THE EFFECTS OF INHIBITION OF PROSTAGLANDIN SYNTHESIS ON THYROTROPIN SECRETION
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Authors Thompson, Martha Ellen, 1947-
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Xerox University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106 76-29,342 THOMPSON, Martha Ellen, 1947- THE EFFECTS OF INHIBITION OF PROSTAGLANDIN SYNTHESIS ON THYROTROPIN SECRETION. The University of Arizona, Ph.D., 1976 Physiology
Xerox University Microfilms, Ann Arbor, Michigan 48106 THE EFFECTS OF INHIBITION OF PROSTAGLANDIN
SYNTHESIS ON THYROTROPIN SECRETION
by
Martha Ellen Thompson
A Dissertation Submitted to the Faculty of the
DEPARTMENT OF PHYSIOLOGY
In Partial Fulfillment of the Requirements For the Degree of
DOCTOR OF PHILOSOPHY
In the Graduate College
THE UNIVERSITY OF ARIZONA
19 7 6 THE UNIVERSITY OF ARIZONA
GRADUATE COLLEGE
I hereby recommend that this dissertation prepared under my direction by Martha Til 1 e>r> Thnmpwnn entitled THE EFFECTS OF INHIBITION OF PROSTAGLANDIN SYNTHESIS
ON THYROTROPIN SECRETION be accepted as fulfilling the dissertation requirement of the degree of Doctor of Philosophy
/9 /May ny<, Di5.5ferta.tion Director Date
After inspection of the final copy of the dissertation, the following members of the Final Examination Committee concur in its approval and recommend its acceptance:»
— /9 T J//
This approval and acceptance is contingent on the candidate's adequate performance and defense of this dissertation at the final oral examination. The inclusion of this sheet bound into the library copy of the dissertation is evidence of satisfactory performance at the final examination. STATEMENT BY AUTHOR
This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to bor rowers under rules of the Library.
Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or re production of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the in terests of scholarship. In all other instances, however, permission must be obtained from the author.
SIGNED: ACKNOWLEDGMENTS
I wish to express my sincere appreciation to Dr. George Hedge
who provided guidance and encouragement in both the research investi
gation and preparation of this dissertation# I would also like to
thank Dr. Gayle Orczyk, Merck Institute for Therapeutic Research, Rail way, New Jersey, 1975 and Dr. Delbert Fisher, Harbor General Hospital,
Torrance, California, for their personal assistance in the radio
immunoassay procedures in 1972. I also wish to thaiik Ms. Sherri Konen,
Arizona Medical Center, Tucson, Arizona, Dr. Marvin Brown, Salk Insti
tute, La Jolla, California, and the NIAMMDD Rat Pituitary Hormone
Distribution Program for their invaluable assistance in the radio
immunoassay procedures and data analysis. Appreciation is due to
Drs. Douglas Stuart, Eldon Braun and Mark Haussler for their critical review of this dissertation. I am particularly indebted to Susan
Dalby, Judy Cardoni and Nancy Hernden for their excellent technical assistance.
iii TABLE OF CONTENTS
Page
LIST OF TABLES vi
LIST OF ILLUSTRATIONS vii
ABSTRACT x
1. INTRODUCTION 1
Hypothalamic Control of Anterior Pituitary Secretion . 1 Corticotropin-Releasing Factor (CRF) . 6 Thyrotropin-Releasing Hormone (TRH) 8 Luteinizing Hormone-Releasing Hormone (LHRH) ... 9 Growth Hormone-Releasing Factor (GRF) and Somatostatin (GIF) 12 Prolactin Release-Inhibiting Factor (PIF) and Prolactin-Releasing Factor (PRF) 13 Specificity of Releasing Factors for the Anterior Pituitary Hormones 15 Regulation of the Pituitary-Thyroid Axis 17 Hypothalamic Regulation of TSH Secretion .. . • • 18 Environmental Stimuli for TSH Secretion ..... 20 Mechanism of Action of TRH 22 Thyroid Hormone Feedback Control of TSH Secretion 25 Circadian Rhythm of TSH Secretion 27 Mechanism of Action of TSH 27 Prostaglandins and the Hypothalamic-Pituitary- Endocrine Systems 28 Structure and Synthesis 29 Metabolism 31 Mechanism of Action 32 Systemic Effects of Prostaglandins 33 Aim of Present Study 39
2. MATERIALS AND METHODS **1
Animals and Care 4l Surgical Procedures 42 Chronic Indwelling Jugular Catheters 42 Thyroidectomy Procedure ...... Stereotaxic Microinjection Procedure 44
iv V
TABLE OF CONTENTS—Continued
Page
Acute Cold Exposure . kk Pharmacological Preparations ...... kk Radioimmunoassay of Plasma TSH, T-j and T^ .. k$ Assessment of Thyroidal Activity ...... k? T^ and T* Half-life 47 Data Analysis .. 49
3. RESULTS 50
Measurement of Pituitary and Thyroid Responses to Various Stimuli 50 Thyrotropin Response 50 Thyroid Hormone Secretion ...... 63
Effect of Inhibition of Prostaglandin Synthesis on the Hypothalamic-Pituitary-Thyroid Axis •• 73 Systemic Indomethacin in Intact Rats . 73 Indomethacin or Aspirin Treatment in Thyroidec- tomized Rats ...... 8k Stereotaxic Microinjection Studies 99
DISCUSSION 103
Comparison of Plasma TSH, T^ and T^ Levels with Previously Reported Values ...... 103 Measurement of Pituitary and Thyroid Response to Various Stimuli 105 TSH Response 105 Thyroid Hormone Secretion 109 Effects of Inhibition of Prostaglandin Synthesis on the Hypothalamic-Pituitary-Thyroid Axis 113 Systemic Indomethacin in Intact Rats ...... Ilk Indomethacin or Aspirin Treatment in Thyroidec- tomized Rats 118 Stereotaxic Microinjection Studies 122 Specificity of Indomethacin and Aspirin 123 Comparison of Results in This Study with Previous Studies Which Used Exogenous Prostaglandins ..... 126 Conclusions 127
LIST OF REFERENCES 129 LIST OF TABLES
Table Page
1. Plasma thyrotropin (TSH) levels in response to ac *. ,* cold exposure with ether or pentobarbital anesthesia .. 51
2m Effect of acute cold exposure on plasma thyrotropin (TSH) concentrations in unanesthetized rats ...... 5^
3. The effect of acute cold exposure on plasma Tj concen trations with ether or pentobarbital anesthesia .... 65
4. The effect of acute cold exposure on plasma Tif. concen trations with ether or pentobarbital anesthesia .... 66
5. Plasma levels in response to TRH with ether or pentobarbital anesthesia 69
6. Plasma T^ concentrations in response to thyrotropin (TSH) with ether or pentobarbital anesthesia ...*•• 72
7. Time schedule of the administration of drugs and sub sequent blood sampling for experiments in which intact rats were used 75 125 124 8. Disappearance rates for I-Tjj. and I-T^ following indomethacin treatment 82
9. Time schedules for surgery, drug (or vehicle) injec tions, T/j. replacement, and collection of blood samples for experiments in which TRH or thyroidectomy were used as stimuli to TSH secretion 85
10. Plasma TSH, T^, and T^ concentrations following TRH and aspirin (^5 mg/injection; Asp) or gelatin (Gel) treat ment in thyroidectomized rats receiving T^ replacement (3-0 /ig/100 g BW/day) 9^
vi LIST OF ILLUSTRATIONS
Figure Page
1. Plasma thyrotropin (TSH) levels in response to acute cold exposure with ether or pentobarbital anesthesia ... 53
2. The effect of acute cold exposure on plasma thyrotropin (TSH) concentrations in unanesthetized rats 55
3. Temperature in environmental chamber during cooling from 32° C 56
k. Plasma thyrotropin (TSH) concentrations of individual rats in response to thyrotropin-releasing hormone (TRH) anesthetized with pentobarbital (^.0 mg/100 g BW) .... 58
5. Plasma thyrotropin (TSH) concentrations in response to thyrotropin-releasing hormone (TRH) (0 - 1250 ng/100 g BW) in ether anesthetized rats 59
6. The effect of thyrotropin-releasing hormone (TRH) on plasma thyrotropin (TSH) concentrations after 10, 20, and 30 minutes '• 6l
7* Plasma thyrotropin (TSH) concentrations in response to thyrotropin-releasing hormone (TRH) in unanesthetized rats 62
8. Plasma thyrotropin (TSH) and thyroxine (T^) levels following thyroidectomy 6*f
9. Plasma triiodothyronine (T^) concentrations 2 hours after thyrotropin-releasing hormone (TRH) 68
10. Plasma triiodothyronine (T^) concentrations in response to thyrotropin (TSH) 71
11. Thyroid prostaglandin F (PGF) levels following the standard indomethacin (I) or gelatin (G) treatment .... 7^
12. Plasma thyrotropin (TSH) concentrations in response to TRH (50 ng/100 g BW, iv) following standard indomethacin (Ind) treatment in rats . 76
vii viii
LIST OF ILLUSTRATIONS—Continued
Figure Page
13- Plasma TSH, T*, and T^ levels following the standard indomethacin ll) or gelatin (G) treatment using ether anesthesia ...... 78
l*f. Plasma TSH, T^, and T^ concentrations after the standard indomethacin tl) or gelatin (6) treatment using pento barbital anesthesia 79 125 15- Disappearance curves of I - from the plasma of rats receiving the standard indomethacin (Ind) or gelatin (Gel) treatment 8l
16. Plasma T3 concentrations and the release of 125JI (response index) in response to TSH administration fol lowing the standard indomethacin (I) or gelatin (G) treatment 83
17. Plasma TSH, T^ and Tl levels following thyroidectomy with T4 replacement (*f.O j/g/100 g BV//day) and the standard indomethacin (Ind) or gelatin (Gel) treatment . 86
18. Inhibition of TSH response to TRH (250 ng/100 g BW, iv) following the standard indomethacin (Ind) treatment in thyroidectomized rats with T^ replacement (*U0 ng/100 g BW/day) . * 88
19- Plasma TSH, Tj and Ti. levels following thyroidectomy with Tjj. replacement (3.0 g/100 g BW/day) and the standard indomethacin (Ind) or gelatin (Gel) treatment • 89
20. Inhibition of TSH response to TRH (250 ng/100 g BW, iv) following the standard indomethacin (Ind) treatment in thyroidectomized rats with T^ replacement (3*0 ptg/100 g BW/day 90
21. Plasma TSH, T^ and TL. levels following thyroidectomy with T/j. replacement (2.0 fig/100 g BV.'/day) and the standard indomethacin (Ind) or gelatin (Gel) treatment . 91
22. Inhibition of TSH response to TRH (250 ng/100 g BW, iv) following the standard indomethacin (Ind) treatment in thyroidectomized rats with T^ replacement (2.0 /ig/100 g BW/day) * 92 ix
Lisa? OF ILLUSTRATIONS—Continued
Figure Page
23. Plasma TSH and T^ levels following thyroidectomy and the standard indomethacin Clnd) or gelatin (Gel) treatment 95
2*f. Plasma TSH and T^ levels following thyroidectomy and aspirin (10, 30 or k5 mg/injection) or gelatin (Gel) treatment 97
25• Plasma TSH and T/j. levels following thyroidectomy and the standard indomethacin (I) or gelatin (G) treatment . 98
26. Plasma TSH response to intrapituitary injection of TRH (1 ng in 1 fil) following the standard indomethacin (Ind) or gelatin (Gel) treatment 100
27. Plasma TSH levels following the sequential intrapitui tary injection of indomethacin (Ind) (10 fig in 1 fil) and TRH (1 ng in 1 fil), or equal volumes of their vehicles (Veh) 102 ABSTRACT
Prostaglandins (PGs) are a ubiquitous class of fatty acid de rivatives whose physiologic function is unclear at this time.
Exogenous prostaglandins have been shown to alter secretion of a number of anterior pituitary hormones jLn vivo and vitro. In this study the role of endogenous prostaglandins in the secretion of the anterior pituitary hormone thyrotropin (TSH) was investigated. The level of endogenous prostaglandins was decreased by administering an effective dose of a known PG synthesis inhibitor, either indomethacin (Ind) or aspirin (Asp), The drug was administered to adult female rats either systemically which resulted in decreased PG synthesis throughout the body, or in local injection directly into the anterior pituitary which resulted in localized inhibition of PG synthesis.
Plasma TSH levels were monitored in rats receiving indomethacin or aspirin to inhibit PG synthesis. TSH secretion was induced by either exogenous thyrotropin releasing hormone (TRH) or by thyroidec tomy, On the basis of preliminary experiments, Ind was found to in hibit thyroid secretion directly. In order to circumvent this problem, thyroidectomized rats receiving thyroxine (T^) replacement (2-^4- fig/100 g BW/day) were used. These replacement regimens were judged to be adequate on the basis of the measurement of plasma triiodothyronine
(T_) and TJt levels, and the lack of a compensatory rise in plasma TSH ' t levels. Plasma T^ and T^ levels were maintained similarly in both
x xi groups during this time. Under these conditions, Ind significantly inhibited, but did not abolish, the TSH response to exogenous TRH (250 ng/100 g BW, iv). Thyroidectomy-induced TSH secretion was abolished by Ind, and could be reversed upon cessation of Ind treatment. Aspirin was also found to significantly inhibit the compensatory TSH rise fol lowing thyroidectomy. These findings suggest that endogenous pituitary
PGs mediate the stimulation of TSH secretion by either TRH or by re duced feedback of thyroid hormones.
An additional series of experiments was performed in order to determine the mechanism by which Ind lowered thyroid hormone levels.
Plasma T^ and T^ levels were found to be significantly decreased in the intact rats receiving the standard Ind treatment. In order to de termine the role of PGs in controlling thyroid hormone levels the thyroid response to TSH and the half-lives of T^ and T^ after Ind treatment were determined. Ind was found to inhibit thyroid secretion in response to TSH (10 or 100 mU/100 g BW, iv) as determined by the 125 release of I (response index) or plasma levels. The half-life of T^ was only slightly decreased by Ind (p <.05), but the half-life of T^ was markedly increased by the Ind (17-6 h vs. 10.h in the con trol group). This suggests that peripheral conversion of T^ to T^ may be enhanced. However, this effect would not tend to decrease overall thyroid hormone levels, but merely shift the T^ to T^ ratio. There fore, it is concluded that the major effect of Ind in decreasing thyroid hormone levels is to inhibit thyroid hormone secretion. XXI
Conclusion: The results suggest that endogenous PGs are in volved at the pituitary level in the secretion of thyrotropin, and at the thyroid level in the secretion of and T^. CHAPTER 1
INTRODUCTION
Hypothalamic Control of Anterior Pituitary Secretion
Very little was known about the regulation of anterior pituitary secretion prior to 1930. During the subsequent 25 years a great deal of anatomical and physiological evidence was generated which supported the neurohumoral hypothesis for control of the anterior pituitary by the hypothalamus. Before this theory was set forth, a number of scien tists investigated the possibility of simple neural control of the gland. Several possible neural pathways to the gland were studied, but no convincing evidence of simple neural control was found. The last 20 years of intensive study has led to the discovery of hypothala mic neurohumoral factors (releasing or release-inhibiting factors) which regulate anterior pituitary function.
Marshall (1936) was one of the first investigators to show that, aside from coitus, a number of environmental factors (e.g., food supply, temperature, and light) could alter reproductive rhythms in animals. He found that ovulation could be induced in the rabbit by electrical stimulation of the brain (Marshall and Verney, 1936).
Marshall concluded therefore that reflex pathways existed whereby sensory stimuli from the environment could act through the central nervous system (CNS) to alter anterior pituitary secretion. Experiments
1 2 with nerve section or pituitary transplants proved, however, that neural integrity was not necessary for anterior pituitary secretion#
Therefore, it was concluded from these studies that a neural reflex pathway did not exist, and an intensive anatomical study of hypothalamic-pituitary connections was begun.
The discovery of a unique blood supply to the pituitary raised a new possibility for CNS regulation of the gland. Popa and Fielding
(1930* 1933) first described the anatomy of the hypophysial portal sys tem and concluded that blood flowed from the pituitary to the hypo thalamus, Subsequently a controversy arose about the direction of blood flow in the portal vessels. This controversy over direction of blood flow in the portal vessels was resolved by Wislocki and King
(1936) who confirmed the anatomy of the portal vessels and established that blood flowed from the hypothalamus to the pituitary. Green and
Harris (19^9) were the first to visually observe that blood in the stalk portal vessels flowed from the hypothalamus to the pituitary.
It has since been shown that in many mammals the affluent blood of the anterior pituitary is supplied entirely by portal vessels. The main blood supply is provided by the long portal vessels whose primary capillary bed lies in the median eminence and pituitary stalk. The short portal vessels which arise in the posterior pituitary provide the remainder of this blood supply (Daniel and Prichard, 1956; 1957). Thus the anatomical evidence was presented which provided the foundation upon which the current theory of hypothalamic control of pituitary secretion is based. 3
The first step toward elucidating the mechanism of regulation of pituitary secretion was made by Hinsey and Markee (1933) who sug gested that there was neurohumoral control of the pituitary gland.
They postulated that the neurohypophysis could respond to certain stimuli by releasing a humoral factor that might influence the secre tion of anterior pituitary. In spite of the fact that the neurohypo physis was incorrectly suggested as the structure regulating anterior pituitary, the hypothesis of neurohumoral control proved to be a step in the right direction. It was Harris (19^8) who finally developed the hypothesis as we know it today; with the hypothalamus being responsible for producing and releasing humoral messengers which regulate the secretion of the pituitary gland.
After finding several contradictory effects of pituitary stalk section, Harris (1950) suspected the stalk sections may not have been complete in all experiments. Therefore, he devised a technique for obtaining complete disruption of the hypophysial blood supply. Uti lizing a small paper plate or other impermeable barrier placed between the cut ends of the stalk, he was able to prevent any possible regen eration of vessels in this region (Harris, 1950). His experiments proved the necessity of vascular integrity for normal pituitary se cretion. Harris and Jacobsohn (1952) transplanted the pituitary to the median eminence or temporal lobe of hypophysectomized rats and obtained good vascularization in both sites. Only the group with median eminence transplants were capable of normal reproductive function, ultimately mating and bearing young. They concluded that the vessels carried k humoral agents whose function was to transmit neural impulses into biochemical responses by stimulating secretion of the appropriate hor mone from the anterior pituitary. As a result of lesion and electrical stimulation studies a "map" of neural sites was drawn up indicating
"excitatory" hypothalamic regions for pituitary hormone control. For example, Martin and Reichlin (1971) identified the medial ventral hypothalamus as an "excitatory" area for TSH release.
Halasz, Pupp, Uhlarik and Tima (1965) described the area of nerve cell bodies and the bulk of neurons destined to secrete the neurohumoral releasing factors as the "hypophysiotrophic area" of the brain. Hypothalamic and neurohypophysial extracts were shown by
Saffran and Schally (1955)t and by Guillemin and Rosenberg (1955) to possess anterior pituitary regulators. On this basis Saffran, Schally, and Benfey (1955) proposed that the term "releasing factor" be used in reference to the neurohumoral factor regulating adrenocorticotrophic hormone secretion. In agreement with common usage, this discussion will use the term "releasing factor" in reference to those hormones as yet unidentified, and "releasing hormone" in reference to those already identified. An intensive search has been conducted during the past 15 years to characterize and identify these postulated factors for re leasing each of the adenohypophysial hormones. However, one major problem presents itself in that only a very small amount of activity is present in any one hypothalamus or median eminence. The methods used to extract the possible releasing factors were those which would chemically extract peptides from the tissue homogenates. Some 5 investigators (Campbell, Feuer and Harris, 196^; Fawcett et al., 1968) felt that the releasing factors might be peptidic in structure since the median eminence was one of three parts of the neurohypophysis — a structure known to release the peptide hormones oxytocin and vasopressin.
Rioch, Wislocki and O'Leary (19^0) first proposed the nomenclature for parts of the neurohypophysis, neural lobe (infundibular process), neu ral stalk (infundibular stem), and the median eminence. In most early investigations releasing factor activity was sought in extracts of the posterior pituitary. The choice of this tissue can probably be attrib uted to the fact that a portion of the blood supply perfusing the an terior pituitary arises from capillaries in the median eminence. Yet in other animals there is no anastomosis between the two lobes and still there exists normal pituitary control. Therefore, subsequent studies concentrated on hypothalamic extracts in trying to identify releasing factor activity. If a substance that controls pituitary secretion could be found in the hypothalamus, this would be the most direct evidence supporting the neurohumoral control theory.
Since neurohumoral transmitter agents are usually found stored in nerve terminals, it was felt that the greatest amount of releasing factor activity per unit weight would be contained in the median eminence where nerve fibers terminate on the capillaries supplying the pituitary. As an extension of Koch's postulates, the following cri teria have been established for identifying a releasing factor as the physiological hormone (Harris, 1955)! a* Activity must be extractable from the hypothalamus, especially from the region of the median eminence.
b. Such an extract must be capable of producing in a dose response fashion an altered secretion of the particular adenohypophysial hormone by direct action on the gland.
c. Evidence must exist for altered release of the factor in situ ations where pituitary hormone release is altered under a variety of experimental and environmental conditions.
d. The factor should be found in greater amounts in the portal than in systemic blood.
