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Effects of sequential acute stress exposure on stress-induced pituitary luteinizing hormone and prolactin secretion
Life Sciences, Vol. 41, pp. 1249-1255 Printed in the U.S.A.
Pergamon Journals
EFFECTS OF SEQUENTIAL ACUTE STRESS EXPOSURE ON STRESS-INDUCED PITUITARY LUTEINIZING HORMONEAND PROLACTIN SECRETION Karen P. B r i s k i and Paul W. Sylvester Department of Anatomical Sciences, State U n i v e r s i t y of New York at Buffalo, Buffalo, New York (KPB), and Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Memorial I n s t i t u t e , Buffalo, New York (PWS). (Received in final form July 2, 1987)
SUMMARY The present study was carried out to determine the e f f e c t s of repeti t i v e acute stress exposure on p i t u i t a r y secretion of both l u t e i n i z i n g hormone (LH) and p r o l a c t i n (PRL). Adult male rats were exposed to sequential episodes of acute novel environment stress separated by i n t e r v a l s of e i t h e r 60 or 120 minutes. Serial blood samples were obtained from animals before, during and a f t e r each stress episode via indwelling i n t r a - c a r d i a c cannulas. The imposition of i0 minute episodes of novel environment stress on an hourly basis eventually rendered the hypothalamic-hypophyseal LH axis r e f r a c t o r y to the s t i m u l a t o r y e f f e c t of stress. I f sequential stress was imposed at 120 minute i n t e r v a l s , LH release was s i g n i f i c a n t l y enhanced during each exposure. A d i f f e r e n t pattern of PRL release was observed during the same sequential stress schedule. After an i n i t i a l increase in hormone release in response to the f i r s t hourly stress episode, PRL l e v e l s were unaltered during the second and t h i r d hourly stress exposures. Thereafter, plasma PRL l e v e l s showed a trend toward a progressive increase in release during each successive episode, and were s i g n i f i c a n t l y elevated above preceding baseline l e v e l s during the fourth and f i f t h hourly stress exposures. In rats exposed to stress every two hours, a s i g n i f i c a n t increase in PRL l e v e l s occurred following the f i r s t , but not the second stress episode. Hormone release was again enhanced in response to the t h i r d exposure to novel environment. The present r e s u l t s demonstrate that the r e p e t i t i v e exposure to acute novel environment stress results in d i f f e r e n t i a l a l t e r a t i o n s in p i t u i t a r y LH and PRL secretion over time, and that the timing of repeated episodes is an important determinant of continued responsiveness to stress, p a r t i c u l a r l y with regard to LH release. These findings suggest that the LH and PRL hormonal responses to at least t h i s s p e c i f i c stressor are mediated by independent neural mechanisms. I t is recognized that exposure to stress r e s u l t s in widespread a l t e r a t i o n s in endocrine function, including changes in the secretion of several a n t e r i o r p i t u i t a r y hormones (1,2). Current evidence suggests that stress exerts a biphasic e f f e c t on p i t u i t a r y l u t e i n i z i n g hormone (LH) release in the i n t a c t r a t (I): plasma LH l e v e l s are temporarily, but s i g n i f i c a n t l y elevated a f t e r a l i m i t e d exposure to any one of several acute stress s t i m u l i (1,3-6), while chronic or prolonged imposition of stress results in the suppression of c i r c u l a t i n g LH below baseline values (7,8). S i m i l a r l y , p i t u i t a r y secretion of p r o l a c t i n (PRL) is enhanced during acute stress exposure (1,3,9-11), but i f stress is continued for several hours, hormone release is not maintained and plasma l e v e l s show a decline toward baseline l e v e l s ( l l ) . Although a l t e r a t i o n s 0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
