EFFECTS OF CHRONIC ACTH STIMULATTON,' GONADAL HORMONES, THYROIDAL HORMCTNE TREATMENT AND AGE 0N ADRENOCORTICAL FUNCTION OF THE RAT Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY GERALD DALE HESS 1970 main t": was. W“; L, , 1 LIBRARY i’ 5: Michigan Since 3 Univcxsxty This is to certify that the thesis entitled EFFECTS OF CHRONIC ACTH STIMULATION, GONADAL HORMONES, THYROIDAL HORMONE TREATMENT AND AGE ON ADRENOCORTICAL FUNCTION OF THE RAT presented by Gerald Dale Hess has been accepted towards fulfillment of the requirements for Ph.D. Physiology degree in gay/D 92¢ Major professor Date a’NO Q 2 / 9 7 C) 0-169 BINDING BY HMS & SBNS' \ {809V mum NE. T. \ ABSTRACT EFFECTS OF CHRONIC ACTH STIMULATION, GONADAL HORMONES, THYROIDAL HORMONE TREATMENT AND AGE ON ADRENOCORTICAL FUNCTION OF THE RAT BY Gerald Dale Hess The primary objective of this study was to gather physiological data related to endocrine function in aged rats with particular emphasis on the adrenal cortex. The effect of chronic adrenocortical stimulation was studied in young (5 month) and old (26 month) male rats. These rats were exposed to daily injections of a long-acting (depo) ACTH preparation for 6 weeks. Adrenocortical response (corticosterone concentration, ug/lOO ml plasma) to ether stress was depressed (50% of control) in young male rats but was not altered in old male rats during the 6 weeks of treatment. Adrenocortical response to acute ACTH stimulation (saline based exogenous ACTH preparation) was elevated in both young and old male rats following depo-ACTH treatment. Adrenocortical response to ether stress was also depressed in young (3 month) but not old (25 month) female rats fol- lowing chronic ACTH treatment, while adrenocortical response to ACTH was elevated in both age groups. These results are Gerald Dale Hess indicative of a marked alteration of the adrenocortical feed- back control mechanism sensitivity (presumably to circulating corticosteroids) with increasing age in the rat. Ovariectomy of young (6 month) and old (24 month) female rats did not affect adrenocortical response of either age group to ether stress or exogenous ACTH. Gonadal hormone replacement therapy with progesterone (800 ug daily) or estradiol (30 ug daily) for 1 week did not alter adrenocor- tical responsiveness of ovariectomized rats. Adrenocortical responsiveness to exogenous ACTH was reduced in young female rats that had been ovariectomized at 30 days of age (response determined 6 weeks later). Castration of young (5 month) and old (24 month) male rats did not reduce adrenocortical reSponsiveness to ACTH. These experiments indicated that gonadal hormone deficiencies are of limited importance with respect to age-related changes which exist in adrenocortical function of the rat. They also suggested that the principal influence of estrogens on adrenocortical function in the female rat occurs by the onset of puberty with little addi- tional influence of physiological dosages of estrogens on adrenal steriodogenesis during the adult lifespan of the female rat. Adrenocortical responses of male and female rats to ether stress were found to change between 23 and 200 days of age. The fact that adrenocortical stress response changes between 23 and 200 days of age in the rat should Gerald Dale Hess be considered when animals are chosen for studies of adreno- cortical functional parameters. Young and old male and female rats were exposed to thyroid hormone treatment (Protamone, .02%, .O4% and .08% of diet) for a period of three weeks. Adrenocortical response (plasma corticosterone levels) was higher in young (3 month) male rats fed the .04% (81 uQ%) and the .08% Protamone diets (106 ug%) than in controls (59 ug%). Adrenocortical responses of old (23 month) male rats were not significantly different from those of non-treated control rats after 3 weeks of Protamone treatment. Adrenocortical response of young (4 month) female rats treated with .08% Protamone was signif- icantly higher (161 ug%) that of controls (110 uq%), but adrenocortical response was not altered by any level of Protamone treatment in old (25 month) female rats. These findings suggested that the pituitary-adrenal axis of aged rats is less sensitive to thyroid hormone stimulation than the pituitary-adrenal axis of young adult rats of both sexes. This conclusion is in agreement with the previous hypothesis concerning age-differences in corticosteroid feedback influences. Biological half—life, distribution volume and pro- duction rate of corticosterone were estimated in young (5 month) and old (27 month) female rats by calculating dis- appearance curves (single compartment model) for plasma radioactivity following rapid injection of H3-cortisosterone. Gerald Dale Hess The estimated biological half-life of corticosterone was longer in old female rats (10.6 minutes) than in young female rats (9.3 minutes), but the calculated corticoste- rone production rate was significantly higher in young (11.4 ug/min) than in old (7.8 ug/min) female rats. These findings suggested that adrenocortical secretory dynamics change with age in the rat. A similar study in young female rats suggested that chronic ACTH stimulation of the pituitary- adrenal axis does not alter the biological half-life and dis- tribution volume of corticosterone. The H3-corticosterone distribution technique was also used to study the influence of Protamone treatment (.08% for 3 weeks) on corticosterone distribution volume in the male rat. Corticosterone distri- bution volumes were significantly lower (36% of body weight) in treated than in control rats (48% of body weight). These data support the hypothesis that corticosterone distribution volume is altered by therid hormone treatment. Resting adrenocortical responses determined in these rats gave evidence of no significant influence of thyroid hormone treatment on this parameter of adrenocortical function. EFFECTS OF CHRONIC ACTH STIMULATION, GONADAL HORMONES, THYROIDAL HORMONE TREATMENT AND AGE ON ADRENOCORTICAL FUNCTION OF THE RAT BY Gerald Dale Hess A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1970 i, \ ACKNOWLEDGME NT 8 I would like to express my thanks to all those who have made the completion of this study a reality. The patient guidance and assistance of Dr. G. D. Riegle has been deeply appreciated. I am grateful to Dr. Riegle and to Dr. J. E. Nellor for the provision of animal and laboratory resources utilized in this study. The counsel of Dr. J. Meites, Dr. E. P. Reineke, Dr. J. B. Scott, Dr. D. A. Reinke and Dr. W. R. Dukelow during my graduate program has also been appreciated. The friendship, encouragement and assistance of the entire Endocrine Research Unit staff have meant much to me during my tenure there. Special appreciation is due Wendell Hofman, Shirley Johnson and Pat Smith in this regard. Finally, to my wife Jan, I offer my sincere thanks for her enduring patience, encouragement and assistance which made the completion of my graduate program a reality. I also wish to eXpress my gratitude to the Department Of Physiology, Michigan State University, and the National Institutes of Health for the predoctoral traineeship which ‘was granted from March, 1967 to June, 1970. ii TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . . . . . . Control of the Pituitary-Adrenal Axis . . . Steroid-Sensitive Feedback Control . . . . Short Feedback Control . . . . . . . . . . Interaction of Stress and Resting Responses Gonadal Hormone Influences on the Pituitary- Adrenal Axis . . . . . . . . . . . . . . Thyroidal Influences on the Pituitary-Adrenal Axis . . . . . . . . . . . . . . . . . . . . Studies of Endocrine Function in Aging Individuals . . . . . . . . . . . . . . . . Aging and the Pituitary-Adrenal Axis . . . Gonadal Function in the Aged . . . . . . . Influences of Aging on Thyroid Function . MATERIALS AND METHODS . . . . . . . . . . . . . . Experimental Animals . . . . . . . . . . . . . Statistical Analysis . . . . . . . . . . . . . Determination of Adrenocortical Response . . . ACTH Stimulation . . . . . . . . . . . . . Stress . . . . . . . . . . . . . . . . . Blood Collection . . . . . . . . . . . . . Resting Corticosterone Levels . . . . . . Fluorometric Assay of Plasma Corticosterone . H3-Corticosterone Disappearance from Plasma . EXPERIMENTAL Experiment I: Effects of Chronic Adrenocorti- cal Stimulation in Young and Old Male Rats . Materials and.Methods . . . . . . . . . . Results . . . . . . . . . . . . . . . . . Experiment II: Effects of Chronic Adrenocorti- cal Stimulation in Young and Old Female Rats Materials and Methods . . . . ... . . . . Results . . . . . . . . . . . . . . . . . iii Page 11 13 17 20 24 24 29 32 36 36 36 36 36 37 37 38 38 39 43 43 45 61 61 61 I E“, w Page Discussion of EXperiments I and II . . . . . . 75 Experiment III: Influence of Gonadal Steroids on Adrenocortical Function in the Rat . . . . . 81 Materials and Methods . . . . . . . . . . . . 81 Results . . . . . . . . . . . . . . . . . . . 83 Discussion . . . . . . . . . . . . . . . . . . 94 Experiment IV: Adrenocortical Stress ReSponse of Male and Female Rats Between One and Six Months of Age . . . . . . . . . . . . . . . . . 98 Materials and Methods . . . . . . . . . . . . 98 Results . . . . . . . . . . . . . . . . . . . 99 Discussion . . . . . . . . . . . . . . . . . . 106 Experiment V: Thyroid Hormone Influence on Adrenocortical Function in Young Adult and Aged Male Rats . . . . . . . . . . . . . . . 108 Materials and Methods . . . . . . . . . . . . 108 Results . . . . . . . . . . . . . . . . . . . 109 Experiment VI: Thyroid Hormone Influences on Adrenocortical Function in Young and Aged Female Rats . . . . . . . . . . . . . . . . . . 119 Materials and Methods . . . . . . . . . . . . 119 Results . . . . . . . . . . . . . . . . . . . 119 Discussion of EXperiments V and VI . . . . . . 130 Experiment VII: The Effects of Age and Chronic ACTH Stimulation on Biological Half-Life, Distribution Volume and Production Rate of Corticosterone in Female Rats . . . . . . . . . 133 Materials and Methods . . . . . . . . . . . . 133 Results . . . . . . . . . . . . . . . . . . . 134 Discussion . . . . . . . . . . . . . . . . . . 138 Experiment VIII: The Influence of Thyroid Hormone Treatment on Corticosterone Dis- tribution Volume in the Male Rat . . . . . . . . 141 Materials and Methods . . . . . . . . . . . . 141 Results . . . . . . . .'. . . . . . . . . . . 142 Discussion . . . . . . . . . . . . . . . . . . 144 GENERAL DISCUSSION . . . . . . . . . . . . . . . . . . 146 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . 150 LITERATURE CITED . . . . . . . . . . . . . . . . . . . 153 APPENDIX I. MSU REGULAR RAT DIET . . . . . . . . . . . . . 166 II. CURRICULUM VITAE . . . . . . . . . . . . . . . 167 iv 10. 11 LIST OF TABLES Adrenocortical responsiveness to stress and ACTH stimulation in young and old male rats Body weight of young and old male rats at bleeding intervals of chronic ACTH experiment . . . . . . . . . . . . . . . . Adrenocortical responsiveness to stress and ACTH stimulation in young and old female rats . . . . . . . . . . . . . . . . . . . Body weight of young and old female rats at bleeding intervals of chronic ACTH eXperiment . . . . . . . . . . . . . . . . Effects of ovariectomy on adrenocortical response to exogenous ACTH in young and old female rats . . . . . . . . . . . . . . Effect of ovariectomy on adrenocortical reSponse to ether stress in young and old female rats . . . . . . . . . . . . . . . . Adrenocortical response of young female rats to ACTH following gonadal hormone treatment . . . . . . . . . . . . . . . . . Adrenocortical response of old female rats to ACTH following gonadal hormone treatment Adrenocortical reSponse of young female rats to ether stress following gonadal hormone treatment . . . . . . . . . . . . . Adrenocortical response of old female rats to ether stress following gonadal hormone treatment . . . . . . . . . . . . . . . . . Effect of castration of adult male rats on adrenocortical response . . . . . . . . . . Page 59 6O 73 74 86 87 88 89 9O 91 92 .- .. a 1‘... lb- : o -. IS. A.‘ Table 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. Effect of prepubertal gonadectomy on adrenocortical response Adrenocortical response ether stress between 26 age 0 O O O O O O O O O Adrenocortical reSponse to ether stress between of age . . . . . . . . Adrenocortical reSponse to ether stress . . . . O O O O O O 0 0 O of male rats to and 154 days of of female rats 23 and 200 days of female rats Comparison of adrenocortical response to ether stress and ACTH stimulation . . . The effect of Protamone adrenocortical function rats . . . . . . . . . The effect of Protamone adrenocortical function Feed consumption of male rats on Protamone treatment . . . . . . . supplement on of young male supplement on of old male rats Body weight of young and old rats of Protamone eXperiment . The effect of Protamone adrenocortical function rats 0 O O O 9 O O O O The effect of Protamone adrenocortical function rats 0 O O O O O O 0 O C O 0 O O O O O 0 supplement on of young female supplement on of old female 0 O O O O 0 G O 0 Feed consumption of female rats on Protamone treatment . . Body weight of young and old female rats of Protamone experiment Functional parameters of adrenocortical response in young and old female rats . vi Page 93 103 104 105 105 115 116 117 118 126 127 128 129 136 Table 26. 27. Page Functional parameters of adrenocortical response in female rats: effect of chronic ACTH treatment . . . . . . . . . . . 137 Effects of Protamone treatment on func- tional parameters of adrenocortical response in male rats . . . . . . . . . . . . 143 vii Figure 1. 10. .11 LIST OF FIGURES Scheme of chronic ACTH experiment . . . Adrenocortical responses of depo-ACTH treated and control old male rats to acute ACTH stimulation . . . . . . . . . Adrenocortical responses of depo-ACTH treated and control young male rats to acute ACTH stimulation . . . . . . . . . Adrenocortical responses of depo-ACTH treated and control old male rats to ether stress . . . . . . . . . . . . . . Adrenocortical responses of depo-ACTH treated and control young male rats to eter stress . . . . . . . . . . . . . . Effect of chronic ACTH treatment on adrenocortical response to ether stress and acute ACTH stimulation in young and old male rats . . . . . . . . . . . . . Adrenocortical responses of depo-ACTH treated and control old female rats to acute ACTH stimulation . . . . . . . . . Adrenocortical responses of depo-ACTH treated and control young female rats to acute ACTH stimulation . . . . . . . . . Adrenocortical reSponses of depo—ACTH treated and control old female rats to ether stress . . . . . . . . . . . . . . Adrenocortical responses of depo-ACTH treated and control young female rats to ether stress . . . . . . . . . . . . . . Effect of chronic ACTH treatment on adreno- cortical reSponse to ether stress and acute ACTH stimulation in young and old female rats . . . . . . . . . . . . . . . . . . viii Page 48 50 52 54 56 58 64 66 68 7O 72 Figure Page 12. Adrenocortical responses of rats between 23 and 200 days of age to ether stress stimulation . . . . . . . . . . . . . . . . . 102 13. Adrenocortical responses of young and old male rats following thyroid hormone treatment . . . . . . . . . . . . . . . . . . 112 14. Adrenocortical response versus level of Protamone treatment in young and old male rats . . . . . . . . . . . . . . . . . . 114 15. Adrenocortical responses of young and old female rats following thyroid hormone treatment . . . . . . . . . . . . . . . . . . 123 16. Adrenocortical response versus level of Protamone treatment in young and old female rats . . . . . . . . . . . . . . . . . 