Corticotropin-Releasing Factor (CIffT
Once it became evident that the rate of corticotropin (ACTH) release from the pituitary was influenced not only by the circulating level of adrenal corticosteroids, but also by external environmental factors, investigators began searching for a chemical agent as a medi ator of such responses. The search for such an agent is made more dif ficult due to the relative ease with which ACTH release is stimulated and the difficulty in measuring the rate of ACTH release. Nevertheless,
Saffran and Schally (1955) demonstrated that a hypothalamic releasing factor, (CRF) for ACTH does indeed exist which was the first time any releasing factor had been found. These initial studies utilized pri marily posterior pituitary powders as a source of releasing factors since hypothalami were not readily available (Schally, Arimura and
Kastin, 1973). 7
One of the first suggested mediators of ACTH release was the posterior pituitary hormone vasopressin. Although several investiga tors (Martini and Morpurgo, 1955; McCann, 1957) found vasopressin does indeed possess CRF-like activity, it was necessary to use large doses to achieve the effect. Furthermore, vasopressin has been shown to stimulate CRF release, riot ACTH release (Hedge, Yates, Marcus and Yates,
1966). This suggestion that vasopressin might be CRF was finally dis carded when it was demonstrated that rats with hereditary diabetes insipidus (i.e., lacking pituitary vasopressin) could still respond to stress with an increase in ACTH release (Arimura, Saito, Bowers and
Schally, 1967).
Repeated attempts have been made to isolate the physiological
CRF and identify its structure. At best, only a partial amino acid sequence has been suggested for the extremely labile CRF (Schally and
Bowers, 1964). Recently it has been discovered that pressinoic acid
(Saffran, Pearlrautter, Rapino and Upton, 1972) has very high potency for releasing ACTH although this has yet to be repeated by other in vestigators (Saffran personal communication, 1972). Another suggestion has been the polypeptides which were synthesized by coupling the dipep- tides Ser-His or His-Ser to the free amino-terminal group of lysine vasopressin (Doephner, Sturmer and Berde, 1963). These compounds have some CRF activity although not as great as that of vasopressin itself.
Deaminolysine vasopressin has CRF activity almost equal to that of lysine vasopressin but a much higher CRF/pressor ratio (Arimura,
Schally and Bowers, 1969)- 8
Thus CRF was the first postulated hypothalamic releasing hor mone but it is as yet unidentified in structure. With the advent of bioassay, radioimmunoassay, and in vitro intact cell techniques for measuring ACTH, the task may be somewhat easier. The measurement of adrenal corticosteroids as an index of ACTH levels has been a major problem in evaluating postulated CRF's in the past.
Thyrotropin-Releasing Hormone (TRH)
The first hypothalamic hormone to be identified was thyrotropin- releasing hormone (TRH). TRH was first demonstrated to exist by Greer
(1951) who found decreased circulating TSH and thyroid hormone levels following lesions made in the median eminence. Elevated levels of TSH have been demonstrated after electrical stimulation in the rat
(D'Angelo, Snyder and Grodin, 196*f; Martin and Reichlin, 1970; Martin and Reichlin, 1972) and rabbit (Averill and Salaman, 1967). On the other hand, Vertes, Vertes and Kovacs (as cited by Martin, 197M have shown that stimulation of the posterior hypothalamus is inhibitory.
Thus it appears that there may be both facilitatory and inhibitory sites in the hypothalamus for TSH release.
Due to the extremely small amount of TRH present in the hypo thalamus, investigators have conducted extraction procedures on a mas sive scale in search of TRH. In 1966, Guillemin, Burgus, Sakiz and
Ward (1966) obtained 3 mg of TRH from 500,000 ovine hypothalami. With such small amounts of TRH available it took many attempts to finally purify TRH (Guillemin, Yamazaki, Jutisz and Sakiz, 1962; Schally,
Bowers and Redding, 1966), identify the three component amino acids, 9
and ultimately to determine its structure (Boler et al., 1969; Burgus,
Dunn, Desiderio and Guillemin, 1969). It now appears from all of the animal extracts examined that human, bovine, porcine, and ovine TRH are the identical tripeptide L-pyroglutamyl-histidyl-proline amide (Schally,
Arimura and Kastin, 1973)- Full activity is dependent upon the pres ence of a prolyl amide residue at the C-terminal and a cyclic pyroglu- tamyl residue at the N-terminal. In an attempt to determine the relationship between structure and activity many analogs of TRH have been synthesized. One analog exhibiting greater activity than natural
TRH (8-10 times as much activity) has a methyl group in the 3-N posi tion of the imidazole ring of histidine (Vale, Rivier and Burgus 1971;
Rivier et al., 1972; Hoffman and Bowers, 1970). Several other analogs with small changes in structure have about the same potency as the biological form (Grant et al., 197*0.
A number of physiological studies have shown that synthetic TRH has the same activity as natural TRH (Bowers et al., 1970; Burgus et al., 1970). Plasma TSH increases in a dose-related manner to TRH when administered by several routes (Bowers et al., 1970; Burgus et al.,
1970). Furthermore, only small amounts (picograms) are necessary to elevate TSH levels in vivo or in vitro (Burgus et al., 1970; Bowers et al., 1970; Redding, Arimura and Schally, 1970).
Luteinizing Hormone-Releasing Hormone (LHRH)
The role of the CNS in the regulation of gonadotropin secretion was first demonstrated by Marshall and Verney (1936) who used electrical 10
stimulation of the heads of estrus rabbits to elicit ovulation and
pseudo-pregnancy in these animals. Furthermore, in 1937 Harris (as
cited by Harris, 1972) showed that electrical stimulation of the tuber
cinerum and preoptic-suprachiasmic region would elicit the release of
gonadotropins. Stimulation of the pituitary stalk or the pituitary
itself was ineffective, indicating that the pituitary itself does not
regulate the release of gonadotropins. This evidence suggested gonado
tropin secretion is regulated by the hypothalamic releasing factor.
Experiments using pituitary stalk section were found to give variable
results until Harris demonstrated that a barrier was necessary to pre
vent partial or complete regeneration of the vascularity in this region.
He found that gonadal activity after stalk section correlated well with
the amount of vascular regeneration (Harris, 1950). Harris (1950) ob
served ovarian atrophy in monkeys with complete disruption of vascu larity# Kamberi, Mical and Porter (1970; 1971a) demonstrated LKRH,
FSH releasing factor (FSH-RF), and prolactin inhibiting factor (PIF)
activity in pituitary stalk plasma although no activity was found in
perfusions of the median eminence of the rat (Kamberi, Mical and
Porter, 1970).
LH-releasing hormone was finally isolated in pure form from
thousands of porcine hypothalami (Schally, Arimura, Baba et al., 1971?
Matsuo et al., 1971)* The pure releasing factor was identified as a
decapeptide, p-glu-his-trp-ser-tyr-gly-leu-arg-pro-gly-amide (Burgus
et al., 1972; Matsuo et al., 1971)* A number of physiologic (in vivo)
and in vitro studies with the synthetic LHRH subsequently proved that 11
the synthetic form had the same activity as the natural LHRH. It was
found that the synthetic LHRH released not only LH but also FSH in in vitro experiments at nanomolar concentrations (Schally, Arimura,
Baba et al., 1971; Schally, Bedding, Matsuo and Arimura, 1972). The decapeptide also had the same mobility as LHRH in thin layer chroma tography and electrophoresis. In addition to stimulating LH release it also elicited FSH secretion in immature rats or castrated rats pre- treated with testosterone (Arimura, Debeljuk and Schally, 1972; Schally,
Baba, Arimura, Redding and White 1971)- In response to this finding,
Schally, Arimura, Kastin et al. (1971) have proposed one releasing factor (LHRH/FSHRH) that is responsible for stimulating pituitary re lease of both LH and FSH. This suggestion is difficult to reconcile with the fact that plasma concentrations of LH and FSH do not always rise or fall together during the human menstrual cycle or animal estrus cycles. If there is only one releasing factor then the quantity of gonadotropin which is released in response to it must be determined by an additional effector. Two factors have been suggested to modify the response: 1, a modifier for the action of the releasing factor on the gonadotropes, such as the circulating level of sex steroids (e.g., estrogen or progesterone); or 2, there are two types of gonadotropes which respond differently to the releasing factor, one type releasing
FSH and the other releasing LH. If there is only one releasing factor controlling the release of both LH and FSH then the regulation of the secretion would have to be exerted at the pituitary level. On the other hand, at least part of the control would reside in the 12 hypothalamus if there are two releasing hormones (Currie et al., 1973;
Bowers et al., 1973; Johansson et al«, 1973; Corbin, Milmore and
Daniels, 1970)*
Growth Hormone-Releasing Factor (GRF) and Somatostatin (GIF)
The first evidence for hypothalamic control of growth hormone
(GH) secretion was presented by Hethrington and Hanson in 19^0. They demonstrated growth retardation in rats following the destruction of the ventral hypothalamus. Direct proof for a hypothalamic releasing factor was provided by stimulation of release of growth hormone by hypothalamic extracts added to pituitaries incubated in vitro (Deuben and Meites, 196*0. Hypophysial portal blood (Wilber and Porter, 1970) and stalk median eminence extracts (Sandow, Arimura and Schally, 1972) also have been shown to contain releasing factor activity. A polypep tide possessing GH-RF activity was finally isolated from 200,000 por cine hypothalami (Schally et al., 1969)- Schally, Baba, Nair and
Bennett, 0-971) reported a possible structure for GRF. Upon character ization of its structure and synthesis the decapeptide was found to have similarity to the $ chain of porcine hemoglobin (Veber et al.,
1971)* Since the discovery of this possible releasing factor a number of in vitro and physiological experiments have failed to fully confirm the synthetic decapeptide as the true physiologic releasing factor.
Both the pure isolated decapeptide and the synthetic decapeptide are capable of stimulating release of bioassayable GH from rat pituitaries
(Schally, Arimura, Wakabayashi et al., 1972). However, the hypothalamic 13
extracts did not alter pituitary content of GH as measured by radio
immunoassay although the bioassay showed that there was pituitary de
pletion of GH (Muller et al., 1972). Since the decapeptide is also
inactive in man (Kastin, Gual and Schally, 1972) it is unlikely to be
the physiological GRF, Wilber, Nagel and White (1971) have demonstrated
an extractable substance with GRF activity which stimulates the release
of immuneassayable GH in vitro. A GRF may, therefore, exist although
its identity remains to be elucidated.
While searching for the releasing factor, another substance was
found which inhibits the release of GH in vitro (Krulich, Dhariwal and
McCann, 1968). This substance has been isolated, identified as a cyclic tetradecapeptide, and named somatostatin (Brazeau et al., 1973).
A linear form was synthesized and demonstrated to inhibit GH release in
vitro and in vivo in rats (Brazeau et al., 1973)* The cyclic tetrade capeptide waff also synthesized (Coy et al., 1973) and shown to inhibit
GH release in human subjects in response to insulin-induced hypogly cemia (Hall et al., 1973).
Prolactin Release-Inhibiting Factor (FIF) and Prolactin-Releasing Factor (PRF)
Prolactin was the first pituitary hormone shown to be primarily under inhibitory hypothalamic control (Everett, 195^)• Transplantation of the pituitary to extracranial sites results in increased prolactin levels with maintenance of the function of corpora lutea and mammary glands. Lesions in the median eminence or pituitary stalk section led Ik to a continuous release of prolactin in rats (Meites, Nicoll and Tal- walker, 1965? Meites and Nicoll, 1966; Welsch et al.t 1971)* Quartered pituitaries incubated jln vitro secreted large amounts of prolactin, and hypothalamic acid extracts added to the pituitary fragments inhibited prolactin secretion (Talwalker, Ratner and Meites, 1963)* Since that time evidence of a similar prolactin release inhibiting factor (PIF) has also been found in a number of domestic animals (Schally, Arimura,
Baba, Nair et al., 1968). Although the structure of PIF has yet to be determined current evidence indicates it is a low molecular weight poly peptide. It seems likely that dopaminergic transmitters are respon sible for the release of PIF since the injection of dopamine (DA) into the third ventricle elicited PIF release, whereas DA injections directly into the pituitary were ineffective (Kamberi, Mical and Porter, 1971b).
On the other hand, several investigators (Shaar and Clemens, 197^;
Smalstig, Sawyer and Clemens, 1971*-) have demonstrated that in vitro
DA does directly inhibit the pituitary release of prolactin, and they have suggested that DA is the PIF. In addition, they found that apo- morphine, a dopamine receptor stimulant, inhibited the normal release of prolactin during proestrus in rats, and reduced the release of pro lactin in response to chlorpromazine treatment or suckling (Smalstig,
Sawyer and Clemens, 197*0•
A prolactin releasing factor (PRF) has also been postulated to exist (Nicoll et al., 1970). Meites, Talwalker and Nicoll (i960) first demonstrated an increase in prolactin levels with hypothalamic extracts in vivo. A purified extract was shown to contain a PRF which was 15
distinct from TEH and also stimulates prolactin release (Valverde-R.,
Chieffo and Reichlin, 1972). Although TRH has been demonstrated to stimulate the release of prolactin in rats (Tashjian, Barowski and
Jensen, 1971)i in humans (Jacobs et al., 1971)t and in sheep (Debeljuk et al., 1973)* it has yet to be established if this effect is a physio logic or pharmacologic phenomenon.
Specificity in Releasing Factors for the Anterior Pituitary Hormones
As releasing factors have been isolated and characterized it has been shown that several of these are not entirely specific. It has already been mentioned that LHRH stimulates the release of not only LH, but also FSH (Schally, Arimura, Baba et al., 1971; Arimura, Debeljuk and Schally, 1972). As previously discussed it is possible that there is a separate FSHRF which is as yet unidentified.
TRH stimulates the release of not only TSH, but also the re lease of prolactin (Jacobs et al., 1971; Tashjian, Barowski and Jensen,
1971; Vale, Blackwell, Grant and Guillemin, 1973; Takahara, Arimura and
Schally, 197*0- Under certain circumstances TRH also increases the release of FSH (in the male ^fortimer et al., 19757)* LH (in the female at the luteal peak j/Franchimont, 19727) and GH (in acromegaly /Takahara,
Arimura and Schally, 197i^)« A decreased ACTH response to stress has been observed following TRH-induced TSH secretion (Sakiz and Guillemin,
1965). Such data have led to the suggestion that there is an inverse relationship between ACTH and TSH secretion (reviewed by Guillemin,
1968). Somatostatin is also a very non-specific hormone in that it in hibits the release of the pituitary hormones GH, TSH, and FSH, and inhibits the release of the pancreatic hormones insulin and glucagon
(Hanson et al., 1973? Siler et al., 1973; Mortimer et al., 197*0 • The significance of these observations is still uncertain. Somatostatin is a potential therapeutic agent for several diseases in which there are excesses of these hormones.
Aside from the observation that a releasing factor elicits the increased release of more than one pituitary hormone, it can be seen that the pituitary cells themselves must lack some specificity. More than one hormone may stimulate or inhibit the pituitary cell to release its particular hormone. For example, prolactin release may be elicited by either a postulated PRF, or by TRH, substances which have been demon strated to be distinct (Valverde-R. e.t al., 1972). TSH release may be regulated not only by TRH but also by somatostatin which will in hibit its release. Therefore, what began as a simple theory of one releasing factor for each of the pituitary hormones is developing into a much more complex set of releasing and inhibiting factors which may or may not act independently of each other. In addition the releasing factors may have effects other than on the pituitary, e.g., behavioral effects. TRH has been demonstrated to relieve depression in some patients (Kastin, Ehrensing, Schalch and Anderson, 1972; Plotnikoff et al., 1972). Another interesting development in this area is the discovery of a wide distribution of releasing factors throughout the brain, and not just localized to the hypothalamus (Oliver, Eskay and 17
Ben-Jonathan, 197*+; Jackson and Reichlin, 1974). Thus the suggestion has been made that, at least TRH may have an extrathyroidal brain func tion as a central neurotransmitter. This could potentially explain the anti-depressive action of TRH in depressed patients. Therefore, the role of releasing and inhibiting factors in the regulation of pituitary is not as clearcut as was once suggested, and remains to be clarified in future experiments.
Regulation of the Pituitary-Thyroid Axis
The pituitary has been known for more than a century to have some relationship to the thyroid gland. In 1851 Niepce (as cited in
Martin and Reichlin, 1971) observed that the pituitary was frequently enlarged in cretins. In 1922, Smith and Smith (as cited by Purves,
1964) first demonstrated that the function of the thyroid gland was dependent upon hormone secretion by the pituitary gland. They found that the atrophic thyroid glands in hypophysectomized tadpoles would hypertrophy following injections of bovine anterior pituitary tissue.
Subsequently, in 1926 Smith (as cited by Purves, 1964) developed the technique for hypophysectomizing rats and using this technique he demonstrated that pituitary extracts had thyroid-stimulating activity.
Aron, van Caulaert and Stahl (1931) identified a reciprocal interac tion between the pituitary and the thyroid gland. An hypothesis for the negative feedback regulation of the pituitary hormone, TSH, by the level of thyroid hormones was first set forth by Hoskins in 1949-
Early studies of this system were based on the measurement of basal metabolic rate, the measurement of thyroid hormones in the plasma,
thyroid gland turnover of iodine, and the bioassay of thyrotropin in
the plasma or urine. The recent development of specific radioimmuno
assays for TSH and thyroid hormones, triiodothyronine (T,) and thyroxine (T^), permits a far more precise evaluation of pituitary-
thyroid regulation. The following description of the regulation of the hypothalamic-pituitary-thyroid axis will deal primarily with hypo thalamic and pituitary portions of this system. A more detailed description of the synthesis, release, and action of thyroid hormones can be found in several excellent reviews (see Dumont, 1971; Field, 1970;
Liberti and Stanbury, 1971; and Larsen 1972a).
Hypothalamic Regulation of TSH Secretion
The ideas and methods associated with the newly discovered hypophysial portal system and chemotransmitter hypothesis (Harris,
19^8) were rapidly applied to the investigation of the pituitary-thyroid axis. The hypothalamus was first implicated as an integral part of thyroid function when Greer (1951) demonstrated that lesions in the anterior hypothalamus of the rat led to severe disturbances in TSH secretion from the pituitary gland. TRH has since been extracted and isolated from the hypothalamus, in particular the median eminence.
With the refined techniques of collecting hypophysial portal blood by
Porter and Smith (1967), and assay techniques of Redding and Schally
(1969), TRH has been identified in portal blood and it increases in concentration upon cold exposure (see discussion above). 19
A number of investigators have now delineated the area of the
hypothalamus which when destroyed results in decreased thyroid function
(Bogdanove and Halmi, 1955; D'Angelo and Traum, 1958; Florsheim, 1958).
Hypothalamic lesions decrease several indices of thyroid function in
cluding uptake of radioactive iodine and release after incorporation
into iodoamino acids (D'Angelo and Traum, 1958; Florsheim, 1958; Kovacs,
Lissak and Endroczi, 1959)• Halasz, Florsheim, Corcorran and Gorski
(1967) have demonstrated by hypothalamic deafferentation that if the
entire medial basal hypothalamus was left intact this hypothalamic
portion alone would maintain near normal basal TSH levels. A number
of experiments using lesions in various hypothalamic regions have demon
strated that the paraventricular nuclei are very important in the regu lation of TSH secretion. Lesions in the anterior hypothalamus in the
paraventricular region led to a marked decrease in plasma and pituitary
TSH levels (Van Rees and Moll, 1968; Martin, Boshans and Reichlin,
1970). Furthermore, it was demonstrated that these rats could still respond to a decrease in thyroid hormone levels (i.e., thyroidectomy)
with increased TSH secretion.
Electrical stimulation is the best method currently available
to establish the dependence of the anterior pituitary on the CNS.
D'Angelo and collaborators (D'Angelo and Snyder, 1963; D'Angelo, Snyder and Grodin, 196*0 found that stimulation of an extensive region in
cluding the preoptic region and median eminence was effective in
eliciting pituitary TSH depletion. Martin and Reichlin (1970) have
confirmed that stimulation of a rather large hypothalamic region is 20
effective in elevating TSH levels. This broad effective area for
eliciting TSH release is not surprising in light of the correspondingly
broad distribution of TRH in the hypothalamus (Brownstein, Palkovits and Saavedra, 197*0 • Also significant quantities of TRH have been
found outside of the hypothalamus in other parts of the brain (Jackson and Reichlin, 197*0• It thus appears that although brief transient increases in TSH levels may be elicited by electrical stimulation, a prolonged stimulation capable of causing significant thyroid responses is not possible. ,
Recently an enzyme for synthesizing TRH, TRH synthetase, has been found in the brain (Reichlin et al., 1972). The increase in TSH synthetase which normally follows cold exposure was abolished with hypothalamic lesions (Reichlin et al., 1972). Administration of norepi nephrine or dopamine to incubated hypothalamic tissue was found to stimulate the synthesis of TRH, indicating that synthesis may be under monoaminergic control (Grimm and Reichlin, 1973). Furthermore, TRH synthesis may be under positive feedback control of thyroid hormones since synthesis was stimulated by the presence of thyroid hormones and was decreased with hypothyroidism in the rat (Reichlin et al., 1972).
