that papers published during the 20th Century can only be used as data sources. The concepts presented in these paper must be rejected except, 40 Neurons & the Nervous System

C for their reinterpretation in the context of more recent laboratory findings, C Acceptance of the Electrolytic Theory of the Neuron (replacing the archaic chemical theory) and C the recognition that coordinate chemistry (not valence chemistry) is the foundation of the hormonal, as well as of the neural, system. The term glycoproteins is frequently used when discussing the hormonal system, often with less than precision. Molecules are frequently labeled glycoproteins when they contain neither a glycol group or a protein group. A glycol group is defined by a non-conjugated carbon atom in a heterocyclic ring structure accompanied by a hydroxyl group. A protein is defined as a chain of at least six peptide residues. While six is an arbitrary number, less than six, but more than one residues, is more appropriately labeled a peptide. Only one stand alone residue isn’t a residue at all; it is a complete amino acid (or in the case of proline, an α-imino acid).

As an example, the active species of the thyroid hormone, T3, contains two conjugated homocyclic carbon rings connected by an ether oxygen but only exhibits one glycol group, whereas two glycol-based heterocyclic rings would normally incorporate at least four hydroxyl groups distributed among the carbon atoms of the rings. The single amino acid in the only aliphatic side chain continues to exhibit both an NH2 group and a COOH group when shown in non-polar electrical form.

There are a large cohort of hormones that are not based on protein chemistry at all. The steroid hormones are lipophilic derivatives of cholesterol. Cholesterol contains no peptide residues (Section 23.4.1.3).

Cooper addressed the state-of-the-art in the operation of the thyroid glandular system in 200344. His position is compatible with the above position. It is difficult to assemble a group of hypothyroid patients when it is not known whether they are suffering from the same or independent forms of hypothyroidism. Cooper cited the concurrent studies of Clyde et al45. Clyde et al. concluded “Compared with levothyroxine alone, treatment of primary hypothyroidism with combination levothyroxine,T4, plus liothyronine, T3, demonstrated no beneficial changes in body weight, serum lipid levels, hypothyroid symptoms as measured by a HRQL (Health-related Quality-of-Life) questionnaire, and standard measures of cognitive performance.” They provide a box, describing an extensive list of symptoms from which the patient could choose to describe their condition.

Smith provides a very early overview to the nucleotide base coding and amino acid replacements in proteins46. His citations 2 through 7, all PNAS articles available over the internet, provide early dissertations on the character of the genetic code. He notes, “The code appears to be universal, that is, it is the same in all species.” Smith points out his analysis was based on limited information. Publication of his analysis was premature. It is a classic case of performing what can be labeled a “Bayesian analysis” without employing a wide enough factual foundation. This leads to, among other errors, publishing that glutamic acid and aspartic acid share the same genetic code. In 1992, Osawa et al. presented an extensive review of the state- of-the-art relative to the genetic code. It took pains to show not only a much more expansive genetic code, with many corollaries associated with each axiomatic statement, but also that

44Cooper, D. (2003) Combined T4 and T3 therapy—Back to the Drawing board JAMA vol 290(22), pp 3002- 3004

45Clyde, P. Harari, A. Getka, E. & Shakir, K. (2003) Comboned levothyroxine plus liothyronine compared with levothroxine alone in primary hypothyroidism JAMA vol 290(22), pp 2952-2958

46Smith, E. (1962) Nucleotide Base Coding and Amino Acid Replacements in Proteins PNAS USA vol 18, pp 677-684 Hormonal-affectors 23- 41 the previous claim of code universality had been falsified47. The changes in Osawa were so major than any published articles prior to 1990 should not be referenced in any serious scientific article. Osawa et al. do publish a much more complete “baseline genetic code” accompanied by all of the footnotes up to that time. By 2016, an additional very significant change in understanding had occurred48. The specification of a specific peptide sequence is clearly a very complex determinant of the material of Osawa et al. and any more recent modifications from Keeling. The task is not to be undertaken by those who lack stamina. 23.4.1.1 Tyrosine & tryptophan as progenitors of many hormones

The organism employs a variety of simple hormones to control the activity level of the overall CNS and to influence the release of a wide range of more complex hormones. These initial hormones tend to be formed from only a few simple amino acids, particularly tyrosine and tryptophan. The hormones are frequently formed by decarboxylation of the amino acid and the addition of one or more hydroxyl groups at very specific locations relative to the aromatic ring. The resulting chemicals are not peptides in the technical sense. They are not readily incorporated into a peptide chain. Hence, they are used as neuromodulators as they stand. The thyroxines are examples of hormones derived from tyrosine. An exception to the above paragraph appears to be the stimulating hormones, commonly labeled tropins in more recent literature, such as the thyroid-stimulating hormone, TSH, and the adrenal corticotropin hormone, ACTH. Others in this class are shown in the above figure, FH, LH, MSH & GH. These hormones are secreted by the hypophysis (pituitary gland) are of much greater molecular weight, but are steroid hormones based on cholesterol and are not protein-based.

23.4.1.2 The origins of the catecholamine-based hormones

Hokfelt et al, writing in Reichlin et al. have provided some interesting early material on the location of the catecholines within the hypothalamus based on the presence of closely associated enzymes of these materials. Figure 23.4.1-2 reproduces their mapping for several hormones. Each frame reports the presence of two materials, where an individual material is located symmetrically around the central axis. The original caption reads, “Some substances (DA, LHRH, SOM, TRH, ENK, ANG, and GAS) are present mainly in the external layer and thus potentially may be released into the portal vessels, whereas others (NA, A, PROL and SP) are found mainly in the internal layer DA; dopamine. NA; noradrenaline (norepinephrine). A; adrenaline (epinephrine).” For further definitions, see other figures and text from Reichlin et al. below. The implication was that these catecholamines are produced within the pedicle of neurons.

47Osawa, S. Jukes, T. Watanabe, K & Muto, A. (1992) Recent Evidence for Evolution of the Genetic Code Microbiol Rev’s pp 229-264 https://mmbr.asm.org/content/mmbr/56/1/229.full.pdf

48Keeling, P. (2016) Genomics: Evolution of the Genetic Code Cur Biol vol 26, pp R838-R858 http://dx.doi.org/10.1016/j.cub.2023.08.005 42 Neurons & the Nervous System

23.4.1.3 The origins of the peptide- based hormones

Long peptide sequences play a major role in hormone chemistry. Many of these sequences include a shorter sequence that appears across the phylogenic tree. Thus, genetic variations cause similar hormones containing the root sequence but with significant variations. In some cases, the root sequence is found among hormones associated with significantly different effects within a single species. In a 1961 paper, Dixon & Li identified a root sequence of a hormone from the pituitary gland (hypophysis) and traced it to both the adrenocorticotropins (ACTH) and the melanocyte stimulating hormone (MSH)49. The Dixon & Li paper appears to be archaic in the light of more recent reporting of Henley et al.

In 1978, Hokfelt et al. writing in Reichlin et al. published patterns of some enzymes and peptides in nerve terminals. The implication was that these peptides are produced within the pedicle of neurons. Figure 23.4.1-2 Distribution of catecholamines, Figure 23.4.1-3 reproduces their figure 1. GABA and some peptides in nerve terminals in the Again, the caption is quite long and only rat median eminence )front section, mid level. portions will be repeated here. TH, Tyrosine Images based on the formaldehyde-induced hydroxylase; DBH dopamine-β-hydrolase; fluorescence technique. The original caption is GAD, glutamic acid decarboxylase; LHRH, quite long. See text. From Hokfelt et al., 1978. lutenizing hormone releasing hormone; TRH, thyrotropin releasing hormone; SOM, somastatin; NF (VP), neurophysin (vasopressin).

Hokfelt et al. go on to discuss the location of these molecules in greater detail.

They note on page 90, the close association of glutamate, GABA, aspartate and glycine; the four molecules critical to the electrostenolytic powering all neurons according to this work (Section 4.2.4). A question of whether glutamate and aspartate are hormones or neurofacilitators as fuels in the electrostenolytic process. GABA and glycine are clearly residues of the electrostenolytic process and act as neuroinhibitors.

The gist of their discussions is that many, if not all, of the hormones found in the hypothalamus originate in the pedicles of neurons. Most of these neurons have their soma at areas of the hypothalamus remote from their terminals in the median eminence. Their speculative discussion on pages 111 to 118 are particularly interesting. Many of the conjectures can be dismissed out of hand based on more recent findings. They also describe the limitations of the techniques available to them in the 1970's.

49Dixon, J. & Li, C. (1961) The Isolation and Structure of β–Melanocyte-Stimulating Hormone from Horse Pituitary Glands Grn Comp Endocrin vol 1, pp 161-169 Hormonal-affectors 23- 43 They note, “Whereas the enzymes studied are large molecules which by themselves have antigenic properties, the peptides are all comparatively small substances. In most cases they have therefore been coupled with a carrier molecule. . .” It is important to recall this fact when reading the literature. The hormone is of specific interest, its carrier, or the complex, are not. 44 Neurons & the Nervous System

Figure 23.4.1-3A Distribution of some enzymes and peptides in nerve terminals (pedicles, dots) and axons (dashes) in the rat hypothalamus based on indirect immunofluorescence microscopy. Hormonal-affectors 23- 45

Figure 23.4.1-4B Distribution of some enzymes and peptides in nerve terminals (pedicles, dots) and axons (dashes) in the rat hypothalamus based on indirect immunofluorescence microscopy. See text. From Hokfelt et al., 1978. 46 Neurons & the Nervous System

23.4.1.4 The origins of the steroid-based hormones EDIT

[xxx no material similar to that of Hokfelt et a. ] Haymaker et al. (1969) did not discuss the steroid hormones. Despite minimal discussion of the actions of the them, Reichlin et al. (1978) did not address the origin and creation of the steroid- based hormones. The Morgane & Panksepp Handbook of the Hypothalamus (1980) does not address the steroid-based hormones. Henley et al., writing in 2005, did describe the “steroid hormones are lipid molecules derived from a common cholesterol precursor (Cholestane, C27). There are four major classes of steroid hormones: progestins, androgens, estrogens, and corticoids, which contain 21,19,18 and 21 carbons respectively.” They provide schematics of production for these classes including identification of the enzymes associated with the transitions between moieties. They describe the chemical synthesis of each of these classes of steroid hormones. They describe the actions of the androgens after their synthesis and secretion following stimulation by the lutenizing hormone, LH. Their figure 4 illustrates the stimulation of the major glands, gonad and adrenal, by the individual parts of the hypophysis

Henley et al. identify endocrine, paracrine, exocrine and autocrine steroid hormones. This work discounts autocrine hormones pending more factual information but does include the category of pericrine to account for the hormones of the hypothalamus released into the portal leading to the hypophysis only.

23.4.1.4.1 The chemical origins of the steroid-based hormones

Henley et al. have provided a comprehensive chapter on steroid hormones50. They note, “Steroids are lipophilic molecules used as chemical messengers. . . Major sites of steroid synthesis and secretion include the ovaries, testes, adrenals, and placenta.” Steroids can be found in each of the subsystem of the Crine (hormonal) system.

Henley et al. become more specific when they note, “Steroid hormones are lipid molecules derived from a common cholesterol precursor (Cholestane, C27). There are four major classes o steroid hormones: progestins, androgens, estrogens, and corticoids, which contain 21, 19, 18 and 21 carbons, respectively.” Figure 23.4.1-5 illustrates the progesterone branch of the cholesterol family steroids.

50Henley, D. Lindzey, J. & Korach, K. (2005) Steroid Hormones In Melmed, S. & Conn, P. eds Endocrinology, 2nd Edition Totowa, NJ: Humana Press chapter 4 Hormonal-affectors 23- 47

Figure 23.4.1-5 The progesterone branch of the cholesterol family of steroids ADD. It should be noted that this branch of the cholesterol family of hormones contains no peptide residues, nor protein moiety, or nitrogen.

They note that as lipophilic molecules, they exhibit low solubility in the bloodstream. As a result, they are accompanied by serum-binding proteins to help transport steroids via the bloodstream to their target tissue. On the other hand, Lehninger (1972, pg 531) indicates they are esterfied to long chain fatty acids at the 3-hydroxyl group. They remain free of protein residues in this configuration.

It is not clear that any form of coupling between the steroid hormones and either proteins or fatty acids is needed when the hormones are secreted into the urine or via sweat glands as pheromones. The requirement to minimize the molecular weight of the in order to achieve maximum volatility and thereby support oskonation at a significant distance, suggest no coupling for the hormone. Henley et al. assert, “The effect of steroids are typically slow in relation to the rapid time course for the effect of second-messenger-mediated peptide hormones. “Though most characterized effects of nuclear hormones are mediated via nuclear receptors and genomic pathways, there are examples of very rapid, “nongenomic” effects of steroids that appear to be owing to membrane-mediated effects.” They also indicate that most of the steroid hormones penetrate the lemma of a cell and reach a nuclear hormone receptor, NHR. Their figure 2 is a typical conceptual cartoon suggesting multiple pathways within a cell. They describe a hypothalamus-pituitary-gonad, HPG axis, using different parts of the hypothalamus, and the pituitary to stimulate the ovaries, testes, adrenals separately from the HPE- axis of Section 23.4.2. 48 Neurons & the Nervous System

23.4.1.4.2Mechanisms of the steroid-based hormones/receptor interface

Part 3 of the Henley et al. paper asserted the potentiality of both surface receptors and nuclear receptors for the steroid-based hormones. They did not further describe the molecular character or mechanisms of these interfaces. They proceeded to report on the genetic level of activity and mechanisms of the steroids. They did provide some genetic sequences for a few steroid hormones, noting their preliminary nature and their potential to exist in deviant forms. 23.4.1.5 Specialized nomenclature

The following discussions will introduce new expressions related to understanding the physiology of the hypothalamus and hypophysis. Some of these expressions are defined here. Neurosecretory neurons– Stage 7 neurons, generally with the secretory function contained within a pedicle, Portal– A point where hormones are introduced into the capillaries of the isthmus between the hypothalamus and hypophysis. The hormones never pass through the heart or lungs. Portal hypophysial vessels from the hypothalamus to the hypophysis (pituitary). This region contains few cells but many nerve endings in close proximity to the capillary loops from which the protal vessels originate.

Tropic hormones– “stimulate the secretion of substances from other glands.” Stage 7 neuroaffectors (neuro-facilitators). “ There are probably a few nerve fibers that pass directly from the brain to the anterior pituitary, but they do not control anterior pituitary functions. Instead anterior pituitary secretion is controlled by chemical agents carried in the portal hypophysial vessels from the hypothalamus to the pituitary (Ganong, page 166).”

Releasing Factors, RF & Inhibiting Factors, IF– Early designations for releasing hormones, RH and inhibiting hormones, IH.

23.4.1.6 Additional annotation of block diagram of the hypothalamus

With the sources of the neurohormones of the hypothalamus becoming clearer, it is possible to combine the neuraaffector neurons with the various subglands of the hypothalamus identified in Section 23.3.1.3 [Figure 23.3.1-3]

Label Behavioral name Molecule TRH GH-RH GH-RIH PRH Progesterone Releasing Hormone PR-IH Progesterone Release Inhibiting Hormone ? Dopamine LH-RH CRH

Knigge et al. (page 74) noted in 1980, “Sufficient direct and indirect evidence may be available to suggest that all the nuero-hormone associated with the adenophyophyseal control are peptides.” Rewording for convenience, they assert the evidence supports all of the releasing hormones of the hypothalamus secreted into the portal are peptides. There is one assertion in the literature of that time that PR-IH is the catecholamine, dopamine. No additional justification or any citation was provided to support that assertion. However, Knigge et al. went on (beginning with page 78) to describe the role of monoamines (as opposed to multiple amine peptides) in secreting hormones, such as dopamine. “There appears to be little doubt that certain monoamines (e.g., dopamine, norepinephrine and serotonin) exert a significant influence Hormonal-affectors 23- 49 either by regulating or influencing the secretion of hormones” into the portal. These molecules are frequently present in the pedicles as intermediaries in the production of catecholamine hormones as documented by Hokfelt et al. below.

23.4.1.6.1 Cytology of the neuroaffectors of the hypothalamus & neurohypophysis

At least some of the “releasing” hormones listed above appear to be created in neuroaffector neurons similar to those used in the neuron/muscular interfaces developed in Section 16.5.3. Thus, Figure 23.4.1-6 is offered as the generic neuron of the hypothalamus and the neurohypophysis. The generic neuron is found within the area labeled “Neural regime” of the hypothalamus; the pedicle is located within the area labeled the “Median eminence;” the target of the hormone is typically a Glandular affector within the “hypophysis.” The neurohormones are propogated via the portal. A glandular affector is typically stimulated by a neurohormone from the hypothalamus and creates a second “stimulating” hormone sent via the vascular circulation to peripheral glands. These glands typically create hormones with specific operational targets. Within the pedicle, XX1 denotes the proto-hormone leading to the XXH hormone. XXn relates to one or more mediators supporting to the transformation of XX1 into the ultimate hormone, XXH. 50 Neurons & the Nervous System

Figure 23.4.1-6 Generic neuroaffector of the neuron/glandular interface of the hypothalamus. This configuration can be used to generate a broad range of hormones, primarily within the hypothalamus. The undefined labels are dependent on the hormone, XXX, to be released by the specific neuron. See text.

In the case of XXM = DOPA and XX1 = tyrosine, there is only one enzyme involved in the production of DOPA, XX2 = tyrosine hydroxylase. In accordance with Hokfelt [Figure 23.4.1-7], the creation of epinephrine involves 3 intermediaries and 4 enzymes. Whether the enzymes, XX2 through XX5 are contained in separate vesicles is not known. Similarly, whether there are separate vesicles for the intermediaries is not known. Until more definitive information becomes Hormonal-affectors 23- 51 available in this area, further definition of the internal structure of the pedicle is not feasible. The sequence involved in converting the proto-hormone to the product, XXH, is not known. The final release of the product, XXH, into the portal should be under discrete control of the signal from the axoplasm of the stage 7 neuroaffector. 23.4.1.6.2 The chemistry of the neuro-hormones (hormone releasing hormones)

This section will provide a partial tabulation of the neuro-hormones released by the hypothalamus. These hormones can theoretically be of three forms; simple organic molecules, peptides and steroids. The simple organic molecules are primarily monoamines. Within these three are a variety of lower chemical classification. The catecholamines, CA, are a particularly important classification. This class contains molecules based on monoamines; dopamine (DA), norepinephrine (NE) and epinephrine (E). The following tabulation will follow the sequence used in the above section. There are typically many possible chemical processing paths from the material listed under “From” to the chemical listed under the “Chemical name.” Only paths determined by laboratory investigation can be considered meaningful.

Hokfelt et al. (1978, page 73) has provided a table of structural characterizations (with references) of many of the neuro-hormones. Morgane & Panksepp, volume 1, have provided similar information. Neuro-hormones of the hypothalamus Label Chemical name From Reference

TRH– a peptide Morgane pg658

GH-RH– GH-RIH– Somastatin, a tetradecapeptide Morgane pg 661

PRH– PR-IH– Dopamine (catacholine)

LH-RH– a decapeptide Morgane pg 660

CRH–

Oxytocin* oxytocin tyrosine vasopressin* arg-vasopressin tyrosine

* hormones secreted by the neurohypophysis from neurons with soma in the hypothalamus

PR-IH has been tentatively defined as dopamine by xxx.

Figure 23.4.1-7 illustrates a baseline processing path from tyrosine to epinephrine presented by Raum in 2005. His discussion and figure is more explicit than a similar presentation by Hokfelt et al. in 1978, based on their laboratory research. Hokfelt et al. note,”Defining the exact distribution of CA nerve terminal systems in the hypothalamus containing the three CAs, DA, NE and epinephrine (E), on the basis of immunohistochemistry of normal animals is connected with considerable difficulties.” Hokfelt et al. describe the general protocols used in immunohistochemistry (page 70) during the 1970's and cite several reviews of that time. 52 Neurons & the Nervous System

Figure 23.4.1-7 Biosynthesis of catecholamines leading to epinephrine as described by Raum, based on extensive immunochemical assay during the 1970's . Note the dashed boxes indicating the change from the previous step. See text. From Raum, 2005 updating a sequence by Hokfelt et al., 1978.

23.4.2 The hypothalamus-pituitary-epinephrine (adrenal), H-P-E, axis

The hypothalamus-pituitary-adrenal axis comprises a very important hormonal system regulated primarily by the stage 7 neurons (Section 23.3.3) accompanied by, integrated with, a variety of internal feedback loops.

Adrenalin is a hormone secreted by the adrenal glands, especially in conditions of stress, increasing rates of blood circulation, breathing, and carbohydrate metabolism and preparing muscles for exertion. Adrenaline, also known as adrenalin or the more recent name of epinephrine, is a hormone that is also used as a medication. Epinephrine is normally produced by both the adrenal glands An overview of epinephrine (adrenalin) is interesting, Formula: C9H13NO3, molar mass: 183.204 g/mol, duration of action: few minutes, onset of action: Rapid, elimination half-life: 2 minutes, excretion: Urine Figure 23.4.2-1 displays the structural formula for adrenaline. An important feature of adrenaline is the precise arrangement of OH and NH. The d-value between the oxygen and nitrogen of this arrangement is critically important to the operation of adrenaline as a hormone. D-value = 2.877 determined using Discovery Suite 4.1 and the Jmol file of the Royal Society of Chemistry (see the disavowal of these files by the RSC in Section 8.6.1.6.3). Until more accurate d-values become Hormonal-affectors 23- 53 available, the d-values obtained from the RSC files will continue to be used in this work, as illustrative if not precise. Epinephrine may form a DACB with PtdTyr employing its alternate d- value near 2.791 Angstrom.

It appears that PtdTyr may be used differently in the external chemical sensory neuron receptors and the internal chemical receptors. It may also be that PtdTyr is used in two different configurations, PtdTyrd=5.534 and PtdTyrd=2.791 to form DACBs with both external and internal chemical moieties. In the case of the internal chemical hormones, PtdTyrd=2.791 appears to be sensitive to epinephrine while PtdTyrd=5.534 is Figure 23.4.2-1 The epinephrine (adrenaline) sensitive to triiodothyronine, T3. molecule Norepinephrine is similar but omits the methyl group, CH3, at the right margin. ADD. Lehninger (1970, pages 500 &558) indicates both epinephrine and norepinephrine are catacholamines derived from the amino acid, tyrosine. The catecholamines are neuro- modulators generally influencing the dendroplasm (or more generally, neuroplasms) of a wide variety neurons (Section 23.1.3.2).

The following paragraph from Section 23.1.3.2.1 is repeated here for convenience.

Quoting an old but typical reference51, “The effector cells innervated by the autonomic nervous system have two types of chemically defined adrenoreceptive sites–namely alpha (α) and beta (β) receptors. The alpha receptors have an affinity for both epinephrine and norepinephrine, while the beta receptors have a selectively stronger affinity for epinephrine. The alpha receptors are generally excitatory but some may be inhibitory.” and “The beta receptors are generally inhibitory but some may be excitatory.” [Italics added for emphasis]

The ambiguity over the effect caused by these neuro-modulators is due to their affect on neurons in general. Under the Electrolytic Theory of the Neuron, all voltage waveforms produced at the axon of a neuron are positive-going voltage-wise if the stimulus applied to its dendritic input is positive-going. The effects of epinephrine and norepinepherin involve their modulating functions. The so-called α-receptors refer to those neuromodulators stimulating the “non-inverting” dendritic inputs to the neuron. The so-called β-receptors refer to those neuromodulators stimulating the “inverting” poditic inputs to the neuron. The glandular input to the β-receptors results in a reduced amplitude output at the axon of the neuron.

23.4.2.1 Past Flow diagrams of the thyroxine system with feedback EMPTY

As shown in Section 23.3.3, the axis of interest is; hypothalamus, the antidiuetic hormone(ADH), hypophysis, adrenocorticotropic hormone (ACTH), adrenal cortex (gland), corticosterone/cortisol (CORT), & along a feedback path to the hypothalamus. Alternate investigators replace the antidiuretic, ADH, with the Corticotropin releasing hormone, CRH. Figure 23.4.2-2 illustrates this sequence within the concept of a feedback loop. It is drawn to resemble the similar loop used within the H-P-T axis for the thyroxines (Section 23.4.3) As noted above, the hypothalamus secretes a variety of hormones. Many individual hormones stimulate a large variety of physiological elements. It has been common to relate the

51Noback, C. (1967) The Human Nervous System. NY: McGraw-Hill pg 116 54 Neurons & the Nervous System corticotropin releasing hormone (CRH) with the H-P-E axis. As noted in the caption, whether the feedback path to the pituitary gland actually exists has not been demonstrated. It is redundant and not required based only on the feedback theory of electrical engineering unless it incorporates a transport delay, bandlimiting or other functional element. If this second path exists, it can be expected to employ the same receptor as in the case of the path terminating at the hypothalamus. - - - - Wikipedia (as of 2018) suggests such a functional element may exist in the feedback loop within the H-P-T axis. It suggests, “many substances secreted within this axis exhibit slow/intermediate and fast feedback-loop activity. . .The latter (secretion of ACTH from the pituitary) is an example of a slow feedback loop, which works on the order of hours to days, whereas the former (secretion of CRH from the hypothalamus) works on the order of minutes. The half-life of ACTH in human blood is about ten minutes.

23.4.2.2 The adrenocorticotropin Figure 23.4.2-2 INCOMPLETE The feedback loops hormones (ACTH) EMPTY of the H-P-E axis ADD. The term CORT is used to designate a family of corticosteroids released by the adrenal cortex. They may not all operate in 23.4.2.2.1 The chemistry of the precisely the same manner in terms of the stimulating hormones of the feedback paths. See text. hypophysis

Stimulating hormones of the hypophysis Label Chemical family From Reference

TSH GH PRL LH FSH ACTH nonatriacontapeptide (39) Witter 1980, pg 308 α-MSH (13) ACTH β-MSH (7) or (18) ACTH

Oxytocin* peptide (9) Witter 1980, pg 326 vasopressin* peptide (9) Witter 1980, pg 326

* hormones secreted by the neurohypophysis from neurons with soma in the hypothalamus. These were defined as peptides by Witter & deWein1980 and by Bichet in 2005-17. Hormonal-affectors 23- 55 Witter (1980) has provided data on the peptides of the anterior and intermediate hypophysis52, primarily ACTH, oxytocin and vasopressin. 23.4.2.3 Functional representation of the hypophysis–CRH in ACTH out

One portion of the hypophysis processes the tropin, CRH, into the hormone ACTH, which is believed to be one of the major hormones affecting the adrenal gland. While the mechanism performing this conversion has not been found in the literature, Otsuka & Inouye have studied the resultant hormone ACTH. They have provided an extensive exploration of ACTH and its derivatives as of 197553. Their conclusions were; In the preceding sections the structure-activity relationships of ACTH have been discussed with regard mainly to the action of ACTH on the adrenal gland. Although some complexities still remain to be solved, the following conclusions may be made. 1. There is no amino acid residue in the ACTH molecule absolutely essential for the steroidogenic activity. The single substitution or modification of any particular amino acid residue does not lead to the complete inactivation of the hormone. 2. The amino acid sequences His-Phe-Arg-Trp (positions 6-9) seems to be more important than any other portions of the ACTH molecule. In view of the considerable evidence as well as the fact that ACTH(5-10)-OH and ACTH(6-24)-OH can stimulate the adrenal steroidogenesis (section 4), whereas ACTH(ll-24)-OH is completely inactive (Seelig et al., 1971), this tetrapeptide portion may be regarded as the 'active site', the essential region for activity, of ACTH.

3. The sequence Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg (positions 11-18) is not essential for activity, but appears to play an important role as a major binding site in the electrostatic interaction with the adrenal receptor. The special importance of lysine 11 should be emphasized (section 5.4).

4. The N-terminal sequence Ser-Tyr-Ser-Met-Glu (positions 1-5) seems to be less important than the above two portions. This pentapeptide portion may act as a binding site for the additional attachment of ACTH to the specific receptor.

5. The C-terminal half of the ACTH molecule (positions 20-39) may not be involved in the process of adrenal stimulation, but may play a role in stabilizing the hormone in the physiological environment. This portion contains the species-specific amino acid residues. However, it is still unclear whether those residues have some immunological significance.

Briefly, their conclusions stress the critical hormonal portion of ACTH is the sequence, His-Phe-Arg-Trp (positions 6-9). 23.4.2.3.2 Structure of adrenocorticotropin-stimulating hormone, ACTH & its receptor

As noted in Section 23.4.1.2, Dixon & Li have explored the structural character of ACTH in conjunction with their study of the β-melanocyte-stimulating hormones isolated from the pituitary of horses. “ACTH consists of 39 amino acids, the first 13 of which (counting from the N-terminus) may be cleaved to form α-melanocyte-stimulating hormone (α-MSH). (This common structure is

52Witter, A. & de Wied, D. (1980) Hypothalamic-pituitary oligopeptides and behavior in Morgane, P. & Panksepp, J. eds (1980) Physiology of the Hypothalamus, vol 2 of Handbook of the Hypothalamus. NY: Marcel Dekker Chapter 5

53Otsuka, H. & Inouye, K. (1975) Structure-activity Relationships of Adrenocorticotropin Pharmac Therap B vol. 1(3), pp. 501-527. 56 Neurons & the Nervous System responsible for excessively tanned skin in Addison's disease.) After a short period of time, ACTH is cleaved into α-melanocyte-stimulating hormone (α-MSH) and CLIP, a peptide with unknown activity in humans. Human ACTH has a molecular weight of 4,540 atomic mass units (Da)”

Their β-melanocyte-stimulating hormone was shown to be a short peptide defined by the genetic code series, H-Asp.Glu.Gly.Pro.Tyr.Lys.Met.Glu.His.Phe.Arg.Try.Gly. Ser.Pro.Arg.Lys.Asp-OH This is a relatively short series. However, as they note, “Although the sequence differs somewhat from that found in any other naturally occurring melanocyte-stimulating pituitary hormone (see Fig. 3), it still contains the non-varying internal heptapeptide sequence… Met.Glu.His.Phe.Arg.Try.Gly… found in each of these hormones.”

23.4.2.4 Potential stage 7 neurons within the peripheral glands

In 2005, two authors54,55 in Melmed & Conn broached the subject of neuroaffectors within the adrenal gland of the endocrine submodality. Their language was conceptual and few details were presented. Bichet briefly described stage 7 neurons within the pituitary. His figure 1 is a poor rendition of a figure from Wilson et al. of 2002. Wilson et al. would primarily concerned with the state of hydration of certain neurons and the genetics of their situation. They were not focused on the location of the parts of stage 7 neuroaffectors within the glandular system.

In many of these papers, the labels argininevasopressin (AVP and oxytocin (OT) were used instead of (VAS) and(OXY) respectively

Two citations were noted by Bichet that appear relevant; Burback et al56, and Vokes & Robertson57. The Burbach et al. article does not support the presence of realistic stage 7 neurons within the hypophysis (pituitary gland). See Section 23.4.2.4.1. It does provide useful information about the histology of the axolemma of stage 7 neurons. Vokes & Robertson provided related data in their analysis (Section 23.4.2.4.2).

Harbuz & Lightman addressed the potential for stage 7 neurons within the adrenal gland. They used the term neuro-hypophysis in a sentence but did not elaborate on the term. The implication was that it was a common term in the physiology community. They did not address the potential presence of neurons in the pituitary or adrenal gland further.

Cazalis et al. used the term neurohypophysis in 1987 when discussing neurosecretions but did not

54Bichet, D. (2005) Posterior pituitary hormones In Melmed, S. & Conn, P. eds Endocrinology, 2nd Edition Totowa, NJ: Humana Press chapter 14

55Harbuz, M & Lightman, S. (2005) The neuroendocrine–immune interface In Melmed, S. & Conn, P. eds Endocrinology, 2nd Edition Totowa, NJ: Humana Press chapter 8

56Burbach, J. Peter, H. Luckman, S. Murphy, D. & Gainer, H. (2001) Gene Regulation in the Magnocellular Hypothalamo-Neurohypophysial System Physiol Rev vol 81, pp 1197–1267

57Vokes, T. & Robertson, G. (1985) Physiology of secretion of vasopressin In Czernichow, P. & Robinson, A. eds. Diabetes Insipidus in Man. Basel: S. Karger Hormonal-affectors 23- 57 address the character and functionality of the putative neurohypophysis58. Their usage of the term suggested it was a morphological or histological term used in studies of the rat. They quoted several papers from the mid-1960's. Douglas & Rubin did not use the term in 1963. Douglas & Poisner did use the term in 1964 in reporting their laboratory investigations.59 “The infundibular lobe of the neurohypophysis, or 'posterior pituitary gland', has long been known to be the site of storage and release of the posterior pituitary hormones. More recently, however, it has come to be recognized that it is not a 'gland' in the strict sense, but is, rather, an aggregate of secretory terminals, or axons, of cells that are lodged in the suipra-optic and paraventricular nuclei of the hypothalamus (Bargman & Scharrer, 1951; Scharrer & Scharrer, 1954). Along with this, there has developed the belief that the various humoral and nervous factors which evoke the release of the posterior pituitary hormones act through these neurosecretory cell bodies in the hypothalamus and not directly on their neurosecretory terminals in the infundibular lobe (Jewell & Verney, 1957; Harris, 1960; Ortmann, 1960). The results obtained in the present experiments on the isolated neurohypophysis are in harmony with this view.” Note the alternate name of 'posterior pituitary gland' for the neurohypophysis. Note also the belief that the cell bodies of the neurons are actually in the hypothalamus. The pharmacology community has long professed that the neurosecretory axons of these neurons are also in the hypothalamus and the hormonal secretions travel to the hypophysis via an isolated portal. S. L. Paley apparently introduced the term hypophysis to Physiology in 1957.