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in LH and PRL release a f t e r single exposure to various acute stressors have been investigated e x t e n s i v e l y , the e f f e c t s of repeated exposure to a given acute stress stimulus on endocrine function is unknown. This information is of p a r t i c u l a r importance since in l i f e , stress does not usually occur as a single episode, but more often as m u l t i p l e events in a series, separated by varying i n t e r v a l s . Therefore, in the present study, we examined the e f f e c t of repeated exposure to novel environment stress on p i t u i t a r y secretion of LH and PRL during successive stress episodes. Sequential acute stress episodes were imposed at two d i f f e r e n t i n t e r v a l s in order to assess the impact of frequency of exposure on p i t u i t a r y hormone release. Methods Animals: Adult male Sprague Dawley rats (325-350g) were obtained from Charles River Breeding Laboratories and maintained at a controlled temperature (24°C ±I°C) under a 14 h l i g h t / l O h dark schedule f o r at least two weeks p r i o r to experimental use. Rats were housed 3-4 animals per cage, and received r a t chow and water a d - l i b i t u m . Experimental Desig_n: 48 hours p r i o r to experimentation, each r a t was s u r g i c a l l y implanted with an indwelling i n t r a c a r d i a c cannula manufactured from s i l a s t i c tubing, under anesthesia induced a subcutaneous ( s . c . ) i n j e c t i o n of ketamine/xylazine (4:1, 0.065 ml/lOOg b.w.; obtained from the Laboratory Animal F a c i l i t y , State U n i v e r s i t y of New York at Buffalo, Buffalo, NY). A f t e r surgery, animals received a 0 . i ml i.m. i n j e c t i o n of the a n t i b i o t i c , Combiotic (procaine p e n i c i l l i n G plus dihydrostreptomiacine s u l f a t e , 200,000 u n i t s / m l , Butler Pharmaceutical, Columbus, OH) and transferred to i n d i v i d u a l cages. Prior to stress exposure, rats were divided into two groups (n=7 per group) and a blood sample was collected via cannula from each animal into a heparinized syringe. Rats in group i were exposed to f i v e consecutive 10 minute episodes of novel environment stress, separated by 60 minute i n t e r v a l s . Animals in group 2 were exposed to three consecutive stress episodes, separated by 120 minute i n t e r v a l s . At the beginning of each stress episode, animals were removed from t h e i r o r i g i n a l quarters and transported on a cart to a second room w i t h i n the animal f a c i l i t y ; a f t e r i0 minutes, animals were returned to t h e i r home quarters. Blood samples were obtained from rats in group i at 10 and 60 minutes a f t e r the onset of each stress episode, while animals in group 2 were bled at 10, 30 and 120 minutes a f t e r each stress period. Throughout the experiment, blood samples were immediately centrifuged a f t e r each c o l l e c t i o n to separate plasma from red blood c e l l s , which were resuspended in s t e r i l e saline and returned to the respective donor animal. Plasma samples were stored frozen at -20°C u n t i l radioimmunossay f o r LH and PRL concentrations. Radioimmunoassay: Plasma LH and PRL l e v e l s were assayed under the d i r e c t i o n s and with reagents from k i t s generously provided by the National Hormone and P i t u i t a r y Program, NIADDK. Values were expressed in terms of the LH-RP-2 and the PRL-PR-2 standards, r e s p e c t i v e l y . Statistical: s t a t i s t i c a l determinations were performed by analysis of v ~ ~ followed by Duncan's M u l t i p l e Range Test. P
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Minutes FIG. 1 E f f e c t of r e p e t i t i v e novel environment stress i n i t i a t e d every 60 minutes (~ : ) or 120 minutes ( ~ - - ~ ) on c i r c u l a t i n g PRL l e v e l s . Data points represent mean plasma PRL concentrations ±S.E.M.obtained from 6-7 animals. The f i v e v e r t i c a l panels i n d i c a t e the onset and duration of f i v e consecutive hourly stress episodes; the f i r s t , t h i r d and f i f t h panels, which contain cross-hatching, i n d i c a t e stress exposures i n i t i a t e d every 120 minutes. * , P
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10,
1987
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Minutes FIG. 2 Figure 2: Effect of r e p e t i t i v e novel environment stress i n i t i a t e d every 60 minutes (t~---o) or 120 minutes ((D---~) on c i r c u l a t i n g LH l e v e l s . Data points represent mean plasma LH l e v e l s ±S.E.M. obtained from 6-7 animals. The f i v e v e r t i c a l panels indicate imposition of f i v e hourly stress episodes; panels containing cross-hatching indicate stress exposures i n i t i a t e d every 120 minutes. *, P