Environmental Stimuli for TSH Secretion
Cold. TSH secretion has been found to vary inversely with ambient temperature (Dempsey and Astwood, 19^3)• This cold response is evident in the human infant at birth and for about kQ hours thereafter, and may be prevented if the infant is kept very warm after birth 21
(Fisher and Odell, 1969; Fisher and Odell, 1971; Wilber and Baum, 1970).
However, no response to acute cold is observed in the adult human (Berg et al., 1966; Hershman et al., 1970)- Acute cold may stimulate a rapid increase in TSH release in the adult rat and rabbit (Reichlin et al., 1972;
Hershman et al,, 1970; Itoh et al., 1966; Fortier et al., 1970). While a decrease in ambient temperature is capable of activating the thyroid, local hypothalamic cooling in the goat, rat, and baboon will also stimulate thyroid secretion. Enhanced TSH secretion has been demon strated after lowering core temperature in animals at constant ambient temperature. These data suggest that the receptors for acute cold ex posure may lie within the brain (Andersson et al., 1962; Reichlin,
196'+). On the other hand, the hypothalamic core temperature showed no change during acute cold exposure, suggesting that the cold response was mediated by peripheral cold receptors (Martin and Reichlin, 1970).
Stress. Inhibition of TSH secretion has been shown to accom pany acute stress which activates the pituitary-adrenal system (Bogoroch and Timiras, 1951; Brown-Grant, Harris and Reichlin, 195^; Ducommun,
Salciz and Guillemin, 1966; Dewhurst et al., 1968). An inverse relation ship between ACTH and TSH was postulated after the additional observa tion that stress-induced ACTH secretion was decreased during TRH- induced TSH secretion (Salciz and Guillemin, 1965)* Glucocorticoids suppress thyroid function at least partly by inhibition of pituitary
TSH secretion in both man and rat (Wilber and Utiger, 1970). The fact that electrical stimulation of the mid- and posterior median eminence resulted in increased thyroid activity only after adrenalectomy (Harris, 22
196^; Harris and Woods, 1958) also suggested that endogenous glucocor ticoids may exert some regulatory influence on the system (Nicoloff,
Fisher and Appleman, 1970), Administration of large doses of dexametha- sone elicited an acute decrease in serum TSH in normal subjects and an even greater decrease in hypothyroid human subjects (Wilber and Utiger,
1970). Since the TSH response to TRH was normal with glucocorticoid treatment, these steroids may be acting at the hypothalamic level to inhibit the release of TSH in both man (Hershman and Pittman, 1970) and the rat (Wilber and Utiger, 1970).
A large number of experiments have resulted in conflicting data regarding the effects of glucocorticoids on the hypothalamic-pituitary- thyroid axis. A recent report by Brown and Hedge (1973) suggests that the apparent discrepancies may be the result of the dose and timing differences in the various experiments. According to their study of
TSH Becretion, the TSH response to TRH and cold stress is a function of the level of glucocorticoids given and the time at which they are administered.
Mechanism of Action of TRH
Plasma TSH concentrations rise when TRH is administered paren- terally (Schally et al., 1968; Bowers et al., 1970), orally, or infused into hypophysial portal vessels (Porter et al., 1971). When given intravenously TRH is effective at nano- or microgram levels and elicits measurable plasma TSH increases within one or two minutes in animals and humans (Guillemin, Burgus and Vale, 1971)• A dose-response 23 relationship between TSH levels attained and TRH administered has been
demonstrated (Schally and Bowers, 1970; Bowers et al., 1970). Esti
mates of the multiplication factor of TRH-induced TSH release in vivo indicate a factor of 1:100,000; i.e., one molecule of TRH may elicit release of 100,000 molecules of TSH (Martin and Reichlin, 1970),
After administering radioactive TRH to mice or rats, the radioactivity is concentrated in the pituitary (Redding and Schally, 1972; Redding and Schally, 1971)- TRH is rapidly inactivated by rat and human plasma
(Labrie et al., 1972; Vale, Burgus, Dunn and Guillemin, 1971; Poirier et al., 1972) primarily by enzymatic cleavage of the amide group at the prolyl end (Nair, Redding and Schally, 1971)• The half-life of TRH in the blood of the rat is about 4 minutes (Redding and Schally, 1971;
1972) with a considerable portion of TRH being excreted in the urine in an unchanged form.
The mechanism by wh'ich TRH stimulates the secretion of TSH has not yet been fully elucidated. The evidence suggests that cellular secretory processes are initiated by the attachment of the TRH molecule to specific membrane receptors on the thyrotropic cell in the anterior pituitary. Actinomycin D, puromycin and cycloheximide, inhibitors of protein synthesis, do not alter the immediate release of TSH in re sponse to TRH, but these inhibitors do prevent thyroid hormone from blocking TSH release induced by TRH. It appears, therefore, that the
TRH causes a discharge of stored TSH from the pituitary, and in order for thyroid hormones to block this effect protein synthesis is required
(Schally and Redding, 1967; Bowers, Lee and Schally, 1968; Vale, Burgus 24
and Guillemin, 1968; Wilber and Utiger, 1968). Specifically, it has
been found that oligomycin and dinitrophenol, drugs which block the
accumulation of high-energy phosphate compounds by mitochondria, block
TSH release in response to TRH. This suggests that energy is required for the release of TSH from the pituitary thyrotropes.
The secretion of TSH appears to occur via the model which
Douglas and Poisner (1964) proposed for stimulus-secretion coupling.
The activity of TRH is dependent upon a critical amount of extracellular calcium (Ca++) ions. Excess potassium ions in the extracellular medium || with sufficient Ca present can simulate the action of TRH and elicit
TSH release from in vitro pituitary cultures or incubations (Valet
Burgus and Guillemin, 1967a; Vale and Guillemin, 196?; Geschwind, 1969).
Excess potassium ions or TRH may lead to depolarization of the pitui tary cell membrane, and thus increase the permeability of the cell to
Ca++ and mediate TSH release as suggested by Milligan and Kraicer
(1971)" They found transmembrane potential reversal in pituitary cells with high potassium ion concentration.
There is a limited amount of evidence suggesting that TRH may stimulate TSH secretion by activating a system to increase cyclic AMP
(cAMP) concentration. TRH stimulates the formation of cAMP (Wilber,
Peake and Utiger, 1969; Zor et al., 1970), and several prostaglandins also stimulate cAMP production, particularly However, in the absence of calcium, cAMP formation could still be stimulated by TRH, while TSH production was inhibited. Increasing intracellular cAMP levels, by blocking phosphodiesterase activity with theophylline, or 25 addition of dibutyryl cAMP to pituitary incubations enhances the pro duction of ACTH, GH, LH in addition to TSH. All of the experiments thus far investigating the mechanism of action of TRH have utilized hetereogenous pituitary cell incubations in which the thyrotropes are merely one of several types of pituitary cells. Thus the effects observed in these experiments are the sum total of the responses of all of these cells* If the specific action of TRH on the thyrotrope is to be identified, techniques of isolating these cells must be refined.
Thyroid Hormone Feedback Control of TSH Secretion
The response to pituitary stalk section or pituitary trans plants in the rabbit were surprising in light of previous studies. The investigators had expected that thyroid function would be virtually abolished if the hypophysial portal system was interrupted. Instead the thyroid secretion was partially reduced, but it was still greater than that observed in the hypophysectomized animal (Brown-Grant, Harris and Reichlin, 1957)- Thus it appeared that the pituitary could main tain some of its secretory capacity even with the isolation from the portal vessels. In addition to those data, the systemic injection of thyroxine was found to produce a transient decrease in thyroid secre tion. Therefore, it was concluded that the hypothalamus could exert tonic control of basal TSH levels, and thyroid hormones could modulate pituitary secretion of TSH by negative feedback at this site.
Current available evidence suggests that the level of plasma
TSH is dependent upon the level of thyroid hormones; plasma thyroid 26 hormones can be viewed as the controlled variable in this feedback sys tem. As mentioned previously thyroid hormones have been shown to inhibit TRH-stimulated TSH release in vitro (Bowers et'al., * 1967; Vale et al., 1967b; Vale, Burgus and Guillemin, 1968; Bowers, Lee and
Schally, 1968) probably through the formation of an inhibitory protein.
Large amounts of TRH may overcome this inhibition which suggests that there may be competitive interaction between TRH and thyroid hormones.
Increasing doses of T^ have been shown to cause progressively greater decreases in plasma TSH levels in both the rat and man (Reichlin et al.,
1970; Reichlin and Utiger, 1967).
The primary site of negative feedback control of TSH release by thyroid hormones is currently believed to be at the level of the pitui tary (Reichlin et al., 1972). However, there is a limited amount of evidence indicating that thyroid hormones act on the brain to alter TRH synthesis and/or release. For instance, crystalline thyroxine im planted into the anterior hypothalamus, but not the cerebral cortex, inhibited thyroidal radioiodine release (Kajihara and Kendall, 1969;
Chambers and Sobel, 1971)* Hypothalamic deafferentation blocked the inhibition of TSH release due to thyroxine pellets which were implanted into the preoptic region (Joseph and Knigge, 1972). Some of these results are questionable because the thyroxine might have been trans ported to the pituitary from the brain by the hypophysial portal ves sels. 27
Circadian Rhythm of TSH Secretion
The diurnal secretory pattern of some pituitary hormones, such as ACTH and GH, is thought to be mediated by rhythmic secretion of the specific hypothalamic-releasing factor. Significant diurnal variation in plasma and pituitary thyrotropin secretion has been found in the rat although there is disagreement as to when the peak actually occurs
(Bakke and Lawrence, 1965; Singh et al., 1967). Studies in the human have not consistently provided evidence for such variations (Hershman and Pittman, 1971; LeMarchand-Beraud and Vannotti, 1969; Webster, Guan- sing and Paice, 1972; Vanhealst et al., 1972; Alford et al., 1973)•
The levels of TSH in rats have only small variations throughout the day. These levels are inversely proportional to the levels of corti- costerone (Bakke and Lawrence, 1965; Singh et al., 1967; Retiene et al., 1968) indicating that ACTH and TSH levels may be inversely related. The possible diurnal variation of TSH in the human may also be inversely related to glucocorticoid levels (Nicoloff, Fisher and
Apple man 1970).
Mechanism of Action of TSH
TSH is a glycoprotein hormone which is synthesized and stored in the thyrotropic cells of the anterior pituitary. It is composed of a and (3 subunits (Pierce, 1971) of which the /3 subunit confers biologi cal specificity to the TSH activity. The a subunit is identical to that found in FSH (Shome and Parlow, 197*0* LH (Pierce, 1971) and HCG
(Pierce, 1971)- A thyroid cell membrane receptor that binds TSH has been isolated (Pastin, Roth and Macchia, 1966). TSH binding to the 28 receptor is the first step in the initiation of a number of effects which TSH has on the thyroid gland. Once bound to the receptor TSH activates adenyl cyclase stimulating the formation of cAMP which acts as the "second messenger" for the hormone in the thyroid cell (Dumont,
1971)- Dibutyryl cAMP mimics TSH stimulation of thyroid secretion demonstrating that TSH acts through adenyl cyclase activation. Wolff and Jones (1971) have suggested that there are separate sites of action for the binding of TSH and the activation of adenyl cyclase. They demonstrated inhibition of TSH-induced iodine uptake by thyroid cells after treatment with such drugs as phenothiazine, some detergents, and cobramine B, whereas fluoride-activated adenyl cyclase was stimulated under these conditions. In addition, TSH stimulation was shown to occur with excess potassium concentration, but not with excess fluoride ions.
Thus it appears that although TSH may have an effect on cAMP levels the mechanism of this effect is still unclear.
Prostaglandins and the Hypothalamic- Pituitary-Endocrine Systems
Prostaglandins (PGs) are a ubiquitous group of long chain hydroxyunsaturated fatty acids with diverse pharmacological actions and unidentified physiological actions. Their activity in animal species is variable but they are active in microgram doses. While searching for epinephrine in animal tissues in the 1930*s von Euler (1937) found a substance extracted from human semen that stimulated smooth muscle preparations and lowered the blood pressure of rabbits. At first the extract was thought to be produced by the prostate gland, hence the 29 name prostaglandins, but later it was found to he secreted by the seminal vesicles* PGs are acidic and lipid-soluble making them dif ferent chemically from other known substances with similar biological actions (e.g., histamine, acetylcholine). Once methods for purifying lipid-soluble substances were developed, Bergstrom and Sjovall (1960a;
1960b) isolated two compounds which behaved differently on partition between ether and phosphate buffer. The one more soluble in ether was named PGE, and PGF was more soluble in the aqueous phosphate buffer.
They demonstrated that PGs were closely related derivatives of pros- tynoic acid, which is not a naturally occurring substance. Vertebrate seminal fluid represents the richest animal source of PGs with human seminal fluid having no less than 13 PGs (Hamberg and Samuelsson, 1966;
Bygdeman and Samuelsson, 1966). Pickles (1957) isolated menstrual stimulants from the female reproductive tract, and later PGE£ and PGF^ were found to be present here.
Structure and Synthesis
PGs are a class of 20 carbon fatty acids with a cyclopentane ring with two aliphatic side chains, one a carboxylic side chain and the other sua alkyl side chain. The PGs are subdivided into groups named by letter (i.e., PGA, PGB, PGC, PGE, PGF) according to the degree of saturation, and the type and position of radicals in the cyclopen tane ring. The degree of unsaturation of the side chains is indicated by a numerical suffix. The suffix a indicates that both hydroxyl groups are on the same side of the ring and that they are on oppo site sides (Shaw and Ramwell, 19&9)- 30
PGs have been identified in most mammalian tissues in small amounts, but only rarely are they found circulating in the blood. Most tissues have the capacity for synthesizing PGs from essential fatty acids, although it is not known what triggers their synthesis and re lease. Bergstrom, Danielsson and Samuelsson (1964) and Van Dorp et al.,
(1964) found that arachidonic acid could be converted to PGEg by a microsomal enzyme system, PG synthetase, in sheep seminal vesicles.
This PG synthetase is found distributed in most mammalian organs.
PGFis also formed from arachidonic acid (Anggard and Samuelsson,
1965)1 while PGF^ and PGF^q are similarly formed from dihomo-5-linolenic acid (an immediate precursor of arachidonic acid). Although the mech anism of synthesis has been reasonably well elucidated the number of component enzymes in the multi-enzyme complex is not known. Mechanical, endocrine and neural stimuli can be effective in stimulating synthesis and release, probably by triggering a membrane bound enzyme associated with the microsomal fraction of homogenates (Lands, Lee and Smith, 1971).
Glutathione enhances the yield of E compounds partly at the expense of
PGF but other sulfhydryl groups are less effective (Hamberg and
Samuelsson, 1967)# Only free unesterified fatty acids can be utilized by PG synthetase (Granstrom, Lands and Samuelsson, 1968; Van Dorp,
1967)* Unsaturated fatty acids are to a large extent incorporated into phospholipids in membranes from which they are released by phospholi- pase A. Free arachidonic acid thus formed may be converted to PGs under the influence of PG synthetase (Hamberg and Samuelsson, 1966;
1967)* PG synthetase can be inhibited by essential fatty acid analogs 31 including tetraynoic acid (Ahern and Downing, 1970), and by several non-steroidal antiinflammatory agents (i.e., aspirin and indomethacin
/Vane, 1971; Smith and Willis, 1971; Ferreira, Moncada and Vane, 19737).
These inhibitors of PG synthesis may provide an important tool for investigating the physiological role of prostaglandins. The activity of prostaglandins can be inhibited by antagonists such as 7-oxa-13- prostynoic acid (Fried et al., 1969)t or polyphloretin phosphate which stabilizes the cell membrane (Beitch and Eakins, 19&9)-
Metabolism
Due to rapid metabolism of PGs in the liver, lungs and other tissues (Hamberg, 197^; Samuelsson et al., 1971) the blood half-life of
PGs in the circulation is only a few minutes (Raz, 1972). More than
8056 of PGE^ is metabolized during a single passage through the liver or lungs (Ferreira and Vane, 1967; Vane, 1969)* probably by prostaglandin dehydrogenase•
The major route of degradation is by prostaglandin dehydrogenase which catalyzes oxidation of the C15 hydroxyl group to form the respec tive ketone (Anggard and Samuelsson, 1966, as cited by Marrazzi and
Andersen, 197*0* A 8"^ reductase which splits the C13 and Cl^ double bond has been found in pig liver and spleen (Larsson and Anggard, 1970).
and <0- oxidations have also been demonstrated in rat liver (Hamberg,
1968) and guinea pig liver (Israelsson, Hamberg and Samuelsson, 1969)* respectively. Numerous metabolic breakdown products of prostaglandins have been found in the urine (Granstrom, 1967)- 32
Mechanism of Action
The first suggestion that PGs may exert some of their effects at the level of the cyclic AMP system was made by Steinberg and associates (196*0 who reported that PGE^ acted as an antagonist of both lipolysis and phosphorylase activation in rat epididymal fat cells stimulated by lipolytic hormones. The results of experiments utilizing intact epididymal fat pads from rats was at first baffling; PGE^ in hibited epinephrine-induced cAMP production, but PGE.^ alone stimulated the production of cAMP. Later it was found that isolated intact adi pocytes derived from epididymal fat pads did not accumulate cAMP in response to epinephrine if PGE^ was added to the incubation medium, or would decrease to control levels if PGE^ were added after epinephrine had already stimulated the cAMP production. The first observation of both effects was apparently due to the cellular heterogeneity of intact fat pads since it was also found that the stromovascular elements were stimulated by PGE^ to produce cAMP. PGs have also been shown to alter other cAMP mediated events. In the toad bladder, PGE^ can block the movement of water and ions which is stimulated by ADH or theophylline
(Orloff, Handler and Bergstrom, 1965). These data thus suggest that the PGs may be acting by altering intracellular levels of cAMP. A number of hormones that increase cAMP formation have also been found to increase the concentration of PGs in a variety of tissues (Flack,
1973)i in addition to stimulating the release of PGs from these tissues
(Flack, 1973). These observations have opened the way for a general investigation of PG effects on the cAMP second messenger system. 33
Systemic Effects of Prostaglandins
Prostaglandins have been demonstrated to have a variety of
effects on the gastrointestinal, cardiovascular, respiratory, renal,
and nervous systems (for review see Ramwell, 1973)# The present dis
cussion of systemic effects of PGs has been limited to an overview of selected anterior pituitary hormones in this rapidly developing field.
Hypothalamic-Pituitary-Ovarian System, The role of prostaglan
dins in reproduction has been pursued ever since von Euler (193*0 first
extracted them from human semen- Thus far PGs have been shown to be
involved in nearly all phases of the regulation of reproduction.
Zor, Kaneko, Schneider and others (1969) and Zor, Kaneko, Schneider and Field (1970)found that ovine hypothalamic extract stimulates the release of LH and stimulates adenyl cyclase activity in male rat pitui- taries _in vitro. Aside from the extract PGE^ was also found to in crease significantly cAMP levels in the rat anterior pituitaries. This was later confirmed by several investigators (Ratner et al., 197^;
Makino, 1973) who also found that PGs would elicit LH release and stimulate cAMP production. In addition, Makino (1973) showed that stimulation of LH secretion by LHHF was decreased by simultaneous treatment with the PG antagonist 7-oxa-13-prostynoic acid. This same
PG antagonist has been shown to inhibit the LH secretion in response to high K+ levels or LHRH. Thus it appears from these in vitro experi ments that there may be a PG receptor involved with the release of LH.
A similar action of PG has been demonstrated in vivo (Spies,
Norman and Campbell, 1973) PGE administered directly into the third 34 ventricle of the rat during proestrus stimulated LH release and induced ovulation. Following direct microinjection of PGE^ or PGE2 into the rat anterior pituitary plasma LH levels were increased (Sato, Hirono,
Jyujo and others, 1975)- Indomethacin, a PQ synthesis inhibitor! has been shown to inhibit basal LH concentrations which returned to con trol levels following treatment with PGPga Jyuj0* Hirono and lesaka, 1975)- Saksena and coworkers (1975) demonstrated that indo- methacin treatment significantly reduces plasma LH and testosterone levels after 30 days of treatment.
Although there are few data concerning the effect of PGs on
FSH the data that are available suggest that PGs may be involved in the regulation of FSH secretion. Pharriss, Wyngarden and Gutknecht
(1968) found no effect on the LH content of the rat pituitary after
PGF2a treatment but they did observe increased FSH content. This effect on FSH is supported by one other report (Sato et al., 197*0 that intravenous PGEg led to increased plasma FSH concentrations in male rats.
So far PGs have not been shown to be involved with early fol licular development but seem to play a direct role in the ovulatory process. Indomethacin and aspirin have been shown to inhibit ovulation in the rat and the effect can be overcome with simultaneous administra tion of PG (Orczyk and Behrman, 1972). This was confirmed by Armstrong and Grinwich (1972) in the rat and rabbit. Orczyk and Behrman demon strated that the interval between the time of expected ovulatory surge and the indomethacin administration was important. If the interval 35
was 3 hours LH could overcome the block (Behrman, Orczyk and Greep,
1972). Thus it has been suggested that both central and gonadal effects
of indoraethacin were observed.
