Regulation of Catecholamine Release from the Adrenal Medulla Under the Physiological Stress Response

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REGULATION OF CATECHOLAMINE RELEASE FROM THE ADRENAL MEDULLA UNDER THE PHYSIOLOGICAL STRESS RESPONSE

BARBARA A. KURI

Submitted in partial fulfillment of the requirements

For the degree of Doctor of Philosophy

Dissertation Adviser: Dr. Corey Smith

Department of Physiology and Biophysics

CASE WESTERN RESERVE UNIVERSITY

January 2010 CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

______

candidate for the ______degree *.

(signed)______(chair of the committee)

______

(date) ______

*We also certify that written approval has been obtained for any proprietary material contained therein. TABLE OF CONTENTS

Table of Contents ii

List of Figures iv

Acknowledgements vi

List of Abbreviations vii

Abstract ix

Chapter 1: Adrenal Medulla Contribution to Release of

Catecholamine Under Stress

1.1 The Adrenal Gland 2

1.2 Stress 7

1.3 Regulation of Catecholamine Release 11

1.4 Cellular Mechanisms Regulating Exocytosis in Chromaffin 18

Cells

1.5 Methods Used to Study Catecholamine Release in Adrenal 22

Chromaffin Cells

1.6 Summary Statement of Thesis Project 32

Chapter 2: Increased Secretory Capacity of Mouse Adrenal

Chromaffin Cells by Chronic Intermittent Hypoxia: A Role

for Protein Kinase C

2.1 Introduction 36

2.2 Materials and Methods 38

2.3 Results 42

2.4 Discussion 53

Chapter 3: PACAP Regulates Immediate Catecholamine Release

from Adrenal Chromaffin Cells in an Activity Dependent

Manner through a Protein Kinase C Dependent Pathway

3.1 Introduction 57

3.2 Materials and Methods 61

3.3 Results 67

3.4 Discussion 91

Chapter 4: Discussion and Future Directions

4.1 PKC 95

4.2 Sodium Calcium Exchange 102

4.3 T-type Calcium Channels 112

2+ 4.4 IP3 Release of Ca from Internal Stores 112

4.5 Epac 113

4.6 Overall Conclusion 114

4.7 Future Directions 116

Work Cited 123

iii

LIST OF FIGURES

Figure 1.1 The biosynthesis of catecholamine 6

Figure 1.2 PACAP is an activator of adenylate cyclase and a potent 16

secretagogue

Figure 1.3 Capacitance recordings provide a high resolution measure of 26

quantal catecholamine release

Figure 1.4 Carbon fiber amperometry quantitatively measures 29

catecholamine release

Figure 2.1 Intermittent hypoxia leads to an increased RRP 44

Figure 2.2 Continuous hypoxia does not lead to an increased RRP 46

Figure 2.3 Superoxide dismutase mimetic blocks the CIH-dependent 48

increase in RRP

Figure 2.4 The CIH-d4-mediated increase in the RRP is due to an 51

increased PKC phosphorylation

Figure 3.1 PACAP stimulation of adrenal chromaffin cells results in an 70

immediate and robust release of catecholamine

Figure 3.2 PACAP evokes catecholamine release under elevated 73

sympathetic input

Figure 3.3 PACAP-dependent secretion is dependent on extracellular 77

Ca2+ and blocked by PLC and PKC inhibition

Figure 3.4 PACAP-dependent Ca2+ signals are dependent on 78

extracellular Ca2+ and blocked by PLC and PKC inhibition

Figure 3.5 Ni2+ -sensitive Ca2+ currents in adrenal tissue slices 82

Figure 3.6 PACAP-dependent catecholamine secretion is blocked by 85

benzamil, voltage clamp and extracellular Ni2+

Figure 3.7 PACAP-dependent Ca2+ signals are blocked by benzamil, 86

voltage clamp and extracellular Ni2+

Figure 3.8 Epac stimulation elicits nickel-sensitive catecholamine 88

secretion

Figure 3.9 Summary diagram for the proposed PACAP signaling 90

pathway

Figure 4.1 Phosphorylated Protein Kinase Cδ is found in mouse 99

adrenal medullary tissue

Figure 4.2 PACAP knockout mice fail to elicit a secretory response 120

under burst-mode stress stimulation

Figure 4.3 PACAP knockout mice express the PACR-1 receptor 121

Figure 4.4 PACAP knockout mice respond to exogenous PACAP 122

Acknowledgements

I would like to thank, first and foremost, my advisor Dr. Corey Smith for his support both professionally and personally. I would like to thank my committee members; Dr Cathleen Carlin, Dr. Carole Liedtke, Dr. Andrea Romani, Dr. Steve

Jones, and Dr. Thomas Dick for their guidance and support. Additionally, I would like to thank my lab-mates Dr. Shyue-An Chan, Dr. Bryan Doreian, and Jackie

Hill for their support, technical assistance, personal support and friendship.

Finally, I would like to thank my family and friends for their unending support.

List of Abbreviations

A Adrenaline

Ach Acetylcholine

ACTH Adrenocorticotropic Hormone

ANOVA Analysis of Variance

Cm Capacitance

CA Catecholamine

CaM Kinase Calmodulin Dependent Protein Kinase cAMP Cyclic Adenosine Monophosphoate

CH Continuous Hypoxia

CIH Chronic Intermittent Hypoxia

DAG Diacylglycerol

DBH Dopamine P-hydroxylase

Epac Exchange Proteins Activated Directly by cAMP

FURA-2 AM Fura-2-acetoxymethyl ester

GIRK G-protein Coupled Inward Rectifying K+ Channel

GPCR G-protein Coupled Receptor

HVA High Voltage Activated Ca2+ Channels

IHC Immunihistochemistry

IP3 Inositol1,4,5 trisphosphate

LVA Low Voltage Activated Ca2+ Channels

MnTMPyP manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride NA Noradrenaline

vii nAChR Nicotinic Acetylcholine Receptor

NCX Sodium Calcium Exchanger

PACAP Pituitary Adenylate Cyclase Activating Peptide

PACR-1 PACAP Receptor- 1

PFA Paraformaldehyde

PKA Protein Kinase A

PKC Protein Kinase C

PLC Phospholipase C

PMA phorbol 12-myristate 13-acetate

PNMT Phenylethanolamine N-methyltransferase

ROS Reactive Oxygen Species

RRP Readily Releasable Pool

TBARS Thiobarbituric Acid Reactive Substances

TRP Transient Receptor Potential Cation Channels

TSH Thyroid Stimulating hormone

TTX Tetrodotoxin

VIP Vasointestinal peptide

XIP Endogenous Exchanger Inhibitory Region

viii

Regulation of Catecholamine Release from the Adrenal Medulla Under the Physiological Stress Response

Abstract

BARBARA A. KURI

Release of catecholamine from the adrenal glands is a highly regulated process.

The chromaffin cells of the adrenal medulla are the parenchymal cells of the

adrenal gland and are capable of releasing catecholamine (adrenaline and

noradrenaline) and neuroactive peptides into the circulation. Under autonomic nervous control, the innervating splanchnic nerve directs the release of

catecholamine. In this study, we show how chronic intermittent hypoxia (CIH), a common form of metabolic stress, affects the release of catecholamine from the adrenal chromaffin cells, in situ. Using electrophysiological capacitance recording techniques we show that exposure to CIH results in an increase of the

amount of catecholamine available for release, upon a given stimulus, through

the recruitment of catecholamine containing granules to the readily releasable

pool. We show that this is possible through the generation of reactive oxygen

species (ROS) and subsequent activation of protein kinase C (PKC). To further address the regulation of catecholamine release under sympathetic stimulation we developed a bipolar stimulation protocol that mimics stimulation frequencies

at rest and under burst mode stress firing. Using an in situ adrenal gland slice

preparation we show that there is an activity dependent regulation of

catecholamine release involving pituitary adenylate cyclase activating peptide

(PACAP) which is released from the splanchnic nerve under conditions

mimicking the burst mode stress firing. We show that PACAP acts through a

novel mechanistic pathway that is independent of the conventional sympathetic

cholinergic pathway. This characteristic of PACAP makes it an ideal

neurotransmitter under high intensity, burst mode sympathetic firing, in that it

does not desensitize like the cholinergic pathway. We show that PACAP acts

through a G-protein coupled receptor to activate phospholipase C (PLC) which in

turn activates PKC. Activation of PKC plays a role in increasing the activity of the

sodium calcium exchanger (NCX) in the forward direction to depolarize the cell

membrane to potentials that increase the open probability of a Ni2+ sensitive, low

voltage activated Ca2+ channel. It is the influx of Ca2+ through this channel to generate the Ca2+ dependent release of catecholamine.

Chapter 1:

Adrenal Medulla Contribution to Release of Catecholamine Under Stress

1.1 The Adrenal Gland

The adrenal gland is the primary peripheral endocrine gland in the sympathetic

stress response. The parenchymal cells of the adrenal medulla are the

chromaffin cells [1]. Belonging to the paraneuron family, chromaffin cells of the

adrenal medulla are of neural crest origin, capable of eliciting action potentials,

and secrete catecholamines under appropriate stimulation [1-3]. Formed during

the early stages of development, there are two separate precursors migrate

suprarenally and eventually differentiate to make up the adrenal gland. The outer layer of the adrenal gland is derived from mesoderm tissue and becomes the

adrenal cortex [4]. The adrenal cortical cells, are not excitable, are not of neural

crest origin but, are secretory and responsible for the production and release of

glucocorticoids: aldosterone, cortisol and androgens [4]. The cortex comprises

approximately 90% of the total adrenal gland mass. The remaining 10% of the

adrenal gland is comprised of the fore mentioned chromaffin cells. The adrenal

medulla is derived of sympathetic ganglion cells from the neural crest during

development. After neural crest cell migration, the chromaffin cells are engulfed

by the cortical cells and subsequently differentiate into neuroendocrine secretory

cells [4]. The chromaffin cells are responsible for the secretion of catecholamines

(noradrenaline (NA) and adrenaline (A)) as well as a host of neuroactive

peptides. Within the adrenal medulla there are two types of catecholamine

containing cells, those that secrete nor-adrenaline and those that release

adrenaline [5-11].

Secretion of both glucocorticoids and catecholamines is highly regulated.

Corticosteroid production and release from the adrenal cortex is regulated by adrenocorticotropic hormone (ACTH) that is released from the pituitary gland.

Catecholamine biosynthesis and secretion from the chromaffin cells is controlled

by sympathetic nerve activity [12]. The adrenal medulla is innervated by

preganglionic sympathetic fibers of the splanchnic nerve [1]. These sympathetic fibers enter the medulla and disperse to innervate adrenal chromaffin cells [6, 7,

10-15]. Upon presympathetic stimulation, chromaffin cells release their contents

into the circulation. Thus, chromaffin cells can are considered to be modified

postganglionic neurons releasing catecholamine into the blood stream rather

than a synaptic junction [16]. In addition to the sympathetic motor efferent

innervation, there are afferent sensory innervations originating in the dorsal root

ganglia cells from T3-L2 regions in the spinal column. These sensory nerve

endings resemble the baroreceptors found in the carotid sinus [12]. It is

suggested that these nerves serve as a monitor of blood pressure (baroreceptor

function) and to monitor the intramedullary levels of catecholamine

(chemoreceptor) [12].

Glucocorticoid and catecholamine secretions are important in regulating the “rest

and digest” state of physiology as well as the “fight of flight” sympathetic stress

response. Glucocorticoids are released from the adrenal cortex in response to

ACTH stimulation. The pituitary gland, under control from the hypothalamus,

3 drives the release of glucocorticoids in response to stress, with the purpose of maintaining homeostasis under chronic stress. Cortisol is the primary glucocorticoid in humans. Upon cortisol binding to its receptor on effector cells, the receptor cortisol complex translocates to the nucleus where it regulates the transcription of the appropriate hormone responsive genes [17]. Additionally, cortisol serves as a negative feedback loop to decrease subsequent release of

ACTH from the pituitary by acting upon both the hypothalamus and pituitary glands [17]. Prolonged exposure to circulating cortisol causes an increase in catabolic metabolism, anti-reproductive effects as well as immunosuppressive effects [17].

While glucocorticoids are involved in the maintenance of the chronic stress response, the primary effects of catecholamine in the sympathetic stress response is to ready the organism for survival under acute stress [16]. Adrenaline is released under stress and results in the dilation of arterioles in skeletal muscle, an increase in the rate and force of heart contractions, and stimulation of the breakdown of glycogen to increase blood sugar providing fuel for muscle action.

All of these responses prepare the organism for “fight or flight” [16]. However, despite their pro-metabolic role in the stress response, catecholamine release at diminutive levels is also important for proper homeostatic regulation under the

“rest and digest” parasympathetic mode and is thus released in to the blood stream to maintain proper vessel tone and drive basic metabolic functions at rest.

Catecholamines are derivatives of dopamine. As shown in Figure 1.1, dopamine formed from 3,4-dihydroxyphenylalanine is converted to noradrenaline by

dopamine β-hydroxylase (DBH). Noradrenaline is then converted to adrenaline

by phenylethanolamine N-methyltransferase (PNMT) [18]. These two effector

signaling molecules are both found in chromaffin cells, but as mentioned prior,

they are located in different cells. Those cells possessing PNMT contain

adrenaline and those without PNMT contain noradrenaline. The secretory cell

type can be distinguished with electron microscopy by the shape and density of

the secretory granules [5]. It was further shown that differential release of noradrenaline and adrenaline occurs in a manner dependent upon stimulus intensity; where adrenaline is the predominant effecter released under acute stress stimulation [9]. The release of adrenaline from chromaffin cells binds primarily to β-adrenergic receptors throughout the body to increase gluconeogenesis, increase insulin secretion, as well as, increase cardiac contractility and heart rate while promoting arteriolar vasodilation in skeletal and cardiac muscle. Noradrenaline primarily binds to the α-adrenergic receptors causing arteriolar vasoconstriction, a decrease in insulin secretion, increases in sweating, and dilation of the pupils [19].

Figure 1.1: The biosynthesis of catecholamines begins with dopamine. In a figure adapted from “The Adrenal Glands” chapter of Physiology, a schematic representation of catecholamine biosynthesis shows that it is a multi-step process [19]. Chromaffin cells of the adrenal medulla are capable of releasing both adrenaline and noradrenaline. These two metabotropic byproducts are not stored within the same cell. The enzyme phenylethanolamine N- methyltransferase (PNMT) is the enzyme responsible for the conversion of noradrenaline to adrenaline. Probing for this enzyme allows differentiation between the different cell types within the adrenal medulla by determining its catecholamine contents. Those positive for PNMT are adrenaline releasing cells.

1.2 Stress

The term “stress” was first used by Hans Selye, an endocrinologist, in the early

20th century, who described it as the physiological response in which an animal fails to react appropriately to emotional or physical threats (stressors) [20]. Stress was later described as “the state of disharmony or of threatened homeostasis that evokes specific and non-specific responses” [21, 22]. Some physiologic responses include an increase in glucose consumption, a greater ability in decision making, a decrease in digestive processes, and a decrease in reproductive capabilities. The parasympathetic and sympathetic nervous systems are highly coordinated to maintain physiological homeostasis and are under the control of the autonomic nervous system. In a term coined by Walter Cannon, activation of the sympathetic nervous system results in the “fight or flight” stress response while down-regulating the parasympathetic nervous system [21-24].

The body is continuously exposed to different stressors and it responds accordingly. Physical stressors (noise, pain, heat/cold), psychological (anxiety, fear, frustration), social stress (unemployment, martial distress, death), and stress to challenge (cardio and metabolic homeostasis, exercise, orthostasis, hypoglycemia, and hemorrhage) are all examples of different types of stressors

[25]. The sympathetic nervous system is the primary responder under exercise, heat/cold, or minor blood loss releasing norepinephrine into the periphery as well as release of epinephrine into the circulation. Greater epinephrine release from the adrenal chromaffin cells is a result of global and metabolic stress (exercise to exhaustion, emotional distress, shock, and hypoglycemia) [24, 25].

Medullary chromaffin cells of the adrenal glands are specialized sympathetic ganglia. While the primary postsynaptic sympathetic neurotransmitter is noradrenaline, the adrenal medulla releases both adrenaline and noradrenaline.

Under basal or rest conditions, it has been shown that the sympathetic nervous system regulates the amount of serum catecholamine through low frequency stimulation of the adrenal medulla via cholinergic release from the splanchnic nerve. Patients who have undergone a splanchnicectomy demonstrate severe hypotension as a consequence due to a lack of catecholamine output from the adrenal medulla [16]. Additionally, disconnect between the sympathetic regulatory response and adrenomedullary control is seen in patients who have undergone adrenalectomy. Adrenalectomized patients demonstrate an increase in serum noradrenaline (the primary neurotransmitter of the sympathetic nervous system) in response to stress, but fail to demonstrate an increase in serum adrenaline under hypoglycemia with insulin shock [25, 26]. These findings suggest that the mechanisms controlling chromaffin cell stimulation are highly regulated at rest as well as under duress. Furthermore, regulation of the sympathetic and adrenomedullary systems is highly important as their primary function is to mediate the body’s response to stress. Prolonged stimulation, however, due to misregulation or chronic stress can manifest in hypertension, cardiovascular events, gastrointestinal disease and carcinoma [25, 27]. A review by Arun et al. discussed that while catecholamine released under the acute stress response is necessary to promote survival, continual or misregulated release of catecholamine can result in chronic disease and even promote death

[28]. Most recently, it has been shown that chronic and repetitive activation of the sympathetic stress response leads to coronary heart disease as well as elevated blood pressure that is unable to be treated with the typical treatments [29, 30]. In today’s society, activation of the stress response is no longer due to confrontations with animals that put our lives in peril. Our bodies are, unfortunately, unable to differentiate between a wild animal chasing us and a looming deadline placed upon us by external or personal social stressors.

Therefore the resulting stress response initiated is the same under both cases.

Because of the multitude of stressors discussed previously, and the body’s inability to recognize those that are truly life threatening, we are in what may appear to be a constant state of stress. Therefore, understanding how stress is presented and how the body responds is of great importance in attempts to combat the very serious consequences of misregulation and over stimulation.