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rats stressed on an hourly basis. Plasma PR[ was again increased during the f i r s t stress exposure, but remained unaltered from baseline values during the second exposure, even though i t was i n i t i a t e d 120 minutes after the f i r s t episode. PRL release was again s i g n i f i c a n t l y elevated after the third stress exposure, i n i t i a t e d at 240 minutes after time zero. The disparity between patterns of LH and PRL release during sequential novel environment stress supports the view that these two hormonal responses may be mediated by independent mechanisms. While i n i t i a l baseline PRL values, as well as hormone levels 10 minutes after i n i t i a t i o n of the f i r s t stress exposure, were greater in group 2 than those detected in group 1, the r e l a t i v e increase in PRL in response to i n i t i a l stress was comparable in both groups: plasma PRL increased approximately 5 fold over baseline values in group i , while a 4 fold increase was observed in group 2. Although subsequent stress-induced PRL release at +120 minutes was comparable between groups, the last stress challenge administered to both groups again revealed a disparity in the magnitude of hormone released. Although these findings suggest that group differences may have had an impact on l a t e r PRL responses under the two stress paradigms, i t should be noted that in both groups, the increase in plasma PRL e l i c i t e d during the last stress episode was comparable to that observed following the f i r s t . The present experiments demonstrate that the timing or frequency of repeated stress exposure~ is an important determinant of continued p i t u i t a r y hormone release during successive stress episodes, particularly in the case of LH. While the mechanisms responsible for the loss of the LH response during hourly stress exposure are not clear, i t is possible that alterations in hypothalamic secretion of luteinizing hormone-releasing hormone (LHRH) may occur during specific conditions of r e p e t i t i v e stress. Recent studies indicate that acute stress exposure enhances the a c t i v i t y of specific hypothalamic neurotransmitter systems known to influence LH release, including adrenergic and noradrenergic neurons (14-17), which are known to exert a stimulatory influence on LHRH and subsequent LH release (18), and also serotonin neurons, which exert an inhibitory influence on LH (19). I t has recently been shown that these particular neurotransmitter responses can be modified during sequential stress exposure, depending upon the interval between stress stimuli (20). At present, the impact of repetitive novel environment stress on hypothalamic neurotransmitter a c t i v i t y is not known, but i t is possible that specific frequencies of exposure may a l t e r neuromodulatory input to LHRH neurons, with resultant alterations in LHRH release during stress. Alternatively, sequential stress may result in altered p i t u i t a r y responsiveness to stress-induced LHRH release. Stress is known to influence the release of several hormones, some of which may potentially influence subsequent p i t u i t a r y LH release. For example, two hormones whose release is stimulated by stress, ie. PRL and corticosterone, can act at the level of the p i t u i t a r y to depress LH-RH stimulated-LH release (21,22). While the present findings indicate the PRL levels are increased only during the i n i t i a l exposure to novel environment stress, the impact of this early elevation in hormone levels on LH release during hourly stress is not clear. At present, the effect of sequential novel environment stress on circulating corticosterone, and resultant influence on hypothalamic-hypophyseal LH responsiveness to successive stress remains to be determined. The present findings also i l l u s t r a t e the development of a refractory state by the hypothalamic-hypophyseal PRL axis to repeated novel environment stress, regardless of whether stress is imposed at intervals of 60 or 120 minutes. Current evidence suggests that stress-related increases in circulating PRL are mediated by neural mechanisms that involve a decrease in release of dopamine by tuberoinfundibular neurons into the hypothalamic-hypophyseal portal circulation (23). Central endogenous opioid peptides (EOPs) are apparently also involved since EOPs can depress hypothalamic dopamine release (25.26), while blockade of opiate receptors can reverse the stimulatory effect of stress on PRL release