Although indoraethacin inhibited ovulation, progesterone syn
thesis was apparently unaffected. Both acute steroidogenic activity
(Grinwich, Kennedy and Armstrong! 1972) and progesterone levels were
found to be normal during the cycle (O'Grady et al., 1972), in spite
of retention of the mature ovum. LeMaire et al. (1973) reported a
direct role of PGs in the follicular rupture process after direct
measurement of follicular PG concentrations in the rabbit following
an ovulatory dose of gonadotrophin. Tsafrir, Koch and Lindner (1973)
demonstrated that the maturation of ova is not impaired by indomethacin.
Pharriss and Wyngarden (1969) demonstrated a luteolytic action
of PGF2a in the rat and suggested that it was due to a collapse of
ovarian venous secretion. Later they modified this suggestion to de
scribe decreased blood flow to the ovary (Pharriss* Cornette and
Gutknecht, 1970). Behrman, Yoshinaga, Wyman and Greep (1971) and Behrman,
Yoshinaga and Greep (1971) were unable to show any change in venous
secretion from the ovary even with markedly reduced progesterone levels
induced by PGFg^ They later demonstrated that PGFacts directly on
the corpus luteum to prevent the gonadotropic activity in the rat
(Behrman, MacDonald and Greep, 1971)•
Hypothalamic-Pituitary-Adrenal System. As early as 1957*
George and Wray demonstrated that lesions of the median eminence 36 completely blocked the adrenal ascorbic acid depletion which normally occurs within one hour after administration of aspirin, a PG synthesis inhibitor. Later PGE^ and PGE^* but not PGF^fl or PGF2QI were found to stimulate ACTH release in the rat (de Wie.d et al., 1969).
Flack, Jeesup and Ramwell (1969) have shown that PGE^ and PGF^ stimulate corticosteroidogenesis in the adrenal. In vitro studies indicated that the latency of the response to PGs is similar to the latency to ACTH and cAMP (Flack and Ramwell, 1972). However, the dura tion of elevated corticosteroidogenesis in response to PGs is much shorter than that observed with ACTH and cAMP (Flack and Ramwell, 1972).
Thirteen-prostynoic acid had no effect in inhibiting ACTH or PGE^ in duced synthesis of corticosteroids (Flack, 1973)- PGE2 and ACTH effects were additive and, thus, they concluded PGE£ may potentiate the action of ACTH. This has been disputed by Gallant and Brownie (1973) who found that in vivo PGE2 and ACTH effects were not additive in hypo- physectomized rats. Using a large dose of ACTH (8 U/rat) PGEg was found to reverse the inhibitory action of indomethacin on ACTH-induced corti- costerone release. PGE^ alone or with ACTH had no effect. Peng, Six and Munson (1970) also found no in vitro adrenal stimulation with PGE^ after hypophysectomy, but did find adrenal activity was stimulated in the intact rat. They concluded on pharmacological bases that the PGE^ must act at a higher level to stimulate ACTH release. This conclusion is supported by Hedge (Hedge and Hanson, 1972; Hedge, 1976) who found
PGE^, F1^, FP/xt A and B were effective in stimulating ACTH when stereo- taxically microinjected into the medial basal hypothalamus, but not 37 in the anterior pituitary. These results suggest that PGs may act to mediate CRF release from the hypothalamus and, in addition, may act at the adrenal to cause corticosteroidogenesis.
Hypothalamic-Pituitary-Thyroid System. Although there is a considerable amount of evidence that PGs play a role in the regulation of thyroid function, the data available on the possible action of PGs at the pituitary in the release of TSH is rather limited. Vale and co workers (1972) have found that PGE,, directly stimulated the release of
TSH from incubated putuitary cells. When administered in vivo directly into the rat anterior pituitary, the PGs alone have no effect on TSH secretion (Brown and Hedge, 197*0 • However, under these same condi tions, all PGs tested were found to increase the TSH response to exogenous TRH (Brown and Hedge, 197*0• This suggests that in the intact hypothalamic-pituitary-thyroid system the PGs may modulate the action of TRH on the pituitary.
Although PGs have been found in high concentrations in the human thyroid (Karim, Sandler and Williams, 1967)1 the role of the PGs in thyroid function is unclear at this time. Exogenous PGs can repro duce several of the effects of TSH on the thyroid, including in vitro stimulation of thyroid glucose oxidation (Burke, 1970? Dekker and
Field, 1970; DeRubertis et al., 1975; Kaneko, Zor and Field, 1969;
Onaya and Solomon, 1970; Rodesch et al., 1969? Zor, Bloom, Lowe and
Field 1969; Zor, Kaneko, Lowe and others, 1969)• colloid droplet for mation (Burke, 1970; Onaya and Soloman, 1970; Neve and Dumont, 1970; 38 1^1 Sato et al., 1972) and I release (Burke, 1970; Onaya and Solomon,
1970; Burke, 197*0• PGE^ activates the thyroid plasma membrane adenyl cyclase (Kowalski, Sato and Burke, 1971*-; Mashiter, Mashiter and Field,
197^; Wolff and Jones, 1970) and the formation of cAMP (Kaneko, Zor and Field, 19^9; Zor, Kaneko, Schneider and others, 19&9), thus pro viding a possible mechanism for the mimicry of TSH action.
Intracellular PGs have been shown to increase 30-80# in the thyroid in response to TSH (Yu, Chang and Burke, 1972) suggesting the presence of PG synthetase in this tissue. This effect has also been observed in rat and mouse thyroid glands (Burke, 1973) and it was hor- monally specific to TSH and reproducible with DBcAMP (Burke, 1973;
Burke, Chang and Szabo, 1973) or phosphodiesterase inhibitors (Burke et al«, 1973)- Aspirin and indomethacin block the stimulatory effect of TSH or DBcAMP on mouse thyroid PG levels (Burke, 1972). These data suggest that the effect of TSH on PG synthesis is mediated through the cAMP second messenger system.
A PG receptor in the thyroid plasma membrane has also been found (Moore and Wolff, 1973)• Various PGs were found to stimulate adenyl cyclase activity as determined by 3H-adenine incorporation into <2 H-cAMP in dog thyroid slices (Ahn and Rosenberg, 1970; Field et al.,
1971).
Dumont (1971) has cited much evidence to support the concept that the effects of TSH on iodide trapping, organification of iodide, endocytosis of colloid, and thyroid hormone release are mediated by the formation of cAMP. In addition to 131I release, has been 39 observed to stimulate the organic binding of iodine (Rodesch et al.,
1969; Madaoui, Rappaport and Nunez, 197^; Ahn and Rosenberg, 1970) and to reduce the thyroid-to-medium ratio for iodine (Ahn and Rosenberg,
1970) which also occurs with TSH or dibutyryl cAMP stimulation. Col loid droplet formation is stimulated by PGE^ in vitro in dog (Neve and
Dumont, 1970) and sheep (Burke, 1970) thyroid slices and in vivo in mice (Melander, Sundler and Ingbar, 1973; Onaya and Solomon, 1970),
It, therefore, has been shown that PGB^ may mimic the action of TSH in stimulating the uptake and incorporation of iodine into iodoproteins and the secretion of these as thyroid hormones.
Several PGs have been reported to stimulate the oxidation of lif 14 C-l-glucose to CO2 which also occurs with both TSH and DBcAMP
(Dekker and Field, 1970; Field et al., 1971; Onaya and Solomon, 1970;
Rodesch et al., 1969; Zor, Bloom, Lowe and Field, 1969; Zor, Kaneko,
Lowe and others, 1969) in dog and sheep thyroid slices. Thus it appears that most of the effects of TSH on iodine metabolism, thyroid hormone secretion, and glucose oxidation may be reproduced by TSH, probably via binding to a specific plasma membrane receptor and acti vation of the cAMP second messenger system.
Aim of Present Study
The evidence for the involvement of PGs in the regulation of
TSH secretion is just beginning to appear. All experiments performed so far have utilized only the administration of exogenous PGs to either in vitro or in vivo preparations. One of these experiments demonstrated bo that although PGs themselves have no effect on TSH secretion, they
potentiate the pituitary responsiveness to TRH (Brown and Hedge, 197*0•
Although these findings suggest that endogenous PGs may modulate the action of TRH on the pituitary, no studies have yet investigated the role of endogenous PGs under physiological conditions. I propose that the manipulation of the endogenous level of PGs, rather than using pharmacological doses, may demonstrate a physiological role for the
PGs in the regulation of TSH secretion in the rat. The level of en dogenous PGs may be decreased by administering a PG synthesis inhibitor, indomethacin or aspirin. The drug will be administered either systemi- cally, which will result in decreased PG synthesis throughout the body, or in a local microinjection into the pituitary. Following the drug treatment, TSH secretion will be stimulated using cold exposure, TRH, or thyroidectomy (i.e., reduced negative feedback by thyroid hormones).
Blood samples will- be assayed for TSH, T^, and T^ concentrations through out the experiments via specific radioimmunoassays. CHAPTER 2
MATERIALS AND METHODS
Animals and Care
Female Sprague-Dawley rats from our breeding colony (Division of Animal Resources, University of Arizona Medical Center, started from
Charles River CD rats) weighing 130-275 g were used throughout these experiments. Generally, the weight range within an experiment was not greater than 60 g. The animals were housed in temperature and humidity controlled quarters with a cycle of 12 h of light and 12 h of dark.
Ordinarily, the animals were fed Purina Rat Chow and given either tap water ad libitum for intact rats, or 2# CaClg drinking water ad libitum following thyroidectomy. However, in one experiment (see Results) they were fed only a glucose solution via stomach tube three times daily
(9:00 a.m., 4:00 p.m., and 9:00 p.m.).
Surgical procedures, intravenous injections, and blood sampling were all performed under ether or sodium pentobarbital (4.0 mg/100 g
BW, i.p.) anesthesia. Intravenous injections were made via chronic in dwelling cannulae in the external jugular, or a lateral tail vein, and blood samples were taken via the indwelling cannulae, cardiac puncture, or decapitation. No anesthesia was used for injections and sampling in fats with chronically implanted jugular catheters.
4l k2
Surgical Procedures
Chronic Indwelling Jugular Catheters
In some experiments rats were prepared with chronic jugular catheters in order to avoid the necessity of anesthesia for intravenous injections and blood sampling. The chronic jugular catheters were placed according to the method of Steffens (1969) as modified by Brown and Hedge (1972). In brief, the method involves the use of silastic tubing (Dow-Corning No. 602-151t I.D. 0.025" O.D. 0.047") with adhesive tape collars to mark the correct insertion length. The catheter tip is notched on opposite sides in order to assure patency when the catheter tip is placed correctly in the superior vena cava just rostral to the heart. The distal end of the catheter is attached to an "L" shaped
1.5 cm length of tubing cut from a 22 gauge disposable needle. This
"L" adapter is tied between two No. 2, 1/4" flat head brass wood screws placed in the parietal bones of the skull. The area around the screws is then filled with dental cement. After placement of the catheter, it is filled with heparinized (500 U/ml) polyvinyl pyrrolidone (PVP) via
P.E. 50 polyethylene tubing and sealed with a 5 mm segment of P.E. 200 polyethylene tubing placed over the "L" adapter and P.E. 50 tube. The rats were given sodium pentobarbital anesthesia (4.0 mg/100 g BW) prior to surgery. Once surgery was completed, a broad spectrum antibiotic
(40,000 U Combiotic, Chas. Pfizer and Co., Inc., N.Y.) was administered, and once awake the rats were placed in individual wire cages where they were housed prior to the day of the experiment. Four days later the ^3 rats were brought to the laboratory and then housed overnight in indi vidual netal waste cans with food and water ad libitum. On the morning of the fifth day a P.E. 50 sampling tube which was filled with hepar- inized saline (150 U/ml) was attached to the "L" adapters after removal of the PVP-heparin solution. The rats were then left undisturbed for two hours prior to the experiment. This arrangement allowed free move ment of the animals with the sampling tube draped over the lip of the can, and permitted blood sampling and intravenous injections without disturbing the rat.
Thyroidectomy Procedure
Rats were thyroidectomized under light ether anesthesia. A
2 cm midline incision was made over the trachea and the thyroid located with blunt dissection by means of hemostats. The two lobes of the thyroid were quickly removed individually using a fine-pointed probe and fine forceps. A minimal amount of bleeding was observed at this time. After surgery the rats were housed and fed as usual except that they were given 2$ CaCl^ drinking water. The 2$o CaCl^ drinking water was found to be necessary when it was determined that the parathyroid gland was inadvertently removed with the thyroid. Although there is no evidence of interaction between thyroid and parathyroid hormones, this could represent a potential interference and remains to be in vestigated. The CaCl^ was not found to alter the plasma TSH levels in response to thyroidectomy or exogenous TRH (data not shown). 44
Stereotaxic Microinjection Procedure
Pituitary microinjections were made stereotaxically in pento barbital anesthetized (4.0 mg/100 g BW) rats according to the method of Hedge and associates (1966; Hedge, 1971). Injections were made through glass cannulae (200-250/* O.D.) at a rate of 1 fil/30 sec.
Following each injection 1 /il of toluidine blue dye was delivered be fore the cannula was removed. At the end of each experiment the ani mals were decapitated, and injection sites located by observing the dye in the pituitaries using a dissecting microscope fitted with a micrometer eyepiece.
Acute Cold Exposure
Rats were exposed to acute cold (4° C) in one of two ways.
Either the animals in their individual cages were quickly and quietly transferred to a cold room at 4° C, or the animals were housed indi vidually in an environmental chamber at 32° C for at least 3 h (to allow animals to become accustomed to the new environment) and the room was rapidly cooled to 4° C without the movement of the animals.
Blood samples were taken immediately prior to, and during cold exposure as described with the results of the individual experiments.
• Pharmacological Preparations
Synthetic thyrotropic releasing hormone (TEH) was a gift from
Dr. Clarence Gantt of Abbott Laboratories. The TRH was diluted to con centrations of 0.04 - 100 fig/ml with 0.9^ saline and used at doses of
0.01 - 25 /ig/100 g BW. Bovine thyrotropin (TSH) was purchased from ^5
Calbiochera and diluted to concentrations of 0.028 - 10 U/ral with 1% BSA
in 0.9% saline. TSH was administered at doses of 7 - 2500 mU/100 g BW.
Thyroxine (T^) was administered via a stomach tube twice a day at total
daily doses of 2, 3, or ^ fig/k ml 2# CaClg in 0.0001 - 0.0002 N NaOH/
100 g BW under brief ether anesthesia. Indomethacin (Ind) and aspirin
(Asp) were purchased from Merck, Sharp and Dohme, and from Whiteworth,
Inc., respectively, and were suspended in 15& gelatin (Gel) for sub
cutaneous injection. Unless indicated otherwise, the Gel, Ind, and
Asp were administered as described in Tables 7 and 8 (pp. 62 and 6*0.
Pure indomethacin for pituitary injection was a gift of Dr. Gayle
Orczyk of Merck Institute for Therapeutic Research, and was adminis
tered at a dose of 50 /ig/5 f*l 0.1 M sodium phosphate buffer, pH 7 A.
Toluidine blue dye was used at a concentration of 16 mg/ml 0.95& saline).
Radioimmunoassay of Plasma TSH, T-,, and T,,
Heparinized plasma samples were frozen and stored until
assayed. TSH was assayed using homologous radioimmunoassay materials
(TSH standard, TSH for iodination, and anti-rat TSH) provided by the
NIAMDD Rat Pituitary Hormone Distribution Program. The assay was per
formed as suggested in the kit using 75 or 100 /i1 of rat plasma, except
that 2& normal rabbit serum NRS) in 0.01 M phosphate buffered saline (PBS) was used in place of the recommended buffer. The TSH standard was added in doses ranging from 2.5 to 1000 mg per tube and run in triplicate. To each of the tubes containing standard or rat
plasma was added 100 /il of 0.1 M ethylenediaminetetraacetate (EDTA) in k6
PBS, 200 of antiserum diluted to 1:10,000 with 2% NRS-PBS, 100 #*1 of
125I-TSH ' diluted to 10-12,000 cpm with 2# NHS-PBS, and enough 2& NRS-
PBS to produce a final volume of 0,8 ml. The tubes were then agitated gently on a Vortex mixer, and incubated at room temperature for one day.
Sufficient anti-rabbit gamma globulin to precipitate maximally the an tibody bound labeled rat TSH was then added, and the tubes incubated at
C for one day. At the end of this period the tubes were centrifuged at 2600 HPM for 20 minutes, the supernatant was aspirated, and the pre cipitate was counted to 10,000 counts in a Nuclear Chicago IO85 gamma counter. The data are expressed as /xg of rat TSH/100 ml of plasma using NIAMDD rat TSH-HP-1 (0.22 U/mg) as standard. Carrier free for iodination of TSH was purchased from ICN Pharmaceuticals, Inc. TSH was iodinated using a procedure that is similar to that suggested in the NIAMDD kit, except that only 2 fig of TSH was iodinated using 0*5 mc
12^I, 50 Mg chloramine T in 0.01 M PBS, 10 fig Na^S^^ in 0.01 M PBS and
20 fil O.k M PBS. Only 20 - 30 seconds was allowed for the reaction to take place before adding the Na^S^O^ and transferring the mixture to the prepared Sephadex column. Specific radioimmunoassay for triiodo thyronine (T^) (Chopra, Ho, and Lam, 1972) and thyroxine (Chopra, 1972) were performed using antisera provided by Dr. D. A. Fisher, Harbor
General Hospital, Torrance, California. ^.oo mC/mg) and
- T, (SA 100 mC/mg) were obtained from Industrial Nuclear Co.,
St. Louis, Missouri, and L, - T^ and Ij - T^ were obtained from Cali- biochem. T^ radioimmunoassay was performed as described by Chopra and associates (1972) except that 100 /il of 0.1 M EDTA in 0.01 M PBS was 47
added, to each of the assay tubes as suggested by Dr. D. A. Fisher (per
sonal communication 1972) in order to chelate calcium which inhibits
interference of the antigen-antibody reaction by complement. In addi
tion, T^-free plasma was prepared by charcoal extraction of thyroid
hormones according to the method of Larsen (1972b). Anti-rabbit gamma
globulin was purchased from TLC antibodies, Tucson, Arizona.
Assessment of Thyroidal Activity
Thyroidal activity was assessed by either thyroid release of 125 T^ and/or T^, or thyroid release of I as previously described (Brown
and Hedge, 1972). In brief, thyroid release of 125I was determined 125 three days following the administration of carrier free I (25 /ic/ 125 rat, sc). The release of I is expressed as a response index (RI)
that is calculated as a percentage of cpm in the blood sample 2 hours
after TSH (10 mU/100 g BW) was administered, as compared to the cpm
in the blood sample prior to TSH.
Tk and T? Half-life 125 Half-lives of T^ were determined by administering I-T^ (5
Hc/rat; SA, 100 mc/mg) and taking sequential blood samples (150 fi1) 125 over the next 30 h via cardiac puncture. .The amount of I-T^ in the plasma samples was determined in an assay by adding 50 yl of plasma to
250 Hi of 1% normal rabbit serum in barbital buffer, pH 8.6, ionic strength 0.075 0$> NRS-BB), and 100 fil (150 fig) of 8-anilo-l-
naphthalene sulfonic acid (ANS) to each of the samples, which were then
mixed gently and incubated at 25° C for 30 minutes. Subsequently 100 kS
yl of rabbit anti-T^ serum diluted 1:100 in 1% NHS-BB was added to each
of the tubes, mixed gently, and incubated at 25° C for one hour. Sheep
anti-rabbit gamma globulin was added, the tubes were mixed gently, and
incubated at C for 2b h. The supernatant was aspirated after cen-
trifuging for 20 minutes at 2000 rpm, and the remaining precipitated
pellet was counted to 10,000 counts in a Nuclear Chicago 1085 gamma
counter. The results are expressed as mean cpm of plasma radioactivity
and are plotted semilogarithmically. From these plots the disappear
ance curves were defined by the method of least-squares fit. The half-
life (t^) of T^ removal was calculated from the slope of the disap
pearance curve.
Half lives of T, were determined by a procedure similar to that
for T^. (10 ptc/rat, SA 100 mc/mg) was administered and blood
samples (150 /il) were taken over the next 26 h via a chronic indwelling 125 jugular catheter. The amount of I-T^ in the plasma samples was de
termined in an assay by adding 100 /i2 of plasma to 600 /i1 of 1# NES-BB,
100 /il (250 fig) of ANS, 100 /il of 0.1 M EDTA in 0.01 M PBS, and 100 fO.
of rabbit anti-T^ serum diluted 1:2000 in 1% NES-BB to each of the
samples, which were then mixed gently and incubated at 1f° C for 20 h.
Subsequently 50 /il of sheep anti-rabbit gamma globulin was added, the
tubes were gently mixed, and incubated at b° C for another 2b h. The remainder of the procedure was exactly the same as that for the deter
mination of the half-life of T^. ^9
Data Analysis
Radioimmunoassay results for T^, T^ and TSH were analyzed on a
CDC 6400 computer utilizing the radioimmunoassay data processing pro gram (Rodbard, 1970) of Dr. David Rodbard, National Institute of Health,
Bethesda, Maryland. For our use this program was modified slightly by
Ms. Sheri Konen, Arizona Medical Center, Tucson, Arizona. The data specified in Figures 1, 6 and 11, and Tables 1, 3» ^ and 6 were calculated with a Hewlett-Packard 9100 B calculator using the linear portion of the curve and the method of least-squares fit. In every experiment, experimental and control samples were run in the same assay.