Chronic intermittent hypoxia (CIH) is one form of reoccurring stress that has been shown to involve excessive activity of the sympathetic nervous system resulting in hypertension. A simple clinical observation in 1984, suggested that those suffering from sleep apnea, a form of CIH, have an increase in sympathetic activity that is responsible for the prevalent hypertension [31]. Those afflicted with

CIH present clinical conditions similar to those who have pheochromocytoma, an adrenal derived tumor responsible for excessive levels of epinephrine in the serum [32]. Studying the physiological response to CIH in humans is difficult and thus, work has turned to animal models where exposure to CIH can be controlled and the effects studied.

In studies using rats, CIH induced hypertension is reduced through sympathetic

denervation and adrenal demedullation [33, 34], supporting the involvement of

sympathetic activation. While researchers have shown that there are many

factors involved in eliciting the physiological response to CIH, the mechanism by

which it acts is still unknown. Chemoreceptors in the carotid body act as O2

sensors and serve a crucial role in initiating the sympathetic stress response.

Prabhakar et al. described the chemoreceptors as a primary sensor that plays a critical role in preventing the effects of hypoxia through activation of the sympathetic nervous system [35]. Additional studies have shown that peripheral chemoreceptor activity increases when exposed to CIH, while exposure to hypercapnia (CO2) or chronic continuous hypoxia (CH) has no effect on carotid

body firing. While they believe that the PO2 sensed in the carotid bodies initiates the sympathetic stress response, researchers are still unsure of the reason that those suffering from sleep apnea show daytime hypertension and how serum catecholamine levels remain elevated for periods after the apneic episodes are

over [36]. One possible explanation is due to the effects of the CIH oxidative

stress on the system that results in the generation of reactive oxygen species

(ROS) [37-40]. At low concentrations, ROS are capable of activating many

signaling cascades that regulate cell growth and differentiation, but at higher

concentrations they result in production of molecules involved in the inflammatory

response such as cytokines, adhesion molecules, NFκB, as well as promoting

cell death [41, 42]. What remains unclear is the role of ROS production in the

release of catecholamine from the adrenal medulla.

While there is an increase in sympathetic firing and ROS generation in CIH, a

study by Hui et al. showed that while there is an increase in sympathetic

activation from the carotid bodies changes are not seen in the superior cervical

ganglia or the adrenal glands [43]. These findings encourage further study on the

mechanism by which CIH results in increased serum catecholamine, especially if

there is not an increase in production of catecholamines from the primary output of the sympathetic nervous system. More importantly it helps to address the possible role that increased sympathetic activation and ROS play in regulating catecholamine release under physiological stress.

1.3 Regulation of Catecholamine Release

Characteristic of the autonomic nervous system, acetylcholine is the primary presynaptic neurotransmitter. Released from the splanchnic nerve, acetylcholine binds to the nicotinic- acetylcholine receptor (nAChR) on the adrenal chromaffin cells. Upon acetylcholine binding, the K+ and Na+ (primarily Na+) conductance

through the receptor channel increases, depolarizing the cell and opening

tetrodotoxin (TTX) sensitive voltage activated Na+ sodium channels. The further

increased influx of Na+ results in an action potential that increases the probability

of opening high voltage gated Ca2+ channels to increase the intracellular calcium

concentration. This rise in intracellular Ca2+ is necessary for release of

catecholamine containing granules of adrenal chromaffin cells. Cholinergic

excitation is regulated at many points. One point of regulation is the enzyme

acetylcholinesterase that is co-localized with sympathetic nerve fibers in the

adrenal medulla and is responsible for the degradation of acetylcholine [44].

Rapid removal of acetylcholine allows for repeated stimulation in rapid

succession and effective transmission. Cholinergic desensitization, however,

occurs at receptor/ligand binding as well as many points past the presynaptic

release of acetylcholine. Some of the neuronal nAChR’s found in the CNS and

autonomic ganglia are composed of the α2/β4, α3/β4 and the α7 subunits of the

known 19 subunits for nAChR [45]. All of those listed above are prone to desensitization which results in a block of synaptic transmission in the presence of agonist under prolonged exposure to acetylcholine [46]. It has been suggested

that intracellular signaling pathways play a role in modulation of the cholinergic

response in both facilitation as well as potential inhibition [45-47]. Cholinergic

desensitization is not attributed completely to receptor desensitization however,

because a similar desensitization phenomenon can be seen in chromaffin cells

stimulated with ringer buffer containing high K+ concentrations [48]. Other

possible explanations of desensitization are a result of inactivation of the voltage-

dependent ion channels (Na+ and Ca2+) or a change in the Ca2+ dependent

processes involved in exocytosis [1]. Additional regulation of catecholamine

release under acute sympathetic stress occurs through the release of ATP from

catecholamine containing granules. ATP binds to the P2γ-purinoceptors

decreasing Ca2+ current providing a potent autocrine/paracrine negative

feedback mechanism and subsequent decrease in cholinergic responsiveness

[49]. Furthermore other studies have shown that while acetylcholine is believed

to be the primary neurotransmitter involved in evoking catecholamine release

from adrenal chromaffin cells, other neuromodulators may have a role as well. It

is thought that while acetylcholine is contained in the small clear vesicles of the

splanchnic nerve terminals, there are also larger granular vesicles which may

release other neuropeptides that play a role in regulating catecholamine release

[1, 15]. These neuromodulators may have a possible role in providing negative

feedback to regulate catecholamine release under sympathetic activation.

Histamine and angiotensin II are released from the splanchnic nerve and both

result in depolarization of the chromaffin cell membrane and subsequent

exocytosis though their mechanisms are not the same as acetylcholine [50].

Binding of histamine to the H1 receptor on chromaffin cells results in activation of

a Gαq pathway. Thus, histamine stimulation activates PLC to form inositol 1,4,5 trisphosphate (IP3) and diacylglycerol (DAG). The observed depolarization is

believed to be through direct interaction of PLC inhibition on the potassium (Im)

current [51]. Block of the (Im) current results in membrane depolarization, making

the cells more excitable. The histamine response also results in an increase in

2+ intracellular Ca through IP3-channels, but studies have shown that this response is not responsible for the evoked exocytosis of catecholamine [51, 52].

Additionally, Angiotensin-II evoked catecholamine release is believed to occur

under periods of hypotension and hemorrhage and results in an increase of

intracellular Ca2+ from spatially restricted sites close to the nucleus, as well as

through a ω-conotoxin sensitive Ca2+ channels (N-type channel) [52].

Neuroactive peptides such as vasointestinal peptide (VIP), neuropeptide Y,

substance P, and pituitary adenylate cyclase activating peptide (PACAP) are also

known secretagogues that may act along with or independent of acetylcholine to regulate catecholamine release [1, 52, 53].

Pituitary Adenylate Cyclase Activating Peptide (PACAP) was first isolated in

1989 by a group who was attempting to describe novel hypophysiotropic

neuropeptides. Using ovine hypothalamus, they were screening for peptides

having a stimulatory effect on adenylate cyclase and isolated one that had a

sequence homology to VIP but was 1000x more effective at activating adenylate

cyclase [54, 55]. PACAP is a member of the glucagon/secretin superfamily of

peptides and shares a 68% amino acid homology with VIP [56]. Other members

of this super family of peptides include: Glucagon, GRF, and secretin [55].

PACAP has two potential isoforms, PACAP-38, the predominant form of PACAP,

and PACAP-27 that corresponds to the N-terminal 27 amino acid sequence of

PACAP-38 [55, 56]. PACAP has a broad range of functions where it can act as a

hormone, neurohormone, Autocrine/Paracrine substance, neurotransmitter,

neuromodulator, neurotrophic factor, and immunomodulator [56]. Some known

physiological effects of PACAP stimulation result in neurotransmitter release,

vasodiltation, bronchodilation, activation of intestinal motility, increase of insulin

and histamine secretion, and stimulation of cell multiplication and differentiation

[55, 56] .

The amino acid sequence of PACAP-38 is His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-

Tyr-Ser-Arg-Tyr-Arg-Lys-Gln- Met-Ala- Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-

Lys-Arg-Tyr-Lys-Gln-Arg-Val-Lys-As n-Lys - NH2. [54]. Its tertiary structure is

14 composed of two helices separated by a disordered region. Its structure is similar to that of VIP and PACAP-27. It is believed that small shifts in position of the formation of the first and/or second helix help contribute to the selectivity of the peptides for their receptors [57, 58]. Figure 1.2 shows the amino acid sequence of PACAP and other peptides in its superfamily, demonstrating the conserved a.a. sequence amongst the family members. Below in Figure1.2 is an image of the secondary structure of PACAP-38 [59].

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10

Figure 1.2: A) The amino acid sequence of PACAP. Originally Isolated from ovine pituitary tissue, PACAP was identified as a potent activator of adenylate cyclase. It is 38 amino acids in length and is approximately 18 kDa [59]. As a member of the secretin family, it is highly conserved amongst other peptides including glucagon and vasoactive intestinal polypeptide. B) Taken from Baurgault et al.; the backbone structure of PACAP and below is the schematic ribbon representation [60].

The mechanistic pathways of PACAP-mediated signaling are as diverse as is its

distribution throughout the central and peripheral nervous systems [55]. PACAP immunoreactive fibers have been located in non-neuronal cells; PACAP is

expressed in sensory neurons of the dorsal root ganglia, as well as in the

sympathetic innervations of the adrenal medulla [53, 55, 61]. A study by Moller,

showed that PACAP immunoreactive nerve fibers were present in both newborn

and adult adrenal medulla [53]. While the evidence suggests that PACAP is

released from presympathetic ganglia under sympathetic activity, it is also of

importance to address the possibility of PACAP as a neurotransmitter released

from sensory fibers of chromaffin cells under stress as well [61]. The study by

Dun et al. shows that the primary innervations of PACAP immunoreactive fibers

in rat adrenal glands are from the nodose and dorsal root ganglia [61]. Though

they recognize that PACAP is released from the splanchnic nerve and the

resolution limitations of their immuno-histochemical techniques they suggest the

possibility of PACAP under the appropriate nerve stimulation may represent an

example of sensory regulation of catecholamine release [61]. Studies probing for

PACAP receptor expression showed that PACAP mRNA is not in the chromaffin cells themselves, suggesting that PACAP stimulation comes from neuronal release onto the chromaffin cells (preganglionic or sensory afferents) [53].

Additional studies showed that surgical denervation of rat adrenals resulted in a lack of PACAP immunoreactive fibers and that VIP innervation of the rat medulla was sparse, but VIP innervation of the outer cortex was more dense.

The mechanism by which PACAP exerts its effects is unclear and appears to be specific to its targets and possibly exhibits species dependence. In this thesis, I provide novel and direct evidence that PACAP acts as a primary neurotransmitter at the sympatho-adrenal synapse and that its primary role is to elicit catecholamine secretion under the sympathetic stress response. Its mechanistic role in catecholamine release from adrenal chromaffin cells will be further addressed in Chapter 3. We will also address the potential physiological

relevance of PACAP regulation of catecholamine release versus the conventional

neurotransmitter acetylcholine. We show that PACAP 1) does not appear to

desensitize under prolonged stimulation, 2) is released from the splanchnic nerve

in a frequency dependent manner, and 3) its pathway is independent of the

cholinergic pathway, and involves either or both adenylate cyclase and

phospholipase C activation, and calcium influx from extracellular milieu.

1.4 Cellular mechanisms regulating exocytosis in chromaffin cells

There are many cellular mechanisms regulating exocytosis of catecholamine.

While all of the second messengers involved and their respective targets have

not been identified, Ca2+ and protein kinase C (PKC) have been recognized as

two major players. First demonstrated by Douglas et al. in 1961, experiments

involving the absence of extracellular Ca2+ showed that Ca2+ influx was

necessary for exocytosis [62]. Furthermore, studies have attempted to isolate the

specific channels through which Ca2+ current is conducted. It is suspected that

depolarization of the membrane from nAChR stimulation results in the opening of

18 voltage sensitive Ca2+ channels. Studies by Artalejo et al. suggest the involvement of three different high voltage activated (HVA) calcium channels the

L-type, N-type and P-type [63, 64]). The primary channel was believed to be L- type Ca2+ channel, but studies show that block with dihydropyridines only partially blocks total Ca2+ current and only attenuates catecholamine release [64].

Furthermore, highly localized rises in calcium facilitate exocytosis suggesting a tight coupling between calcium channels and the catecholamine containing granules [65]. Further characterization of calcium channels in chromaffin cells is needed to address the role of Ca2+ influx during both basal and stress stimulation. Studies by Chan et al. and others have shown that channel subtype location, as well as, stimulus firing frequency can effect catecholamine release.

While under square pulse depolarization L-type channels have the greatest peak current, under more physiological stimulation, action potential waveform stimulation, P/Q subtypes show a greater influence on regulation of exocytosis

[66]. These findings suggest that under physiological stress a channel subtype that activates quickly by an action potential and is able to recover from inactivation equally as quickly would provide better synaptic efficiency than one with slower kinetics that it is capable of passing greater current. Recently another calcium channel subtype is being recognized as a stress response channel. The

T-type channel is a rapidly activating, and rapidly inactivating calcium channel.

These channels are involved in physiological functions such as generation of cardiac pacemaking activities, muscle contraction and hormone release [67].

These channels are unique because they are low voltage activated (LVA)

channels that activate and inactivate at more negative voltages with a large

window current [67]. Recent evidence suggests that T-type channels play a role

in the release of catecholamine in adrenal chromaffin cells. A study by

Giancippoli demonstrated that T-type channels are coupled to exocytosis with the same efficiency as the HVA channels when exposed to cAMP and that this response can be attenuated by NiCl2 (a blocker of T-type channels) [68]. The study goes on to discuss that recruitment of T-type channels significantly lowers the firing threshold allowing for greater synaptic efficiency under stress, similar to the suggested role of P/Q channels in the paper by Chan et al. [66, 68]. It is the kinetics of the T-type channels and their expression characteristics that suggest a coupling of these channels to catecholamine release under the physiological stress response.

As suggested by Douglas; the influx of Ca2+ plays a pivotal role in exocytosis, yet

its direct role is still under investigation. It was shown by Neher and colleagues that there is a Ca2+ gradient generated near that plasma membrane that may

reach 1-10 µM concentration before catecholamine release occurs [69]. It is also

known that there are certain cytosolic proteins as well as sub-plasmalemmal

proteins that must be recruited or activated in a Ca2+ dependent manner for

membrane fusion to occur, thus driving exocytosis [65]. Syntaxin and

synaptotagmin are associated with the SNARE complex and are involved in

vesicle docking and fusion and are active in a Ca2+ dependent manner. It has

been suggested that synaptotagmin is the Ca2+ sensor driving release of

catecholamine containing vesicles upon reaching the critical Ca2+ concentration

[70]. Upon stimulation, the calcium dependent release of catecholamine is rapid

but studies where synaptotagmin has been mutated show that exocytosis occurs,

though it happens on a much slower time scale [71]. Taken together these

results suggest that there are other Ca2+ dependent processes regulating catecholamine release. Rapid release of catecholamine however, requires Ca2+

binding to synaptotagmin to interact with the SNARE complex [70, 71]. Ca2+ is also shown to be a regulator of signaling second messengers. Calcium binding to calmodulin activates calmodulin dependent protein kinase (CaM kinase). CaM kinase is involved in both acceleration of vesicle mobilization and maintenance of the secretory pool [72]. Protein Kinase C (PKC) is another Serine/threonine protein kinase that may be activated by an increase in intracellular Ca2+ [73, 74].

Protein Kinase C is a versatile signaling molecule with over ten isoforms. PKC

has a large number of target substrates. The isoforms can be divided into three

groups, those belonging to the conventional subgroup, those of the novel

subgroup and those of the atypical subgroup. The conventional isoforms (α, β,

and γ) are activated through a Ca2+ and DAG dependent process. Upon binding of these two substrates the PKC isoform translocates to the cell membrane where is becomes enzymatically functional [75]. The novel isoforms (δ, ε, η, and

θ) are similar to the conventional isoforms except they rely only on DAG for activation and are Ca2+ independent. Lastly, the atypical isoforms (ξ and ι) act

independently of both DAG and Ca2+ [75]. Many studies have implicated PKC in

exocytosis. PKC acts at many different steps along the secretion pathway to

facilitate catecholamine release. It has been shown that Ca2+ dependent

recruitment of PKC to the plasma membrane is a key regulator of fast release of

catecholamines from chromaffin cells [74, 76-78]. Studies show that PKC may

enhance the Ca2+ sensitivity of the final steps of membrane fusion through its

interaction with Munc-18 [73]. Other studies suggest that PKC activation is

involved in the recruitment of catecholamine containing granules to the readily

releasable pool (RRP) thus resulting in a greater functional response under

stimulation [74, 77]. It is believed that PKC phosphorylation of SNAP-25 is responsible for increasing the rate of RRP recruitment [73, 79]. Interestingly, it is assumed that PKC isoforms in the above mentioned mechanisms are conventional isoforms based on the Ca2+ sensitivity of their response, but there is

recent evidence to suggest that the novel isoforms also play an important role in

catecholamine release through second messenger signaling pathways utilizing

phospholipases and DAG. Phospholipase and DAG are both capable of

activating conventional and novel PKC isoforms through g-protein coupled receptor (GPCR) mediated responses [80, 81]. Because cells express several

PKC isoforms, it is believed that their function is determined by location within the cell and specific target proteins; this will be further addressed in chapters 2, 3 and the discussion.

1.5 Methods used to study catecholamine release in adrenal chromaffin cells

An important part in studying catecholamine release is the preparation of the chromaffin cells themselves. In this study, two different preparations are utilized.

An isolated cell preparation, where the surrounding connective tissue, glial cells, nerves and epithelial cells from the vasculature have been enzymatically removed, leaving behind spherical isolated chromaffin cells in primary culture that are available for experiments up to 3 days following the preparation. Isolated cells are ideal for immunohistochemistry as well as fluorescent imaging experiments. The second preparation used is the in situ slice preparation. In this case, adrenal glands (medulla and cortex) are removed from the animal, embedded in agar and sliced into 200 µm thick slices using a vibratome. This preparation is ideal when studying physiological signaling and function because the chromaffin cells are left in as physiological context as possible, leaving innervation and cell architecture intact [37, 82]. Studies have also shown that chromaffin cells in the slice preparation maintain more synchronous secretion with greater coupling to action potential stimuli [83]. Both preparations serve as equally important models when studying catecholamine release from chromaffin cells.