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(27). Circulating PRL levels are increased following a single i n j e c t i o n of the exogenous opiate morphine, but the hypothalamic-hypophyseal PRL axis is subsequently characterized by diminished responsiveness to f u r t h e r opiate stimulation (28). This e f f e c t is apparently mediated in part by tuberoinfundibular neurons since the opiate-induced decrease in dopamine release is followed by a rebound-related increase in dopaminergic neuronal function. On the basis of these findings, i t is tempting to speculate whether stress-induced increases in endogenous opiate a c t i v i t y r e s u l t in a temporary desensitization of the hypothalamic-hypophyseal PRL axis to f u r t h e r stress stimulation. Enhanced corticosterone release r e s u l t i n g from the f i r s t exposure to stress may also play a r o l e ; this hormone has been implicated thus far in mechanisms r e s u l t i n g in the diminished PRL release in response to stress and opiate receptor stimulation (29) observed during prolonged imposition of stress. Nevertheless, the exact i n t e r r e l a t i o n s h i p between hypothalamic EOP and neurotransmitter a c t i v i t y and c i r c u l a t i n g levels of adrenal steroid hormones in the mediation of PRL responses to sequential stress is not c l e a r l y understood. In l i g h t of increasing evidence for the heterogeneity of molecular forms of PRL, and demonstrations of d i s p a r i t i e s bewteen bioassay and radioimmunoassay measurements of c i r c u l a t i n g PRL concentrations under a v a r i e t y of physiological and experimental conditions (30-33), i t is worth considering the p o s s i b i l i t y that r e p e t i t i v e stress stimulation may have an impact on the r a t i o of bioactive versus immunoreactive forms of PRL released during successive stress episodes. Although Owens et al. (33) reported that the r a t i o of bioactive to immunoreactive PRL is not s i g n i f i c a n t l y altered a f t e r a single acute exposure ether stress, the e f f e c t of repeated imposition of stress on this r a t i o is not known. The present results indicate that less immunoreactive PRL is secreted during middle time points of stress exposure, but do not preclude the p o s s i b i l i t y that other, non-immunoreactive molecular forms may continue to be secreted. Further work is necessary to ascertain whether the apparent development of a t r a n s i t o r y r e f r a c t o r y state by PRL release mechanisms can be explained upon the basis of d i f f e r e n t i a l release of s p e c i f i c forms of PRL With d i f f e r e n t assay a c t i v i t i e s . In conclusion, the present results demonstrate a clear dissociation between radioimmunoassayable p i t u i t a r y LH and PRL release during successive exposures to novel environment stress, and indicate that the frequency of repeated stress exposures is an important determinant of continued p i t u i t a r y responsiveness, p a r t i c u l a r l y with regard to LH release. These findings suggest that separate neuroendocrine mechanisms are involved in the mediation of the LH and the PRL hormonal responses to r e p e t i t i v e novel environment stress. ACKNOWLEDGEMENTS The authors wish to thank Dr. Harold Brody, Department of Anatomical Sciences, SUNY at Buffalo, for his encouragement and support of the present research project. REFERENCES 1. 2. 3. 4. 5. 6. 7.
L.KRULICH, E.HEFCO, P. ILLNER and C.B. READ, Neuroendocrinology 16 293-311 (1974). R. COLLU, Y.TACHE AND J. R, DUCHARME, J.Steroid Biochem. 11 989-1000 (1979). J.S.EUKER, J.MEITES and G.D. RIEGLE, Endocrinology 96 85-72 (1975). R.A.SIEGEL, J.WEIDENFELD, S.FELDMAN, N.CONFORTI and I.CHOWERS, Endocrinology 108 2302-2307 (1981). K.P.BRISKI, K. QUIGLEY and J.MEITES, Life Sci. 34 2485-2493 (1984). G.LESCOAT, D. LESCOAT and D. GARNIER, Can J. Pharm~acol. 62 1231-1233 (1984). G.E.GRAY, E.R.SMITH, J.R.L.EHRENKRANTZ and J.M. DAVIDSON, Neuroendocrinology 25 247-256 (1978).
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8. 9. 10. II. 12. 13. 14. 15.
16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
26. 27. 28. 29. 30. 31. 32. 33.