Data from separate assays were pooled only if the results of aliquots of a plasma pool run in each assay were not significantly different and within one standard error of the mean (SEM) of each other. Results are expressed as means accompanied by their standard errors. On a given figure, the number of animals in each group is indicated either parenthetically on line graphB, or given within the appropriate bar on histograms. Students' t-test was used to establish the significance of a difference between two groups. The paired t-test was used to establish the significance of a difference in two sequential samples from the same animal. The two-tailed test was used in all statistical com parisons. CHAPTER 3
RESULTS
Measurement of Pituitary and Thyroid Responses to Various Stimuli
Because the TSH, T^» and T^ radioimmunoassay procedures are such new techniques, the magnitude of the hormone response to accepted thyroid stimuli was initially studied. The effects of PG synthesis inhibitors can only be observed if the control group responds to the stimulus. Therefore, a variety of stimuli, e.g., acute cold exposure,
TRH, TSH, and thyroidectomy, were tested for their ability to elicit
TSH and/or T^ and T^ secretion. With these data then available, the appropriate stimuli and timing could be used to assess the effect of
PG synthesis inhibitors on the system. Therefore, the first section of results will deal with preliminary attempts to elicit TSH and/or T^ and T^ secretion.
Thyrotropin Response
Acute Cold Exposure. The first series of experiments utilized acute cold exposure as a stimulus for thyrotropin secretion. The re sults of a series of experiments using cold exposure in ether or pento barbital (PB) anesthetized rats are given in Table 1. In none of these experiments did TSH levels rise significantly in response to cold exposure, although mean TSH levels did tend to increase slightly after
50 Table 1. Plasma thyrotropin (TSH) levels in response to acute cold exposure with ether or pento barbital anesthesia. — Rats were acclimated to 32° C in an environmental chamber for 7 days prior to placing in 4° C cold chamber. Bats in experiments 30A. and 31A were acclimated to 32° C in an environmental chamber for 7 and 21 days, respectively, prior to cooling the chamber to 4° C. Data given for experiments 17 and l8 were calculated using the Hewlett-Packard 9100B calculator as described in the Materials and Methods section. Ether anesthesia was used in all experiments except 17 in which pento barbital was used.
Length of Cold Exposure (min) Plasma TSH (fig/100 ml) Expt. n Treatment 0 15 30 45 60 75 90 105 120
17 6 None 42.5 62.5 .73.7 .95-5 68.4 -12.5 -ho A 149.6 -27.4 1 9.5
18 5 None x59.4 +54.9 .54.2 .57-9 ^50.2 +5i.9 .,59.9 - 5-5 - 2.9 1 4.2 - 5.3 1 6.2 1 3.9 1 5.3
23 4 7 da, 32° 38.3 68.4 113.2 102.7 20.6 115.9 *12.3 ± 6.9 -13.0 tl9.4 ± 6.8 134.2
25 4 None 123-7 42.4 .57.2 44.2 45.0 45.0 -15-2 -11.7 -11.2 ill.8 1 3.1 1 5.2
30A 4 7 da, 32° 77-5 128.8 161.0 131.7 100.6 106.1 4.57.8 58.0 -21.9 - 7.4 131.7 -20.9 135.2 120.6 ll2.8 111.2
31A 3 21 da, 32° .55-5 90.8 114.6 104.3 43.3 36.9 59.0 77.1 -29.1 131.9 I27.8 144.9 1 5.2 I23.O 1 5.7 129.7 52
30 minutes of cold esqjosure (Fig. 1). Even acclimazation to 32° C for
7 or 21 days prior to cold e:q>osure did not increase the sensitivity
of the system enough to permit a rise in plasma TSH levels (Fig 1,
Table 1). Since mean TSH concentrations did increase in most of the
experiments but the individual animal variation was great, the anes
thesia was considered to be a possible inhibitory influence on the cold response. Therefore, subsequent experiments were performed in unanesthetized rats with chronic indwelling jugular catheters for blood sampling (Table 2). The results of the first experiment (Fig. 2) using this technique indicated a slight but significant increase (as deter mined by the paired J>test) in plasma TSH levels after 3 hours of warm acclimazation at 32° C and subsequent cold exposure in an environmental chamber. But when this experiment was repeated with a larger number of rats the magnitude of TSH rise did not change (Fig. 2, second experi
ment). In this second experiment mean plasma TSH levels were greater than that observed throughout the first experiment, probably due to interassay variation in the TSH radioimmunoassay.
The pattern of the response was similar in both experiments although the TSH levels were significantly increased (p <.02) at 90 minutes in the first, and significantly increased (p <.003^ at 60 min utes in the second. In spite of the rather large increase in the mean value at 120 minutes during the first experiment, the increase was significant only at the .05 level as determined by the paired Jt-test.
The temperature profile for the chamber is shown in Figure 3. As shown in the figure, the fall in temperature is greatest during the first 60 ANESTHETIC: •—• Ethar (4) O—o PB (6)
DURATION OF COLD EXPOSURE (min.)
Figure 1. Plasma thyrotropin (TSH) levels in response to acute cold exposure with ether or pentobarbital anesthesia. — The results for pentobarbital- treated (PB) rats were taken from one group of 6 rats. The results for ether-treated rats were pooled from four groups of 4 rats each, which were each sampled twice during the 120 minutes of cold exposure follow ing 7 days of warm-acclimation at 32° C. Plasma TSH levels for experi ments using pentobarbital were calculated using a Hewlett-Packard 9100B calculator as described in the Materials and Methods section# Table 2. Effect of acute cold exposure on plasma thyrotropin (TSH) concentrations in unanes- thetized rats. — Rats were acclimated to 32° C in an environmental chamber for 3 h, 7 da, or 21 da prior to cooling the chamber to 4° C.
Length of Cold Exposure (min) Plasma TSH (ug/l.00 ml) Expt. n Treatment 0 30 45 60 75 90 105 120
29 6 3 h, 32° 24.9 52.5 58.4 ,50.6 .77.4 i 3.0 - 7.4 -11.5 - 5.6 -19.6
30B 6 7 da, 32° J*.o 59.2 50.8 >7.7 40.0 47.3 40.9 58.6 i 7.5 il2.2 t 6.4 - 4.9 t 5.8 t 6.2 - 5.1 -11.0
31B V 21 da, 32° 28.2 .,70.2 68.8 .75.5 84.2 80.0 69.6 121.4 ^28.9 -15.2 -17.5 126.2 -30.1 ±30.1 -25.8 ^36.0
75, 12 3 h, 32° 50.9 65.5 72.0 69.6 59.8 77 - 7-8 - 9.4 - 6.5 ill.2 -13.5 Experiment: 100 -1 First (6) Second (12)
E 75- o 0 N CD 3 1 CO 50- I- < p<: .05 5 c/) 0- 25 -
0 J r~ T ~r~ ~~i 30 0 30 60 90 120 150 DURATION OF COLD EXPOSURE (min.)
Figure 2. The effect of acute cold exposure on plasma thyrotropin (TSH) concentrations in un- anesthetized rats. ~ The results are from two separate experiments in which the rats were acclimatized to 32° C for 3 h prior to acute cold exposure in an environmental chamber. The temperature of the chamber during this period is shown in Figure 3» 35
30
25-
20-
15-
10-
5-
0J 30 45 60 75 90 105 120 Time (min)
Figure 3- Temperature in environmental chamber during cooling from 32° C, 57 minutes of cold exposure. Therefore, it was not surprising to observe somewhat elevated TSH levels during this time.
Table 2 gives additional results for experiments in which the first group of rats were exposed to cold after 7, and after 21 days of warm-acclimazation (32° C). Neither period of acclimazation led to a greater increase in the TSH response to cold. Thus acute cold exposure was not found to be a reliable stimulus for eliciting increased TSH secretion. In the next series of experiments, TRH was chosen as a stimulant for TSH secretion.
Thyrotropin-Releasing Hormone. Although TRH was known to elicit TSH secretion in a dose-response fashion (as discussed in Chap ter 1), it was unknown what the level of TSH response would be in our laboratory as determined by our radioimmunoassay. In addition, the TSH response using several different anesthetics and blood sampling pro cedures were unknown. A dose of TRH was desired which would give a non-maximal TSH response, thus allowing either stimulatory or inhibi tory effects to be observed. Using pentobarbital anesthetized rats, the TSH response to 50 ng TRH was found to vary among five individual rats (Fig. Thus, it was decided to use another anesthetic, ether, in order to determine if the pentobarbital was a contributing factor
t to scattered results. Plasma TSH concentrations in response to 0 -
1250 ng TRH/100 g BW using ether anesthesia and cardiac puncture for blood sampling are shown in Figure 5« As seen in this figure, the 50 and 250 ng TRH doses gave significant, but non-maximal TSH responses at 10 minutes. These data are shown in semi-logarithmic form in 800-
o 600 H o*•» o 3
« 400 H
CO 5 0_ 200-
0 J 0 5 10
TIME AFTER TRH (min.)
Figure Plasma thyrotropin (TGH) concentrations of individual rats in response to thyrotropin-releasing hormone (TRH) anesthetized with pentobarbital ragA°0 g BU), — ftnta were calculated using a Hewlett-Packard 9100B calculator as described in Materials and "ethods.
\ji CO Figure 5- Plasma thyrotropin (TSH) concentrations in response to thyrotropin-releasing hormone (TRH) (0 - 1250 ng/100 g BW) in ether anesthetized rats.
Data were calculated with a Hewlett-Packard 9100B calcu lator as described in Materials and Methods. 59
ng TRH/100 g: 0 (4) 1200- 10 (4) 50 (4) A—A 250 (4) 1250 14) 1000-
o 800 - o n D) =1
CO t- 600 - < 2 CO 5 Q. 400 -
200 -
0 J r I I 0 10 20 30 TIME AFTER TRH (min.)
Figure 5. Plasma thyrotropin (TSH) concentrations in response to thyrotropin releasing hormone (TRH) (0 - 1250 ng/100 g BW) in ether anesthetized rats. 6o
Figure 6. Since a characteristic sigmoid dose-response curve is ob served at 10 minutes, this time was chosen to observe peak TSH responses in future experiments. The response at 30 minutes can also be used as an indication of the rapidity of decline in the TSH release. There were still large variations in TSH levels among the individual rats.
Therefore, the experiment was repeated using unanesthetized rats which were given the same doses of TRH, except there was no 10 ng dose (Fig.
7)• In this experiment the rats could be sampled frequently through the chronic indwelling jugular catheters without stressing the rats.
The peak TSH response occurred at 5 minutes following TRH administra tion, and was not significantly different from that observed previously at 10 minutes using ether anesthesia (Fig. 7). Also, the responses to the 50 or 250 ng doses of TRH at 10 minutes were not significantly dif ferent. using unanesthetized or ether-anesthetized rats. But at 10 minutes the TSH level after 1250 ng dose with no anesthesia was sig nificantly less than that observed with ether anesthesia. Thus it was decided that since the responses are quite similar with either ether anesthetized or unanesthetized animals, unless a large number of blood, samples are required, ether anesthesia would be used in future experi ments during TRH administration. In addition, the 50 and 250 ng doses of TRH were determined to be sufficient but sub-maximal stimuli for TSH secretion.
Thyroidectomy. Rats were thyroidectomized in order to evoke
TSH secretion in response to reduced negative feedback of thyroid Min. after TRH: 1000
_ 800-
O O O) 600- 3
c/> H 400- (/) a Q_
200- -i
0 -J I 50 250 1250 TRH (ng/100 g) Figure 6. The effect of thyrotropin-releasing hormone (TRH) on plasma thyrotropin (TSH) concen trations after 10, 20, and 30 minutes. Rats were anesthetized with ether and given 0 - 1250 ng TRH/100 g BW, iv. Data were calculated as described in Figure 5» 1200 ng TRH/100 g: 0 (6) 50 (9) 1000 - 250 (5) 1250 (2) o 0 800 - D) 3 1 600 - 03 I—
CO 400 - 5 Q-
200 -
0 J r t—r i r~ nr 1 o 5 10 20 30 45 60 75 TIME AFTER TRH (min.)
Figure 7. Plasma thyrotropin (TSH) concentrations in response to thyrotropin-releasing hormone (TRH) in unanesthetized rats. — Rats were sampled via chronic indwelling jugular catheters and given 0 — 1250 ng TRH/100 g BW via the same catheter# hormones. Plasma TSH and Tj^ levels following thyroidectomy are shown in Figure 8. As shown in the figure, plasma TSH levels were increased significantly at 2 days after thyroidectomy and remained increased throughout the rest of 8 days during which samples were taken. During this period of time (2 to 8 days) plasma concentrations fell dra matically and remained significantly decreased throughout this period.
Thyroidectomy was thus determined to be another sufficient stimulus for TSH secretion.
Thyroid Hormone Secretion
After finding that TRH and thyroidectomy were adequate stimuli for TSH secretion, the next series of experiments was concerned with selecting stimuli for thyroid hormones, T^ and T^, secretion. Stimuli chosen had previously been shown to elicit thyroid hormone secretion as determined by techniques other than radioimmunoassay (see Chapter 1).
Acute Cold Exposure. Plasma T^ and responses to acute ex posure are shown in Tables 3 and *f, respectively. Using pentobarbital anesthesia (Experiment 10), there was virtually no increase in plasma
T^ or T^ during the k hours following 30 minutes of cold exposure. In subsequent experiments rats were anesthetized with ether and exposed to cold throughout the blood sampling period. During 75 minutes of cold exposure (Experiment 25) there was no change in either plasma T^ (Table
3) or T^ (Table 4) levels. There was also no increase in T2 or T^ levels during 6 hours of cold exposure as shown for experiments Ik and
22 on Tables 3 and *f« The 75 minute and 6 hour periods of cold 250-i r 10
200- p < .001 ? i -8 A o o 150-i -6 O \ O O) N 3 O) 3 x 100- -4
50- CO CO •5- -2 '—5 5a. p < .001 (13) 0- r T nr ~T~ 1 Lo 0 2 4 6 8 TIME AFTER THYROIDECTOMY (days)
Figure 8. Plasma thyrotropin (TSH) and thyroxine (T/j.) levels following thyroidectomy. — Rats were anesthetized with ether for surgery and blood sampling via cardiac puncture.
o^ -p* Table 3. The effect of acute cold exposure on plasma T3 concentrations with ether or pento barbital anesthesia. — Results for experiment 10 were calculated using a Hewlett- Packard 9100B calculator as described in Materials and Methods. Rats were anesthetized with ether for all experiments, except 10 in which pentobartibal anesthesia was used.
Length of Cold Exposure (h) Plasma (ng/lOO ml) Expt. n Conditions Exposure 0 2 3 4 5 6
10 4 None 30 min .36.5 .32.7 a35.4 ± 7.6 ±11.0 -10.9
14 5 None 6 h 72.8 ,7°.9 56.1 54.6 ^11.2 ±16.3 - 9.2 ± 8.7
21 7 da, 32° 6 h 26.4 37.4 26.6 41.1 15-4 36.6 ± 4.0 t 8.0 ±15.9 ±22.3 ± 4.9 ± 9.6
22 4 None 6 h 41.8 ^38.2 .39.5 >9-1 .29.5 19.8 ± 4.0 ± 8.8 ± 4.8 ±10.0 ± 5-8 ± 5.3
45 60 75 (min)
23 4 7 da, 32° 75 min 65.0 u.75.8 .51-0 ± 0.8 - 5.4 ±29.5
25 4 None 75 min ,79.3 114.8 105.8 ±16.5 ±11.7 ± 9.9 Table 4. The effect of acute cold exposure on plasma Ti+ concentrations with ether or pento barbital anesthesia. — Results for experiments 10 and 22 were calculated using a Hewlett-Packard 9100B calculator as described in Materials and Methods. Rats were anesthetized with ether anesthesia in all experiments except 10 in which pento barbital anesthesia was used.
Length of Cold Exposure (h) Plasma (ug/100 ml) Expt. n Treatment Exposure 0 2 3 4 5 6
10 5 None 30 min 5.3 5.4 + 9'3 + + - 1.2 1.7 1.1
14 5 None 6 h 8.0 7.1 7.0 5.4 6.0 6.7 + + + + + ± 0.7 0.6 0.6 0.5 0.03 0.2
21 4 7 da, 32° 6 h 3-5 5.1 4.3 3.5 7.0 + + + + + +- 0.5 0.2 0.2 1.1 0.3 1.7
22 4 None 6 h 10.0 8.2 4.3 6.9 6.6 4.1 + + + + + - 1.0 2.2 1.3 0.7 0.9 1.4
0 15 30 40 60 75 (min) 23 4 7 da, 32° 75 min 4.6 6.8 3.8 3.0 6.5 3.0 + + + + + - 0.2 1.1 0.6 0.6 0.4 0.7
25 4 None 75 min . 7.6 10.0 7.5 5.5 5.6 9.5 + + + + t 0.4 - 1.1 0.4 0.8 0.9 2.0 67
exposure were similarly repeated with rats which had been acclimated
to 32° C for 7 days (Tables 3 and Experiments 25 and 21). Once
again there was no significant increase in either plasma T^ or T^.
In this case the plasma T^ level appears slightly, but insignificantly
increased at 6 hours after cold exposure in Experiment 21 but this is
probably due to the inordinately low T^ levels during the previous 6 hours of sampling. Since all experiments using cold exposure were un successful in eliciting either T^ or T^ secretion, these experiments were not continued.
Thyrotropin-Releasing Hormone. Plasma T^ responses to TRH are shown in Figure 9* As much as **00 ng TRH/100 g BW failed to give a significant increase in T^ concentrations during 6 hours following the
TRH (Fig. 9* bottom panel). Subsequently much larger doses of TRH
(up to 25 us) were administered to pentobarbital-anesthetized rats.
These rats were sampled only once 2 hours following the TRH (Fig. 9t upper panel). Even these much larger doses of TRH failed to elicit a significant increase in T^ levels, although there was a trend of in creased mean T^ levels with progressively higher TRH doses. Plasma T^ responses to these same injections of TRH are shown in Table 5- In every case there was no significant increase in response to TRH at either the nanogram or microgram level of TRH administration. Thus it appeared that TRH could not be used as a stimulus for thyroid hormone secretion as determined in this laboratory. Figure 9* Plasma triiodothyronine (T^) concentrations 2 hours after thyrotropin-releasing hormone (TRH).
Hats were anesthetized with pentobarbital for the 0 - 400 ng/100 g BW, iv doses (lower figure). Ether was used as an anesthetic in the upper figure (0 - 25 fig/100 g BW, iv). Tj data in the upper figure were calculated using a Hewlett-Packard 9100B calcu lator as described in Materials and Methods. 68
80 n
oO 60- N O) c n 40 < w 20 5 a.
0 1 5 25 DOSE OF TRH (jug/100 g)
ng TRH/100 g: 100n •—o 0 (3) o--o 100 (4)
80- A—• 200 (5) A—A 400 (5)
60 -
40 -
J 0 r T T "I 0 2 4 6 TIME AFTER TRH (hrs.)
Figure 9» Plasma triiodothyronine (T^) concentrations 2 hours after thyrotropin-releasing hormone (TRH). Table 5» Plasma Tlj. levels in response to TRH with ether or pentobarbital anesthesia. — Rats were anesthetized with pentobarbital (PB) for the 0 to MDO ng/100 g BW range, and with ether (E) for the 0 to 25 pg/100 g BV range of TRH doses.
Time (h) Dose Plasma Tt] (us/100 ml) (per 100 g)nO 2 3 ^ 5 6
Veh (PB) 5 6.5 - 0.6 5.5 - 0.6 6.3 z °-2 5A - OA 5.5 * 0.3
100 ng 5 7.2 t o.8 7.2 i 0.7 7.2 ± 0.5 6.9 - 0.7 6.8 ± 0.3 7.0 ± 1.2
200 ng 5 6.0 i oA 8.0 i 0.8 6.9 - 0.3 6.8 t o.b 5.5 - 0.5 6.5 - 0.6 too ng 5 7.0 t 0.5 7.6 - 0.7 6.8 ± 0.7 6.8 i 0.8 5.8 ± 0.2 6.1 t 0.3
Veh (E) 3 3.9 - 0.5 3.8 - 0.2
1/ig 3 3.3 - 1.2 3.8 t 0.9
5 us 3 3.5 t 0.2 2.7 ± OA
25 /zg 3 3.7 - 0.6 5.8 t 1.6 70
Thyrotropin. Plasma T^ and T^ concentrations following thyro tropin administration are shown in Figure 10 and Table 6, respectively.
Using pentobarbital anesthesia, plasma T, levels tended to increase at
2 hours following 20, 40, or 80 raU TSH although the levels were not significantly increased (Fig. 10, lower panel). Interestingly, the mean response to ho mil was greater than that in response to 80 raU TSH.