125 ix llAI y- I “I '-,v. cw . INTRODUCTION Although progress in experimental gerontology has been less rapid than that in many other fields of biology, a number of significant studies dealing with the biology of aging have been reported in recent years. A comprehensive review of progress in experimental gerontology was presented by Comfort (1970). A detailed search for individual error processes, presumably at the cellular level, has constituted an important facet of recent gerontological investigation. DeSpite the recent emphasis upon studies of aging at the cellular and molecular level, the importance of studies at the systemic level is also recognized. Sacher (1968) criticized the molecular approach in experimental gerontology because of its persistent lack of consideration for certain systemic aSpects of aging and death. The molecular approach implies that the aging pro- cess can be explained purely on the basis of physical phenomena. The fact that a mouse is senile at three years tvhile a man is senile at 90 years of age, although both are almost identical at the molecular level, illustrates the Problem with this assumption. Sacher (1968) concluded that a greater emphasis must be placed upon the systemic aspects 0f aging if experimental gerontology is to develOp as a complete discipline. Studies of aging at the systemic level are required to produce such an emphasis. Frolkis (1968), Comfort (1970) and others regard the aging process as a gradual loss of homeostasis in highly complex organisms. Many reasons can be given to explain this homeostatic decrement. Curtis (1964) prOposed that spontaneous mutations in the somatic cells are involved in the aging process. The organs of the body become full of defective cells and the resulting loss of efficiency con- stitutes the deterioration known as aging. According to Bjorksten (1968) the progressive cross-linkage of protein and nucleic acid molecules is responsible for the aging of biological organisms. The resulting molecular alteration leads to a progressive deterioration of physiological per— formance and the eventual death of the organism. Age- related changes in the control mechanisms of the central nervous and endocrine systems were examined by Frolkis (1968) and co-workers. They obtained evidence that endocrine func— tional activity diminished with increasing age. The sensi- tivity of tissue to hormones, determined by finding the lowest dose of hormone capable of producing metabolic and functional changes, was found to increase with age. In- creased tissue sensitivity to hormones was thought to represent an adaptative element to compensate for decreased .functional activity of the aging endocrines. Included in this; adaptation was an increased sensitivity of endocrine glanxis to hormones produced by other endocrine glands. On the other hand, the same group (Frolkis, 1968) found that tissue reactivity to hormones, i.e., the potential range of metabolic and functional changes taking place in response to large doses of hormones, decreases with increasing age. Endocrine homeostasis can be maintained in old age because of these "allometric" changes which occur in the endocrine control systems. However, the onset of patholog- ical or other extreme conditions in aged individuals may upset the adapted regulatory mechanisms and cause the collapse of homeostasis. Studies of the biological aging process have been limited despite the recent surge in basic biological re- search. Many theories have been prOposed to account for the aging process, but these have suffered from a lack of the experimental evidence needed to substantiate them. The loss of homeostatic capacity is a well-known characteristic of biological aging. Because of its importance in maintaining body homeostasis as well as its control by the nervous system, the endocrine system is a logical prospect for in- volvement in the aging process, whether this involvement be cause or effect of the aging phenomenon. The primary objective of this study was to gather pkwsiological data related to endocrine function in aged rats with particular emphasis on the adrenal cortex. To 13118 author's knowledge, few if any studies dealing with funrztional measurements of the pituitary-adrenal axis in aged rats have been reported heretofore other than those from our laboratory. In several studies undertaken for this dissertation, experiments in aged animals were prefaced by considerable investigation of the systems involved in young adult rats. LI TERATURE REVIEW Control of the Pituitary-Adrenal Axis Steroid—Sensitive Feedback Control The existence of a steroid sensitive feedback mecha- nism for controlling pituitary ACTH release and synthesis is well documented. MacKay and MacKay (1926) demonstrated that unilateral adrenalectomy induces compensatory hypertrOphy of the remaining adrenal cortex. De Groot and Harris (1950) and Hume and Wittenstein (1950) working independently, showed that the hypothalamus influences ACTH secretion. Studies by Saffran gt EL. (1955) and Guillemin and Rosenberg (1955) suggested the existence of a corticotrOphin-releasing factor (CRF) of hypothalamic origin. Other investigators also showed that the hypothala- mus is involved in the control of pituitary ACTH release. .Bogdanove and Halmi (1953) and McCann (1953) found that lesions of the medial basal hypothalamus (MBH) may cause adrenal atrOphy. Lesions of the MBH blocked the compensa- ‘tOTy hypertrOphy which normally follows unilateral adrenal- GHZtOmy in several studies (Ganong & Hume, 1954; Endréczi & Mess, 1955; Fulford & McCann, 1955) . Implantation of corticosteroids in the MBH has been used in a number of more recent studies to show that the brain is involved in control of the pituitary-adrenal axis. Endr6czii_gl_l. (1961) inhibited both resting adrenal secre- tion and stress induced increases in adrenal secretion with cortisone implants into the MBH of normal cats and rats. Smelik and Sawyer (1962) confirmed these results in rabbits bearing median eminence implants of cortisol. Corbin §§__l. (1965) found that plasma and adrenal corticosterone levels and adrenal weight could be reduced significantly by median eminence implants of Dexamethasone. They also showed that a similar but less extensive effect could be produced by implants of cortisol. Dexamethasone implants into the pituitary or cerebral cortex did not affect the parameters studied. Median eminence implants of either cortisol or corticosterone blocked adrenocortical stress responses in studies by Davidson §£_§l. (1965). These implantation studies have shown rather clearly that the MBH is involved in the control of the pituitary-adrenal axis, but have failed to determine whether the MBH merely conveys signals from other brain structures to the anterior pituitary or exerts a controlling influence itself. Demonstration of changes in the CRF content of the 1median eminence in reSponse to pituitary-adrenal activation INES provided additional evidence for central nervous system inVTDlvement in the control of adrenocortical function. Vernikos-Danellis (1965) demonstrated that median eminence CRF activity closely parallels or precedes stress induced changes in blood and pituitary ACTH concentrations. Expo- sure of female rats to ether stress, sham adrenalectomy or true adrenalectomy increased the CRF content of the median eminence while the median eminence CRF content was reduced and the stress induced rise of median eminence CRF was completely blocked by cortisol pretreatment. This study was important in validating the concept that CRF's are physiologically involved in the synthesis and release of ACTH . The medial basal hypothalamus (MBH), also called the hypOphysiotrOpic area, appears to be the source of the neural factors which control anterior pituitary function. Halasz gt_gl, (1962) showed that the normal function and structure of the anterior pituitary was maintained following tranSplantation to the MBH region. Transplantation of pitu- itaries to other hypothalamic or extrahypothalamic areas resulted in a loss of normal structure and function, hence the name hypOphysiotrOpic area. The significance of the hypOphysiotrOpic area in adrenocortical control was studied further following neural isolation of this region. Using an ingenious knife assembly mounted in a stereotaxic apparatus, Iialasz and Pupp (1965) were able to completely sever the hlqfliphysiotrOpic area from the rest of the brain without breaking its contact with the pituitary. Although vascular regeneration occurred subsequent to this procedure, neural isolation endured for the course of the experiments. Halasz 31; 511, (1967) found that complete deafferen- tation of the hypOphysiotrOpic area does not alter compensa- tory hypertrophy of the remaining adrenal following uni- lateral adrenalectomy. Similarly, Palka Jig. (1968) showed that neural isolation of this region did not prevent dexamethasone suppression of non—stress plasma corticoste- rone levels. Even more surprising is the finding by several groups (Matsuda e_t_§_l__., 1964; Halasz _e_t_ a1” 1967; Palka SE 31:, 1968) that a normal adrenocortical response to ether stress can still be produced following neural isolation of the hypOphysiotropic region. Voloschin g1; _a_l_. (1968) also fOlmd that the pituitary-adrenal axis was capable of respond- ing to ether stress after neural isolation of the MBH, but the observed response was at a submaximal level. Matsuda at \ _l_. (1964) found no response to traumatic stress in MBH iSolated rats despite a normal response to ether stress in tl’lese animals. Upon reviewing studies of adrenocortical function fOllowing isolation of the hypOphysiotropic area conducted in several laboratories including his own, Halasz (1969) QOncluded that adrenocortical responses to ether stress, immobilization and corticosteroid feedback are mediated tI‘Lrough the MBH. Studies of adrenocortical function based on neural isolation of the hypOphysiotrOpic area do not pre— Qlude the possibility that the MBH maintains normal anterior n.» I e R‘- h pituitary structure and function, with the stress response and corticoid feedback being mediated at least partially by the anterior pituitary. Since MBH neural isolation blocks some stress responses while leaving others intact, Halasz (1969) concluded that several factors activate ACTH release by different mechanisms. The release of vaSOpressin follow— ing ether (Turner, 1966) could represent one such mechanism. EXperimental evidence now available suggests that steroid sensitive receptors active in controlling pituitary- adrenal function exist outside of the hypothalamus. Endrbczi ._£,_1. (1961) and Corbin gt a1. (1965) found that cortico- steroid implants into the midbrain region depressed pituitary- adrenal function in rats. Davidson and Feldman (1963) reported that midbrain implants of cortisol were partially effective in blocking adrenal compensatory hypertrOphy after unilateral adrenalectomy. Slusher (1966) inhibited diurnal changes in adrenal and plasma corticosterone levels of rats with cortisol implants into the hippocampus or midbrain. Kendall §£_§1, (1969) found that systemically ineffective amounts of corticoids could reduce ACTH secretion when placed in certain areas of the ventricular system. They concluded that previous reports of extrahypothalamic feed- back receptor sites in the rat forebrain might be explained by tranSport of corticoids from their implantation site to a single feedback site located in the basal hypothalamus or the anterior pituitary via the ventricular system. o.y- - 5". C :a' an. L- n- cv. u.. - ‘ho 10 Evidence from other studies suggests that the pitu- itary gland may contain receptor sites for the adrenal steroid feedback mechanism. De Wied (1964) found that dexamethasone pretreatment prevented pituitary ACTH release by hypothalamic extracts containing CRF while Chowers gt_al. (1967) showed that dexamethasone implants into the median eminence or pituitary reduced pituitary ACTH content. Only median eminence implants reduced CRF content (Chowers gt_a1., 1967). Arimura 2; a1. (1969) found that dexamethasone sup- presses the action of CRF at the pituitary level, although this blockade does not develOp immediately. Dexamethasone injected bilaterally into the anterior pituitary by Russel ._§.;l. (1969) prevented pituitary ACTH release in response to both exogenous and endogenous CRF. Similar results were obtained when dexamethasone was injected into the median eminence or into the septal region of the brain. Using a fluorescent dye, Russel gt al. (1969) found that materials injected into the median eminence or septal region spread very rapidly to the pituitary. They concluded that previous evidence for hypothalamic feedback sites for pituitary— adrenal control resulted from corticoids which had traveled to the pituitary and exerted an effect there. The fact that other investigators did not demonstrate a local inhibitory effect of dexamethasone at the pituitary was attributed to inadequate exposure of the pituitary to dexamethasone. A recently reported study of ACTH release in dogs (Gonzalez- Luque §£_§l,, 1970) also suggested that adrenal steroid 11 feedback control resides in the pituitary. Although this group as well as others have inhibited the release of ACTH with dexamethasone at the pituitary, this effect has not been shown for physiological levels of natural corticoids. Short Feedback Control The pituitary gland is regulated by two different types of feedback mechanisms. The peripheral target gland hormones serve as feedback signals for the classic control system while the pituitary hormones themselves are involved in a short feedback control system. Short feedback control systems have been demonstrated for all the anterior and intermediate lobe pituitary hormones, including prolactin, growth hormone and melanocyte stimulating hormone which have no peripheral target glands and hence are not regulated by the classic feedback system (Motta §£_gl., 1969). The short feedback receptors are located primarily in the brain (hypothalamus) (Mess & Martini, 1968; Motta _£._1,, 1969). The pituitary hormones may reach the brain by either the general circulation or by transport up the pituitary stalk. TOrOk (1964) demonstrated a vascular system passing from the posterior surface of the anterior pituitary to the capillary complex. Several pituitary hormones have been isolated where this vascular system ends in the basal hypothalamus (Guillemin et al,, 1962; Schally t al., 1962; Johnson & Nelson, 1966). u. ‘n -.v.. V “q a ‘1‘ I.Q- \ .uou ago- - Vu'i n... n '¢b~ ..', '60: .._ I.‘ fie “- . ‘b dd. n ‘~ IN .. ’v 'Q I". I. 12 Early evidence that ACTH influences its own secre— tion by feedback on CNS structures was reported by Hodges and Vernikos (1958, 1959). They found that stressful stimuli induced a greater secretion of ACTH in adrenal— ectomized rats with low initial plasma ACTH levels than in those with high initial levels. Apparently, high levels of circulating ACTH reduced the reactivity of the hypothalamic- pituitary-adrenal axis. Kitay §§_al. (1959) increased pitu- itary ACTH stores in prOportion to the dose of exogenous ACTH administered in adrenalectomized rats. Similarly, Kitay gt a1. (1959) blocked the stress induced fall of pituitary ACTH in adrenalectomized rats with exogenous ACTH. Further support for short feedback control of ACTH is pro- vided by studies of Vernikos-Danellis and Trigg (1967) utilizing ACTH secreting pituitary tumors. In the presence of such tumors, stress induced increases of plasma ACTH in adrenalectomized rats were lower than usual and the eleva- tion of plasma and pituitary ACTH normally seen after adrenalectomy was absent. Dallman and Yates (1968) considered the effects of chronic ACTH pretreatment on stress induced ACTH secretion in intact rats. Response to stresses such as noise and ether applied 24 hours after the last ACTH injection were partially inhibited but response to other stresses (e.g., scald, laparotomy) was not inhibited. They concluded that ACTH can block stress induced rises in ACTH secretion, although the inhibition appears to depend on the type of 13 stimulus employed. In response to the argument that an accumulation of endogenous corticoids might be producing the blocking effects, the inhibitory effects of ACTH pretreatment were studied in intact animals whose classic feedback recep- tors had been saturated by dexamethasone administration. Histamine and laparotomy caused ACTH release even after high doses of dexamethasone, but this response was blocked when animals received both dexamethasone and ACTH simultaneously. Vernikos~Danellis (1965) and Motta gt_gl. (1968) reported increases in the hypothalamic CRF concentration following adrenalectomy. A further increase in hypothalamic CRF content above adrenalectomy levels occurred when animals were both adrenalectomized and hypOphysectomized (Motta gt g1,, 1968). The fact that CRF has been found in the circu- lation after hypOphysectomy (Brodish and Long, 1962), sug- gests that eliminating the ACTH feedback signal activates both synthesis and release of CRF. Treatment of adrenal- ectomized-hypophysectomized animals with exogenous ACTH (Motta §£_gl,, 1968) reduced CRF stores to the level found in animals adrenalectomized only, but would not lower them to pre-adrenalectomy levels. Interaction of Stress and Resting Responses Experimental evidence substantiates the fact that the pituitary-adrenal axis is controlled by negative feed- back mechanisms which are sensitive to either ACTH or corticosteroids. This control mechanism is thought to 14 regulate adrenocortical function under ”resting" conditions. The role of negative feedback control during stress activa- tion of the pituitary-adrenal axis is not completely under- stood. The concepts of stress and stress activation of the pituitary—adrenal axis were popularized by the General Adaptation Syndrome of Selye (1950). Ganong (1963) has reviewed the role of the many different non-Specific stimuli termed stresses in activation of the pituitary- adrenal axis. Some authors have attempted to classify stressors according to the different sites and modes of action involved in stimulation of the pituitary—adrenal axis. Mangili _E _l. (1966) proposed that the stimulating power of stressors might be a more valid basis for classification than their modalities of action. Diurnal fluctuations of "resting’l plasma corticoste- rone levels have been observed in many different species including humans and the rat (Liddle g£_§l,, 1962; Critchlow t 1., 1963; Ganong, 1963). Sayers and Sayers (1947) and Yates _£_§1, (1961) proposed that pituitary ACTH secretion is closely regulated by a mechanism sensitive to circulating plasma corticosterone levels. According to their hypothesis, stress stimulation of the pituitary-adrenal axis resulted in a "resetting" of the control mechanism so that higher plasma corticosteroid levels were attained, but precise control by the negative feedback mechanisms was still maintained. 