In this work, the neurohypophysis of the rat (and of other animals) will be described as the posterior pituitary gland. It will be assumed to receive its signals in electronic form extending to the secretory pedicles from the soma remaining in the hypothalamus. These stage 7 neuron pedicles secrete arginine vasopressin and oxytocin. 23.4.2.4.1 The Burbach et al, definition of a hypothalamus-neurohypophysis system, HNS

Although, Burbach et al. indicated they were taking a physiological perspective instead of a pharmacological perspective, their physiology did not show how the system of the rat worked as a unitary system. Burbach et al. define the focus of their work as the Hypothalamo- Neurohypophysial System, HNS, in a 47 page article. The paper contains less than five flow diagrams or schematics. These are all highly conceptual. The goal of the paper was stated as, “This review is an inventory of what we know about genes expressed in the HNS, about the regulation of their expression, and about their function. Genes relevant to the central question include receptors and components that receive and process the message that the organism is in demand of a neurohypophysial hormone.” It is interesting that this goal does not address the neurons described in the title of their article. While using the label “Neurohypophysial,” the paper focuses on the neurons of the hypothalamus, not the hypophysis. Based on this situation, the title of the article should be transposed to the Neurohypothalamo- hypophysis system, NHS.

Burbach et al. have provided considerable morphological and histological data relative to their interpretation of what are neurons. However, they do not review the cytology of such neurons or how they are interconnected as part of the neural modality. They provide 911citations but few quotations are figures from those citations. They do not appear to interpret the literature, but only discuss it in a loose plausible framework based on the unstated principles of the chemical theory of the neuron After a review of a great amount of the literature from a histological perspective, and mentioning

58Cazalis, M. Dayanithi, G. & Nordmann, J. (1987) Hormone Release from Isolated Nerve Endings of the Rat Neurohypophysis J Physiol vol 390, pp 55-70

59Douglas W. & Poisner, A. (1964) Stimulus-secretion Coupling in a Neurosecretory Organ: The role of Calcium in the Release of Vasopressin from the Neurohypophysis J Physiol vol 172, pp 1-18 58 Neurons & the Nervous System their reliance on light microscopy, they proceed into the area of hormonal flow and hormonal receptors without sketching the schematic of the crine (hormonal) modality. They then dive into genetics and gene expression (page 1212), again without presenting significant graphic material. They close their article by asserting, “This research puts emphasis into two directions. On the one hand it focuses on the physiological integration of the HNS in the control of peripheral functions by the brain, and on the other hand it aims to understand how this integration is accomplished mechanistically at the level of the cell and the molecules involved. This review has attempted to highlight the latter direction by reviewing what we know about HNS-expressed genes that play a role in receiving and transducing signals from the brain and periphery, and connecting them to the machineries responsible for transcription of the VP and OT genes and for manufacturing and releasing the hormonal output of the HNS.” The article has a strongly academic flavor. It is truly a review of the work of others. Virtually no discussion of laboratory results or system synthesis is present. As an archive of 20th Century thought and discovery, it is of considerable value.

23.4.2.4.2 The Vokes & Robertson description of xxx EMPTY

Vokes & Robertson presented a brief article that . . . [xxx find Vokes & Robertson, footnote 82 ] 23.4.2.5 Adrenal family of hormones–epinephrine, norepinephrine, etc.

The adrenal family of hormones has been studied for many decades. Recently, a name change has occurred. Adrenaline is now known as epinephrine. This change will take a long time to implement in both the academic and medical literature. The family includes both epinephrine, norepinphrine and a large host of their derivatives. These hormones are of the catacholamine class of pharmaceuticals. 23.4.2.5.1 The catacholamines

Three major neuro-affectors are derived from tyrosine by way of L-dopa as a result of enzymatic action as shown in Figure 23.4.2-3 (Ganong, page 149 and Stacy, page 203). The differences between the three neuro-affectors shown along the bottom of the figure are remarkably small. They all contain a dihydroxyphenyl ring and a two carbon side chain ending with a nitrogen. This common structure probably plays an important role in their interaction with receptor sites. These agents are frequently labeled catecholamines because the side chain, containing two carbons and ending in a nitrogen, could be derived from choline. As shown in the upper part of the figure, there are alternate sources for the required side chain. Hormonal-affectors 23- 59

Figure 23.4.2-3 Synthesis of the principle catecholines. Either phenylalanine or p-tyrosine are readily available amino acids that can be used to create members of the adrenaline family, the lower row of chemicals in this figure. Pharmaceutically introduced Dopa passes easily through the blood-brain-barrier. From Ganong, 1975.

Dopa and dopamine are formed within the cytoplasm of the cell. The dopamine then enters the granulated vesicles within which it is converted to norepinephrine. The norepinephrine is catalyzed to epinephrine within these same granulated vesicles.

23.4.2.5.1 Potential DACB between epinephrine and a PtdTyr receptor on any cell.

By inspection, the most likely DACB involving epinephrine and a receptor site on/in a neuron is that involving the OH and NH along the aliphatic chain of epinephrine. The distance (d-value) between these two chemical constituents is 2.877 Angstrom. The 3D rendition used Discovery Suite 4.1. Looking for a phosphatidyl-amino acid exhibiting this d-value, a potential candidate is a simple or modified form of tyrosine. Tyrosine exhibits two potential DACB sites of interest; one at d = 2.791 Angstrom and one at d = 5.534 Angstrom. For discussion purposes, a cellular receptor formed of a type 2 region of outer cell lemma consisting of a block of phosphatidyltyrosine, PtdTyr molecules oriented to expose the d= 2.791 Angstrom arrangement as a receptor should be considered. No data has been located on how closely the d-values of the stimulant and the receptor must be to be effective. As used in the gustatory (Section 8.5) and olfactory modalities (Section 8.6), the match need not be perfect. These modalities use the receptors in an analog mode, where the degree of coupling is a useful parameter. 23.4.2.5.2 The sensitivity of physiological cells to epinephrine in the bloodstream

Based on the DACB joining a epinephrine molecule with any cell on a short term basis and then releasing the epinephrine molecule unchanged, the cells containing the putative PtdTyr receptor are sensitive to the average concentration of epinephrine in the bloodstream and matrices 60 Neurons & the Nervous System serving all cells in the body exhibiting suitable receptors. The short lifetime of epinephrine within the system is a controlling factor in epinephrine utilization. 23.4.2.5.4 Summary of hormone & receptors along H-P-E axis

Figure 23.4.2-4 shows a tabulation of the hormones, receptors, and receptor locations along the H-P-E axis.

Figure 23.4.2-4 Hormone tabulation & receptor locations along H-P-E axis ADD. All receptors are an amino acid of the lipid, phosphatidic acid. The receptor PtdTyr is tentative, while awaiting confirmation of the DACB value. ACTH consists of 39 amino acids. The designation CORT refers to a variety of corticosteroids (or glucocorticoids). See text.

As noted in the caption, there is no unique definition of the label CORT at this time. It is used differently by different investigators to indicate a group of hormones released by the adrenal cortex upon instruction from the pituitary gland. The receptor locations for CO RT is described as the hypothalamus (with respect to the feedback path). There may also be a feedback path to the pituitary gland as well. In fact one or more of the CORT hormones stimulates nearly every cell in the organism when all combinations of the CORT are considered. As noted in the text, even the definition of ACTH lacks precision. Although ACTH is described as a 39 residue peptide, it is recognized that this peptide can be cleaved at different points to create shorter length peptides that are in their own right hormones. Similar tables to the above figure can be assembled for other axes of the hypothalamus as well as for the external chemical sensory receptors. A comprehensive summary of all of the receptor molecules consisting of a combined with an amino acid by condensation, regardless of modality in which used, will be found in Appendix ZA, Table of Phosphoglycerides. A proposed flow diagram for the H-P-E axis can not be presented at this time. It will be similar to that for the H-P-T axis in Section 23.4.3.6. However, because of the variety of corticosteroids secreted by the pituitary in conjunction with the H-P-E axis, it may need to be more general or shown to vary with the specific corticosteroid involved. Hormonal-affectors 23- 61 23.4.3 The hypothalamus-pituitary-thyroid, H-P-T, axis

When discussing the hormones related to thyroxin, the global designation, thyroxine(s), is frequently used in the literature to avoid longer and more complex individual names. Designations are critically important within the H-P-T axis; serum thyroxine, T4, and free thyroxine, FT4, are distinctly different. https://www.endocrineweb.com/conditions/thyroid/thyroid-function-tests provides the current definitions of these terms. The hypothalamus-pituitary-thyroid axis comprises a very important hormonal system relates to the thyroid and its regulation by the stage 7 neurons of the hypothalamus, and related hypophysis (pituitary) glands (Section 23.3.3). The complexity of these operating elements and the cardiovascular system remains a very difficult problem in the academic/clinical and medical communities. A recent mini-review by Costa-e-Sousa & Hollenberg contains no diagrams at all and is filled with generic or anecdotal material along with many citations60. The ninth edition of Werner & Ingbar’s “The Thyroid” counters this long-standing lack of specifics61. It is a significant rewrite of an authoritative text. The 9th Edition contains a great deal more data than earlier editions but begins to suffer from lack of detailed textual discussion. Fortunately, the 9th is not just a re-write of earlier editions. For example, chapters 3, 8 & 9 provide more discussion and in some cases, more meaningful graphics than the 9th. The 3rd Edition also introduced Ingbar as a co-editor. Some of this material will be introduced below.

23.4.3.1 Past Flow diagrams of the thyroxine system with feedback

The operation of the hormones related to thyroxin have been a subject of intense study for most of the 20th Century; however, the current level of understanding remains unsatisfactory. Figure 23.4.3-1 presents a very simple illustration of the problem62. The system involves a nesting of servo loops in a manner that has been virtually outlawed in software languages because of the difficulty of understanding their operation. The loops are not hierarchal, one within another. The dashed path actually interconnects areas inside one loop and outside of that same loop.

60Costa-e-Sousa. R. Hollenberg, A. (2012) Minireview: The Neural Regulation of the Hypothalamic-Pituitary-Thyroid Axis Endocrinology vol 153, pp 4128–4135

61Braverman, L. & Utiger, R. eds. (2005) Wermer & Ingbar’s, The Thyroid: A fundamental & Clinical Text, 9th Ed. NY: Lippincott Williams & Wilkins

62Rose, G. (1976) A current interpretation of the anatomy of the mammalian cell In Jamieson, G. & Robinson, D. Mammalian Cell Membranes NY: Butterworth-Heinemann pp 1-30 https://doi.org/10.1016/B978-0-408-70722-0.50005-X 62 Neurons & the Nervous System

Figure 23.4.3-1 The feedback loops between the thyroid, pituitary and hypothalamus. TRF, thyroid-releasing factor, also known more recently as TRH, thyrotrophin-releasing hormone. Modified from Rose, 1976.

When the more detailed operation of the hormones related to thyroxin, such as T3 and T4 are factored into the overall analysis, the problem is doubly difficult to solve. Hormonal-affectors 23- 63 An alternate to the above figure by Orlander63 in 2018 is shown in Figure 23.4.3-2 . This variant shows positive/negative feedback by symbols in the slightly different paths than above. It shows T3 and T4 explicitly.

63Orlander, P. (2018) Hypothyroidism https://emedicine.medscape.com/article/122393-overview 64 Neurons & the Nervous System

Figure 23.4.3-2 Alternate feedback loops of the hypothalamus-pituitary-thyroid axis. This variant shows positive/negative feedback by symbols in the slightly different paths than above. Note the assertion that T4 is also converted to T3 in peripheral tissue, where the molecules are employed. Modified from Orlander, 2018 Hormonal-affectors 23- 65 This is significantly different than in the previous figure. Both T3 and T4 are explicitly shown as present in both feedback paths. This situation is well documented as of 2018. Much of the current medical literature remains superficial in treating the thyroid gland. It speaks of this gland from a global perspective, assuming the entire gland is healthy, failing or already failed. It does not consider the possibility that some of the receptors associated with the manufacturing function, such as the chemical receptors in the feedback paths termination at the hypothalamus and pituitary, might have failed while the manufacturing functions within these two glands might remain completely functional. - - - - As a matter of standardization of nomenclature, the literature has tended toward all hormones released by the hypothalamus being labeled, releasing hormones. All hormones secreted by the hypophysis (pituitary gland) are being labeled stimulating hormones. As and example, the hypothalamus secretes the corticosteroid releasing hormone, CRH, while the associated hypophysis secretes the thyroid stimulating hormone, TSH. - - - - The two distinct feedback paths in the figure are redundant in the absence of additional information suggesting one of the paths exhibit/encounter additional signal processing mechanisms. Section 23.4.2.1 includes a poorly sourced comment from Wikipedia suggesting such a difference within the H-P-E axis. Such a mechanism is incorporated in the summary flow diagram of Section 23.4.3.6. 23.4.3.2 Functional features of the hypothalamus

ADD xxx

One of the better discussions of TRH appears in the Brittanica64. “Thyrotropin-releasing hormone, simplest of the hypothalamic neurohormones, consisting of three amino acids in the sequence glutamic acid–histidine–proline. The structural simplicity of thyrotropin-releasing hormone is deceiving because this hormone actually has many functions. It stimulates the synthesis and secretion of thyrotropin (thyroid-stimulating hormone) by the anterior pituitary gland. Given in high doses by injection, it stimulates the secretion of prolactin from the pituitary gland, although it does not appear to regulate the secretion of prolactin. Thyrotropin-releasing hormone is also found throughout the brain and spinal cord, where it is thought to serve as a neuromodulator.

Thyrotropin-releasing hormone appeared very early in the evolution of vertebrates, and, while its concentration is highest in the hypothalamus, the total amount of thyrotropin-releasing hormone in the remainder of the brain far exceeds that in the hypothalamus. The nerve cells that produce thyrotropin-releasing hormone in the hypothalamus are subject to stimulatory and inhibitory influences from higher centres in the brain and from serum thyroid hormone concentrations, with low concentrations stimulating and high concentrations inhibiting the production of thyrotropin-releasing hormone. In this way, thyrotropin-releasing hormone forms the topmost component of the hypothalamic-pituitary-thyroid axis. Deficiency of thyrotropin-releasing hormone is a rare cause of hypothyroidism. Straub et al. have presented their investigations into TRH and its potential receptor on the pituitary65. Their perspective is that of the geneticist. They note, “Thyrotropin-releasing hormone (TRH) is an important extracellular regulatory molecule that functions as a releasing factor in the anterior pituitary gland and as a neurotransmitter/neuromodulator in the central and peripheral

64Britannica (as of 2018) Thyrotropin-releasing hormone https://www.britannica.com/science/thyrotropin-releasing-hormone

65Straub, R. Frecht, G. Joho, R. et al. (1990) Expression cloning of a cDNA encoding the mouse pituitary thyrotropin-releasing hormone receptor Proc. Natl Acad Sci USA vol 87, pp 9514-9518 66 Neurons & the Nervous System nervous systems. Binding sites for TRH are present in these tissues, but the TRH receptor (TRH-R) has not been purified from any source.” Based on their work, they followed the conventional wisdom of the time and assert the receptor is a G protein-coupled receptor. “The 3.8-kilobase mouse TRH-R cDNA encodes a protein of 393 amino acids that shows similarities to other guanine nucleotide-binding regulatory protein-coupled receptors.” This assumption is quite contrary to the other external chemical sensory receptors that are characterized by a simple phospholipid attached to an amino acid. It is quite likely the GPCR they have identified in the mouse pituitary is near or actually in the sensory neuron, but it is not likely to be the actual receptor. The actual receptor portion of the internal chemical sensory neuron is more like a phosphoaminoacid.

23.4.3.2.1 Configuration options in the neurons of the hypothalamus

It appears there are a number of options relating to the topology of the input neurons of the hypothalamus related to the H-P-T axis. These options affect whether these neurons are described as stage 7 neurons or some other hybrid designation. The key is whether these input neurons support both a sensory role at their poditic (inverting or subtractive) input terminal while simultaneously supporting a neural input from a stage 6 neuron at their dendritic (non-inverting) input terminal (Section 2.3.2 and Section 6.4 for additional details), or whether there are separate neurons supporting these different functions. In the later case, the initial sensing of these two different signals would be supported by separate neurons, one a typical stage 1 sensory neuron and one a typical stage 2 signal processing neuron. The differencing function would then be supported by a typical stage 2 signal processing neuron. This latter configuration would suggest the hypothalamus (as a whole) operates like a mini-brain within the H-P-T axis and possibly other axes as well.

Figure 23.4.3-3 shows the options discussed above. The lower frame describes a conceptual hybrid neuron of the hypothalamus. It totally integrates the functions of neural signal (processing), internal chemical sensing, and the production and releasing of TRH. TRH is presumed to be produced in the mitochondria, or other structures within the homeostatic portions of the cell separately from the neural compartments.

In the 9th edition of Werner & Ingbar, whole chapters are dedicated to explaining how various thyroxines and associated molecules are prepared based entirely on the chemical theory of the neuron. These chapters include multiple complex caricatures and frequently refer to the related chapters in the 8th edition (written by different contributors) and hundreds of citations for additional support. Among these discussions are many detailed discussions involving statements like, “The biochemical mechanisms resulting in H2O2 generation are controversial.”

In the 3rd Edition of Werner & Ingbar, a briefer discussion of these topics is presented that appears more understandable to most investigators familiar with the subject matter.

Actual understanding of the actions of the thyroxines in coordination with the neural and glandular receptors requires employment of The Electrolytic Theory of the Neuron and the concepts of coordinate chemistry. The TRH is released by the pedicle of the axon. The stage 6B decoder (Section 14.3 of PBV for a complete discussion of the 3B form) and Section 9.6 of this work for a discussion limited to the 6B designation) on the left is a conventional neural circuit providing an analog signal to the positive (non-inverting or dendritic) input to the hybrid. A more positive analog signal increases the output of TRH at the pedicle. The sum of the T3 and T4 concentrations in the blood stream is sensed at the negative (inverting or poditic) input to the hybrid. The sensing process involves the individual T3 and T4 molecules forming a temporary dual antiparallel coordinated bond (DACB) with a molecule presumed at this time to employ a second DACB binding site of phosphatidylTyrosine, PtdTYRd=5.534 (Section 23.4.3.5). This bond changes the electrical potential of the poditic terminal. An increase in the amount of T3 + T4 bound to the PtdTYRd=5.534 at any given instant reduces the amount of TRH released by the pedicle into the capillary channel extending to the hypophysis (pituitary gland). Hormonal-affectors 23- 67 The upper frame describes the same functional capability using discrete neurons for each stage involved in accepting a neural stimulus via stage 6B and a chemical feedback signal at a stage 1 internal chemical sensory neuron. The chemical feedback signal may be processed further via an optional stage signal processing engine before being delivered to the inverting (or negative feedback) terminal of the stage 7 neuro affector neuron. This stage 7 neuron accepts only electrical stimuli but generates and emits the TRH hormone at its pedicle into the capillary structure common to the hypothalamus and hypophysis (pituitary). There are an endless variety of circuit configurations that can be illustrated between the upper and lower frames. Only detailed histology and electrophysiology can confirm the precise circuit configuration used within the physiological context of Chordata.

] Based on the molecular similarity of T4 and T3 described in Section 23.4.3.5, it is likely that both molecules can form a DACB with PtdTyr d=5.534. Thus, their sum total concentration in the blood stream would affect the amount of TRH and TSH released.

TRH is transmitted from the hypothamus to the hypophysis (pituitary) via the capillaries within the ithmus between these two morphological entities. 68 Neurons & the Nervous System

Figure 23.4.3-3 Topological options for the neurons of the hypothalamus, specifically addressing the H-P-T axis. Top; Discrete neuron option. Bottom; Totally integrated hypothalamic neuron option serving the H-P-T axis. Intermediate options not shown explicitly. Other similar neuron topologies may be serving other axes beginning at the hypothalamus. See text. Hormonal-affectors 23- 69

23.4.3.2.2 Structure of thyrotropin-releasing hormone, TRH

Figure 23.4.3-4 illustrates the structure of TRH. TRH contains no glycol groups and no carboxylic group. It does contain three heterocyclic ring structures. It is clearly not a glycoprotein. Based on the common framework for all of the external chemical sensory neuron receptors in mammals (Section 8.4), it is very likely that the sensory neuron receptor of the pituitary employs a phospholipid that can form a DACB with TRH. The molecules of the proposed phospho-amino acid would likely compose a region of the outer leaf of the external type 2 lemma of a sensory neuron. The receptor molecule would be desribed as phosphatidylproline, PtdPro. Proline is usually considered one of the “standard” amino acids used to form proteins. However, it is the only such amino acid where the nitrogen atom is part of a five member ring. That fact makes it actually an α-imino acid instead of an α-amino acid. There are a great many alternate pairs of an oxygen atom and a nitrogen atom in the TRH molecule that could participate in a DACB with PtdPro. Such DACB bonds are coordinate bonds that only exist for a limited period of time. When broken, they leave the two participants in their original configurations. This property makes it difficult to demonstrate the existence of such DACBs in the laboratory. Phosphatidylproline is very likely to be the molecule Straub et al. described as TRH-R in 1990. It is neither a protein nor is it defined by a cDNA sequence of 393 amino acids.

Ganong (page 267) has labeled TRH a tripeptide, (pyro)GLU-His-Pro-NH2, after considerable rearrangement. 23.4.3.3 Functional features of the hypophysis (pituitary)

Sandman et al, writing in chapter 1 of Hrdina & Singhal66 have noted, “Several peptides- based hormones (MSH, ACTH and endoorphins) can be derived from a larger neuronal glycoprotein of about 31,000 daltons.” The strikeout has been added because of lack of relevance and age of the assertion. “Further, MSH (which share a behaiourally acive segment with ACTH) and the endorphins are contained within the structure of the 90-amino-acid-chain lipotropin, LPH. It is logical to assume the hypophysis contains individual elements that extract the individual named hormones from the prohormone, LPH. The result suggests each of these extracted hormones consist of less than 90 peptide residues. This would suggest the figure of Labrie et al. in Section 23.3.3.1 should be expanded to show the Figure 23.4.3-4 TRH, the thyrotropin-releasing creation of protohormones in the hypophysis hormone. The distance between the oxygen before developing the hormones, to be atom and the nitrogen in the 5 sided ring at lower secreted, from these protohormones. right is 2,739 Angstrom. This is a good match for a DACB of L-proline with a spacing between a Wiegant & Wied provide the code for these similar pair of atoms of 2.749 Angstrom. molecules in chapter 2 of Hrdina & Singhal. Alternately, there are a variety of such pairs available with similar distances between them in TRH. See text. 23.4.3.3.1 Functional representation

66Hrdina, P. & Singhal, R. (1981) Neuroendocrine regulation and altered behavior. London: Croom Helm 70 Neurons & the Nervous System of the hypophysis–TRH in, TSH out

The fabrication of TSH as controlled genetically is addressed by Cohen & Wondisford (Chapter 10A of Werner & Ingbar, 9th Edition). It infers a structure for TSH based on the chemical theory of the neuron. The knowledge base relating to TSH has not changes significantly since the 3rd Edition of Werner (chapter 10). It is reported to be a very complex glycoprotein (molecular weight ~28,000 to 30,000) without any significant details. Vassart & Costagliola address the TRH receptor (Chapter 10B of Werner & Ingbar, 9th Edition). The chapter infers a structure for TRH based on the chemical theory of the neuron. The reaction of TRH at the surface of the hypophysis is not addressed in their discussion. TRH is described as a much more complex glycoprotein than shown in the above figure. Chapter 10B does not address how their TRH causes the hypophysis to produce TSH; no equations are presented for the process. - - - Discussion of actual control of TSH fabrication. xxx ADD The thyroid-stimulating hormone, TSH, is also known as a thyrotropin. The fabrication of TSH as controlled genetically is addressed by Cohen & Wondisford (Chapter 10A of Werner & Ingbar, 9th Edition). It infers a structure for TSH based on the chemical theory of the neuron. The chapter infers a structure for TSH based on the chemical theory of the neuron. It does not address how TSH causes the thyroid to produce T4; no equations are presented for the process. The TSH molecule they describe is at least an order of magnitude higher in molecular weight than the TSH molecule shown in figure xxx. Cohen & Wondisford describe TSH as a trans-lemma G-protein.

The knowledge base relating to TSH has not changes significantly since the 3rd Edition of Werner of (1971, chapter 10). It is reported to be a very complex glycoprotein (molecular weight ~28,000 to 30,000) without any significant details. The 3rd edition of Werner & Ingbar (figure 12-1) does provide a more detailed rendition of the outputs of the hypophysis (pituitary) than presented in Section23.3.3.1 of this work.

Figure 23.4.3-5 illustrates the potential functional arrangement for each axis of the hypophysis. It is illustrated for the H-P-T axis. The unique situation that stands out is the potential that the hypophysis receives input via two distinct chemical channels and secretes a chemical output into a chemical channel while simultaneously exhibiting the internal operations of a neuron. For purposes of discussion this is labeled a stage 8 hybrid neuron.

The output of a stage 8 neuron is characteristically and functionally a stage 7 neuroaffector. Simultaneously, its non inverting (dendritic) input is that of a stage 1 sensory neuron, incorporating all of the features of a sensory neuron receptor. This situation brings into focus the inverting (poditic) inputs of the stage 7 hypothalamus and the stage 8 hypophysis. The poditic inputs resemble the conventional stage 1 chemical sensory receptor input structures. How should the hypothalamus be considered in the hierarchy of stages assigned to the neurons? As in the case of the hypothalamus of the earlier figure, there is not sufficient information available related to the hypophysis to define its internal functional characteristics uniquely. Thus both a potential “totally integrated” configuration is described at the bottom of the figure and a discrete neural configuration is shown at the top of the figure. Additional configuration can be described that span the range between these two configuration. These configurations are all compatible with the signal flow diagrams presented in Section 23.4.3.1. One of the features of the neural/glandular interface is highlighted in these figures; 1) The -tropin family of hormones generated by the hypothalamus all travel to the hypophysis via the portal capillaries rather than the more general bloodstream. This distinction brings into question whether these two entities are functionally separate or are just morphologicaly separate, and 2) the fact that the stimulating hormones, i.e., TSH etc, are more complex molecules than found in a majority of both the external and internal chemical stimulants.. The more complex molecules of the stimulating hormones appear to require a more Hormonal-affectors 23- 71 complex receptor than the typical phospholipid amina acid utilized for all of these other stimulants. 72 Neurons & the Nervous System

Figure 23.4.3-5 The potential Hypophysis of the H-P-T axis as an exemplar. As in the above description of the hypothalamus, the literature does not provide sufficient information to define a unique internal arrangement for the hypophysis. The bottom frame of this figure shows a potentially totally integrated hypophysis. The top frame shows an alternate configuration where individual neural functions associated with one axis of the hypophysis are shown separately. A series of intermediate configurations can be defined. The selection of the correct configuration awaits additional data. The unique combination of chemically sensitive input structures and the secretion of hormones by its output structure causes the individual portions of a hypophysis (pituitary gland) to be considered a uniques stage 8 hybrid neuron. See text. Hormonal-affectors 23- 73

A second question arises, as initially surfaced in Section 23.4.3.1, is there a significant difference in the apparently redundant feedback paths proceeding to the hypothalamus and the hypophysis? The discrete hypophysis configuration at the top of the figure accommodates such a possibility. It is shown here as a potential electrical circuit within a stage 2 signal processing that provides a band limiting capability that would normally be associated with a time delay in this circuit. An option would be that this capability is provided, outside of the neuron complex, by the caliber of the cardiovascular elements providing the feedback paths to either the hypothalamus or hypophysis.

23.4.3.3.2 Structure of thyroid-stimulating hormone, TSH & its receptor

The literature has not addressed the thyroid stimulating hormone, TSH, in detail as of 2018. The focus of the last decade has been conceptual in character and has not resolved how the hormone or its receptor work. Szkudlinski et al67. have provided a major review in 2002. “—This review focuses on recent advances in the structure-function relationships of thyroid-stimulating hormone (TSH) and its receptor.” The complete review is based on the chemical wisdom of the 20th Century and based on the chemical theory of the neuron. It describes TSH as a large protein with a molecular weight of 28,000 to 30,000 Da. They note on page 485, “The TSHR together with LH and FSH receptors are related members of rhodopsin/-adrenergic receptor family in the seven-transmembrane domain, GPCR superfamily. The thyroid-stimulating hormone receptor, TSHR, is critical in the development, growth, and function of the thyroid. In contrast to many other subfamilies of GPCRs, the TSHR and other glycoprotein hormone receptors contain a large 300- to 400-amino acid-long extracellular domain with at least 8 highly conserved Cys residues, involved in formation of extracellular domain tertiary structure that appears important in both ligand binding and inactive receptor conformation. Only selected particularly new aspects of TSHR are discussed here. Several other aspects of TSHR studies, including its relation to Graves’ disease, are summarized in several comprehensive reviews (six citations).”

They then proceed to describe the short-comings of their baseline TSH and TSHR,

“There are several important functional differences between TSHR and other glycoprotein hormone receptors. First, TSHR is more frequently activated by gain-of-function mutations than gonadotropin receptors. Second, TSHR is unique among glycoprotein hormone receptors in that some mature receptors on the cell surface are cleaved into two subunits. Third, unlike the single-chain gonadotropin receptors, TSHR is considered to be ‘noisy,’ transducing a signal via adenylate cyclase even in the absence of ligand (17). Fourth, similar to the thrombin receptor, proteolytic degradation or small deletion in the extracellular domain of TSHR can lead to its activation. Although the LH receptor seems to be activated by proteolytic enzymes as well, TSHR seems to be much more prone to such activation. Fifth, unlike many other GPCRs, including the FSHR where guanine nucleotides reduce agonist binding, TSH binding to TSHR did not change in the presence of a nonhydrolyzable GTP analog. Finally, (Sixth)our studies on hTSH superactive analogs, including analogs with multiple mutations, demonstrate that the increase in receptor binding activity and in vitro bioactivity is generally parallel, suggesting that there is no clear distinction between recognition and activation binding sites in the TSHR. (Seventh), in contrast to the LH and FSH receptor, there are two unique insertions in the TSH primary structure. The first 8-amino acid insertion near the NH2 terminus (residues 38 – 45) was shown to be important for TSH and stimulating antibody binding, whereas a second 50-amino acid residue insertion (residues 317 – 366) has no apparent effect on TSHR function. (Eighth) The cluster of four cysteine residues at positions 24, 29, 31, and 41 was recently shown to be involved in formation of highly conformational epitope for thyroid stimulating antibodies. Two highly conserved cysteines in extracellular loops 1 and 2 of TSHR are also predicted to form a disulfide bond as identified in bovine rhodopsin structure.”

67 Szkudlinski, M. Fremont, V. Ronin, C. & Weintraub, B. (2005) Thyroid-Stimulating Hormone and Thyroid-Stimulating Hormone Receptor Structure-Function Relationships Physiol Rev vol 82, pp 473–502; 10.1152/physrev.00031.2001 74 Neurons & the Nervous System

This number of difficulties with their proposed hormone and receptor suggest alternate theories might be worthy of exploration! Their eighth problem might help highlight alternate concepts. The focus on the size of their TSH molecule and its 8 highly conserved Cys residues might suggest their proposed hormone plays a role similar to thyroglobulin in the generation of the thyroxines. In thyroglobulin, a number of tyrosine residues exhibit a similar prominence leading to their iodonation. After iodonation, the various thyroxines are assembled as described in Section 23.4.3.4. Under a section labeled “V(C). Naturally Occurring TSHR Mutations,” they note additional difficulties yet to be resolved with respect to their TSH. They note a wide variety of genetic mutations that have been associated with their TSH/TSHR interface but have not defined the specific structural problems in either of these molecules. Under a section labeled “V(E). Other Concepts of TSH-TSHR Interaction” they note,

“Despite more than 20 years of intensive studies, the nature of TSH interaction with TSHR and the mechanism of transmembrane signaling remain largely unknown in part due to the absence of information regarding receptor structure. . . . However, the exact spatial relationships of ligand and receptor are still unknown, and different models have been proposed (97). Similarly, there is no consensus regarding whether the extracellular loops of the receptor participate in direct hormone-receptor interaction.”

Under a section labeled “V(D), Suppressive Effect of Extracellular Domain: The ‘Two-State’ Model” they sought to introduce an alternate model to their baseline but continued the conceptual framework of the genetics community. Within that section, they noted,

“In our study, constitutive activity has been demonstrated for the first time for truncated TSHR lacking 98% of the extracellular domain (citing a paper by members of the same team68 .” This finding should be enough to cause the team to seek a different TSHR structure than a member of the GPCR family.

Under sections “VI( C). Perspectives in the Engineering of TSH” and “VII. New Concepts and Technologies”, Szkudlinski et al. discuss a variety of paths to pursue in broad terms and largely conceptual form.

- - - -

The above comments from Szkulinski et al. do not provide a satisfactory understanding of the portion of the H-P-T axis related to the thyroid-stimulating hormone, TSH, or its receptor associated with the pituitary gland.