Plasma T^ levels during this time were also not significantly in creased, but just as noted for T^, the mean response to the 20 and *t0 mU TSH doses were increased at 2 hours. In the top panel, plasma T^ levels at S hours following 0 - 2500 mfl TSH/100 g BW using ether anes thesia are shown. None of the doses of TSH elicited a significantly greater concentration than that observed in the vehicle group. Only the response to the 100 mTJ dose was nearly significant. In a subsequent experiment 100 mU elicited a significant increase (t> <.01) in plasma T, j levels as determined by the paired ^-test (see Fig. 1(5 later in Re sults). Plasma T^ levels in response to 0 - 2500 mTJ TSH are shown in
Table 6. Hats were sampled at 2 and k hours following TSH but no in creases in Tj^ concentrations were elicited.
From all of the results obtained, it was found that it is very difficult, if at all possible, to elicit increased T^ and/or T^ secre tion. Subsequent experiments would, therefore, have to rely on the measurement of T^ release in response to TSH as an indication of thy roid activation. Figure 10. Plasma triiodothyronine (T_) concentrations in response to thyrotropin (TSH).
In the lower panel, rats were anesthetized with pento barbital anesthesia and were given 0 - 80 mU TSH/100 g BW, iv. In the upper panel, ether anesthetized rats were given 0 - 2500 mU TSH/100 g BW and blood samples were taken 2 hours after TSH. 71 '
o 100- o
Ui c CO I- 50- i i cn 5 a.
7 35 100 175 500 2500 DOSE OF TSH (mU/100 g)
mU TSH/100 g: 150 n 0 (3) o—o 20 (4) o A-A 40 (5) o A—A 80 (4) 100 ir
if) 50- 5a.
J 0 r T ~r i 0 2 4 6 TIME AFTER TSH (hrs.) Figure 10, Plasma triiodothyronine (Tjj) concentrations in response to thyrotropin (TSH)- Table 6. Plasma concentrations in response to thyrotropin (TSH) with ether or pentobarbital anesthesia. ~ Data from experiments in which ether (E) was used as an anesthetic (*) were calculated using a Hewlett-Packard 9100B calculator as described in Materials and Methods.
Time (h) Plasma T^ (fig/100 ml) (mU/100 g) n 0 2 3 b 5 6 + + Veh *(E) 6 5.0 0.9 3.6 0.7 + + + + + + Veh (PB) 3 6.8 0.7 6.8 1.0 5.6 0.7 7.7 0.8 5.3 O.Jf 7.3 0.*f + + 7* bA 0.6 **.5 0.3 + + + + + + 20 6.7 0.5 7.5 OA 6.2 0.*f 7A 0.2 6.3 0.6 7.2 0.6 + + 35* 6.0 1.0 6.8 0.8 + + + + + + ko 5 7.2 0.5 9.0 1.1 7.2 1.5 7.9 1.2 7.^ 0.5 8.5 1.1 + + + + + + 80 b 7.2 0.3 7-3 0.8 6.8 0.5 9-3 1.5 6A 0.7 8.5 1.3 + + 100* 10.9 2.8 7.8 1.0 + + 175* 6.7 lA 7.5 0.9 + + 500* 8.7 1.6 11.1 2.5 + + 2500* 8.9 2A 9.^ 3.0 73
Effect of Inhibition of Prostaglandin Synthesis on the Hypothalamic-Pituitary- Thyroid Axis
The previous section of data has described the determination of appropriate stimuli for activating the hypothalamic-pituitary-thyroid axis. Thyrotropin-releasing hormone and thyroidectomy were each found to be sufficient stimuli for TSH secretion. Thyrotropin was found to stimulate a significant increase in T^ levels. These various stimuli can now be used to assess the role of endogenous prostaglandins in this neuroendocrine axis.
Systemic Indomethacin in Intact Rats
The indomethacin and aspirin treatments used in these experi ments have previously been shown by Behrman, Orczyk and Greep (1972) to markedly decrease hypothalamic and pituitary PGF levels. Measure ment of hypothalamic and pituitary PGF levels by Dr. Gayle Orczyk
(personal communication 1975) confirmed this reduction (12.8$ and
*H.6?S of control, respectively). In addition, in these experiments, thyroidal PGF concentrations were found to be significantly (p <.001) reduced by the Ind (Fig. 11). Therefore, the drug treatment was suf ficient to lower endogenous PGF levels throughout the hypothalamic- pituitary-thyroid system. The time schedule for the administration of drugs and subsequent blood sampling in the next series of experiments is given in Table 7.
Response to TRH. The release of TSH in response to 50 ng TRH is shown in Figure 12. As shown in the figure TSH levels increased p < .001
—i— —i 0 500 1000 1500 2000 2500
PGF (pg/mg thyroid tissue)
Figure 11. Thyroid prostaglandin F (PGF) levels following the standard indomethacin (I) or gelatin (G) treatment — Thyroids were quickly removed under pentobarbital anesthesia (3»5 ng/100 g BW, ip)» snap frozen on dry ice, and stored at 0° C until assayed.
-o -r 75
Table 7- Time schedule of the administration of drugs and subsequent blood sampling for experiments in which intact rats were used. — All experiments were begun between 8:00 and 10:00 a.m.
Response Thyroid Response Thyroxine Time to TRH to TSH Half-life (h) (pages 73-77) (pages 80-84) (pages 77-80)
-2k 125^. for HI
0 Ind Ind Ind
7 Ind Ind Ind
125 2k Ind Ind Ind + I-Tk
28 Sample
35 Sample
36 Sample
48 Sample + TEH Sample + TSH Sample
50 Sample
5k Sample 76
450 -i Gel (7) Ind (6)
400 -
350 -
| 300 - o 0 p < .01 1 250-
X in 200 -
150 - p< .01 OL p <.05
100
50
10 30 TIME AFTER TRH (min.) Figure 12. Plasma thyrotropin (TSH) concentrations in response to TRH (50 ng/100 g BW, iv) following standard indomethacin (Ind) treatment in rats. 77 significantly (p <.01) in the Ind group at 10 minutes although the Gel group responded less significantly (p <.05). Not only was the TSH level greater at 10 minutes in the Ind group, but also at 30 minutes the TSH concentration was significantly greater (p C.01) than that in the Gel group. This response was surprising until the basal and T^ levels were measured. Both plasma T^ and T^ levels were found to be markedly decreased in this experiment in the indomethacin (I) group in spite of normal TSH levels (Fig. 13)• In this experiment plasma T^ levels were found to be lower than normal in this laboratory in the gelatin (G) group due to a variation in the radioimmunoassay. There fore, the thyroid hormone levels were determined in another experiment
(Fig. 1*0. Again the indomethacin was found to significantly (p <.001) reduce plasma T^ and T^ concentrations as compared to normal levels in the Gel group. Under these conditions of normal basal TSH levels, the markedly decreased thyroid hormone levels in the Ind group could be due to either a reduced responsiveness of the thyroid to TSH (i.e., thyroid hormone secretion rate is decreased), or to an increased clearance rate of thyroid hormones. In order to elucidate the site of action of the indomethacin in decreasing thyroid hormone levels, the effect of Ind on both clearance and secretion of thyroid hormones was assessed.
Thyroxine and Triiodothyronine Half-lives. The disappearance 125 of I-T^ was followed during 30 hours immediately following the standard Ind injections (see Table 7)- During this time the half-life for T^ was determined to be 12.9 h in the Ind-treated group and 17.2 h PLASMA TSH PLASMA T3 PLASMA T4 fojg/100 ml) (ng/100 ml) Oig/100 ml) NS p< .01 p< .001
100 1 40n 4-i
50 - 20- 2-
(5) 0 0 G I G I
Figure 13. Plasma TSH, T^, and T/j. levels following the standard indomethacin (I) or gelatin (G) treatment using ether anesthesia.
-n3 03 PLASMA TSH PLASMA T3 PLASMA T4 {jjg/100 ml) (ng/100 ml) (Mg/100 ml) NS p <.001 P< .001 100 -T 50 i 10 i * i
50 - 25 - 5 -
14 14 0 0 0 G I G I G I
Figure 14, Plasma TSH, Tx, and Tif concentrations after the standard indoraethacin (I) or gelatin (G; treatment using pentobarbital anesthesia.
-o \o 8o
in the Gel group (Fig. 15 and Table 8). The decrease in half-life of
T^ was small (*f.5 h) and only slightly significant (p <.05). On the 125 other hand, the half-life of I-T^ was determined to be significantly
(p <.01) increased during this time. Ind and "^^I-T^ were administered 125 as indicated for Ind and I-T^ on Table 8, and blood samples were taken at 28, 30, 3^i ^7i and 50 h. Indomethacin treatment increased the half-life of T^ by 7-2 h which would tend to elevate the T^ plasma level during the time of drug treatment. Indomethacin has thus altered the half-life of both thyroid hormones in this study.
Thyroid Response to TSH. Thyroid secretion rate was measured 125 by the release of I or T^ in response to exogenous TSH administra tion. In the first experiment, TSH (10 mU/100 g BW) was administered 125 to rats following I and the standard Ind treatment (see Table 7). 125 The release of I in response to the TSH is shown in Figure 16 and is expressed as response index (see Materials and Methods for calcula- 125 tion). Blood I levels increased 23# (p C.OOl) in the Gel group in 125 response to the TSH, whereas blood I levels increased only 6# in the
Ind-treated rats which is not a significant response. The Ind, there- 125 fore, inhibited thyroidal release of I in response to TSH.
Another parameter for determining thyroid secretion is to measure plasma levels of thyroid hormones. Due to the lack of measur able T^ response to TSH only plasma T^ levels were determined in this experiment. As shown in the left-hand panel of Figure 16, the Ind- treated group had no significant T^ response to the exogenous TSH 81
7.5-1
7.0- •—•Gel (7) O- -O Ind (6)
\
6.5-
E 6.0- •.u c
5.5-
. 5.0-
0 4 9 1213 24 30 TIME (hrs.) 125 Figure 15« Disappearance curves of I - Tif from the plasma of rats receiving the standard indomethacin (Ind) or gelatin (Gel) treatment. 82 125 12^f Table 8. Disappearance rates for I-Tj, and I-T3 following indomethacin treatment. — See Table 7 for the schedule of drug administration and blood sampling.
Half-life (h) Drug N T3
Gel 7 17.2 i ljf lO.Jf t 0.9
Ind 6 12.9 - 1.3 17.6 t 1.8 p <-05 p <.01 83
PLASMA T3 RESPONSE INDEX 70 -i (ng/100 ml) <%) p<.01
60 - 130-1 p<.001
50 -
40 - 120 -
30 - NS
20- 110 - NS (7) (5) 10-
15 100
125 Figure 16. Plasma Tj concentrations and the release of I (response index) in response to TSH administration following the standard indomethacin (I) or gelatin (G) treatment. — Rats were anesthetized with ether and blood was collected immediately -prior to and 2 hours following TSH. TSH was administered at doses of 100 mU/100 g BW, ivt for response and 10 mU/100 g BWf iv, for response index (see Materials and Methods for description of data analysis). 8k
(100 mU/100 g BW). The Gel group responded significantly (p <.01) with
a mean increase in plasma T^ levels of b3 ng/100 ml. In this experi
ment the higher dose of TSH (100 mU) was necessary in order to elicit 125 a significant thyroid response in control rats. The release of I
apparently was a more sensitive parameter requiring only 10 mU TSH to
obtain a significant response.
The indomethacin treatment in intact rats, therefore, was found
to increase the magnitude of TSH response to TRH due to reduced nega
tive feedback of thyroid hormones. Because of this resulting change
in the level of negative feedback, one would not know whether an ob
served effect should be attributed to an effect of the drug on the
pituitary, or to the altered feedback. In order to circumvent such
problems in the next experiments, the responsiveness to TRH was as
sessed using thyroide'ctomized animals receiving T^ replacement.
Indomethacin or Aspirin Treatment in Thyroidectomized Rats
The time schedule for surgery, drug injection, T^ replacement,
and collection of blood samples is indicated in Table 9.
Response to Exogenous TRH with Tk Replacement. The thyroid
hormone and TSH levels in thyroidectomized rats receiving the 4 fig dose
of T^ are shown in Figure 17. As shown at the top of the figure, T^ levels in the Ind group at ^8 h following thyroidectomy are slightly,
but significantly (p <.01), lower than in the control. At the end of
the experiment, plasma T^ levels in the two groups were not 85
Table 9« Time schedules for surgery, drug (or vehicle) injections, Ti|. replacement, and collection of blood samples for experi ments in which TRH or thyroidectomy were used as stimuli to TSH secretion. — Terminal, blood samples were obtained by decapitation but all other samples (.3 ml each) were taken by cardiac puncture* All thyroidectomies and TRH stimula tions were performed between 8:30 and 11:30 a.m. Indo- methacin was used at a dose of 3 rag/0.5 ml/injection, and aspirin was used at doses of 10, 30, ^5» or 60 mg/ml/ injection.
Time of Stimulus Time (h) TRH Thyroidectomy
0 Sample + thyroidectomy Sample + thyroidectomy + drug + T^ + drug
7 Drug + T^ Drug
2k Drug + T^ Drug
31
48 Sample + TRH Sample 86
? 12i Gel (14) o o Ind (14) s U) 8 - 3
h- 4 - 2— ~
V) i
0 J
o r 40 o I O) t - 20 CO h*
L 0 CO s 0L £ 150 n o
a> 100 •I
U) h- 50 -
W 5 0 J Q. r 1 0 48 TIME AFTER THYROIDECTOMY (hrs.) Figure 17- Plasma TSH, T3, and T4 levels following thyroidectomy with T4 replacement C+.O fig/XOO g BW/day) and the standard indo- methacin (Ind) or gelatin (Gel) treatment. — See Discus sion section for comment on TSH data in this figure. 87 significantly different. Samples for T^ analysis were taken only at the time the rats were killed because of the rather large volume of plasma (250 ftl) required for this assay. Apparently the overall rate of the replacement was not less than physiological since in neither group was there evidence of compensatory TSH secretion ^8 h after thy roidectomy (Fig. 17* lower panel). The TSH response following the TRH stimulation is shown in Figure 18. The indomethacin group responded significantly to the TRH but its peak value was significantly (p <.02) lower than that of the gelatin group.
Since the TRH responsiveness in the Gel group of the proceeding experiment was slightly less than normally observed in this laboratory, the T^ replacement was lowered to the 3 jig dose for the following ex periment (Fig. 19)• Even with this lower dose, the amount of re placed was sufficient to prevent compensatory TSH secretion. Under these conditions, indomethacin was once again observed to significantly
(p <.01) inhibit the TSH response to TRH (Fig. 20). As in the first case, the TSH response of the Gel group was approximately twice that of the Ind group and in this case it was quite similar to the response that is normally observed in intact rats.
Finally, when the T^ replacement was reduced further to the
2 fig dose, the replacement was still sufficient to prevent a compen satory rise in TSH concentrations (Fig. 21). In this case, the stan dard Ind treatment again decreased the TSH response to TRH, but this time to a lesser degree (p <.05 /^ig- 227). The response of the Ind group was similar to that observed in the previous experiment, Figure 18. Inhibition of TSH response to TRH (250 ng/100 g BW, iv) following the standard indomethacin (Ind) treat ment in thyroidectomized rats with TV replacement (4.0 [xg/100 g BW/day).
Time zero on this figure is coincident with 48 h on Figure 17- 88
300
Gel (14) Ind (13)
250
g 200 o o o> 3 150 p< .02
50
0 30 TIME AFTER TRH (mih.) Figure 18. Inhibition of TSH response to TRH (250 ng/100 g BV/, iv) following the standard indomethacin (Ind) treatment in thyroidectomized rats with Tjj. replacement ('f.O fxg/100 g BW/day)• 89
? 12 n Gel (14) o o Ind (13) s o> 8- 3
h < 4- 2 CO s Q_ 0-J
40 o o I O) 3 -20 h? *< 5 L0 CO 5 o. 150 n O O ob 100 3
*2 50- < i i 2 CO
0 J 0 48 TIME AFTER THYROIDECTOMY (hrs.)
Figure 19• Plasma TSHt T3 and levels following thyroidectomy with Tif replacement (3.0 ^g/100 g BW/day) and the standard indomethacin (Ind) or gelatin (Gel) treatment. 90 350 -i
Gel (14) 300 - Ind (14)
250 - p<.01 E o o 200-
co 150 - 5 CL
100-
50-
0 10 30 TIME AFTER TRH (min.) Figure 20. Inhibition of TSH response to TRH (250 ng/100 g BW, iv) following the standard indomethacin (Ind) treatment in thyroidectomized rats with T£f replacement (3*0 fxg/100 g BW/day). ~ Time zero on this figure is coincident with *+8 h on Figure 19- 91
^ 12 n Gel (8) o o Ind (8) O) 8 - 3
H < 4- CO —5 5 Q- 0 J
o r 40 o o> i c - 20 CO § H
L 0
CL
§ 100-1
U) 3 5 5cH I-
c/) 0 J r 0 48 TIME AFTER THYROIDECTOMY (hrs.) Figure 21. Plasma TSH, T3 and levels following thyroidectomy with Tjj. replacement (2»0 jxg/100 g BW/day) and the standard indomethacin (Ind) or gelatin (Gel) treatment# Figure 22. Inhibition of TSH response to TRH (250 ng/100 g BW, iv) following the standard indomethacin (Ind) treatment in thyroidectomized rats with Tr replacement (2.0 /ig/100 g BW/day).
Time zero on this figure is coincident with 48 h on Figure 21. 92
300 n Get (8) Ind (8)
250 -
200 - o o O) 5 150 - w H
C/) 5 CL 100 -
50-
0 -J
TIME AFTER TRH (min.)
Figure 22. Inhibition of TSH response to TRH (250 ngAOO g BW, iv) following the standard indomethacin (Ind) treatment in thyroidectomized rats with replacement (2.0 fig/100 g BW/day)• 93
although the magnitude of the TSH response in the Gel group was lower.
This may have contributed to a less significant decrease in TRH respon
siveness due to indomethacin treatment.
Using the 3-0 MS replacement dose of T^, a smaller amount of
indomethacin (3 mg/rat, given only once, 2k h prior to TRH) had no
effect on the response to TRH. The TSH concentration in the Gel group
increased from 38-2 - 12.1 to 222.6 - 31.k US a*1** the Ind group in
creased from 50-7 - 12.9 to 30^-9 - 59-3 (*g % of TSH at 10 minutes
after TRH. The TSH response to TRH was also unaffected after aspirin
(^5 mg/injection) treatment in rats receiving the 3.0 fig replacement
dose (Table 10). This may have been due to significantly lower plasma
Tj and T^ levels in the Asp group at the time of TRH administration.
Response to Thyroidectomy. In the next series of experiments
the effect of PG synthesis inhibition on TSH secretion in response to
reduced negative feedback induced by thyroidectomy was studied. In the
first such experiment, the effect of our standard indomethacin treat
ment on thyroidectoray-induced TSH secretion was measured.
As shown in Figure 23» thyroxine levels dropped sharply in both
groups as expected following thyroidectomy. Indomethacin was adminis
tered during the first 2k h following thyroidectomy as described in
Table 9i and it was found to have a reversible inhibitory effect on
TSH release. At day 2 after thyroidectomy, the gelatin group had
reached a maximal TSH level while the indomethacin group showed vir
tually no increase. On day three days after the treatment was 9k
Table 10. Plasma TSH, T^, and Tconcentrations following TRH and aspirin (^5 mg/injection; Asp) or gelatin (Gel) treatment in thyroidectomized rats receiving Ti± replacement (3*0 /ig/100 g BW/day). — TRH (250 ng/100 g BW, i.v.) was administered using ether anesthesia. Plasma hormone con centrations are given for TSH immediately, prior to (Pre-TRH), and at 10 minutes after TRH (Post-TRH), and for Tj and T^ at 30 minutes after TRH.
TSH Tk Drug (fig/100 ml) (f*g/100 ml)
Pre-TRH Post-TRH
Gel 10 bk.5 t 6.6 181.9 - 18.1 9-0 - 5.1 10.0 t 1.2
Asp 5 46.6 ± 11.2 232.4 - 27.8 0.2 - 0.2 6.1 t 1.7 95
8 - • • Gel (12) o—-o Ind (11)
O O 6 -
OS 3 4 - H
2 - cn s 1 Q_ -5 0 - ~i
Gel (13) 200 - '-° Ind (17) o o 150 - O) 3 x CO 100 -
50 - CO / ¥ 5 «• CL 0 - i ~1 2 4 TIME AFTER THYROIDECTOMY (days)
Figure 23• Plasma TSH and Tlevels following thyroidectomy and the standard indomethacin (Ind) or gelatin (Gel) treatment. 96 stopped, the TSH concentration in the indomethacin group had risen to a level that was essentially the same as the control level.
The response to thyroidectomy was also studied in rats receiving aspirin treatment (10, 30» 45 or 60 rag/injection). At the 60 mg dose the mortality rate was quite high (70$) and therefore the results were considered invalid. The mortality rates were considerably less in the cases of the 10, 30 and 45 mg doses (12.5^1 31-3$ and 30.C# respec tively), and the results of experiments using these three doses are presented in Figure 24. As expected, T^ levels fell drastically in all groups during the 48 h period. The compensatory increase in TSH secre tion was inhibited by the aspirin; this effect was statistically sig nificant at the 30 and 45 mg doses. Although aspirin is known to inhibit PG synthesis, these mortality rates warn that the effect of this drug on TSH secretion might be partially due to some non-specific effect of the drug on the health of the rats.
During the course of some of these experiments, indomethacin- treated animals were observed to eat and drink less than gelatin groups.