To effectively study the stimulus-secretion function of chromaffin cells under conditions mimicking physiological conditions it is necessary to employ techniques with high resolution to quantitatively demonstrate the differences between rest and digest (parasympathetic control) and fight or flight (sympathetic control) responses. Catecholamine, as well as, other neuroactive peptides

(enkephalins, chromogranins A/B, ATP, and dopamine) are contained in small secretory granules within the chromaffin cells [84]. Upon stimulation, these granules fuse with the cell membrane and release their contents into the

circulation. The maturation of these secretory granules is highly regulated and

undergoes multiple processing steps before they are ready for release. There are at least two pools of secretory granules in the chromaffin cells. Those granules ready for immediate release have undergone processing and are considered to be the readily releasable pool (RRP). A second reserve pool of catecholamine containing vesicles also exists that contains granules that are able to migrate to the membrane after depletion of the RRP [85, 86]. The catecholamine containing

vesicles of the RRP are docked and primed at the plasma membrane [87]. The

RRP releases catecholamine under physiological stimulation conditions and the

reserve pool only becomes significant under strong synaptic stimulation [87]. To

estimate the size of the RRP or number of release competent granules electrical

capacitance measurements are used.

Time resolved capacitance (Cm) measurements rely on the relative size of the

cell and the surface area of the cell membrane [88, 89]. As secretory granules

fuse with the cell membrane during exocytosis, the surface area of the plasma

membrane increases and capacitance increases in direct correlation with each

fused granule. Debus and colleagues showed that through the use of electrical

capacitance recordings they were able to detect capacitance changes from the

fusion of a single secretory granule [90]. Capacitative recordings rely on an

equivalent circuit which can be described by a simple model seen in Figure 1.3.

By clamping a cell with a sinusoidal wave and applying a stimulus, measuring the

phase shift of the resulting current sinusoid provides an estimate of Cm [91]. By measuring the capacitance changes during stimulation and calculating the

number of vesicles fusing during a stimulus event it is possible to measure the

size of the readily releasable pool. Measuring the size of the RRP [85] under stimulation mimicking that of parasympathetic control or of sympathetic stress allows for examination of the effect of stress on the stimulus-secretion function of adrenal chromaffin cells. This paradigm is further addressed in chapter 3.

Figure 1.3: Capacitance recordings provide a high resolution measure of quantal catecholamine release. As catecholamine containing granules fuse with the plasma membrane, its area increases in direct proportion to the size of the

granule. By monitoring the capacitance (Cm) (direct correlation to surface area of the cell membrane) one can resolve the number of granules fusing with the membrane with a given stimulus. By clamping a cell and using a sinusoidal voltage stimulus, capacitance can be estimated by the change in phase and magnitude of the resultant current sinusoid.

While measuring the number of secretory granules available for release under different stimuli provides information on the stimulus-secretion function, these results are best paired with the measurement of catecholamine release to not only show that granules are fusing with the membrane under stimulation, but that exocytosis of catecholamine occurs as well. To quantitatively measure the release of catecholamine carbon fiber amperometry is used. Catecholamines released from the secretory granules of a chromaffin cell are readily oxidizable.

Oxidation of catecholamines occurs by placing a carbon fiber electrode in close proximity to the cell. By clamping the electrode at potentials above the redox potential of adrenaline, the fiber oxidizes the catecholamine and 2e- are released in the reaction and transfer to the voltage clamped carbon fiber resulting in a current that can be measured. Carbon fiber amperometry is very sensitive and capable of detecting catecholamine released from the fusion of a single secretory vesicle in the chromaffin cells [92, 93]. It is possible to determine the total catecholamine release per stimulus event by taking the integral of the recorded raw amperometric trace. Figure 1.4 provides a schematic of the oxidative reaction that occurs upon catecholamine release and the resultant amperometric signal in isolated chromaffin cells. Each individual spike is considered to be a quantal unit of catecholamine released [94, 95], thus providing high temporal resolution of catecholamine release upon stimulation. Use of carbon fiber amperometry in situ, while providing accurate measurement of catecholamine release does not always show distinct amperometric spikes, yet shows a distinct rise in baseline. This difference between amperometric recordings in isolated

chromaffin cells and in situ slices is due to diffusion of catecholamine through the connective tissue, glial cells and epithelial cells of the blood vessels maintained in adrenal slices. While the fiber is placed near the chromaffin cell, diffusion of catecholamine changes the time course in which it is oxidized by the fiber.

Greater amounts of catecholamine are oxidized at a given time due to summation of catecholamine released from many secretory granules. This event

causes the measured catecholamine to be represented by a change in baseline

rather than visualization of individual spikes in the amperometric recording. Total catecholamine release is still calculated by integration of the amperometric signal. Stimulation of the innervating splanchnic nerve by bipolar stimulation

(discussed in chapter 3) results in catecholamine release, measured by carbon

fiber amperometry. The integrated amperometric traces appear to continue to

release catecholamine after stimulation has ceased. This is believed to be a

result of the fore mentioned diffusion of catecholamine during stimulation in situ.

Carbon fiber amperometry, used alone or together with capacitative

measurements, is a very useful tool to study the mechanisms involved in

catecholamine release.

Figure 1.4: The oxidative reaction between catecholamine and a charged carbon fiber electrode. A carbon fiber electrode placed in close proximity to a cell without perturbing it, is held at a constant voltage (650 mV). As catecholamine containing granules are released from the chromaffin cells, the catecholamine oxidizes resulting in quinone and the release of 2e-. This current can be measured. By integrating the area under the spikes it is possible to determine the total catecholamine release over time (pC).

To further evaluate the mechanisms of catecholamine release, molecular techniques as well as imaging techniques are employed. Capacitance and

amperometric techniques are useful in showing a physiological response and

molecular and imaging techniques are useful in showing the proteins involved in

regulating this evoked response. To probe for specific proteins involved in the

exocytic process, western blotting or immunohistochemistry (IHC) are two

functional techniques. They both rely on the use of specific antibodies against the

protein in question. After primary antibody binding to the molecule of interest, a secondary antibody is used to amplify and visualize the signal.

Immunohistochemistry utilizes fluorescently tagged antibodies which allow for visualization of the target protein in the context of the intact tissue. While its resolution is limited to that of light microscopy, it does help identify the location of the protein of interest and can be co-stained with other proteins of choice to show

“co-localization” or possible protein-protein interactions, giving further insight into

the molecular scaffolding within the cell that regulates exocytosis. Western

Blotting allows for visualization of the protein of interest in regards to protein expression and can be quantified.

One other fluorescence technique that proves useful while studying exocytosis is

the measure of intracellular [Ca2+] by Fura-2-acetoxymethyl ester (FURA-2 AM)

imaging. At rest the internal [Ca2+] resides in the low 100 nM concentration

range, but upon stimulation Ca2+ concentrations rise through the release of calcium from internal stores as well as an influx of Ca2+ from voltage dependent

Ca2+ channels in the cell membrane. FURA-2 AM is a membrane permeable

ratiometric calcium indicator. The use of a ratiometric dye was specifically

designed to measure the changes in intracellular calcium, while cancelling out any variability due to the size of the cell, dye photobleaching, or loading concentration of effective dye [96]. The isosbestic point of FURA-2 AM buffer is

360 nm. Measurements at this wavelength serve as a control to account for cell

size, dye loading and photobleaching. As FURA-2 binds to intracellular Ca2+ its

absorbance spectra shifts to 390 nm and its intensity decreases in a calcium dependent manner. By measuring the ratio of FURA fluorescence at its isosbestic point to that of the Ca2+ -bound FURA-2 AM ( λ 360/390), it is possible to accurately monitor the changes in intracellular Ca2+ in a time dependent

fashion [96]. The Ca2+ sensitive properties of FURA-2 make this an ideal tool for measuring the gradients of Ca2+ within the cell as well as measuring

concentrations at basal levels, micro-molar levels and above as a result of

stimulation and activation of intracellular signaling cascades [96, 97]. Studies

employing FURA-2 AM have shown that the Ca2+ gradient in chromaffin cells is

highest near the plasma membrane with stimulation by action potentials [97].

Additional work by Augustine and Neher has also shown that the increase in calcium concentrations necessary for exocytosis are most likely a result of influx from voltage gated calcium channels and that the release of calcium from internal stores plays less of a role [69].

1.6 Summary Statement of Thesis Project

Chromaffin cells of the adrenal medulla release catecholamine into the circulation under the control of the sympathetic nervous system. Acute stress causes sympathetic activation which greatly increases transmitter output from the adrenal medulla. The increased circulating serum catecholamine acts to regulate vasculature and metabolic functions to prepare the organism for defense or escape. However, the mechanism by which elevated catecholamine output occurs remains unclear. For example, it is not known whether the increased catecholamine release from chromaffin cells is simply a function of increased sympathetic input, or an inherent alteration in the stimulus-secretion function of the chromaffin cells themselves, or both.

Episodic or chronic intermittent hypoxia (CIH) occurs in patients suffering from sleep apnea and is due to breathing obstruction. CIH activates the stress response of the sympathetic nervous system. If left untreated, CIH leads to

pathologies such as hypertension, vascular disease and diabetes. Recently I

exposed mice to multiple hypoxic conditions. I then measured the exocytic capacity from single chromaffin cells in situ with perforated patch electrophysiological capacitance recordings. Data from these experiments indicate that exposure to intermittent hypoxia results in an increase in the RRP

(number of catecholamine-containing secretory granules available for release).

An increased RRP leads to a greater secretory quantal content and thus elevated catecholamine output in response to a given stimulus. Furthermore, I report that this effect is specific to intermittent hypoxia and is not triggered by continuous

hypoxic conditions. Lastly, I provide a cellular mechanism for this CIH-mediated secretory facilitation that involves a reactive oxygen species (ROS)-dependent increase in the level of active protein kinase C (PKC), leading to the enhanced availability of secretory granules.

In further on-going studies, I am investigating other stress-mediated regulators of

catecholamine output from the adrenal medulla. It has been established that

acute stress causes chromaffin cell stimulation through cholinergic innervations

from the sympathetic splanchnic nerve. This signaling pathway, however,

desensitizes under sustained activity, yet chromaffin cells continue to secrete

transmitter in the stress condition. I investigated the possibility of a secondary

sympathetic transmitter that results in the release of catecholamine under the

acute stress condition. Pituitary Adenylate Cyclase Activating Peptide (PACAP)

is a potent chromaffin cell secretagogue and is packaged within the splanchnic

nerve terminal. I investigated the potential mechanism for PACAP induced

catecholamine release. I have data that demonstrate PACAP excitation of

catecholamine release includes activation of PLC and subsequent activation of

PKC, which in turn activates the Na/Ca exchanger (NCX) to depolarize the cells

to potentials at which low-voltage-activated T-type calcium (α1H) channels open.

Calcium influx through this path then elicits catecholamine release. Furthermore I

have data suggesting the differential release of acetylcholine and PACAP under

basal and stress firing stimuli. I hypothesize that PACAP is released in response

to sympathetic stress and subsequent sustained splanchnic firing. The release of

PACAP represents the predicted secondary synaptic transmitter system for

33 chromaffin cell stimulation that bypasses the desensitized nicotinic signaling path but utilizes PKC to facilitate release.

Chapter 2:

Increased Secretory Capacity of Mouse

Adrenal Chromaffin Cells by Chronic

Intermittent Hypoxia: A Role for Protein

Kinase C

BA Kuri, Khan SA, Chan SA, Prabhakar NR, Smith CB. Journal of Physiology. 2007 Oct 1; 584(Pt 1):313-9.

2.1 INTRODUCTION

Humans with sleep-disordered breathing manifested as recurrent apnoeas

exhibit considerable cardiovascular morbidity. A major advance in the field of

apnoea research is the discovery that chronic intermittent hypoxia (CIH) rather

than chronic intermittent hypercapenia is a major contributing factor for evoking

the cardiovascular morbidity [35]. Studies in both humans and experimental

models have shown that CIH leads to hypertension as well as increased plasma

and urinary catecholamines (CA) [98-100]. It has been further shown that

adrenalectomy prevents CIH-induced elevations of serum CA as well as

hypertension. These data suggest that CA secretion from the adrenal medulla

plays a critical role in CIH induced cardiovascular pathologies [101]. We recently

reported that catecholamine secretion from adrenal medullae in response to

acute hypoxia is markedly enhanced by prior exposure to CIH. This facilitation

involves reactive oxygen species (ROS)-mediated signalling [38]. The cellular mechanism for the effects of CIH on chromaffin cell secretion, however, is not known.

Chromaffin cells in the adrenal medulla release catecholamines through the

Ca2+-dependent fusion of large dense core secretory granules. The amount of

catecholamine released is in part regulated by the number of fusion-competent

secretory granules that make up the readily-releasable granule pool (RRP) [102].

Thus, regulation of the number of granules that comprise the RRP represents a

major control point for regulating the catecholamine secretory capacity from

chromaffin cells [103]. Previous studies have shown that the number of granules

in the RRP is regulated by several separate mechanisms, including the direct

Ca2+-mediated as well as protein kinase C (PKC)-dependent increase in the rate at which granules are recruited to the RRP, thus increasing its size [85, 104].

Here we examine whether CIH increases the RRP of chromaffin cells, and if so,

by what mechanism(s). Our results show that in mouse adrenal tissue slices, CIH

but not continuous hypoxia increases the number of catecholamine-containing

secretory granules in the RRP. This increased secretory capacity involves ROS–

mediated protein kinase C (PKC) activity.

*The work in the following manuscript was completed in part by BA. Kuri; The TBars

assay was completed by SA. Khan. SA. Chan provided analytical support and aid in

capacitative measurements.

2.2 MATERIALS AND METHODS

General methods: Experimental protocols were approved by the institutional

animal care and use committee (IACUC) of Case Western Reserve University.

Studies were performed on male mice (C57/BL6; 6-10 weeks age) that were

exposed to: 1-4 days of CIH; i.e. alternating episodes of hypoxia (5% O2 nadir for

15 s) and normoxia (21% O2 for 5 min) 9 episodes/h for 8 h/day as described

previously [105], chronic hypoxia (CH); either 2.5 hours or 4 days of hypobaric

hypoxia (0.4 ATM), or 1-4 days of alternating cycles of room air (normoxia)

served as controls. In experiments where the effect of reactive oxygen species

was examined, mice were given manganese (III) tetrakis (1-methyl-4-pyridyl)

porphyrin pentachloride (MnTMPyP; ALEXIS Biochemicals, CA, USA) at 5

mg/kg/day; I.P. MnTMPyP is a membrane-permeable superoxide dismutase

mimetic that traps superoxide anion radicals without generating H2O2. Control

and experimental mice were injected with vehicle (saline) or MnTMPyP every day

during the 4 days of CIH exposure. All experiments were performed within 8h of

terminating the final day of CIH exposure.

Adrenal medulla slice preparation and electrophysiological recording: Animals were anesthetized by isoflurane (Abbott Laboratories, Abbott Park, IL, USA) and sacrificed by decapitation. Adrenal glands were removed and 200 μm thick slices

were prepared for experimentation as previously described [106]. Adrenal tissue

slices were super-fused at 1 ml per minute with normal-Ca2+ BBS containing (in

mM): 140 NaCl, 2 KCl2, 3 CaCl2, 2 MgCl2, 26 NaHCO3, 10 Glucose and gassed

with 95% O2/5% CO2. Electrophysiological and capacitance measurements were

performed as previously described [107].

Western blot analysis of PKC levels in adrenal medulla: Adrenal glands were

collected from control, CIH, or CH treated mice. The medulla was dissected from

the cortex and placed in 150 μL protein extraction reagent (Pierce, Rockford IL,

USA) with 1.5 μL protease inhibitor cocktail (Roche Diagnostics, Mannheim,

Germany) and 0.150 μL phosphatase inhibitor cocktail (Calbiochem, La Jolla CA,

USA), homogenized and centrifuged at 10,000x g for 5 min. Protein

concentration of the homogenate was determined against BSA standards (Bio-

Rad, Hercules CA). Protein was subjected to gel electrophoresis (12% Tris-

Glycine Polyduramide gels; Cambrex, Rockland, ME, USA) and transferred to

nitrocellulose membrane. Membranes were blocked in TBS (20 mM Tris-buffered

saline, pH 7.6) containing 4% BSA and probed with a polyclonal anti-phospho-

pan-PKC antibody (Cell Signalling Tech. Inc. Danvers MA, USA) or with a

monoclonal anti β-actin antibody in TBS containing 4% BSA at 25o C overnight.

Protein bands were visualized by enhanced chemiluminescence (Peirce,

Rockford IL, USA).

Measurements of thiobarbituric acid reactive substances (TBARS): Medullary

tissue was homogenized in 10 volumes of 20 mM phosphate buffer (pH 7.4) at 4°

C and centrifuged at 500x g for 10 minutes at 4° C. TBARS were analyzed in

supernatant as described previously [38, 108]. Briefly, 100 μL of sample or the

calibration standard was added to 50 μL of 8.1% (W/V) SDS, 375 μL of 20%

(V/V) acetic acid and 375 μL of 0.8% (W/V) thiobarbituric acid. The samples were

heated for 60 min followed by incubation on ice for 10 min and centrifuged at

3,000x g for 15 min. The supernatant was removed and the absorbance of the

solution was monitored at 532 nm. Malondialdehyde (MDA) was used as a

standard, and the level of TBARS was reported in nmol of MDA formed per mg of

protein.

Data analysis: All data are expressed as means ± S.E.M. Statistical significance was evaluated by Student's unpaired t-test or one-way ANOVA for repeated

measures. P values less than 0.05 were considered significant.

2.3 RESULTS

Intermittent hypoxia increases the RRP in chromaffin cells. All

electrophysiological experiments were performed in freshly prepared adrenal

medullary slices because they allow measurements of chromaffin cell function in

the native cell context (including cell morphology and cell-cell contacts). Slices

also permit separation of chromaffin cell secretion from sympathetic input.