15 Subsequent evidence from other investigators suggested the existence of a more complicated control mechanism. The failure of large corticosteroid doses to block ACTH release following acute stress suggested a lack of precise negative feedback control under certain conditions (Hodges & Jones, 1963; Smelik, 1963). Zimmerman and Critchlow (1967) pro- posed that mechanisms regulating acute pituitary-adrenal response to stress and those regulating the diurnal rhythms of the non-stress response are independent of one another. Slusher (1964) had previously reported a dissociation of stress and non—stress mechanisms following studies on male rats bearing hypothalamic lesions. In more recent studies, Zimmerman and Critchlow (1969a, 1969b) have shown that relatively low doses of dexamethasone injected subcutaneously or implanted intra- cerebrally selectively suppress non-stress levels of corti- costerone without blocking acute plasma corticosteroid responses to stress. In a third study (Zimmerman & Critchlow, 1969c), intravenous corticosteroid injections which produced elevated (but physiological) plasma corticosteroid levels blocked non—stress levels without affecting acute stress reSponses. A double bleeding technique was used in these studies. The plasma corticosterone level of the initial plasma sample, obtained within three minutes of handling of the rat, was considered a resting response, while the plasma corticosterone level of a second sample, obtained 15 minutes after ether anesthesia, was considered to be a stress response. 16 Although Zimmerman and Critchlow's definition of a "resting" response can be criticized, these findings suggest that control of stress and non-stress responses of the pituitary- adrenal axis is under separate neural mechanisms. In contrast to the earlier hypotheses of Sayers and Sayers (1947) and Yates _§__1. (1961), the findings of Zimmerman and Critchlow (1969a, 1969b, 1969c) indicated that adequate stress stimuli are capable of overriding the control mechanism which Operates under resting conditions. These findings were in conflict with those of other investigators (Hedner & Rerup, 1962; Mangili t al., 1965; Dallman & Yates, 1968; Davidson gt.al., 1968) in which pretreatment with dexamethasone blocked acute adrenocortical response to ether in the rat. Several reasons were given by Zimmerman and Critchlow to account for the discrepancies in blocking of the stress response. Differences in the degree of stress may have been responsible. According to this argument, the stress used by investigators reporting an apparent block of acute stress reSponse was inadequate to overcome the blocking effect of dexamethasone pretreatment. Complications of Nembutal anesthesia and differences in the time interval between stress and blood sampling are given as additional explanations for the results of Zimmerman and Critchlow as Opposed to those of the conflicting studies. Results of experiments and computer simulation studies reported by Yates §£_g1, (1969) indicate that the capacity of corticosteroids to inhibit stress activation of 17 the pituitary—adrenal axis reaches a maximum; beyond which further increases in the stress intensity will activate the system despite increased corticosteroid dosage. Yates g§_al. (1969) found that the feedback inhibition prOperty of the pituitary-adrenal axis has non-competitive saturation char- acteristics. Because of this saturation characteristic, many investigators fail to appreciate the capacity of corti- costeroids to inhibit the stress response, according to Yates §£_gl, (1969). This implies that experiments in which corticosteroids did not block the stress response involved stresses whose stimulus strength exceeded the saturation level of the feedback system. Inhibition would have been observed had a lower stimulus strength been used. Gonadal Hormone Influences on the Pituitary-Adrenal Axis Gonadal steroids, particularly the estrogens, exert a significant influence on adrenocortical function. The well recognized sex difference of adrenocortical function in the rat is at least in part a consequence of this gonadal steroid influence. Kitay (1961) reported that ether stress stimulation induced higher and more persistently elevated plasma corticosterone levels in female rats than in male rats. Exogenous ACTH stimulation gave similar results. This study was prompted by previous studies of Troop (1959) and Urquhart _t_gl, (1959) which had described sex differ- ences in the rate of corticosterone and cortisone metabolism by rat liver tissue in vitro. 18 Kitay (1961) also demonstrated that corticosteroid metabolism, both ig_yiyg.and ig_vitro, occurs at a faster rate in female rats than in male rats. In a subsequent study, young adult male and female rats were treated with exogenous estrogen for two weeks (Kitay, 1963a). Following this treatment, stimulated (ether stress or exogenous ACTH) plasma corticosterone levels of intact male rats were sig- nificantly higher than levels of non-treated controls. Both ig_yiyg_and ig_vitro corticosteroid metabolism were also increased. The same level of estrogen treatment did not significantly alter stimulated plasma corticosteroid levels of intact female rats, but it decreased the rate of hepatic corticosteroid metabolism in_yiyg_and in_vitro. The effects of gonadectomy on adrenocortical func- tion have also been studied by Kitay (1963b). Rats were gonadectomized at 30 days of age and adrenocortical re- sponses were tested six weeks later. Both resting and stimulated (ether stress or exogenous ACTH) plasma corti- costerone levels were reduced in ovariectomized female rats but castration did not affect these parameters in male rats. Corticosteroid metabolism was increased in castrated male rats and decreased in ovariectomized female rats. Estradiol added directly to adrenal slices from ovariectomized rats increased ig_vitro corticosterone production, thereby sug- gesting that estrogenic influences upon adrenocortical function result at least in part from a direct estrogen effect on the adrenal cortex (Kitay, 1963a). 19 Evidence for estrogenic stimulation of adrenocorti— cal function at the hypothalamo-hypophyseal level has been reported. Kanematsu and Sawyer (1963) and Telegdy (1964) found that estradiol implants in the hypothalamic area increased adrenocortical function. Richard (1965/66) found basal levels of plasma corticosterone were increased by implantation of estradiol-17'B into either the accurate nucleus, the anterior pituitary or the lateral mammillary area of ovariectomized rats. The exact site of estradiol stimulation of adrenocortical function at the hypothalamo- hypophyseal level is not known. Coyne and Kitay (1969) demonstrated that estradiol stimulates ACTH secretion by increasing pituitary responsiveness to CRF-like activity and by increasing pituitary ACTH synthesis. The effects of estradiol on the hypothalamo—hypOphyseal system are thought to occur independently of corticosteroid feedback (Coyne and Kitay, 1969). Estrogens are known to cause increased levels of corticosteroid binding protein in the circulation. Westphal _§_‘l. (1962) have demonstrated the presence of transcortin activity in rat plasma. Tait and Burstein (1964) obtained evidence in humans, however, that increased transcortin binding of corticoids under estrogen stimulation had little effect on levels of free circulating corticoids. Since the free plasma corticoids are thought to be the primary feed- back signal for regulation of resting pituitary-adrenal 20 function (Tait and Burstein, 1964) it is likely that estrogen stimulation of transcortin has little effect upon adrenocortical function. It is reasonable to conclude that estrogenic stim— ulation of the pituitary-adrenal axis occurs at multiple sites, including the hypothalamus, the pituitary, and adrenal cortex and the liver. The relative importance of estrogenic stimulation at these sites under physiological conditions remains to be determined. Thyroidal Influences on the Pituitary-Adrenal Axis The thyroid gland is known to exert an influence on adrenocortical function (Sayers, 1950; Wallach & Reineke, 1949). Wallach and Reineke (1949) studied adrenal response to prolonged thyroxine stimulation in rats. After receiving daily injections of thyroxine ranging from 5 to 80 ug thyroxine (T4)/100 g for 28 days, adrenal weight and ascorbic acid content of treated rats were increased above those in non-treated controls at all dose levels above the 5 ug treat— ment. In a similar study, dosages of Protamone, a thyro- active iodinated protein, ranging from 0.01% to 0.16% of the diet produced no significant change in adrenal response after 4 weeks of treatment. In a separate experiment Wallach and Reineke (1949) were able to prevent the thyroxine induced increase in adrenal weight and ascorbic acid content by con- current administration of adrenocortical extracts. This was 21 taken as evidence that the adrenals are in a state of hypersecretion in a hyperthyroid animal. The enlarged adrenal glands of hyperthyroid animals were interpreted as evidence for an increase in ACTH secretion. Steinetz and Beach (1963) found increased adrenal gland size in rats made hyperthyroid by feeding a diet con- taining 0.15% USP thyroid for eight days. Thyroidectomized rats maintained on the same diet showed similar results. Thyroid treatment was not effective in increasing adrenal weights in hypOphysectomized or prednisolone pretreated rats. D'Angelo and Grodin (1964) confirmed the lack of adrenocortical response to thyroid hormone treatment in hypophysectomized rats. D'Angelo and Grodin also found that administration of tri-iodothyronine (T3) increased adrenal weight of treated rats although adrenal ascorbic acid concentrations were consistently reduced at higher T3 dose levels. Decreased adrenal ascorbic acid concentration was offset by adrenal hypertrOphy, so that vitamin C content of the enlarged male adrenals was significantly increased. The increased ascorbic acid content of the enlarged adrenals in hyperthyroid animals confirmed the earlier work of Wallach and Reineke (1949). I Kawai (1962) also reported a significant increase in adrenal weight of hyperthyroid rats. By way of contrast, "resting" plasma corticosterone levels (blood from rapid decapitation) were similar in hypothyroid, euthyroid and I‘JYperthyroid rats, suggesting that if increased ACTH 22 secretion rates actually exist under basal steady state conditions they must be balanced by changes in other param- eters which determine plasma corticosterone levels. Pitu- itary ACTH depletion 15 minutes after stress was greater in hyperthyroid than in euthyroid rats (Kawai, 1962) although pre-stress pituitary ACTH concentrations did not differ between these groups. Steinetz and Beach (1963) measured plasma cortico- sterone levels one hour after injection of ACTH into intact rats. Plasma corticosterone levels were elevated in thyroid— treated rats in comparison to non-treated controls. Similar results were observed in thyroidectomized rats following treatment with thyroid hormone. Thyroid prefeeding did not alter plasma corticosterone levels after ACTH injections in hypophysectomized rats. D'Angelo and Grodin (1964) also reported increased plasma cortocosterone levels in rats following thyroid treatment. Interpretation of their results is complicated by possible inadequate standardiza- tion of the stress involved in obtaining blood samples. Bohus gt a1. (1965/66) studied the effects of thyroxine implantation into the brain on adrenocortical response. Bilateral implantation of thyroxine led to an enhancement of adrenal function when implants were located in the mid- posterior part of the arcuate nucleus. A weaker activation of the adrenals was evident when implants were placed into the anterior median eminence. When thyroxine implants were placed in other areas of the hypothalamus or in the anterior 23 pituitary there was no change in the corticosterone output of the adrenals. The results of this study suggested that hypothalamic CRF release was stimulated by the thyroxine implants if prOperly placed in the median eminence. Steinetz and Beach (1963) demonstrated a 50% reduc- tion in the apparent distribution volume of corticosterone in hyperthyroid rats. Distribution volume was determined following intravenous injection of unlabelled corticosterone. Additional experimental evidence showed that plasma volume, red cell binding of corticosterone and ”capillary permeabil- ity" were not sufficiently altered to account for the reduc- tion in distribution volume. In_vitro eXperiments in which adrenal corticosterone production was not markedly influenced by thyroid treatment also suggested that distribution volume was reduced in hyperthyroid rats. In hypOphysectomized rats, ig_yiy9.plasma corticosterone levels one hour after ACTH were higher in animals treated with both thyroid and repos- itory ACTH than in animals treated only with reSpository ACTH. These results were attributed to the 50% reduction in corticosterone distribution volume of hyperthyroid rats. In spite of increased adrenal size in hyperthyroid animals, Steinetz and Beach (1963) found no marked differ- ence between normal and hyperthyroid rats when ig_vitro adrenal steroid production was compared. However, stimu- lation of adrenals from both groups by equal amounts of ACTH ig_vitro could reflect inadequate ACTH for maximal stimula- tion of hyperthyroid adrenals. Thyroxine administration to 24 rats ig_yiyg_caused increased activity of enzymes associated with several adrenocortical biochemical pathways including glycolysis and NADPH production and resulted in increased adrenal size (Freedland &:Murad, 1969). They suggested that increased activities of the NADPH producing enzymes may be related to the increase in corticoid production following thyroid treatment that has been reported by other investi- gators. Yates §§_gl, (1958) reported a close correlation between adrenal size and the in_vitro capacity of the liver to inactivate steroid hormones in hyperthyroidism. Total hepatic capacity for in_vitro reduction of ring A of corti— sone was increased by 38% in male and female rats following thyroid treatment. Increases of total steroid hydrogenases and NADPH availability in the liver were thought to account for this increase. McGuire and Tomkins (1964) found that the increased rate of steroid reduction observed in rats treated for three days with thyroxine resulted from in- creased levels of reduced NADPH in liver homogenates. Studies of Endocrine Function in Aging Individuals Aging and the Pituitary—Adrenal Axis The concept of age-related changes in adrenocortical function originated with studies by JackSon (1919) on post- natal develOpment of the rat adrenal gland. There was evi— dence of a general increase in the adrenal parenchymal cell 25 size from one to ten weeks of age with little change there- after. Blumenthal (1945) reported a gradual diminution in the number of mitoses occurring in the guinea pig adrenal gland with increasing age. Other studies (Dribben & Wolfe, 1947; Jayne, 1953) provided evidence of changes in connec- tive tissue structure of the adrenal glands of aging rats. Studies on histochemical and degenerative changes in the adrenal cortex of the aging rat by Jayne (1957) indicated that cellular degeneration may substantially decrease the steroid producing ability of adrenocortical parenchymal cells. Friedman _E._l. (1965) showed that administration of adrenal and neurOphypophyseal hormones significantly prolonged the life span of 24 month old rats and visibly improved general body condition. The results of this study suggested that hormonal inadequacies may facilitate the aging process. Early studies of adrenocortical steroid output indicated that l7-ketosteroid excretion decreases with increasing age (Hamilton & Hamilton, 1948; Pincus, 1955). These compounds are primarily androgen rather than gluco- corticoid metabolites. Borth et a1. (1957) showed that there are definite correlations between urinary excretion of both l7-ketosteroids and l7-hydroxycorticosteroids and age. They concluded that l7-ketosteroid excretion was more dependent upon age than was l7-hydroxycorticosteroid excre- tion. Their findings suggested that glucocorticoid produc- tion by the adrenal cortex does change with advancing age. 26 On the contrary, Romanoff _t._l, (1957) reported that increasing age produced no change in the urinary excretion of the more characteristic corticosteroids (e.g., tetra- hydrocortisone, tetrahydrocortisol, cortisone and cortisol). Romanoff §£_gl, (1957) reported a 10% decrease in the number of detectable l7-ketosteroids in the urine of elderly sub— jects. This became a 20% decrease, as compared to young subjects, following ACTH administration. In a subsequent study Romanoff t al, (1958) deter— mined excretory levels of tetrahydrocortisol, 3d-allotetra- hydrocortisol and tetrahydrocortisone in the urine of young and old men and women. The mean quantitative excretory levels of the three metabolites were significantly lower in young and old women and in old men than in young men. They found that the differences in quantitative excretion of these metabolites disappeared when urinary excretion was expressed as a function of creatinine excretion. Further investigation (Romanoff _t__l., 1959) showed that B-cortolone, a regularly occurring metabolite found in the urine of young and old subjects of both sexes, also