Absent additional laboratory-based information, little can be provided here about the more likely case of an amino acid-based TSH and TSH receptor. However, it can be stated that all other external sensory neural receptors (gustation, olfaction, oskonation), and all other internal sensory neural receptors of mammals (and specifically humans) explored by this investigator have as of 2018 relied upon sensory receptors involving simple derivatives of amino acids, with a molecular weight of less than 500, joined to a phospholipid that does not by itself penetrate the outer lemma of a sensory neuron, See Appendix ZA, Table of Phosphoglycerides. [xxx is ACTH an exception due to the genetics community. Tuncel has published a paper concerning potential thyroid cancer and the role of the thyroid-

68Zhang, M. Tong, K. Fremont, V. Chen, J. Arayan, P. Puett, D. Weintraub, B. & Szkudlinski, M. (1999) The extracellular domain suppresses constitutive activity. . . Endocrinology vol 141, pp 3514–3517 Hormonal-affectors 23- 75 stimulating hormone receptor from the medical perspective69. While providing a large bibliography, he does not develop the structure of TSH or the TSH receptor. 23.4.3.4 Functional features of the Thyroid 23.4.3.4.1 Functional representation of the thyroid– TSH in T4/T3 out

Arvan & Di Jeso have addressed the formation of T4, and/or T3 from thyroglobulin in Chapter 5 of Werner & Ingbar, 9th Edition. Thyroglobulin, Tg, is a very large glycoprotein, with a “molecular mass of its monomeric structure (~330 kDa). Approximately 10% of this mass (i.e., ~30 kDa) consists of covalently bound carbohydrates, the result of post translational modification as Tg passes along the intracellular secretory pathway of thyroid follicular cells (thyrocytes).” “The percentage of tyrosine residues in Tg that become iodinated normally varies with the dietary iodine supply, but it is not unusual for 20% of them to contain iodine, with three quarters of these as residues of monoiodotyrosine (MIT) and diiodotyrosine (DIT) that are not used for the formation of T4 or T3.” Although not stated, the preceding assertion relates to a given pass of Tg along the follicular cells. MIT and DIT are precursors of T3 and T4 within the thyroid gland. “Thus, Tg protein serves as the primary internal reservoir of recycling iodine in the body, upon which future biosynthesis of thyroid hormones is based.”

Arvan & Di Jeso give a complete genetic code for Tg of mice. They also annotate this code with a series of mutations or substitutions. They do not address the reason for these changes. The reasons are probably beyond the detailed understanding of the genetic code as of 2005. Page 81 describes many details of the Tg decomposition and the initial folding of the excised proto-hormones that have been observed. The remainder of the chapter are observational in character with several cartoons showing potential mechanisms and alternatives.

Tropp gives a very complete review of the genetic code, its operation and potential modifications as of 200870. However, even this edition is out of date with respect to epigenetics. It devotes only one sentence to epigenetics (Page 741). 23.4.3.4.2 Structures of major thyroid molecules

[xxx need to add text to accompany these figures

T4, thyroxine is not the major active output of the thyroid gland, T3 is as shown in the next figure. However both T3 and T4 may be produced by partial decomposition of Tg and stored within the thyroid gland. Both T3 and T4 are derivatives of the amino acid tyrosine and not glycoproteins; see the discussion in Section 23.4.1 for more on this question of definition.

By merging one molecule of MIT and one molecule of DIT, as these molecules are described by Arvan & Di Jeso, followed by condensation of these two moieties at their hydroxyl groups, and then replacement of the alanine, associated with the MIT by a hydroxyl group, a molecule of T3 is obtained directly. Figure 23.4.3-6 illustrates the major molecules of thyroxine. Tyrosine is the amino acid precursor for this molecule. It is called tyrosyl when referred to as a functional group or side chain. It is widely available in high protein foods. An alternate means of initiating the preparation of T4 beginning with the condensation of two DIT molecules is offered above the hashed line for discussion purposes. It is immensely simpler than the synthesis described conceptually from thyroxoglobulin in Chapter 7 of Werner & Ingbar, 9th Ed. Following condensation, the left; most amino acid, alanine, is removed via an alanine hydroxylase. The positions of the iodine atoms are not shown precisely.

69Tuncel, M. (2017) Thyroid Stimulating Hormone Receptor Mol Imaging Radionucl Ther. Vol 26(Suppl 1), pp 87–91 doi: 10.4274/2017.26.suppl.10

70Tropp, B. (2008) Molecular Biology: Genes to Proteins, 3rd Ed. Boston, MA: Jones and Bartlett 76 Neurons & the Nervous System

The left-most phenol is added to the tyrosine by condensation. Removing the specific iodine at C5', to achieve the active species, T3, would appear to be a challenge for the appropriate enzyme wherever it might be encountered within the mammalian body. [xxx edit re T2 & revolving part around single bonds See also sec 23.4.3.5.3 & rationalize ] Since the major portion of the T3 and T4 molecules are formed from the amino acid, tyrosine, it is to be expected that tyrosine and either T3 or T4 can easily form a DACB with a receptor described as phosphotidyltyrosine, PtdTyr. A complication arises from the figure. All four of the molecules shown can form DACBs with PtdTyr d=5.524. This situation can complicate evaluation of the operation of the servo loops relating to the hypothalamus as well as the pituitary. The PtdTyrd=5.524 receptors will respond to the topical application of T4, T3, reverse T3 and even T2 (3,3- diiodothyronine). This complication will not arise in other areas of the mammalian physiology as the receptors specific for the active form of thyroxine, T3, will involve other DACB relationships and not be sensitive to T4, reverse T3 or T2. Iodine is a high molecular weight halogen with more than enough electrons in its outer shell to form a complete octet. As such it can form coordinate bonds easily, like oxygen, nitrogen, sulphur and occasionally phosphorus. Its role in coordinate chemistry is well known particularly in conjunction with the metals and phosphorus71, but also with oxygen and the benzenes. The coordinate bonds that might give T3 its unique hormonal properties will not be pursued here, but the unique position of the iodine atoms and the oxygen atoms, with respect to coordinate chemistry, can not be ignored. As Lee noted, “The term hypervalent was established in1969 for molecules with elements of groups 15-18 bearing more electrons than an octet in their valence shell.” The electron shell arrangement of any atom is usually found in any physical chemistry text72. She did not use the expression coordinate bond in her extensive paper.

Brent has provided a geneticists view of how T3 operates as a hormone73. His explanations are probably over complex due to their reliance on the chemical theory of the neuron. He notes,”The broad range of genes whose expression is modified by thyroid hormone status makes studying the effect of thyroid hormone action a daunting challenge.” Recognition of the potential role of iodine and oxygen atoms of T3 in coordinate chemistry reactions is likely to simplify the interpretation of the role of T3 as an active hormone.

The active T3 molecule is assembled in the order, MIT + DIT + Alanine. The inactive, i.e. reverse T3 is assembled in the (reverse) order DIT + MIT + Alanine. Ganong74 presents a figure similar to the above as his 18-6. He also present a Table 18-4 showing the efficacy of these molecules in different situations

Figure 23.4.3-6 shows the numbering method used for the thyroxines.

71Lee, S. (2003) The chemistry of hypervalent iodine MacMillan Group Meeting https://www.princeton.edu/chemistry/macmillan/group-meetings/SL-iodine.pdf

72Maron, S. & Lando, J. (1974) Fundamentals of Physical Chemistry. NY: McGraw-Hill Table 3-7

73Brent, G. (2012) Mechanisms of thyroid hormone action J Clin Invest vol 122(9), pp 3035–3043 doi: 10.1172/JCI60047

74Ganong, (1973) Review of Medical Physiology, 7th Ed. Los Altos, CA: Lange Medical Publications Hormonal-affectors 23- 77

Figure 23.4.3-6 Numbering pattern used for thyroxine molecules. The angle between the plane of the benzene ring β and the plane of the benzene ring, α, is 110 degrees in the actual molecule. Note the amino acid designation at lower right compared to Alanine. Tyrosine is the parent of the thyroxines. The upper designation recognizes the origin of the β-ring is phenol and not a glycogen. Note the resonance indicator (circle) surrounding the labels, α and β. See text. From Braverman & Utiger, 2005

Figure 23.4.3-7 Major molecules of thyroxine & potential original condensate. D-labels correspond to individual enzymes. Potential source is shown above the hashed line (without regard to the position of the iodine atoms). See text. Modified from Braverman & Utiger, 2005.

23.4.3.4.3 Synthesis of glandular thyroxine & structure of thyroxoglobulin

The synthesis of thyroxine is still being studied as of 2018. Chapter 4B in the 9th Edition of Werner & Ingbar is dominated by an overly complex caricature, figure 4B.1, attempting to rationalize many individual concepts. Figure 4B.11 shows one proposed method of creating thyroxine within the structure of thyroxoglobulin,Tg. The concept displayed involves the iodization of two tyrosine residues along the peptide chain of Tg before their condensation into one molecule of T4. How the molecule of T4 is freed from the parent Tg molecule is not discussed but a referral is offered th to the 8 Edition of Werner & Ingbar. Frame 4 shows a permanently changed form of Tg. Both the 3rd & 9th Editions describe the ioination of Tg in words. The question is whether the iodination of the tyrosine residues along the peptide chain is a statistical event, with some tyrosine molecules acquiring two iodine atoms thereby forming diiodotyrosine, DIT, and others acquiring only one thereby forming mono-iodotyrosine, MIT. In the 3rd Edition, Tong wrote on page 27, “the Tg polypeptide consists of 5650 residues as a protein of which about 125 are tyrosyl units (possibly only 66 in human Tg based on Arvan & Di Jeso (page77).” Tong further notes “However, the iodine content of Tg is quite variable and is closely related to the iodine intake of the subject. When the Tg contains one percent by weight of iodine, only about 10 fo the tyrosyl residues occur 78 Neurons & the Nervous System in the form of MIT an six in the form of DIT. Under these conditions, the Tg will contain not more than five T4 residues per molecule.” The terminology in the chapter by Arvan and Di Jeso, writing in Werner & Ingbar’s Thyroid, 9th Edition, is distinctly different from that of other recent papers, and other chapters in the same edition. Arvan & Di Jeso do provide significant statistical information relating to thyroxoglobulin,Tg, page 77. They assert, “Tg protein serves as the primary internal reservoir of recycling iodine in the body, upon which future biosynthesis of thyroid hormones is based.”

At one point in chapter 7 of Werner & Ingbar’s 9th Edition, it is noted that the basic structure of T4 appears to be formed from a dimer of the amino acid tyrosine. Tyrosine is one of the 20 or 22 standard amino acids but is not an “essential” dietary amino acids. More likely, T4 is derived from two tyrosine molecules by condensation at the location of the hydroxyl groups followed by the replacement of one of the amino groups by a hydroxyl group. This is a much simpler synthesis of T4 than discussed in connection with the cited figure. The specific sequence and any intermediate steps in the synthesis may depend on energy considerations. A question appears to remain unanswered, are the peptides of iodonated tyrosine freed from the Tg prior to their condensation into a two benzene ring structure or do they remain within the Tg molecule until they have formed a complete moiety consisting of two benzene rings and two alanine residues as well? While it is obvious that Tg is significantly restructured following release of the thyroxine molecule, it is possible that one alanine residue remains in position to replace a tyrosine residue. While Arvan & Di Jeso note (page 86), “It is nearly impossible to study iodinaton and hormonogenesis in human Tg in vivo.”, They do proceed to describe a variety of options.

th Kopp (9 Edition, page 67) provided a “Proposed scheme for the formation of T4 within the Tg th protein before the extraction of T4 leaving a denatured Tg in his frame 4. He cites the 8 Edition, page 61as the source of the graphic. Kopp’s own graphic (9th Edition, figure 4B.1, of thyroid hormone synthesis is a hopelessly complex cartoon. 23.4.3.4.4 Synthesis of T3 from T4 in peripheral tissue

As noted by Braverman & Utiger, only about 20% of the active hormone T3 is secreted from the thyroid gland. The remainder is formed from T4 in the peripheral circulation. The process involves a series of enzymes as noted by the designations, D1 through D3 in [Figure 23.4.3-6]. Section 23.4.3.4.5 define these enzymes 23.4.3.4.5 The deiodinases enzymes– D1, D2, & D3

Bernal, writing in The Encyclopedia of Hormones (2003)– page 392, develops the properties of the deiodinases in considerable detail. The Abstract to that entry asserts,

“Deiodination, the most important metabolic pathway of thyroid hormones, is catalyzed by deiodinases, which contain the amino acid selenocysteine in their active center. Three types of deiodinase have been identified in vertebrate tissues: D1, D2 and D3. Deiodinase activity is regulated during development and by physiological factors including the thyroid hormones. D1 and D2 are expressed postnatally and in the adult; D3 is expressed at high levels in placenta, pregnant uterus, and fetal tissues. Deiodinases appear to play a role in regulating intracellular T3 levels in a tissue-specific manner and in coordinating vertebrate growth and development.” Much of the text involves detailed discussion of the many metabolites involved, including their genetic origins. However, there is little proof that the various production sequences are actually those used within the living animal. Table 1 of the entry in the Encyclopedia of Hormones is valuable. Figure 2 is virtually the same as [Figure 23.4.3-6]. The entry closes with its own Glossary.

23.4.3.4.6 Clearance rates for T3 and T4 in humans

Oppenheimer et al. have provided a dated study of the clearance rates of the thyroxines for Hormonal-affectors 23- 79 different organs of the body75. Generally, the clearance rate of T3 is 2.4 times faster than for T4. However the relationship is not a fixed number. The applicable models are complex. As an example, L-thyroxine is typically 6-7 days for euthyroid individuals, but 3-4 days for hyperthyroids and 9-10 days for hypothyroids (Section 23.4.4.5).

23.4.3.5 Structure-function-relationship of the thyroxines

Chapter 9 of Wegner & Ingbar’s Thyroid, 9th Edition is devoted to the discussion of the thyroid hormone structure-function relationship, SFR. On page151, Cody notes, “Although thyroid hormones elicit a multitude of biologic responses, the specific nature of their actions remains unclear.” The geometry of the family of hormones is then described in considerable detail. During a review of th material, no discussion of potential coordinate binding of a hormone and a target was found. Other texts and papers discuss the structure-activity-relationship, SAR, in physiology versus the SFR. The difference appears to be one of convenience. The term SAR will be used subsequently in this work. Based on the proposal and likelihood that TRH acts through a DACB coupling at the pituitary gland internal chemical sensory neuron receptor (section 23.4.3.2.2), it is proposed that T3 acts through a DACB when interacting with the broadly dispersed molecules of the physiological system (with no restriction that the cells be neurons.

T3, Triiodothyronine, a.k.a. liothyronine has the following specifications, liothyronine

Molecular Formula C15H12I3NO4 Average mass 650.974 Da Monoisotopic mass 650.790039 Da ChemSpider ID 5707

Figure 23.4.3-8 shows Two variants of 3D representations of the thyroxines. Neither of these representations is to scale.

75Oppenheimer, J. Schwartz, H. Shapiro, H. et al. (1970) Differences in Primary Cellular Factors Influencing the Metabolism and Distribution of 3,5,3'-L-Triiodothyronine and L-Thyroxine J Clin Invest vol 49, pp 1016- 1025 80 Neurons & the Nervous System

Figure 23.4.3-8 3D representations of the thyroxines ADD. From Cody et al., 1986. Top; Forms of T4, T3 & reverse T3 displayed in ball and stick form. T3 is the only active hormonal form. Bottom; a more complete set of thyroxines in filled form. T3 remains the only active hormonal form. I3' and OH4' are the only active atoms within T3 that can form a DACB with other molecules. When present, it is proposed that I5' effectively shields O4' from participating in such a DACB. See text. From Cody et al., 1986. Hormonal-affectors 23- 81

The 3D configuration of T3 as presented by Discovery Studio 4.1 software based on the JSmol file of the Royal Society of Chemistry, RSC, does not resemble the configuration described in the above figure and in tabular form in Werner & Ingbar, 9th Edition. The caution presented in Section 8.6.1.6.3 and in Section 23.4.2, related to the JSmol files of the RSC applies here). The distance between the OH4' group and I3' is given as 3.123 Angstrom based on a flat six-sided ring rather than a more precise 3.233 Angstrom expected in a true representation. This value was obtained from a 3D representation described in the next sub-paragraph. While both the ball and stick and the space-filled representations of T3 & T4 in the above figure do show a twist of 110 degrees between the two benzene rings, the similar representations, figure 7-2 and 7-3 in the 3rd Edition of Werner & Ingbar are much more definitive. Specifying the dimensions of an atom is difficult and depends on the method used. The value is usually determined indirectly by calculating the distance between two atoms of the same type in a covalent molecular arrangement. Based on this method, the diameter of the iodine atom (2.66 Angstrom) is much larger than the oxygen atom (1.48 Angstrom)76. The carbon-iodine bond length is given as 213 pm or 2.13 Angstrom but this may vary for a carbon involved in a six-sided non-conjugated ring. 23.4.3.5.1 Potential DACB between T3 and a PtdCys receptor on any cell.

Jorgensen77, as reported in the 3rd Edition of Werner & Ingbar (figure 7-6), has presented the historical assumption concerning the throxines and a putative receptor site. It is based loosely on the conventional chemical theory of the neuron and valence chemistry. These concepts are archaic and should be discarded in favor of The Electrolytic Theory of the Neuron and the DACB binding concept from coordinate chemistry

By inspection, the most likely DACB involving T3 and a receptor site on/in a neuron is that involving I3' and OH4'. Using 2-iodocyclohexanol_158842, C6H11IO, as a template, the distance (d-value) between these two chemical constituents is 3.233 Angstrom. The 3D rendition used Discovery Suite 4.1. Looking for a phosphatidyl-amino acid exhibiting this d-value, a potential candidate is a simple or modified form of cysteine. The unmodified form exhibits a d-value = 3.219 Angstrom. For discussion purposes, a cellular receptor formed of a type 2 region of outer cell lemma consisting of a block of phosphatidylcysteine, PtdCys, molecules should be considered.

No data has been located on how closely the d-values of the stimulant and the receptor must be to be effective. As used in the gustatory (Section 8.5) and olfactory modalities (Section 8.6), the match need not be too perfect. These modalities use the receptors in an analog mode, where the degree of coupling is a useful parameter. 23.4.3.5.2 The sensitivity of physiological cells to T3 in the bloodstream

Based on the DACB joining a T3 molecule with any cell on a short term basis and then releasing the T3 molecule unchanged, the cells containing the putative PtdCys receptor are sensitive to the average concentration of T3 in the bloodstream and matrices serving all cells in the body exhibiting suitable receptors.

23.4.3.5.3 Potential DACB receptor compatible with both T3 and T4

[xxx rationalize the d-values here and in Section 23.4.3.4.2 ] As shown in the servo loop figures, Section 23.4.3, both T3 and T4 are known to be able to suppress the output of the hypothalamus and of the hypophysis (pituitary). The most likely location of a

76Barrow, G. (1966) Physical Chemistry, 2nd Ed. NY: McGraw-Hill page 390

77Jorgensen, E. (1964) Stereochemistry of thyroxine and analogues Mayo Clinic Proc vol 39, page 560 82 Neurons & the Nervous System

DACB between both the T3 and T4 molecules and an internal chemical receptor appears to be the two iodine atoms at I3 and I5. While these two atoms would be expected to have the same outer shell configuration, the shells are not complete when joined to the six-sided ring. It is therefore possible for one iodine atom to contribute an electron to a DACB while the other atom accepts an electron. One of these atoms could combine with a hydrogen atom to support the anticipated hydrogen bond expected of the T3 and/or T4 moieties in a DACB. The distance (d- value) between the two iodine atoms would be expected to be 5.783 Angstrom (based on Ipodate_5051 with a conjugated benzene ring instead of a fully saturated cyclohexane ring). A phosphatidyl amino acid supporting this d-value in a DACB might be phosphatidyltyrosine, PtdTyr, with a d-value of 5.534 Angstrom. While the d-values are only close, the precision of the numbers is not good. Future 3D models of the thyroxines should give more accurate d-values. 23.4.3.6 Summary of hormones, receptors & flow diagrams of the H-P-T axis

Figure 23.4.3-9 shows a tabulation of the hormones, receptors, and receptor locations along the H-P-T axis.

Figure 23.4.3-9 Hormone tabulation & receptor locations along H-P-T axis. All receptors are an amino acid of the lipid, phosphatidic acid. The receptor PtdTyr is tentative, while awaiting confirmation of the DACB value involved in sensing both T3 & T4 in the feedback portions of the servo loops of the H-P-T axis. T4 is generally circulating in the cardiovascular system until it is captured at a location capable of converting it to the active form, T3. T3 is a major hormone used to stimulate a great variety of cells in the physiological system of Chordata, and possibly other Classes of taxonomy as well. See text.

Similar tables to the above figure can be assembled for other axes of the hypothalamus as well as for the external chemical sensory receptors. A comprehensive summary of all of the receptor molecules consisting of a phospholipid combined with an amino acid by condensation, regardless of modality in which used, will be found in Appendix ZA, Table of Phosphoglycerides.

Figure 23.4.3-10 presents a proposed new and more precise representation of the Flow Diagram of the thyroxine system. It includes the receptors and their locations within the diagram. Hormonal-affectors 23- 83

Figure 23.4.3-10 2018 Flow Diagram of the H-P-T axis with receptors and their location ADD. The dashed feedback path to the pituitary gland can not be confirmed based on the available literature. It appears to be unnecessary from the feedback theory of electrical engineering. There does not appear to be creditable information in the literature concerning the structure of TSH. This situation prevents the TSH receptor within the thyroid gland to be defined precisely. See text.

The figure starts with a synapse between the stage 6B decoding neuron on the left and the stage 7 neuroaffector neuron within the hypothalamus. This neuroaffector is shown as an integrated functional element as shown in the lower portion of the figure of Section 23.4.3.2.1.

On theoretical grounds, the feedback path can deliver a range of thyroxines to the hypothalamus and the hypophysis. This is based on the fact that many thyroxines can interact with a receptor with a d-value on the order of 5.534 Angstrom. This value assumes the thyroxines containing a diiodotyrosine structural moiety can form a DACB with a PtdTyr as shown. The d- value of the diiodotyrosine moiety is not known precisely because of the theoretical rotational freedom between the benzene ring and the aliphatic chain as indicated in [Figure 23.4.3-7]. The DACB is assumed to involve either I3 or I5 (or a rotational average position for both) and nitrogen 1 2 attached to C8. The preferred angles, χ and χ under biological conditions must be determined before a definitive choice of receptor can be made.

Note the feedback path delivers a broader range of molecules compatible with the PtdTyr d=5.534 Angstrom receptor than shown in earlier flow diagrams of Section 23.4.3.1. This is plausible since the efficacy of the additional thyroxine, T2, is known to be quite low. As a result, its participation in the feedback process may have been overlooked. As noted in the caption, whether the feedback path to the pituitary gland actually exists has not been demonstrated. It is redundant and not required based only on the feedback theory of electrical engineering unless it incorporates a transport delay, bandlimiting or other functional element, Z. If this second path exists, it can be expected to employ the same receptor as in the case of the path terminating at the hypothalamus. - - - - Wikipedia (as of 2018) suggests such a functional element may exist in the feedback loop within the H-P-T axis. It suggests, “many substances secreted within this axis exhibit slow/intermediate and fast feedback-loop activity. . .The latter (secretion of ACTH from the pituitary) is an example of a slow feedback loop, which works on the order of hours to days, whereas the former (secretion of CRH from the hypothalamus) works on the order of minutes. The half-life of ACTH in human blood is about ten minutes. This short lifetime would suggest the H-P-E axis probably employs a different circuit configuration for the feedback applied to the pituitary. 23.4.4 Diseases of the H-P-T axis

When reviewing the vast medical and academic literature about diseases of the thyroid gland, the literature has not changed much since the 1960's (from the perspective of this author and recent suffer). While the complex closed loop(s) structure of the thyroid gland system is becoming 84 Neurons & the Nervous System better documented, the trend is to assert such diseases are probably of the auto-immune type. This label is only marginally stronger than asserting the disease is idiopathic, of unknown cause! While many investigators have listed over 100 citation in support of their papers, most of the papers reflect a “case study” character that leads to a narrow view of the overall functional and dis-functional nature of the disease being reported. It becomes very difficult to distill a single consistent model of the overall H-P-T axis. [xxx Append E Disease.wpd may be supportive here, even in its primitive form ] It is probable that the general classification of thyroid disease contains more than the conventional labels, hyperthyroid, hypothyroid etc. While each of these labels is subdivided, particularly in the clinical arena, a consistent framework and set of symptoms associated with each category is particularly difficult to define. In some cases, a particular set of symptoms may point to a distinct disease outside the conventional framework. Circumscribed pretibial myxedema appears to be such a separate though related disease. It is associated with both hyperthyroid and hypothyroid disease. Some authors have treated it as a distinct disease under the designation of primary myxedema. When considering the potential cause of the various thyroid diseases, it becomes important to introduce the subject of goiter versus non-goiter forms of the disease. These considerations call for an expanded flow diagram beyond the nominal, steady-state situation introduced in the previous figure of Section 23.4.3.6. Figure 23.4.4-1 provides such an expansion. This figure has been expanded to accommodate the presence of lymphocytes. Based on the literature of Kraiem et al., the lymphocytes appear to play an important role in controlling the movement of the constituents and final products of the thyroid.

Figure 23.4.4-1 Expansion of the thyroid elements of the H-P-T axis relative to disease ADD.

The label, lymphocytes, is not clearly defined in most papers because of their ubiquitous nature. Quoting the National Library of Medicine (USA), “ A lymphocyte is a type of white blood cell that is part of the immune system. There are two main types of lymphocytes: B cells and T cells. The B cells produce antibodies that are used to attack invading bacteria, viruses, and toxins. The T cells destroy the body's own cells that have themselves been taken over by viruses or become cancerous.” Recently a third designation has been appearing, “natural killer cells,” Lymphocytes (a traditional name) are not associated with the lymphatic system. The separate and distinct lymphatic system is poorly defined in most papers relating to the auto-immune system. The lymphatic system is fundamentally a scavanging system designed to recover blood plasma provided to body tissue by the arterial system and not fully recovered by the venous system. The lymphatic system collects this plasma, accumulates it in a cistern and then returns it to the venous system. How and why the lymphatic system generates lymphocytes is not generally addressed. For purposes of discussion, five additional paths have been introduced that could accommodate the flow of lymphocytes (characterized by the wavy lines in the figure) to different locations along the flow diagram. In addition, a path 6 has been included that may feedback signals of Hormonal-affectors 23- 85 unknown type. The reasoning for examining these paths, is that it is reasonable to assume a goiter involves the buildup of excessive stores of one intermediate product or another due to the failure of the next element of the system to accept the intermediate product at an acceptable rate. There are at least three locations where signals are applied to the block diagram to control its operation, 1) There is the signal fed back to the hypothalamus, controlling its output with a time constant of minutes 2) The signal fed back to the Hypophysis (pituitary), with a time constant of hours to days . 3) a signal fed back to an undefined element by a AITD labeled LATS, with a time constant measured in days or longer. This signal may control the quiescent level(s) of thyroxine in the physiological system. It is possible the time constant associated with LATS may not exist; it may be that the length of the presumed time constant actually relates to the quiescent point of thyroid concentration set by the primary feedback loop (currently undefined) of the H-P-T axis. Figure 23.4.4-2 summarizes the normal range of values related to a clinical blood test focused on the H-P-T axis. Note the variability in the units in the right column. The derivation of the range is seldom stated; the values are commonly the plus and minus two sigma values about the mean.

Figure 23.4.4-2 Nominal blood test values focused on the H-P-T axis. Note the variation in units in the right column. The cholesterol values are included because their relationship to the thyroid values are a matter of current debate. Some of the labels and names are give to ease discussions related to the literature; they are largely archain in the USA.

Long acting thyroid stimulator, LATS, is frequently associated with hyperthyroidism but may also be present in hypothyroidism. It will be addressed below under hypothyroidism. The symptoms 86 Neurons & the Nervous System associated with this disease are illustrated in figure 34-1 of Werner & Ingbar, 3rd Edition. Topical treatment of the symptoms have been less than successful. Topical treatment with concentrated corticosteroid creams (0.2% fluocinolone acetonide) under an occlusive dressing has been found helpful. Fatourechi et al. reviewed 150 cases in 199478. They suggested a more appropriate name might be “thyroid dermopathy.” Fatourechi , writing in Werner & Ingbar, 9th Edition discusses pretibial myxedema along with another variant involving the big toes and shown in Figure 23.4.4- 3. This figure resembles the disease of gout suffered by the author over many years and controlled by the medication, allopurinol.

Pretibial myxedema is likely due to an auto- immune thyroid disease, AITD. In that case, the LATS may take over control of the thyroid from the pituitary and the thyroid stimulating hormone, TSH, may no longer be indicative of H-P-T axis operations (Section 23.4.4.3).

Bianco & Larsen in Werner & Ingbar, 9th Edition introduced new information regarding the conversion of the protohormone, T4, into the active form of the hormone triiodothyronine, T3. This conversion is a significant activity in the understanding of hypothyroidism. The material is extremely dense academically and quite dense Figure 23.4.4-3 Thyroid dermopathy involving the editorially. It has generated an immense first toes. Note the similarity to the condition amount of follow-on research. caused by gout. From Fatourechi, 2005. Figure 23.4.4-4 expands on the recent chapter by Bianco & Larsen. These authors have updated the state of knowledge concerning the H-P-T axis considerably by extending the analyses into the peripheral tissue. As noted by Bianco & Larsen, the accuracy of the demarcation between D1 & D2 activity is limited in humans .

78Fatourechi, V. Pajouhi, M. & Fransway, A. (1994) Dermopathy of Graves Disease (Pretibial Myxedema) Medicine vol 73(1), pp 1- 7 Hormonal-affectors 23- 87

Figure 23.4.4-4 Daily T3 production in humans & rats. “Dotted lines in the cylinder representing human extra-thyroidal production reflect the uncertainty about the contributions of type 1 deiodinase (D1) and type 2 deiodinase (D2) to this pool. Values given are based on studies cited in the text.” From Bianco & Larsen in Braverman & Utiger, 2005.

They expand dramatically on the understanding of how the three deiodinases participate in the mechanisms relating to the conversion of Tg into T3. As an initial observation, in conjunction with the figure of Section 23.4.3.1 from Orlander, the thyroid initially secretes 20% of the eventual total available T3. The suggestion is that neither D1 or D2 is necessary in the cardiovascular system if the organism is able to operate near normally on a concentration of T3 at about 2.3 pg/ml. The normal range of Free T3 is 2.2-4.2 pg/ml.

As a summary, they present a final step converting T4 to T3 using the deiodinase they label D1. Figure 23.4.4-5 describes their proposed method of conversion of T4 to T3 in the peripheral tissue79. The definition of RSSR and RSH are not addressed in the Abstract to their paper. The terms are usually addressed in Mechanical Engineering activities exploring the kinematics of multi-body motions.

79Leonard, J. & Rosenberg, I. (1978) Thyroxine 5'-deiodinase activiy of rat kidney-.observations on activation by thiols and inhibition by propylthiouracil Endocrinology vol 103, pp 2137- 88 Neurons & the Nervous System

Figure 23.4.4-5 Mechanism of type 1 deiodinase (D1)-catalyzed thyroxine, T4, conversion to triiodothyronine, T3. The steps in the enzymatic reaction cycle at which iodoacetic acid, IAc, and propylthiouracil, PTU, are thought to inhibit catalysis are indicated. From Leonard & Rosenberg, 1978.

Two important papers by their team followed the Bianco & Larsen chapter. Christoffolete is the lead author on both papers80,81. The earlier paper, dealing with rats, introduces several new feedback relationship not previously discussed in the literature and extends the discussion down to the gene level. The later paper, dealing with mice, begins with an introductory paragraph,

“T4 IS THE MAJOR product secreted by the thyroid gland, yet T3 is the ligand for thyroid hormone receptor (1). Thus, T4 must be converted to T3 by the type 1 or 2 iodothyronine deiodinases (D1 or D2) to mediate its biological effects (reviewed in Ref. 2). In humans, approximately 80% of daily T3 production is derived from extrathyroidal D1- and D2-mediated T4 to T3 conversion, whereas in rats, approximately 60% of their daily T3 production is through this pathway.” [Emphasis in the original.] The paper discusses very complex relationships between multiple mice strains, some with significant abnormalities in their H-P-T axis. The paper does not present any significant findings but

80Christoffolete, M. Ribeiro, R. Singru, P. et al. (2006) Atypical Expression of Type 2 Iodothyronine Deiodinase in Thyrotrophs Explains the Thyroxine-Mediated Pituitary Thyrotropin Feedback Mechanism Endocrinology vol 147(4), pp 1735–1743

81Christoffolete, M. Arrojo e Drigo, R. Gazoni, F. et al. (2007) Mice with Impaired Extrathyroidal Thyroxine to 3,5,3'-Triiodothyronine Conversion Maintain Normal Serum 3,5,3'-Triiodothyronine Concentrations Endocrinology vol 148(3), pp 954–960 Hormonal-affectors 23- 89 considerable data of value to ongoing experiments. Whether T4 replacement therapy can be totally satisfactory or whether a combination of T3 and T4 therapy is a better choice has been controversial for decades. In 2008, Jonklaas et al82. readdressed this question by surveying a cohort who experienced total thyroidectomies due to a variety of diseases. They tentatively concluded (because of their limited cohort size) that relying only on ingestion of T4 led to adequate levels of T3, once the correct dosage was determined. They did not address the role of the deiodinases in the conversion of T4 into T3. They did provide a large bibliography, including many post 2000 papers relating to the T3 dosage question. The optimum medication combination for thyroid illnesses was also reviewed by Cooper in 2003. He found no clear requirement to employ a mixture of T3 and T4 over just T4 in treating hypothyroidism, Section 23.4.4.2.