Regulation of the readily-releasable pool (RRP) of secretory granules represents

a key control point for setting the overall transmitter release from chromaffin

cells. The RRP dual-pulse depression protocol is based on a previous study in

isolated chromaffin cells [77] and has been used extensively to quantify the

number of granules resident at defined steps along the secretion path [85, 103,

109, 110]. Briefly, two 100 ms square pulse stimuli were delivered at a 100 ms

interval between pulses (Figure 2.1Ai). Cell membrane capacitance was

monitored to provide an index of granule fusion. The evoked capacitance

response for each pulse was measured and their magnitudes were used to

calculate the total number of release-ready granules. The first pulse causes

granule fusion and depletion of the RRP, leaving fewer granules for release

during to the second pulse. This results in a depletion-dependent secretory

depression in response to the second pulse. By measuring the ratio of the

second response to the first, the total size of the RRP can be calculated (see

Figure 2.1 legend and Gillis et al. (1996) for a full description of the technique). In

CIH exposed cells, the magnitude of the readily-releasable pool was greater than

the control chromaffin cells (Figure 2.1A). Average data of the time course of CIH

revealed that the RRP increases to a significant level by day 2 and remained

elevated on the 4th day of CIH exposure (* = P< 0.05). In contrast, CIH exposure decreases the magnitude of depolarization-evoked Ca2+ influx (Figure 2.1B), with significance reached after 3 days of CIH exposure.

Continuous hypoxia increases the RRP in chromaffin cells. To determine whether a comparable, cumulative duration of continuous hypoxia (CH) also increases

RRP, mice were exposed to 2.5 h of hypobaric hypoxia (0.4ATM), equivalent to the cumulative duration of hypoxia experienced during 4 days of CIH-treatment.

As shown in Figure 2.2, exposure to 2.5 h of CH affected neither cell capacitance

(Ai) nor Ca2+ influx (Aii). It is possible that a single exposure to CH may not be

adequate in evoking changes in RRP. Therefore, to test whether multiple

exposures to CH can induce hypoxic sensitivity, another group of mice were

exposed to 4 days of CH. Cells taken from mice exposed to CH for 4 days also

failed to show any difference in secretory capacity (RRP) or Ca2+ influx. These

results suggest that comparable cumulative duration of CH for either 2.5 hours or

4 days were ineffective in evoking changes in secretory behaviour as well as

Ca2+ influx in chromaffin cells (pooled data did not show significance and are not

shown).

Reactive oxygen species (ROS) scavenger prevents the CIH-induced increase in

the readily-releasable pool. CIH increases reactive oxygen species (ROS),

measured as thiobarbituric acid reactive substances (TBARS; Ramanathan et al.

2005), an index of increased protein oxidation. A recent study suggests that

ROS–mediated signalling plays a critical role in CIH-induced changes in adrenal

medullary secretion [38]. To assess the potential role of ROS in CIH-evoked changes in chromaffin cell secretory capacity, we monitored the TBARS levels in adrenal medullae harvested from CIH and normoxia exposed mice. As shown in

Figure 2.3A, TBARS levels were significantly elevated in CIH adrenal medullae

.- compared to controls. Systemic administration of MnTMPyP, a scavenger of O2 ,

(5 mg / 1 kg/ day/-I.P.) prevented elevations in TBARS in CIH exposed adrenal

medulla. MnTMPyP tends to decrease the size of the RRP in control cells

(p>0.05), whereas it completely blocked CIH-induced increase in RRP as well as

the decrease in evoked Ca2+ influx (Figure 2.3D; pooled data did not show

significance from control and are not shown). These results taken together

suggest that CIH elevates ROS in chromaffin cells and ROS-mediated signalling plays a role in CIH-evoked increase in RRP.

CIH increases the RRP through protein kinase C (PKC). It has been shown that

PKC represents a key regulatory element in setting the secretory capacity in chromaffin cells [77]. Furthermore, ROS has been shown to activate PKC [111].

We monitored phosphorylation of PKC at Thr514, which denotes catalytically

active enzyme [112], as an index of PKC activity. A representative Western blot

and pooled data are presented in Figure 2.4 A. CIH increased phospho-PKC

levels in adrenal medulla relative to control by 210% (p <0.01). The CIH-induced elevations in phospho-PKC were abolished by co-treatment with MnTMPyP

(Figure 2.4Aii). In contrast, CH for 4 days had no effect on the phosphorylation status of PKC (Figure 2.4Aii). Thus, these results suggest CIH leads to increased pan-PKC activation.

Figure 2.1: Intermittent hypoxia leads to an increased RRP. (Ai) Cells were voltage clamped at -80 mV and stimulated with a pair of 100 ms depolarizations. Cell capacitance (Cm) is proportional to the cell surface area, which increases with granule fusion and catecholamine release. Thus stimulus-evoked jumps in Cm provide an index of the number of granules fusing. In the example recording provided, the first pulse resulted in a greater capacitance increase than the

second pulse (Cm1 and Cm2 respectively). Formally, the RRP can thus be 2 quantified as RRP = S( 1-R ) where S = the sum of Cm1 and Cm2 and R is the

ratio of Cm2 to Cm1. It is assumed that a large fraction of catecholamine containing granules are released upon stimulation of the first pulse and that a fraction of the remaining fraction of granules is released upon the second pulse. These two capacitative jumps provide points from which the size of the RRP can be extrapolated. Only cells that exhibit strong depression (Cm2 is small compared to Cm1) provide an accurate estimate of the releasable pool size by the dual pulse method. For this reason only cells that exhibited a depression ratio

(Cm2/Cm1) of 0.7 or less were included for further analysis. Example capacitance records recorded from control (black trace) and 4-day CIH-exposed mice (gray trace) are provided. Evoked responses from CIH-exposed cells are larger than those recorded from control cells. (Aii) Data pooled from control and mice exposed to CIH for 1 to 4 days are shown (n = 5, 7, 9, and 12, respectively). This time course shows that the CIH-evoked increase in RRP reaches significance (* = p < 0.02) within 2 days. (Bi) Representative evoked currents during the dual- pulse protocol show that CIH-treatment causes decreased Ca2+ influx. (Bii) Pooled data (n = 12) show the time course of CIH-exposure on evoked Ca2+ influx and demonstrate that the decrease reaches significance (* = p < 0.02) by day 3.

Figure 2.2: Continuous hypoxia does not lead to an increased RRP. Mice were exposed to continuous hypoxia to either match the accumulated total time of exposure under CIH (CH – 2.5 hours) or to match the total days of exposure (CH – day 4). Tissue slices were prepared and the RRP was measured by dual-pulse excitation. (Ai) Representative examples of capacitance recordings as well as calcium currents (Aii) evoked from CH-2.5 hour treatment are provided and do not exhibit the enhanced capacitance response or decreased Ca2+ influx measured under the CIH day 4 condition. Likewise, evoked capacitance jumps (Bi) as well as calcium currents (Bii) under CH-day 4 also failed to display the facilitation measured in the CIH-treated mice. (C) Data pooled for all conditions (n = 12, 15, 7, 9) show that only cells taken from mice treated with intermittent hypoxia (CIH – d4) show a significant difference in the size of the RRP as assayed by dual pulse stimulation (* = p < 0.02).

Figure 2.3: Superoxide dismutase mimetic blocks the CIH-dependent increase in RRP. General oxidative status of cells was determined by a TBARS assay. (A) TBARS was measured in control adrenal medullary tissue and in CIH-d4 tissues with and without concurrent injection with superoxide dismutase mimetic (MnTMPyP). Pooled data (n = 3, 5 and 5) show that administration of MnTMPyP blocks the increased oxidative stress observed in CIH-d4 treated mice. (B) Likewise, representative recordings show that co-treatment with MnTMPyP blocked the CIH-d4-mediated increase in evoked capacitance jumps. (C) Pooled data show that MnTMPyP treatment decreased the RRP measured in control cells. Though this failed to reach significance, it may indicate a small basal level of ROS-mediated signalling even in the control recording condition. MnTMPyP co-treatment did abolish the significant CIH-dependent increase in RRP from CIH-d4 cells (n = 7, 5, 7 and 6 respectively). (D) MnTMPyP treatment also reversed the CIH-dependent decrease in evoked calcium influx.

Next, we examined whether PKC inhibition impacted the CIH-induced increase in

chromaffin cell RRP. We tested both Ro-31-8220 (100 nM) and Gö 6983 (100

nM), members of the bisindolylmaleimide family of PKC blockers. Both PKC

inhibitors provided identical results. Therefore the data were pooled. As shown in

Figure 2.4Bi, pharmacological blockade of PKC resulted in smaller evoked

capacitance responses in CIH-treated cells. Likewise, PKC inhibitors restored

Ca2+ influx in CIH-treated cells to control levels. Furthermore, phorbol 12-

myristate 13-acetate (100nM; PMA), a potent activator of PKC, increased the

RRP in control chromaffin cells to a level comparable with that seen in CIH-

exposed cells (Figure 2.4 Bii). Taken together, these data indicate that CIH- treatment alters stimulus-secretion function in chromaffin cells through a ROS- mediated regulation of PKC. Here we show that local application of PKC inhibitor is able to attenuate the CIH induced capacitative jumps suggesting that CIH-4d

exposure results in an increase in PKC as well as phospho-PKC. By blocking

PKC during the in situ studies we are assuming that activated PKC plays a role in

recruiting vesicles to the RRP and that release of these vesicles is a PKC

dependent process and can be attenuated by blockade of PKC at the point of

stimulation rather than during CIH exposure. Additional studies are needed to

address the role of PKC blockade under CIH exposure, similar to that of SODM

administration.

Figure 2.4: The CIH-d4-mediated increase in the RRP is due to an increased PKC phosphorylation. (Ai) Adrenal tissue collected from control and CIH-d4 mice were evaluated for relative levels of phospho-PKC with a pan-phospho-PKC polyclonal antibody. β-actin served as a control for protein loading. (ii) Phospho- PKC levels were quantified from 3 blots by densitometry and show that CIH-d4 treatment resulted in an approximate 210% increase in total PKC phosphorylation over control levels whereas the Phospho-PKC levels in CIH-d4+ MnTMPyP and CH showed no change. (Bi) Representative recordings show that treatment with protein kinase inhibitors blocks the CIH-d4-mediated augmentation of evoked-capacitance jumps. (Bii) Pooled data from control and CIH-exposed cells with and without 5-minute bath applied pre-treatment with PKC inhibitors (n=5, 12 and 7 respectively) show that block of PKC restores Ca2+ influx to control levels. (C) Quantified data (n=5, 5, 12 and 7 respectively) show that treating control cells with PMA (100 nM) causes the RRP to grow in magnitude to match that observed in CIH-d4-treated chromaffin cells (“*” denotes significance at p < 0.02). Likewise, block of PKC in CIH-d4 cells causes the RRP to shrink in size to match that measured in control cells.

2.4 DISCUSSION

The amount of catecholamine release from adrenal medullae is, in part, set by

regulating the number of release-competent granules; a population that

comprises the ‘ready-releasable pool’ (RRP; [102]). The present results

demonstrate that CIH increases the RRP in adrenal chromaffin cells. The effects

of CIH on RRP were time-dependent in that one day exposure was ineffective

and required a minimum of two days of CIH. Previous work demonstrated that

PKC plays a fundamental role in regulation of secretory capacity by increasing

the rate of granule recruitment to the RRP [85]. The following lines of evidence

suggest CIH-evoked increase in RRP requires PKC activation: 1) CIH increased

the phosphorylation of PKC at Thr514, which generates catalytically active

enzyme [112] and 2) PKC inhibitors prevented the effects of CIH on RRP. In striking contrast, continuous hypoxia (CH) either for 2.5h (equivalent to hypoxic

duration accumulated over 4d of CIH) or for 4 days did not increase RRP. These

findings are consistent with a previous report that CIH, but not CH augments

catecholamine secretion from adrenal medulla in rats [38]. Unlike CIH, CH did

not result in increased PKC activation as evidenced by the probe of PKC

phosphorylation at Thr514. Therefore, the absence of RRP increase by CH is

conceivably due to its inability to activate PKC. Several isoforms of PKC have

been identified. However, which of the PKC isoforms are affected by CIH and

how they contribute to alteration in RRP requires further investigation.

Our results provide evidence for the involvement of ROS-mediated signalling in

CIH-evoked increase in the secretory capacity of adrenal chromaffin cells. First,

.- MnTMPyP, a scavenger of O2 anions prevented CIH-evoked increase in RRP.

Second, CIH increased TBARS levels in the adrenal medulla. Third, MnTMPyP,

.- a membrane permeable scavenger of O2 anions prevented CIH-induced

increases in TBARS, suggesting that elevated ROS leads to changes in TBARS.

How might ROS contribute to increases in RRP by CIH? ROS is a potent

activator of PKC [113, 114]. Because CIH resulted in PKC activation and PKC

inhibitors prevented increases in CIH-evoked increases in RRP, we suggest that

ROS mediates CIH-induced PKC activation. Further studies, however, are needed to establish that CIH-induced increase in PKC is a result of direct activation of PKC by ROS or concomitant inhibition of phosphatases, or both.

Chromaffin cells of the adrenal medulla release catecholamines through Ca2+-

mediated fusion of secretory granules with the cell surface. Several groups

document that PKC activation decreases depolarization-evoked Ca2+ influx in

chromaffin cells [77, 115]. This is further demonstrated in our data showing that

CIH attenuated the stimulus-evoked Ca2+ currents in chromaffin cells. PKC

inhibitors restored CIH-dependent decrease in stimulus-evoked Ca2+ influx. Ca2+

plays multiple roles in the regulation of secretion from chromaffin cells. Not only

does it act to trigger the final step of granule fusion and transmitter release, it

also regulates steps upstream from the final fusion event. In order to sustain

prolonged catecholamine release, chromaffin cells must recruit reserve granules

to the RRP. This recruitment has been shown to be dependent on Ca2+ [104]

through PKC-dependent and independent mechanisms [85], thus any

perturbation to the influx of Ca2+ may act to alter secretory granule recruitment to

the RRP. It is likely that CIH acts through a mechanism that is either insensitive

to decreased Ca2+ influx or overcomes this potential limitation. Alternatively, CIH may act to increase the RRP by elevating cytosolic Ca2+ levels independent of

voltage-gated calcium entry. For example, CIH may act to increase cytosolic Ca2+

by decreasing the extrusion of Ca2+ through Na+/Ca2+ exchange. Further experimentation is required to determine the effects of CIH on cell calcium mobilization and to determine its potential influence on the stimulus-secretion function of the adrenal medulla.

In summary, the present study demonstrates that CIH increases RRP in adrenal chromaffin cells via ROS-mediated activation of PKC. Previous studies on rodents have shown that CIH results in elevated circulating catecholamines, which involves increased sympathetic activity and subsequent secretion from adrenal medulla [101]. The present results suggest that CIH can directly affect the secretory capacity of chromaffin cells and contribute, in part, to the sustained elevated catecholamine levels, independent of sympathetic stimulation.

Chapter 3:

PACAP Regulates Immediate

Catecholamine Release from Adrenal

Chromaffin Cells in an Activity Dependent

Manner through a Kinase-C Dependent

Pathway

BA Kuri, Chan SA, Smith CB. J Neurochemistry 2009 Aug; 110(4):1214-25.

3.1 INTRODUCTION

The body responds to stress by increasing sympathetic firing. A major

consequence of heightened sympathetic activity is increased catecholamine

release from the adrenal medulla into the general circulation. Sympathetic control

of the adrenal medulla takes place at cholinergic synapses between innervating

splanchnic neurons and neurosecretory chromaffin cells [6]. Acetylcholine

released from the presynaptic terminal binds to postsynaptic ionotropic nicotinic

acetylcholine receptors (nAChR) on the chromaffin cells. Ligand-gated

conductance through the nAChR leads to chromaffin cell depolarization, firing of

an action potential and opening of voltage operated calcium channels. Resultant

increases in cytosolic Ca2+ lead to fusion of transmitter-containing large dense core secretory granules and exocytosis of their transmitter cargoes into the bloodstream [1, 6]. It has been shown, however, that this cholinergic response rapidly desensitizes at multiple stages along the exocytic pathway and that frequent stimulation or exposure to maintained levels of ACh at the sympathoadrenal synapse results in a decreased efficiency of evoked catecholamine release from chromaffin cells [1, 116, 117]. Physiologically, however, it is known that under the acute stress response, or heightened splanchnic firing, catecholamine release persists and elevated serum catecholamine levels are observed [9, 29, 38, 118, 119]. Thus, a secondary synaptic excitation mechanism other than ACh is likely to exist.

Pituitary Adenylate Cyclase Activating Peptide (PACAP) is a member of the secretin family and is implicated in regulating the release of many peptide

transmitters from different secretory cells, including insulin from pancreatic cells,

thyroid stimulating hormone (TSH) in the pituitary, and renin from granular cells

in the kidney [120-124]. PACAP is also a known secretagogue for catecholamine

release from adrenal chromaffin cells [125-130]. Splanchnic nerve terminals in the adrenal medulla have been shown to contain PACAP while chromaffin cells express the high affinity PACAP receptor, PACR-1 [126]. Previous studies have shown that prolonged (20 min) PACAP exposure results in a robust persistent catecholamine release from clonal PC-12 cells [131] and isolated adrenal chromaffin cells [129, 130, 132]. As indicated by the name, PACAP exposure is thought to evoke catecholamine release through activation of adenylate cyclase, subsequent cyclic adenosine monophosphate (cAMP) generation and activation of protein kinase A (PKA) [133, 134]. However, there are conflicting data describing the complete mechanism of action by which PACAP elicits its secretory response. Work in the PC-12 cell line showed 20 minute exposure to

PACAP elicits catecholamine secretion that is sensitive to inhibition of phospholipase C (PLC) and evokes Ca2+ influx through a mechanism that does

not include L-type channels [131]. Additional studies performed in primary

cultures from rat adrenal medulla have shown PACAP stimulation to directly

inhibit L-type channel activation [135] while other studies have shown that

PACAP causes an increased L-type conductance [136]. Furthermore, PACAP- evoked Ca2+ influx has been shown to depend upon external Na+ and Na+ influx through a route that is not sensitive to blockers of voltage gated Na+ channels

[137, 138]. These data suggest an alternate route of Na+ influx independent of

action potentials but that may result in depolarization. Other studies point to

PACAP-mediated Ca2+ release from internal stores to evoke secretion [134].