shows an age related decline in excretory levels when expressed as mg/hr. This difference was also eliminated when cortolone excretion was expressed in terms of mg/g creatinine excretion. In order to confirm their previous findings, Romanoff §£_gl, (1961) determined the resting adrenal secre— tory rates of eight young and eight old men. The 24-hour 27 secretion rate of cortisol in old men was only 75% of that in young men, regardless of the metabolite on whose excre- tion the determination was based. Secretion rates were the same for young and old subjects when expressed as mg/g creatinine/24 hr. This study (Romanoff §t_gl., 1961) con- firmed previous findings that apparently neither cortisol metabolism nor cortisol secretion by the adrenal differed with age when the muscle mass (based on creatinine excretion index) of the subject was considered. Tyler §t_§1, (1955) compared plasma l7—hydroxycorti- steroid levels of young adults and geriatric patients follow- ing a 6 hour ACTH infusion. They reported that adrenal response to ACTH and hepatic metabolism of corticosteroids did not change with increasing age although corticosteroid distribution volumes and turnover rates were decreased. West gt a1. (1961) found a progressive decrease in the rate of cortisol removal from the circulation with increasing age but no difference in the distribution volume of the infused cortisol. Suggestive evidence for an age- related decrease in the cortisol production rate under resting conditions was also reported. Samuels (1956) demonstrated an increased biological half-life of cortisol in aged human subjects. The apparent distribution volume of cortisol was also lower in old than in young subjects. Because of these changes, the secretory rate required to maintain normal resting plasma cortisol levels was much lower (about 50%) in old men than in young men. This 28 implied that the adrenals of older persons were less func- tional than those of younger individuals. Moncloa gt QL. (1963) showed that l7-hydroxycorti— costeroid excretion/24 hr decreased progressively with increasing age in healthy men ranging from 20—85 years of age. They concluded that decreased production and increased biological half-life of cortisol accounted for these changes. Standard tests for liver function performed on their sub— jects indicated no abnormalities. This implies that de- creased hepatic metabolism was not the reason for increased half-life. Moncloa _t__l, (1963) also found that l7-hydroxy- corticosteroid production was lower in old subjects than in young subjects in reSponse to multiple levels of ACTH infusion. The conclusion by Samuels (1956) and Moncloa _t_al. (1963) that adrenocortical response to ACTH decreases with increasing age was also supported by the findings of Riegle and Nellor (1967). Following ACTH infusion, plasma gluco- corticoid levels in bulls showed a marked and progressive decrease with age, indicative of a decreased adrenocortical responsiveness to ACTH. Plasma ACTH levels were higher in old animals than in young ones. Histological studies revealed varying degrees of cortical degeneration with age which were positively correlated with the relative insensi- tivity to exogenous ACTH infusions. Apparently the increased levels of plasma ACTH were required in older animals to main- tain normal plasma glucocorticoid levels. Subsequent studies 29 in goats (Riegle _t__l,, 1968) also revealed an age-related decrease in adrenocortical responsiveness to ACTH. Age related changes in adrenocortical function have also been reported in guinea pigs (Fajer & Vogt, 1963) monkeys (M, mulatta) (Bowman & Wolf, 1969), and mice (Grad, 1969). Recent studies in our laboratory have shown that adrenocortical reSponsiveness to ether stress or exogenous ACTH is decreased in both male and female rats with in— creasing age (Hess & Riegle, 1970). This age-difference in adrenocortical function in the rat did not exist in the absence of ether stress or ACTH stimulation. Gonadal Function in the Aged The concurrent progression of the aging syndrome and changes in gonadal function observed in humans, produced the theory that gonadal hormone changes caused aging to occur (Sobel, 1967). Considerable experimental evidence suggested that a sudden decrease in estrogen production occurred in human females at menOpause, while a gradual decrease in l7-ketosteroid production occurred with advancing age (Gherondache g£_§1,, 1967). The cessation or diminution of gonadal steroid production with increasing age disrupts the fine balance between hormones which is normally found in young adults. Following gonadal changes in aged humans, the pituitary gland secretes increased amounts of gonadotrOpins (Gherondache §t_g1,, 1967). The develOpment of endocrinol- ogy was expected to provide the necessary knowledge for 3O reversing or at least controlling the aging process. It is now apparent that the aging phenomenon is much more complex than can be explained by hormonal changes with increasing age. This does not mean that gonadal hormone changes are strictly a consequence of the aging process, however. Evidence now available suggests that the causative factors behind reproductive failure in the aging human female are quite different from those in aged rats. At menopause the human female suffers the loss of ovarian steroids, which is attributed to the absence of follicular cells (Engle, 1955). Krohn (1955) reported that three phenomena associated with the onset of menOpause in women are a disappearance of ovarian oocytes, a decreased produc- tion of sex hormones and increased gonadotropin levels in blood and urine. Becker and Albert (1965) found that in post-menOpausal women follicle—stimulating hormone (FSH) and luteinizing hormone (LH) secretions (based on urinary excre- tion of these gonadotrOpins) were increased 11 and 9 fold, respectively, compared to normal men. Apparently the meno- pause develOps in women because the ovary is no longer capable of responding to gonadotrOpins (Krohn, 1955). This is not the case in aged rats and mice, in which ovarian function can be restored by gonadotropin treatment (Krohn, 1955). Eckstein (1955) concluded that the decline in ovarian function of aged rats results from anterior pituitary failure rather than from ovarian senescence. 31 .More recently, Ascheim (1965) showed that regular estrous cycles could be induced in aged constant estrous rats by LH injections. The loss of reproductive function in lower animals occurs so gradually that its final stages may not be reached before the end of life. For example, there may be primor- dial and Graafian follicles and even corpora lutea present in aged rats, although in reduced numbers (Eckstein, 1955). It appears that ovarian function declines slowly in old rats and that pituitary failure rather than ovarian senescence alone accounts for this change. These findings imply that some gonadal hormone production could persist in rats beyond the cessation of normal reproductive capability. Clemens §§_gl. (1969) investigated the possibility that hypothalamic changes may account for the loss of normal reproductive function in old rats. Constant estrous, aged rats (20 month) were exposed to treatment with drugs, hor- mones, or direct electrical stimulation of the preOptic area of the brain. Ovulation, confirmed by laparotomy, was induced by progesterone, epinephrine and electrical stimu— lation of the preOptic area, suggesting that changes in brain function are at least partially responsible for the decline of ovarian function. Although this study did not rule out the possibility of ovarian senescence, the observa- tion that old rat ovaries can be reactivated suggested that ovarian failure is caused by changes at a higher level. 32 Changes in brain neural activity are implicated in the loss of normal ovarian function and subsequent constant estrous of old rats. This concept is supported by the finding (Clemens, 1968) that hypothalamic LRF content is very low and FSHRF content is very high in old constant estrous rats compared with 3-month-old cycling rats during estrous. Influences of Aging on Thyroid Function For a number of years there has been evidence that thyroid function is decreased in aged subjects. Turner (1948) reported this phenomenon in poultry, while more recently Long 2E.él- (1952) reported similar findings in cattle and Henneman gt a1. (1955) presented evidence that thyroid secretion rates are lower in old sheep than they are in young sheep. Age—related changes in thyroid function of the rat have been reported by Grad and Hoffman (1955), and Wilansky §£_§1, (1957) while Flamboe and Reineke (1959) observed a progressive decline in thyroid secretion rate of aging goats. Grad and Hoffman (1955) found that thyroid secretion rate (estimated by the goitrogen technique) was at least 25% lower in old (29 month) female rats than in young (4% month) female rats. Wilansky g; al. (1957) found that thyroid secretion rates (estimated by the thyroxine degradation technique) were 20% lower in old (24-25 month) rats than in young (4-5 month) rats. Wilansky t 1. found no difference 33 in circulating PBI levels between the two groups. Gregerman (1963) measured PBI levels in young and old female rats and found that they were lower in 12 month than in 24 month old animals. The thyroid hormone distribution Space per unit body wt was greater in old rats than in young rats, however. Gregerman (1963) also found that the rate of thyroxine degradation was 50%.higher in old rats than in young rats. This finding contradicted previous findings of Grad and Hoffman (1955) and Wilansky §£_§l. (1957) in aged rats. Narang and Turner (1966) estimated thyroid secretion rate in rats at 30 day intervals between weaning and 4 months of age and found a progressive decrease in secretion rate dur- ing this time. Studies of thyroid secretion rate with aging do not give a complete picture of the physiological effects of thyroid hormone in aging rats. For example, Gregerman (1963) pointed out that the exact contribution of tri- iodothyronine (T3) to the metabolic effect of thyroid hormone in the rat is presently undefined as is the rela- tionship between age and T3 secretion rate. A complete understanding of thyroid function in the senescent rat is dependent Upon acquisition of this information. Prelimi- nary experiments conducted by Wilansky gt_§l, (1957) sug- gested that a greater rise in oxygen consumption occurred in old rats than in young rats for the same dose of thyrox- ine per unit metabolic mass. Increased responsiveness to 34 thyroxine in aged rats could be indicative of a homeostatic mechanism whereby senescent rats are able to compensate for a reduction in thyroid secretion rate. Grad (1969) also attempted to determine if target tissue requirements for thyroid hormone are changed in senescent animals. Basal metabolic rate (BMR), based on oxygen consumption, was used as an index of tissue responsiveness to thyroid hormone. Although BMR was higher in old female rats than in young female rats, thyroidectomy caused BMR to drOp to the same extent in both young and old rats. Thyroxine treatment increased the BMR of both young and old rats but the effect was more pronounced in the old animals than in the young. This was interpreted as a greater responsiveness of target tissues to the same dose of thyroxine given in relation to metabolic mass in the senescent rats (Grad, 1969). According to Frolkis (1968), tissue reSponsiveness to hormones is increased in senescent animals. Since this increased sensitivity of tissues to hormones occurs at a time when functional activity of the endocrines is declining, it represents an adaptive reSponse to changes which occur in body control systems with aging. As an example of this change, Frolkis (1968) reported evidence from a study where thyroid stimulating hormone (TSH) treatment produced only insignificant changes in the thyroid glands of mature rats while a similar dose of TSH given to aged rats produced a 35 27% increase in oxygen consumption of thyroid tissue, a 69% increase in thyroid weight and a 66% increase in the height of thyroid epithelium. MATERIALS AND METHODS Experimental Animals Experimental animals were rats of the Long-Evans strain bred and reared in the Endocrine Research Unit rat colony. They were maintained at approximately 220 C and subjected to illumination between 7:00 AM and 7:00 PM. All rats received MSU regular rat diet (Appendix I) and water gg_libitum during the course of eXperimentation. Statistical Analysis The significance of plasma corticosterone differ- ences between treatment groups was determined by the Stu- dents' ("pooled") t-test, except in experiments V and VI where Duncan's Multiple Range Test was used subsequent to‘ Analysis of Variance (Steel and Torrie, 1960). Determination of Adrenocortical Response ACTH Stimulation Exogenous ACTH stimulation of the adrenal cortex was conducted similar to the procedures of Moncloa, Peron and Dorfman (1959). Subcutaneous ACTH injections (1-2 units/100 g body wt) were found to produce a maximal increase in plasma 36 37 corticosterone concentration 60-70 minutes after ACTH (Depo—ACTH, The Upjohn Company, Kalamazoo, Michigan) admin- istration. Rats were lightly anesthetized with diethyl ether (Mallinckrodt Chemical Works, St. Louis, Missouri) immediately before bleeding. Stress Exposure to ether vapors is a recognized adreno— cortical stressing agent in the rat (Zimmerman and Critchlow, 1967). Duration of ether exposure was employed as a stan- dardizing criterion in these experiments. In order to insure uniform maximal stimulation of the pituitary-adrenal axis experimental animals were exposed to 40 minutes of periodic ether vapor eXposure. (The rats were anesthetized at 40 minutes, 10 minutes and again just before blood sam- pling.) Blood Collection Peripheral blood samples (0.5 to 1.5 ml whole blood) were collected into dry heparinized beakers by orbital sinus puncture with a capillary tube in lightly anesthetized rats. The red blood cells were centrifuged out and plasma samples collected as soon after bleeding as possible. Plasma sam- ples not subjected to corticosterone extraction following centrifugation were stored at -150 c until they could be assayed. 38 Resting Corticosterone Levels Rats were rapidly anesthetized with ether so that blood samples could be obtained by orbital sinus puncture within two minutes or less from the time the animal was initially disturbed in its cage. Fluorometric Assay of Plasma Corticosterone Corticosterone concentration in rat plasma was measured by the fluorometric assay procedure of DeMoor and Steeno (1963). Fluorescence was measured with a Turner #110 fluorometer equipped with narrow band, high transmit— tancwailters (excitation wave length 470 mu, emission wave length 530 mu). Corticosterone was measured in duplicate plasma samples of different volumes (normally 0.1 and 0.2 ml plasma). The corticosterone concentration of each plasma sample was calculated by comparing its fluorescence intensity to that of corticosterone standards run concurrently with each group of plasma samples. The reliability of the fluorometric technique for corticosterone quantitation in rat plasma was tested by measuring the plasma corticosterone concentration of a pooled plasma sample by both the simple fluorometric assay and the colorimetric blue tetrazolium reaction. The blue tetrazolium technique of Elliott §t_§l, (1954) was used following purification and chromatographic separation of corticosterone as described by Riegle and Nellor (1967). 39 The validity of the fluorometric technique for measurement of plasma corticosterone in plasma from adrenocortically stimulated rats was verified by the close agreement of the results of the two methods (plasma cortociosterone concen- tration from the fluorometric analysis, 32.4 i 0.6 ug/lOO ml; and from the blue tetrazolium reaction, 32.0 ug/lOO ml). The precision of the fluorometric technique was ascertained in the following manner. A total of 21 samples (0.2 ml each) from a pooled plasma supply were assayed fluorometrically with a variation (standard error of the mean) of 0.40 for the series (58.1 i 0.40 ug/lOO ml). H3-Corticosterone Disappearance from Plasma Procedures similar to those of Glenister and Yates (1961) and Saroff and Wexler (1969) for measuring distribu- tion volume, biological half-life and production rate of corticosterone in the rat were followed in measuring these same parameters in this study. Serial blood samples taken at regular intervals following intravenous injection of radioactive corticosterone were used to calculate a dis- appearance curve for plasma radioactivity with increasing time. Corticosterone -l, 2-H3 (New England Nuclear Corp.) (1.00 millicurie, 0.0115 milligram in 1 ml ethanol-benzene) was diluted to a total volume of 5 ml and served as the labelled hormone used in these studies. 