Guerrero et al. have provided details relating to the characterization of type II thyroxine 5'- deiodinase (D2) activity in rat Harderian gland83. 23.4.4.1 Hyperthyroidism EMPTY

Excessive thyroid activity, Hyperthyroidism, is not of interest to this work. Werner & Ingbar, 9th Edition, covers this subject extensively. Two chapters of part VI in Werner & Ingbar, 9th Edition are devoted to Goiter. The first to pathogenesis. The second to clinical manifestations. Refetoff, writing in chapter 81 of Werner & Ingbar, 9th Edition, discusses a special topic, the resistance to normal hormone levels leading to hyperthyroidism. 23.4.4.2 Hypothyroidism

The paper of 1973 by Evered et al. provides some of the best data available relating to the operation of the hypothalamus-pituitary-thyroid axis and the relative concentrations of T4, T3 &TSH under stressful conditions84. They note, “hypothyroidism is a graded phenomenon. Hypothyroidism may therefore be defined as those conditions which result from sub-optimal circulating levels of one or both thyroid hormones.” Introducing the expression, “or,” into a definitions is obviously a poor practice. Figure 23.4.4-6 provides their definitions of hypothyroidism and statistical values for the serum TSH levels associated with them. “Patients with overt hypothyroidism had obvious clinical features of hypothyroidism and abnormal results from routine tests of thyroid function.” TRH is the thyrotrophin-releasing hormone from the pituitary gland. The bottom category is described more fully in the text, “All 17 of the patients in Group 4 were asymptomatic, gave a normal response to conventional tests of thyroid function, and had a normal serum TSH concentration.” They therefore asserted, “The values recorded in group 4 were by definition normal.”

82Jonklaas, J. Davidson, B. Bhagat, S. & Soldin, S. (2008) Triiodothyrnine levels in athyreotic individuals during levothyroxine therapy JAMA vol 299(7), pp 769-777

83Guerrero, J. Puig-Domingo, M. Vaughan, G. & Reiter, R.et al. (1987) have provided details relating to the Characterization of type II thyroxine 5'-deiodinase activity in rat Harderian gland Life Sciences vol 41(9), pp 1179-1185 https://doi.org/10.1016/0024-3205(87)90638-2

84Evered, D. Ormston, B. Smith, P. Hall, R. & Bird, T. (1973) Grades of Hypothyroidism Brit Med J vol 1, pp 657-662 90 Neurons & the Nervous System

Figure 23.4.4-6 Serum TSH levels before and 20 minutes after 200 micro-grams of TRH intravenously (Mean ± S.D.). The standard deviations are indicative of the sample size, and are very large relative to the Means. See text. From Evered et al., 1973.

Part V of Werner & Ingbar, 9th Edition, is dedicated to hypothyroidism, short chapters numbered 46 to 67.

Hormonal-affectors 23- 91

Ross. writing in the 2005 time period85, expanded the definition of overt hypothyroidism to include short term increases in serum TSH for a period of one or more weeks, due to traumatic injury and potentially including concussions to the head, Figure 23.4.4-7 provides nearly the only available data on TSH levels versus the recognized degrees of hypothyroidism. Cooper readdressed the subject of T4 therapy alone versus combined T3 and T4 therapy in 200386. He reviewed the conventional wisdom concerning hypothyroidism over the preceding 40 years. “After more than a century of successful treatment of hypothyroidism, it is fascinating that such seemingly simple hormonal replacement therapy would be the subject of so much continuing controversy. Before another century elapses, let us hope that the optimum treatment for all hypothyroid patients will be revealed.” As noted following the next paragraph, the situation within the medical community has not changed with regard to subclinical hypothyroidism. Ross has noted, “The term ‘subclinical hypothyroidism’ was first introduced in the early 1970's coincident with the introduction of serum thyrotropin, TSH, measurements. Subclinical hypothyroidism is defined biochemically as a high serum TSH concentration and normal serum free thyroxine, T4, and triiodothyronine, T3, concentrations. Figure 23.4.4-7 Serum TSH concentrations in Samuels et al. (2007) provided an alternate hypothyroidism. Note the European style decimal set of data involving induced point in the TSH values. TSH values exceeding 10.0 hypothyroidism in patients already known to 87 μU/ml are seldom discussed in the literature. See be on thyroxine replacement therapy , text. From Evered, et al., 1973. “Nineteen subjects on L-T4 therapy for primary hypothyroidism participated in the study.” The goal was to evaluate cognitive skills among the subjects. “Nineteen women, aged 20–75 yr, with adult-onset primary hypothyroidism were recruited. Thirteen subjects had autoimmune hypothyroidism and six had received I-131 therapy for Graves disease. Men were not intentionally excluded, but the few male volunteers did not pass the screening tests.” Figure 23.4.4-8 presents their data. “Subjects were then randomized to receive either their usual doses of L-T4, (euthyroid arm) or lower doses of L-T4, calculated to lead to a serum TSH level of 10–20 mU/liter (subclinical hypothyroid arm). For the subclinical hypothyroid arm, we targeted TSH levels of 10–20 mU/liter because studies have showed more consistent clinical effects at these TSH levels.” The baseline values were the result of initial tests on this small cohort. “Twelve weeks after the baseline visit, subjects returned for an extended visit.”

85Ross, D. (2005) Subclinical hypothyroidism In Braverman, L. & Utiger, R. eds. Werner & Ingbar’s Thyroid, 9th Ed. pg 1070

86Cooper, D. (2003) Combined T4 and T3 therapy: back to the drawing board JAMA 290(22), pp 3002-3004

87Samuels,M. Schuff, K. Carlson, N. et al. (2007) Health Status, Mood, and Cognition in Experimentally Induced Subclinical Hypothyroidism J Clin Endocrin Metab vol 92(7), pp 2545–2551 92 Neurons & the Nervous System

Figure 23.4.4-8 Clinical parameters and thyroid function tests at the end of each arm of the study (euthyroid and subclinical hypothyroid ADD. Note the maximum induced TSH level was only 17.37 mU/Liter. The baseline values were obtained at the beginning of the study for the small cohort involved. See text. From Samuels et al., 2007.

To treat or not to treat subclinical hypothyroidism remains controversial to this day. A paper by Vega88 was release on December 7, 2018 for “Continuing Education” credit within the medical profession asserts, “The issue of whether to treat subclinical hypothyroidism is highly controversial.” After reviewing many clinical papers and statements of various committees and government agencies, Vega noted, “Treatment of subclinical hypothyroidism failed to improve any outcome vs the control condition in the current meta-analysis.” This is virtually the same situation presented by Tucker reporting in 2017 through the same information outlet89. 23.4.4.2.1 Thyroid disease in rats

Abrams & Larsen have presented a comprehensive paper on thyroid deficiencies in rats in 197390. They provided very detailed analyses and presented considerable data. They noted that rats could be clinically euthyroid while exhibiting high TSH values. In one situation, this was related to a iodine deficiency. Page 2529 is particularly valuable if also applicable to humans.

23.4.4.2.2 Thyroid disease in humans

[xxx meld into other material within 23.4.4.2 ] The medical community has struggled to understand the diseases of the thyroid for the entire 20th Century. While the 9th Edition of Werner & Ingbar, contains much more data than the 8th Edition, the subject is still difficult to comprehend. This is because of the complexity of the thyroid system and the diverse symptoms reported by individuals.

Currently the diseases of the thyroid are broken into multiple major categories. These are then broken into multiple subcategories as shown in Figure 23.4.4-9 for hypothyroidism according to Ross.

88Vega, C. (2018) Treating subclinical hypothyroidism: What is the benefit? https://www.medscape.org/viewarticle/905690

89Tucker, M. (2017) No benefit of treating subclinical hypothyrodism in older people. https://www.medscape.com/viewarticle/878136

90Abrams, G. & Larsen, P. (1973) Triiodothyronine and thyroxine in the serum and thyroid glands of iodine- deficient rats J Clin Invest vol 52(10), pp 2522-2531 Hormonal-affectors 23- 93

Figure 23.4.4-9 Grades of hypothyroidism. Note the lack of numbers in this table. See text. From Ross, 2005.

The two upper designations in the figure are considered “clinical.” While no numbers appear in this table, the values are usually based on the statistics of current blood tests for the larger population. [xxx include current ranges from my medical records and compare values of Section 23.4.4.2 ]

23.4.4.2.3 The apparent euthyroid hypothyroid sufferer

The author recently experience an abnormal situation with a TSH (3rd gen.)reading of 160 μU/ml while exhibiting his normal (for decades) low normal T3 and T4. On a repeat assay, his blood showed levels of TSH =149 μU/ml. The medical community warned of the eminence of myxedemic coma when I was only aware of a poorer than normal stability along the vertical axis while standing, but routinely walking two miles a day. No perception of vertigo was involved. However a loss of grasping strength in the hands and diffilulty in maintaining the lower leg bones perpendicular to the ground was perceived. The situation was overcome by treatment, ramping up to 125 μg/day of L-thyroxine over a 3.5 month interval. TSH,T3 and T4 all returned to the medically desired range.

Abrams & Larsen address this condition in their work with rats. “iodine-deficient rats have elevated TSH levels that lead to the goiter and increased radioactive iodine uptake characteristic of this condition. It can be argued that such animals are, by definition, hypothyroid. Nevertheless, as previously pointed out, they appear to be ‘clinically’ euthyroid.” “If it can be demonstrated that such animals are euthyroid and T3 levels remain normal with markedly decreased serum T4, the results would be consistent with the interpretation that the TSH elevation in these animals is a compensatory response designed to maintain the animal in a euthyroid state.” Abrams & Larsen appear to be correct in this case. 23.4.4.3 Iodine deficiency in hypothyroidism

Chapter 7 of Wegner & Ingbar’s Thyroid, 9th Edition is devoted to the discussion of iodine addition to the T4 molecule, reduction of T4 to the active form, T3, and to inadequate availability of iodine. The Chapter addresses the properties of the various enzymes, labeled deiodinases, described in the figure of Section 23.4.4.2 required to activate T3. . D1 & D2 are described as “integral mebrane proteins oriented with a small amino-terminal extension in the extracellular space (D1) or lumen of the endoplasmic reticulum (D2), and a single transmembrane domain exiting the membrane at about position 40. This put the active center of both D1 and D2 in the cytosol.” “The deiodination of iodothyronines by D1, D2 and D3 is a reductive dehalogenation that in vitro requires a reducing agent, such as dithiothreitol, as cofactor. An endogenous cofactor capable of sustaining multiple rounds of iodothyronine deiodination has not yet been identified.” Mpre simply stated, it is not known how the deiodination is achieved in vivo! “Because thyroidal secretion accounts for only about 20% of daily T3 production in humans, the illness-associated decrease in serum T3 concentration must be caused, largely if not completely, by decreased T4 to T3 conversion by D1 or D2 or by increase T3 clearance by D3.” 94 Neurons & the Nervous System

“The relative contributions of the two sources of T3, thyroid secretion and extrathyroidal deiodination of T4, can be quantified by determining the T4 to T3 conversion rate, which is , on average, about 35% to 40% in normal humans. Hence, with a normal T4 production rate of approximately 110 nmol/day (85 micro grams/day), approximately 40 nmols ( 25 micro grams) of T3 are produced by peripheral deiodination of T3 and the remaining 10 nmol (6 micro-grams) are released by the thyroid (page 121).” Page 121 discusses iodine deficiency and hypothyroidism. Quoting Werner & Ingbar, 9th Edition, page 122, “Thus, the hallmarks of iodine deficiency, and the early phase of primary hypothyroidism, are low serum T4, normal serum T3, and high serum TSH concentrations91,92.” The 1973 paper by Abrams & Larsen93 explores the ratios between T3 and T4 and the value of TSH on page 2529. They discuss the euthyroid condition under iodine deficiency. This early paper provides a good review of earlier work and suggests many possible areas of future study. They note, “As indicated above, iodine-deficient rats have elevated TSH levels that lead to the goiter and increased radioactive iodine uptake characteristic of this condition. It may be argued that such animals are, by definition, hypothyroid. Nevertheless, as previously pointed out, they appear to be "clinically" euthyroid. Whether, in fact, these animals are euthyroid in all respects awaits the results of more careful in vitro studies of tissue thyroid status. If it can be demonstrated that such animals are euthyroid and T3 levels remain normal with markedly decreased serum T4, the results would be consistent with the interpretation that the TSH elevation in these animals is a compensatory response designed to maintain the animal in a euthyroid state. In addition, it would support the above-mentioned hypothesis that T4, in the normal rat, is primarily a precursor of T3.”

The condition described appears to fit the condition of the author of this work, except there is no sign of current or growing goiter. it is now recognized that T4 is in fact a prohormone and precursor of the active hormone, T3. The study of the conversion of T4 to T3 by enzymatic activity has been updated to 2005 by Bianco & Larsen. The findings have been presented in 2008 by Jonklaas et al. (Section 23.4.4).

Zimmermann has provided a much more comprehensive study of iodine deficiency around the World94.

- - - - 23.4.4.4 Auto immune diseases of the H-P-T axis

Davies,writing in Werner & Ingbar, 9th Edition discusses a potential auto-immune thyroid disease, AITD, that typically is associated with hyperthyroid disease, but in this author’s case may be associated with hypothyroidism. The disease came to be known as “long-acting thyroid stimulator, LATS, disease. 23.4.4.4.1 LATS, long-acting thyroid stimulator disease

The disease appeared in the academic literature in the 1950's. Kriss and associates presented

91Riesco, G. Taurog, A. & Larsen, P. (1976) Variations in the response of the thyroid gland of the rat to different low-iodine diets: correlation with iodine content of diet Endochrinology vol 99, pages 270-

92Greer, M Grimm, Y & Studer, H. (1968) Qualitative changes in the secretion of thyroid hormones induced by iodine deficency Endochrinology vol 83, pages 1193-1198

93Abrams, G. & Larsen, P. (1973) Triiodothyronine and Thyroxine in the Serum and Thyroid Glands of Iodine-Deficient Rats J Clin Invest vol 52(10), pp 2522-2531 https://doi.org/10.1172/JCI107443

94Zimmermann, M. (2009) Iodine Deficiency Endocrine Reviews vol 30, pp 376–408 Hormonal-affectors 23- 95 two well researched papers in the 1960's95,96. Shewring & Rees Smith expanded the available data on the hormone, TSH97. Kraiem et al. extended the disease to include the hypothyroid diseases known as Hashimoto’s disease, and primary myxedema98. They also introduced the subject of lymphocytes in the peripheral blood as important in the study of LATS. This has required expansion of the baseline flow diagram associated with the H-P-T axis in Section 23.4.3. Kriss and associates noted the circumscription of pretibial myxedema to the lower part of the shin in one or both legs. Fatourechi et al. identified three types of dermopathy associate with LATS. Their first type most closely describes this author’s condition; “1) Nonpitting edema accompanied by hyperkeratosis, pigmentation, and pinkish, brownish red or yellowish discoloration of the skin.” They noted, Thyroid dermopathy occurs in 0% to 4.3% of patients with Graves thyrotoxicosis and in 15% of patients with Graves ophthalmopathy but can also occur in Hashimoto thyroiditis (a hypothyroid condition). “Fourteen patients were never clinically hyperthyroid; spontaneous hypothyroidism had developed in 11 in the group (of 150 cases reviewed).” Figure 23.4.4-10 shows typical cases of pretibial myxedema. Werner & Ingbar, 9th Edition includes a wide variety of images of pretibial myxedema. Topical application of 1% hydrocortisone twice a day gave remarkable improvement in the appearance of the dermatitis of the author.

95Kriss, J. Pleshakov, V. & Chien, J. (1964) Isolation and Identification of the Long-Acting Thyroid Stimulator and Its Relation to Hyperthyroidism and Circumscribed Pretibial Myxedema J Clin Endocrin vol 24, pp1005- 1027

96Kriss, J. Pleshakov, V. Rosenblum, A. & Sharp, G. (1967) Therapy with Occllusive Dressings of Pretibial Myxedema with Flucinolone Acetonide J Clin Endocrin vol 27, pp595-603

97Shewring, G. & Rees Smith, B. (1982) an Improved Radioreceptor Assay for TSH Receptor Antibodies Clin Endocrin vol 17, pp409-417

98Kraiem, Z. Lahat, N. Glaser, B. et al. (1987) Thyrotrophin Receptor Blocking Antibodies: Incidence, Characterization and In-vitro Synthesis Clin Endorcrin (Oxford), vol 27, pp 409 96 Neurons & the Nervous System

Figure 23.4.4-10 Pretibial myxedema associated with abnormal levels of TSH, T3 and T4 in the blood RESCAN from source. The condition in frame B rsembles this author’s symptoms. See text. Credited to Malkinson, 1963, in Wegner & Ingbar, 9th Edition but not actually in the Malkinson paper.

Kriss et al., 1967, identified fluocinolone acetonide, a corticosteroid, as an effective treatment of primary myxedema. 23.4.4.4.2 TSH receptor blocking–auto immune

Kraiem et al. opened their summary by noting, “The prevalence and characteristics of TSH receptor blocking activity were examined in patients with different thyroid disorders.” They go on, “TSH receptor blocking activity was only detected in primary hypothyroidism with the following characteristics: (i) Such activity was present in only 16% of the patients (both goitrous, i.e. Hashimoto’s thyroiditis, and non-goitrous, i.e. primary myxoedema), and in three patients with previously active Graves’ hyperthyroidism who had become hypothyroid. (ii) Blocking activity seems to be associated with the Ig fraction of serum as indicated by protein A adsorption. (iii) The block-positive samples did not bind 125I-TSH which seems to rule out an antibody directed against TSH. (iv) The specificity of the blocking activity seems to be directed towards the TSH-(thyroid stimulating immunoglobulin, TSI) receptor-mediated cAMP response since no inhibition of EI –stimulated cAMP production was found. Moreover, all cases in which an inhibitory effect was demonstrated towards TSH also exhibited blocking of TSI-stimulated cAMP, with a high correlation between the degree of inhibition of the TSH to that of the TSI Hormonal-affectors 23- 97 response (r = 0.89, P < 0.00 I, n = 1 1). cAMP– “Cyclic adenosine 3',5'-monophosphate (cAMP) was the first second messenger to be identified and plays fundamental roles in cellular responses to many hormones and neurotransmitters. The intracellular levels of cAMP are regulated by the balance between the activities of two enzymes: adenylyl cyclase (AC) and cyclic nucleotide phosphodiesterase (PDE)99.” (Sassone-Corsi, 2001) It is described in terms of cartoons.

Kraiem et al. distinguish between, Hashimoto’s thyroiditis: clinical signs of hypothyroidism, goitre, increased TSH and elevated titres of antimicrosomal or anti-thyroglobulin antibody. Primary myxoedema: Same as Hashimoto’s thyroiditis except that no goitre was palpable. “In order to exclude the possibility of autoantibody against thyrotrophin rather than against the receptor to TSH, the sera of the block-positive patients were incubated with 125I-hTSH and phosphate buffer followed by polyethylene glycol precipitation, according to Kajita et al. (1983).” “Blocking activity seems to be associated with the immunoglobulin fraction of serum as indicated by the protein A adsorption experiment. The antibody nature of the blocking factor was strengthened by the in-vitro synthesis of TSH receptor blocking antibody by lymphocytes.”

These remarks are quite clear and suggest strongly that it is the TSH receptor cells that are affected by the immunoglobulin fraction of serum. This suggests path P1 in the expanded flow diagram introduces the immunoglobulin fraction as a suppressor of the activities of the TSH receptors. How the receptor cells might be affected remains unknown.

As noted in Section 23.4.3.3.2, the TSH hormone secreted by the hypophysis (pituitary gland) is not a simple derivative of an amino acid, like T3 and T4. It is a full blown protein of high molecular weight on the order of 28,000–30,000. As a result, there is no known theory of how the TSH receptor actually works at levels more detailed than the cartoons found in the late 20th Century literature. 23.4.4.4.3 deiodinase related problems

The operation of the three deiodonase enzymes, D1, D2 &D3, are possible contributors to or subjects of auto-immune disorders.

These enzymes play important roles in the conversion of T4 to T3, both within the thyroid gland and in the peripheral cardiovascular system. These enzymes are generally considered surface-bound proteins of high molecular weight. Their conformations have been studied. They appear to be subsets of a larger protein.

The presence of these surface bound enzymes is believed to be ubiquitous within the walls of the cardiovascular system. This includes the portions of that system physically present within the many morphologically identified elements bridging the arterial and venous portions of that system. It is the presence of the enzyme labeled D2 that is of primary interest in Section 23.4.4.5 based on the conversion of relatively passive T4 to the active T3, as described by Bianco & Larsen in Section 23.4.3.4.2.

23.4.4.4.4 Mood & other intellectual problems related to hypothyroidism

Samuels & Schuff have discussed problems related to the subjects mood, memory and other

99Fimia GM, Sassone-Corsi P 2001. Cyclic AMP signaling. J Cell Sci vol 114, pp 1971–1972 98 Neurons & the Nervous System measurable intellectual characteristics when in an induced hypothyroid state100,101. The first paper provides useful statistical data. The experiments employed only 19 subjects so the precision of the data is not great. The evaluations used a set of standard psychological tests. Their conclusions were summarized as, “We found mild decrements in health status and mood in L-T4-treated hypothyroid subjects when subclinical hypothyroidism was induced in a blinded, randomized fashion. More importantly, there were independent decrements in working memory, which suggests that subclinical hypothyroidism specifically impacts brain areas responsible for working memory.” [xxx ADD ] In the second paper, 23.4.4.5 Thyroid hormone clearance times vs the LATS concept

Researchers began exploring the clearance times of the thyroidal hormones during the 1970's following the early findings that T4 was not necessarily a true hormone. It was appearing to be a proto-hormone, or prohormone within the language of the clinical community. By the turn of the 20th Century into the 21st, the isolation of the enzymes supporting the conversion of T4 into the active hormone, T3, had occurred. Oppenheimer et al. provided significant information about the clearance times of the thyroxines (Section 23.4.3.4.5), but possibly without adequate specificity concerning the performance of the enzyme, D2. In their Introduction they noted,

“It is widely recognized that 3,5,3'-L-triiodothyronine (T3) has a larger space of distribution and a more rapid fractional turnover rate than L-thyroxine (T4) both in man and the rat. Although these differences can be attributed, at least in part, to the fact that T4 is more firmly bound to plasma proteins than T3, no specific studies have been undertaken to determine to what extent intrinsic cellular factors may be involved. . .Our findings indicate significant differences in the primary cellular determinants of T3 and T4 metabolism, factors which may be related to differences in the metabolic potency of these hormones.”

Figure 23.4.4-11 summarizes their findings without any definitive discussion of how these ratios are mediated by various enzymatic mechanisms. Note the definition of more ratios than typically reported earlier. The ratio of metabolic clearance rate between T3 and T4 is significantly high. It suggests the low clearance rate, MCR, of T4 indicates the T4 circulating in the cardiovascular system acts as a reservoir upon which the mechanism converting T4 to the active but lower MCR, T3 can rely upon.

100Samuels, M. Schuff, K. Carlson, N. et al. (2007a) Health Status, Mood, and Cognition in Experimentally Induced Subclinical Hypothyroidism J Clin Endocrin Metab vol 92(7), pp 2545-2551

101 Samuels, M. Schuff, K. Carlson, N. et al.(2007b) Health Status, Psychological Symptoms, Mood, and Cognition in L-Thyroxine-Treated Hypothyroid Subjects Thyroid vol 17(3), pp Hormonal-affectors 23- 99

Figure 23.4.4-11 Differential Cellular Metabolism of T3 and T4 in Man ADD. From Oppenheimer et al., 1970.

Oppenheimer et al. assert, “In each subject the plasma binding ratio of T4 and T3 was determined by simultaneous equilibrium dialysis of T4-131I and T3-'I. The ratio of the metabolic clearance rates of T3/T4 averaged 23.5; the ratio of plasma protein binding T4/T3 averaged 9.94; and the ratio of the cellular metabolic clearance rates averaged 2.40. Thus, in man as well as the rat, differences in the metabolic clearance between T3 and T4 cannot be attributed exclusively to differential plasma protein binding. The cellular compartment metabolizes T3 more rapidly than T4.”

Oppenheimer et al. continue in their discussion, “Comparison of the cellular metabolism of T3 and T4 in man. The results of studies in the rat prompted us to determine whether differences also exist between the cellular metabolism of T3 and T4 in man. The opportunity to obtain multiple plasma samples in man facilitated adoption of a non-compartmental kinetic analysis.” Their Discussion contains many key measurements that they suggest be investigated further.

Oppenheimer et al. employed two different radioactive isotopes of iodine, 125I and 131I, to label their T3 and T4. This allowed them to track the concentrations of T3 and T4 simultaneously through the human cardiovascular system. “All radioactive samples were counted to a statistical accuracy ±2% in a two-channel Autogamma Spectrometer (Packard).” “The cellular metabolic clearance rate (= free hormoneclearance rate) of T3 exceeds that of T4 by a factor 1.8 in the rat. The corresponding ratio in man, 2.4, was determined by noncompartmental analysis of turnover studies in four individuals after the simultaneous injection of T4–125I and T3–131I. The greater cellular metabolic clearance rate of T3 both in rat and man may be related to the higher specific hormonal potency of this iodothyronine.” The ability of the cardiovascular system to “store” T4, compared to the rate of clearance for T3, suggests this mechanism alleviates the necessity of explaining the role of the putative long-acting thyroid stimulator, LATS, identified conceptually by Kraiem et al. in 1987 (Section 23.4.4.4.1). On the other hand, Cavalieri et al. recognized the complexity of the clearance time of the proto- 100 Neurons & the Nervous System hormone, T4, but was not able to provide a flow diagram accounting for this complexity102. They did assert that a simple single-compartment kinetic model of T4 operation was inadequate. They considered a multiple compartment model a necessity in follow on studies after 1971. “Thus, although the conventional, single-compartment model seems to yield a reasonable approximation of MCR in normal individuals, it tends to give an overestimation in Graves' disease, especially in thyrotoxic patients. The deviation from single-compartment kinetics in the latter group may be related to the alterations in distributional kinetics of T3 reported previously. In any case, it is apparent that the single-compartment kinetics model of T3 metabolism is inadequate. Such studies must take into account variations of tracer distribution in various thyroid states.” Currently, the clearance rates of T4 among human patients are also known to vary among those who are euthyroid (6-7 days), hyperthyroid (3-4 days) and hypothyroid (9-10 days). See more details related to euthyroid patients suffering non-thyroid diseases in Wiersinga, Chapter 11D of Werner & Ingbar, 9th Edition. Figure 23.4.4-12 provides a flow diagram for the formation and release of both T4 and T3 and their utilization for initial discussion. It is a compartmental model using an electrical analog of a global cardiovascular system. The analog is similar to that used to represent long distance electrical transmission lines that are periodically interrupted by shunting with lumped capacitances to correct the power factor, and hence efficiency, associated with the lines.

In this analog, the concentration of T3 and T4 are represented by equivalent electrical charges. The analog uses two types of symbology; the first using distributed elements to represent the cardiovascular ducts themselves, as represented by the combination of a distributed resistance and a distributed capacitance shown along the left edge of the figure, and discrete resistances and capacitances as represented by the individual lumped resistors and capacitors. The individual capacitors are represented by the rectangular boxes. These boxes represent large elements of the physiological system that may contain long lengths of cardiovascular paths within the particular element (such as the liver). Each of the discrete resistance and its associated capacitance exhibit a distinct time constant.

The figure is meant to be compatible with the flow diagram in Section 23.4.4 but expanded. It is also compatible with the simpler figure of Meier & Burger on page 230 of Werner &Ingbar, 9th Edition and in Chapter 11D, by Wiersinga in the same 9th Edition. Page 255 of Wiersinga addresses the variation in hormone levels in the presence of non-thyroidal disease (pages251-157. Page 255 presents a different, largely conceptual, but compatible representation of the physiological system involving T3 and T4.

The starting point of this analog assumes thyroglobulin, Tg, is already present in the thyroid gland. The release of a mixture of T3 and T4 is controlled by the valve labeled TSH (thyroid stimulating hormone). The nominal ratio of concentrations of T3 versus T4 is shown to the right of the valve.

102Cavalieri, R. Steinberg, M. & Searle, G. (1971) Metabolic Clearance Rate of L-Triiodothyronine in Man: A Comparison of Results by Single-Injection and Constant Infusion Methods J Clin Endocrine Metab vol 33(4), pp 624-629 Hormonal-affectors 23- 101 As noted below, this ratio is only nominal. It varies with the health of the individual subject. Without adequately considering the health of a small cohort of individuals, various ratios between the concentrations of T3 and T4 have been reported in the literature, and without specifying where in the cardiovascular system the relevant blood samples were obtained. The compartmented analog can be used to address both the transient and steady-state performance of the actual cardiovascular system. As an example, each of the stacked layers, with the tip layer labeled “Liver: exhibits a distinctly different rate of conversion of T4 to T3. Each layer exhibits a distinct time constant. Thus, at point A, the concentration of T3 is enriched over its relative concentration a the TSH valve. Following the simultaneous release of T3 and T4 at the valve, the transient increase in T3 concentration relative to T4 concentration at A follows a complex temporal response because of the difference in connvesion efficacy and time constant. of each physiological element. This complex demonstrated response justifies the assertion of Oppenheimer et al. that a compartmented model of the system is required.

Following the concentration of T3 at the junction, A, both hormones are used to stimulate the tissue targets at different locations within the physiology of the subject. Here again, the stimulation efficacy and associated time constants need to be recognized when evaluating the efficiency of the stimulus at any given element of the tissue targets. Figure 23.4.4-12 T4 to T3 conversion and utilization flow diagram for initial discussion. The The challenge in this area are three, cardiovascular network is described using a C to perfect the analog, combination of geometric shapes and distributed C to develop a conversion table between impedances represented by the symbols along the concentrations of the actual hormones the right edge. The cardiovascular network is and the equivalent parameters of the described as a distributed network. The content analog, and of major elements of the physiological system is C to quantify the parameters of the analog. represented by lumped constant elements. See text. The model can be used for both transient Only after the above steps can a protocol and steady-state analyses. for evaluating the physiological system be investigated in the laboratory. 23.4.5 A case study–potential change in enzymatic activity EMPTY

[xxx Discuss source of D1, D2 & D3 [xxx discuss similarity of D2 and Creon in terms of Figure xxx It is important to determine whether the most important of these three enzymes, D2, is denatured during the process of removing an iodine atom from T4. 102 Neurons & the Nervous System

23.5 (Reserved) Hormones of the peripheral endocrine system EMPTY

Section 23.4 provides a superficial description of the peripheral endocrine system served by the hypophysis (pituitary). The thyroid gland has been addressed in Section 23.4.3. 23.5.1 Hormones of the adrenal gland EMPTY

Raum103 has provided a description of the catecholamines and peptides of the adrenal medulla. He notes, “The adrenal gland is actually two distinct glands combined in a common capsule. The cortex is derived from mesoderm and secretes steroid hormones. The medulla is derived from ectoderm chromaffin tissue and secretes catecholamines. In lower animals, such as fish, the cortex and medulla present as separate organs.” Raum discusses the role of “The process of amine precursor uptake and decarboxylation (APUD) is a feature common to a varety of endocrine and neuroendocrine tissues that are embryologically related and secrete polypeptide hormones, hormone precursors and catecholamines.” 23.5.2 Hormones of the pancreas EMPTY

It is important to note the pancreas receives commands from both the glandular and the neural system of the CNS. While the time delay associated with the vascular circulation may be in the order of multiple seconds, the neural system commands can be received within milliseconds of their generation. 23.5.3 Hormones of the kidney EMPTY

Mukoyama & Nakao104 have provided an extensive discussion of the hormones of the kidney in 2005. They summarize the role of the kidneys succinctly in their introduction. “The kidney plays an essential role in the maintenance of like in higher organisms, not only through regulating the blood pressure and body fluid homeostasis and clearing the wastes, but also by acting as a major endocrine organ.” They identify the hormone renin (1) and then focus on “the renin- system (RAS) that leads to the production of a potent pressor hormone angiotensin, and produces the following hormones and humoral factors: (2) kallikreins, a group of serine proteases leading to the peptide bradykinin; (3) erythopoietin (EPO), a peptide hormone essential for red blood cell (RBC) formation by the bone marrow; and (4) 1,25-(OH)2 vitamin D3, the active form of vitamin D essential for calcium homeostasis, . .” The text requires close study.

23.6 The secretions of the exocrine system

Section 23.3.4 of this work introduces the elements of the exocrine system. The following subsections address the subjects addressed there in greater detail

23.6.1 The gastric juices of the exocrine submodality EMPTY

23.6.1.1 What are enzymes? EMPTY

103Raum, W. (2005) Adrenal medulla (catecholamines and peptides) in Melmed, S. & Conn, P. eds Endocrinology, 2nd Edition Totowa, NJ: Humana Press Chapter Chapter 22

104Mukoyama, M. & Nakao, K. (2005) Hormones of the kedney in Melmed, S. & Conn, P. eds Endocrinology, 2nd Edition Totowa, NJ: Humana Press Chapter Chapter 23 Hormonal-affectors 23- 103

23.6.2 The pheromones of the exocrine submodality EMPTY 23.6.2.1 What are pheromones?

Peter Karlson and Martin Lüscher first proposed the word "pheromone" in 1959, referring to a chemical cocktail emitted by an animal and detected and responded to by other creatures of the same species. That same year, researchers reported the identification of the first pheromone (called bombykol) in silk moths. A pheromone is a secreted (in the perspiration) or excreted (in the urine) chemical factor that triggers a social response in members of the same species. Pheromones are chemicals capable of acting outside the body of the secreting individual to impact the behavior of the receiving individual at a distance. Pheromones are typically volatile. They are primarily selected members, or variants, of the stimulating agents found in olfaction (Section 8.4) and more specifically oskonation (Section 8.xxx). They are frequently associated with an auxiliary olfactory channel. There are alarm pheromones, food trail pheromones, sex pheromones, and many others that affect behavior or physiology. A pheromone to one species is merely an odorant, or even an irritant, to other species.

A variety of pheromones have been identified based on behavioral experiments in insects. Those identified are typically specific stereo-meres of aliphatic alcohols.