Lastly, studies conducted in isolated rat cells or PC-12 cells indicate PACAP acts as an acute secretagogue eliciting exocytosis within seconds through a pathway that requires Ca2+ influx independent of the major high voltage activated Ca2+

channels [128, 131]. Thus, conflicting interpretations of the action of PACAP in

the adrenal medulla exist in the literature.

We set out to study the physiological response to native PACAP release on

adrenal chromaffin cells in situ. We directly stimulated the innervating splanchnic

nerve to elicit catecholamine release. The nerve was paced at low frequencies

designed to match sympathetic tone and with higher frequency bursts designed

to match the sympathetic stress response. We provide data that demonstrate

PACAP acts as a native chromaffin cell secretagogue specifically under

sympathetic burst mode firing. We further show that PACAP-dependent secretion

does not depend on PKA signaling. Rather it evokes a rapid activation of PLC,

protein kinase C (PKC) and activation of the Na+/Ca2+ exchanger (NCX) to

depolarize the cell membrane. Membrane depolarization evokes Ca2+ influx

through a NiCl2 and mibefradil-sensitive conductance to evoke a sustained catecholamine secretion. We place this signaling mechanism in the context of the current literature summarized above and propose a single mechanism for

PACAP-evoked secretion. We provide data that potentially reconcile both the

PACAP-mediated cAMP and PKC signaling paths described in the literature.

Exchange Proteins Activated directly by cAMP (Epac) has been shown to be activated by PACAP [139] and has been shown to activate PKC [140]. We

provide direct data demonstrating that Epac activation elicits secretion from

chromaffin cells nd is blocked by NiCl2 in same manner that that NiCl2 blocks

PACAP-dependent secretion.

*The work from this manuscript was primarily performed by BA Kuri. SA Chan is

responsible for the Ni2+-sensitive current isolation and current clamp recordings in

adrenal tissue slices demonstrated in Figure 3.5.

3.2 MATERIALS AND METHODS

Slice preparation: Mouse adrenal tissue slices were prepared for experimentation as previously described [106]. Adult C57BL/6 mice (8-10 wk old; Jackson

Laboratories, Bar Harbor, ME) were used in this study. All anesthesia and euthanasia protocols were reviewed and approved by the institutional animal care and use committee (IACUC) of Case Western Reserve University, an accredited oversight body (Federal animal welfare assurance No. A3145-01).

Briefly, animals were deeply anesthetized by isoflurane inhalation (Abbott

Laboratories, Abbott Park, IL) and sacrificed by decapitation. Adrenal glands

were immediately removed and placed in ice-cold, low calcium bicarbonate

buffered saline (BBS) containing in mM: 140 NaCl, 2 KCl, 0.1 CaCl2, 5 MgCl2, 26

NaHCO3, and 10 glucose that was bubbled with 95% O2/ 5% CO2. Osmolarity of

the BBS was 320 mOsm. All chemicals were acquired from Fisher Scientific

(Hanover Park, IL) unless otherwise noted. The adrenal glands were trimmed for

excess fat and connective tissue and embedded in 3% low gelling point agarose

(Sigma, St. Louis, MO) that was prepared by melting agar in low calcium BBS at

110o C followed by equilibration to 35o C. The agarose block containing the

adrenal glands was trimmed into 3 mm cubes and glued to a tissue stand of a

vibratome (WPI, Sarasota, FL). The adrenal glands were sectioned into 200 µm

slices in ice-cold bubbled BBS. Sections were cut along the major axis and

parallel to the maximum width to preserve native splanchnic innervation for in situ

stimulation.

Immunohistochemistry: Adrenal glands were removed and prepared as previously described. The adrenal glands were immersion fixed by placement in

2% phosphate buffered paraformaldehyde (PFA) solution for 2 hours at 4oC.

They were post-fixed and stored overnight in 30% sucrose solution containing

2% PFA. The glands were embedded in OCT (Ted Pella, Inc., Redding, CA),

cryosectioned into 16 µm sections and mounted on slides. To probe for PACAP

or the PACR-1 receptor, the adrenal sections were washed three times with PBS

and blocked with a solution of 10% donkey serum in PBS containing 0.1% Triton

X-100 for 20 minutes at room temperature. The sections were incubated with

goat-anti PACAP (1:50) and rabbit-anti PACR-1 (1:50) (Santa Cruz

Biotechnology, Inc. Santa Cruz, CA) overnight at 4oC. The sections were washed

six times each with PBS and incubated with donkey-anti goat-Alexa 498 (1:100)

and donkey anti-rabbit-Alexa 594 (1:100; Invitrogen, Carlsbad, CA) for 1 hour at

room temperature. Sections were washed six times with PBS and mounted with

immuno-mount (Thermo Scientific, Pittsburgh, PA) under a cover slip.

Fluorescence signals were visualized on an Olympus IX81 microscope at 100x

magnification. Image stacks were captured at pixel-width z steps (square voxels) and deconvolved with a constrained-iterative algorithm built into SlideBook

(Intelligent Imaging Innovations, Denver, CO) image processing software.

Electrophysiology: Adrenal chromaffin slices were visualized using an upright microscope (Olympus, Melville, NY) equipped with a 40x water-immersion objective. Slices were held in place by sliver wires placed over the agar margin

and constantly superfused (1 ml/minute) with normal Ca2+ BBS containing in mM:

140 NaCl, 2 KCl, 2 MgCl2, 26 NaHCO3, 10 glucose, 3 CaCl2 bubbled with 95%

O2 / 5% CO2. Recordings were performed in the perforated patch configuration of

the patch-clamp technique. Patch pipettes were pulled from borosilicate glass (4-

5 ΜΩ). They were partially coated with molten dental wax and polished by a

microforge (Narashige, Tokyo, Japan). The perforated patch pipette solution

used for voltage clamp contained in mM: 135 CsGlutamate, 10 HEPES-H, 9.5

NaCl, 0.5 TEA-Cl, and 0.53 amphotericin B. The pH was adjusted to 7.3 and

osmolarity was adjusted with mannitol to 320 mOsm. The solution for current clamp contained in mM: 145 potassium glutamate, 10 HEPES-H, 8 NaCl, 2

MgATP, 1 MgCl2, 0.1 EGTA and 0.53 amphotericin B. The amphotericin was

prepared as a 100x stock solution in DMSO (Sigma, St. Louis, MO) and diluted

into the pipette solution. The pipette was backfilled without tip-dipping. Cells were

allowed to perforate to a series resistance of no more than 30 ΜΩ and held at -80

mV. For current clamp configuration, series resistance was compensated at 80%

and 100 μs. Records were acquired at 20 kHz through an EPC-9 voltage-clamp

amplifier (HEKA Elektronik, Lambrecht, Germany) under the control of Pulse

software (v 8.40, HEKA Elektronik). In the I-V protocol, cells were held at -100

mV and depolarized to potentials between -80 and 15 mV in 5 mV increments.

Each depolarization was 100 ms in duration and separated from the previous by

45 s to allow for complete recovery from inactivation. The peak Ni2+ sensitive

Ca2+ conductance was isolated by the subtraction of currents recorded in the above recording BBS containing 50 µM NiCl2 from currents recorded in the

absence of NiCl2. All records were corrected for junction potential. Data were

analyzed using IGOR Pro (Wavemetrics, Lake Oswego, OR).

Bipolar electrical stimulation: The splanchnic nerve was stimulated by a parallel

bipolar stimulator fitted with two platinum electrodes (250 µm spacing) (FHC,

Bowdoin, ME) and connected to an ISO-Stim 01-D stimulator (ALA Scientific

Instruments, Westbury, NY) and controlled by TTL triggers generated by the

EPC-9 through the Pulse software. The adrenal gland slice was placed between the two electrodes and visualized using an upright microscope (Olympus,

Melville, NY) equipped with a 40x water-immersion objective. The innervating splanchnic nerve was stimulated to mimic either basal tone firing conditions or stress firing conditions. To compare relative cholinergic and PACAP-evoked secretion, stimulation protocols were balanced to evoke quantitatively identical amounts of catecholamine under cholinergic control (i.e. blocking the PACAP signaling path under stress firing elicits the same nicotinic component as elicited by basal stimulation; see Figure 2C). Under basal stimulation the nerve was stimulated for 10 pulses at a frequency of 0.2 Hz. For stress stimulation, 4 bursts of 15 pulses were delivered at 2 Hz frequency with an inter-burst interval of 15 s.

All stimuli were delivered at constant voltage (35 V) with 10 µs duration.

Amperometry: Carbon fiber electrodes with 5 µm diameter tips were used for amperometric detection of catecholamine release (ALA scientific, Westbury, NY,

USA). Fibers were cut fresh daily and again whenever debris accumulated on the

tip. A +650 mV potential was placed on the fiber. After placement into the bath,

the initial fiber oxidation current was allowed to relax to a steady value. For

recordings, the fiber was placed as close to the cell of interest without physically

distorting the cell. PACAP was puffed on the slice with a micro-perfusion system

(Warner Instruments; Hamden, CT). Oxidative amperometric currents, indicating

catecholamine release, were recorded using a dedicated VA-10 amperometry

amplifier (ALA Scientific). Experiments were conducted primarily with either a

500 MOhm or 1 GOhm feedback head stage and currents were calibrated prior

to pooling. The current was filtered at 1.5 kHz and sampled at 20 kHz. All

pharmacological blockers used to isolate the PACAP signaling pathway were

added to the BBS and were puffed on the slice for 5 minutes prior to, and during

PACAP stimulation. Acute PACAP-evoked catecholamine secretion was

determined as the integrated amperometric current 60 s after PACAP exposure.

The concentration of pharmacological blockers is as follows: U73122 (10 µM),

U73433 (10 μM), Gö6983 (100 nM), Benza mil (10 µM), NiCl2 (50 µM), 8-pCPT-

2’-O-Me-cAMP (100 μM). The Ca2+-free BBS was prepared as above with an

equimolar substitution of MgCl2 for CaCl2.

FURA recording: Intracellular Ca2+ dynamics were measured in cells loaded with the calcium indicator Fura-2 penta-acetoxymethyl ester (Fura-2 AM) (Molecular

Probes, Eugene, OR). Isolated chromaffin cells were prepared as described in

the literature [141] and incubated in 2 µM FURA (50 µg Fura2-AM was dissolved

in DMSO and diluted to 1 mM) at 37oC for 15 minutes. After loading, the cells

were washed for 5 minutes by superfusion with HEPES-buffered Ringer solution containing in mM: 150 NaCl, 10 HEPES, 10 glucose, 2.8 CaCl2, 2.8 KCl, 2

MgCl2, pH 7.4. Cells were visualized with an inverted microscope (Olympus IX81 microscope; Tokyo Japan) with a UV-optimized 40x water-immersion objective.

Ratiometric measurements of intracellular Ca2+ were recorded by FURA dye

excitation with light alternating between 360 nm and 390 nm using a fast

monochromator-based system (TILL Photonics, Eugene OR). Emission was

measured at >510 nm. All fluorescence signals were recorded in the Pulse X-

Chart program and analyzed in IGOR.

3.3 RESULTS

PACAP has been shown to act as both a both a neuromodulator and a secretagogue for catecholamine secretion from adrenal chromaffin cells.

Heterogeneous effects of PACAP exposure may be encoded by the multi-potent

HOP cassette of the PACR1 receptor that activates both Gs and Gq-mediated

signaling cascades [131, 133, 137]. Much attention has been focused on

understanding the long term modulatory effects of exogenous PACAP exposure

while the mechanism responsible for the direct secretagogic action is not yet

resolved. Evidence points to a variety of potential mechanisms for acute PACAP-

evoked Ca2+-dependent secretion including influx through high voltage activated

(HVA) L-type Ca2+ channels [136], influx through non HVA Ca2+ channels [128]

and release from intracellular stores [131]. In order to understand which of these mechanisms, if any, are responsible for physiological catecholamine release under the sympathetic stress response, we initiated a series of experiments in an acute tissue slice preparation from mouse adrenal medulla. We introduce a native splanchnic-chromaffin stimulation method to show for the first time that splanchnic-chromaffin signaling under elevated burst-mode sympathetic firing occurs through a largely PACAP-dependent mechanism. We test a variety of signaling paths potentially responsible for the secretion in situ. We present data defining a mechanism that includes activation of PLC, PKC, Na+/Ca2+ exchange

and Ca2+ influx through a voltage-dependent, nickel and mibefradil-sensitive

conductance to evoke catecholamine release.

PACAP is localized in the splanchnic synaptic terminals: The experimental

configuration is shown in Figure 3.1A. A bright-field image (left) of the in situ slice

preparation with a 5 μm amperometric carbon fiber electrode placed near the

chromaffin cell to measure catecholamine release. Immunohistochemistry (right)

shows that PACAP, stained green, is located peripheral to the chromaffin cells,

but not in the chromaffin cells themselves. These data are consistent with the

literature [53, 126], showing PACAP expression in the innervating splanchnic

terminals. The high affinity PACAP PACR-1 receptor, stained red, is expressed

over the surface of the chromaffin cells [53, 142, 143]. Next, we employed single cell carbon fiber amperometry to directly measure catecholamine release under

PACAP stimulation. Carbon fiber amperometry has been a valuable tool to measure catecholamine release on the quantal level [93, 144]. Briefly, a carbon fiber is placed near a cell and held at a constant positive potential (+650 mV). As catecholamine molecules are released from the cell, they are oxidized in the local electric field near the fiber tip, resulting in a current in the fiber. A representative amperometric recording shows that local application of exogenous PACAP (1

μM) results in an immediate and robust signal (Figure 3.1B). Each spike represents the fusion of a single catecholamine-containing granule. In order to compare PACAP-evoked secretion to intracellular calcium dynamics, we measured relative intracellular Ca2+ in freshly isolated FURA2-AM loaded cells

stimulated with 1 μM exogenous PACAP. Relative Ca2+ levels are reported as

ΔF/F0 (see methods for recording and analysis). As demonstrated in the

representative recording (Figure 3.1C), PACAP evokes a rapid and long-lasting

elevation in cytosolic Ca2+. Thus, based on immunostaining, we confirm that

PACAP is found peripheral to chromaffin cells and that chromaffin cells express

PACR1 receptors. Additionally, chromaffin cells exhibit a rapid secretory

response to focal PACAP stimulation in situ.

Figure 3.1: PACAP stimulation of adrenal chromaffin cells results in an immediate and robust release of catecholamine. (A) Left image: A bright-field image of the in situ adrenal slice preparation demonstrates the amperometric recording configuration used to measure catecholamine release from chromaffin cells. Right image: Immunohistochemical staining shows that PACAP (green) is localized peripheral to chromaffin cells. The PACAP specific receptor (PACR-1, red) is expressed in chromaffin cells. (scale = 10 μm) (B) An amperometric trace recorded from a chromaffin cell in situ that has been stimulated by focal perfusion with a Ringer containing 1µM PACAP causes an immediate catecholamine exocytosis. (C) Ratiometric FURA signal from a freshly isolated cell demonstrates an immediate rise in intracellular Ca2+ upon PACAP stimulation.

PACAP specifically evokes catecholamine release under elevated sympathetic

input: As demonstrated in Figure 3.1, PACAP is localized peripheral to single

chromaffin cells in the medulla; chromaffin cells express the PACR1 receptor.

PACAP stimulation causes a rapid elevation in cytosolic Ca2+ and subsequent

exocytosis of catecholamine. Yet, no studies to date have demonstrated that

endogenous splanchnic PACAP release stimulates chromaffin cells. We

developed an intact in situ splanchnic-adrenal preparation to deliver native synaptic stimulation to single chromaffin cells. Briefly, tissue slices were cut and placed on the microscope recording chamber. A platinum bipolar stimulator was placed peripheral to the medulla and vertically spanned across the adrenal cortex. Low frequency pulses were delivered to the bipolar stimulator while a carbon fiber electrode was moved within the proximal adrenal medulla until a responsive chromaffin cell was identified. After establishing functional neuronal-

chromaffin cell coupling, the nerve was pulsed at either a low periodic frequency

to match sympathetic tone or at a higher frequency bursting pattern to mimic

sympathetic activation (see Methods). Cumulative stimulus-evoked catecholamine release from the cell was quantified by integrating the raw amperometric current. Example integral traces for basal and stress firing stimulus patterns are provided in Figure 3.2A. Basal firing elicited a modest catecholamine release compared to burst mode firing. To determine PACAP-dependent secretion under each condition, we bathed slices in the competitive PACAP inhibitor PACAP(6-38) (10 µM), a PACAP receptor antagonist [145].

Pretreatment with PACAP(6-38) did not affect the total catecholamine release

71 under basal stimulation but attenuated release under burst mode stimulation to the level observed under basal stimulation (Figure 3.2B). These data show that

PACAP acts as a transmitter under the bursting firing pattern. Furthermore, the data show that PACAP represents the magnitude difference between catecholamine secretion under conditions designed to match basal sympathetic tone and those designed to match the sympathetic stress response. Combined data from each of the above stimulation protocols were combined and are plotted

(Figure 3.2C). These data show that PACAP is the predominant synaptic transmitter under burst mode splanchnic firing.

Figure 3.2: PACAP evokes catecholamine release under elevated sympathetic input. (A) Bipolar stimulation of the splanchnic nerve (indicated by the black bar above the sample recording) evokes catecholamine release from chromaffin cells. Sample traces for total catecholamine release (integrated amperometric current) under firing rates that mimic basal sympathetic firing (dotted line) and bursting stress firing patterns (dashed line). These sample traces indicate that elevated frequency burst mode firing elicits greater catecholamine release than basal firing. Inlaid are raw amperometric recordings of the basal (bottom) and burst mode (top) splanchnic stimulations. Individual amperometric spikes are recorded as well as a change in amperometric baseline is observed. The rise in baseline is indicative of catecholamine release that must diffuse through epithelial cells of the vasculature, connective tissue as well as cellular debris (as discussed in chapter 1) before being oxidized by the carbon fiber. (B) Catecholamine release as a function of PACAP signaling was measured under basal and burst mode firing. Inhibition of the PACR1 receptor by bath application of the PACAP antagonist, PACAP (6-38), decreased catecholamine release under burst mode firing but not under basal firing patterns. (C) Total catecholamine release was measured and pooled for each protocol indicated in panels A and B and are plotted (n = 11, 10, 4 and 4, respectively). The data show that the PACAP-mediated catecholamine release occurs under burst mode firing, but not under basal firing conditions (* = p < 0.02; paired Student’s t-test).