In order to convince ourselves that the effects we had seen were not simply due to this factor an experiment was repeated with controlled food and water intake. Indomethacin-treated and control rats were fed the same amount of food (5.8 g glucose/day) and water (15 ml/day) during the 48 h following thyroidectomy. As seen in Figure 25i the indomethacin once again inhibited the compensatory rise in TSH secre tion. Figure 24. Plasma TSH and T^ levels following thyroidectomy and aspirin (10, 30 or ^5 mg/injection) or gelatin (Gel) treatment.
Inadvertently, no samples were collected for Tif assay in the experiment in which the 30 mg dose was used. Normal TSH and T^ levels are indi cated by the horizontal bars which represent the means and standard errors of samples taken from all of these rats just prior to surgery and drug treatment. Vertical bars indicate TSH and T^ levels at 2 days following thyroidectomy and initial drug treatment. PLASMA T4 PLASMA TSH (ug/100 ml) 250 n (jjg/100 ml)
8-1 200- NS 6- 150- I p<.02 p<.01 4- 100- P 0 0 2- i 50- (11) |{19) 0J u M 10 45 mg 10 30 45 Gel Aspirin Gel Aspirin
Figure 2*f* Plasma TSH and levels following thyroidectomy and aspirin (10, 30 or ^5 mg/injection) or gelatin (Gel) treatment.
vo •vl Figure 25- Plasma TSH and levels following thyroidectomy and the standard indonethacin (I) or gelatin (G) treatment.
In this experiment the indomethacin-treated rats were given the same amounts of food and water as were the control rats. Other than this, the protocol for this experiment was exactly the same as described in the right hand panel of Table 9- See legend to Figure for explanation of the horizontal and vertical bars. 125 1 12 - M44 E s- 100 o E o o o O) 8 - S 75 H 3.
H P<.05 l—CO 50crt CO 4 - ds 3 w Q. 3a. 25
0) (9) ;(8) J 0 J I
Figure 25. Plasma TSH and T. levels following thyroidectomy and the standard indomethacin (I) or gelatin (G) treatment. 99
Stereotaxic Microinjection Studies
Because the systemic indomethacin treatment has such widespread
effects throughout the body, several experiments were performed in
which the TRH, or the Ind and TRH were administered locally into the
pituitary. These local injections could thus indicate the effect of
the drug when its distribution is confined to the area of interest, the
anterior pituitary.
Systemic Indomethacin Treatment and TRH Microinjection. In the
first series of such experiments, indomethacin was administered sys-
temically as previously described in Table 7« TRH was then locally
injected into the anterior pituitary in order to assess the possibility
of reduced delivery of the TRH to the pituitary in the previous experi
ments.
Results of this procedure are shown in the left panel of Figure
26. There was no significant difference between the two groups in the
TSH response to TRH. Previously, the TSH response in the Ind group
was potentiated after intravenous TRH. Due to the dramatically de
creased thyroid hormone levels (as previously observed in Figure l*f),
this experiment was repeated with thyroidectomized rats receiving the
3.0 fig replacement dose of T^. The results of this experiment are
shown in the panel on the right in Figure 26. As shown in the figure,
the TSH responses of the Ind group was decreased at both 10 and 20
minutes after TRH microinjection, but significantly reduced (p C.01)
only at 20 minutes. Gel (7) 300-i Gel (10) Ind (10) Ind (12)
250-
§ 200- O) 3 x 150- c/) P<.01 H < S CD 100- 5Q_ 50-
0J 1 I 1 10 20 10 20 TIME AFTER TRH (min.) TIME AFTER TRH (min.)
Figure 26. Plaema TSH response to intrapituitary injection of TRH (1 ng in 1 fil) following the standard indoraethacin (Ind) or gelatin (Gel) treatment. « The panel on the left shows the response of intact ratsf and the panel on the right, results of thyroidectomized rats receiving the 3 fig replacement dose of T^. 101
Intrapituitary Microinjection of Indomethacin and TRH. The next series of microinjection experiments utilized local pituitary injections of both Ind and TRH. With Ind so restricted, the effects of Ind only at the pituitary could be observed. The TRH was adminis tered immediately thereafter through the cannula in the same position so that the TRH perfused the identical portion of the pituitary as did the Ind, However, under these conditions, no inhibition of the re lease of TSH by Ind was observed (Fig. 27). The interval between Ind and TRH microinjections was either 30 seconds or 5 minutes, and in both cases there was no inhibition. Perhaps the Ind requires more time to inhibit PG synthesis, but it is not possible to delay injection of TRH through the cannula for more than 5 minutes. Figure 27. Plasma TSH levels followine: the sequential intrapituitary injection of indomethacin (Ind) (10 fig in 1 pi) and TRH (1 ng in 1 pi), or equal volumes of their vehicles (Veh).
In the first three cases listed in the key the time in terval between Veh or Ind and TRH (or saline, Sal) was 30 seconds, whereas it was 5 minutes in the last case. 102
300 VEH-SAL (7) o VEH-TRH (10) o--o IND-TRH (14) Ar-A. lND-5'-TRH (8)
250
200
150
100 Q_
50
0 20 TIME AFTER TRH (min)
Figure 27• Plasma TSH levels following the sequential intrapituitary injection of indomethacin (Ind) (10 pg in 1 til) and TRH (1 ng in 1 fil), or equal volumes of their vehicles (Veh)• CHAPTER
DISCUSSION
Just as the results of this study were presented in two major sections, the interpretation of this data will be dealt with in a like
manner. Less emphasis will be placed on the first portion of this study in which appropriate stimuli were selected, and the bulk of this dis cussion will deal with the effects of the PG synthesis inhibitors on the hypothalaraic-pituitary-thyroid system.
Comparison of Plasma TSH, T^ and Ti<. Levels with Previously Reported Values
The plasma concentrations of TSH, T^, and T^ can vary with several conditions aside from the experimental ones imposed in this study. First of all, all three hormones were determined by specific
RIAs which are very new techniques for measuring these hormones. The values determined for these hormones may not be the same as those pre viously determined by heterologous RIA or by bioassay. Adams, Kennedy, and Utiger (1972) have compared the measurements of serum TSH in humans by bioassay and immunoassay techniques and found that there was good agreement at high TSH levels but at basal levels the immunoassay tech nique may overestimate TSH levels. In the Adams study basal levels of
TSH were nearly undetectable and generally serum had to be concentrated for TSH measurement. This concentration procedure may have led to the
103 lOff
differences found between the bioassay and the immunoassay. Plasma TSH levels in male Wistar rats were found to be lower when measured by bio assay (Mornex et al., 1973) as compared to that by radioimmunoassay of plasma TSH levels in Sprague-Dawley rats (Reichlin et al., 1970)•
Secondly, plasma TSH, T^» and concentrations may differ with the sex of the animal used. Female rats have been reported to have sig nificantly lower levels of TSH and T^ as determined by a specific homologous RIA (Rapp and Pyun, 197*0 • Mean plasma TSH levels were
J2.1 fig/100 ml in female Vister rats versus 70-8 jig/100 ml in male rats (Rapp and Pyun, 197*0 » and mean plasma T^ values were 6.65 /ig/100 ml in females versus 7-^3 fig/100 ml in male rats. Kieffer, Mover,
Ferico and Maloof (1976), on the other hand, found that plasma T^ levels were 12% higher in female rats. These hormone levels for female rats correspond closely to the basal levels determined in Sprague-
Dawley female rats in this study. On the other hand, Wilber and Utiger
(1967) found no difference in basal TSH levels of male and female
Sprague-Dawley rats (15-66 ^U/ml) as determined by a heterologous radioimmunoassay. The discordant findings for plasma TSH levels in male and female rats may be the result of differing HIA techniques utilized by the three groups. The problem of differing basal plasma levels in the two sexes was avoided by using female rats throughout this study.
Kieffer, Mover and Maloof (1975) and Kieffer, Mover, Ferico and Maloof (1976) have shown that there are only minor fluctuations in plasma TSH and T^ concentrations during the female estrous cycle. 105
Therefore, it v/as considered unnecessary to determine the stage of
estrous for the individual rats in the present study.
A third factor which may affect plasma TSH levels is the diur
nal variation which has been previously observed. A number of inves
tigators have found this variation in plasma TSH concentrations
although the time of peak and trough levels is disputed. Bakke and
Lawrence (1965) have found peak TSH levels occur at 8:00 a.m. in male rats as determined by bioassay. Schindler and coworkers (1965) and
Ducommun, Sakiz and Guillemin (1966) also suggest a zenith in the
morning and a nadir in the late afternoon or early evening in male and
female rats although specific times were not indicated for the first
study. On the other hand, several reports have suggested that the
plasma TSH levels are low during daylight hours and reach a peak before dark (Singh et al., 1967; Retiene et al., 1968). Thus, although a controversy exists over the time of zenith and nadir the data do sup port a diurnal variation in plasma TSH levels. This potential problem has been avoided in the current study by performing experiments of a group to be pooled at the same time of day.
Measurement of Pituitary and Thyroid Response to Various Stimuli
TSH Response
Acute Cold Exposure. Plasma TSH concentrations were not con sistently increased by acute cold exposure. While ether or pento barbital anesthetized rats showed no significant increase in plasma io6
TSH, unanesthetized rats had significantly increased TSH levels at 60 or 90 minutes in two experiments (Fig. 2). This increase in plasma
TSH level is less than that previously reported (1^7 //g/100 ml) for female rats (Leppaluoto et al., 197*0 after 15 to 60 minutes of cold exposure. Adult male rats have also been shown to have increased plasma TSH levels from a basal of 15-30 fig/100 ml to. 80-200 pg/100 ml following 30 minutes of cold exposure with (Tuomisto et al., 1975) or without (Hershman and Pxttman, 1971) one week of warm acclimation.
Plasma TSH levels were found to be quite variable among individual rats in this present study and in other experiments (Tuomisto et al., 1975)-
In the present study the individual variation contributed to a large calculated SEM at each time the rats were sampled. Therefore, when the samples taken prior to cold exposure were compared to those taken after cold exposure the differences were insignificant by Student's t-test.
In all of the previously reported results discussed above the rats were exposed to *f-5° C for various periods of time ranging from
10 minutes to one hour. The degree of cold exposure also has been found to determine the magnitude of the rise in plasma TSH levels. At
15° the plasma TSH levels were unchanged from the TSH levels in rats maintained at room temperature (20°), but when rats were exposed to
10° or 5° for 30 minutes plasma TSH levels were significantly increased
(Leppaluoto et al., 197*0• The TSH level in the group exposed to 5° was increased, but not significantly, over the TSH level in the group exposed to 10°. Although Brown-Grant (1956) suggested that extreme cold (0°) could inhibit TSH secretion (as measured by thyroidal 107 radioiodine release), Itoh and coworkers (1966) could find no depen dence of TSH levels on the degree of coldness* They examined the TSH response to 15°, 8°, and 0° C exposure and found a similar rise in TSH levels from 30 to 120 minutes in all cases. In contrast to the Leppa- luoto study (197*0 t the rats were housed at 28° in the study done by
Itoh which could account for the response to the cold stress at 15°•
Thus in both laboratories a cold stress of 10° or more lower than nor mal room temperature is sufficient to enhance TSH secretion (Itoh et al., 1966; Leppaluoto et al., 197*0•
Thyrotropin-releasing Hormone* The 50 and 250 ng doses of
TSH were found to elicit increased, but sub-maximal TSH secretion using ether-anesthetized or unanesthetized rats. Montoya, Seibel and
Wilber (1975) found a maximal plasma TSH level of 46 mTJ/lOO ml fol lowing 100 ng TRH (in rats weighing 175-200 g) which corresponds closely to the plasma TSH levels following 50 ng TRH/lOO g BW in this study. When the data in the present study are expressed as mU TSH/
100 ml plasma, the peak TSH response was 57 and 51 mU/100 ml for the ether anesthetized and unanesthetized rats, respectively. Azizi,
Vagenakis, Bollinger, Reichlin, Bra verm an and Ingbar (197*0 found a similar magnitude of peak TSH response to 50 ng TRH/lOO g BW at 10 and 20 min utes after TRH. Blake (197*0 reported an increase of plasma TSH levels from 46.2 £ 8.1 jxg/100 ml before 100 mg TRH to 207.0 i 48.4 at 2 min utes and 398.0 - 20.9 at 10 minutes after TRH. The plasma TSH level at 2 minutes is quite similar to the peak response observed at 5 minutes in unanesthetized rats after 50 ng TRH/lOO g BW in the present study, although in their study the actual peak TSH occurred at io8
10 minutes. There is no apparent explanation for this delay in peak response since most reports observe that TSH levels do peak prior to
10 minutes after i.v. injections of TRH. The plasma TSH levels fol lowing the 50 ng dose were also quite similar to those observed by
Leppaluoto and coworkers (197*0 but the levels after the 250 ng dose were somewhat greater throughout the first 30 minutes after TRH.
Leppaluoto and coworkers (197*0 used *f,0 mg/kg pentobarbital anesthesia in that experiment, and found somewhat greater variation about each mean for 5 rats than that observed in the present study. The peak TSH response was found at 5 minutes after 10 or 50 ng TRH and 10 minutes after the 250 ng dose. In the current study when pentobarbital was used as an anesthetic during administration of 50 ng TRH, the peak re sponse occurred at 5, 10 or30 minutes depending upon the individual rat.
In unanesthetized rats, the plasma TSH concentration at 5 minutes was consistently greater than that at 10 minutes with every dose of TRH
(50-1250 ng). The pentobarbital anesthetic may have contributed to the inconsistent results in these experiments. Martin and Reichlin
(1972) found that the peak TSH response to TRH was prolonged with pen tobarbital as compared to ether anesthesia. Blake (197*0 recently reported that ether alone had no effect on plasma TSH levels for one hour following a six minute period of ether exposure.
Thyroidectomy. Following thyroidectomy plasma TSH levels were found to be significantly increased by 2 days and reached a peak at 6 days, during which time plasma T^ levels were significantly decreased 109 to nearly undetectable levels. Although there are few data previously reported for the TSH levels immediately following thyroidectomy, one recent report by Mornex and coworkers (1973) indicated that plasma TSH concentrations at 10 days after thyroidectomy (18-129 mU/100 ml) were comparable to those determined at 8 days in this experiment (191 /ig/
100 ml or kZ mTJ/100 ml). On the other hand, Salaman (1964) examined the acute rise in plasma TSH concentration during 10 days after thy roidectomy and found a "triphasic" response. At 2 days TSH levels had risen from 87 to 201 mU/100 ml, had fallen again to 6k by 5 days, and by 10 days had risen again to 119 mU/100 ml. The large difference be tween the TSH levels measured by Salaman and the present results may be due to a bioassay using jin vitro thyroid slices which was used to de termine TSH levels. Plasma from 2 to b rats had to be pooled for assay and this may have further contributed to the large differences observed.
Another study by Reichlin and coworkers (1970) showed that plasma TSH levels were 175 - 15*7 mU/100 ml in control male rats. Thus the re sults in the current study are in agreement with those most recently reported but are significantly less than those reported earlier by
Salaman (1964).
Thyroid Hormone Secretion
The basal levels of thyroid hormones determined in this study are similar to those previously reported. Plasma T^ concentrations were determined to be 52.0 - 4.1 ng/100 ml in female rats (Kojima et al., 1975)- Plasma T^ levels in adult female rats have been previously 110 reported to be 5*1 - 0.2 jig/100 ml (Kieffer et al., 1976). Plasma T^ and T^ concentrations in male rats are not significantly different from those levels found in female rats (Nejad et al., 1975; Abrams and
Larsen, 1973)-
Acute Cold Exposure. Although no significant increase in either plasma T^ or secretion was found in this study, several in vestigators have reported increased thyroid secretion in rats follow ing cold exposure. Jobin and coworkers (1975) recently found that plasma T^ levels rose to a peak of 50# increase over basal levels 2 hours following cold exposure. In all other studies enhanced thyroid secretion after cold exposure was determined by such parameters as the increase in colloid droplets in the thyroid (Kotani, Onaya and Yamada,
1973? Kajihara et al., 1972), increase in radioactive iodine in the thyroid (Williams, Jaffe and Kemp, 19^9)» or release of radioactive iodine from the thyroid (Williams et al., 19^9; Brown-Grant, 195&).
Thus although various metabolic or cytologic effects of acute cold on the thyroid can be observed, there is little evidence that significant elevation of plasma and occurs. It has been suggested that the increase in thyroid secretion which may occur might be "balanced by a commensurate utilization and disposal of the hormone at the peripheral tissue" (D'Angelo, 1963).
Thyrotropin-Releasing Hormone. Plasma thyroid hormones did not rise significantly in response to any of the doses of TRH (0.1 - 25 fig/
100 g BW). There are no other reports of the measurement of thyroid Ill
hormones in response to a series of TEH doses in the rat. Azizi,
Vagenakis, Bollingert Reichlin, Bush and Braverman (197*0 have found a
significant (70#) increase in the level of plasma T^ at one hour after
100 fig TEH (sc) with a peak level of 63 - 10 /ig/100 ml at 2 hours.
The basal plasma T^ level was rather low (28 £ 5 ng/100 ml) in compari
son to the levels determined in the present study which may have con
tributed to falsely elevated T^ levels after TRH. One hour after 100 fig TRH the plasma T^ level had risen only slightly (*f.3 - 0.3) and at
3 hours a peak level was reached (5*7 - 0,3 fig/100 ml). Once again the | basal T^ was rather low (3-7 - 0.2 fig/100 ml) in comparison to most studies. Nakagawa (1975) recently reported a slight but insignificant rise in plasma in two of three experiments after 50 fig TRH (ip) in
male Wistar rats weighing 300 - 350 g. Even in human subjects a rise
in plasma but not T^ has been found after 100 y.g TRH (iv) (Hollander
et al., 1972). Azizi and coworkers (1975) have found that a higher
dose of TRH (2 mg, im) elicits a maximum individual increment of plasma
T^ ranging from 0.5 - ^-0 /ig/100 ml (mean of 2.*f - 0.7) at 4 or 5 k after TRH in normal human subjects. When the peak plasma T^ levels were pooled, irrespective of the length of time after TRH* the increase in plasma T^ was found to be significant. Obviously even in humans the rapidity and magnitude of the response to TRH is not consistent in all individuals.
Thyrotropin. Thyroid hormone secretion was not significantly
increased by any of the doses of TSH (7- 2500 mU/100 g BW) in the pre
liminary experiments (Fig. 10). On the basis of a nearly significant 112
Tj response to the 100 mU dose of TSH (Fig. 10), in a later experiment this dose was tried again and this time gave a significant response as calculated by the paired _t test. In no case did any TSH dose stimulate the release of T^. This is in sharp contrast to the report by Kieffer,
Mover and Maloof (1975) who found increased T^ levels in male (5.9 i
0.*f) and female rats (8.2 * 0.*f jig/100 ml) at 2.5 hours after TSH (333 mU/100 g). These levels in each group of six rats were compared to groups of untreated controls (h.O - 0.2 and 5*1 - 0.2, respectively) with 21 and 22 rats per group. These data may be questioned just on a statistical basis of reasonable comparisons. In this study the mean plasma T^ level at 2 hours after TSH was frequently increased but the individual variation was so great that the calculated t values for the difference between the pre-samples and 2 hour samples was very small and thus insignificant.
Several other parameters of thyroid activity have been measured previously and interpreted as indications of increased thyroid hormone secretion. Rosenfield and Rosenberg (1966) found that 50-5000 fig of
TSH enhanced thyroid uptake of radioactive iodine and protein-bound iodine formation at 3 hours after TSH administration (i.v.)* Sasson and Rosenberg (1963) measured thyroidal iodine before and after TSH (3
USP units, i.v.) administration and detected a substantial decrease in thyroidal ^*^"1 3 hours after TSH.
Although TSH can be demonstrated to effect a number of meta bolic variables in the thyroid, it can not be demonstrated to elicit a substantial increase in plasma thyroid hormone levels. The small ele vations that have been observed may be indicative of either a very 113
small amount of thyroid hormones being released in response to TSH, or
a simultaneous increase in the degradation of thyroid hormones occur
ring in conjunction with enhanced secretion. This degradation of thy
roid hormones may be primarily a peripheral conversion at the tissue
level. In addition, only very small increases in circulating thyroid
hormones may be necessary to respond to a stress physiologically and,
therefore, the system may not be designed to respond with a large out
put of thyroid hormones.