Identification of animal pheromones has suffered from an excess of behavioral experiments and a lack of adequate applied research on the chemical structure of these putative materials. The available evidence suggests either 14 to 20 carbon aromatic ring structures with specific additional features, or undefined complex proteins.

As of 2009, and following investigations over decades, “the hunt for a human pheromone has come up short105. However, since that time, significant progress has been made (Section 8.7.6). 23.7 The hormone/receptor interface

23.7.1 Receptors of hormones

Mayo106, writing in 2005, has provided an extensive survey of the types of hormonal “receptors” that also includes a discussion of means of transporting hormones through the lemma of cells. He notes in his Summary, “In most of the examples considered in this chapter, our knowledge of the signaling pathways from hormone-receptor interaction to biologic response is still at best rudimentary.” He goes on, “. . .there are relatively few examples for which a complete response pathway has been elucidated, . .”

His section 2.2 begins, “Substances that act as signaling molecules have extremely diverse structures and chemical properties.” He does define his version of signal transduction as, “The consequence of hormoe-receptor interaction is the activation of pathways that lead to an appropriate biologic response in the target cell.” He also defines a second messenger as, “. . cell surface receptors transduce a signal to the cell interior, directly or indirectly altering the activity of proteins within the cell. This action often involves the enzymatic generation of a second messenger, . . “ He asserts, “Small lipophilic molecules such as the steroids, retinoids, calcitrol, and thyroid hormone are able to enter cells directly by diffusion across the lipid bilaer, . . “The thyroid hormone, T3 has a molecular weight of 650 and many steroids are even larger. Mayo does not

105 www.livescience.com/3233-sexual-pheromones-myth-reality.html January 14, 2009

106Mayo, K. (2005) Receptors: molecular mediators of hormone action in Melmed, S. & Conn, P. eds Endocrinology, 2nd Edition Totowa, NJ: Humana Press Chapter 2 104 Neurons & the Nervous System provide any justification or citations supporting this assertion. The section continues in a very philosophical manner providing many conceptual options but little specific information. He does provide a conceptual cartoon comparing surface and intracellular capture of a hormone that is almost certainly overly complex. His section 2.4, titled, “Cellular Signal Transduction Themes,” does not focus on either potential cell surface receptors or intracellular receptors but focuses on subsequent activity within a . Mayo does not arrive at a viable cellular surface or nuclear transduction model for hormone/receptor interaction. Part 3 further explore surface receptors including section 3.9 addressing potential transport receptors. Part 4 explores intracellular receptors addressed in this work in Section 23.2.2.2. Part 5 relates to the disease aspects of hormonal or hormone/receptor abnormalities. Part 6 is a summary of Mayo’s paper. Besides the two quotation reproduced in the introduction of this section, he provides several assertions. However, like an earlier assertion concerning olfaction by Axel & Buck in 1991, Mayo suggests a boundless number of different receptors involved in the hormone/receptor interface. As in the olfaction case, where a combinatorial approach was found to be employed, ultimately the number of receptors will be quite finite and probably in the low hundreds (in line with the number of identifiable hormones). 23.7.1.1 Cell surface receptors & the hormone/receptor interface [xxx consider a three level section for this material. ]

As noted above, Mayo did not address the hormone/receptor interface at the molecular or process level in 2005. He only spoke conceptually at that time.

A team led by Evans has been working to clarify the hormone/receptor interface for several decades.

Evans107, wrote an extensive paper in 1988. It provided a synopsis of earlier ideas and then presented a comprehensive concept he perceived at that time. In a section labeled, Mechanisms, he reviewed the questions he sought to answer,

“What are the molecular interactions between the ligand and the receptor that lead to activation? Once activated, how does this molecule find a particular binding site and what is the detailed nature of the DNA-protein interaction? What is the molecular interaction between the receptor and the transcriptional machinery? How do receptors and their potential interaction with other transactivators cause RNA polymerase II to initiate transcription? It must be emphasized that steroid and thyroid hormones can repress gene expression as well as activate it. It is important to determine whether repression and activation are mediated by the same types of DNA sequences and whether other protein factors are involved.” He did not explore answers to the first question! He immediately jumps to the following questions starting with, “Once a receptor is bound to DNA, how does it activate transcription? As a result, his writings in this paper more appropriately belong in Section 23.2.1.2 on intracellular receptors.

[xxx ]

107Evans, R. (1988) The steroid and thyroid hormone receptor superfamily Science vol. 240(4854), pp. 889-895 DOI: 10.1126/science.3283939 Hormonal-affectors 23- 105 Giguere et al108. wrote in 1988, “In this study, using the DNA-binding domain of the human oestrogen receptor cDNA as a hybridization probe, we have isolated two cDNA clones encoding polypeptides with structural features suggestive of cryptic steroid hormone receptors which could participate in a new hormone response system.” [xxx ] Evans et al. received a patent in 2006 after filing in 1995. The patent focuses on nuclear receptor sites for the receptors. “A number of receptor proteins are known, each specific for steroid hormones e.g., estrogens (estrogen receptor), progesterones (progesterone receptor), glucocorticoid (glucocorticoid receptor), androgens (androgen receptor), aldosterones (mineralocorticoid receptor), Vitamin D (vitamin D receptor), retinoids (e.g., retinoic acid receptor) or thyroid hormones (e.g., thyroid hormone receptor. Receptor proteins have been found to be distributed throughout the cell population of complex eukaryotes in a tissue specific fashion. Molecular cloning studies have made it possible to demonstrate that receptors for steroid, retinoid and thyroid hormones are all structurally related and comprise a Super family of regulatory proteins. These regulatory proteins are capable of modulating specific gene expression in response to hormone stimulation by binding directly to cis-acting elements.” The last sentence implies they are talking about nuclear receptors. The more functional description of a may be that of the T3, thyroxine hormone receptor presented in Section 23.4.3. The proposed T3/receptor interface involves a dual antiparallel coordinate bond (DACB) of coordinate chemistry rather than a conventional valence bond. Such a bond has a very low energy and is readily broken as posited in the reversibility property of hormone/receptor interface assumed by Mayo on page13. 23.7.1.1.1 Crystal structure of the TSH receptor

Sanders et al109, members of the Evans team provided significant data on the TSH receptor in 2007. Their Abstract notes,

“Objective: To analyze interactions between the thyroid-stimulating hormone receptor (TSHR) and a thyroid-stimulating human monoclonal autoantibody (M22) at the molecular level. Design: A complex of part of the TSHR extracellular domain (amino acids 1–260; TSHR260) bound to M22 Fab was prepared and purified. Crystals suitable for X-ray diffraction analysis were obtained and the structure solved at 2.55 Å resolution. Main outcome: TSHR260 comprises of a curved helical tube and M22 Fab clasps its concave surface at 90/ to the tube length axis. The interface buried in the complex is large (2500 Å2) and an extensive network of ionic, polar, and hydrophobic bonding is involved in the interaction. “ [xxx resolve whether they are discussing a cell surface or nuclear receptor site. ] The bonding is possibly the key to understanding the bonding found in many if not all hormones to their respective receptors.

[xxx expand the bonding considerably ]

23.7.1.1.2 A scenario for the cell surface glandular receptor & interface

By assembling the available information in this work, it is possible to describe a potential hormone/receptor interface of general application. The concept draws upon; 1. The work of Shellenberg et al. and other colleagues working on the super sweeteners of the 1970s (Section 8.5.1.6.7).

108Giguère, V. Yang, Na. Segui, P. & Evans, R. (1988) Identification of a new class of steroid hormone receptors Nature vol 331, pages 91–94

109Sanders, J. Chiradze, D. Sanders, P. et al. (2007) Crystal Structure of the TSH Receptor in Complex with a Thyroid-Stimulating Autoantibody Thyroid vol 17(5), pp xxx https://doi.org/10.1089/thy.2007.0034 106 Neurons & the Nervous System

2. The dual antiparallel coordinate bond (DACB) of coordinate chemistry (Section xxx). 3. The description of the bonding encountered within the xxx (bitter) channel of gustation (Section 8.xxx) 4. The sensitivity of the Activa within each neuron controlled by the impedance of the base, a.k.a. poditic, terminal (Section xxx). 5. The flexibility of employing the hormones as either direct signals or indirect (parametric) signals as defined previously for the modulator terminals of neurons. 6. The measured data of the bonding arrangement between the TSH hormone and its receptor, TSHR, provided by Sanders et al. As a starting point consider the generic neuron schematic of Figure 23.7.2-1. This circuit has great flexibility. It can act as a simple amplifier (inverting through or non-inverting through Vin(1)). The most frequently used signal path follows the dashed line, resulting in a unity voltage gain amplifier with a lower impedance in the output circuit. It can act as a differential input device, Vin(1)-- Vin(2). A key factor in operating as a differential amplifier is that the amplification factor associated with both input paths be equal. The voltage gain through the base terminal of an Activa is typically 200:1. To adjust the voltage gain of the inverting input to 1:1, to match the non- inverting input, a voltage divider is introduced where the value of the impedance, RG is normally much higher than RP. In this figure, a modulator terminal is shown conceptually. As shown, it can affect the bias point of the Activa and/or it can act as a shunt in parallel with RP, thereby lowering the voltage gain associated with the Vin(2) signal path. In other configuration, the voltage gain can be increased by reducing the impedance, RG. Thus, the modulator terminal can significantly change the operation of the circuit on a transient or long term basis.

If the modulator terminal shown should be configured like a gustatory sensory neuron input terminal, it would exhibit a DACB capability with a d-value appropriate for a specific hormone. A large d-value is associated with the bitter taste in gustation at 4.74 Angstrom, a putrid odor in olfaction at 8 Angstrom, and an undefined reaction in oskonation (the channels sensitive to pheromones, d-values above 8.5 Angstrom). These values are illustrated in Section 8.5.1.6 for gustation and Section 8.6.2 for both olfaction and oskonation.

Figure 23.7.2-1 Fundamental neuron schematic with modulator terminal. The modulator terminal is shown symbolically. Its precise mode of operation has not been determined. However, its electrical effect can involve both long term and short term effects and can raise or lower the gain of the Activa. Hormonal-affectors 23- 107

The sensing associated with a bitter taste in gustation is shown in Figure 23.7.2-2 reproduced from Section 8.5.4.5. It illustrates the complete picric acid molecule. It is proposed that this picrophore has a d-value optimized for stimulating the picric channel sensory neuron that employs phosphatidyl-asparagine (PtdAsn) as its receptor. The stimulation involves a DACB. Taking picric acid as the premier member of the bitter tasting chemical family, there are two potential ligands of interest. They appear simultaneously and in multiple instances. The first ligand of interest is O=N-C-=C-=C- N=O and it is planar. Note the backbone includes two nitrogen atoms instead of an all carbon backbone. The notation C-=C is used to indicate a resonant linkage between the two carbons due to the parent ring structure. This gustaphore occurs three times in this (gustant) molecule In each case, the best available d-value is 4.746 Angstrom. The second ligand of interest is the N-C-=C- =C-N sequence alone. The nitrogen atoms as configured in picric acid continue to have an unshared pair of electrons capable of supporting a dual antiparallel coordinate bond but would need to acquire a hydrogen from the solvent through Figure 23.7.2-2 The premier gustant of the bitter or rearrangement of the structure. The d-value P-channel of gustation. Distances in Angstrom. for these pairs remains 4.746 Angstrom. See text. From a PDB file on the Boston University web site, 2012. Note the complexity of the picric acid molecule. Note also the linear distance between the paths highlighted. Any molecule with this d-value ± a currently unknown tolerance between orbitals as defined in this work (Section xxx) that are accessible to the exterior of the molecule will taste form a DACB with the picric gustatory sensory neuron and taste bitter. It can be a simple aliphatic alcohol with two oxygen atoms with the appropriate d-value, a more complex phenyl alcohol, or a very large peptide.

More complex configurations can introduce a super-bitter perception based on the three-point interface based on the work of Shallenberger et al. and a colleague, Beets (Section 8.5.1.6). This molecular arrangement between gustaphore and sensory material requires an appropriate DACB plus a third critical stereo-electrolytic position in the gustaphore that aligns with a similar position in the sensory molecule.

The resulting configuration between stimulant molecule and receptor joined by a minimum of one DACB can be applied to any type 2 cell lemma. The type 2 lemma is fundamentally an electrolytic element. It can be biased to operate as an Activa or as what is called in electronics an active diode (Section xxx). When the polarity of the stimulating molecule, i.e., the difference in polarity between the orbitals contributing to the DACB due to their location, is algebraically summed with the polarity of the lemma, the result is typically different from the polarity alone. This net polarity will result in a change in the electrical potential at the surface of the interior bilayer of the cell lemma. This change in potential, or in association with an organic catalyst, an enzyme, can be considered a “second messenger” within the cell. The liquid crystalline outer bilayer of the type 2 lemma may consist of a single phospholipid or in the case of a three-point interface between the stimulant and the bilayer may consist of a mixture of phospholipids. Even more than three points may be involved. Sanders et al. has described the multiple bonds between the TSH hormone and its receptor, TSHR, in Section 23.7.1.1.1. 108 Neurons & the Nervous System

23.7.2.2 Intracellular receptors EMPTY

23.8 Chemistry of Crine modality receptors EMPTY

Section 23.10 and Section 23.11 accumulates the available information about the hormones and hormonal receptors of the Crine modality [xxx edit next two paragraphs ] Virtually nothing substantive has been reported concerning the chemistry of the hormonal receptors associated with the hypothalamus, hypophysis and peripheral glands. See Section 23.9.1. Section 23.3.1 identifies the major hormones flowing between these hormonal elements but nothing about the receptors they stimulate. Similarly, virtually nothing has been reported concerning the chemistry of the hormonal receptors of the glands of the peripheral glandular system. Section 23.3.1 identifies the major hormones flowing between the hypophysis and these peripheral hormonal elements but nothing about the receptors they stimulate.

23.8.1 Chemistry of the surface receptors of a cell

Discussing the chemistry of the surface hormonal receptors is complicated by the fractured character of the bioscience community working in this area. Describing scientific progress in the area by a vertical column, the progress related to understanding the cell lemma is not substantial. Work in the area of the lemma by cytologist looking at the lemma is in total conflict with the work of the pharmacology/immunology sectors. Using a multiple stove pipe analogy, these two specialties are completely isolated from each other and their models of the cell lemma are in total conflict. The work of Fambrough, and the associated specialists shows the lemma in the area of the receptors is a tightly packed of phospholipids liquid crystal without voids. On the other hand Missias et al. and associated specialists, using low resolution immunofluorescent analytical techniques, continue to describe the receptors as forming a pore in the llemma To date, no one has documented (presented an image of) a pore in a lemma large enough to pass a hydrated sodium ion much less a larger hormone. Such documentation of a pore should be achievable based on electron-microscopy, where individual atoms on a surface have been documented.

More than a decade ago, scientists of IBM arranged individual atoms on a substrate to spell IBM. They then took electron micrographs of the ensemble to demonstrate their capability.

In 1999, Deamer, Kleinzeller & Farmbrough 110 published a book on membrane permeability. Chapters 2, 3 & 4 are particularly relevant to this discussion. McIntosh discussed the Structure and physical properties of lipid membrane in Chapter 2. He noted the immense range of diffusibilities of [xxx ADD ] In chapter 4, Paula & Deamer discussed the problems with the standard equations when applied to a highly structure lemma of a biological cell. They note these problems gave way to the concept of a pore through the membrane. They go on to note, “Although the solubilty-diffusion mechanism is satisfactory for most permeants, there are a few instances in which it clearly fail. These mismatches are encountered in thinner bilayers if the permeants are positively charged

110Deamer, D. Kleinzeller, A. & Farmbrough, D. eds. (1999) Membrane Permeability. NY: Academic Press Hormonal-affectors 23- 109 ions, such as potassium in liposomes made from phosphatidylcholines.” The important work in Deamer, Kleinzeller & Farmbrough will be reviewed more fully in Section 23.8.2. As recently as 2018 and in the absence of any physical evidence, Wang et al111. have been working diligently on a mathematical theory of the voltage-gated pores in a lemma. They present an extensive mathematical analysis, using cartoons of their pore, but no image of an actual pore supporting their model or validating their model. They did not discuss the practical diameter of their predicted pores. Their analysis did not consider the physical chemistry of the lemma of a cell. 23.8.1.1 Chemistry of the exocrine system surface receptors of a cell

The chemistry of the exocrine system receptors are the receptors of the oskonation modality developed and described in Section 8.7. In general, they sense the pheromones released into the environment by other animals of the same species.

23.8.1.2 Chemistry of the paracrine system surface receptor for @N=O There are only two recognized hormones of the paracrine system, AN=O and acetylcholine. AN=O is known to diffuse through the lemma of most cells. Alternately, it may pass through a type 3 lemma pore or be otherwise restricted to passing through type 3 lemma. This area has not been adequately researched, however, it appears unlikely that AN=O could readily pass through a type 1 or type 2 lemma. In any case, AN=O does not employ a cell surface receptor. 23.8.1.3 Chemistry of the paracrine system surface receptor for acetylcholine

Section 23.3.2 reviews the histology of the acetylcholine receptor but does not address the the precise molecular structure of the acetylcholine receptor, sometimes labeled AChR.

Recent papers on the AChR include; [xxx may want to omit in favor of 21st Century ]

Farnbrough112 noted the very great density of AChR compared to the remaining surface of the lemma in 1979. “AChRs are present at high concentrations (>104/micron2) in the postsynaptic membrane, but are nearly absent (<10/micron2) from the remaining 99% of the muscle fiber surface.” The value of >10 4 implies a linear spacing of >102/micron 2 or a molecular pitch of <10 nanometers. The precision of this concentration is not adequate to draw any conclusions. The diameter of a single phospholipid has been estimated at 9,76 Angstrom or 0.976 nanometers; this value would support a concentration of ~106 molecules/micron 2.

Mishina et al113. xxx New & Mudge114 xxx

111Wang, H. Wang, J. Thow, X. & Lee, C. (2018) The first principle of voltage gated ion channel and the general probability of circuit probability theory bioRxiv doi: http://dx.doi.org/10.1101/343905

112Fambrough, D. (1979) Control of acetylcholine receptors in skeletal muscle Physiol Rev’s vol 59(1), pages xxx-xxx https://doi.org/10.1152/physrev.1979.59.1.165

113Mishina, M. Takai, T. Imoto, K. et al. (1986) Molecular distinction between fetal and adult forms of muscle acetylcholine receptor Nature vol 321, pages 406–411

114New, H. & Mudge, A. (1986) Calcitonin gene-related peptide regulates muscle acetylcholine receptor synthesis Nature vol 323, pages 809–811 110 Neurons & the Nervous System

Missias et al115. in 1996 performed an elaborate immunological and genetic study using transgenic mice. They asserted the receptor was a pore in the lemma of mouse muscle. “The distribution of AChRs was assessed by an immunofluorescent method.” No figure was provided describing the dimensions of their conceptual pore or how acetylcholine passed through it. Kaminski, Kusner & Block116 even describe isoforms of the AChRs of the extraocular muscles of mice based in immunofluorescent studies. But they do not claim to have ever verified the presence of their AchRs. As noted in Section 23.8.1, the documentation of a pore in the lemma of any cell should be accomplishable the technologies presently available, specifically electron-microscopy. In the 21st Century, Gill-Thind et al117. have written on [xxx I have ] 23.8.2 Permeability of in-vivo lemma by diffusion and/or pores

A large part of the bioscience community has been led to believe that many types of molecules enter the cell through pores in the cell wall based more on concept than data. Addressing the question of how large molecules might pass through the combination of two bilayers that form a lemma is a difficult one from the view of instrumentation as well as fundamental knowledge of the laws of physical chemistry that apply. As the 1999 paper118 by Paula and Deamer note, “To cross a single bilayer membrane, the permeating particle must dissolve in the hydrophobic region, diffuse across, and leave by re-dissolving into the second aqueous phase.” In the real case, this process must be repeated twice. At the molecular level, the total permeation must be achieved in a liquid crystalline environment wherein the middle aqueous phase is itself highly structured (Section xxx).

Until the work of Paula and various colleagues in the 1990's, there had been little progress in this field in several decades. Their work was still limited to various small ions moving through a single man made bilayer of phospholipid material. 23.8.2.1 Permeability of single bilayer in-vitro by diffusion and/or pores

Paula & Deamer provided a comprehensive study of the permeability of a single bilayer in 1999 based heavily on two papers, Paula, et al. of 1996119 and Paula, Volkov & Deamer of 1998120, by addressing single layers of a phospholipid surface. The surface self assembles into a liquid crystal as noted by Kucerka et al121. more recently in 2008. In their Introduction, they note the liquid- crystalline character of the di-phophatidic molecules, “Biological function is intrinsically linked to

115Missias, A. Chu, G. Klocke, B. Sanes, J. & Merlie, J. (1996) Maturation of the Acetylcholine Receptor in Skeletal Muscle: Regulation of the AChR γ-to-ε Switch Develop Biol vol 179(0253), pp 223–238

116Kaminski, H. Kusner, L. & Block, C. (1996) Expression of Acetylcholine Receptor Isoforms at Extraocular Muscle Endplates IOVS vol. 37(2), pp 345-351

117Gill-Thind, J. Dhankher, P D’Oyley, J. et al. (2015) Structurally Similar Allosteric Modulators of α7 Nicotinic Acetylcholine Receptors Exhibit Five Distinct Pharmacological Effects J Biol Chem vol 290(6), pp. 3552–356

118Paula, S. & Deamer , D. (1999) Membrane Permeability Barriers to Ionic and Polar Solutes In Deamer, D. Kleinzeller, A. & Farmbrough, D. eds. (1999) Membrane Permeability. NY: Academic Press Chapter 4

119Paula, S. Volkov, A. Van Hoek, A et al. (1998) Permeation of Protons, Potassium Ions, and Small Polar Molecules Through Phospholipid Bilayers as a Function of Membrane Thickness Biophysical J vol 70, pp 339-348

120Paula, S. Volkov, A. & Deamer , D. (1998) Permeation of Halide Anions through Phospholipid Bilayers Occurs by the Solubility-Diffusion Mechanism Biophysical J vol 74, pp 319–327

121Kucerka, N. Nagle, J. Sachs, J. et al. (2008) Lipid Bilayer Structure Determined by the Simultaneous Analysis of Neutron and X-Ray Scattering Data Biophys J vol 95, pp 2356–2367 Hormonal-affectors 23- 111 membrane structure. The structural basis of biomembranes arises from fluid phase lipid bilayers with almost liquid-like conformational degrees of freedom, so that the structure is best described by broad statistical distributions rather than the sharp δ-functions typical of crystals.”

It is not clear that any of the models used in the Paula et al. papers properly recognize the added constraints imposed by the liquid crystalline state. Paula et al. varied the molecular length of their phospholipids to provide their data points. They did not address the length of the hydrophobic lipids in natural phospholipids of a bilayer. 23.8.2.1.1 Volkov & Deamer on bilayer physical chemistry

Volkov & Deamer122 published a paper in 1997 providing supporting material to the above papers. It provided the size of a long list of both ions and their hydrated forms. They did not address larger molecules the size of most hormones. in Figure 23.8.2-1 provides their Table 2 of hard to find data, with citations. They also provided surface tension values for the oil/water and liquid/air interfaces at 20°C for a variety of simple organic molecules.

122Volkov, A. & Deamer, D. (1997) Two mechanisms of permeation of small neutral molecules and hydrated ions across phospholipid bilayers Bioelectrochem Bioenergetics vol 42, pp 153-160 112 Neurons & the Nervous System

Figure 23.8.2-1 Bare and hydrated ions. Dimensions in nanometers. See text. From Volkov & Deamer, 1997.

The findings of Volkov & Deamer are worth summarizing, Hormonal-affectors 23- 113 “In this paper, we discuss two possibilities that in a sense represent alternative hypotheses. The first hypothesis is that ion or dipole permeation can be understood in terms of the energy related to the partitioning of ions into the non-polar phase of the lipid bilayer, and that electrostatic considerations, including the Born energy, are primary concerns. The second hypothesis is that high dielectric defects (transient pores) occur in the bilayer and allow permeating ions or dipoles to bypass the partitioning energy barriers. The two hypotheses were tested by experimental measurements of proton and potassium flux across lipid bilayers of varying thickness, and the results are described in detail in a paper by Paula et al. (1996).” Partitioning within solutions involves a parameter of Nernst’s Distribution Law of dilute solutions. The partition coefficient, the ratio, at equilibrium, in which a solute separates into two immiscible solvents based on the molar concentrations is typically a temperature sensitive function. It is not clear how Nernst’s Distribution Law applies to the penetration of a 2-bilayer lemma by large protein hormones, and when they are combined with another protein to raise their solubility in blood serum. The potential for these materials, with molecular weights exceeding 1000 Da, to penetrate a natural lemma appears to be marginal to infinitesimal. “Although the partitioning model can describe the barrier properties of lipid bilayers under specified conditions, certain assumptions regarding the hydrated radius of the permeating species are required. We now consider an alternative hypothesis, in which fluctuations in the bilayer structure produce rare transient defects that allow solutes to bypass the electrostatic and so|vophobic energy barriers. Two types of pore structure in the lipid hilayer are possible, which can be roughly classified as hydrophobic and hydrophilic defects. During the formation of a tran- sient hydrophobic defect, lipid molecules are moved apart by thermal fluctuations so that the membrane hydrophobic core makes contact with and is penetrated by the aqueous bulk phase. Hydrophilic defects are formed when the lipid molecules are tilted into the transient defects, so that they are lined with lipid polar head groups. In both hydrophobic and hydrophilic defects, pore formation results from the dynamic properties of the lipid bilayer, and the equilibrium pore distribution is relatively constant over time.”

“In testing the two models described above, an important variable under experimental control is the bilayer thickness, which can be changed by choosing lipids with longer or shorter hydrocarbon chains. In the results discussed in this section, our approach was to measure the proton and potassium flux across liposomes composed of phospholipids with chain lengths varying from 14 to 24 carbons.” They reasoned that, in lipids with short-chain fatty acids, non-polar forces would not be sufficient to stabilize bilayer structures. However, as the chain length increased, at some point stable bilayer vesicles would be formed providing a permeability barrier to the ionic flux. If only the partitioning energy was involved, the ionic flux would be reduced to a minimum when stable bilayers were formed and would not change significantly when longer chain lipids were used. However, if transient defects were the primary factor regulating the ionic flux, shorter chain lipids would have many more defects, leading to much higher ionic permeability than longer chain lipids. The results expressed as proton and potassium permeability coefficients are shown in Figure 23.8.2-2. In 1970, Lehninger asserted that the hydrophobic portion of natural bilayers usually have an even number of carbons between 12 and 22, and that cell lemma consisting of 2-bilayers, of different species varied significantly between 60 and 100 Angstrom thick. 114 Neurons & the Nervous System

We found that the permeability decreased logarithmically as the bilayer thickness increased in the short chain lipids, following the slope of the line predicted by the transient pore mechanism. However, the permeability tended to level off in the thicker bilayers (C16 and C18 lipids), approaching the lines predicted by the partitioning model. Although more data are needed to reach a conclusion, the results suggest that the mechanism of ionic permeation may depend on the bilayer thickness: thinner bilayers have many transient defects that allow rapid permeation of small ionic species, whereas in thicker bilayers defects become so rare that partitioning mechanisms dominate the ionic flux.” “Earlier work has shown that the permeability of lipid bilayers to protons is five to six orders of magnitude greater than to other monovalent cations. This result is consistent with the partitioning model only if every proton carries at Figure 23.8.2-2 Comparing the dependence of K+ least four waters of hydration into the and H+ permeability on diameter with the length bilayer phase.” of the hydrophobic lipid chain at 30°C. See text. from Volkov & Deamer, 1997. It was noted in one of the Paula papers that using the radius pf am H+ ion into their equations gave absurd results.

There was no mention of whether their pore or partition models are compatible with a bilayer in the liquid- crystalline state of matter. 23.8.2.1.2 Paula et al. papers in bilayer physical chemistry

After reviewing these papers, it should be noted they only addressed single molecular bilayers. It would take two bilayers back-to-back to represent a lemma. The single layers were made of phosphatidylcholine, not a phosphatidylamine, suggesting a type 1 (symmetrical and highly electrically insulating) lemma.

The thickness of a given laboratory prepared phospholipid surface varies significantly based on the measurement technique used, from about 50.75 Angstrom reported by Suwalsky (Section 2.1.4) to 41.3 ± 3 Angstrom using a atomic force microscope by Lind et al. (Section 2.1.4.6.1) Suwalsky provided his measurement for DLPE, L–α–dilauroylphosphatidylethanolamine exhibiting 10 CH2 groups in each fatty acid chain. He also provided higher values for two other molecules, DMPE at + 5 Angstrom greater than DLPE and DPPE at + 10 Angstrom greater than DLPE. The 1996 paper’s Abstact define their study, “Two mechanisms have been proposed to account for solute permeation of lipid bilayers. Partitioning into the hydrophobic phase of the bilayer, followed by diffusion, is accepted by many for the permeation of water and other small neutral solutes, but transient pores have also been proposed to account for both water and ionic solute permeation. These two mechanisms make distinctively different predictions about the permeability coefficient as a function of bilayer thickness. Whereas the solubility-diffusion mechanism predicts only a modest variation related to bilayer thickness, the pore model predicts an exponential relationship. To test these models, we measured the permeability of phospholipid bilayers to protons, potassium ions, water, urea, and glycerol. Bilayers were prepared as liposomes, Hormonal-affectors 23- 115 and thickness was varied systematically by using unsaturated lipids with chain lengths ranging from 14 to 24 carbon atoms.” The Abstract closes with, “The results for protons and potassium ions in shorter-chain lipids are consistent with the transient pore model, but better fit the theoretical line predicted by the solubility-diffusion model at longer chain lengths.” There was no discussion of the length of phospholipids in natural bilayers in vivo. The summary of their 1996 paper is informative, “In conclusion, pores seem to be the dominant permeation mechanism for ions, but only if the membrane is sufficiently thin. Ion permeation by partitioning and diffusion seems to become of greater importance as membrane thickness increases, because the number of pores in the membrane produced by thermal fluctuations certainly decreases as the bilayers become increasingly stable. This transition from one mechanism to another is observed at chain lengths between 16 and 18 carbons for potassium ions and at chain lengths between 20 and 22 carbons for protons. On the other hand, because of their high solubility in a hydrocarbon phase, neutral molecules apparently cross exclusively by the solubility-diffusion mechanism, regardless of membrane thickness.”

There was no mention of whether their pore or partition models are compatible with a bilayer in the liquid- crystalline state of matter.

Their 1998 Abstract defines that study,

“ Two alternative mechanisms are frequently used to describe ionic permeation of lipid bilayers. In the first, ionspartition into the hydrophobic phase and then diffuse across (the solubility-diffusion mechanism). The second mechanism assumes that ions traverse the bilayer through transient hydrophilic defects caused by thermal fluctuations (the pore mechanism). The theoretical predictions made by both models were tested for halide anions by measuring the permeability coefficients for chloride, bromide, and iodide as a function of bilayer thickness, ionic radius, and sign of charge. To vary the bilayer thickness systematically, liposomes were prepared from monounsaturated phosphatidylcholines (PC) with chain lengths between 16 and 24 carbon atoms.”

The conclusion drawn by Paula, Volkov & Deamer was quite important. They asserted,

“We conclude from our analysis that the solubility-diffusion mechanism better describes permeation of halide ions across phospholipid bilayers than the pore mechanism. We base our argument on the experimentally observed dependence of the permeability coefficients on the sign of the ionic charge, on the bilayer thickness, and on the ionic size. In each case, the comparison of the experimental evidence to theoretical predictions supported the solubility-diffusion mechanism and was inconsistent with permeation through pores.” This finding did not even consider the more complex back-to-back bilayers of an actual biological lemma. Paula & Deamer (1999) go on to discuss the [xxx need for two figures to account for data ] [xxx edit for two figures ] Two figures ultimately from Paula in chapter 4123 provides their estimates of permeability through a membrane based on their measured values in related experiments is reproduced as Figure 23.8.2-3.

123Paula. S. & Deamer. D. (1999) Membrane Permeability Barriers to ionic and Polar Solutes in Deamer, D. Kleinzeller, A. & Farmbrough, D. eds. (1999) Membrane Permeability. NY: Academic Press Chapter 4 116 Neurons & the Nervous System

Figure 23.8.2-3 Permeability coefficients of potassium ions and halides [experimental data from Paula et al. (1998)] Solid lines are solubility–diffusion mechanism and dashed lines are pore mechanism. See text. From Paula & Deamer, 1999.

Figure 23.8.2-4 shows the equally important graph of permeability as a function of ion radii. Hormonal-affectors 23- 117

Figure 23.8.2-4 Calculated permeability coefficient for a single bilayer of a lemma (membrane) of a cell. See text. From Paula & Deamer, 1999.

In the close of the Paula, Volkov & Deamer, paper they assert “We conclude from our analysis that the solubility-diffusion mechanism better describes permeation of halide ions across phospholipid bilayers than the pore mechanism. We base our argument on the experimentally observed dependence of the permeability coefficients on the sign of the ionic charge, on the bilayer thickness, and on the ionic size. In each case, the comparison of the experimental evidence to theoretical predictions supported the solubility-diffusion mechanism and was inconsistent with permeation through pores.” 118 Neurons & the Nervous System

124 In 1999, Paula, Akeson & Deamer provided more data on the passage of water, H2O, through “biological membranes.” Their Introduction begins with “The bacterial toxin α-hemolysin secreted by Staphylococcus aureus damages cells by forming large pores in the cell membrane. The subsequent leakage of molecules and ions from the cell interior leads ultimately to cell disruption.” α-hemolysin channels were incorporated into rabbit erythrocyte ghosts at varying concentrations, and water permeation was induced by mixing the ghosts with hypertonic sucrose solutions.” Their procedure for preparing ghosts is complex and includes centrifugation at 12000X g for 8 minutes. They did provide useful data on the water permeability of xxx. Typical examples for single-channel permeability coefficients reported in the literature for water are 9.6 x 10--15 cm3 /s for gramicidin A, 5.6 x 10–14 cm3/s for certain water transporters found in toad bladders, 6 x 10–14 cm3/s for the water channel CHIP 28, 1.5 x 10–14 cm3/s for double-length nystatin, 1 x 10–13 cm3/s for single-length nystatin, and 4.5 x 10–14 cm3/s for amphotericin B. These numbers are up to two orders of magnitude smaller than the single-channel permeability coefficient estimated for α-hemolysin which is consistent with the much larger pore size of α-hemolysin.