PACAP stimulation acts through a PLC and PKC dependent process: The mechanism by which PACAP acutely elicits catecholamine release is unclear.

Our effort to define the mechanism responsible for catecholamine release in situ

initially focused on whether PACAP-evoked Ca2+ signals originate from release

from internal stores or through influx from the extracellular space. Examples

collected under diverse stimulation protocols for each route exist in the literature

[146, 147]. In this set of experiments we chose to stimulate cells through focal

superfusion with exogenous PACAP (1 μM) to isolate its specific mechanism

independent from other presynaptic transmitters (i.e. acetylcholine, adenosine and Ca2+). To test for the requirement of external Ca2+, we bathed tissue slices in

a Ca2+-deficient Ringer solution and stimulated with PACAP. As in Figure 3.1,

catecholamine secretion was measured by carbon fiber amperometry and

quantified by integrating the amperometric current. The top left panel of Figure

3.3A shows a rapid and robust catecholamine release in response to stimulation

under control conditions. The top right plot shows that Ca2+-free Ringer did not support PACAP-evoked catecholamine release. In paired experiments, PACAP-

evoked cytosolic Ca2+ increases were also shown to be dependent on

extracellular Ca2+ (Figure 3.4). Thus, acute PACAP stimulation requires

extracellular Ca2+ for catecholamine exocytosis.

Next, we considered the route of PACAP-evoked Ca2+ entry. Previous studies provide a variety of signaling cascades initiated by PACAP excitation, as would be expected through the multi-potent HOP cassette of the PACR1 receptor. First,

we considered PKA-mediated modulation of L-type HVA calcium conductance

described in clonal PC-12 cells [131]. We challenged cells with pharmacological inhibitors that target this pathway. We pre-incubated slices in H-89 (1 μM), a PKA inhibitor, and in nifedipine (1 μM), an L-type channel blocker. Neither of these reagents had any effect on acute PACAP-dependent catecholamine release

(data not shown). We then considered the prospect of a phospholipase C- dependent PACR1-mediated pathway being responsible for secretion [131, 135].

Cells were pre-treated with the pharmacological PLC blocker, U73122 (10 µM).

Pre-treatment with the active U73122 PLC-blocker completely inhibited

catecholamine release in situ upon PACAP stimulation (Figure. 3.3A, lower left

plot). The inactive U73122 analog, U73433 (10 μM), had no significant effect on

PACAP secretion compared to PACAP stimulated untreated control cells (97.8 ±

20.7 pC [n=9] for U73433 + PACAP, 140.3 ± 46.5 pC [n=10] for PACAP alone

and 9.1 ± 3.1 pC for U73122 + PACAP [n=10]). Again, in paired experiments,

Ca2+ transients measured in freshly isolated cells showed the same behavior,

U73122 pretreatment blocked PACAP-evoked Ca2+ signals (Figure 3.4). Thus,

acute stimulation with PACAP in situ elicits catecholamine secretion through a

PLC-dependent pathway.

Figure 3.3: PACAP-dependent secretion is dependent on extracellular Ca2+ and blocked by PLC and PKC inhibition. (A) Carbon fiber amperometry was used to measure total catecholamine release (integral of the amperometric current) from single chromaffin cells in situ. Cells were stimulated by focal perfusion with PACAP (1 μM, as indicated by the bar above each sample recording). Sample single recordings for PACAP stimulation alone (top left) and PACAP in the presence of a Ca2+-free Ringer (top right), PACAP in the presence of the PLC inhibitor U73122 (10 μM, bottom left) and PACAP in the presence of the PKC inhibitor Gö6983 (100 nM, bottom right). (B) Pooled data from 10 experiments for 2+ each condition shown in panel A demonstrate that extracellular Ca , PLC, and PKC, are all necessary components of the immediate PACAP secretory mechanism in adrenal chromaffin cells (* = p < 0.02; paired Student’s t-test).

Figure 3.4: PACAP-dependent Ca2+ signals are dependent on extracellular Ca2+ and blocked by PLC and PKC inhibition. (A) Cells were loaded with FURA-2AM and relative intracellular Ca2+ was measured at 360 and 390 nm excitation as indicated in Methods. Cells were stimulated by focal perfusion with a Ringer with PACAP (1 μM, as indicated by the bar above each sample recording). Sample single traces of time-resolved FURA ratios are plotted. Single representative recordings for PACAP stimulation alone (top left) and PACAP in the presence of a Ca2+-free Ringer (top right), PACAP in the presence of the PLC inhibitor U73122 (10 μM, bottom left) and PACAP in the presence of the PKC inhibitor Gö6983 (100 nM, bottom right). (B) Pooled data from 10 experiments for each 2+ condition shown in panel A show that extracellular Ca , PLC, and PKC, are all necessary components of the immediate PACAP-dependent cytosolic Ca2+ elevations (* = p < 0.02; paired Student’s t-test).

PLC activity results in generation of two second messenger molecules, inositol

2+ triphosphate (IP3) to release intracellular Ca stores and diacylglycerol (DAG) to

activate protein kinase C (PKC). PACAP-evoked secretion requires influx of

2+ external Ca , thus IP3-mediated release of internal stores does not significantly

contribute to the immediate catecholamine secretion. Moreover, previous studies

2+ have shown that PACAP-evoked Ca mobilization is not due to IP3 signaling, but

is dependent on PKC signaling [138]. For this reason we tested DAG-mediated activation of PKC as an effector for secretion. We pretreated tissue slices with

Gö6983 (100 nM), a blocker of both classical and novel PKC isoforms, and then stimulated by focal PACAP perfusion. PACAP-dependent catecholamine release was blocked by Gö6983 pretreatment (lower right plot, Figure 3.3A). As above, paired experiments showed that Gö6983 treatment had a similar effect of blocking evoked Ca2+ signals in FURA2-loaded cells (Figure 3.4). Combined data

from each of the above protocols were pooled and are presented in Figure 3.3B

and show that the immediate PACAP-dependent catecholamine secretion in situ

is blocked by PLC and PKC inhibition and requires extracellular Ca2+. In order to

confirm that native synaptic PACAP signaling also included a PKC-mediated

signaling mechanism, we repeated the basal and burst mode firing patterns

delivered by direct bipolar stimulation presented in Figure 3.2 in the presence of

Gö6983. Data from these experiments confirmed that elevated catecholamine release under burst mode firing is sensitive to PKC inhibition (143.4 ± 40.1 pC

[n=10] versus 46.3 ± 10.0 pC [n=4], untreated cells versus Gö6983-treated cells respectively).

PACAP stimulation depolarizes the cell but does not fire an action potential:

PACAP exposure has been shown to cause a sub-threshold membrane

depolarization that is dependent upon external Na+ [138]. We tested if PACAP stimulation of chromaffin cells in situ expressed the same behavior. We held chromaffin cells in perforated patch current clamp and focally perfused 1 μM

PACAP. A representative recording is provided in Figure 3.5A and demonstrates

a sub-threshold depolarization upon PACAP exposure (+12.4 ± 2.8 mV from a

resting potential of -69.6 ± 1.9 mV; n= 7). Next, we tested the possibility that this sub-threshold depolarization may evoke the influx of Ca2+ through activation of

low voltage activated (LVA) Ca2+ channels.

Zinc selectively inhibits currents through LVA T-type Ca2+ channels relative to currents through HVA channels [148, 149]. One study noted a Zinc sensitivity for

PACAP-evoked Ca2+ signaling, with Zn2+ being more effective than other Ca2+

channel blockers at inhibiting PACAP excitation [128]. Other groups have shown

that isolated adrenal chromaffin cells recruit the LVA T-type Ca2+ channels to

regulate a rapid and long lasting catecholamine release in response to acute

stress [67, 150]. We set out to determine if there is also a pronounced T-type

Ca2+ current in adrenal slices and to determine its potential role in PACAP

signaling. NiCl2 has been shown to be a potent and more selective T-type

blocker at low concentrations than Zn2+ [148]. We tested if there was a Ni2+- sensitive current component in adrenal chromaffin cells in situ. We depolarized chromaffin cells to potentials that match the measured PACAP-evoked

80 depolarization and are expected to evoke low voltage activated current. Sample records are provided in Figure 3.5B and show the activation of a Ni2+-sensitive influx at -55 mV, a potential and pharmacological profile indicative of LVA T-type currents. Furthermore, an analysis of Ni2+-sensitive currents provides an I-V relationship matching that for T-type currents (Figure 3.5C). The mechanistic role for Ni2+-sensitive currents as a component of PACAP-evoked secretion will be considered in the context of a signaling cascade below.

Figure 3.5: Ni2+ -sensitive Ca2+ currents in adrenal tissue slices. (A) A sample recording from a chromaffin cell held in the current clamp mode of the perforated- patch configuration in situ. Focal perfusion with 1 μM PACAP (bar) elicits a 12 mV depolarization. (B) To test for a Ni-sensitive component in adrenal chromaffin cells, a chromaffin cell was voltage clamped in situ in the perforated patch configuration. The cell was depolarized from -80 mV to 15 mV in normal Ringer and Ringer containing 50 μM Ni2+. The nickel-sensitive component was determined by subtraction of currents recorded in these two Ringer solutions. Sample currents recorded at relevant membrane potentials are shown; -70 mV (the resting potential of chromaffin cells in situ) and -55 mV (the mean potential recorded after PACAP stimulation). (C) The peak Ni2+-sensitive current was measured in response to depolarization as above from 5 recordings and are plotted as an I-V relationship. 82

PACAP-evoked stimulation involves Na+/Ca2+ exchange: We tested a potential

signaling event that could link the requirement for external Na+ and PKC

activation (Figure. 3.3) in PACAP-evoked catecholamine secretion. It has been

previously shown that the electrogenic Na+/Ca2+ exchanger (NCX) activity is

upregulated by PKC [151-154]. Increased NCX activity would therefore provide

an inward Na+ flux to depolarize the cell membrane and increase T-type

conductance. We tested for the involvement of the NCX in the PACAP-evoked

secretory response. Adrenal chromaffin slices were bathed in a NCX blocker,

benzamil (10 μM), prior to the application of PACAP. A sample recording is

provided in Figure 3.6 (top right plot) and demonstrates that PACAP-evoked

catecholamine release is sensitive to NCX inhibition. We conducted parallel

experiments in FURA2-loaded cells and showed that benzamil also blocked

PACAP-evoked increases in cytosolic Ca2+ (Figure 3.7). We further tested if

membrane depolarization is required for rapid PACAP-evoked catecholamine

release. Chromaffin cells were held in the perforated patch voltage clamp

configuration and the membrane potential was clamped at -80 mV to block

activation of any voltage-sensitive processes. Cells that were clamped at -80 mV

prior to PACAP stimulation did not show PACAP-dependent catecholamine

secretion (Figure 3.6A, lower left plot). Again, parallel experiments showed that

voltage clamp at -80 mV also blocked PACAP-evoked cytosolic Ca2+ increases

(Figure 3.7). In order to confirm that PACAP-mediated membrane depolarization is dependent on NCX activity, we bathed cells in benzamil as above and measured membrane potential in response to PACAP stimulation. We found that

benzamil significantly inhibited the depolarization observed under PACAP

stimulation (3.4 ± 0.5 [n=4] in PACAP + benzamil versus 12.4 ± 2.8 mV [n=7] in

PACAP alone). Lastly, to test for involvement of Ni2+-sensitive T-type calcium channels in the immediate PACAP response, adrenal slices were bathed in 50

µM NiCl2 prior to PACAP stimulation. This again blocked catecholamine

secretion (Figure 3.6A, lower right plot) and Ca2+ signals (Figure 3.7). Pooled

data for each of these data sets is provided in Figure 3.6B and paired pooled

data demonstrating similar results for PACAP -dependent cytosolic Ca2+ signals

is provided in supplemental data (Figure 3.7B). We bathed cells in 10 μM mibefradil, another blocker specific to T-type channels at this concentration [155].

We found mibefradil to also block catecholamine release and Ca2+ elevations in a

manner statistically identical to Ni2+ (data not shown).

Figure 3.6: PACAP-dependent catecholamine secretion is blocked by benzamil, voltage clamp and extracellular Ni2+. (A) Carbon fiber amperometry was used to measured total catecholamine release under PACAP stimulation as indicated in Figure 3.3. Sample single recordings for PACAP stimulation alone (top left) and PACAP in the presence of a benzamil (10 μM, top right), PACAP stimulation under perforated patch voltage clamp at -80 mV (bottom left) and PACAP stimulation in the presence of NiCl2 (50 μM, bottom right). (B) Pooled data from 10 experiments for each condition shown in panel A show that Na+/Ca2+ exchange and membrane depolarization are required for PACAP-dependent catecholamine release. They also show that that PACAP-secretory response is sensitive to extracellular Ni2+ (* = p < 0.02; paired Student’s t-test).

Figure 3.7: PACAP-dependent Ca2+ signals are blocked by benzamil, voltage clamp and extracellular Ni2+ (A) Cells were loaded with FURA2-AM and stimulated with PACAP as in Figure 3.4. Sample single traces of time-resolved FURA ratios at 360 nm and 390 nm excitation are plotted. Single representative recordings for PACAP stimulation alone (top left) and PACAP in the presence of benzamil (10 μM, top right), PACAP stimulation under perforated patch voltage clamp at - 80 mV (bottom left) and PACAP in the presence of Ni2+ (50 μM, bottom right). (B) Pooled data from 10 experiments for each condition in panel A show that that Na+/Ca2+ exchange and membrane depolarization are required for PACAP-dependent increases in cytosolic Ca2+ and that the Ca2+ influx is sensitive to block by extracellular Ni2+ (* = p < 0.02; paired Student’s t-test).

As described above, previous studies have reported divergent potential signaling paths for PACAP-evoked catecholamine secretion, including cAMP-mediated

[128] and PKC-mediated [138, 156] pathways. These divergent data have been interpreted as due to a multi-potent HOP cassette of the PACR1 receptor eliciting multiple signaling cascades. However Epac, a signaling molecule indicated to play a modulatory role in secretion from a variety of cell types [136, 157, 158], may provide a simplification of these data. Epac acts to stimulate PLC through a non-canonical cAMP-dependent pathway [140]. In a last set of experiments, we tested a potential that Epac is involved in PACAP signaling. No specific Epac inhibitors are yet available, rather we turned to the Epac activator 8-pCPT-2’-O-

Me-cAMP (8-pCPT) [158, 159]. As demonstrated by the raw data in Figure 3.8A and the pooled data in 3.8B, acute exposure to 8-pCPT elicits a statistically identical catecholamine release as PACAP stimulation. Also reminiscent of

PACAP stimulation, 8-pCPT stimulation is sensitive to block by NiCl2.

Figure 3.8: Epac stimulation elicits nickel-sensitive catecholamine secretion. (A) Carbon fiber amperometry was used to measure total catecholamine release under 8-pCPT exposure in (100 μM, top) and 8-pCPT + NiCl2 exposure (100 μM and 50 μM, respectively, bottom trace). (B) Pooled data show that 8-pCPT elicits catecholamine release identical in magnitude to PACAP stimulation (dotted line) and that this secretion is sensitive to block by NiCl2 (* = p < 0.02; paired Student’s t-test).

Taken together, we demonstrate that PACAP stimulation in situ is a potent and rapidly-acting secretagogue. We show that PACAP is natively released from the innervating splanchnic nerve under elevated burst stimulation that mimics the sympathetic stress response. As summarized in Figure 3.9, we show that this immediate PACAP response mechanistically involves PLC and PKC activation and that the electrogenic Na+/Ca2+ exchange depolarizes the cell allowing LVA T- type Ca2+ channels to evoke catecholamine release.

Figure 3.9: Summary diagram for the proposed PACAP signaling pathway. Key components of the proposed PACAP-mediated signaling path as described in the text are shown.

3.4 DISCUSSION

Prolonged release of ACh from the splanchnic terminals results in a rapid

desensitization of the cholinergic response [160]. Yet, elevated levels of

catecholamine are maintained in the blood stream under acute sympathetic

stress. This phenomenon suggests that excitation of the adrenal medulla must

occur by another mechanism under sympathetic stress. Here we demonstrate that pacing native neuronal input elicits an activity-dependent PACAP-evoked catecholamine release from adrenal medullary chromaffin cells. We show that this signaling path is selectively active only under burst mode firing and does not appreciably contribute to medullary stimulation under low frequency splanchnic firing. PACAP stimulation could potentially be through activation of three different receptors. PACAP has a relatively lower binding affinity to vasointestinal peptide receptors 1 and 2 (VPAC1 and 2) and a higher affinity for PACR1. This is a significant difference in that the VPACR1 signals through a Gs-coupled path, leading to adenylate cyclase activation and ultimately cAMP-mediated signaling.

VPACR2 signals through a Gq-coupled path leading to PLC activity and PKC signaling while the PACR1 receptor, with the HOP cassette signals through both pathways [137]. Thus, identification of the relevant receptor for PACAP signaling in the medulla could help define the signaling path for evoked catecholamine secretion. Previous work has shown that all three potential receptors are expressed in adrenal chromaffin cells [53, 142, 143]. The data contained in this study are consistent with either signaling through both VPAC receptors or through the single PACR1 receptor. Work in human adrenal chromaffin cells

showed that block of the VPAC receptors does not block PACAP-mediated catecholamine release [143] indicating that the PACR1 receptor is likely the

relevant receptor for PACAP-evoked secretion. Further studies will be necessary to confirm this conclusion in the mouse model.

Closer examination of the data set provides for mechanistic description of

PACAP-evoked excitation in situ. We show that sequentially, PKC activation is

required for elevated cytosolic Ca2+, placing PKC activation temporally prior to

the Ca2+ response. In combination with the absolute requirement for external

Ca2+, this point implies that PKC is not being activated in a Ca2+-dependent manner (as might be expected from conventional PKC isoforms). It also implies that acute PACAP-evoked secretion does not include Ca2+ mobilization from

internal stores (as might be expected by PLC activation and subsequent IP3

production). The lack of release from internal stores may be due to the fact that

the majority of the IP3 receptors in chromaffin cells are located on the nuclear

envelope, rather than the endoplasmic reticulum [161]. This location distal to the

site of granule fusion may attenuate its efficacy. Rather, the nuclear location of

IP3 receptors may facilitate the role of long-term PACAP exposure in gene

regulation.