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Y.TACHE, P.DuRUISSEAU, J.R. DUCHARMEand R. COLLU, Neuroendocrinology 26 208-219 (1978). G.D. RIEGLE and J. MEITES, Proc. Soc. Exp. Biol. Med. 152 441-448 (1976). M.G.SUBRAMANIANand R.R. GALA, Proc. Soc. Exp. Biol. Med. 157 415-417 (1978). M. KAWAKAMI, T.HIGUCHI and M.MATSURRA, Neuroendocrinology 29 262-269 (1979). M. DOBRAKOVOVAand J.JURCOVICOVA, Exp. Clin. Endocrinol. 83 21-27 (1984). P.DuRUISSEAU, Y.TACHE, P.BRAZEAU and R. COLLU, Neuroendocr~nology 29 90-99 (1979). K.A. ROTH, I.M.MEFFORD and J.D. BARCHAS, Brain Res. 239 417-424 (1982). M.GOLDSTEIN, A.SAUTER, K. UETA and K. FUXE, Effect o f ~ t r e s s on central catecholamine levels. In: Catecholamines and stress: Recent advances. E.Usdin, R. Kvetnansky, and l.J.Kopin (Eds.). Elsevier/New York (1980), pp.47-52). E.A. STONE, J.Neurochem. 21 589-601 (1973). R. NAKAGAWA, M.TANAKA, Y.KOHNO, Y.NODA and N.NAGASAKI, Pharm. Biochem. Behav. 14 729-732 (1981). C.A.BARRACLOUGHand P.M.WISE, Endocrine Rev. 3 91-119 (1982). G.P.MEULLER, C.P.TWOHY, H.T. CHEN, J.P.ADVIS and J.MEITES, Life Sci. 18 715-724 (1976). E.B.DeSOUZA and G.R.VanLOON, Brain Res. 367 77-86 (1986). D.M.BALDWIN, Endocrinology 105 120-128 ( ~ 9 ) . A.GARCIA, L. HERBON, A. BARKIN, S.PAVAVASILIOU, J.C.MARSHALL, Endocrinology 117 954-959 (1985). K.T.DEMAREST, K.E.MOORE and G.D. RIEGLE, Neuroendocrinology 41 437-444 (1985). M. CRONIN, J.ROBERTS and R.WEINER, Endocrinology 103 302-309 (1978). L.FERLAND, K. FUXE, P.ENEROTH, J.A.GUSTAFFSON and P.KLETT, Eur. J. Pharmacol. 43 89-90 (1977). G.A.GUDELSKY--ANDJ.C. PORTER, Life Sci. 25 1697-1702 (1979). D.A.VanVUGT, J.F.BRUNI and J.MEITES, Life Sci. 22 85-90 (1978). G.A.GUDELSKY, E. PASSARO and H.Y.MELTZER, J. Pharmacol. Exp. Ther. 236 641-645 (1986). M.I.K. FEKETE, B.KANYICSKA, T.SZENTENDREI, A.SIMONYI AND E.STARK, J. Endocrinol. I01 169-172 (1984). M.WALLIS, M. DANNIELS and S.A.ELLIS, Biochem. J. 189 605-614 (1980). D.M.LAWSON and R.W.STEVENS, Life Sci. 27 1489-1494 (1980). D.M.LAWSON, N.SENSUI, D.H.HAISENLEDER a~d R.R.GALA, Life Sci. 31 3063-3070 (1982). R.E.OWENS, F.F. CASANUEVA and H.G. FRIESEN, Mol. Cell. Endocrinol. 39 131-140 (1985).
Pergamon Journals
EFFECTS OF SEQUENTIAL ACUTE STRESS EXPOSURE ON STRESS-INDUCED PITUITARY LUTEINIZING HORMONEAND PROLACTIN SECRETION Karen P. B r i s k i and Paul W. Sylvester Department of Anatomical Sciences, State U n i v e r s i t y of New York at Buffalo, Buffalo, New York (KPB), and Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Memorial I n s t i t u t e , Buffalo, New York (PWS). (Received in final form July 2, 1987)
SUMMARY The present study was carried out to determine the e f f e c t s of repeti t i v e acute stress exposure on p i t u i t a r y secretion of both l u t e i n i z i n g hormone (LH) and p r o l a c t i n (PRL). Adult male rats were exposed to sequential episodes of acute novel environment stress separated by i n t e r v a l s of e i t h e r 60 or 120 minutes. Serial blood samples were obtained from animals before, during and a f t e r each stress episode via indwelling i n t r a - c a r d i a c cannulas. The imposition of i0 minute episodes of novel environment stress on an hourly basis eventually rendered the hypothalamic-hypophyseal LH axis r e f r a c t o r y to the s t i m u l a t o r y e f f e c t of stress. I f sequential stress was imposed at 120 minute i n t e r v a l s , LH release was s i g n i f i c a n t l y enhanced during each exposure. A d i f f e r e n t pattern of PRL release was observed during the same sequential stress schedule. After an i n i t i a l increase in hormone release in response to the f i r s t hourly stress episode, PRL l e v e l s were unaltered during the second and t h i r d hourly stress exposures. Thereafter, plasma PRL l e v e l s showed a trend toward a progressive increase in release during each successive episode, and were s i g n i f i c a n t l y elevated above preceding baseline l e v e l s during the fourth and f i f t h hourly stress exposures. In rats exposed to stress every two hours, a s i g n i f i c a n t increase in PRL l e v e l s occurred following the f i r s t , but not the second stress episode. Hormone release was again enhanced in response to the t h i r d exposure to novel environment. The present r e s u l t s demonstrate that the r e p e t i t i v e exposure to acute novel environment stress results in d i f f e r e n t i a l a l t e r a t i o n s in p i t u i t a r y LH and PRL secretion over