4O Adequate volumes of this diluted isotOpic cortico- sterone were added to a solution of unlabelled corticoste- rone (200 ug/ml) in saline (0.9%) to provide final concen- trations of 1.2 or 2.0 uC/ml. The volume injected into each rat was 0.25 ml (0.3 or 0.5 uC H3-corticosterone, 50 ug of unlabelled corticosterone). Rats were anesthetized with Nembutal approximately 10—15 minutes before isotope injection (3.5 mg/100 body wt injected ip). The femoral vein injections (0.25 ml) were facilitated by small skin incisions over the injection region which were closed with surgical clips following injections. A 15 minute equilibration period was allowed between isotope injection and the start of blood sampling. Blood samples of 0.5 to 0.75 ml were collected by the orbital sinus route at 15, 20, 25, 30 and 35 minutes post-injection in male rats. Because of the more rapid plasma corticosterone disappearance rate in female rats blood samples were collected by the orbital sinus route at 15, 19, 23, 27 and 31 minutes post-injection. Plasma was separated by centrifugation and 0.1/m1 fresh plasma samples were prepared for methylene chloride extraction. The remain- ing plasma was stored at -150 C until assayed fluorometri- cally for corticosterone concentration. Methylene chloride extraction and alkali wash of plasma samples were conducted by the same procedure used in fluorometric corticosterone determinations. A 4 m1 aliquot of the methylene chloride 41 extract (5 m1 original volume) was transferred to a scintil- lation vial and the contents evaporated under a constant air flow at room temperature. Following addition of 10 m1 of a toluene based counting solution the radioactivity was counted in a Nuclear Chicago.Mark.I liquid scintillation spectrometer. In order to confirm that plasma radioactivity was associated with corticosterone, 0.1 ml plasma samples were obtained from an adult female rat 15 minutes after injection of a large dose (15 uC in 0.25 ml) of H3-corticosterone. Following chromatographic isolation of corticosterone accord- ing to methods described by Riegle and Nellor (1967), the percentage of total radioactivity eluted from the chromato- gram associated with corticosterone was determined. Calculations for parameters of adrenal secretory dynamics were patterned after those used by Saroff and Wexler (1969) and reviewed in detail by Tait and Burstein (1964), for the single compartment model. The single com- partment model was assumed to adequately represent adrenal secretory dynamics for the purposes of the present studies. The least squares line for (natural) log CHM (5 points/animal) with increasing time was calculated for the results from each rat. Extrapolating this curve to zero time, one obtained the volume of distribution, provided mixing had occurred instantaneously (the intercept of this line on the ordinate represented log CPM/O.l ml plasma, from which the value of CPM/ml plasma was readily obtained). 42 Using dilution techniques, the appropriate volume was calculated. = Total CPM injected CPM/m1 at zero time distribution volume (ml) The lepe of this least squares line (disappearance curve) was used to calculate corticosterone turnover rate (biological half-life): . _ .69315 t% (min) _ lepe Metabolic Clearance Rate (M.C.R.), the volume of plasma completely cleared of corticosterone per unit time, was calculated for subsequent use in calculating corticoste- rone production rate. Total CPM's injected x slope CPM/m1 plasma M.C.R. (ml plasma/min) = Production Rate (P.R.) of corticosterone, the amount of corticosterone required per unit time to maintain plasma corticosterone levels (assuming steady state conditions), was calculated in the following manner: P.R. (pg/min) = M.C.R. x plasma corticosterone concentration The corticosterone concentration of each serial blood sample was determined fluorometrically. Average corticosterone concentration (pg/100 ml plasma) of individual animals dur- ing the blood sampling period was used in calculating P.R. values. EXPERIMENTAL EXperiment I: Effects of Chronic Adrenocortical Stimulation in Young and Old Male Rats Materials and Methods The purpose of this eXperiment was to study the effects of chronic adrenocortical stimulation on the ability of rats to respond to subsequent exogenous ACTH or stress. Interest in these studies was in two areas: (1) the capac- ity of chronically stimulated adrenal cortices of young and old rats to respond to exogenous ACTH, subsequent to chronic stimulation and (2) the effect of continuous elevation of endogenous corticosterone on the ability of stressors to mobilize hypothalamic-pituitary ACTH release. Plasma cor- ticosterone levels following adrenocortical stimulation were used as an index of adrenocortical responsiveness. The experimental scheme used to test these objec- tives is shown in Figure 1. Five month and 26 month old male rats were grouped into treated and control groups, the former receiving daily injections of a depo-ACTH preparation while controls received daily injections of the gelatin vehicle alone. During the first 2 weeks of treatment all treated rats received 10 U ACTH/day in 15% gelatin. During 43 44 the last 4 weeks of treatment the rats received 2 U ACTH/100 g B.W./day in order to compensate for differences in body weight among the experimental subjects. In most animals this dosage amounted to 10 U ACTH/day. Adrenocortical response was tested at five intervals throughout the course of the experiment: prior to the start of chronic ACTH treatment, after 2 weeks, 4 weeks and 6 weeks of chronic ACTH treatment and after 2 weeks recovery from chronic ACTH treatment (Figure 1). Two types of adrenocortical responses were tested at each interval of the experiment: response to a standardized ether stress procedure and response to levels of exogenous ACTH adequate to maximally stimulate adrenocortical steroid- ogenesis. The latter parameter will be referred to as acute ACTH stimulation to distinguish it from depo-ACTH treatment. Plasma samples were obtained by orbital sinus puncture and corticosterone levels were determined fluorometrically. Individual body weights of experimental animals were recorded at each bleeding interval. Adrenocortical response differ- ences between depo-ACTH treated and control rats of the same age, sex and mode of acute adrenocortical stimulation at each interval of the experiment were statistically analyzed by Student's t-test (Steel & Torrie, 1960). 45 Results _Adrenocortical responses to acute ACTH stimulation in old male rats receiving daily depo—ACTH injections are pre— sented in Table l and Figure 2. Adrenocortical response is represented as the mean plasma corticosterone level :_S.E.M. Only after 6 weeks of chronic ACTH stimulation did treated old rat response differ (p < .05) from control group old rat response. The effect of chronic adrenocortical stimulation on adrenocortical responses of young male rats to acute ACTH stimulation is presented in Table 1 and Figure 3. Although there was no difference between treated and control responses initially, there was a significant increase of response in treated young rats over that of control young rats after 2 weeks (p<.05), 4 weeks (p<.01) and 6 weeks (p<.01) of chronic ACTH treatment. Response of treated young rats did not differ from that of non-treated young controls after a 2 week recovery period. Adrenocortical responses of depo- ACTH treated young male rats to acute ACTH stimulation were significantly higher than those of depo-ACTH treated old male rats to acute ACTH stimulation after 2 weeks (p< .01) and 4 weeks (p<:.01) of depo-ACTH treatment. The ether stress response of both treated and con- trol old male rats receiving chronic depo-ACTH injections is shown in Table l and Figure 4.. The ether response of treated old rats did not differ from that of control old 46 rats at any interval of the experiment. This is in contrast to the ether stress response of young rats (Table l and Figure 5). The ether stress response of young treated rats was significantly lower than young control group response after 2 weeks (p<<.01), 4 weeks (p< .05) and 6 weeks (p<<.01) of chronic ACTH treatment. After 2 weeks recovery, treated and control group responses were not significantly different. In a composite representation (Figure 6), this data is presented as change of treated response from control response at each interval of the experiment. Change is represented as ug corticosterone/100 ml plasma above or below control response. Group body weight data (mean :_S.E.M.) for each interval of the experiment is presented in Table 2. Al- though mean body weight of both young and old depo-ACTH treated rats dropped during the 6 weeks of treatment, these changes were not significant in either young or old rats. 47 .ucoE lummnu meumlomop Eoum >um>ooou mxmm3 m Houmm new unoEumoHu mBumlommn mo mxwmz w cam mxooz v .mxooz m Houwm .Anmmum co H mm Umumcmemocv ucoEumoHu msumlommp mo unmum wcu oHOMoQ onB mam>umucfl mmona .ucoEHHmmxo on» mo Hm>uoucw some umwoocHEHouop mm3 ASQ oouv ocm ooum cmo3uon omcHEHouoov coHumHsEHum mevm ouoom ou can Acooc new 24 ooum comBDmn cogeauoumpv mmwuum Hosum ou Amao>oa ocououmooauuoo mammamv oncommon Hmuwuuooocouom .mmooum Houucoo pom pmumouu Opus po©e>ep Sumo oHoB mumu 0H0 cam mcdow .ucoefluomxo £804 oncouno mo mamnom .H musmam 48 H mesmem .8280 Bo 69.60: .20 .828 ocao> 23m 23¢ .xBN $2.04 0.20m10L 22.3 mmIFm 369: 0:30» 22$ I._.o< 49 .uGMEumoHu mews Iomop Eoum >Ho>ooou mxooz N Houmm cam DQQEumoHu mBOmIOQop mo mxmo3 o kuum pom mxom3 v Houmm .mxooB m Houmm .Anmmnm :0 H mm pmumcmflmmov ucmEumoHu meom Iomop mo uumum ocu ouomon omcHEHmuoo ouoB noncommmu Hmoeuuooocouos .ocoam cflumamm mo mooHuowmcH ©o>HoUoH maouucoo oEHu noecz cognac .mxmo3 0 mo oceuwm m How cflumamm.XmH an maom mo Ahaamp D oav mCOeuooncH moomcmusonsm ©m>amumu mumu pmummuu meoslommn .mmocm>amcommmu Hmonuuooocoupm mo xmpce Gm mm coma onm3 AHE ooa\m1v mao>oa mcououmoofluuoo mammam poceaumuop maamowuumeononam .mCHHQEmm GOOHQ mscfim Hmuenuo Op Hoflnm mmuscfla on AusmwoB >609 miooa\D NV coHuomncH maom comma ocHHmm m om>aoomu mumu Ham .coflumHSEeum meofl ousom On many mama Anacoe omv oao mo noncommwn Hmoauuooocmupm mucwmoummu sumo mane .co«umHoEHum macs ousom ou mumu mama pao Houucoo pom poummuu maumlomop mo noncommmu Hmoeuuouocmnps .N musmflm 50 m enumem 22:89:. 1.54 mzocoooxw 9.33 A m m v N H .9250 § Ih0< oEoEo DU 8:235 1,84 934 to 585 mm ON ..n .b O? o/o m cm U... m. o om we «a m 8. w ON_ 03 51 .ucmEumouu maom Iomoo Eoum mum>oomn mxooB m Houmm pom ucoEummHu mammIOQoo mo mxom3 m Hmumm Cum mxmm3 V Houmm .mxmmz N Hmumm .Anmmnm :0 H mm pmumcmflmmpv unmEummHu EBUG tomoc m0 uumum may muommn pocHEHoumU mHoB noncommmu Hmoauuoooconpfl .ocon GaumHmm mo mcofluoonce vo>nmoou maouucoo nuanz meanso .mxmmB 0 mo poHme m How cHumHom Xma Ge.meom mo Amawmp D oav mcoHuomflcH msoocmusonsm ©o>HmUmH mumu poumouu maumlommn ..mmmco>amcommon Hmofluuoooconom mo xmpca am mm poms muo3 AHE ooa\m1v mao>oa odououmouaunoo mammam pmaeEHouop adamoenumeouosah .mcHHmEmm Uooan msch Hmuflnno ou HOHHQ mouscae on Aucmflo3 moon 0 ooa\D NV coHuoomcH macs comma mcwamm m po>HonH mumu Ham .mumu mama ASDCOE mv masom CH coHumassauw mews wusom o» noncommmu HmoHuHOUOGmH m mucommumon mump mane .coHumasaflum macs musom ou mums came mecca aouucoo cam poumouu meomlomop mo noncommou Hmowuuooocoupm .m mesmem 52 m musmam 29:68... 1.54 maocmooxm 232, A.) m m N H 8) WE, .228 g :84 2:88 flu ceasfa :54 284 to 555 m cm w 04 o/o . 0 oo m . 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Om m. J 1 AU U .00_ 9 . ON. .9200 g . 1.54 0:525 D . OS 3:831 385 35m Me 5.86 m 57 Figure 6. Effect of chronic ACTH treatment on adrenocorti- cal response to ether stress and acute ACTH stimulation in young and old male rats. Adrenocortical responses (plasma corticosterone levels in ug/lOO ml) of depo-ACTH treated young and old male rats are presented as change from the comparable control response at each interval of the eXperiment. Responses to both ether stress and acute ACTH stimulation determined before the start of depo-ACTH treatment (designated as I on graph), after 2 weeks, 4 weeks and 6 weeks of depo-ACTH treatment and after 2 week recovery period from depo-ACTH treatment are presented here. Depo-ACTH treatment consisted of daily injections of ACTH (10 U/rat) in a 15% gelatin preparation. Ether stress response consisted of ether anesthe- tization 40 minutes, 10 minutes and just prior to orbital sinus blood sampling while acute ACTH stimulation consisted of exogenous ACTH adminis- tration (2 U/lOO g body weight) 70 minutes before orbital sinus blood sampling. Young rats averaged 5 months and old rats averaged 26 months of age at the start of the experiment. 4O 30 -30 -40 1 I 58 Chronic ACTH Experiment A Corticosterone ,q Acute ACTH r Q-‘ ,” \ I: ~-v’ v;———————5monthd / A \ I ,’ »;-L———26 monrho' I I \\ I ,’ \\\ II I’K \\ I I I I l d I}? I I ’ Ether Stress I (X 26 month (3' 5 month (i g 1 l l J I 2 4 6 R —> Weeks Exogenous ACTH Treatment Figure 6 59 .mo. v Q .Houwac umNHomuomsm 05mm mcfi>mn ncmozr .Ho. V Q .Howqu unauumuomsm mama mew>mn annexe .mnoum mom oEwm an msHDEwum oEmm ecu acumen mucommou Houucou new oncommou mBU4romop “w” W m“: .moHQEmm ocean ouomon mamsoocmunonam pouumnca madamm ca Am ooa\D NV 18044 .chpooHn mo menu on» an new ouONon mouscae 0H .ououon mouscae ov um ousmomxo nomm> nonumm .cflumaom xaco po>emoou mumu Houucoo .>m©\cNumHom cu mamsomCmusunsm whom muacs 0A oo>aooou mumu pwummuBN .z.m.m_fl some QDOum mm poucomoum mammam HE ooa\mcoumumouwuuoo mia A4. H 0.3 NS H 0.3 m6 H To... Cm H 53 or. H m.mm 33:8 SN H m6... AH H v.3 nee H TS. mm... H 0.3 m.~ H T3 Sucrose“. ensue 0H0 ASH To... mSH Ewe m.mH We... o.oH~.om RAH RS. 33:8 m.m H Tm... To H CT. Wm H ode m6 H WT. Wm H ine Ecuomoe mumnuo m.~ H 5.3 m.m H new m.m H 12 or. H ~65 m4 H new. 83:8 NS H 5S 1%.... H «.8 stir. H 1mm 26K H 063 T... H ~.mm Eocuommo «mega manor TN H 93 54 H 13 o.m H «.3 m6 H «.3. m4. H ~86 33:8 Rm H CE :56 H «.3 .66 H new «1...». H 9.0m m6 H «.8 $84.33 mums»... >uo>ooom o e N ucoEumouB macho maaaawum omd muouom usuaumoue NuamEumoua 1904 no axons mumu came use can maze» cw coNUMA:E«um mfiud can mmwuun ou .a manna A mmoco>dncommmu Hmowuuouocouvc 6O oo>eoowu mumu HOHDCOO .maemp Ceumaom CH DHU4 mums: OH po>HoUoH mumu omummue .2.m.m.fl Anamum new usmwoz moon msouo .qumamm haco N a Hm H.omm km H.Hsv mm.H «we ov.H mme me.H Ham Houuaoo 6H0 mm H.o~e mm H.0He mm H.mmv mm H.mee mm H.mmv emummsp meomuomme mH.H one mH.H 4mm 4H.H Ham on H.Gmm om.H mom Homecoo I. masow AH + mme me + ham ea + mam on + mow me + mmv emummuu msomuommo >Ho>ooom o v N ucmEumoHB msouw ucwEumoHB om4 muommm mucmsummue sees no memes ucmaaummxm D804 oesouso mo mam>umuca manomoan um mumu came pao new masom mo unmam3 upon .N magma H 61 Experiment II: Effects of Chronic Adrenocortical Stimulation in Young and Old Female Rats Materials and Methods The objective and procedures followed in Experiment II were similar to those of Experiment I except that 3 month and 22 month old female rats were used as experimental sub- jects. Female rats received daily injections of 6 U ACTH in 15% gelatin throughout the 6 week treatment period. Results Adrenocortical responses of both chronic ACTH treated and control old female rats to acute ACTH stimula— tion at each interval of the eXperiment are presented in Table 3 and Figure 7. Adrenocortical response is repre- sented as the mean plasma corticosterone level :_S.E.M. Adrenocortical response of chronic ACTH treated old rats to acute ACTH stimulation was significantly higher (p<:.05) than that of non—treated old controls after 4 weeks and 6 weeks of chronic ACTH treatment. The adrenocortical re— sponse of chronic ACTH treated and control young female rats to acute ACTH stimulation is presented in Table 3 and Figure 8. Only after 6 weeks of chronic ACTH treatment was the adrenocortical response of chronic ACTH treated young rats to acute ACTH stimulation higher (p‘<.01) than that of non- treated young controls. Adrenocortical response of depo- .ACTH treated young female rats to acute ACTH stimulation was significantly higher than that of depo-ACTH treated old 62 female rats to acute ACTH stimulation after 6 weeks (p<1.05) of depo-ACTH treatment. The ether stress response of both chronic ACTH treated and control old female rats is given in Table 3 and Figure 9. After 6 weeks of treatment, the ether stress response of chronic ACTH treated old animals was signifi— cantly higher (p<<.05) than that of old controls. By con- trast, the ether stress response of chronic ACTH treated young female rats (Table 3 and Figure 10) was significantly lower (p:<.01) than young control response after 2 weeks, 4 weeks and 6 weeks of chronic ACTH treatment. After 2 weeks recovery, treated and control reSponses to ether stress were not different in either young or old female rats. Figure 11 is a composite representation of this data, in which the data is presented as change of treated response from control response at each interval of the eXperiment. Change is represented as ug corticosterone/100 m1 plasma above or below control response. Group body weight data (mean :_S.E.M.) for each interval of the experiment is presented in Table 4. Body weight changes of treated animals closely parallel those of non-treated controls for both age groups. 63 .ucmsummuu meomuommo some muo>oomu mxomB N Houmm cam ucoEumoHu D804lomop mo mxooz o Hmumm new mxooB 4 Houmm .mxom3 N Hmumm .Acmmum CO H mm woumcmemoov ucoEumoHu D904Iomo© m0 pumum mnu cucumn UocHEHouoo mHoB noncommmu Hmofluuooocoup4 .ocon Caumaom mo mcofluowncD ©o>eooou maouucoo oEHu £UH£3 oceuop .wxom3 0 mo ocenom m How Cwumamm CH 3804 m0 Amawmp D we chNuuoth msomcmusonsm ©m>amomu mumu topmouu D904Iomom .mmmco>emcommou Hmoeuuooocmuom mo xmocn am no poms mHoB AHE ooa\m1v mam>ma oconoumooauuoo mammam UmceEHmump adamoflnquOHODHm .mcHHmEmm oooHQ mscem Hmuenno ou HOHHQ noon H Au3 moon m ooH\D av cofluooflce $904 comma mafiamm m ©o>eooou mumu HH4 .coHumaseeum $804 canon on menu mHmEow Anucoe NNV oao mo noncommou Hmowuuooooocmupm mucommumon mump mane .coHumHnEHum 4504 ousom Cu mumu onEmm pao Houucoo pom topmouu m904lomoo mo noncommou HmoHuHoUocoHp4 .h ousmflm 22 month 3 Acute ACTH Stimulation Chronic ACTH iZZZI Control CI I40r - l20" 64 V r / V I r V V r V / r V v R r/i / (I, 52/,” r" / .7]; V / '/ / i / / I'll / , I / /,/ y A K A V A I i’ / ' / i Weeks Exogenous ACTH Treatment \\ \“ \\\\\\: ~ \\\: \ \x\ ‘ \\ \‘.,\\'\" l“_r‘:r‘:~ \\\\\: \\\\\\\; \\\\ \\ \ \ \‘\\;\ 1‘ 1 \\\ q- .