23.8.2.2 21st Century research into lemma of two bilayers

23.8.2.2.1 Shinoda paper in bilayer physical chemistry

Shinoda presented a paper in a special issue of Biochimica et Biophysica Acta vol 1858 in 2016. His focus was on medical and pharmaceutical applications. He notes, “Experimental permeability measurements have been performed for many solute molecules through various lipid membranes. Permeability or leakage of a small molecule should give an experimental measure of the structural stability of the membrane because it should reflect lipid packing in the membrane core. However, experimental approaches cannot provide adequate information on the mechanism of passive transportation at the molecular level. Understanding of the regulation and/or mechanisms of molecular transport by lipid membranes requires a detailed estimation of interactions between permeants and lipid membranes.”

He also notes, “Molecular simulation can prove to be very useful for probing the molecular mechanism of membrane permeation processes. However, a brute-force molecular dynamics (MD) simulation is not straightforwardly useful because the typical time range of MD is too short to directly observe the complete permeation process through the membrane. Thus, an alternative approach based on a permeation model is needed.

Figure 23.8.2-5 demonstrates clearly that Shinoda was working with lemma consisting of two bilayers back-to-back.

Shinoda has provided 162 citations.

In discussing his approach, he notes, “Although several models have been developed to explain the possible permeation mechanisms, in this review, we focus on two major mechanisms for membrane permeation, namely, the solubility-diffusion mechanism and transient pore formation mechanism. Permeation of small neutral or polar molecules across lipid bilayers with different membrane thicknesses (lipid chain lengths of 14–24 carbon atoms) has been systematically investigated by Paula and associates, and their results have shown that the solubility-diffusion model works well for modeling permeation of small molecules as long as the membrane under consideration is thick. Additionally, most MD studies evaluating membrane permeability use this model; therefore, we have primarily focused on this model. However, when permeation of a polar small molecule through a thin lipid membrane is considered, transient pore-like defects in the membrane could explain the permeability better. In this case, the permeation process is very different, and pore formation itself is a time-limiting step for the permeation.”

124Paula, S. Akeson, M. & Deamer, D. (1999) Water transport by the bacterial channel α-hemolysin Biochim Biophys Acta vol 1418, pp 117-126 Hormonal-affectors 23- 119 “Overton observed that the membrane permeability coefficient of a solute is correlated with its oil/water partition coefficient. This leads to a simple model wherein the lipid membrane is treated

Figure 23.8.2-5 Snapshots of CER2 bilayers in ethanol–water mixtures after at least 100 ns of MD simulation. The ratio of ethanol:water molecules is shown in the bottom right corner of each snapshot. CER2 molecular are colored in cyan, water molecules in blue, and ethanol molecules in orange. Reprinted from Thind et al., 2015. as a simple homogeneous oil slab. In the solubility-diffusion model (also known as the Meyer – Overton rule), the intrinsic permeability coefficient, P , is written as P . KD/d where K , D ,and d are the oil/water partition coefficient, the solute's diffusion coefficient in the oil slab, and membrane thickness, respectively. As shown by many experimental and computational observations, the lipid bilayer membrane is quite heterogeneous along the bilayer normal, Z. Thus, an inhomogeneous solubility-diffusion model was proposed to provide a more realistic description of membrane permeation.”

“Although the probability of spontaneous pore formation in an ordinary lipid bilayer by thermal fluctuation is quite low, once it happens, there is no doubt that the pore will provide a readily permeable pathway for hydrophilic molecules to cross the membrane. Therefore, in this case, analysis of pore formation itself is critical. Studies have also shown that transient pore formation can be induced through several methods, including electroporation, antimicrobial peptides, cationic polymers, external stress, shock waves, and sonoporation (with citations). A more complete, broad review on defect- mediated transport can be found in Gurtovenk et al., in Marrink et al. and in Bennett et al.” The Bennett125 paper is very useful here. Figure 23.8.2-6 xxx

125Bennett, W. & Tieleman, D. (2014) The Importance of Membrane Defects—Lessons from Simulations Acc Chem Res vol 47(8), pp 2244-2251 120 Neurons & the Nervous System

Figure 23.8.2-6 Snapshots of the tyramine/POPC systems, with tyramine near the bilayer center. Tyramine is a derivative of tyrosine. A; The unprotonated tyr permeates without any water accompaniment, and with no obvious orientational preference. B; The protonated tyr+ never loses the solvation shell around the amine group, and as such always orients along its long axis with the phenolic group leading and the amine trailing. Reprinted from Holland et al, 2015.

23.8.2.3 Permeability of lemma by hydrogen ion–A special case

Paula & Deamer (1999) have described the permeability of H+ with respect to the biological lemma as a Special Case. They note,

“The permeability of membranes to solutes is a fundamental property of all cellular life. Living cells depend on membranes that provide a substantial barrier to ionic solutes such as protons, sodium, potassium and calcium in order to maintain the ion gradients necessary for bioenergetic functions, nerve cell activity, and regulatory processes. Paradoxically, the same membranes must offer much lower barriers to the flux of water, oxygen and carbon dioxide, which need to move rapidly into and out of the cell.” [xxx continue here ]

We now know that despite being approximately the same size, solutes such as sodium ions, hydrogen ions, water , and oxygen permeate the lipid bilayer at rates that vary by 12 orders of magnitude. How could such a vast difference in permeation rates by imposed by a seemingly simple bimolecular layer of amphiphilic molecules? Surprisingly, hydrogen ions (protons) penetrate the same bilayer at rates 5 orders of magnitude faster than other monovalent cations. How might such a large anomaly arise? Among all the permeants that are of biological interest, protons should be regarded as a special case!” Following these remarks, Paula & Deamer review several optional models to the solubility-diffusion model and the transient pore model and find they give unrealistic results. They address one model as follows, “However protons can diffuse by a special mechanism that is not available to other cations, which involves hopping along hydrogen-bonded chains of water. One example of this hopping is the translocation that occurw along the single strand of water molecules in the gramidcidin channel (with citations)”. Unfortunately, they did not address the most simple and likely mechanism that is foreign to the background of most bio-scientists, even in 1999. It is not necessary for a proton to actually Hormonal-affectors 23- 121 penetrate the lemma for a free proton to appear on the other side and immediately combine + + with water to form H3O or H9O4 . The method is known as “hole transport” through a crystalline or liquid-crystalline material that is known to be a semiconductor. The process is discussed in detail in any textbook on semiconductor physics. The phospholids of a membrane contain long chain fatty acids which can give up an electron easily and produce what is called a “hole.” A hole is the absence of a negative charge; equivalent to a positive charge or a.k.a., the equivalent of a proton. This apparent positive charge is observed to travel through the crystal by a hopping process; an electron fills the hole (a vacancy in the crystalline lattice) resulting in creating another hole. The process continues until the hole reaches the edge of the lattice and passes into a different semiconducting material such as water. There, the hole is annihilated by combining with an electron. The result is the appearance of a positive charge where the + electron came from. This charge is most easily associated with a molecule of water to form H3O . + Thus a molecule of H3O on one side of a bilayer can be transferred to the others side of the lemma at a velocity slower than that of an electron but faster than any other ionic species. The heuristic argument that a hole behaves as a free positive charge carrier may be justified by quantum mechanics and demonstrated in the laboratory. The above mechanism is the key to to the operation of all man made transistors and the Activa defined as the biological transistor in this work (Section 2.2.1.3). In doped silicon transistors, the nominal mobility of holes is 500 cm2/V-s where the electrical field within the semiconductor is given by V/cm. and the velocity is in cm/s. The mobility of electrons in the same material is 1300 cm2/V-s.

The conductivity of biological semiconductors is an important area of research at this time. In organic, and biological, chemistry, the double bond of carbon, specifically the p-bond, can act as a donor type of dopant and act as a source of electrons within a liquid-crystalline structure. All phospholipids associated with a cell lemma contain at least on unsaturated fatty acid.

The hole transport model of semiconducting phospholipids in a cell lemma provides a much more acceptable solution to the permeability of bilayers to hydrogen ions than any other model.

Note the voltage gradient across the membrane is a significant factor in the motion of both electrons and holes in semiconductors like the phospholipids in the liquid crystalline state. As a result, measurements of the lemma or individual bilayers in a pH environment that is the same on both sides of the sample, like those of Nichols & Deamer. Their equation 1 does not include an electrical bias across their sample as normally encountered in both neurons and simple cells.

During the 1980's, additional efforts were made to account for the proton conductivity of lemma.

In 1986, Perkins & Cafiso126 made additional attempts to account for the high permeability of phospholipids to hydrogen ions using a different protocol. After reviewing the literature and their experiments, they note, “The molecular basis for this conductivity is presently unknown.”

Investigations slowed significantly in the 1990's and have remained sporadic up to the current time. It should be noted that it was only in the late 1980's that investigators of hydrogen ion transport began speaking of the conductance (an electrical engineering term) rather than permeability (a chemistry term) of lemma and individual bilayers127. [xxx add here ] 23.8.3 Chemistry of the intracellular receptors of a cell EMPTY

The premise of this section is that hormones are able to pass through type 3 lemma of a cell, a

126Perkins, W. & Cafiso, D. (1986) An electrical and structural characterization of H+/OH– currents in phospholipid vesicles Biochem vol 25, pp 2270-2276

127Gutknecht, J. (1987) Proton/hydroxide conductance and permeability through phospholipid bilayer membranes PNAS vol 84,p 6663-6446 122 Neurons & the Nervous System highly ordered but apparently dynamic, liquid crystalline bilayer membrane, by either common diffusion or via a pore. After passing through the lemma, the hormone is able to stimulate a receptor either on the surface of the nucleus, or on another element within the cytosol of the cell. 23.8.3.1 Chemistry of the nuclear receptors EMPTY

23.8.3.2 Chemistry of receptors of elements in the cytosol EMPTY Hormonal-affectors 23- 123

23.9 Interplay of the neural-glandular-muscular modalities in animal physiology.

This section is duplicated in Section 16.6. The interplay of the neural-glandular-muscular modalities offers an immense capability, and flexibility, to the physiology of any animal. The neural modality and the glandular modalities effectively operate in quadrature. The result is that the capabilities of each modality are multiplied together in order to describe the overall potential capability. Figure 23.9.1-1 illustrates in block form the options for combining the neural, muscular and glandular modalities. Frame A illustrates a neuron as displayed previously but with a defined modulator receptor site. The receptor at this location is primarily to accommodate glandular inputs, such as epinephrine or norepinephrine. The concept (applicable to all three frames of the figure) is that there can be separate receptors for one or more glandular inputs to a neuron besides the required electrolytic receptor, neurite receptors of neural (electrical) signals and chemical inputs if required (to support stage 7 neurons). These defined receptor locations are distinct from those required for the cell to maintain homeostasis.

Frame B illustrates a muscle or gland in block form. [xxx consider separating ] If representing a muscle, the same supporting receptors are shown with the output in the form of a mechanical force. The input is shown as two alternatives; a positive input causing a striated muscle to contract when stimulated by acetylcholine, ACh, and a negative input causing a smooth muscle to relax when stimulated by @NO. If representing a gland, the same supporting receptors are shown with the output in the form of a hormone. The input can be either a neural signal or a hormonal stimulus applied to the positive input terminal. Again, the chemical supplies are those provided in addition to those required for homeostasis.

In both cases represented by frames A & B, the modulator receptors are sensitive to and selective for hormonal stimulants. Their selectivity may allow multiple hormones at each location (such as both thyroxine and either epinephrine or norepinephrine.) The potential enzyme protecting the receptor might even accommodate a master key system allowing different hormones to stimulate multiple cells in selected groups. 124 Neurons & the Nervous System

Figure 23.9.1-1 Options for the interplay of neural, muscular & glandular ADD modalities. The input signals for the neurons of stage 1 through stage 7, frame A, typically enter the cells via type 2 lemma as part of a tight (electrical) synapse (typically without the need for any receptor). The receptors for the modulators and the electrolytic supplies also utilize type 2 lemma but with a distinctive receptor molecule present. The chemical supplies may enter the cell via type 3 lemma. See text. Hormonal-affectors 23- 125 The modulator and energy supply terminals in A, and probably in B, do not employ protein receptors of the GPCR family. See Section 3.2.4. The receptors at these locations are typically amino-phospholipids. The receptor for the electrolytic supplies is known to be phosphatidylserine molecules in a liquid crystalline array forming the outer bilayer of a region of type 2 lemma of the cell involved. With the flexibility offered by the multiple neural signal inputs accompanied by possible modulator inputs, stage 7 neurons can exhibit great flexibility. The flexibility is much lower for stage 3 and stage 6 neurons who generally operate in the pulse mod of signaling. Most stage 1 neurons contain their own mechanism for controlling their input signals and providing automatic gain control (Section 3.xxx). They do not need the flexibility of modulators and their input signals are controlled by the environment. Frame B illustrates a muscle in block form. The same supporting receptors are shown with the output in the form of a mechanical force. The input is shown as two alternatives. The upper, positive, input causes a striated muscle to contract when stimulated by acetylcholine, ACh. The lower, negative input causes a smooth muscle to relax when stimulated by @NO. Again, the chemical supplies are those provided in addition to those required for homeostasis. Frame C illustrates a gland in block form. If representing a gland of the hypothalamus or hypophysis, the same supporting receptors are shown with the output in the form of a another hormone. The input is shown as two alternatives. The upper, positive, input causes an increase in hormonal output. The lower, negative, input causes a reduction in hormonal output (regardless of what hormone stimulates this negative input terminal. The same supporting receptors are shown with the output in the form of a hormone. The input can be any hormone that can stimulate the receptor associated with the appropriate receptor. Again, the chemical supplies are those provided in addition to those required for homeostasis.

If frame C represents a peripheral gland. The potential input terminals, the electrostenolytic, and chemical supply terminals operate the same. However, the output can be considered to be hormones, or be given a different functional name, like gastric juices, perspiration, etc.

In the cases represented by frames A, B & C, the modulator receptors are sensitive to and selective for hormonal stimulants. Their selectivity may allow multiple hormones at each location (such as both thyroxine and either epinephrine or norepinephrine.) The potential enzyme protecting the receptor might even accommodate a master key system allowing different hormones to stimulate multiple cells in selected groups. The neurons, muscles and glands can each take advantage of the flexibility offered by the modulator channel. 126 Neurons & the Nervous System

The modulator and energy supply terminals in A, and probably in B & C, do not employ protein receptors of the GPCR family. See Section 3.2.4. The receptors at these locations are typically amino-phospholipids. The receptor for the electrolytic supplies is known to be phosphatidylserine molecules in a liquid crystalline array forming the outer bilayer of a region of type 2 lemma of the cell involved.

23.9.1 A hair follicle as an exemplar

[xxx show figure of follicle used twice in this chapter see also 23. 7.2.1.1 ] Figure 23.9.1-2 illustrates a typical hair follicle of animals (including humans). [xxx add ]

When discussing the joint capability of this combination of modalities, it is useful to discuss the individual hair follicle of skin. The hair follicle consists of a single semirigid hair that is supported and moved by a muscle. This muscle is driven routinely by a stage 7 neuron, supporting a localized chemical interface between the two. Simultaneously, the muscle, and/or the stage 7 neuron is susceptible to a variety of individual hormones.

For hormonal action to affect the stage 7 neuron, the neuron may support regions of type 2 outer lemma that constitute receptor sites for one or more distinct hormones. The hormones are transported to the neuron by the global cardiovascular network. An individual receptor site may be sensitive to only one or one combination of hormones. Alternately, the type 2 outer lemma may support multiple types of receptor sites. These sites may be present on the dendritic lemma and/or the poditic lemma of the neuron. Similarly the neural inputs to the Figure 23.9.1-2 Complex hair follicle showing same stage 7 neuron may converge on muscular, glandular and neural elements. See either the dendritic (non-inverting) or the text. poditic (inverting) terminal of the neuron. Each synapse can be considered a local stimulus affecting only one neuron. In fact, the synapses associated with either the dendritic or poditic terminals may be associated with on the order of 1000 individual antidromic neurons. Without greater knowledge of the signaling format used within the neural system, little more can be said about the messages delivered by these large groups of synapses. The important point is that the architecture of the physiological system provides a maximum number of combinations of potential positive stimuli, and/or negative stimuli (suppressants) applied to a given stage 7 neuron and associated muscular cell at a given instant is virtually unlimited. It is up to the specific architecture of the physiological system to determine how many of these potential combinations are implemented in order to satisfy the requirements of the ecological niche where the animal resides and thrives. The specific architecture is far beyond our current ability to comprehend. 23.9.2 [Reserved]

23.9.2.1.1 The formation and release of pheromonesin sweat ? EMPTY Hormonal-affectors 23- 127 See Figure 8.7.1-4 for an overly complex figure on a hair follicle and surrounding tissue. [xxx somatosensory art/ skin mod.wpg ]

23.9.2.1.2 The formation and release of pheromones in urine ? EMPTY [xxx ]

- - - -

23.10 Cataloging hormones by chemical structure

Historically, there has been little progress in organizing knowledge concerning hormones based on their molecular structure. After the turn of the 21st Century, several texts have appeared that still reflect the conventional 20th Century views of the community based on valence chemistry and the chemical groups of organic chemistry. These texts have largely failed to describe the operation of hormones or their familial relationships. Coordinate chemistry and the spatial relationship between the “active atoms” within a molecule hold the key to understanding the operation of the hormones. The active atoms consist of oxygen, nitrogen, sulfur and occasionally phosphorous along with various bonds of aliphatic chains and ring structures that can share electrons in a coordinate bond relationship.

Section 23.1.3.1 has introduced a familial structure for hormones that simplifies the above problem.

23.10.1 Background

To support a major effort to determine the pertinent structural features of hormone receptors, it is important to have an orderly understanding of the potentially different types of hormones. Any attempt to organize the various types of hormones must necessarily be arbitrary. However, it is important to begin somewhere.

A common problem noted in 1976, in a preface to the book by Levey appears to remain true today128. “Traditionally a ‘receptor’ has been difficult to define in pharmacologic terms.” This assertion appears to be based on the chemical theory of the neuron (and other cells) current during the 20th Century. The primary difficulty is that, at least the simpler hormonal receptors are not chemically based physiological features. They are fundamentally electrical features with some steric constraints best explained in terms of the Electrolytic Theory of the Neuron. The bonding (binding) does not employ valence bonds but is based on coordinate (London) bonds. It should be noted that nitric oxide, @N=O, was not recognized as a relaxant of smooth muscle until late in the 20th Century.

A second problem from the 1975 time period were the requirements stated by Cuatrecasas129. He essentially repeated his assertions of 1974 in the 1976 Levey compilation, “C One, the hormone interaction with the receptor should conform to known steric and structural specificity. C Two, the binding sites should be finite in number and therefore, saturable. C Three, hormone binding should have tissue specificity consistent with biological specificity. C Four, hormone binding should be of high affinity and consistent with its physiologic concentrations. C Five, the binding of hormone to its receptor should be reversible.”

128Levey, G. ed. (1976) Hormone-receptor interaction: molecular aspects. Volume 9 of Modern Pharmacology- Toxicology NY: Marcel Dekker

129Cuatrecasas, P. (1975) xxx Adv Cyclic Nucl Res vol 5, pp 79-xxx 128 Neurons & the Nervous System

These are sensible requirements within the knowledge base of the early 21st Century but were considered within a set of largely constrained set of inadequately defined Bayesian ground rules in the 20th Century. Levey adopted a figure from 1967 for the cover of his book. The figure is at best conceptual under what is known now about the surface chemistry relating to the lemma of a cell. As a specific challenge at that time, “binding” was not well defined in the context of hormone-receptor interaction. Section 23.10.2 provides a proposed update of the Levey cover art. Lefkowitz, chapter 14 in Levey, has made intriguing comments about the receptors for the catecholamines and a methodology for identifying those receptors. This apparently has not occurred to the level of the critical molecular structural features as of 2018. A complicating factor in trying to identify hormone receptors is their wide distribution within the physiological systems of an animal (or plant). This chapter is restricted to, and will only address, the neural/glandular and glandular/glandular interfaces. [xxx is this correct? Move to start of chapter ]

23.10.2 Defining framework circa. 2018

The study of the complete hormonal system has not been named within the physiology community. For purposes of this work, the broad study of all areas of the hormonal system shall be labeled Crinology.

While the field of crinology is dominated within the academic and medical field by a focus on endocrinology, it is much broader than that. As noted in Section 8.3, it also includes paracrinology and exocrinology as well as endocrinology. This broader area is illustrated in a figure in Section 23.10.2.1.

Belfiore & LeRoith have recently published a major treatise on endocrinology as the term is used in the medical community130.

ADD

Belfiore & LeRoith, in their preface, attempt to broaden the traditional medical field of endocrinology to include a broader framework.

“We are proposing this series as the manifesto for Endocrinology 2.0, embracing the fields of medicine in which hormones play a major part but which, for various historical and cultural reasons, have thus far been “ignored” by endocrinologists.”

Reviewing their table of contents, it is clear that their proposed Endocrinology 2.0 still does not address paracrinology or exocrinology adequately. It does not address what are generally considered important hormones in physiology; nitric oxide and acetylcholine are considered important hormones critical to the local stimulation of muscle tissue. They also do not address exocrinology in a broad context; they do not address the pheromones, that are of major importance in the continued existence of virtually any animal species. Therefore, the first edition of their endocrinology 2.0 might be considered a call to arms within the medical community, but not a comprehensive review of the broad field of crinology as used in this work and the physiology community. Leung & Farwell, provided chapter 1 in Belfiore & LeRoith. Unfortunately, the material is significantly dated and not appropriate for the editors’ objective of an Endocrinology 2.0. Leung & Farwell have provided a figure 1 on page 5 that ostensibly describes their perception of the endocrine system. It is little more than a cartoon. An extensive table on their page 25 provides an organized introduction to their hormone types. However, the table does not include

130Belfiore, A. & LeRoith, D. (2018) Principles of Endocrinology and Hormone Action. NY: Springer https://doi.org/10.1007/978-3-319-44675-2_1 Hormonal-affectors 23- 129 nitric acid or acetylcholine as paracrine hormones. The table also omits the exocrine hormones, the pheromones. Since they have not included a definition of a hormone, the inclusion of retinoic acid as a steroid hormones in the table appear unjustified. “Steroid hormones are lipid molecules derived from a common cholesterol precursor (Cholestane, C27)131.” Retinoic acid is typically considered a terpene derivatives. Leung & Farwell introduce the subject of hormone receptors on page 27 but the discussion is cursory and does not include any substantive material. Figure 2 on that page can be expanded to include the processing of paracrine and exocrine hormones. However, such an expansion requires a major rewrite of their paragraphs under the heading “Hormone–Receptor Binding” as well as many other sections within chapter 1. Malandrino & Smith presented chapter 2 in Bellfiore & LeRoith. They primarily addressed the formation of peptide hormones based on the genetic code of DNA and mRNA. They provided little specific information on the structural features of the peptide hormones that made them function as hormones. Acconcia & Marino presented chapter 3 in Bellfiore & LeRoith. They primarily addressed the formation of steroid hormones. They provided a tabulation of steroid hormones but without providing any precise definition of a steroid. They do not address the process by which steroid hormones interact with the appropriate receptor molecules in order to create a neural signal. They do note that, “Vitamin D and its metabolites are not technically steroids in the strict chemical sense, as the B ring of cholesterol is opened (secosteroid). Nevertheless, these sterols are derived from cholesterol, assume shapes that are very similar to steroids, and bind to a nuclear receptor. They do not address how binding to a nuclear receptor is accomplished.

Their figure 6 describes the feedback associated with the H-P-E axis )their H-P-A axis) and a proposed pancreatic-insulin loop. Both frames lack any discussion of the servo loop parameters usually associated with such operations.

In discussing the different crine (hormonal) subsystems, they do note, “A type of hormonal communication system does not involve the circulatory system at all. In paracrine systems, hormones secreted from the steroidogenic cells interact with their cognate receptors in neighboring cells, which are reached by diffusion. As with endocrine subsystem, the nearby target cells may be all the same type or may differ from each other.” They also introduce an intracrine submodality and note, “Finally, some cells both produce the same hormone and respond to it. This type of system is referred to as autocrine for all class of hormones excluding steroid hormones.” They do not elaborate on the reason for this exclusion or how the autocrine interfaces function.

With regard to steroid- binding proteins, they note, “Most steroid hormones have limited solubility in plasma due to their intrinsic hydrophobic nature; accordingly, steroid hormones are largely (99%) bound to specific plasma transport proteins, which are synthesized in the liver. All steroid hormones, except one, have their cognate plasma-binding protein. The exception is aldosterone; 50% of aldosterone is believed to circulate as the free steroid in the plasma compartment. Each transport protein has a specific ligand-binding domain for its cognate hormone, which displays little amino acid sequence homology with the ligand binding of the cognate receptors. The Kd of a steroid hormone for its plasma transport is always “looser,” e.g., 1–100 X 10--8 M, than that of the Kd of the nuclear receptor. Thus, the tighter binding of the steroid hormone to its target receptor when it has arrived at a target tissue allows the hormone to be concentrated inside the target cell. The current view is that it is the “free” form of steroid hormones and not the complex of the hormone with its plasma transport proteins that interacts with receptors in or on the target cells to begin the sequence of steps that result in the generation of a biological response.” Koibuchi presented chapter 4 in Bellfiore & LeRoith. He primarily addressed the synthesis and secretion of the thyroid hormones. The material was not significantly different from what is currently in this work. He did reiterate, “The thyroid hormone synthetic pathway is quite unique, in that, although it is a relatively small molecule comprising two iodinated phenol rings, it is

131Henley, D. Lindzey, J. & Korach, K. (2005) Steroid Hormones In Melmed, S. & Conn, P. eds Endocrinology, 2nd Edition Totowa, NJ: Humana Press chapter 4 130 Neurons & the Nervous System synthesized by the breakdown of a large protein, TG. Furthermore, most synthetic process occurs in the extracellular space (follicular lumen).” Part III of Bellfiore & LeRoith address “Hormone Receptors and Signal Transduction Processes.” However, the entire chapter 5, by Pincas et al. is devoted to the putative G-protein coupled receptor. The GPCR’s have not played a significant role in the development of this work and within the Electrolytic Theory of the Neuron. The remaining chapters of Belfiore & LeRoith will be addressed in Section 23.11.1. Figure 23.10.2-1 shows a proposed extension of the hormone/receptor interface dating from Levey during the 1967-1976 time period. The cell membrane is formed as a bilayer of two phospholipid. When the two phospholipids are identical, the membrane is labeled type 1, When they differ, the membrane is labeled type 2. In the case of sensory receptor tissue, the outer phospholipid appears to be a phosphatidyl-amino acid. This appears to be true of virtually all sensory receptor tissue, whether involved in taste, smell, pheromone or internal chemical sensing. This proposal is supported by much of the literature, dating from the compilation of papers edited by Levey in 1976 forward. The catalyst is shown in conceptual form. It is defined as adenylate cyclase, an enzyme in modern usage. No discussion was presented as to how this enzyme controlled the ATP to cAMP transition.

The coupler is in fact a thin layer of water molecules in the liquid crystalline state coupling the two phospholipid pairs at the molecular level (Section 2.1.4 and Section 2.2.2.7). The resulting phospholipid pairs are assembled with other similar molecules to form a large two-dimensional bilayer. Two such bilayers are joined at their planar surfaces to form a lemma. If the lemma is used as the outer lemma of a cell, it is usually described as a cell membrane. However, it is common for such lemma to occur within a given cell. Hormonal-affectors 23- 131

Figure 23.10.2-1 Proposed generic hormone/receptor interface extended from Levey. If both phospholipids in a phospholipid pair are phosphatidylcholine, the membrane is labeled type 1 and forms a near perfect electrical insulator. If the two phospholipids in a phopholipid pair are different, the membrane is labeled type 2 and the membrane can exhibit semiconducting electrical characteristics. See text. Extended from Levey, 1976.

To understand the total field of crinology requires an ever more precise definition of the subject matter. See Section 23.10.2.3. 23.10.2.1 Potential cellular locations of hormonal receptors

The papers in Levey, 1976, begin to focus on the location of the hormonal receptors via relatively simple experiments appropriate for that time period. They did differentiate between receptors located on the external lemma of the cell and the alternate situation where the receptors are either located on the external surface of the nucleus (and possibly other locations within the cytosol of the cell). [xxx add ] The group of papers in Levey only address a few types of hormonal receptors. A problem in the protocol frequently used in discussing receptors, is the failure of most investigators to show their receptor of interest is exclusively senstive to a single hormone or its close derivatives. There is a considerable chance that many hormones may interface with a given receptor that is also sensitive to other hormones (with a substantially different efficacy between the two hormones.

The potential for multiple hormones to interface with a given receptor with different efficacies suggests that the hormones may employ either 2-point of 3-point coordinate bonding as found 132 Neurons & the Nervous System among some of the gustatory receptors (particularly the sugars and sugar substitutes (Section 8.xxx).

23.10.2.2 Potential categories of hormones based on structural complexity

Initially, it is important to establish the relative complexity of different hormones affecting the neural, muscular, and lower level glands of the physiology of an organism. Figure 23.10.2-2 provides a simple attempt to organize the fundamental characteristics of hormones, their region of operation. [xxx ADD ] Hormonal-affectors 23- 133

Figure 23.10.2-2 Preliminary table characterizing hormones ADD. The gastric juices are considered hormones in some academic circles even though they do not communicate between distinct living cells. See text.

The labeling of the gastric juices as hormones may stretch the definition given above. They clearly originate in glandular tissue but their destination is not another biologically living cell. Gastric juices were probably the origin of the largely archaic term ducted hormones. They are generally conveyed from the pancreas to the digestive tract of the alimentary canal. As time went by, the other hormones were found to be delivered ductlessly, via the cardiovascular system. Currently, the hormones (using the above definition) of the hypothalamus designed to 134 Neurons & the Nervous System travel to the hypophysis (Pituitary gland) are the only major hormones traveling through a dedicated duct, known as a portal. 23.10.2.3 Definition of a hormone and hormonal system, circa. 2018

In 2005, Conn provided an update on the definition of a hormone132, “Substances that provide the chemical basis for communication between cells are called hormones. This word coined by Bayliss and Starling, was originally used to describe the products of ductless gland released into the general circulation in order to respond to changes in homeostasis. Hormone has taken on a broader usage in recent years. Sometimes hormones are released into portal (closed) circulatory systems and have local actions. The word paracrine is used to describe the release of locally acting substances. This word also describes local hormone action as the diffusion of gastric juice acts on neighboring cells. Hormonal substances release by an animal that influence responses in another animal are referred to as pheromones.” Conn goes on to expand the above definition marginally, “On occasions, growth factors are (appropriately) called hormones, because they mediate signaling between cells. In recent years, the word has become a catchall to describe substances released by one cell that provoke a response in another cell even when the messenger substance does not enter the general circulation.”

The two paragraphs of Conn’s definition can be combined into a more succinct version based on the above figure. Specifically, C Pheromones are elements of the exocrine subsystem that do not involve the general cardiovascular circulation of either animal. The pheremones are found to support external chemical sensing similar to that used in the olfactory modality (Section 8.5). The actual sensing of Pheromones follows the same sensory processes as the olfactory modality, and is defined as the oskonatory modality in Section 8.6. Their mode of sensing is highly suggestive of the character of various sensory receptors used throughout the physiology of an animal. C There is no defining characteristic that separates the (previously defined) growth factors from other hormones. Their molecular structures are included in one or more categories of the above figure. C The question of whether the hormones released into the digestive tract are endocrine or exocrine hormones is open to discussion. Technically, the digestive tract is external to the animal physiology, even though the animal tissue surrounds this channel. The channel is open to the external environment at both ends. Only muscular contractions isolate the digestive tract temporarily and intermittently from the external environment. In this work, digestive juices are elements of the exocrine subsystem. C Many of the previously defined growth factors are actually hormones by definition; they do mediate signaling between cells beginning with the hypothalamus. They generally communicate with the hypophysis (pituitary gland) via a portal. C All of the hormones investigated as part of this research program (circa. 2018) communicate with their target receptor via dual antiparallel coordinate bonding, DACB. C There are recent references in the academic literature to an autocrine facility133. The autocrine facility consists of a hormone secreted from a cell and targeting a receptor on the surface of the same cell. This is a degenerate condition. The resultant circuit may perform similarly to the

132Conn, P. (2005) Introduction to endocrinology In Melmed, S. & Conn, P. eds Endocrinology, 2nd Edition Totowa, NJ: Humana Press page 3

133 Pandit, N. (2007). Introduction To The Pharmaceutical Sciences. p. 238. ISBN 978-0-7817-4478-2. Hormonal-affectors 23- 135 “active diode” form of synapse (Section xxx). In the active diode, the collector (post synaptic terminal) is connected directly to the base of the Activa forming the synapse. The electrolytic circuit is used to form a “perfect diode” which prevents any signaling component from traveling from the post synaptic terminal to the presynaptic terminal of the synapse. It is necessary to understand what other signals may be stimulating the cell before accepting an autocrine function within the hormonal system, C There are recent references in the academic literature to a juxtacrine facility134 , 135. The entry in Wikipedia is not attributed to anyone and lacks any citation. It is accompanied by a cartoon but no other substantial data. Quoting that source, “juxtacrine signaling (or contact-dependent signalling) is a type of cell / cell or cell / extracellular matrix signaling in multicellular organisms that requires close contact.” This quotation sounds very similar to the local paracrine type of hormonal activity defined above. A juxtacrine facility will not be addressed further in this work. A consolidated definition of hormones and the hormonal system, as of 2018, can be stated as, Substances that provide the chemical basis for communication between cells are called hormones. The term hormone includes the previously labeled “.” Hormones support a large modality within the physiology of animals given the name hormonal system. The hormonal system can be subdivided into the paracrine, endocrine and exocrine subsystems. The paracrine subsystem supports local hormonal (chemical) signaling, primarily between stage 7 neuroaffector neurons and muscle cells within the physiology of the animal. The exocrine subsystem supports chemical communications between hormonal cells within the body of the animal and receptors associated with cells external to the body of the animal. The cells external to the body of the animal may be within the confines of the digestive tract or they may be intended to reach target cells associated with a separate animal. In the former case, the hormones have been labeled gastic juices. In the latter case, the hormones have been labeled pheromones. The endocrine subsystem supports chemical communication over an extended distance within the confines of the skin of an animal, usually by a gland secreting hormones that travel via the cardiovascular system, but occasionally by a more limited portal system, until reaching a chemical receptor on another cell of the same physiological system. The hormonal interface between a hormone and a receptor employs coordinate bonding between the two entities. The bonding is typically of the dual antiparallel coordinate bond, DACB, type.