time, and that the timing of repeated episodes is an important determinant of continued responsiveness to stress, p a r t i c u l a r l y with regard to LH release. These findings suggest that the LH and PRL hormonal responses to at least t h i s s p e c i f i c stressor are mediated by independent neural mechanisms. I t is recognized that exposure to stress r e s u l t s in widespread a l t e r a t i o n s in endocrine function, including changes in the secretion of several a n t e r i o r p i t u i t a r y hormones (1,2). Current evidence suggests that stress exerts a biphasic e f f e c t on p i t u i t a r y l u t e i n i z i n g hormone (LH) release in the i n t a c t r a t (I): plasma LH l e v e l s are temporarily, but s i g n i f i c a n t l y elevated a f t e r a l i m i t e d exposure to any one of several acute stress s t i m u l i (1,3-6), while chronic or prolonged imposition of stress results in the suppression of c i r c u l a t i n g LH below baseline values (7,8). S i m i l a r l y , p i t u i t a r y secretion of p r o l a c t i n (PRL) is enhanced during acute stress exposure (1,3,9-11), but i f stress is continued for several hours, hormone release is not maintained and plasma l e v e l s show a decline toward baseline l e v e l s ( l l ) . Although a l t e r a t i o n s 0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
1250
Sequential
Stress
on LH and PRL Release
Vol.
41, No.
i0, 1987
in LH and PRL release a f t e r single exposure to various acute stressors have been investigated e x t e n s i v e l y , the e f f e c t s of repeated exposure to a given acute stress stimulus on endocrine function is unknown. This information is of p a r t i c u l a r importance since in l i f e , stress does not usually occur as a single episode, but more often as m u l t i p l e events in a series, separated by varying i n t e r v a l s . Therefore, in the present study, we examined the e f f e c t of repeated exposure to novel environment stress on p i t u i t a r y secretion of LH and PRL during successive stress episodes. Sequential acute stress episodes were imposed at two d i f f e r e n t i n t e r v a l s in order to assess the impact of frequency of exposure on p i t u i t a r y hormone release. Methods Animals: Adult male Sprague Dawley rats (325-350g) were obtained from Charles River Breeding Laboratories and maintained at a controlled temperature (24°C ±I°C) under a 14 h l i g h t / l O h dark schedule f o r at least two weeks p r i o r to experimental use. Rats were housed 3-4 animals per cage, and received r a t chow and water a d - l i b i t u m . Experimental Desig_n: 48 hours p r i o r to experimentation, each r a t was s u r g i c a l l y implanted with an indwelling i n t r a c a r d i a c cannula manufactured from s i l a s t i c tubing, under anesthesia induced a subcutaneous ( s . c . ) i n j e c t i o n of ketamine/xylazine (4:1, 0.065 ml/lOOg b.w.; obtained from the Laboratory Animal F a c i l i t y , State U n i v e r s i t y of New York at Buffalo, Buffalo, NY). A f t e r surgery, animals received a 0 . i ml i.m. i n j e c t i o n of the a n t i b i o t i c , Combiotic (procaine p e n i c i l l i n G plus dihydrostreptomiacine s u l f a t e , 200,000 u n i t s / m l , Butler Pharmaceutical, Columbus, OH) and transferred to i n d i v i d u a l cages. Prior to stress exposure, rats were divided into two groups (n=7 per group) and a blood sample was collected via cannula from each animal into a heparinized syringe. Rats in group i were exposed to f i v e consecutive 10 minute episodes of novel environment stress, separated by 60 minute i n t e r v a l s . Animals in group 2 were exposed to three consecutive stress episodes, separated by 120 minute i n t e r v a l s . At the beginning of each stress episode, animals were removed from t h e i r o r i g i n a l quarters and transported on a cart to a second room w i t h i n the animal f a c i l i t y ; a f t e r i0 minutes, animals were returned to t h e i r home quarters. Blood samples were obtained from rats in group i at 10 and 60 minutes a f t e r the onset of each stress episode, while animals in group 2 were bled at 10, 30 and 120 minutes a f t e r each stress period. Throughout the experiment, blood samples were immediately centrifuged a f t e r each c o l l e c t i o n to separate plasma from red blood c e l l s , which were resuspended in s t e r i l e saline and returned to the respective donor animal. Plasma samples were stored frozen at -20°C u n t i l radioimmunossay f o r LH and PRL concentrations. Radioimmunoassay: Plasma LH and PRL l e v e l s were assayed under the d i r e c t i o n s and with reagents from k i t s generously provided by the National Hormone and P i t u i t a r y Program, NIADDK. Values were expressed in terms of the LH-RP-2 and the PRL-PR-2 standards, r e s p e c t i v e l y . Statistical: s t a t i s t i c a l determinations were performed by analysis of v ~ ~ followed by Duncan's M u l t i p l e Range Test. P