+. 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EEA V. . E, \ VEE 1 VE.E.EEE,\A \. A \.x\\ Y. . A EEEE E, E. \\.EA EEEEW E 4 ON V EE E. EA .. ,. \EE. \ ,. VVWE\ E. \A \\\\\A VVE\ x. A l E. E E, A .EE .. xxE \A \E\ EEEEE \ EE\\ . \EE. E, E 1 0V V,.. .A V .EA EE\E\ EE , EA VE. VA \EEEEEEA \\....\..\ \,EEE..EEA 1 A“: \‘E \AA vwE ...\ Ex\ \9 .pE lAuw \\EEEE V\EA \x..\ Y.\\.\. fix E E. A V. E ,A V x A \ . A V E, E. ,. \ .\ , V .\.E. ,.EE \ 1 E \ E ..E. .1 EEE . EEE .E A \ .\ E. fiE \ E.A “EVA \ .EE\A , 1 om \ ,.\ , \ .E.A \\ AWL “ml 1 \EE.\ E+ \EE. E.,EA \ . . rm». E 00. .928 mm low. 1642820 U E L nvv_ omcoammm $25 .95.». 5:9: m auwaysooguoo % brf Figure 11. 71 Effect of chronic ACTH treatment on adrenoCorti- cal response to ether stress and acute ACTH stimulation in young and old female rats. Adrenocortical responses (plasma corticosterone levels, ug/lOO ml) of depo—ACTH treated young (3 month) and old (22 month) female rats are presented as change from the comparable control response at each interval of the experiment. Responses to both ether Stress and acute ACTH stimulation determined before the start of depo- ACTH (designated as I on graph), after 2 weeks, 4 weeks and 6 weeks of depo-ACTH treatment and after a 2 week recovery period from depo-ACTH treatment are presented here. Depo-ACTH treat- ment consisted of daily injections of ACTH (6 U/rat) in a 15%.gelatin preparation. Ether stress responses consisted of ether anestheti- zation 40 minutes, 10 minutes and just prior to orbital sinus blood sampling while acute ACTH stimulation consisted of exogenous ACTH admin- istration (l U/lOO g body weight) 1 hour before orbital sinus blood sampling. 4O 3O 20 -30 -40 -50 i 72 Chronic ACTH Experiment A Corticosterone Acute ACTH “f I I & I I \r—\ 3 month 2 \ 1’ ’,0" O \\/ A I I \ / ‘3- 9 \ x,” ‘\\ l’z’ \ Ether Stress E I], \ 0 A “ 22 month 9 O ‘ 3month 3 \ A \ A A A I 2 4 6 R Weeks exogenous ACTH Treatment Figure 11 73 .mo. v m .umuufio uQHHUmHmQSm mamm wca>mn mammZM .msoum mom mEmm CH mnaseflum mEmm How umwwflo mmcommmu Houucoo can oncommmu meoclommv Ho. V m*« 3. v 9. .mcHamEmm voodn mquwn H505 H hamsomCmusonsm nouomnca madamm CH Am ooa\D Hy maome .OCHUmmHQ mo QEHu mnu um can OHOMwn mQuDCHE 0A EmHOMwn mmuDCHE ov um musmomxo Homm> umSumM .cHumem xaco ©m>umomu mumu Houucoo .>m©\cwumamm CH mamdowcmusonsm mfiofi muwcs m om>wmomu mumu kummusm .z.m.m.fl cams msoum mm nousommum .mEmmHm HE ooa\mc0umumooauuou mna 54 H m.~m N6 H 9: m.~ H «.3 ed H 5.8 5». H mas m 3380 m.~ H m.mm mica H v.3 «06 H 12: mg H 0.2: m.m H :0 o 5.04-38 e52 30 mg. H man im H m.~m m.m H mam j. H 93 o.~ H mas 0 H8380 Wm H Eniom 3.6. H «.mm m6. H 9mm 1m H 18 m6 H m.~n m 52.33 mumfio o.m H mam 9v H 93 m.~ H Tom m.~ H m.om m.m H mas o 33:8 320 H 5.: 32.0 H mag o.m H MKS ox“. H 12: 9v H 0.2 w 504.33 venom manor mic H 0.2: m.~ H m.mm EHN H 5.03 We H 0.8 v.~ H «.mm o Houucoo Wm H m.~m 256 H 5.2 :9... H 93 :95 H 93 in H «.8 m mfizuomoc mums»... >Hm>oumm m e N unusumoua : macho msaseaum mod muommm unusummua Nucmaumwua :804 no mxowz mum» mHmEmw oao new meso> ca coHuMH5EHum whom new mmwuum Ou mewcm>fimcommwu Hmoauuouocouo< .m magma 74 .cHumem maco ©m>Hmomu wumH Honucoo EmHHmo cHumHmm QH $904 muflcs w ©w>Hmomu mumu omummna m .z.m.m.H AmEmHm :HV uanm3 moon msouwH S H EN 3 H mom 2 H SN 3 H HR 3 H «R Honucoo nao OH.H mom HH.H Hmm oa H.vmm ma H.emm OH H.mmm omummnu mBUHtommo NH.H 5mm HH.H com HH.H mmm HH.H mew OH H.mmm Houucoo masow om + onm mm + Hmm ma + omm mH,H.mm~ ma H.mm~ omummuu maoHm>oumm o v m quEummHB msouo ucmEummHB 0mm mHOMmm «unusummua meom mo mHmmS. page.“ ummxm 50¢ UHCOHSU mo mam>umucH mchmmHQ um mumu mamaww oao cam mado> mo Hunmflm3 moom .w manme 75 Discussion of Experiments I and II Chronic ACTH stimulation of adrenocortical function in young and old, male and female rats.--Although the role of the adrenal cortex in the stress reSponse of animals is not completely understood, functional adrenal cortices are essential for survival in the face of many stressful stimuli. An index commonly used to measure adrenocortical function related to stress response is the functional reserve capac- ity of the adrenal cortices (the capacity of the stimulated adrenal cortices to secrete glucocorticoids in excess of normal resting steady state levels). Studies in aged cattle (Riegle and Nellor, 1967) and in aged goats (Riegle §§_§l,, 1968) provided evidence that adrenocortical functional reserve capacity was lower than in young animals. Decreased adrenocortical responsiveness to exogenous ACTH stimulation has also been observed with increasing age in rats (Hess and Riegle, 1970). These findings in cattle, goats and rats suggested that exhaustion of adrenocortical function might occur in old rats if they were exposed to prolonged periods of adrenocortical stimulation. This possibility was investi- gated in this study by eXposing both young and old rats to chronic depo—ACTH treatment for 6 weeks. Actual experimen- tal results did not support the hypothesis of adrenocortical exhaustion in either young or old rats after 6 weeks of Chr on ic de po—ACTH tre atment . 76 Adrenocortical response of chronically stimulated adrenal glands to an acute ACTH injection (sufficient ACTH to maximally stimulate the adrenal cortex) was elevated in young and old male and female rats following long-term daily injections of depo-ACTH (depo-ACTH concentrations sufficient to maintain elevated plasma corticosterone levels for at least 8 hours). Although adrenocortical responsiveness to acute ACTH stimulation increased in both young and old' groups the response was significantly higher in young than in old rats of each sex. Adrenocortical response to ether stress stimulation represents adrenocortical stimulation by endogenously released ACTH and hence reflects the influence of feedback control effects on hypothalamic-pituitary regulation of adrenocortical function. Ether stress results of the pres- ent study suggested that there was a marked age—related difference in feedback sensitivity of the adrenocortical control system. In old depo—ACTH treated male rats, plasma corticosterone levels following ether stress were not sig— nificantly different from those of controls after 2 weeks, 4 weeks and 6 weeks of depo-ACTH treatment. In young male rats, plasma corticosterone levels of depo—ACTH treated animals were significantly lower than those of controls after 2 weeks, 4 weeks and 6 weeks of treatment. In old female rats, adrenocortical response to ether Stress was higher in depo-ACTH treated than in control rats after 6 weeks of treatment although treated and control 77 responses did not differ after 2 weeks and 4 weeks of treat— ment. In young female rats, ether stress response of depo- ACTH treated animals was lower than that of control rats after 2 weeks, 4 weeks and 6 weeks of treatment. The increased responsiveness of young and old rat adrenal cortices to acute ACTH stimulation and the markedly decreased responsiveness to stress in young rats suggest that the hypothalamic-pituitary corticotrOpin control center (5) of the young rats are responding differently to the elevated blood corticoid levels following prolonged adreno- cortical activation than those of the aged animals. Direct observation of age-related differences in the feedback sensitivity of endocrine control systems is rela- tively new to experimental gerontology. Some observations of endocrine functional changes with aging support this concept. Ascheim (1965) showed that regular estrous cycles could be induced in aged constant estrous rats by injections of LH, suggesting that hypothalamic control of the pituitary failed in aging rats. Clemens §§_§l, (1969) investigated the possibility that hypothalamic changes may account for the loss of normal reproductive function in aged rats. Ovulation was induced in aged constant estrous rats by electrical stimulation of the preOptic area as well as by treatment with epinephrine or progesterone. These findings Saggested that reproductive failure in aged rats was not due tC> ovarian tissue senescence alone. Although observed in this gonadal rather than the adrenocortical system, the 78 findings of Ascheim and Clemens §£_al. provide suggestive evidence for age-related changes in endocrine control systems. The present data implies that hormone sensitivity of the feedback control system is changed in aged rats° Recognizing the similarities of control system dynamics between gonadal and adrenocortical systems, it is assumed that similar altered sensitivities to gonadal steroid feed- back could at least partially explain the data of Ascheim and Clemen's §£_§;, The anatomical site or sites of the age change in adrenocortical feedback sensitivity cannot be established from the data of the present study. Evidence from other investigators suggests at least two mechanisms of adreno- cortical feedback control that could have been involved. One possibility is that exogenous ACTH acted at short loop feedback receptor sites to exert a feedback control effect on adrenocortical function. Hodges and Vernikos (1958, 1959), Vernikos-Danellis and Trigg (1967) and Motta §£_§l. (1968) have demonstrated such a feedback effect of ACTH on adrenocortical function in rats. In these studies, plasma ACTH levels were markedly elevated by adrenalectomy or ACTH secreting pituitary tumors in order to produce the observed effects. Although injected depo-ACTH may have elevated plasma corticotrOpin levels enough to produce hypothalamic inhibition of the ether stress response in the present study, the slow release of ACTH from the gelatin and the short 79 biological half-life of ACTH (Syndor and Sayers, 1953) suggest that the increase would have been of small magnitude. Although suppression of the adrenocortical stress response by ACTH acting at short 100p feedback receptors remains a possibility, it seems unlikely that exogenous ACTH was directly responsible for the age-related difference in feed- back sensitivity observed in the present study. A more likely possibility is that chronically elevated plasma corticosterone levels, produced by the slowly released exogenous ACTH, were responsible for sup- pression of the adrenocortical stress response in young male and female rats. Halasz §§_§l, (1967), Zimmerman and Critchlow (1969b), Smelik (1969) and others have shown that the corticosteroid sensitive feedback control of the pitu- itary-adrenal axis is located primarily in the hypothalamus. More specifically, Halasz g;_al, (1967) have shown that corticosteroid sensitive feedback control of the adrenocor— tical stress response is located in the median eminence area of the hypothalamus. Whether suppression of the adrenocor— tical stress response in young male and female rats was caused by elevated plasma corticosteroid levels or by direct action of exogenous ACTH on short 100p feedback receptors, the age difference in feedback sensitivity remains apparent. The physiological significance of the age-related difference in corticosteroid feedback sensitivity is diffi- cult to assess. Zimmerman and Critchlow (1969a) concluded 80 that no level of corticoid treatment will inhibit the adrenocortical stress response, provided that an adequate stress stimulus is employed. This concept was supported by Yates et al. (1969), who described the saturation character- istics of the steroid sensitive adrenocortical control mechanism. Although this relationship may hold under the conditions of their acute experiments, it is reasonable to conclude that the ether stress response can be suppressed by long-term elevation of corticosterone levels, as in the present study. The apparent age-related change in hormone sensi- tivity suggests that the adrenocortical control mechanism has undergone alterations which in turn have compromised the precision of adrenocortical regulation. Such changes could represent an important link in the progressive deterioration of homeostasis with increasing age. Changes in the ether stress feedback control imply that the control system for "resting" adrenocortical function probably also becomes less sensitive to corticosteroid feedback with increasing age. Similar conclusions concerning the corticosteroid feedback sensitivity of adrenocortical control systems have been reached in studies (Riegle, Wallace and Hess, unpub— lished observations) in which young and old rats were exposed to exogenous corticosteroid (Dexamethasone) treat- ment for 2 weeks. Apparently, feedback sensitivity to both endogenous and exogenous corticosteroids decreases with increasing age in rats° 81 Experiment III: Influence of Gonadal Steroids on Adrenocortical Function in the Rat Materials and Methods The purpose of this experiment was to test the hypothesis that age-related changes in adrenocortical func- tion arise from changes in gonadal steroid production. Gonadal hormones (particularly the estrogens) are known to influence adrenocortical function in the rat (Kitay, 1963b) and increasing age is known to alter gonadal function in the rat (Eckstein, 1955; Krohn, 1955). Two methods were used to assess the influence of gonadal steroids on adrenocortical response to both ether stress and exogenous ACTH stimulation in young adult and aged rats. Both young adult and aged rats were gonadectomized, to determine if the loss of endog- enous gonadal hormone production would alter adrenocortical responses. Subsequently, the influence of exposure to a gonadal hormone treatment regime (utilizing exogenous hormones) on adrenocortical responses was determined in young and old rats. The first experiment employing this design was con- ducted in a group of young (6 month) female rats and another group of old (24 month) female rats. Adrenocortical response to both ether stress and exogenous ACTH was tested in both young and old female rats prior to ovariectomy, 4 days post- ovariectomy and 2 weeks post—ovariectomy. Adrenocortical reSponses of intact control young and old female rats were aLso determined at each of these intervals.- During the 82 third week of the eXperiment all animals received daily in- jections of progesterone (800 ug in 0.1 ml olive oil, s.c.) for a total of 7 days. Adrenocortical responses to both ether stress and exogenous ACTH were determined at the end of progesterone treatment. During the 4th week of the experiment, young and old control rats and one group each of young and old ovariectomized rats received estrogen replacement therapy for 7 days (30 ug estradiol in 0.1 ml olive oil, s.c., daily). A second group of young ovariec- tomized rats and a second group of old ovariectomized rats received injections of the olive oil vehicle (0.1 ml, s.c., daily) alone during the 4th week of the experiment. The treatment regimes of the 4th week were reversed during the 5th week, so that all of the experimental animals were exposed to both estradiol and oil injections during the course of the experiment. Adrenocortical responses to ether stress and exogenous ACTH were determined in all rats at the end of the 4th and the 5th week of the experiment. A similar experiment was conducted in young adult (5 month) and aged (24 month) male rats. Adrenocortical response to exogenous ACTH stimulation was determined prior to castration, after 2 weeks of castration and after 6 weeks of castration. Adrenocortical responses of intact controls of both age groups were also determined at these three intervals throughout the experiment. During the 7th and 3th week of the experiment, all of the rats were treated Witfln testosterone prOpionate (1 mg daily in olive oil, s.c.). 