Effects of Inhibition of Prostaglandin Synthesis on the Hypothalamic- Pituitary-Thyroid Axis
Although exogenous PGs have been shown to alter the pituitary
responsiveness to TRH in vivo and thyroid response to TSH in vitro,
there is no evidence regarding the possible role of endogenous PGs in
TSH and thyroid hormone secretion. There are several approaches pos sible for investigating the role of endogenous PGs. As mentioned be
fore in Chapter 1, PG levels may be altered by the use of essential
fatty acid analogs such as tetraynoic acid (Ahern and Downing, 1970),
which compete as a substrate for PG synthetase, or by PG synthetase
inhibitors, such as indomethacin or aspirin (Vane, 1971). Another alternative would be to inhibit the action of PGs by using an antago nist such as 7-oxa-13-prostynoic acid (Fried et al., 1969). Consider ing the difficulties encountered previously by other investigators
UBing the PG antagonists (Hedge and Hanson, 1972) and the little amount known about the in vivo effects of the essential fatty acid analogs, the PG synthetase inhibitors, aspirin, and indomethacin were chosen as the preferred technique for lowering endogenous PG levels. In addition, 11^
it seemed important to inhibit synthesis in order to determine the role of endogenous PGs in light of the work by several investigators which suggests that the action of PGs is dependent upon, not the release of stored PGs, but the synthesis of new PGs (Silver and Smith, 1975)*
Another approach which might shed light on the importance of endogenous
PGs would be to elevate in vivo levels of PGs by administration of a substrate such as arachidonic acid. Although this seems to be an inter esting possibility, few investigators have attempted such experiments
(Anggard and Larsson, 197^5 Seyberth et al., 1975? Danon, Heimberg and
Oates, 1975)* This approach was not attempted in the present study since the doses required to increase endogenous PG levels have not been worked out and could in itself be very time consuming. Instead indo- methacin and aspirin were used at dosages previously shown to lower in vivo hypothalamic and pituitary levels of PGF.
Systemic Indomethacin in Intact Rats
Indomethacin was demonstrated in this study to significantly decrease the endogenous hypothalamic, pituitary, and thyroidal PGF con centrations. This confirms the previous findings of Behrman, Orczyk and Greep (1972) that indomethacin significantly reduces the hypo thalamic and pituitary PGF concentrations. In addition, Behrman and coworkers (1972) found that aspirin at the same doses as used in this study, inhibits PG formation in these same tissues. PGE^ and ^GF^ levels have been decreased in rat thyroid in vivo (Richman et al., 1975) and in vitro (Burke, 1972) using other doses of indomethacin. Thus, the Ind was effective in producing the desired effect, that of reducing endogenous PG levels. 115
Response to TRH. Although the plasma TSH response to TRH was enhanced in intact rats following Ind treatment basal plasma TSH levels were unchanged, in spite of dramatically decreased circulating thyroid hormones. This suggested that the Xnd could block TSH secretion in response to reduced feedback. In order to examine the effect of Ind on the TSH response to TRH the experiments were continued in animals with identical thyroid status, i.e., both groups receiving the same T^ replacement after thyroidectomy.
Thyroxine and Triiodothyronine Half-lives. Although the T^ half-life was slightly decreased in the Ind-treated group (12»9 h) as compared to the control group (17»2 h) this would not contribute sig nificantly to measurements of thyroid secretion made over a 2 hour period such as that in the TSH stimulation test. However, this slight increase in the rate of disappearance may contribute to the markedly reduced plasma T^ levels observed in the intact rat receiving indo- methacin. The half-life measured in the Gel-treated group (17.2 h) is quite similar to the 16 h half-life reported for male rats by Mor- nex and coworkers (1973) and Salaman (1964), and is also similar to the l8.6 h reported for female rats by Turner (1969).
The half-life of T^ was markedly increased (17«6 h) by Ind in this study as compared to the controls (10.4 h). The rate of disap pearance of T^ in control rats (10.4 h) was similar to the 10.3 h which has been previously reported (Turner, 19^9). 125 125 Since a11** were measured as hormone precipitated from plasma, the actual volume of distribution (V^) cannot be 116 calculated. However, since the amount of isotope injected was constant and is equal to the amount injected divided by the intercept at zero time, the intercept can be used as an index of the V^. In the case of
T^, the fractional rate of disappearance (k) was slightly increased after Ind treatment, and the was unchanged (intercept 101? - 139 vs.
11?8 - 5° in controls). Thus the metabolic clearance rate (MCR) also may have been slightly increased (MCR = k x V^). The fractional rate of disappearance of T^ was markedly decreased after Ind treatment, whereas the estimated was increased (intercept 908 £ 70 vs. 135^ -
128 in controls, p <.02). Since k decreased and increased, these effects would tend to cancel each other resulting in an unchanged MCR.
From such deductions, it appears that the clearance rate of T^ may be slightly increased (to the extent of the increased fractional rate of disappearance) and that of T^ was near normal after Ind treatment.
Indomethacin has been observed to displace T^ from the plasma binding proteins in vitro and in vivo (Tsukui et al., 197*0* If this were an important factor in the present experiments the increased amount of free T^ circulating in the blood could inhibit TSH secretion due to increased negative feedback at the pituitary. TSH secretion in response to TRH has been observed to significantly decrease after Ind treatment in thyroidectomized rats receiving T^ replacement. This in hibitory effect was attributed to a direct effect of the Ind on the pituitary, but it could also be due to increased negative feedback, i.e., an increased amount of circulating free T^. However, the clear ance of T^ from the blood has not been increased after Ind treatment 117
as might be expected due to increased amounts of free T^ resulting from
decreased plasma protein binding (Sterling, 1970). Thus it is unlikely
that binding has been altered in this study, but rather Ind has
directly inhibited pituitary secretion.
Thyroid Response to TSH. In spite of normal plasma TSH con
centrations found in Ind-treated rats, plasma thyroid hormone concen
trations were reduced to levels observed in thyroidectomized rats after
2 days. Since this suggested that the thyroid was not responding to
endogenous TSH, the response to exogenous TSH was assessed after Ind treatment. The thyroid response to TSH as measured by either release
of 125I (response index) or by plasma T^ levels was inhibited by Ind. 125 Although 10 mU TSH was sufficient to stimulate thyroidal I release, it was found that 100 mU TSH was necessary to elicit significant T^ secretion in normal rats. No significant T^ response could be obtained with any of the TSH doses used (7-2500 mU/100 g BW). Indomethacin treatment thus significantly inhibited thyroid secretory response to the TSH as measured by two parameters.
PG synthesis inhibitors have previously been observed to in hibit the thyroid response to TSH in dog thyroid slices (Boeynaems,
Van Sande, and Dumont, 1975) and mouse thyroid cell preparations (Burke,
1973; Burke, 1972). Indomethacin inhibited TSH induced release of iodine (BEI) from the thyroid, although the lactate production and ATP concentration in the dog thyroid were unaffected (Boeynaems et al.,
1975)- Burke has found that the PG concentrations increase in thyroid cell preparations with TSH or dibutyryl cAMP stimulation (Burke, 1973; 118
Burke, 1972). PG synthesis inhibitors, indomethacin and aspirin, inhibit the increase in PG levels due to TSH stimulation (Burke, 1972;
Burke, 1973) or dibutyryl cAMP stimulation (Burke, 1973)- Not only these in vitro experiments but one other in vivo experiment is in agreement with our finding of PG involvement in TSH stimulation of the thyroid, TSH has been found to stimulate ornithine decarboxylase (ODC) activity in the rat thyroid (Richman et al., 1975)* This increased
ODC activity is inhibited by pretreatment of the rat with indomethacin
(1-5 mg), which also elicits a 10-fold decrease in thyroid PG levels.
Therefore, the several reported experiments utilizing Ind in in vivo or ^in vitro experiments have supported the contention that TSH stimu lation of thyroid hormone release necessitates the formation of PGs.
Indomethacin or Aspirin Treatment in Thyroidectomized Rats
Response to TRH. In order to circumvent the direct thyroid effects of Ind which were observed in the intact rat, pituitary sensi tivity to TRH was tested in thyroidectomized rats receiving T^ replace ment. The mean T^ levels 48 hours after thyroidectomy with periodic
T^ replacement were consistently somewhat lower than the normal pre- thyroidectomy level. However, since the T^ levels were not signifi cantly lower than pre-thyroidectomy levels, and since TSH concentrations showed no compensatory rise, the overall replacement must have main tained thyroid hormones near the normal physiologic level. 119
The levels in Ind-treated rats were usually slightly lower
than in control rats at the time of TRH stimulation. This slightly lower T^ level could be due to an increased rate of removal from the
circulation, or to a decreased rate of absorption from the gut. In
any case, this factor does not invalidate our results since decreased
T^ levels in the Ind group would result in decreased negative feedback at the pituitary and would allow an increased response to TRH, In fact, a decreased response to TRH following Ind treatment was found in spite of slightly lower T^ levels.
It is of interest that T^ levels were maintained near normal in spite of the fact that only T^ was administered. The peripheral deiododination of T^ to T^ outside of the thyroid is well known to exist and has been demonstrated to occur in both man and rats (Braver- man, Ingbar, and Sterling, 1970; Surks et al., 1973)* Indomethacin or aspirin treatment has apparently not impaired this deiodination.
Basal TSH levels prior to surgery and drug treatment were 0 -
76 jig/100 ml which is typical for the range of plasma TSH levels nor mally observed in this laboratory when considering the samples were not all run in one radioimmunoassay. I have no explanation for the undetectable plasma TSH level in Figure 21 except that the radioim munoassay in this case may have been less sensitive and thus the very low basal levels were undetectable. The assay in this case was not particularly different by the statistical parameters which are moni tored in this laboratory. 120
The 50 ng dose of TRH was previously determined to be a suf ficient dose to stimulate a non-maximal TSH response* However, this dose was found to be inadequate for eliciting a significant rise in plasma TSH levels in thyroidectomized rats receiving T^. The 250 ng dose of TRH was subsequently found to be a sufficient dose for a non- maximal TSH response in these rats. Pituitary sensitivity did increase with decreasing T^ doses which suggests that the decreased responsive ness to TEH was related to the T^ replacement therapy. This effect was not surprising in light of previous work which has demonstrated that thyroxine inhibits pituitary TSH secretion. This decrease in pituitary sensitivity in the control would only tend to attenuate any effect Ind treatment may have on the response to TRH since the response would be compared to a control group which has been somewhat reduced itself.
Thyroidectomy. The Xnd-induced inhibition of TSH secretion observed on day 2 following thyroidectomy was completely reversible upon cessation of treatment; the TSH level had risen to the control level 3 days following the last Ind injection. On the other hand, the effect of Ind on the responsiveness to exogenous TRH was apparent 10 minutes, but not at 30 minutes after the TRH. At first glance, this finding suggests that the effect of Ind on TRH-induced TSH secretion is rather transient in nature. However, from the temporal patterns of these responses, it seems more likely that the drug increases the half- life of TSH in the circulation as well as decreasing the secretion in response to TRH. TSH half lives were not measured in these experiments 121
but this may be the most likely interpretation of the data in Figures
18t 20 and 22. This suggestion is also consistent with the changing
TSH levels in the Ind-treated rats depicted in Figures 19, 21 and 23.
On the other hand, the data shown in the right-hand panel of Figure 26
indicate that the Ind may have increased the TSH half-life. However,
the data on the left-hand panel in this figure again supports the pre
vious thesis. Thus it would require the measurement of TSH half-lives
in such experimental animals in order to clarify this issue. This re
mains to be done at this time.
Several side effects of Ind treatment were observed during the
course of treatment: decreased food and water intake, mild diarrhea,
and visible vasoconstriction in the extremities. The possibility that
altered food and water intake might be responsible for the effect which
was attributed to Ind was eliminated when the drug was found to be com
pletely effective when food and water intake were controlled. Both
groups received glucose water three times during the day from 9:00 a.m.
- 9i00 p.m. Although rats are nocturnal and normally eat at night,
both groups were fed at the same time so that the time factor may be
disregarded in considering differences between the two groups. The TSH response to TRH following thyroidectomy in the control group receiving limited food and water (Fig. 25) was slightly lower than the increase in TSH observed in the control group receiving food and water ad libitum (Fig. 23). The limited food and water intake may have led to the slightly decreased TSH response in the control group, but it cer tainly did not attenuate the effect of Ind. 122
In addition to the altered feeding behaviour, cardiovascular
effects were considered to be a possible complicating factor in these
experiments. PGs are known to have either vasocontrietor or vasodila-
tory properties when administered _in vivo or in vitro depending upon
which PG is used and what parameter is being observed (Weeks, 1969;
Staszewska-Barcsak and Vane, 1975)* In an attempt to eliminate this problem
the distribution of the drug Ind was restricted to the region of in
terest, the pituitary, in some of the experiments. In other experi
ments TRH was administered stereotaxically in order to examine the
possibility that delivery of TRH had been decreased and, therefore, resulted in decreased TSH secretion after Ind treatment and TRH.
Stereotaxic Microinjection Studies
Systemic Indomethacin Treatment with TRH Microinjection. Ind- treatment once again inhibited TSH secretion following intrapituitary injections of TRH. Thus the suggestion of decreased TRH delivery to the pituitary is not supported. It is of interest though that plasma
TSH levels were reduced but not significantly at 10 minutes as they were at 20 minutes. This pattern of results suggests that TSH clear ance may have been altered.
Intrapituitary Microinjection of Indomethacin and TRH. By restricting the distribution of Ind to the pituitary, other factors such as timing are also necessarily confined. With the technique used, a maximum of a 5-minute delay between the injection of Ind and TRH was 123
possible in order to assure that both drugs were injected at the same
site of the pituitary. Using such a procedure Ind was found to have
no effect on the TSH response to TRH. Although this experiment failed
to provide evidence for endogenous PGs regulating TSH secretion, it
certainly does not disprove the suggestion since there is no way of
knowing the length of time or amount of Ind required to significantly
decrease endogenous PG levels. The possibility that the cardiovascular
effects of Ind may have contributed to the observed inhibitory effects
on TSH secretion cannot be ruled out, therefore, at this time.
Specificity of Indomethacin and Aspirin
Indomethacin is recognized to be the more potent inhibitor of
PG synthetase of the two drugs by a ratio of about 10 or 15 to 1. In
addition to inhibiting PG synthetase, several other enzymes are known
to be inhibited by indomethacin. These enzymes are PG dehydrogenase
(Hansen, 197*0 i phosphodiesterase (Flores and Sharp, 1972), dopa de
carboxylase (Skidmore and Whitehouse, 1966a), histidine decarboxylase
(Skidmore and Y/hitehouse, 1966b), collagenase (Brown and Pollock, 1970)
and various enzymes involved in oxidative phosphorylation (Whitehouse
and Haslam, 1962). Fortunately the concentrations of these drugs re
quired to inhibit PG synthetase are generally much lower than that
required to inhibit these other enzymes. Aspirin is also known to
inhibit PG dehydrogenase (Hansen, 197^)• In the case of this enzyme,
inhibition of its action would attenuate the desired effect of lowered
endogenous PG levels. Since PGF levels were found to be significantly 12b decreased in these experiments this factor can be disregarded as a pos sible interference. The one enzyme which is particularly sensitive to indomethacin is phosphodiesterase (PDE) and thus requires more careful consideration.
Flores and Sharp (1972) have reported that Ind (1 jig/ml) in creased ADH induced water flow in the toad bladder and that of dibu- tyryl cAMP, and thus attributed the effect to an effect on the cAMP generation, and not an effect on adenyl cyclase. They also tested 0.1,
1, and 10 /ig/ml Ind on PDB activity and found from 8-2096 inhibition of
PDE activity depending upon the dose of Ind and the concentration of cAMP used. Albert and Handler (197*0 found a stimulation of water per meability response to ADH by Ind with a change of 50-30&& using 0.7 or
1*10 /*M Ind. This was reversed by 0.33 /*M PGE^ but was still present with cAMP which was stimulated by 300$. Polyphloretin phosphate (PPP) also could stimulate ADH, and this effect was reversed by PGE^. Simi larly cAMP was stimulated by both PPP and ADH, but PPP was only known to inhibit PDE at 10 times the concentration used (50 jig/ml). They concluded that the effects of Ind and PPP could not be taken as evi dence that PGs inhibit the action of ADH. Flores et al., (1975) has more recently demonstrated that PGE^ stimulates osmotic water flow in the toad bladder.
The importance of these findings would be the potential of ele vated levels of cAMP. The concentration required to inhibit PDE activity appear to be larger than may exist in vivo with my drug regi men, but assuming that it is not, the effects would not correlate with 125 my findings. Pituitary cAMP levels are thought to increase with pitui tary secretion and cAMP is known to mediate thyroid hormone release at the thyroid. Thus with increased cAMP levels possible with Ind inhibi tion of PDE one would expect increased secretion of both TSH and thyroid hormones. But the present findings are in direct opposition to this. Ind was found to decrease thyroid hormone release, and to decrease TSH secretion in response to TRH or to decreased negative feedback by thyroid hormones due to thyroidectomy. Thus it appears that this effect on PDE could only act to dampen the observed effects of Ind in the pituitary-thyroid axis. If actual plasma concentrations of Ind were known it would be much easier to determine if any of these effects on other enzymes could be possible.
It is possible that any Ind or aspirin in the rat plasma may interfere with the RIA used to determine TSH, T^, or T^ and give falsely low values. Once again it would be helpful to know actual plasma concentrations of Ind or Asp. Hucker et al. (1966) has studied
Ind metabolism in vivo and determined a half-life of about H hours in the rat. Twenty-four hours after administering indomethacin (1.0 mg/
100 g, iv or po), the highest plasma Ind concentration was 3-7 - 0-8 pig/ml. The dose administered in this study was 1.5 times as much as
Hucker used, although it was administered via a different route in this study (sc). Thus on the basis of Hucker's measurements, plasma
Ind levels may be as high as 5-5 /ig/ml. In order to determine if the
RIA procedure is affected by the presence of Ind in the plasma, one could simply add this amount of Ind to the plasma and assay the plasma 126
sample with and without Ind. There are presently no reports available
concerning this issue, Hucker has also mentioned the procedure for
assaying Ind in the plasma by extraction and spectrophotometric deter
mination. This may be done in the future in order to determine any
RIA interference.
Comparison of Results in This Study with Previous Studies Which Used Exogenous Prostaglandins
There is not yet a consensus regarding the effect of PGs on
pituitary TSH secretion. Pituitary cells in culture have been shown -5 to increase their release of TSH in response to 10 M at 2.5 h
although tfPGE2 produced dramatic changes in the morphological appear
ance of the cells" (Vale et al., 1972). In contrast, Tal, Szabo and
-5 Burke (197*0 found that 5 x 10 M PGE^ or PGFla failed to elicit TSH secretion from whole pituitary glands incubated ^n vitro. In this same
study TRH (1 fig/ml) stimulated release of TSH. 7-oxa-13-prostynoic
acid (P"X^), a PG antagonist, had no effect on TRH stimulation of TSH release and did not modify TSH release in the presence of PGs. A vari ety of PGs (H fig) has also been found to be ineffective in stimulating
TSH secretion in vivo upon intrapituitary administration (Brown and
Hedge, 197*0• However, the same amount of these PGs increased the re sponsiveness to subsequent TRH (Brown and Hedge, 197*0* suggesting that
PGs do play some role in the modulation of TSH secretion.
The data that are presented in the current study, showing that reduced endogenous PGs inhibit TSH secretion in response to TRH and thyroidectomy, further support the contention that PGs play a role in modulating TSH secretion. Although the precise role of PGs in this system is as yet unknown some conclusions may be made from the data thus far accumulated. First, the PGs themselves do not stimulate the release of TSH and, therefore, probably do not mediate TSH secretion directly. Second, since basal TSH levels were unaffected by a marked reduction in the PG level, thyrotrope secretion is not completely de pendent upon PG synthesis. Third, the PGs probably are important to the normal physiological regulation of TSH secretion as demonstrated by the inhibition of TSH secretion in response to TRH or thyroidectomy when endogenous PG synthesis is inhibited. This suggests that PGs may be necessary for the thyrotropes to respond to an.alteration in the system.
With PG synthesis intact, thyrotropes may be relatively sensitive to altered thyroid hormone levels and/or increased TRH release, but in the absence of normal PG synthesis, the system may become very insensitive to changes. The present data do suggest that PGs participate in the regulation of TSH secretion, but are not obligatory intermediates.
Conclusions
The present study indicates that endogenous prostaglandins par ticipate in the regulation of TSH secretion. After treatment with a
PG synthesis inhibitor, indomethacin or aspirin, a number of effects on the hypothalamic-pituitary-thyroid axis were observed.
First, the responsiveness to exogenous TRH in intact rats was increased due to the markedly decreased negative feedback, i.e., re duced levels of circulating thyroid hormones. The markedly reduced ia8
plasma thyroid hormone levels were due to direct thyroid inhibition by
the drug, Ind treatment caused significant inhibition of the thyroidal 125 response to exogenous TSH as measured by the release of I or by
plasma levels.
In order to circumvent this problem, TRH responsiveness was
assessed in rats after thyroidectomy and replacement during Ind or
Asp treatment. With similar thyroid hormone levels, Ind and Asp were
observed to decrease the TSH response to exogenous TRH. Thus endogenous
PG synthesis is necessary for normal pituitary secretion in response to TRH.
Indomethacin or aspirin treatment were both observed to inhibit compensatory TSH secretion in response to reduced negative feedback, i.e., thyroidectomy. This effect was shown to be reversible in the
case of Ind. Thus endogenous PG synthesis is also necessary for normal
pituitary secretion of TSH in response to decreased negative feedback.
When Ind treatment in the thyroidectomized rat receiving T^ replacement was followed by stereotaxic microinjection of TRH the re lease of TSH was inhibited. Therefore, the effect of inhibition of
TSH secretion appears to be a direct effect on the pituitary, and not an inhibitory effect on delivery of the TRH to the pituitary.
These data thus suggest that endogenous PG synthesis is neces sary for normal TSH secretion in response to exogenous TRH and thy roidectomy. LIST OF REFERENCES
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