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1251
20 _1
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Minutes FIG. 1 E f f e c t of r e p e t i t i v e novel environment stress i n i t i a t e d every 60 minutes (~ : ) or 120 minutes ( ~ - - ~ ) on c i r c u l a t i n g PRL l e v e l s . Data points represent mean plasma PRL concentrations ±S.E.M.obtained from 6-7 animals. The f i v e v e r t i c a l panels i n d i c a t e the onset and duration of f i v e consecutive hourly stress episodes; the f i r s t , t h i r d and f i f t h panels, which contain cross-hatching, i n d i c a t e stress exposures i n i t i a t e d every 120 minutes. * , P
1252
Sequential
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Minutes FIG. 2 Figure 2: Effect of r e p e t i t i v e novel environment stress i n i t i a t e d every 60 minutes (t~---o) or 120 minutes ((D---~) on c i r c u l a t i n g LH l e v e l s . Data points represent mean plasma LH l e v e l s ±S.E.M. obtained from 6-7 animals. The f i v e v e r t i c a l panels indicate imposition of f i v e hourly stress episodes; panels containing cross-hatching indicate stress exposures i n i t i a t e d every 120 minutes. *, P
Vol. 41, No. i0, 1987
Sequential Stress on LH and PRL Release
1253
rats stressed on an hourly basis. Plasma PR[ was again increased during the f i r s t stress exposure, but remained unaltered from baseline values during the second exposure, even though i t was i n i t i a t e d 120 minutes after the f i r s t episode. PRL release was again s i g n i f i c a n t l y elevated after the third stress exposure, i n i t i a t e d at 240 minutes after time zero. The disparity between patterns of LH and PRL release during sequential novel environment stress supports the view that these two hormonal responses may be mediated by independent mechanisms. While i n i t i a l baseline PRL values, as well as hormone levels 10 minutes after i n i t i a t i o n of the f i r s t stress exposure, were greater in group 2 than those detected in group 1, the r e l a t i v e increase in PRL in response to i n i t i a l stress was comparable in both groups: plasma PRL increased approximately 5 fold over baseline values in group i , while a 4 fold increase was observed in group 2. Although subsequent stress-induced PRL release at +120 minutes was comparable between groups, the last stress challenge administered to both groups again revealed a disparity in the magnitude of hormone released. Although these findings suggest that group differences may have had an impact on l a t e r PRL responses under the two stress paradigms, i t should be noted that in both groups, the increase in plasma PRL e l i c i t e d during the last stress episode was comparable to that observed following the f i r s t . The present experiments demonstrate that the timing or frequency of repeated stress exposure~ is an important determinant of continued p i t u i t a r y hormone release during successive stress episodes, particularly in the case of LH. While the mechanisms responsible for the loss of the LH response during hourly stress exposure are not clear, i t is possible that alterations in hypothalamic secretion of luteinizing hormone-releasing hormone (LHRH) may occur during specific conditions of r e p e t i t i v e stress. Recent studies indicate that acute stress exposure enhances the a c t i v i t y of specific hypothalamic neurotransmitter systems known to influence LH release, including adrenergic and noradrenergic neurons (14-17), which are known to exert a stimulatory influence on LHRH and subsequent LH release (18), and also serotonin neurons, which exert an inhibitory influence on LH (19). I t has recently been shown that these particular neurotransmitter responses can be modified during sequential stress exposure, depending upon the interval between stress stimuli (20). At present, the impact of repetitive novel environment stress on hypothalamic neurotransmitter a c t i v i t y