83 Adrenocortical reSponse to exogenous ACTH was again deter- mined at the end of the treatment period. A third experiment was conducted to test the effect of prepubertal gonadectomy on adrenocortical function in male and female Long-Evans rats. Ovariectomy of female rats and castration of male rats was performed at 30 days of age. Sham Operations were also carried out on control male and female rats at 30 days of age. Adrenocortical responses to exogenous ACTH stimulation were determined 6 weeks post— surgery. Body weights were recorded at the time of surgery and at the time of bleeding, six weeks later. Experimental results of each part of this study were analyzed by Student's t-test. Results Ovariectomy of young adult female rats did not affect adrenocortical reSponse to exogenous ACTH or ether stress stimulation either 4 days or 2 weeks post-surgery (Tables 5 and 6). Similarly, no significant changes in adrenocortical response to ether stress or exogenous ACTH were observed 4 days or 2 weeks following ovariectomy of old female rats (Tables 5 and 6). Both ovariectomized and intact rats of both age groups were eXposed to gonadal hormone replacement during the 3rd, 4th and 5th week post-surgery° All rats received pIOgesterone treatment (800 ug daily) during the 3rd week. BOth young and old intact rats and a group each of young and 84 old ovariectomized rats received estradiol treatment (30 ug/day) during the 4th week and received oil vehicle injections during the 5th week of the experiment. _The treatment regime was reversed in the second group of young and old ovariectomized rats. These rats received the oil vehicle injections during the 4th week and estradiol (30 ug/day) during the 5th week of the experiment. The only effect of progesterone treatment in young and old female rats was reduced adrenocortical responsive- ness (p<1.05) to exogenous ACTH stimulation in old intact rats compared to the corresponding pretreatment response. However, the adrenocortical response of old intact rats following progesterone treatment was not different from that of old ovariectomized rats following progesterone treatment (Table 8). Following estradiol treatment (30 ug/day for 7 days), adrenocortical response to exogenous ACTH was significantly lower than the similar response prior to the gonadal hormone treatment regime in young intact (p (.05) and old intact (p‘<.Ol) rats (Tables 7 and 8). Estradiol treatment did not alter adrenocortical response to exogenous ACTH in either young or old ovariectomized rats (Tables 7 and 8). Adreno- cortical response to ether stress following estradiol treat- ment was loWer than the ether stress response prior to the gonadal hormone treatment regime in young intact rats (p <.05), but was not altered by estradiol treatment in old intact and young and old ovarectomized rats (Tables 9 and 10). 85 Injections of oil vehicle alone elevated the ether stress adrenocortical response (p‘<.05) of one group of young ovariectomized rats (Table 9), but had no significant effect on adrenocortical responses of any other groups. In a related study, adrenocortical response to exogenous ACTH stimulation following castration of young and old male rats was considered (Table ll). Although adrenocor- tical responses of young male rats were higher (p<<.05) than those of old male rats 6 weeks after castration, adrenocorti- cal responses of young and old male rats 2 weeks and 6 weeks after castration were not significantly different from pre- castration responses. Testosterone replacement therapy (1 mg/day) was given during the 7th and 8th weeks of the experiment. After 2 weeks of testosterone, adrenocortical response of young male rats was lower (p<:.01) than the 6 week response but was not significantly different from the 6 week reSponse in old male castrate rats (Table 11). Adrenocortical response to exogenous ACTH was also studied in prepubertally ovariectomized female rats. Plasma corticosterone levels 6 weeks after ovariectomy were signif— icantly lower (p‘<.Ol) than those of sham-Operated control female rats (Table 12). In male rats, adrenocortical response was not different between castrate and intact animals 6 weeks after prepubertal castration (Table 12). 86 Table 5. Effects of ovariectomy on adrenocortical response1 to exogenous ACTH2 in young and old female rats Before 4 Days 2 Weeks n Ovariex Ovariex Ovariex Ovariex 9 lO9.6;:3.7 lll.0;t4.4 ll0.0j;5.7 Control 4 99.1165 100.4i4.8 114.4365 Ovariex 9 90.2:6.9 73.7:7.4 80.8:6.3 Control 4 89.9:5.3 82512.6 87.1:3.6 1Plasma corticosterone (ug/lOO m1 : S.E.M.). 21 U ACTH/100 g 1 hour before blood sampling. 3Ovariectomized rat response did not differ from comparable control response at any interval of the experiment. 87 Table 6. Effect of ovariectomy on adrenocortical reSponsel in young and old female rats to ether stress Before 4 Days 2 Weeks n Ovariex Ovariex Ovariex Ovariex 9 97.8:;5.7 98.9j;5.7 97.1;15.2 Young Control 4 89.0-+3 2 97.5:;5.3 95.8;13.9 Ovariex 9 88.8'+4 9 74.9 17.4 77.4j;7.7 Old3 Control 4 88.8;t5.6 84.4 12.8 86.2;t8.8 4 1Plasma corticosterone (ug/lOO ml i_S.E.M.). 2Exposed to ether anesthesia 40 minutes, and 0 minutes before blood sampling. 10 minutes, 3Ovariectomized rat response did not differ from comparable control response at any interval of the experiment. 4n = 8. 88 Table 7. Adrenocortical response of young female rats to ACTH1 following gonadal hormone treatment2 Ovariex3 Intact Ovariex4 n 5 4 Pretreatment (2nd week 105.418.9 ll4.4i6.5 llS.8:6.6 3rd week 109.8 _-t_-_5.8 91313.4 123.6:5.7 4th week 95.7:6.9 89.8:2.0* 123.5:S.1 5th week 129.618.8 lO8.6_-t4.7 106.7 16.4 lPlasma corticosterone (ug% :_S.E.M.) 1 hour after ACTH injection (1 U/lOO g body weight). 2 1 week: Progesterone (800 ug/day) Estradiol (30 ug/day). 3Treatment regime as follows: Progesterone, 3rd week Estradiol, 4th week Oil vehicle, 5th week. 4Treatment regime as follows: Progesterone, 3rd week Oil vehicle, 4th week Estradiol, 5th week All rats were exposed to the following hormones for *Response differs from pretreatment (2nd week) response of same group (p<:.05). 89 Table 8. Adrenocortical response of old female rats to ACTH1 following gonadal hormone treatment2 Ovariex3 Intact3 Ovariex4 n 4 4 4 Pretreatment (2nd week) 76.4: 9.2 87.1:3.6 85.1:9.4 3rd week 65.4i6.l 67.0 :6.3* 64.8:6.3 4th week 63.7:6.3 61.01 3.3** 76.4:6.l 5th week 74714.1 87.414.75 74.1:12.o lPlasma corticosterone (ug% i_S.E.M.) 1 hour after ACTH injections (1 U/lOO g). 2All rats were eXposed to the following hormones for 1 week: Progesterone (800 ug/day) Estradiol (30 ug/day) 3Treatment regime as follows: Progesterone, 3rd week Estradiol, 4th week Oil vehicle, 5th week. 4Treatment regime as follows: Progesterone, 3rd week Oil vehicle, 4th week Estradiol, 5th week. 5n = 3. *Response differs from pretreatment (2nd week) response of same group (p< .05). **Response differs from pretreatment (2nd week) response of same group (p< .Ol). 90 Table 9. Adrenocortical reSponse of young female rats to ether stressl following gonadal hormone treatment Ovariex3 Intact3 Ovariex4 n 5 4 4 Pretreatment (2nd week) 89.2:6.4 95.8:3.9 107.0: 6.1 3rd week 95.6;t3.3 89.2:;5.7 102.21;3.3 4th week 86.2;16.4 79.Sj;4.l* 103.2 17.4 5th week lO7.9:4.8* 99.9 15.2 94.7:5.5 lPlasma corticosterone (ug%.i S.E.M.) following exposure to ether anesthesia 40 minutes, 10 minutes and 0 minutes before bleeding. 2All rats were exposed to the following hormones for 1 week: Progesterone (800 ug/day) Estradiol (3O ug/day). 3Treatment regime as follows: Progesterone, 3rd week Estradiol, 4th week Oil vehicle, 5th week. 4Treatment regime as follows: Progesterone, 3rd week Oil vehicle, 4th week Estradiol, 5th week. *Response differs from pretreatment (2nd week) response of same group (p<<.05). **Response differs from pretreatment (2nd week) response of same group (p< .Ol). 91 Table 10. Adrenocortical response of old female rats to ether stressl following gOnadal hormone treatment2 Ovariex3 Intact3 Ovariex4 n 4 4 4 Pretreatment (2nd week) 73.1:13.4 86.2:8.8 81.7 :9.3 3rd week5 57.1 i 6.9 67.0 :6.3 56.7:8.8 4th weeks 46.6 i 8.9 69.2i 3.5 74.1 16.6 5th week5 49.1i8.5 79.o_+_7.16 70.5;I-_8.8 1 Plasma corticosterone (ug%.:_S.E.M.) following exposure to ether anesthesia 40 minutes, 10 minutes and 0 minutes before bleeding. 2All rats were exposed to the following hormones for 1 week: Progesterone (800 ug/day) Estradiol (30 ug/day). 3Treatment regime as follows: Progesterone, 3rd week Estradiol, 4th week Oil vehicle, 5th week. 4Treatment regime as follows: Progesterone, 3rd week Oil vehicle, 4th week Estradiol, 5th week. 5Treatment means did not differ from 2 week (pre— treatment) responses. n = 3. 92 Table 11. .Effect of castration of adult male rats on adrenocortical response Young2 n Old3 n Before castration 65.4 i 2.9 6 55.8 i_3.2 5 2 weeks castrate 55.0 i 4.7 6 49.1 i 1.9 5 6 weeks castrate 66.1 i 2.9 6 53.3 i 4.7 5 2 weeks testosterone 48.4 i 5.6** 3 38.7 + 5.7 3 l . . Adrenocortical responses reported as plasma corti- costerone levels (ug% :_S.E.M.), determined 70 minutes after injection of ACTH (2 U/lOO g). 25 months old. 324 months old. 41 mg/day, testosterone prOpionate. **Mean differs from 6 week castrate response, (p<:.Ol). 93 Table 12. Effect of prepubertal gonadectomyl on adrenocor- tical response n ug% Corticosterone Body Weight3 Ovariex 12 71.0 i 3.5 225 9 Female Intact 11 92.4 : 6.0a 180 g Castrate 3 66.6 i 4.9 223 9 Male Intact 3 59.0 i 5.3 270 g lGonads removed at 30 days of age. 2Response to exogenous ACTH 6 weeks post surgery, 1 U ACTH/100 g in female, 2 U ACTH/100 g in male, adrenocor- tical response reported as plasma corticosterone level (ug i_S.E.M.) 1 hour after ACTH. 3Average weight of group (grams). aMeans with the same superscript differ, (pmH mcoumumOOHuHoo mammam .coHumadeHum mmmuum Hmnum ou mmcommmu HmoHuHOOocmupm mucwmmnmmu mum: noncommum mumn .coHumHsEHum mmwnum Hmnum Ou mmm mo mwmp oom paw mm cmm3uwn mumn mo noncommmu Hmowuuooocmupm .NH onomnm 102 nXuN Rum_ NH mnemnm 1| mac .6 960 ON. 09 Aum_ Avg. Om 0m 0? ON 1 a q 1 d u d 5 q d J) J .oe .06 too on $01 now .gum tnxg l e O _ _ $25 55m 2 oncoomom 62:86:23. 103 Table 13. Adrenocortical response of male rats to ether stress between 26 and 154 days of age1 Body Age Plasma Weight (days) Corticosterone (g) 26 40.2 i 2.2a'b 54.6 i 2.2 13 44 67.4 : 2.4a"C 115.4 : 3.5 13 58 62.3 i 2.9 161.9 i_6.4 13 76 53.8 : 1.61"C 226.2 2“. 8.9 13 95 53.2 i 1.1 274.6_: 9.7 13 115 49.3 :_l.8 326.2 1 9.8 13 154 53.2 i 1.1 373.1 i_10.l lPlasma corticosterone (ug% :_S.E.M.) following ether stress. a’b’cMeans with same superscript are significantly different, (p < .01). 104 Table 14. Adrenocortical response of female stress between 23 and 200 days of rats to ether age Body Age Plasma Weight (days) Corticosterone (g) n 23 50.4 : 1.8a 59.2 .t 1.7 12 31 57.7 i 1.8 85.8 _t 2.2 12 44 75.3 i 3.6 132.5 i_3.4 12 58 82.8 i 3.0 194.2 .t 3.4 12 76 97.4 i 3.5 230.0 : 3.2 11 95 104.0 1: 5.6 240.9 1 6.7 11 115 106.2 2“. 5.16"]0'C 262.3 1 6.2 11 160 96.8 i. 5.9C 289.9 1. 9.5 11 200 87.5 13.9b 295.5 :_8.2 11 1Plasma corticosterone (ug% i_S.E.M.) following ether stress. a’bMeans with the same superscript are different, (p < .01). TMeans with the same superscript are different, (p < .05). significantly significantly 105 Table 15. Adrenocortical response of female rats to ether stress Plasma Age at n Corticosterone Puberty Experimental rats 11 104.0 1 5.6 41.8 Control rats 12 103.8 + 2.5 40.6 1Ether stress adrenocortical response of experimental group at 95 days of age compared to adrenocortical response of control group exposed to ether stress for first time at 90 days of age. 2ug/lOO ml plasma i_S.E.M. 3Average age of onset of puberty (day of vaginal Opening). Table 16. Comparison Of adrenocortical response1 to ether stress and ACTH stimulation Ether2 ACTH3 n Response Age Response Age Female 11 87.5 i 3.9 200 day 81.9 i_4.5 203 day Male 13 53.2 i 1.1 154 day 50.2 i 1.6 158 day 1Response of same rats given as ug corticosterone/100 m1 plasma :_S.E.M. 2Exposed to ether anesthetization 40 minutes, 10 minutes and just before bleeding. 31 U ACTH/100 g body weight in female, 2 U ACTH/100 g body weight in male, 1 hour before bleeding. 106 Discussion Studies of adrenocortical responsiveness to ACTH and stress in the rat have used a substantial range of animal ages. The present experiment was undertaken to determine if calendar age of the maturing rat was involved in adreno— cortical responsiveness. The data presented in Figure 14 illustrates that adrenocortical response to other stress stimulation does change between 23 and 200 days of age in the rat. Adrenocortical response in male rats reached a peak at 44 days Of age. Adrenocortical responses were similar in male rats between 76 and 154 days of age. To be sure that these results were not just reflecting accommoda- tion to ether stress, adrenocortical response to exogenous ACTH was tested at 158 days of age. The close similarity of this response to the 154 day ether stress response indicated that the animals were not accommodating to ether stress. Although it has been shown that the effect of ovarian secretion on adrenocortical function occurs by puberty in the rat, male-female difference in adrenocortical responsiveness was not different until 58 days of age. The peak response of adrenocortical function in female rats occurred at 115 days of age. The possibility that rats were accommodating to stress with repeated sampling was tested by measuring adrenocortical response of a group of female rats bled for the first time following ether stress stimulation 107 at 90 days of age. The similarity of stress response of this group of non—accommodated rats to the experimental group and the parallel response of the experimental group to ACTH stimulated response at 203 days of age indicated that the female rats were also not accommodating to the experimental procedure in this study. Although adrenocor- tical responsiveness was reduced at 160 and 200 days in these female rats, the response remained within the range of response for adult female rats (Hess, 1968). The results Of this study suggested that the sex difference in adrenocortical response to ether stress in the rat is closely associated with the onset of puberty. In addition, these results emphasize the fact that adrenocor- tical response to ether stress does change in the maturing rat and should be considered when studies of adrenocortical function are conducted in rats with eXperimental animals chosen accordingly. Young mature male and female rats chosen for the studies of this thesis were in the age ranges characterized by small changes in the adrenocortical response curves of Figure 12. 108 Experiment V: Thyroid Hormone Influences on Adrenocortical Function in Young Adult and Aged Male Rats Materials and Methods This experiment was designed to test the effect Of different levels of thyroid hormone (Protamone) stimulation on adrenocortical function in male rats. Quantities of Protamone equivalent to .02%, .04% and .08% of the diet were carefully mixed with the standard rat diet used in our rat colony. In the initial phase of the experiment, 24 three month old male rats were divided into four groups (six rats/group). One group was maintained on each level of Protamone and a fourth group was maintained on the normal diet for a period of 3 weeks. Rats were caged in groups of three. Feed consumption of each group was estimated daily and body weights were taken at weekly intervals throughout the experiment. Adrenocortical response (plasma cortico- sterone level) to a standardized ether stress was determined after three weeks of treatment. A similar experiment was conducted in a group of male rats averaging 23 months of age. Because of antici— pated variation in the adrenocortical response of old rats, response to ether stress was also determined prior to the start of Protamone treatment. The rats were divided into four treatment groups (4-6 rats/group), each of which received one of the three Protamone levels or the regular rat diet. Rats were housed two or three per cage. Daily 109 food consumption was estimated and body weights were deter- mined at weekly intervals. Adrenocortical response to ether stress stimulation was determined after three weeks of Protamone treatment. Results Ether stress adrenocortical reSponses of young male rats treated with different levels of Protamone are given in Table 17 and Figure 13. Plasma corticosterone levels follow- ing ether stress were compared between the four experimental groups by Duncan's Multiple Range Test subsequent to Analysis of Variance. Adrenocortical responses Of rats receiving the .O4% and .08% diets were significantly different from those of non-treated controls and from one another (p < .01). Adrenocortical response of old male rats to ether stress was determined prior to the start of Protamone treat- ment as well as at the end of the 3 week treatment period. The resulting data is given in Table 18 and Figure 13. Adrenocortical responses of the four experimental groups of Old male rats were not significantly different either before the start or after 3 weeks of Protamone treatment. Plots of adrenocortical response versus log-dose Protamone for both young and Old rats suggested that adreno- cortical response was related to the level of Protamone treatment in young but not in Old male rats (Figure 14). Average food consumption for all treatment groups of young and Old male rats is presented in Table 19. Feed consumption 110 was similar for all treatment groups of young and old male rats. Group body weight averages (: S.E.M.) of young and Old male rats are given in Table 20. Body weight after Protamone treatment was not significantly different from pre-treatment body weight in any of the eight treatment groups of young and old male rats. 111 .mmcommmH HMUHHHOOOCOHOM mo :oHumcHEHmump ou HOHHQ mxmm3 m How Amv OGOEmuoum.Rmo. mchHmucoo ume m can sz mcoamuoum Rec. mcHCHmucoo DOHU m .AHV OCOEmuoum XNO. mchHmucoo umHO m .AUV uwHO HmHsmmH mnu pom OHOB 30H53 mHmEHcm ucmmwummu msoum 0mm some EH mmsoum HSOM one .ucuEumeu OCOEmDOHm mo mxmmz m Hmumm mmsommmu HmOHuHooocmupm mucmmwummu wumn may mo aoHuHom ammo gnu OHH£3 unmEummHu OCOEmuoum ou HOHHQ mmcommwu HMOHuHooocmupm mucmmmumwu mumn 0:» mo coHunom pmnoumn mmouo one .ucmaumouu OGOEmuoum mo mHm>mH HQOHOMMHO Op mumu mHmE ASHGOE mmv 0H0 mo uncommon HmOHuHooocmupm mucmmmummu Hound uanH any mHan ucmEummHu moosmponm mo mHm>mH ucmHOMMHp ou mumH mHmE ASDGOE mv 0:50» mo mucommmu HmoHuHooocwHOm unnammnmmu Hmsmm pmmH one .maHHmEmm OOOHQ msch HmanHo muommn umsfl new mouscHE OH .mmuscHE o¢ GOHHMNHuwnummcm H0300 Op ommomxm muwz mumm .GOHHOGSM HMOHuHooocmem mo xmch am no poms mum3 AHE OOH\m1v mHm>wH mcououmooHuHou mammHm .coHumHsEHum mmmuum Hmnum on mumu mHmE mo uncommon HmoHuHoooamem musmmwummu mum: OmucwmmHm mump was .ucmEummnu OCOEHOS OHouxnu mcHBOHHOM mumu mHmE OHO cam mono» mo noncommmu HmOHuHooocmHom .3 0.53m 112 .86 I c 6v _2 E E M0 So 8v ma onsoem I 3v 2 6v .f.11 1"..