Initially, the data presented here seemingly remain inconsistent with elements of

the existing literature. However, closer consideration provides a potential

common interpretation. As outlined above, different studies have provided a

complex and sometimes conflicting mechanism for PACAP-signaling in adrenal chromaffin cells. One possibility is due to the multi-potent HOP cassette of the

PACR1 receptor [137] that initiates PKA and PKC activation. It is likely that these paths represent the divergent physiological effects of PACAP stimulation, ranging from immediate transmitter exocytosis [128] to long term regulation of gene transcription [162, 163]. Another intriguing possibility is that the immediate

PACAP-evoked secretion may represent a non-canonical cAMP/PLC signaling mechanism. Specifically, one study stimulated cells with PACAP and showed an immediate secretory effect that was dependent on influx of extracellular Ca2+, but

this Ca2+ influx was not through HVA Ca2+ channels. Influx was also shown to be

blocked by LVA Ca2+ channel blockers [128]. Furthermore Przywara et al.

showed that secretion was blocked by rp-cAMPs, a blocker of cAMP signaling.

Based on this result the authors interpreted that the secretion was elicited

through a PKA-mediated mechanism. We did not see any effect of direct PKA

inhibition on PACAP-evoked secretion in situ. Moreover, exchange proteins

activated directly by cyclic AMP (Epac) have recently been recognized as a non-

canonical signaling effecter for cAMP [164]. Studies have shown a signaling path

through cAMP-dependent Epac activation that then stimulates PLC activity.

Indeed, when challenged with the Epac activator, 8-pCPT, we found an evoked

catecholamine release identical in magnitude and nickel sensitivity to that

measured under acute PACAP stimulation. This represents a potential

convergence point where cAMP signaling may initiate PKC-dependent processes

[140, 159]. The presence of Epac activation by cAMP and its ability to initiate a

PLC signaling suggests that while cAMP formation is required for the PACAP

response, the cAMP may in fact act through PLC and PKC activation and

subsequent influx of Ca2+ through a LVA to elicit the robust catecholamine release in situ (Figure 3.9).

In a further link between Epac and PACAP signaling, work conducted in isolated rat chromaffin cells showed that increased cAMP levels elicit Epac-mediated increases in low-voltage activated T-type Ca2+ current [165]. We have shown that

the low voltage activated Ca2+ channels play an integral role in the immediate

PACAP response. Influx through these channels is necessary to generate the

Ca2+ dependent exocytosis of catecholamine. As shown by the Carbone group,

hypoxic stress induces the increased expression of α1H T-type Ca2+ channels

[150]. Together, these findings further support the growing body of evidence that

T-type channels are involved in catecholamine release under stress [166]. This emerging hypothesis matches well with the idea that PACAP acts as a stress transmitter in the adrenal medulla. Further studies will need to be conducted on the systems, cell and molecular level to determine the activity-dependent roles of

Epac and PLC/PKC/NCX on PACAP-evoked catecholamine release

Chapter 4:

Discussion and Future Directions

4.1 PKC

It has been shown that the acute stress response elicits catecholamine release from adrenal chromaffin cells in an activity dependent manner and can be regulated by multiple neurotransmitters as well as intracellular signaling pathways involving protein kinase C (PKC) and other second messengers. While

many of the past studies evaluating the role of PKC were focusing on phorbol 12-

myristate 13-acetate (PMA) driven activation of PKC, the specific isoforms

involved were never directly addressed. It is assumed that in the regulation of

fast exocytosis, Ca2+ dependent activation of PKC is involved in increasing the

RRP [74]. While work with broad spectrum PKC inhibitors would enforce the

belief that conventional PKC isoforms are the primary players, it is still possible

that novel PKC isoforms are involved. PKC has been shown to play a pivotal role

in the release of catecholamine as discussed in chapters 2 and 3. It is interesting

to note that while a conventional isoform may be involved in increasing the RRP

in mice exposed to chronic intermittent hypoxia; because PKC activity precedes

Ca2+ influx in the PACAP mediated pathway, a novel PKC isoform may be more

likely to drive the activation of the sodium/calcium exchanger and subsequent

release of catecholamine. Or, because is it speculated that PKC is activated in a

reactive oxygen species dependent manner under CIH, it may be either a novel

PKC isoform or an atypical PKC isoform utilized to regulate catecholamine release. These phenomena require further evaluation.

PKC consists of two major domains, the regulatory and the catalytic domains.

The regulatory domain of the novel PKC isoforms has a C1 binding domain,

similar to that also found in the conventional PKC isoform. This site has been

shown to be the binding site for the PMA and/ or diacylglycerol (DAG), both

known activators of PKC [75]. To better understand the impact that each PKC

isoform may have on exocytosis, it is important to further characterize the PKC

isoforms by studying their enzymatic properties at the level of protein sequence.

Novel PKC isoforms can be further grouped into two subtypes; the θ and δ and ε

and η [75]. Recent studies have shown that PKC δ and ε are two major isoforms

involved in exocytosis [80, 167-169]. A study by Uchida et al. showed that not

only does PKCδ interact with and phosphorylate munc-18, but by knocking out

PKCδ in pancreatic β cells, exocytosis is greatly attenuated [169]. In that same

study they showed that the presence of conventional PKC isoforms, α and β,

played a less significant role in exocytosis [169]. While these experiments were

done in a different organ system, it opens up the possibility that PKCδ could also

play an important role in adrenal chromaffin cells.

To further address the role of PKC in the physiological stress response, we

looked at a link between generation of reactive oxygen species and the increase of phosphorylated PKC on Thr 514. Catecholamines released by chromaffin cells

are capable of oxidizing under physiological conditions to form quinone and 2e-.

This reaction occurs at a modest rate, but oxidation of catecholamine in the presence of reactive oxygen species is thought to occur much faster [170]. The oxidation of catecholamines by ROS was initially believed to be protective, in that it resulted in the removal of the radical oxygen species, but recent evidence is emerging to suggest that the reduced adrenaline products result in cellular toxicity as well. A study by Costa et al. showed that adrenaline in the presence of

ROS results in a feed forward mechanism; using cardiac myocytes, his group showed that adrenaline in the presence of ROS, resulted in the generation of free radicals that in turn increased the rate of oxidation of the remaining adrenaline

[171]. The question then becomes; at what concentration does ROS promote cytotoxicity? There is an abundance of literature suggesting that at low concentrations, ROS are emerging as an important second messenger signaling molecules. They are capable of promoting cell proliferation, as well as initiating gene transcription and have the ability to increase intracellular Ca2+ levels [172].

As proposed in chapter 2, the generation of ROS in mice exposed to CIH

appears to serve as a second messenger yet; the direct signaling mechanism between ROS and PKC remains to be determined. There is a lack of literature

providing a definitive response to the PKC isoforms present in adrenal chromaffin

cells, though the current literature suggest that PKCα, ε, and ζ are the

predominant chromaffin cell isoforms [173-176]. It can be proposed however, that

the novel PKC isoforms PKCδ or PKCε or the atypical PKC isoforms are

responsible for the increased RRP in our cells. ROS generation may result in

increased phosphorylation of PKCδ [113]. This hypothesis can be further

supported by our work that shows Ca2+ levels appear un-changed; suggesting

Ca2+ independence in PKC activation additionaly we propose that PKC is

activated by ROS suggesting that this isoform may also be independent of DAG,

these findings would suggest that CIH activated PKC may be that of an atypical

isoform. Turning to the literature, we found a substantial amount of literature

showing the involvement of PKCδ and ε in regulating exocytosis [169, 175, 177,

178] as well as a preliminary western blot showing an increase in phosphorylation of PKCδ using a specific phospho-antibody. Figure 4.1 shows

mouse adrenal medulla tissue in control and those exposed to 4-days CIH

express phospho-PKCδ. The antibody used is a pan-phospho antibody against

PKCδ and does not indicate where phosphorylation occurs. One could assume

that it is on a Thr or Ser residue, but there has been recent evidence suggesting

that PKC activation may also be regulated through phosphorylation on Tyr

residues under oxidative conditions [113, 114, 179, 180]. While our initial study

was done to show the possible mechanism for increased catecholamine

secretion under a physiological stress response, determination of the specific

PKC isoform could prove useful in understanding the associated signaling

pathways involved in CIH facilitation of catecholamine release as well as aid in

designing pharmacological treatment for those suffering with CIH and the associated pathologies.

Figure 4.1: Phosphorylated Protein Kinase Cδ is found in CIH and control mice. A western blot analysis probing for phosphorylated PKCδ revealed that there is phospho-PkCδ in mouse adrenal gland. While statistical analysis has not been performed it is quite possible that this protein may express higher levels of the phospho-protein under CIH conditions than in control samples. A β-actin staining was performed as a loading control.

While it is believed that PKCδ is the possible isoform responsible for the

facilitation of exocytosis of catecholamine under CIH exposure, it is not clear if

the PKC isoform in regulating exocytosis under PACAP stimulation is different. It is quite possible that the PKC isoforms are the same, but as mentioned earlier, due to the multiple isoforms expressed in cells, spatial compartmentalization may determine which PKC is responsible for an elicited response. As addressed in chapter 3, PACAP elicits a frequency dependent release of catecholamine, via

splanchnic firing. The CIH mediated response is also dependent on splanchnic

innervation, but the PACAP mediated mechanism involves G-protein coupled

receptors (GPCR) and the CIH response relies on the involvement of ROS as a

signaling molecule, thus suggesting two independent signaling pathways. As

proposed in chapter 3, it is possible that PKCδ or ε is the primary PKC isoform involved in the PACAP response. This novel PKC isoform is believed to be involved based on the FURA-2 analysis as well as carbon fiber amperometry which both place PKC activation before Ca2+ influx in PACAP mediated exocytosis [81], suggesting that PKC activity precedes Ca2+ influx and that the

2+ PKC isoform is independent of Ca . Studies show that PKCε is capable of

regulating exocytosis in chromaffin cells and in other cell types as well [167, 168,

181].

Interestingly, it is the lack of evidence in identifying specific isoforms responsible

for exocytosis in chromaffin cells that continues to fuel the notion that cellular location of a specific isoform is directly related to its function on respective

targets. It was long perceived that classical isoforms were involved in driving

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exocytosis because 1) exocytosis is a Ca2+ dependent process, 2) PMA is capable of activating multiple isoforms of PKC (both classical and novel) and 3)

Isolated chromaffin cells incubated in PMA result in increased recruitment of catecholamine containing granules to the readily releasable pool [74, 77]. A brief analysis on the role of classical PKC isoforms in chromaffin cells would prove useful.

In an early paper by Gillis et al., they proposed that classical isoforms were responsible for the increase in granule recruitment, based on the assumptions stated above and the use of a pharmacological blocker GÖ6983 that blocked the

PMA induced increase [77]. While identification of a classical isoform is a likely conclusion, characterization of the inhibitor GÖ6983 shows that it is able to block

not only the classical isoforms (α and β), but it is also capable of blocking the

novel isoform PKCδ as well [182]. A study by Chan et al. shows a PKC

dependent role in endocytosis involving classical PKC isoforms. This is an

interesting, yet fitting finding. Exocytosis is a Ca2+ dependent process and the

concentration of Ca2+ at the plasma membrane at the initiation of exocytosis was

calculated to be approximately 600nM, a concentration capable of activating PKC

[183]. Therefore, one could speculate that after stimulation of catecholamine

release when Ca2+ levels are relatively high, activation of classical PKC isoforms

would promote endocytosis and vesicle recycling whereas the novel PKC

isoforms are capable of granule recruitment to the membrane prior to Ca2+

signaled release. Further investigation into the possible link between PKCδ/ε

regulated exocytosis under the physiological stress response (chapters 2 and 3)

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and the involvement of the PKCα as a modulator of endocytosis under

physiological stimulation would be interesting. One would hope to determine 1)

the localization of each isoform, 2) the stimulus dependent activation of each

type of isoform and 3) the time course of activation, where novel isoforms may be

activated initially, but a secondary wave of classical activation occurs, driving

endocytosis. The use of specific PKC inhibitor peptides or specific message

knock-down would be useful in addressing these questions as well as capacitive

measurements to study exo/endocytosis.

4.2 Sodium Calcium Exchange

In chapter 3 we are proposing a novel pathway where PACAP is a frequency

dependent neurotransmitter released under the physiological stress response.

This pathway may prove to be controversial for many reasons: 1) membrane

depolarization through activation of the NCX in the forward direction, 2) a

significant T-type current to facilitate exocytosis, and 3) the involvement of

exchange proteins activated directly by cAMP (Epac) to activate PLC and PKC

via adenylate cyclase generation of cyclic adenosine monophosphate (cAMP).

Each one of these points will be addressed below in support of the pathway provided in Chapter 3.

The sodium calcium exchanger (NCX) shuttles Na:Ca2+ across the membrane

with a 3:1 ratio, respectively. When active in the forward direction, Na+ is shuttled

across the plasma membrane in to the cell while Ca2+ is transported to the

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extracellular milieu. The direction of NCX activity is determined by the driving force of ion concentrations intra and extracellularly. Activity in the forward direction is a common mechanism to remove Ca2+ from the cell; preventing Ca2+

2+ 2+ toxicity after [Ca ]i rises due to the opening of voltage dependent Ca channels

or intracellular Ca2+ mobilization from internal stores. This exchanger is also

capable of working in the reverse mode, seen primarily in cardiac smooth muscle

during ischemia/reperfusion injury [184]. The NCX is composed of 9 transmembrane domains with a large regulatory loop between TM 5 and TM 6.

This loop contains Ca2+ regulatory domains as well as the endogenous

exchanger inhibitory peptide region (XIP) [185]. NCX activity appears to be regulated in many different ways yet it is a bit elusive in terms of exact mechanism. Studies have shown that NCX activity can be up-regulated with

phosphorylation by PKA and PKC [154, 185, 186]. A study by Iwamoto et al.

showed that NCX1 is regulated by PKC phosphorylation, specifically at the S250

and that this site as well as the full cytoplasmic loop is necessary for proper NCX

regulation and function [187]. The NCX1 isoform is present in bovine chromaffin cells. A study by Soma et al. suggests that when exposed to low extracellular

Na+; the resulting Ca2+ influx enhances catecholamine release, most likely

regulated by NCX activity in the reverse mode [188]. Furthermore, they showed

that this response can be blocked by using the NCX inhibitors SEA0400 and KB-

R7943, but that their inhibitory action was not limited to only the NCX [188]. Thus

additional studies are necessary to address the NCX function in the physiological

context using other possible NCX blockers in mouse chromaffin cells. The

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newest generation of NCX inhibitors is mostly directed at blocking the NCX in the reverse mode and a majority of studies examining NCX function rely on a Na+

dependent Ca2+ influx, where cells are loaded with Na+ and then later placed in

solutions with 0Na+ to change the driving force, activating the NCX in reverse

mode. [154, 185-187, 189-191]. While these functional studies give support to

the role of phosphorylation regulation, addressing the possibility of differential

regulation between forward and reverse mode would certainly further the field of study. This new group of NCX inhibitors has been shown to be more specific for

NCX blockade, but they too have their drawbacks similar to the older generation

of amiloride derivatives. Benzamil is one older generation NCX inhibitor. Some of

the unspecific inhibitory effects are; binding that results in the block of INa, ICa, Ik,

Ito, Iach, and IKNa [189].

Our study employed benzamil, a known blocker of NCX in the forward direction.

A study using intact heart suggests that benzamil causes a dose dependent

block of NCX activity [192]. Data from this paper suggests that 10µM benzamil

causes sufficient block of NCX activity; thus we chose this concentration for our

in situ adrenal slice model. To further investigate the functional properties of

benzamil, we turned to a comprehensive review by Kleyman et al.. The review

looks at the differences in amiloride and its derivatives on the efficacious block of

Na+ transport. They look at the effects of amiloride and its derivatives on ENaC

(Na+) channels, Na/H exchanger, Na/Ca exchanger, and Na/K-ATPase [193]. In brief summary; they show how the addition of a hydrophobic moiety to amiloride

(to generate benzamil) can greatly enhance its efficacy in block of NCX activity

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as well as Ca2+ channel current [193]. In this paper, benzamil was shown to have an IC50 of 10 µM against the NCX and ENaC [193]. The ENaC channel,

however, is not expressed in chromaffin cells and therefore is not a possible

“unspecific” target in our study [193]. Other suggested non-specific targets of benzamil are Na+-coupled transporters as well as voltage gated calcium

channels [185]. One specific Na+ coupled transporter is the Na/H exchanger.

While is it possible that small amounts of protons maybe generated activating the

Na/H without damaging the cell. Further study into the role of Na/H exchange in

chromaffin cells is required and could reveal a role for Na/H mediated Na+ influx

during the PACAP mediated response.

Another target of benzamil is Ca2+ channels. In our experiments, the effect of

benzamil acting on voltage gated calcium channels can be ruled out because the

2+ concentrations used in our study are below that of the IC50 to block Ca current

[193]. The review by Kleyman, also suggested that a derivative of benzamil is

able to block the inward rectifier channel, though no mention was made if

benzamil is also capable of acting on this channel [193]. Additionally, an intense

literature search was done examining the effects of benzamil on chromaffin cell

function, and it was difficult to find any reports suggesting that benzamil use

results in these undesired responses. Despite this important point, it is

recognized that the need for an alternative interpretation or NCX inhibitor may be

necessary in future studies.