Conn proceeds to introduce the field of endocrinology using informal language. In section 3 of chapter one, he implies that glucose is a hormone used to signal the pancreas to release more insulin. In this work, the pancrease contains a (internal) sensory receptor site that is sensitive to the presence of glucose in the blood stream. However, glucose is primarily a fuel. Glucose is also used as a precursor to an enormous variety of chemicals used within the physiology of any animal. It is not a hormone created within the hormonal system. In his section 6, Conn asserts, “Endocrinology continues to be a lead science.” This unsupported assertion does not recognize that endocrinology is only one facet of the larger un-named field of hormonal science. See the preface to the 2018 volume by Belfiore & LeRoith endeavoring to introduce endocrinology 2.0, a field more broadly based than that defined by Conn (Section 23.10.2). 23.10.2.4 Potential mode of hormone/receptor interaction

It appears that most of the hormonal/receptor interaction parallels that of interaction between chemical sensory neurons and their stimulants. This is especially true of receptors located on the external lemma of a cell. As an example, the catecholine hormones appear to interface with the receptors on the external lemma of most endocrine targets. Some of the papers in Levey suggest indirectly that the many, if not all, hormone/receptor interfaces may rely upon dual antiparallel coordinate bonding, DACB. Lefkowitz (page 338) notes his laboratory has investigated multiple 125I labeled derivatives of several potent β-

134Gilbert, S. (2001) Juxtacrine signaling in Developmental Biology, 6th Edition. Sunderland, MA: Sinauer Associates

135Vander, A. (2008). Vander's Human Physiology: the mechanisms of body function. Boston: McGraw-Hill Higher Education. pp. 332–333 136 Neurons & the Nervous System adrenergic catecholamine agonists that bear a hydroxyphenyl moiety on the amino N of a molecule, giving “Cc 34" as an example. The top of Figure 23.10.2-3 shows his representation of this molecule. While the chemical nomenclature of the period might be different than today, it appears the molecule exhibits two hydroxyphenyl moieties. Cc 34 is remarkably similar to other important catecholines. The number of possible DACB between any of the catecholines and a receptor is significant. Determining which of these potential DACBs is potentially important as part of the hormone/receptor interface is, on the surface, quite difficult. For a molecule as complex as Cc 34, all of the known forms of a DACB must be considered. Thee include simple DACB emploing only dimers of carboxylic acid groups, alternate involving a paairing of nitrogen and hydroxyl groups, and pairings of any phenyl group with any other potential orbitals (Section xxx). The lower frame of Figure 23.10.2-2 shows two of the catecholines from that section (staggered to align the figures with that of Cc 34. The left half of Cc 34 is functionally identical to epinephrine while the right half is functionally very similar to p-tyrosine. As Section 23.4.2.5.1 showed, p-tyrosine can be used as the progenitor of a wide variety of catecholines, it is therefore likely that many of the catecholines explored by Lefkowitz were functional analogs of the simpler catecholines of Section 23.4.2.5.1. The likelihood that Cc 34 relies upon the same DACBs as those catecholines appears obvious.

Figure 23.10.2-3 “Cc 34" as a potential stimulant for the β-adrenergic receptors. The di- hydroxyphenyl on the left is particularly attractive for forming a DACB with the receptor. The portrayals of the two adjacent hydroxyls on the left in the top and bottom frames are equivalent. The stick representation is not adequate for illustrating the potential d-value of the two adjacent hydroxyl groups or one hydroxyl group and the phenyl ring. The ultimate bonding may Involve a 2-point or 3-point bond. See text.

The Fischer (stick) representations shown in this figure are not adequate for determining the d- value of the potential DACB. Later in Lefkowitz’s discussion, he suggests that propranolol may compete with his hydroxyphenyl derivatives for a β-adrenergic receptor. Propranolol_4777 is not a simple catecholine. However, it could accommodate a variety of DACBs with the target receptor. The precise d-values provided by propranolol_4777 can not be determined precisely because of the problems of the Royal Society of Chemistry, RSC, database described in Section 8.6.1.6.3. Hormonal-affectors 23- 137

As is clear from the above figure, the hyroxyphenyl portion of Cc 34, resembling p-tyrosine, is not the active portion of the molecule; the active portion is the di-hydroxyphenyl by analogy to the complete epinephrine molecule. It is possible the di-hydroxyphenyl portion of the molecule may form a 2-point or 3-point binding to the receptor. If it employs a 2-point binding, the best available d-value would be d = 2.887 Angstrom between the two hydroxyl groups or 2.791 Angstrom between the phenyl ring and one of the hydroxyl groups. This would be similar to the proposed binding of the catecholines of Section 23.4.2.5.1. In analogy to channel 2 of the external olfactory modality (Section 8.6.2.8), it is likely Cc 34 and the majority of catecholines relies upon the phenyl ring and one of the hydroxyl groups to form a DACB with PtdTyr of that sensory receptor. PtdTyr exhibits a central d-value of 2.791 Angstrom. If a 3-point binding was employed, the 2-point DACB would likely be accompanied by an additional binding between the N of the catecholine and another site associated with PtdTyr (similar to the concept of Kier in Section 8.5.3.2). The result would be an augmented efficacy for the hormone compared to the 2-point binding.

As Lefkowitz notes (page 339), “Thus, the binding results described above merely indicate that the agent also binds to other sites as well which, under a number of different assay conditions, The above analyses demonstrates that a variety of catecholines can cause heightened activity within a given neuron. The β-adrenergic receptors are not susceptible to only one hormone. As developed in Section 23.1.3.2 and Section 23.3.1.2.1, the label adrenergic is meaningless in terms of the functional structure of a neuron. It only suggests heightened activity by the neuron. [xxx confirm the above paragraph ]

[ Xxx working here to develop the sites of receptors on/in cells . alt. see sec. 23.10.3 ]

23.10.2.5 Review of guidelines of Cuatrecasas, circa. 1975

The guidelines of Cuatrecasas, published in 1975 (Section 23.10.1) are generally met by the above definition of hormones and the hormonal system (modality). His guidelines are repeated here with relevant discussion points below each entry. C One, the hormone interaction with the receptor should conform to known steric and structural specificity. CC The interaction between the hormone and its receptor, in all cases documented as of 2018, involve the 3D distance between the active atoms of the hormone and receptor being matched within very close tolerance. The present state-of-the-art in modeling molecules is limited in its ability to confirm the steric and structural specificity but for simple hormones, the accuracy of the relevant distances is known to be better than 1%. C Two, the binding sites should be finite in number and therefore, saturable. CC It is not clear why this guideline was included; it appears to lack precise wording. If he meant the binding sites at the molecular level for a single hormone type, the sites are finite by definition (the total surface area of the cell). If he meant the response to the hormone is saturable, he has not indicated how to separate the saturability of the hormone-receptor combination from other mechanisms within the cell. What activity or mechanism did he propose to measure?

C Three, hormone binding should have tissue specificity consistent with biological specificity. CC For all of the situations examined in this work, the type 2 tissue employed by receptors involves a specific phospholipid, typically a phosphatidyl ligand joined to an amino acid, The outer bilayer of the lemma of virtually all animal cells consist of a phosphatidyl ligand joined to an auxiliary structure, frequently an amino acid. The amino acid generally provides the selectivity required to capture a specific hormone. This selectivity may be augmented by an enzyme attached to the surface of the lemma. 138 Neurons & the Nervous System

C Four, hormone binding should be of high affinity and consistent with its physiologic concentrations. CC As noted in item three, the affinity of the receptor site for a given hormone is controlled by the specific amino acid present in the phospholipid of the outer bilaye of the cell external lemma. It may be further mediated by the presence of one or more enzymes. The combination of affinity and selectivity is a field of current evaluation in the exocrine receptors of the oskonatory modality dealing with pheromones (Section 8.6). C Five, the binding of hormone to its receptor should be reversible. CC The defined coordinate bond mechanism of binding via a DACB is totally reversible. No actual reaction occurs as envisioned under the valence bond concept of chemistry. No residues are formed and there is no need to prevent any residue from escaping the binding area. The energy associated with the DACB is very low, typically less than 5 kilocalories/mol. Thus, the DACB is easily broken wherein the hormone is released in its original steric form. 23.10.3 Selected discussions related to individual hormones

Levey, 1976, edited papers addressing only a few hormone/receptor situations. The following table includes the annotated hormones of Levey. [xxx the following table is very incomplete and is becoming immense. ]

HORMONE RECEPTOR SITES

Hormone Abbreviation Receptor Effect of Location Hormone

Insulin Outer lemma of cell increase utiliz. of glucose Corticotropin-releasing CRH Thyrotropin-releasing TRH Stimulating Growth hormone releasing GH@RH Growth hormone release-inhibiting GH@RIH* Inhibiting Prolactin releasing PRH Stimulating Prolactin inhibiting PR@IH Inhibiting lutenizing hormone releasing LH@RH Stimulating

Adrenocirtucitropin ACTH Growth GH Stimulatory Lutenizing LH

Thyroid-stimulating TSH Stimulatory Follicle-stimulating FSH Prolactin PRL Inhibitory Outer lemma of cell Dopamine Epinephrine α-recept. on xxx Norepinephrine α-recept. on xxx Thyroid, T3 T3 cell nucleus Thyroid, T4 T4 Acetylcholine ACh *aka, somastatin Hormonal-affectors 23- 139 23.10.3.1 Insulin–controller of glucose level in bloodstream

Quoting Voet & Voet, and Stryer from Wikipedia, “Insulin (from Latin insula, island) is a peptide hormone produced by beta cells of the pancreatic islets; it is considered to be the main anabolic hormone of the body. It regulates the metabolism of carbohydrates, fats and protein by promoting the absorption of carbohydrates, especially glucose from the blood into liver, fat and skeletal muscle cells.” The active form of human insulin contains 51 amino acids, and has a total molecular mass of 5808 Da. It is a protein rather than a peptide chain in this work by definition . The 3-dimensional structure of insulin was determined by X-ray crystallography in Dorothy Hodgkin's laboratory in 1969136 (PDB file 1ins). Quoting Wikipedia, Insulin is produced by beta cells of the pancreatic islets; it is considered to be the main anabolic hormone of the body. It regulates the metabolism of carbohydrates, fats and protein by promoting the absorption of carbohydrates, especially glucose from the blood into liver, fat and skeletal muscle cells. In these tissues the absorbed glucose is converted into either glycogen via glycogenesis or fats (triglycerides) via lipogenesis, or, in the case of the liver, into both. Insulin is produced and stored in the body as a hexamer (a unit of six insulin molecules. with a total molecular weight of ~35,000, while the active form is the monomer. The hexamer is an inactive form with long-term stability, which serves as a way to keep the highly reactive insulin protected, yet readily available.

Insulin receptors are distributed throughout the body according to the above paragraphs. Their specific character and molecular formula remain unknown.

23.10.3.1.1 The Insulin receptors

Jacob & Cuatrecasas presented a paper in 1976 describing their understanding of the insulin receptors137. They confirmed that the receptor was located on the outer surface of the external lemma of fat cells. They noted at that time, “Much progress has been made in identifying, characterizing, and isolating the insulin receptor. Less is known about the process through which insulin-receptor binding leas to a meaningful biological response.” In brief, at that time little was known about the insulin/receptor interface! They did note there are about 10,000 high-affinity receptor sites per fat cell. They also noted that insulin binds reversibly to its receptor.

“The human insulin protein is composed of 51 amino acids, and has a molecular mass of 5808 Da. It is a dimer of an A-chain and a B-chain, which are linked together by disulfide bonds. Insulin's structure varies slightly between species of animals.” Insulin has been studied in great delail and its structure is well documented. Chain A has 21 amino acids and chain B has 30 amino acids.

“The insulin receptor (IR) is a transmembrane receptor that is activated by insulin, IGF-I, IGF-II and belongs to the large class of tyrosine kinase receptors.” Within this generic description, the operation of controlling xxx can be described as follow, “Cells throughout the body are fueled largely by glucose that is delivered through the bloodstream. A complex signaling system is used to control the process, ensuring that glucose is delivered when needed and stored when there is a surplus. Two hormones, insulin and glucagon, are at the center of this signaling system. When blood glucose levels drop, alpha cells in the pancreas release glucagon, which then stimulates liver cells to release glucose into the circulation. When blood glucose levels rise, on the other hand, beta cells in the pancreas release insulin, which promotes uptake of glucose for metabolism and

136 Blundell, T. Cutfield, J. Cutfield, S. et al. (1971) Atomic positions in rhombohedral 2-zinc insulin crystals Nature vol 231(5304), pp506–11

137Jacobs, S. & Cuatrecasas, P. (1976) The insulin receptor In Levey, G. ed. Hormone-receptor interaction: molecular aspects. Volume 9 of Modern Pharmacology-Toxicology NY: Marcel Dekker Chapter 2 140 Neurons & the Nervous System

storage. Both hormones are small proteins that are recognized by receptors on the surface of cells.” While the current literature describes the insulin receptor as a large molecule, it has not been demonstrated that the identified large molecule is in fact the functional receptor. The recent review of de Meyts provides the best available data138. Under the section titled, Mechanism of Insulin Receptor Activation by Ligand Binding, he notes, “Despite the considerable recent progress in the structures of the extracellular and kinase domains of the insulin (and to a lesser extent IGF-I) receptors, no structure of the complete unliganded and liganded receptors is available, and therefore we lack the details of the precise mechanism whereby extracellular ligand binding results in approximation and activation of the kinase domains.” If, as indicated earlier by Jacobs & Cuatrecasas, the receptor is in fact, “located exclusively at the outer surface of the fat cell membrane,” the proposal of a transmembrane receptor in de Meyts is inappropriate. Their proposed transmembrane receptor would be, by far, the largest known hormone receptor molecule in the human physiology.

23.11 Nominal Crine (hormonal) system operations

This section will introduce many terms possibly foreign to the readers vocabulary. Section 8.6.11.3 will review many of the terms used in this section. The place where other terms are defined will be cited in the text. Alternately, the large glossary associated with this text (or website) can be accessed (http://neuronresearch.net/neuron/pdf/glossary.pdf)

In the course of the investigations prior to preparing this exposition, very little data was found in the literature supporting the definition of hormonal receptors except in the case of the pheromone receptors as they were extrapolated from the olfactory receptors (Section 8.6 extending into Section 8.6.11). Because of this situation, Section 23.11.2 and Section 23.11.3 remain largely empty at this time.

The crine system involves three major sub-systems with clearly differentiated methods of operating. The endocrine sub-system is the most intensely studied and written about. It involves the generation of hormones that rely upon the vascular system to circulate to all parts of the body. It is thus been described as the ductless portion of the crine system. The paracrine sub- system operates differently, the hormones generated by the pedicles of stage 7 neuroaffectors need only travel very short distances to reach their target muscular tissue and do not require any duct as part of their operation. Nitric oxide, a major “hormone” of the paracrine subsystem, is also very short lived. In general, the paracrine sub-system does not involve closed loop feedback mechanisms.

The exocrine sub-system is the least studied from a an overall physiological perspective. It involves two separate operating modes that differ from those of either the endocrine or paracrine subsystems. The most obvious operating mode is that related to digestion. The hormones created by the pancreas and related glands are in general enzymes that support the breakdown of large proteins in the digestive part of the alimentary canal. The second operating mode is assigned the task of helping the organism find partners of the same species for the primary purpose of reproduction. It also plays a role in the post natal search of the newborn in finding the sources of nourishment provided by their mother.

23.11.1 Background– Receptors of the Crine (hormonal) system

In the course of the investigations prior to preparing this exposition, very little data was found in the literature supporting the definition of hormonal receptors. Repeating the

138De Meyts, P. (2016) The Insulin Receptor and Its Signal Transduction Network In De Groot LJ, Chrousos G, Dungan K, et al., editors. Endotext [Internet] www.endotext.org Available from https://www.ncbi.nlm.nih.gov/books/NBK378978/ Hormonal-affectors 23- 141 quotation of Levey in Section 23.10.1 is appropriate, ““Traditionally a ‘receptor’ has been difficult to define in pharmacologic terms.” A receptor has remained difficult to define at the detail level within physiology as well. A possible exception is the case of the pheromone receptors as they were extrapolated from the olfactory receptors (Section 8.6 extending into Section 8.6.11). Because of this situation, Section 23.10.1 is very brief and Section 23.10.3 remain largely empty at this time. Three different classes of receptors have been defined as part of this work. They are external receptors, nuclear receptors and cytosol receptors . The external receptors are on the outer surface of the lemma of a cell. The nuclear receptors are on the surface of the nucleus of a cell. The cytosol receptors are associated with one or more of the structures within a cell but outside of the nucleus. Until the turn of the 21st Century, virtually no knowledge of the receptors used within the hormonal system at the molecular level was available. Mayo addressed this situation in 2005 but in a limited manner139. Most of Mayo’s figures consist of cartoons suggesting various flow paths for general categories of hormones. In his summary, he notes, “ In most of the examples considered in this chapter, our knowledge of the signaling pathway from hormone-receptor interaction to biologic response is still at best rudimentary. . .It seems certain that substantial progress is needed to better characterize the signaling processes that are already known, and to uncover the novel receptor activation and signal transduction pathways that surely await discovery.”

In his figure 1, Mayo only addresses protein hormones (presumably of more than 10 peptide residues) and steroid hormones. He does not address the paracrine or exocrine hormones or their receptors. He does mention nitric oxide in passing but does not appear to address acetylcholine. The paracrine receptors are crucially important as they mediate both striate and smooth muscle activity in animals. The figure asserts that protein hormones are associated with surface receptors of cells. It also asserts that steroid hormones diffuse across the lemma of a cell in order to reach a cytoplasmic or nuclear receptor. The output of multiple flow paths is always labeled “Biological output” without further specificity.

Mayo does not mention the large number of organic molecules used in the exocrine submodalities of the crine (hormonal) system that are not peptides or peptide based (Section 8.6.11.9).

When discussing hormone-receptor interaction, he does suggest that both homodimer and hetrodimer situations may be involved. Many, most or all dimers involve dual antiparallel coordinate bonds, DACBs.

Mayo describes the initial hormone-receptor interaction through biological output as signal transduction. Mayo does introduce many “themes” of cellular (both surface and nuclear) signal transduction, but mostly in conceptual terms. His first three themes involve, C receptors based on G protein-coupled receptors, GPCR, ostensibly the largest family of receptors, C activation of tyrosine kinase or serine/threonin kinase receptors or receptor-associated proteins by growth factor and receptors, and C activation of protein proteolysis or cell localization pathways by developmentally important receptors. After discussing these potential themes, he discusses in Section 3.6, receptors with protein tyrosine phosphatase activity. “A phosphatase is an enzyme that uses water to cleave a phosphoric acid monoester into a phosphate ion and an alcohol. Because a phosphatase enzyme catalyzes the hydrolysis of its substrate, it is a subcategory of hydrolases.” This definition can be easily extended to include a phospholipid in place of a phosphoric acid and the resulting phosphate ion. Schulman has presented material concerning phosphatase activity that is worthy of studying

139Mayo, K. (2005) Receptors In Melmed, S. & Conn, P. eds Endocrinology, 2nd Edition Totowa, NJ: Humana Press chapter 2 142 Neurons & the Nervous System what it does not say as well as what it does say140. Mayo notes in section 3.9, “A significant number of cell-surface receptors may not have classic signaling function but, rather, serve to transport substances into the cell from the plasma.” Are these entities legitimately included in the hormonal receptor category? Many molecules are associated with the transport of other molecules through the type 3 lemma of cells. Mayo also introduces significant material concerning “intracellular receptors” that may or may not refer to crinology per se. They generally relate to what are labeled steroid hormones. He notes, “In response to hormonal binding, these receptors undergo a conformational change and become competent to bind to specific DNA elements that act as hormone-dependent enhancers of gene expression. (with a citation to chapter 4 of Byrne et al. by Schulman). A question arises, are the conformational changes in the receptor reversible? If not, it is not clear that these steroid/ activities relate to hormonal activity as defined in Section 23.10.2.2. As of 2005, there is virtually no detailed information about molecular chemistry of the hormonal receptors, except in the case of the exocrine receptors. The exocrine receptors can be described in detail based on the extension of the olfactory receptors into the oskonatory receptors of Section 8.6.11 of this work.

In the 2018 text by Belfiore & LeRoith141, only limited discussion of the hormonal receptors was provided. Part III was dedicated to this subject. In chapter 5 only the potential role of GCPR receptors were discussed extensively. In chapter 6, White reviewed the putative tyrosine kinases and the insulin signaling system. His effort was focused on the genetics leading to the conceptually defined receptors. In chapter 7, Brooks et al. addressed putative cytokine receptors. These are described as nuclear receptors using cartoons. In chapter 8, Klinge reviewed the putative steroid hormone receptors and signal transduction processes. The receptors discussed were primarily of the nuclear type. However, the description of how the steroid hormones interact with the nuclear receptors was minimal at the molecular level. Klinge did provide an extensive table describing the steroid hormones and providing citations to the most recent reviews for each hormone. Here again, retinoic acid is included as a steroid (derivative of cholesterol) when in fact it is a terpene derivative.

As of 2018, there remain little information in the literature on how the receptors of the crine (hormonal) modality function, with the possible exception of the exocrine receptors described in this work (Section 8.6). See Section 23.10.2.4 for the current situation.

23.11.2 The exocrine sub-system

While it is quite difficult to determine the precise character of the hormone/receptor interface among the endocrine and paracrine portions of the crine (hormonal) system, it is possible to describe the exocrine subsystem in considerable detail. This is primarily due to the limited complexity of the pheromones of the subsystem and the apparent similarity of the receptors to their counterparts in the olfactory modality. The receptors of the olfactory modality operate over a d-value range from about 2.0 to 8.5 Angstrom with the receptors of the oskonatory modality continue seamlessly to operate over the d-value range from 8.5 Angstrom up to at least 23.0 Angstrom.

23.11.2.1 Exocrine, hormonal channels related to species identification [xxx ]

140Schulman, H. (2014) Intracellular Signaling In Byrne, J. Heidelberger, R & Waxham, M. eds. Molecules to Networks, 3rd Edition NY: Elsevier Chapter 4, pp 119-148 https://doi.org/10.1016/B978-0-12-397179-1.00004-X

141Belfiore, A. & LeRoith (2018) Principles of Endocrinology and Hormone Action. NY: Springer Hormonal-affectors 23- 143 23.11.2.1.1 The hormonal channels based on their spectrum

Figure 23.11.2-1 reproduces the proposed mapping of the olfactory and oskonatory d-value space from Section 8.6.11. Note, the definition of 10 discreet oskonatory receptor channels, VR 1 through VR 10. While all 9 of the olfactory receptor molecules have been identified, only a few of the oskonatory receptors, VR1 and VR9, have been identified (at the bottom of the vertical dashed lines) from the available literature as of 2018.

Figure 23.11.2-1 The proposed VR effectivities versus d-value based on moth data. The moth, Manduca sexta exhibits three vodorophores at d = 13.128, 14.685 & 23.352 Angstrom. The silk moth, Bombyx mori exhibits two vodorphores at d = 13.198 and 15.600 Angstrom with the latter more effective. The effectivities are shown as of constant width regardless of center values. The center line values of the VR’s are based on a linear expansion of the scale for VR’s 1 through 3 and the odorophores defined in this work. See Section 8.6.11 for an explanation of the terms in this figure.

The pheromones have been identified to a much more complete level than have the receptors. A variety of exocrine pheromones are shown in this figure. Some of the pheromones appear to be steroids, based on their names, but the majority are aliphatic alcohols or carboxylates. More data mining should identify additional external receptors of the exocrine sub-modality.

The exocrine system involves primarily, C the secretion of volatile steroids, short peptide (less than10 residues) or none-peptide-based organic pheromones. Thes molecules must be both soluble in water-based environments and volatile in air at biological temperatures. These pheromones can remain airborne for significant periods, and travel considerable distances with the wind. C An arrangement of receptors on the surface of the target species that are in easy communications with the surrounding pneumatic environment. In many species, contact between the pheromones and the receptors is enhanced by the breathing mechanism of the animal. C The efficient capture of the pheromones at the pheromone/receptor interface. C The efficient transduction of the presence of the pheromone of a given structural character into a neural signal (stage 1, signal generation) that can be delivered to the central nervous system, CNS. Typically, each pheromone exhibits only one d-value. However, this is not a necessity. Many exhibit multiple d-values that are sensed individually through the process of dual antiparallel coordinate bonding, DACB, with individual receptors connected to individual neural signaling paths. C A stage 2, 3 & 4 neural system supporting the stage 1 sensing process that can form a perception of the matrix of pheromones received in a timely manner for presentation to the stage 5 cognition neurons.

23.11.2.2 Exocrine hormone emitters 144 Neurons & the Nervous System

Exocrine hormone emitters are of two types and three varieties. The first major type consists of the digestive juices prepared primarily by the pancreas and delivered via ducts into the digestive portion of the alimentary canal (the stomach). Th second major type consists of the hormones designed for attracting other members of the same species primarily for purposes of procreation. These hormones are frequently identified as pheromones. There are three easily defined hormones of the pheromone group. Two are emitted in conjunction with perspiration and the other is emitted in conjunction with urination. The two hormones associated with perspiration frequently consist of a low molecular weight variety (typically with molecular weights less than 500) able to evaporate and travel significant distances (meters to kilometers depending on species) with the winds; and a higher molecular weight variety only effective over distances of about a meter. Like the chemicals associated with taste and smell, the pheromone hormones are processed in a matrix manner to result in a specific neural sensation. The pheromones excreted in conjunction with urine are frequently of high molecular weight since they are not designed to travel long distances in the air. They are frequently of adequate volatility to be effective over a meter or so. 23.11.2.3 Typical exocrine hormone reception–Pheromone receptors

A selection of pheromones are described by name in Section 23.11.2.1.1. They are described in greater detail in Section 8.6.11.

The pheromone receptors have been historically associated with the vomeronasal region of the nasal passages. In this work, the pheromones, and their receptors, are associated with a modality defined as the oskonatory modality.

As noted earlier, the potential for the exocrine receptors to be amino-phospholipids based on the similarity, and possible extension of the receptors from the set employed in the olfactory modality cannot be overlooked. Section 8.6.11 will illustrate these possibilities.

23.11.2.3.1 The exocrine hormonal receptors of species selection

The receptors can be described in considerable detail based on the Electrolytic Theory of the Neuron; this theory is the primary and underlying subject of this work (Chapter 2 and Chapter 3).

The fundamental finding of the Electrolytic Theory of the Neuron is that every neuron contains, and every interneuron synapse consists of and active electrolytic amplifier. The stage 1 signal generating neurons actually contain two amplifiers in what is known as a cascode circuit arrangement. The first amplifier in this configuration exhibits considerable signal amplification to ensure maximum sensitivity to the appropriate stimulant. The selectivity of the sensory neuron to a specific pheromone is determined by the physical chemistry of the individual receptor preceding the first amplifier in the transduction process. 23.11.2.3.2 Typical circuit diagram of pheromone receptors

Figure 23.11.2-2 reproduces a portion of [Figure 8.6.5-3] from Section 8.6.5.2.1 where the circuitry is discussed in detail. It is proposed that the pheromone receptors are identical to the receptors of the olfactory modality, except for their sensitivity range defined by their d-values. Thus the label OR (for odorant receptor) can be replaced by the label VR (for vodorant receptor). The first amplifier circuit of the stage 1 signal generating neuron, represents a very high input impedance electrometer. In the absence of any stimulation, the voltage at “circle E” is a quiescent value. Upon stimulation, the voltage at “circle D” rises positively. This voltage is amplified by about 200 times by the 1st amplifier. The 2nd amplifier acts as an impedance lowering device allowing the output at “circle E” to be connected to a large number of synapses serving a variety of stage 2 neurons. Hormonal-affectors 23- 145

Figure 23.11.2-2 Nominal exocrine sensory receptor neuron with a receptor molecule in position. st OR; the receptor molecular array between the two electrodes of the 1 amplifier input circuit. CT; the capacitance of the receptor molecular array. Dashed arrow; points to the battery representing the electrostenolytic power supply of the 1st amplifier. Circle D; the voltage developed by the OR in tens of microvolts. Circle E; the analog output voltage following amplification in millivolts. Curving dashed line; portion of circuit to the left of the line is exposed to the air in the nasal passage. The portion to the right is immersed in the fluid surrounding the neural matrix. See text and Section 8.6.11.

The stimulating (v)odorant couples to the phospholipid liquid crystalline array of the OR via a DACB with a d-value matching the OR.

The label DACB refers to a dual antiparallel coordinate bond. The DACB, the key feature in the formation of any biochemical hetrodimeric configuration, appears to be at the very center of virtually all biochemical interfaces, whether powering individual neurons or sensing stimulants, and/or whether internal or external chemical sensing is involved. Until falsified, this work demonstrates this critical requirement with regard to gustation, olfaction, oskonation and providing electrical power to insure a quiescent negative internal voltage to each individual neuron. The development of the various sensory molecules of chemical sensing and the formation of the hetrodimeric structure between any stimulant and its matching receptor is presented in Section 3.2.2 and Section 8.3 of this work. The stage 1 signal generator neuron is fabricated (grown) biochemically by conventional means that involve a wide variety of biomolecules. However, non of these biomolecules is directly involved in the signal path from receptor to output synapse(s). 146 Neurons & the Nervous System

In the context of this work and the Electrolytic Theory of the Neuron, what is known as the vomeronasal area of the nasal passages in animals is considered a distinct area of exocrine activity. As such the sensing of pheromones from other animals is defined as oskonation. Oskonation functions in parallel with olfaction but with a distinctly different ecological purpose. Whereas olfaction is primarily designed to locate and evaluate food while avoiding the ingestion of toxic substances, oskonation is primarily designed to locate and discriminate among members of the same species (Section 8.6.11.3). It is likely that most pheromones consist of low molecular weight substances (aliphatic alcohols and carboxylates are likely candidates) that are soluble in water-based fluids and are volatile at biological temperatures (See Section 23.10.1). This categorization includes small peptides but not large proteins. Some pheromones may consist of proteins of higher molecular weight where physical contact between two members of the same species is entertained. In that case, the volatility of the pheromone is not a requirement. Some pheromones of higher molecular weight proteins may also be present in urine used to mark territories. In that case, the volatility of the pheromone may be marginal but adequate for short range oskonation (well characterized by activity of dogs). 23.11.2.3.3 Pheromone symbiosis–elephants and flies

The symbiosis between elephants (particularly Asian elephants) and certain families of moths, xxx, is well documented based on behavioral studies. In recent times, it has been possible to chemically isolate the various pheromones associated with these animals and insects. The data has shown a remarkable similarity in the pheromone distribution between these two members of different phyla. While the upper d-value of the pheromones of the oskonation modality has not been determined precisely, it can be said that these two families share at least a major degree of commonality in their pheromone distribution matrices within their oskonatory modalities (Section 8.6.11). Section 8.6.11.9.1 cites the extensive library of moth pheromones.

Wyatt has described a pheromone (vodorant) that contains two distinct vodorophores that are shared between the elephant (Elephas maximus) and 140 species of moth! The vodorant is (Z)-7- dodecen-1-yl acetate, a primary linear aliphatic aldehyde with a cis condition at carbon 7 (Section 8.6.11.7). Rasmussen et al. (1997) closes with the remark, “That (Z)-7-dodecen-1-yl acetate is the major component of the Asian elephant urinary sex pheromone is indeed an unexpected finding of fundamental importance to the field of mammalian [exocrine] chemical communication.” 23.11.2.4 Exocrine hormones associated with digestion [xxx drop if the proposed definition of a hormone exclude the enzymes of digestion ]

The hormones associated with the digestive process have been broadly identified across a wide range of animals. The reader is directed to other writings on this subject. The hormnes released from the pancreas and other “ducted glands” operate primarily as enzymes supporting the breakdown of large protein and fat molecules. This breakdown is necessary for the materials to pass through the wall of the alimentary canal. As an example, the failure of the pancreas to produce and deliver a hormone known commercially as Creon to the digestive tract leaves the digestive system unable to breakdown fats with a backbone of more than 8 carbons. As a result, the organism is unable to digest butterfat, avocado fat and banana fat, etc. The author suffered from this condition for over 50 years before a day came when apparently a duct that had been constricted or blocked suddenly cleared. After that date, the author could eat copious quantities of ice cream and bananas without encountering serious diarrhea.