- .o 932 OW Om on o\. 94 00_ ON. Figure 14. 113 _Adrenocortical response versus level of Protamone treatment in young and old male rats. Adrenocortical response (ug% plasma corticoste- rone) to ether stress is plotted as a function of the log-dose of Protamone (9/100 g) contained in the regular rat diet. The least squares line fitting the three points of each age group is given. 114 Adrenocortical Response to Ether Stress I20 " O—O young J t El--E] old! 0 . § IOO ~ 0) 1,3- . CD .9 t 80F O L) o\,, _. CD 1 60 ’ 40L 1 l L 1_ 1 J l J .Ol .02 .04 .06 .08 °/o Protamone in diet Figure 14 115 .EHo. v o oco EOHM DCOHOMMHU mechHMHcmHm mum umHHomHmmsm 08mm gnu mcHHmmn mcmoz V HOSDOCm m\@§osn\m .ucoEumonu OCOEmuOHm mo mxom3 m Houum .mmmuum Hmnum mcHBOHHom A.E.m.m.fl HE OOH\m1v Ho>mH ocoumumooHuHou mammHm .Uomm CH OCOEmuoum X mm cm>Hm mmmmOO .DOHO HmHsmmH Op nmpnm OGOEmHOHm .oeo neueoe m m N H @ . . m.m.H ~.ooa . . m.m.H m.om . ~.m_H m.mm . O Q m u m U U Q m HmuHu o m o o H.m H.o.mm mmmcommou Houocoupm C .Xmo. x00. KNO. 0 NOCOEmuonm mo H0>0H Hmumu OHME masom mo :oHuucsm HMUHHHOOOCOHUO co ucmEmHmmsm OCOEMDOHm mo Hummmm one .hH mHnt .116 .ucmummMHp mHDCmUHMHcmHm uoc 0H03 OGOEmuoum Hmumm momma ucmEumOHBm .ucoEumouu OGOEmuoum mo 6x003 m Houmm .mmouum Hmnuo mcHonHom A.S.m.m.fl HE OOH\m1V ozonmumOUHuHou mEmmHmH6 .pcmEummHu mcoEmuonm mo unmum ou HOHHQ .mmonum Hmnuw mcHBOHHom A.2.m.m.fl HE OOH\m1v maououmouHuHou mammHm m .600“ CH OCOEmuoum 8 mm cm>Hm mmmmoo .DOHU HmHsmOH O» @0606 OCOEmuOHmN .pHo mnucoe mmH 6.6.H 6.65 n.m.H 6.66 6.HH H.6.6n 6.6.H 6.66 oeoEnnoum Houmm oncommon mEvauHuHouocoun¢ 6.6 + 6.66 6.6 + 6.66 6.8 H.~.nm «.6 + 6.66 oeoEououm muomon uncommon MHmuHuHouocOHpm m 0 v m a Xmo. $60. KNO. 0 choEmuoum mo Hw>mq Hmumu mHmE OHO mo coHuuasm HmUHuHouocmHUm co quEOHQQSm OGOEmuoum mo uummmm one .mH mHnt 117 Table 19. Feed consumption of male rats on Protamone treat— ment Level of Protamone2 O .02% .04% .08% Young 19.7 19.9 18.7 21.6 Old 21.1 22.6 21.7 23.7 1Average feed consumption (grams/rat/day), during 3 week treatment period. 7% Protamone contained in feed. 118 Table 20. Body weight1 of young and Old male rats Of Protamone experiment Level of Protamone2 o .02% .04% .08% Initial3 300 316 296 330 .119 .:24 .110 .113 Young Final 348 365 328 343 .121 .126 .114 ,:11 Initial3 482 530 445 541 .:22 “:36 .133 :16 Old 4 Final 488 525 449 533 .123 :38 ,:29 .:16 1Average weight of group in grams : S.E.M. 7% Protamone in diet. 3Weight prior to start Of Protamone treatment. 4Weight after 3 weeks of Protamone treatment. 119 Experiment VI: Thyroid Hormone Influences on Adrenocortical Function in Young and Aged Female Rats Materials and Methods This experiment was designed to test the effect of different levels of thyroid hormone (Protamone) stimulation on adrenocortical function in young adult and aged female rats. Protamone treatment levels were .02%, .05%, and .08% of the diet. A group of 23 young adult rats averaging 4 months and a group of 22 old rats averaging 25 months Of age were used in this study. Each age group was divided into four treatment groups (5 or 6 rats/group), with each receiv- ing one of the three Protamone treatment levels or the regu- lar rat diet. Rats were housed in groups of two or three with group food consumption being estimated daily. The rats were weighed at weekly intervals throughout the course of the 3 week treatment period. Adrenocortical response to ether stress was determined prior to Protamone treatment and at the end of the 3 week treatment period. Results Ether stress adrenocortical responses Of young female rats treated with different levels of Protamone are given in Table 21 and Figure 15. Plasma corticosterone levels between the four experimental groups after 3 weeks of Protamone treatment were compared by Duncan's Multiple Range Test subsequent to Analysis of Variance. Adrenocor- tical responses of rats fed the .08% Protamone diet were 120 significantly different (p < .01) from those of non-treated controls. Adrenocortical responses of these four experimen- tal groups to ether stress were not significantly different prior to the start Of Protamone treatment. Ether stress adrenocortical reSponses of Old female rats treated with different levels of Protamone are given in Table 22 and Figure 15. Adrenocortical reSponses of the four experimental groups of old female rats were not signif— icantly different either before the start or after 3 weeks of Protamone treatment. Plots of adrenocortical response versus log-dose Protamone for both young and old female rats suggested that adrenocortical response was related to the level of treat- ment in young but not in old rats (Figure 16). Although the response of Old rats showed a linear relationship to log- dose Protamone, adrenocortical responses to .02%, .04%, and .08% Protamone treatment were not significantly different from one another. On the other hand, adrenocortical response to .08% Protamone was significantly higher (p<<.01) than the response to .02% Protamone in young female rats. Average feed consumption for all treatment groups of young and old female rats is given in Table 23. Feed consumption differ- ences between young and old rats were not considered large enough to have influenced the experimental results. Average body weights (i S.E.M.) for each treatment group of young and Old female rats at the start and at the end Of Protamone 121 treatment are given in Table 24. Post-treatment body weights did not differ from pretreatment body weights for any treat- ment group of either young or old rats. .122 .omcommOH HmOHuHouocmHOO mo coHumcHEHouop ou HOHHQ 6x003 m How Hmv OCOEmuOHm_me. mchHmucou DOHO m can .ASV OCOEmuOHm XQO. mchHmucou DOHU m .AHV wcoEmuoum,RNO. mchHmucou ume m .Hov uOHp HmHsmou mnu pom ouo3 nuH£3 mHmEHcm poomoumou msoum 0mm 3060 CH mmsoum usom one .ucoEummHu mcoamuoum mo 6x003 m Houmm oncommou HmOHuHouocmupm mucomoummu mumn gnu mo aoHuHom ammo on» 0HH£3 ucmsummuu OGOEmuOHm on HOHHO uncommon HMUHuHooocmupm muammoumou mnmn on» No GOHHHOQ ponuumn mmouu one .ucoauwouu OGOEmuoum mo mHo>0H DQOHOHMHO on menu OHmEmm Anacoe ONO UHO mo uncommon HmuHuHouocwHOm mucwmoumwu Hound uanH mnu 0HH£3 ucmEumonu OCOEmuoum mo me>mH ucmHOMMHO ou mumu 0HmEmm finance my munch mo uncommon HmOHUHOUOCOHOm mucmmmummu Hmem HMOH one .mcHHmEmm OOOHQ msch HmuHQHO ouOmon umsn new wouOGHE OH .mmuscHE Ov eoHumNHuonumOCm Honum Ou Oomomxo muoB mumm .GOHuucsm HmOHuHooocoupm mo xOOcH am no poms 0Ho3 AHE OOH\O1V mHm>OH maoumumOUHuHoo mammHm .coHumHOEHum mmmuum Hmnuo on mumu mHmEom mo uncommon HmOHuuouocoupm mucmmmumou who: noncommum pump one . .ucmEumouu ocosuon oHoumnu mcH3OHHOH mumu onEow OHO cam masom mo noncommou HmuHuHouocmupm .6H onomnm 123 mH musmwm m me be me me me no me Elwe 06 cm Om OO_ o\o 01 ON_ 03 Om. Om. Figure 16. 124 Adrenocortical response versus level of Protamone treatment in young and Old female rats. Adrenocortical response (ug% plasma corticoste- rone) to ether stress is plotted as a function of the log-dose of Protamone (g/100 9) contained in the regular rat diet. The least squares line fitting the three points of each age group is given. 125 Adrenocortical Response to Ether Stress '60' 0-0 young 3 O _ EH3 old 3 8 I40— 8 Q) '23 T 0 O C) ’E IZO' o o 0 #— 6" 3100- x43" - H’fl”’ B ” ” 80L 7 Cl .02 .04 .06 .08 °/o Protamone in diet Figure 16 126 .HHO. v mv unonmmep mHucmOHstmHm mum umHHomHmmsm 0866 on» mcHume mommsr.m Houmm .mmmuum Hmnum OGHBOHHOM A.2.m.m + HE OOH\O1V mcououmOUHuHou mammHm HOHHO .mmmuum Hmnum msH3oHHOm H.2.m.m + H8 OOH\O1V moonoumOOHuHou mEmMHm .Umom CH mcoEmuoum .ucoEummHu OGOEmuoum mo 6x003 m 6 .ucoEummHu OGOEOHOHm mo uumum Op m 8 mm co>Hm mommmOO .umHo HmHsmmH Ou pmppm OCOEmDOHmN .6Ho meoeoe 6H m.O©H m.hOH + 0.0mH 6.6 + H.mHH mm.6 + 0.0HH OGOEmuonm Hmumm mmaommwu vauHuHooocouom + m.hOH m.m + O.mOH m.m + 6.MHH OCOEMDOHO ouommn oncommmu mHMUHuHouocmH©< 6 m 6 E. e xmo . X00. KNO. 0 N mflOEm UOHm MO H0>0~H Hmumu mHmEmm venom mo soHuocsm HmuHuHouoconpm co DGQEOHmmsm OOOEmuoum mo uommmo one .HN 0Hnt 127 .ucmHOMMHO hHucmoHMHcmHm poo mums OCOEmuoum Houwm mamoE unmEumOHBm .ucoEumouu OCOEmuoum m0 6x003 m Hmumm .mmmnum Hmzum mGHBOHHOM H.2.m.m + H8 OOH\m1V occumumOUHuHOU mEmmHmv .ucoEumouu OGOEmuoum mo uumum OD HOHHQ .mmonum Honuo mcH3oHHom A.S.m.m.H HE OOH\mjv mcoumumouHuHou mammHm m .pmmm OH mcoEmuoum X 66 co>Hm mommmOU .DOHO HmHsmmu Ou poppm OOOEmuoumm .OHO mnucoe mNH H.6H H 6.66 6.3 H 6.66 93 H 6.66 6.6 H 6.66 6.886636 Houmm uncommon 60H Hooocou m.6H .u pm 6.6 H 6.66 26 H 6.66 6.6 H H66 6.6 H 6.66 2.586636 muomwn uncommon MHmoHuHOoocoHO< 6 O m m a .6660. $60. nHomo. O NOGOEmuoum mo Hm>0H Hmumu meEmm OHO mo coHuucsw HmUHuHouocmHUm so HGQEOHmmsm OGOEmuonm Mo uommmo one .Nm mHnt 128 Table 23. Feed consumption of female rats on Protamone treatmentl Level of Protamone2 0 .02% .04% .08% Young 15.9 15.5 17.2 16.9 Old 14.4 14.3 12.2 12.9 1Average feed consumption (grams/rat/day), during 3 week treatment period. 2% Protamone contained in feed. 129 Table 24. Body weight1 of young and old female rats of Protamone experiment Level of Protamone2 o .02% .04% .08% Initial3 253 249 261 253 + 9 + 9 1:10 .:11 Young Final 272 262 277 270 .112 + 8 ‘113 .113 Initial3 322 319 293 305 _:24 .:24 ,:14 .:15 Old 4 Final 314 309 275 278 .:22 .:25 .:14 .:14 lAverage weight of group in grams (i_S.E.M.). 2%Protamone in diet. 3Weight prior to start of Protamone treatment. 4 Weight after 3 weeks of Protamone treatment. 130 Discussion of Experiments V and VI Thyroid hormone influence on adrenocortical function in young adult and aged male and female rats.--The interac- tion of thyroidal and adrenocortical function in the rat has been reported by a number of investigators including Wallach and Reineke (1949), Kawai (1962), Steinetz and Beach (1963) and D'Angelo and Grodin (1964). The present study was designed to consider the effect of different levels Of thyroid hormone treatment on adrenocortical function in young and old rats. Protamone, a thyro-active protein, mixed in the normal rat diet at concentrations Of .02, .04 and .08%.of the diet, was used to induce hyperthyrodism in the experi- mental subjects. Protamone contains thyroxine (1%.by weight) of which approximately 33%.is absorbed when administered by the oral route (E.P. Reineke, personal communication). For an average feed consumption of 5 g/100 g body weight/day, rats fed the .02% diet absorbed approximately 3 pg thyroxine/ 100 g body weight/day. Reineke and Singh (1955) reported a daily thyroid secretion rate of 2.21—2.56 pg L-thyroxine/lOO g in adult female rats and 2.15 pg L-thyroxine/lOO g in adult male rats. According to this data (Reineke and Singh) for thyroid secretion rate, the .02% diet provided a near physiological level of exogenous thyroxine on a daily basis. This level of treatment was expected to induce only a mini- mal level of hyperthyroidism during the 3 week treatment period. The .04% diet supplied approximately 6 pg thyroxine 131 100 g body weight daily and was expected to induce a moder- ate degree Of hyperthyroidism. The .08% diet provided approximately 12 pg thyroxine/100 g body weight daily and was expected to induce a definite state of hyperthyroidism in the rat. The .08%.diet represents approximately four times the normal thyroxine secretion rate in the rat. This approaches the limit for a non—toxic dose in the rat (E.P. Reineke, personal communication). A plot of adrenocortical response versus log-dose thyroxine treatment (Figure 14) suggested that adrenocorti- cal response was related to the level of thyroid hormone treatment in young male rats. Following 3 weeks of thyroid hormone treatment, adrenocortical response to ether stress was greater than that Of non-treated controls in rats which had received the .O4%.and the .08% diet. Adrenocortical response of rats on the .08%.diet was also significantly different from that Of rats receiving the .04% diet. From these results it was concluded that thyroid hormone stimula- tion of the pituitary-adrenal axis occurs in the young male rat, provided that a sufficient level of hyperthyroidism was achieved. Similar Protamone levels in Old male rats did not alter the adrenocortical response to ether stress. Adreno- <:ortical response in this study showed no relationship to 'the level of Protamone used (Figure 17). EDifferences in .feed consumption (Table 16) between young and old rats were 132 not considered to be sufficient to influence the eXperimen— tal results. A similar study was conducted to consider the effect of different levels of thyroid hormone treatment on adreno- cortical function in young and Old female rats. In young female rats treatment with the .08%.Protamone diet for 3 'weeks increased adrenocortical response to ether stress above the response of non—treated controls. Adrenocortical response to ether stress in old female rats was not altered ‘by 3 weeks of Protamone treatment at any of the levels included in this study. Although plots of adrenocortical response versus log-dose Protamone (Figure 17) suggested that adrenocortical response to thyroid treatment was dose— .Ielated in both young and Old female rats, adrenocortical .Iesponses to different levels Of Protamone were signifi- <:antly different only in young female rats. Feed consump— tzion differences between young and old female rats were not (:cmsidered to be sufficient to account for the age-related diifferences in the eXperimental results (Table 16). The mechanism of thyroidal stimulation of the pitu— i.t:ary-adrenal axis in the young rat has not been investi- ‘Eiéated in the present study. .Evidence from other investi- SBNErtors suggests that thyroidal stimulation exerts its effect '5’1: several sites in the pituitary-adrenal axis. Kawai ( J6962) reported that pituitary ACTH depletion following 8 t:l:‘ess appeared greater in hyperthyroid than in control rats. 133 D'Angelo and Grodin (1964) concluded that adrenocortical stimulation in hyperthyroid rats is mediated primarily by way of ACTH secretion. Other changes such as increased steroidogenic capacity of the adrenal cortices (Freedland and.Mrad, 1969) and reduced corticosterone distribution volume (Steinetz and Beach, 1963) have also been reported in hyperthyroid rats. The results of this study suggested that the pitu- itary-adrenal axis of aged rats is less sensitive to thyroid hormone stimulation than that of young rats. This conclusion is in agreement with the previous hypothesis concerning age differences in corticoid feedback influence. Experiment VII: The Effects of Age and Chronic ACTH Stimulation on Biological Half-Life, Distribution Volume and Production Rate of Corticosterone in Female Rats Materials and Methods The first part Of this study was conducted to com- pare functional data on adrenocortical response exclusive of ,plasma corticosterone levels in young (5 month) and Old (27 tnonth) female rats. Biological half—life, distribution \rOlume and production rate of corticosterone in young and old female rats were estimated by the H3-corticosterone disappearance technique. In the second part of this study, biological half- 1ife, distribution volume and production rate Of corticoste— rone were estimated in depo-ACTH treated young adult (5 134 month) female rats and compared to these same parameters of adrenocortical function in non—treated control rats. The H3-corticosterone disappearance technique was used to estimate these parameters of adrenocortical function in both treated and control rats. Rats in the treated group were exposed to 3 weeks of depo-ACTH treatment (9 U ACTH daily) in 15% gelatin while control rats received the gelatin vehicle alone prior to determination of adrenocortical func- tional parameters. Adrenocortical responses to ether stress and acute ACTH stimulation were determined before the start and after 2 weeks of depo-ACTH treatment in both treated and control rats. Experimental results of both parts of this study were analyzed by Student's t—test. Results In the first part of this study, H3-corticosterone disappearance curves were obtained for a group of young (5 month) and a group of Old (27 month) female rats. Chroma— tographic isolation Of radioactive corticosterone showed ‘that 75% of the extractable radioactivity in the circula- tzion 15 minutes after H3-corticosterone injection was Imelated to cortisterone. Disappearance curves were calcu— 1.ated for the disappearance of total radioactivity from the pfilasma. Biological half—life, distribution volume and pro- lection rate of corticosterone, based on the disappearance Cllrve data, were compared between young and Old rats. The 135 single compartment model of steroid dynamics was used in calculating these parameters of adrenocortical function. The estimated biological half-life of corticosterone in young rats was less than that Of old rats (p < .05) but estimated corticosterone distribution volumes (Table 25) showed no age difference in this study. The calculated production rate of corticosterone (pg corticosterone/minute) in young rats was significantly greater (p < .01) than that of old rats. Plasma corticosterone levels during the period of blood sampling were significantly higher in young (p