The next question related to the involvement of the NCX in the forward direction

is its ability to depolarize the cell membrane to the activation potential of the low

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voltage activated Ni2+ sensitive calcium channels. We have shown in Chapter 3,

that PACAP stimulation depolarizes the membrane and is able to elicit

catecholamine release in an action potential independent mechanism. The observed depolarization is 12.5 mV. As suggested in the paper, this depolarization is sufficient to activate a Ni2+ sensitive Ca2+ current which is quite possibly a result of activation of the NCX. To transport the necessary amount of

Na+ into the cell only a very small percentage of Ca2+ would have to be

transported out of the cell (7%). This was calculated using the formula Q=C*V,

where the net charge is directly related to the capacitance of the cell and the voltage jump to affect the open probability of our Ni2+ sensitive current. In this

case, the average chromaffin cell is approximately 9.2 pF, and the voltage jump

is 12.5 mV [81]. The net flow necessary for a 12.5 mV depolarization is about

718.6 x103 cations. The size of the cell can be calculated from the measured

capacitance and 1 µF/1 cm2. From determining the size of the cell and the

number of cations that must flow, one can calculate the amount of Ca2+ that

would leave the cell under NCX activity in the forward direction. The Ca2+

necessary for transport, assuming 70% accessible volume due to intracellular

organelles; and a Ks 50, where 1:50 calcium ions are not being sequestered or

buffered, would be an approximately 11.3 nM Ca2+ from its resting [150-200 nM]

[194]. Furthermore, this Ca2+ would rapidly be replenished with the opening of

the first T-type channels. It is important to account for ionic leak to accurately

calculate the current flow through the NCX. Thus, we can average the input

resistance from our recordings, and measure the PACAP dependent change in

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voltage (ΔV) travel across the membrane to calculate the total current passed

through the NCX.

While it is believed that the NCX exchanger plays a necessary role in the

proposed PACAP mechanism, it is important to recognize other possible sources

of GPCR induced depolarization. Studies in sympathetic ganglia suggest that

activation of second messenger signaling pathways result in 1) inhibition of Im

current (a voltage dependent K+ current) and 2) an inward current with reversal

potentials similar to those of Na+ [195]. Additionally, the inward current has been shown to be TTX insensitive, removing the involvement of voltage sensitive Na+

channels [195]. Furthermore, additional studies in sympathetic ganglia cells

suggest that peptidergic stimulation results in excitatory postsynaptic potentials

(EPSP) resulting in a greater frequency of spontaneous action potentials [196].

While these studies provide potential responses to the PACAP mediated

depolarization it is important to note that adrenal chromaffin cells under PACAP

stimulation do not result in action potentials. Interestingly, a study involving

PACAP stimulation of sympathetic ganglia showed that PACAP acts similar to

the peptidergic stimulation suggested in the Jones papers. PACAP stimulation of

rat sympathetic dorsal root ganglia resulted in a depolarization through inhibition

+ of Im current as well as a Na influx [197]. The findings from this paper are consistent with our observations that extracellular Na+ is necessary for the

PACAP evoked response in chromaffin cells. They propose that a possible source of this current is through non-selective cation channels.

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One possibility of the observed inward current is through transient receptor

potential cation channels (TRP). These channels have been shown to be non-

selective cation channels that may carry Na+ and Ca2+ and are also regulated by

DAG as well as PKC [191, 198-200]. The TRPC or classical family of TRP

channels has two different channel types, those that are activated due to a

depletion of intracellular Ca2+ and those that are activated in response to

receptor signaling [201]. TRPC 3 and 6 are two such receptors involved in

receptor operated current and are mostly associated with a calcium influx

through activation by DAG [198, 201]. Studies performed in rat ventricle, HEK

293 expression systems, and in drosophila retinal cells show a link between

TRPC3 movement of Na+ to activate the NCX in reverse mode to drive Ca2+

influx [198, 200, 201], yet they don’t show the extent of Na+ influx or amount of

depolarization. While it is possible that this coupling occurs in adrenal chromaffin

cells as well, there has been no definitive characterization of TRP channels in

chromaffin cells. A study has been performed using an over-expression of

TRPC3 in adrenal chromaffin cells, but it was not clear if there was endogenous

protein present [202]. Pheochromocytoma (PC12) cells, a neuroendocrine cell

derived from adrenal tumors have been shown to express TRPC1, 3, and 6

[198]. One dominant factor argues against TRP channels playing a role in the

PACAP mediated mechanism; the PACAP mediated mechanism is a GPCR

mediated pathway involving the activation of PKC through PLC. While PLC

activation results in the generation of DAG, a key regulator of TRPC channels;

PKC is also activated. PKC is a major signaling molecule in the PACAP

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mechanism and it has been shown to inhibit TRPC current [203]. PKC-dependent

inactivation is counter-active to the discussed behavior of the PACAP pathway.

Thus this finding would rule out TRP channels as those responsible for the

necessary sodium influx resulting in the 12.5 mV depolarization observed in

PACAP stimulated cells.

Another possibility that may contribute to the PACAP mediated membrane

depolarization is through another non-selective cation channel, the calcium

activated non-selective cationic channels (Ican). These channels are activated by

a depolarizing stimulus that is generally a result of L-type Ca2+ HVA current [204].

While these channels are equally permeable to Na+ and K+, and are voltage independent, they are activated after an influx of Ca2+ which would make these channels an unlikely player in the PACAP mediated mechanism, where depolarization occurs before the Ni2+ sensitive influx of Ca2+ [81].

Thirdly, it has been shown in synaptic transmission that G-protein coupled inward

rectifying K+ channels (GIRK) channels play a role in modulating neuronal

function by affecting cell excitability [205]. These channels are regulated through

GPCR activation, more specifically through the resultant disassociated βγ subunit

of the Gαi/o receptor activation. Binding of the βγ subunit to the target GIRK

channel results in an increase in current resulting in depolarization of the

membrane [205]. Consequently, activation of Gαq signaling cascades results in a

decrease in GIRK current through a PLC dependent pathway most likely

involving both PIP2 and PKC [205, 206]. While activation of these channels is

capable of membrane depolarization the I-V curve shows that this current is

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inactivated at the potentials observed in the PACAP mediated pathway [207].

Furthermore, because the PACAP receptor is a Gαq or Gαs, it is unlikely that

activation of this receptor would result in an increase in GIRK current to promote

depolarization of the membrane, rather it would result in inhibition of current.

Thus, these channels, while present in adrenal chromaffin cells, are an unlikely

player in the PACAP mediated membrane depolarization [207].

Another potential source of inward current would be in the block of Ca2+ activated

K+ (BK) channels. These channels are activated at depolarized potentials in

response to high intracellular levels of Ca2+. They are expressed in chromaffin

cells and have been shown to be regulated by second messengers. However,

studies have shown that BK activity is positively regulated by PIP2, to drive

hyperpolarization, rather than inhibit its activity; thus eliminating their role in the

PACAP mediated depolarization [208, 209]

Potassium leak current plays an important role in maintain membrane potential in

cells. By modulating the current through these channels, the membrane potential

can be affected. A large family of K+ leak channels is the KCNK family. These

tandem pore domain K+ channels are mainly mechano-sensitive or pH sensitive.

Additionally, only one isoform, the TREK channel, has been isolated in adrenal

gland, though it is primarily located in the adrenal cortex [210-212]. Further investigation is necessary to address the role of K+ leak current in PACAP

evoked depolarization and exocytosis from adrenal chromaffin cells.

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A relatively recent field of study has emerged addressing the role of persistent

Na+ current and facilitation of cell excitation. The NALCN channel is a non-

selective, voltage independent channel that has been shown to be a pacemaker

cell in the pre-boetzinger complex regulating respiration [213]. Little is known of

how these channels are regulated. They appear to be modulated by substance P

but independently of GPCR mediated pathways. This suggests that they are not

involved in PACAP mediated depolarization and exocytosis [214, 215].

Finally, acid sensing ion channels (ASIC) are non-selective that primarily pass

Na+ and Ca2+. These channels are members of the ENaC channel family and are activated by a decrease in extracellular pH [216]. Activation of these channels

increase Na+ conductance and are believed to play a functional role in synaptic transmission. Our studies use a BBS solution that is under constant bubbling with 95% O2/ 5% CO2 to maintain a physiological neutral pH, thus activation of

these channels under our conditions is highly improbable. Furthermore, a brief

literature search provides little evidence of the presence of these proton sensitive

channels in chromaffin cells.

4.3 T-type Calcium Channels

The second critical part of the PACAP pathway is the activation of a NI2+

sensitive, low voltage gated Ca2 channel. While we propose that they are of the

T-type isoform as supported by Carbone et al. [67], we do not rule out the

possibility of an R-type Ca2+ current which exhibits Ni2+ sensitivity as well. A

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deeper look into R-type Ca2+ channels reveals that its activation and inactivation kinetics are much different from those of T-type channels. A study using cerebellar granule neurons and undifferentiated NG108-15 cells revealed that activation of α1e (representative of R-type channels) occurs at -35 mV whereas the activation of T-type channels of this paper as well as our own occur at -55 mV [81, 155]. While this does not completely eliminate the possible role of R-type

current in Ca2+ evoked catecholamine release, it does suggest that the Ni2+

sensitive current responsible for the Ca2+ influx is initially T-type.

2+ 4.4 IP3 Release of Ca from Internal Stores

As discussed in chapter 3, PACAP evoked secretion requires activation of PLC.

PLC activation acts on PIP2 to form IP3 and DAG. We provide Fura-2 evidence

and functional amperometric evidence to suggest that the PACAP mechanism

requires activation of PKC yet it does not involve release of Ca2+ from internal

stores through IP3 receptors. One possible explanation of this phenomenon is in our study we are looking at a very initial time point in the PACAP evoked

2+ response. We believe that IP3 mediated Ca release must occur, but does so at

a time point not included in our studies. This is possible do to the location of the

IP3 receptors in adrenal chromaffin cells. Studies have suggested that IP3

receptors are located on ER membrane that is contiguous with the nuclear

2+ membrane and that diffusion of IP3 to these sites of Ca storage could cause a

delay the rise in intracellular Ca2+ from internal stores [52, 65, 161]. Studies

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involving PACAP does show that release of Ca2+ from internal stores occurs

[138] though we did not see this in our studies and believe that this may be due to a possible species difference. Further studies are required to address 1) the cellular location of IP3 receptors within chromaffin cells and 2) the occurrence of

2+ IP3 release of Ca and its role in PACAP evoked secretion of catecholamine.

4.5 Epac

Exchange proteins activated directly by cAMP (Epac) are emerging as multi- function proteins involved in many regulatory pathways including Ca2+ handling,

vesicle trafficking, secretion, ion transport and neuronal signaling [217]. Epac

proteins have been involved in the regulation of exocytosis, not only in facilitating

the recruitment of granules to the RRP, but in increasing the probability of

granule release as well [218, 219]. Studies have also shown that the Epac

mediated pathways are PKA independent and have even been linked to

activation of PKC though a new PLCε isoform [217, 219]. While its exact signaling

mechanism is unclear, there continues to be evidence supporting its possible role

in the PACAP mediated mechanism, including the presence of Epac2 in adrenal

chromaffin cells, the expression of PLCε and PKCε in adrenal chromaffin cells and the role of Epac in regulating fast exocytosis in similar neuronal and endocrine cell types [219]. Activation of Epac occurs through the binding of cAMP to the cAMP binding domain, which is analogous to the cAMP binding domain of the PKA regulatory subunits [159]. Subsequent activation results in

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activation of signaling pathways including the activation of RAP (a GTPase of the

RAS family). Activation of RAP results in multiple cellular processes ranging from

integrin-mediated cell adhesion, to hormone gene expression, to activation of

PLCε [159]. In our studies we show that activation of Epac with a specific

activator results in release of catecholamine at levels similar to that of PACAP

driven release. Additionally we show that we can block the Epac mediated

exocytosis with use of PKC inhibitor Gö6983. It is possible that Epac may act on other molecules within the chromaffin cells to aid in the secretory response. This is something that requires further study.

4.6 Overall Conclusion

Catecholamine release from the adrenal chromaffin cells is highly regulated under the sympathetic stress response. Protein kinase C appears to be a major regulatory signaling molecule involved in many processes including granule recruitment to the readily releasable pool to the actual fusion event when release of catecholamine takes place. We have demonstrated that PKC activation can be the direct result of splanchnic firing or through generation of reactive oxygen species due to oxidative stress. Chronic intermittent hypoxia activates the sympathetic stress response resulting in increased levels of serum catecholamine. We show in mouse chromaffin cells in situ, that activation of PKC results in the recruitment of catecholamine containing granules to the readily releasable pool. Furthermore we show that this response can systemically be

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blocked through intra-peritoneal injection of a free radical scavenger. Additionally

in our studies we probed the activity dependent regulation of catecholamine

release with the involvement of two different neurotransmitters released from the

splanchnic nerve. Through the development of a bipolar stimulation protocol we

are able to show that PACAP is the primary neurotransmitter released under the

physiological burst mode stress firing. The physiological relevance of two

neurotransmitters released under different frequency splanchnic firing suggests

that PACAP stimulation results in more efficient synaptic transmission under

stress that results in rapid and prolonged catecholamine release from chromaffin

cells. We show that release of PACAP from the splanchnic nerve results in acute

catecholamine release through a novel PKC dependent pathway involving not

only the sodium calcium exchanger, but a nickel sensitive Ca2+ channel that has also be associated with stress evoked catecholamine release. Both of these studies contribute to a greater understanding of the stress evoked sympathetic response. From these findings it is possible to further understand how different stressors promote catecholamine release and how one could potentially regulate this release in those individuals suffering from chronic stress.

4.7 Future Directions

The obvious question that stems from the findings reported in chapters 2 and 3 is if and how PACAP may be involved in mediating catecholamine release under chronic intermittent hypoxia, a known physiologically relevant stress that results

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in an increase in serum catecholamine. We have shown that PACAP is released

from the splanchnic nerve under stress mediated firing and that it regulates

catecholamine release through a PKC dependent mechanism. We show that CIH

too, involves a PKC mediated mechanism to increase the size of the readily

releasable pool. The first and logical step to probe for a possible link between these two pathways is to isolate the PKC isoform involved in each paradigm. As discussed above; for both circumstances, it is most likely a novel PKC δ, or ε.

Additionally, it would be interesting to evaluate the effect of PACAP on the readily releasable pool. One would think that if the isoforms involved in the PACAP response and the CIH mediated response are the same, then not only would

PACAP serve as a secretagogue in a direct signaling pathway to activate the

NCX, but the active PKC would also serve to increase the RRP. Thus, measuring the RRP with PACAP stimulation could provide further insight into the PACAP mediated secretory response and potentially provide a link between CIH mediated catecholamine release and PACAP as the regulatory neurotransmitter in this paradigm as well.

Thirdly, studies involving PACAP knockout mice have shown that these animals

are unable to properly respond to environmental or metabolic stress [126, 220].

PACAP knockout mice present an increased incidence of sudden death between

1-3 weeks postnatal, but those that survive have no major physical deformities

and resemble their wild type littermates [126, 220]. In a study by Cummings et

al., they showed that PACAP knockout mice have an inability to respond to

hypoxic conditions as demonstrated by a decrease in circulating catecholamine

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[220]. Additionally, in a study by Hamelink et al., she was able to demonstrate that PACAP null mice are unable to properly respond to metabolic stress; insulin shock in these animals resulted in hypoglycemia and death [126]. Under normal physiological conditions, high levels of circulating insulin result in increased splanchnic firing to drive catecholamine release. It is the high levels of circulating catecholamine that signal gluconeogenesis in the liver to compensate for the low plasma glucose levels. It can then be suggested that the lack of PACAP under this metabolic stress would then explain the high rate of morbidity, where there a resultant inability to release catecholamine to compensate for the administered insulin bolus.

I have conducted preliminary studies using PACAP null mice to study catecholamine release from the adrenal chromaffin cells with our bipolar stimulation protocol. I wanted to evaluate if adrenal slices were able to respond to stimuli mimicking both basal stimulus firing and that of burst mode firing.

Figure 4.2 shows that under basal firing conditions, catecholamine is released at levels similar to those of the wild type control. Under burst mode stress firing conditions however, the secretory response is attenuated, as expected. Since, I have just demonstrated that PACAP is released from the splanchnic nerve under burst mode stress firing [81], and that PACAP null mice are unable to respond to this stimulation protocol (Figure 4.2) the question arose if PACAP null mice were able to respond to exogenous PACAP. It was hypothesized that because of the lack of PACAP there would be a compensatory down regulation of the specific

PACR-1 receptor and thus an inability to respond to exogenous PACAP. Figures

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4.3 and 4.4 show that the PACAP null mice express the PACR-1 receptor, yet they are less sensitive to PACAP stimulation (1µM) compared to the wild type controls.

The next step in characterizing the stress response in PACAP null mice would be to expose them to chronic intermittent hypoxia. Exposure to an insulin bolus is a strong metabolic stress with a high mortality rate. It would be interesting to examine if CIH results in the same high mortality or if mice possess a compensatory mechanism that promotes survival even though this stress also activates the adrenomedullary hormone system. Conducting studies of this nature would also address the involvement of PACAP as a sensory neurotransmitter. Studies have shown that denervation attenuates the CIH mediated response. If our results, using PACAP knockout mice, show a link between PACAP and CIH mediated increases in catecholamine release; we can then link the release of PACAP from the splanchnic nerve rather than from sensory neurons further supporting the role of PACAP as a neurotransmitter under the sympathetic stress response.

Continuing research into stress mediated catecholamine release from adrenal chromaffin cells will contribute greatly to the field of endocrinology and provide the opportunity of exploring the regulation of catecholamine release in the context of transitional biology. As discussed in the introduction, there is a variety of stressors that the body is exposed to on a daily basis. The body is unable to differentiate between mortal danger and emotional stressors leaving the body in a state of constant stress. Physiologically, this state of stress will result in

118 pathologies such as hypertension, cardiac, cancer and death. By understanding how the body responds to stress, it may become possible to target and regulate catecholamine release under different stressors in an attempt to combat these devastating diseases.

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Figure 4.2: PACAP knockout mice fail to elicit a secretory response under burst- mode stress stimulation compared to its wild type control. Adrenal gland slices from PACAP knockout mice were stimulated with a bipolar stimulator at basal tone stimulus conditions and those of the burst mode stress firing conditions. Basal firing catecholamine release from knockout mice is similar to that of the wild type adrenal slices. The knockout animals however show a significant attenuation in burst mode stimulation secretion.

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Figure 4.3: PACAP knockout mice express the PACR-1 receptor. Western blot analysis of the PACAP Knockout mouse adrenal glands show that the PACAP specific PACR-1 receptor is still expressed in the adrenal chromaffin cells. A β- actin blot was used as a loading control.

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Figure 4.4: PACAP Knockout mice respond to exogenous PACAP (1 µM). Local perfusion of 1 µM PACAP resulted in a secretory response in situ. Adrenal gland slices from a PACAP knockout mouse release catecholamine in response to PACAP stimulation but the total catecholamine release is significantly less. (p<0.05).

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