23.11.2.4.1 Gastric juices

The gastric juices are a mixture of inorganic and organic chemicals. Only the organics, primarily pepsins and lipases can be considered organic hormones from the perspective of their secretion. Hormonal-affectors 23- 147 The gastric juices secreted by the pancreas and other glands into the digestive tract can be considered exocrine hormones of the ducted variety. Although not used for purposes of communication within the body of an animal, like most endocrine hormones. or for purposes of longer distance communications like other exocrine hormones, they appear to be formed in a manner similar to hormones.

23.11.2.5 and are tropic hormones

Gastrin is a tropic hormone secreted into the blood stream to cause the release of gastric juices by the pancreas and other glands. Secretin is also a tropic hormone secreted into the blood stream to control water homeostasis throughout the body. Its major targets are the liver and pancreas.

23.11.3 The paracrine sub-system NEARLY EMPTY

[xxx need words regarding acetylcholine and its surface receptor on the lemma of a striated cell. ]

Acetylcholine and nitric oxide are hormones are secreted by stage 7 neurons, neuroaffectors, within the paracrine environment.

- - - -

The ability of NO to pass through the lemma of a cell suggests, it does not depend on an external receptor associated with the cells lemma. It is believed to penetrate into the intracellular space of a smooth muscle cell. It also appears to stimulate structures other than the nucleus directly. 23.11.3.1 Paracrine hormone emitters

23.11.3.2 Paracrine receptor

The paracrine receptors are primarily associated with the surface of muscle tissue involved at the muscle/stage 7 neuron interface. While the morphology of these interfaces have been sketched repeatedly over the years, the actual molecules forming the receptors remains unreported in the literature. As a result the subsections of this section are necessarily brief. 23.11.3.2.1 Potential nitric oxide, @N=O, receptor Spindel has provided information concerning the paracrine receptors that may belie the title of his chapter142. Paragraphs 4 and 5 address the nitric oxide and acetylcholine receptors respectively. Paragraph 4.1 suggests the receptor for nitric oxide may be a guanylate cyclase, GC. “The soluble GC that acts as the NO receptor is a heterodimer of Mr =151,000.” The last sentence may suggest there is also a non-soluble GC that would be necessary to form a long life receptor on the surface of a cell. Spindel closes with the statement, “However, activation of GC likely does not explain all of NO’s actions, and other @NO signal transduction mechanisms remain to be determined.” His paragraph 4.2 offers a largely different role for GC. Spindel’s presentation is primarily via cartoons of possible metabolic processes. Little specific information is presented that can be verified by experiment. Other investigators have noted the ability of CNO to pass through the lemma of cells and have

142Spindel, E. (2005) Second-messenger systems and signal transduction mechanisms In Melmed, S. & Conn, P. eds Endocrinology, 2nd Edition Totowa, NJ: Humana Press chapter 3 148 Neurons & the Nervous System proposed it stimulates a cytosol receptor, or cytosol constituent, directly. It appears nitric oxide does not require an external membrane receptor because of its ability to pass through the lemma of a cell and reach a receptor within the cytosol of the cell. It is likely that a receptor molecule of much lower molecular weight than Spindle suggested is actually employed in the @N=O receptor. Whether the receptor binds to the @NO via a DACB or whether it takes advantage of the “free radical” character of @N=O is not known. The reversibility requirement for any hormone/receptor interface favors the DACB. 23.11.3.2.2 Potential acetylcholine receptor

In Spindel’s paragraph 5, he addresses the potential role of acetylcholine receptors using primarily symbolic language. It does not advance our knowledge of the subject matter. Potter has provided a range of properties associated with the acetylcholine receptors, but without identifying the actual receptors at the molecular level143. Unfortunately, the language of 1976 is quite archaic and needs reinterpretation within the Electrolytic Theory of the Neuron.

Interpreted within the framework of the Electrolytic Theory of the Neuron, Figure 23.11.3-1 shows acetylcholine is capable of forming two or more different DACB. Therefore, the possibility that the acetylcholine receptors of the paracrine submodality may be amino-phospholipids on the surface of target tissue (primarily striated muscle) must be considered. The function of muscle end-plates must be examined in the context of such potential amino-phospholipid receptors.

The potential amino-phospholipids closest to the d-values of acetylcholine are PtdSer (d = 2.276 Angstrom), Ptd4Hi (d = 3.508 Angstrom). An amino-phospholipid near d = 4.849 Angstrom has not been identified. See Section 8.6.2.

23.11.4 The endocrine sub-system EMPTY Figure 23.11.3-1 Acetylcholine showing three 23.11.4.1 Endocrine hormone possible DACB, with d-values of 4.849, 3.729 and emitters EMPTY ADD 2.279 based on Discovery Suite 4.1 software. See text.

23.11.4.2 Non-neural endocrine hormone receptors ADD

This section will be used as a repository of hormonal receptors when they are found in the literature and are pertinent to the discussions in this work. The endocrine hormones are of two major types, those that are peptide-based and those that are cholesterol-based.

As is the case for endocrine hormones, their receptors can be categorized in multiple ways. Here, they will be categorized initially by their ability to act as receptors for tropic and non-tropic hormones (Section 23.xxx).

143Potter, L. (1976) Acetylcholine receptor-channel molecules In Levey, G. ed. (1976) Hormone-receptor interaction: molecular aspects. Volume 9 of Modern Pharmacology-Toxicology NY: Marcel Dekker chapter 18 Hormonal-affectors 23- 149 23.11.4.2.1 Tropic hormone receptors ADD

As noted above, the tropic hormones are primarily secreted tp control the secretions of other hormones by the various glands. The tropic hormones vary greatly in their complexity and molecular weights. Some are mere peptides. Others are proteins of significant molecular weight.

23.11.4.2.2 Non-tropic hormone receptors EMPTY

23.11.4.2.3 Nuclear receptors

The glucocorticoid receptor (GR, or GCR) also known as NR3C1 (nuclear receptor subfamily 3, group C, member 1) is the receptor to which cortisol and other glucocorticoids bind. The GR is expressed in almost every cell in the body and regulates genes controlling the development, metabolism, and immune response. Because the receptor gene is expressed in several forms, it has many different (pleiotropic) effects in different parts of the body.

GR is embedded in a very large protein. In humans, the GR protein is encoded by NR3C1 gene which is located on chromosome 5 (5q31).

A label, MR, for mineralocorticoid acheved some use near the end of the 20th Century. The mineralocorticoid receptor (or MR, MLR, MCR), also known as the aldosterone receptor or nuclear receptor subfamily 3, group C, member 2, (NR3C2) is a protein that in humans is encoded by the NR3C2 gene that is located on chromosome 4q31.1-31.2.[5]

MR is a receptor with equal affinity for mineralocorticoids and glucocorticoids. It belongs to the nuclear receptor family where the ligand diffuses into cells, interacts with the receptor and results in a signal transduction affecting specific gene expression in the nucleus.

The roles of the GR and MR appear to overlap considerably and may not be separate receptors functionally. They may employ different protein moieties, with that of the MR being the smaller protein. 150 Neurons & the Nervous System

Table of Contents 5 January 2019

23 Glandular–affectors & the hormonal system ...... 1 23.1 Introduction...... 1 23.1.1 xxx Organization of the stage 6 & 7 neuro-affector neurons ...... 3 23.1.1.1 Xxx Role of neuro-affectors in overall system ...... 4 23.1.1.2 Xxx Role of glandular-affectors in overall system EMPTY.....5 23.1.2 Common inter-modality functional blocks ...... 5 23.2 Background in literature ...... 7 23.2.1 Developing a modern definition of a hormone ...... 8 23.2.1.1 Expanding on the 6 aspects converging on the definition of a hormone EMPTY ...... 9 23.2.1.2 Proposed working definition of a hormone ...... 9 23.3 Anatomy of the neuro-glandular interface ...... 10 23.3.1 The differentiation between neuro-affectors and hormones ...... 15 23.3.1.1 The definition of a neuro-hormone ...... 15 23.3.1.2 The dual character of the efferent neural system...... 16 23.3.1.3 Introductory schematics from the stage 7neurons to their targets ...... 16 23.3.2 The anatomy and physiology of the paracrine system EDIT ...... 21 23.3.2.1 The physiology of the neuroaffector-smooth muscle interface EMPTY...... 23 23.3.2.2 The physiology of the neuroaffector-striate muscle interface EMPTY...... 23 23.3.3 The anatomy and physiology of the endocrine system...... 24 23.3.3.1 The unique physical position of the hypophysis ...... 25 23.3.3.1.1 Hypophysis & part of the hypothalamus are outside the BBB...... 30 23.3.3.2 The configuration of the neuroendocrine signal paths ...... 30 23.3.3.3 Background on oxytocin & arginine vasopressin ...... 30 23.3.3.3.1 Potential formation of AVP–by Bichet...... 32 23.3.3.3.2 Critique of Bichet mechanism for AVP production . . 35 23.3.3.4 Endocrinolgy of aging...... 35 23.3.4 The anatomy and physiology of the exocrine subsystem MAY DUPLICATE ...... 36 23.3.5 Terminology EDIT...... 36 23.4 Neuro-affectors releasing endocrine hormones–Global perspective ...... 36 23.4.1 Background relative to the hormonal system ...... 39 23.4.1.1 Tyrosine & tryptophan as progenitors of many hormones . . . 41 23.4.1.2 The origins of the catecholamine-based hormones...... 41 23.4.1.3 The origins of the peptide-based hormones ...... 42 23.4.1.4 The origins of the steroid-based hormones EDIT ...... 46 23.4.1.4.1 The chemical origins of the steroid-based hormones ...... 46 23.4.1.4.2Mechanisms of the steroid-based hormones/receptor interface...... 48 23.4.1.5 Specialized nomenclature...... 48 23.4.1.6 Additional annotation of block diagram of the hypothalamus ...... 48 23.4.1.6.1 Cytology of the neuroaffectors of the hypothalamus & neurohypophysis ...... 49 23.4.1.6.2 The chemistry of the neuro-hormones (hormone releasing hormones) ...... 51 23.4.2 The hypothalamus-pituitary-epinephrine (adrenal), H-P-E, axis ...... 52 23.4.2.1 Past Flow diagrams of the thyroxine system with feedback EMPTY ...... 53 23.4.2.2 The adrenocorticotropin hormones (ACTH) EMPTY ...... 54 23.4.2.2.1 The chemistry of the stimulating hormones of the hypophysis ...... 54 23.4.2.3 Functional representation of the hypophysis–CRH in ACTH out Hormonal-affectors 23- 151 ...... 55 23.4.2.3.2 Structure of adrenocorticotropin-stimulating hormone, ACTH & its receptor...... 55 23.4.2.4 Potential stage 7 neurons within the peripheral glands .....56 23.4.2.4.1 The Burbach et al, definition of a hypothalamus- neurohypophysis system, HNS ...... 57 23.4.2.4.2 The Vokes & Robertson description of xxx EMPTY . . . 58 23.4.2.5 Adrenal family of hormones–epinephrine, norepinephrine, etc...... 58 23.4.2.5.1 The catacholamines...... 58 23.4.2.5.1 Potential DACB between epinephrine and a PtdTyr receptor on any cell...... 59 23.4.2.5.2 The sensitivity of physiological cells to epinephrine in the bloodstream...... 59 23.4.2.5.4 Summary of hormone & receptors along H-P-E axis ...... 60 23.4.3 The hypothalamus-pituitary-thyroid, H-P-T, axis ...... 61 23.4.3.1 Past Flow diagrams of the thyroxine system with feedback . . 61 23.4.3.2 Functional features of the hypothalamus ...... 65 23.4.3.2.1 Configuration options in the neurons of the hypothalamus ...... 66 23.4.3.2.2 Structure of thyrotropin-releasing hormone, TRH ....69 23.4.3.3 Functional features of the hypophysis (pituitary)...... 69 23.4.3.3.1 Functional representation of the hypophysis–TRH in, TSH out...... 70 23.4.3.4 Functional features of the Thyroid ...... 75 23.4.3.4.1 Functional representation of the thyroid– TSH in T4/T3 out ...... 75 23.4.3.4.2 Structures of major thyroid molecules...... 75 23.4.3.4.3 Synthesis of glandular thyroxine & structure of thyroxoglobulin ...... 77 23.4.3.4.4 Synthesis of T3 from T4 in peripheral tissue ...... 78 23.4.3.4.5 The deiodinases enzymes– D1, D2, & D3 ...... 78 23.4.3.4.6 Clearance rates for T3 and T4 in humans...... 78 23.4.3.5 Structure-function-relationship of the thyroxines ...... 79 23.4.3.5.1 Potential DACB between T3 and a PtdCys receptor on any cell...... 81 23.4.3.5.2 The sensitivity of physiological cells to T3 in the bloodstream...... 81 23.4.3.5.3 Potential DACB receptor compatible with both T3 and T4 ...... 81 23.4.3.6 Summary of hormones, receptors & flow diagrams of the H-P-T axis ...... 82 23.4.4 Diseases of the H-P-T axis...... 83 23.4.4.1 Hyperthyroidism EMPTY...... 89 23.4.4.2 Hypothyroidism...... 89 23.4.4.2.1 Thyroid disease in rats ...... 92 23.4.4.2.2 Thyroid disease in humans ...... 92 23.4.4.2.3 The apparent euthyroid hypothyroid sufferer ...... 93 23.4.4.3 Iodine deficiency in hypothyroidism...... 93 23.4.4.4 Auto immune diseases of the H-P-T axis ...... 94 23.4.4.4.1 LATS, long-acting thyroid stimulator disease ...... 94 23.4.4.4.2 TSH receptor blocking–auto immune...... 96 23.4.4.4.3 deiodinase related problems...... 97 23.4.4.4.4 Mood & other intellectual problems related to hypothyroidism...... 97 23.4.4.5 Thyroid hormone clearance times vs the LATS concept .....98 23.4.5 A case study–potential change in enzymatic activity EMPTY...... 101 23.5 (Reserved) Hormones of the peripheral endocrine system EMPTY ...... 102 23.5.1 Hormones of the adrenal gland EMPTY ...... 102 23.5.2 Hormones of the pancreas EMPTY...... 102 23.5.3 Hormones of the kidney EMPTY ...... 102 23.6 The secretions of the exocrine system...... 102 152 Neurons & the Nervous System

23.6.1 The gastric juices of the exocrine submodality EMPTY...... 102 23.6.1.1 What are enzymes? EMPTY...... 102 23.6.2 The pheromones of the exocrine submodality EMPTY ...... 103 23.6.2.1 What are pheromones? ...... 103 23.7 The hormone/receptor interface...... 103 23.7.1 Receptors of hormones...... 103 23.7.1.1 Cell surface receptors & the hormone/receptor interface . . 104 23.7.1.1.1 Crystal structure of the TSH receptor...... 105 23.7.1.1.2 A scenario for the cell surface glandular receptor & interface...... 105 23.7.2.2 Intracellular receptors EMPTY...... 108 23.8 Chemistry of Crine modality receptors EMPTY...... 108 23.8.1 Chemistry of the surface receptors of a cell ...... 108 23.8.1.1 Chemistry of the exocrine system surface receptors of a cell ...... 109 23.8.1.2 Chemistry of the paracrine system surface receptor for @N=O .....109 23.8.1.3 Chemistry of the paracrine system surface receptor for acetylcholine...... 109 23.8.2 Permeability of in-vivo lemma by diffusion and/or pores...... 110 23.8.2.1 Permeability of single bilayer in-vitro by diffusion and/or pores ...... 110 23.8.2.1.1 Volkov & Deamer on bilayer physical chemistry . . . 111 23.8.2.1.2 Paula et al. papers in bilayer physical chemistry . . 114 23.8.2.2 21st Century research into lemma of two bilayers...... 118 23.8.2.2.1 Shinoda paper in bilayer physical chemistry ...... 118 23.8.2.3 Permeability of lemma by hydrogen ion–A special case . . . 120 23.8.3 Chemistry of the intracellular receptors of a cell EMPTY ...... 121 23.8.3.1 Chemistry of the nuclear receptors EMPTY ...... 122 23.8.3.2 Chemistry of receptors of elements in the cytosol EMPTY . . . 122 23.9 Interplay of the neural-glandular-muscular modalities in animal physiology. . . . 123 23.9.1 A hair follicle as an exemplar...... 126 23.9.2 [Reserved]...... 126 23.9.2.1.1 The formation and release of pheromonesin sweat ? EMPTY ...... 126 23.9.2.1.2 The formation and release of pheromones in urine ? EMPTY ...... 127 23.10 Cataloging hormones by chemical structure ...... 127 23.10.1 Background ...... 127 23.10.2 Defining framework circa. 2018 ...... 128 23.10.2.1 Potential cellular locations of hormonal receptors...... 131 23.10.2.2 Potential categories of hormones based on structural complexity ...... 132 23.10.2.3 Definition of a hormone and hormonal system, circa. 2018 ...... 134 23.10.2.4 Potential mode of hormone/receptor interaction ...... 135 23.10.2.5 Review of guidelines of Cuatrecasas, circa. 1975 ...... 137 23.10.3 Selected discussions related to individual hormones...... 138 23.10.3.1 Insulin–controller of glucose level in bloodstream ...... 139 23.10.3.1.1 The Insulin receptors ...... 139 23.11 Nominal Crine (hormonal) system operations ...... 140 23.11.1 Background– Receptors of the Crine (hormonal) system ...... 140 23.11.2 The exocrine sub-system...... 142 23.11.2.1 Exocrine, hormonal channels related to species identification ...... 142 23.11.2.1.1 The hormonal channels based on their spectrum ...... 143 23.11.2.2 Exocrine hormone emitters ...... 143 23.11.2.3 Typical exocrine hormone reception–Pheromone receptors ...... 144 23.11.2.3.1 The exocrine hormonal receptors of species selection ...... 144 23.11.2.3.2 Typical circuit diagram of pheromone receptors ...... 144 Hormonal-affectors 23- 153 23.11.2.3.3 Pheromone symbiosis–elephants and flies ...... 146 23.11.2.4 Exocrine hormones associated with digestion...... 146 23.11.2.4.1 Gastric juices ...... 146 23.11.2.5 Gastrin and secretin are tropic hormone...... 147 23.11.3 The paracrine sub-system NEARLY EMPTY...... 147 23.11.3.1 Paracrine hormone emitters...... 147 23.11.3.2 Paracrine receptor ...... 147 23.11.3.2.1 Potential nitric oxide, @N=O, receptor...... 147 23.11.3.2.2 Potential acetylcholine receptor...... 148 23.11.4 The endocrine sub-system EMPTY ...... 148 23.11.4.1 Endocrine hormone emitters EMPTY ADD...... 148 23.11.4.2 Non-neural endocrine hormone receptors ADD ...... 148 23.11.4.2.1 Tropic hormone receptors ADD...... 149 23.11.4.2.2 Non-tropic hormone receptors EMPTY...... 149 23.11.4.2.3 Nuclear receptors ...... 149 154 Neurons & the Nervous System

Chapter 16 List of Figures 1/5/19 Figure 23.1.1-1 The stage 7 neuro-affector neurons within the overall neural system ...... 5 Figure 23.1.2-1 Common inter-modality functional blocks ADD ...... 6 Figure 23.3.1-1 The classifications of the hormonal system used in this work ADD...... 13 Figure 23.3.1-2 A caricature of the neural-glandular system UPDATE xxx...... 14 Figure 23.3.1-3 Top Block diagram of the glandular modality, without feedback ...... 16 Figure 23.3.1-4 Schematic of stage 7 neuroaffectors and their targets UPDATE xxx...... 19 Figure 23.3.3-1 Sagittal view of the left human hemisphere focused on the brainstem...... 26 Figure 23.3.3-2 The two divisions of the neuro-endocrine system in this parasagittal view....27 Figure 23.3.3-3 Schematic of the hypothalamohypophysis complex...... 29 Figure 23.3.3-4 Comparison of OT and AVP by Bichet ...... 31 Figure 23.3.3-5 Family of OT & AVP hormones...... 32 Figure 23.3.3-6 Potential mechanism of vasopressin production...... 34 Figure 23.4.1-1 Schematic diagram of the target organs of the pituitary hormones ...... 39 Figure 23.4.1-2 Distribution of catecholamines, GABA and some peptides in nerve terminals ...... 41 Figure 23.4.1-3A Distribution of some enzymes and peptides in nerve terminals...... 44 Figure 23.4.1-4B Distribution of some enzymes and peptides in nerve terminals...... 45 Figure 23.4.1-5 The progesterone branch of the cholesterol family of steroids ADD...... 47 Figure 23.4.1-6 Generic neuroaffector of the neuron/glandular interface of the hypothalamus ...... 50 Figure 23.4.1-7 Biosynthesis of catecholamines leading to epinephrine ...... 52 Figure 23.4.2-1 The epinephrine (adrenaline) molecule Norepinephrine is similar but omits the methyl group, CH3, at the right margin. ADD...... 53 Figure 23.4.2-2 INCOMPLETE The feedback loops of the H-P-E axis ADD...... 54 Figure 23.4.2-3 Synthesis of the principle catecholines ...... 59 Figure 23.4.2-4 Hormone tabulation & receptor locations along H-P-E axis ADD ...... 60 Figure 23.4.3-1 The feedback loops between the thyroid, pituitary and hypothalamus .....62 Figure 23.4.3-2 Alternate feedback loops of the hypothalamus-pituitary-thyroid axis...... 64 Figure 23.4.3-3 Topological options for the neurons of the hypothalamus...... 68 Figure 23.4.3-4 TRH, the thyrotropin-releasing hormone ...... 69 Figure 23.4.3-5 The potential Hypophysis of the H-P-T axis as an exemplar...... 72 Figure 23.4.3-6 Numbering pattern used for thyroxine molecules ...... 77 Figure 23.4.3-7 Major molecules of thyroxine & potential original condensate...... 77 Figure 23.4.3-8 3D representations of the thyroxines ADD...... 80 Figure 23.4.3-9 Hormone tabulation & receptor locations along H-P-T axis ...... 82 Figure 23.4.3-10 2018 Flow Diagram of the H-P-T axis with receptors and their location ADD ...... 83 Figure 23.4.4-1 Expansion of the thyroid elements of the H-P-T axis relative to disease ADD . . 84 Figure 23.4.4-2 Nominal blood test values focused on the H-P-T axis ...... 85 Figure 23.4.4-4 Daily T3 production in humans & rats...... 87 Figure 23.4.4-5 Mechanism of type 1 deiodinase (D1)-catalyzed thyroxine, T4, conversion to triiodothyronine, T3 ...... 88 Figure 23.4.4-6 Serum TSH levels before and 20 minutes after 200 micro-grams of TRH ...... 90 Figure 23.4.4-7 Serum TSH concentrations in hypothyroidism...... 91 Figure 23.4.4-8 Clinical parameters and thyroid function tests...... 92 Figure 23.4.4-9 Grades of hypothyroidism...... 93 Figure 23.4.4-10 Pretibial myxedema associated with abnormal levels of TSH, T3 and T4 in the blood RESCAN from source ...... 95 Figure 23.4.4-11 Differential Cellular Metabolism of T3 and T4 in Man ADD ...... 99 Figure 23.4.4-12 T4 to T3 conversion and utilization flow diagram ...... 100 Figure 23.7.2-1 Fundamental neuron schematic with modulator terminal...... 106 Figure 23.7.2-2 The premier gustant of the bitter or P-channel of gustation...... 107 Figure 23.8.2-1 Bare and hydrated ions. Dimensions in nanometers...... 112 Figure 23.8.2-3 Permeability coefficients of potassium ions and halides...... 116 Figure 23.8.2-4 Calculated permeability coefficient for a single bilayer...... 117 Figure 23.8.2-5 Snapshots of CER2 bilayers in ethanol–water mixtures ...... 119 Figure 23.8.2-6 Snapshots of the tyramine/POPC systems...... 120 Figure 23.9.1-1 Options for the interplay of neural, muscular & glandular ADD...... 124 Figure 23.9.1-2 Complex hair follicle showing muscular, glandular and neural elements . . . 126 Hormonal-affectors 23- 155 Figure 23.10.2-1 Proposed generic hormone/receptor interface extended from Levey....131 Figure 23.10.2-2 Preliminary table characterizing hormones ADD...... 133 Figure 23.11.2-1 The proposed VR effectivities versus d-value based on moth data...... 143 Figure 23.11.2-2 Nominal exocrine sensory receptor neuron ...... 145 Figure 23.11.3-1 Acetylcholine showing three possible DACB ...... 148 156 Neurons & the Nervous System

SUBJECT INDEX (using advanced indexing option) 3D...... 59, 79-82, 137 50%...... 129 98%...... 74 99%...... 109, 129 acetylcholine.....1, 2, 6, 12, 15, 19, 21-23, 32, 37, 38, 109, 110, 123, 125, 128, 129, 138, 141, 147, 148 Activa ...... 1, 35, 106, 107, 121, 135 active diode...... 107, 135 adrenergic ...... 1, 6, 73, 136, 137 AITD...... 85, 86, 94 allosteric ...... 110 amplification ...... 106, 144, 145 arginine ...... 2, 8, 12, 18, 28, 30, 31, 57 arginine vasopressin ...... 8, 18, 28, 30, 31, 57 attention...... 1 axon segment ...... 5 axoplasm ...... 51 Bayesian ...... 40, 128 BBB...... 13, 24-27, 30 bilayer...... 7, 107, 110, 111, 113-115, 117-122, 125, 126, 130, 137 bilayer membrane...... 110, 119, 122 blood-brain barrier ...... 11 Bombyx mori...... 143 cAMP...... 96, 97, 130 catecholamine ...... 41, 48, 49, 136 Central Nervous System ...... 8, 143 cholinergic ...... 1, 6, 21, 26 Circle of Willis ...... 30 coma...... 93 computational...... 119 confirmation...... 60, 82 consciousness...... 24 coordinate bond...... 1, 76, 105-107, 127, 135, 138, 145 coordinate chemistry ...... 1, 40, 66, 76, 81, 105, 106, 127 Crine modality...... 1, 5, 8-10, 13, 15, 19, 37, 108 DACB...... 1, 53, 59, 60, 66, 67, 69, 76, 79-83, 105-107, 134-138, 143, 145, 148 database ...... 136 decoder ...... 66 dendrolemma ...... 35 determinants ...... 98 diencephalon ...... 11, 32 diode...... 1, 107, 135 DNA...... 8, 17, 104, 105, 129, 142 dopamine...... 14, 23, 25, 37, 41, 42, 48, 51, 59, 138 double bond ...... 121 EDRF...... 12, 15 Electrolytic Theory of the Neuron ...... 1, 15, 35, 36, 40, 53, 66, 81, 127, 130, 144, 146, 148 electrometer ...... 144 electrostenolytic process...... 2, 8, 36, 42 electrostenolytics...... 10 endocrine . . . 1-3, 5, 8-15, 17, 19, 20, 24, 25, 27, 30, 35, 36, 46, 56, 94, 100, 102, 128, 129, 134, 135, 140, 142, 147, 148 epigenetics...... 75 equilibrium ...... 99, 113 evolution...... 32, 41, 65 expanded...... 3, 5, 15, 18, 28, 39, 69, 84, 91, 95, 97, 100, 129 feedback ...... 1, 15, 16, 27, 29, 52-54, 60-65, 67, 73, 82-85, 88, 129, 140 Hormonal-affectors 23- 157 flavor...... 58 GABA...... 2, 9, 10, 22, 41, 42 genetics ...... 56, 58, 74, 142 glutamate...... 1, 2, 9, 10, 42 glycol...... 40, 69, 97 glycosylation ...... 17 GPCR...... 7, 35, 66, 73, 74, 125, 126, 141 g-protein...... 35, 70, 130 halogen...... 76 heterodimer...... 147 hippocampus ...... 7 hole ...... 15, 121 hole transport...... 121 homogeneous ...... 119 hormone . 3, 7-10, 12-20, 23-25, 27-29, 31, 33-37, 40-43, 46-57, 60, 62, 65, 67, 69, 70, 73-76, 78, 79, 82, 86, 88, 89, 94, 95, 97, 98, 100, 102-108, 122, 123, 125, 127-131, 134-144, 146-149 hydrogen bond...... 82 hypophysis . . 1, 7-11, 13-20, 24, 25, 27-30, 32, 34-38, 41, 42, 46, 48, 49, 53-57, 61, 65-67, 69, 70, 72, 73, 82, 83, 85, 97, 102, 108, 125, 134 hypothalamus . . 1, 7-10, 12-14, 16-20, 25-30, 32, 34, 36-39, 41, 42, 44-55, 57, 60-62, 64-68, 70, 72, 73, 76, 82, 83, 85, 89, 108, 125, 133, 134 ice ...... 20, 146 in vitro ...... 9, 22, 73, 93, 94 in vivo ...... 9, 10, 78, 93, 115 inhomogeneous ...... 119 insulin...... 129, 135, 138-140, 142 internal feedback ...... 52 interneuron...... 1, 144 inverting ...... 53, 66, 67, 70, 106, 126 in-vitro ...... 110 in-vivo ...... 110 Jmol...... 52 LATS ...... 85, 86, 94, 95, 98, 99 liquid-crystalline...... 1, 111, 121 lysine ...... 55 Medulla...... 11, 24, 102 metabolites...... 78, 129 microtubule ...... 34 modulation...... 9 multiple probe...... 1 NANC ...... 1, 37 neurite...... 123 neurites ...... 1 neuromodulator ...... 11, 24, 65 neurotransmitter...... 3, 11, 65 neuro-affector ...... 1, 3-5 neuro-affectors ...... 1-5, 15, 24, 27, 36, 37, 58 nicotinic ...... 21, 110 nitrergic...... 1 nitric oxide ...... 1, 1, 2, 6, 12, 15, 19, 23, 127, 128, 140, 141, 147, 148 nitroergic...... 1 non-inverting ...... 53, 66, 106, 126 n- ...... 107 orbitals...... 107, 136 oskonation ...... 47, 74, 103, 106, 109, 145, 146 oskonatory ...... 36, 134, 138, 142-144, 146 oxytocin ...... 8, 16, 18, 20, 25, 27, 28, 30-35, 37, 39, 51, 54-57 paracrine ...... 1, 2, 9-13, 15, 19-21, 24, 30, 36, 46, 109, 129, 134, 135, 140-142, 147, 148 parametric...... 9, 106 parvocellular ...... 31-33 phenotype ...... 22 pheromone ...... 47, 103, 130, 140, 141, 143, 144, 146 phylogenic tree...... 42 158 Neurons & the Nervous System picrophore ...... 107 pituitary gland . . 1, 11-14, 18, 26-29, 31, 32, 36, 37, 41, 42, 54, 56, 57, 60, 65, 66, 72, 74, 79, 83, 89, 97, 134 poditic...... 53, 66, 70, 106, 126 protocol ...... 101, 121, 131 PtdCys...... 81 PtdPro ...... 69 PtdSer ...... 148 reading...... 43, 93 residue...... 40, 55, 60, 73, 78, 138 resonance...... 77 saliency map ...... 23 SAR...... 79 second messenger ...... 97, 103, 107 servo loop...... 81, 129 sleep ...... 23 smooth muscle ...... 2, 3, 6, 19, 21, 23, 123, 125, 127, 141, 147 spinal cord ...... 65 stage 1 ...... 1, 1, 2, 6, 35, 36, 66, 67, 70, 124, 125, 143-145 stage 2 ...... 66, 73, 143, 144 stage 3 ...... 5, 30, 125 stage 4 ...... 23 stage 5 ...... 143 stage 6 ...... 1-4, 14, 20, 32, 66, 125 stage 6b ...... 66, 67, 83 stage 7 . . . 1, 1-7, 12-15, 19, 20, 25, 27, 28, 30, 32, 35-37, 48, 51, 52, 56, 57, 61, 66, 67, 70, 83, 123- 126, 135, 140, 147 stage 8 ...... 1, 70, 72 stress...... 52, 55, 119 surface tension...... 111 synapse...... 1, 2, 4, 12, 13, 15, 83, 124, 126, 135, 144, 145 T3 ...... 24, 27, 29, 36, 40, 53, 62-67, 75, 76, 78-82, 86-89, 91, 93-95, 97-101, 103, 105, 138 T4 ...... 24, 27, 29, 36, 40, 61-67, 70, 75-82, 86-89, 91, 93-95, 97-101, 138 telencephalon...... 32 thalamus...... 11, 25, 32 top block...... 16 topology...... 66 transduction...... 57, 103, 104, 130, 140-144, 147, 149 translation...... 32 trans- ...... 70 TSH...... 1, 14, 17, 24, 27, 29, 37, 41, 54, 65, 67, 70, 73-75, 83, 86, 89-97, 100, 101, 105-107, 138 type 1 ...... 2, 87, 88, 109, 114, 130, 131 type 2 ...... 2, 7, 35, 59, 69, 81, 87, 88, 107, 109, 124-126, 130, 131, 137 type 3 ...... 2, 109, 121, 124, 142 type 4 ...... 2 type II...... 89 vodorant...... 144, 146 Wikipedia ...... 8, 29, 54, 65, 83, 135, 139 working memory ...... 98 xxx .... 2-5, 14, 19, 35, 50, 51, 58, 65, 70, 101, 103, 105-107, 109, 110, 118, 119, 125, 127, 132, 135- 139, 146, 148 [xxx . . 1, 2, 5, 11, 28, 30, 46, 58, 74-76, 81, 84, 92, 93, 98, 101, 104, 105, 108-110, 115, 120, 121, 123, 126-128, 131, 132, 137, 138, 142, 146, 147