is not known, but i t is possible that specific frequencies of exposure may a l t e r neuromodulatory input to LHRH neurons, with resultant alterations in LHRH release during stress. Alternatively, sequential stress may result in altered p i t u i t a r y responsiveness to stress-induced LHRH release. Stress is known to influence the release of several hormones, some of which may potentially influence subsequent p i t u i t a r y LH release. For example, two hormones whose release is stimulated by stress, ie. PRL and corticosterone, can act at the level of the p i t u i t a r y to depress LH-RH stimulated-LH release (21,22). While the present findings indicate the PRL levels are increased only during the i n i t i a l exposure to novel environment stress, the impact of this early elevation in hormone levels on LH release during hourly stress is not clear. At present, the effect of sequential novel environment stress on circulating corticosterone, and resultant influence on hypothalamic-hypophyseal LH responsiveness to successive stress remains to be determined. The present findings also i l l u s t r a t e the development of a refractory state by the hypothalamic-hypophyseal PRL axis to repeated novel environment stress, regardless of whether stress is imposed at intervals of 60 or 120 minutes. Current evidence suggests that stress-related increases in circulating PRL are mediated by neural mechanisms that involve a decrease in release of dopamine by tuberoinfundibular neurons into the hypothalamic-hypophyseal portal circulation (23). Central endogenous opioid peptides (EOPs) are apparently also involved since EOPs can depress hypothalamic dopamine release (25.26), while blockade of opiate receptors can reverse the stimulatory effect of stress on PRL release
1254
Sequential Stress on LH and PRL Release
Vol. 41, No. i0, 1987
(27). Circulating PRL levels are increased following a single i n j e c t i o n of the exogenous opiate morphine, but the hypothalamic-hypophyseal PRL axis is subsequently characterized by diminished responsiveness to f u r t h e r opiate stimulation (28). This e f f e c t is apparently mediated in part by tuberoinfundibular neurons since the opiate-induced decrease in dopamine release is followed by a rebound-related increase in dopaminergic neuronal function. On the basis of these findings, i t is tempting to speculate whether stress-induced increases in endogenous opiate a c t i v i t y r e s u l t in a temporary desensitization of the hypothalamic-hypophyseal PRL axis to f u r t h e r stress stimulation. Enhanced corticosterone release r e s u l t i n g from the f i r s t exposure to stress may also play a r o l e ; this hormone has been implicated thus far in mechanisms r e s u l t i n g in the diminished PRL release in response to stress and opiate receptor stimulation (29) observed during prolonged imposition of stress. Nevertheless, the exact i n t e r r e l a t i o n s h i p between hypothalamic EOP and neurotransmitter a c t i v i t y and c i r c u l a t i n g levels of adrenal steroid hormones in the mediation of PRL responses to sequential stress is not c l e a r l y understood. In l i g h t of increasing evidence for the heterogeneity of molecular forms of PRL, and demonstrations of d i s p a r i t i e s bewteen bioassay and radioimmunoassay measurements of c i r c u l a t i n g PRL concentrations under a v a r i e t y of physiological and experimental conditions (30-33), i t is worth considering the p o s s i b i l i t y that r e p e t i t i v e stress stimulation may have an impact on the r a t i o of bioactive versus immunoreactive forms of PRL released during successive stress episodes. Although Owens et al. (33) reported that the r a t i o of bioactive to immunoreactive PRL is not s i g n i f i c a n t l y altered a f t e r a single acute exposure ether stress, the e f f e c t of repeated imposition of stress on this r a t i o is not known. The present results indicate that less immunoreactive PRL is secreted during middle time points of stress exposure, but do not preclude the p o s s i b i l i t y that other, non-immunoreactive molecular forms may continue to be secreted. Further work is necessary to ascertain whether the apparent development of a t r a n s i t o r y r e f r a c t o r y state by PRL release mechanisms can be explained upon the basis of d i f f e r e n t i a l release of s p e c i f i c forms of PRL With d i f f e r e n t assay a c t i v i t i e s . In conclusion, the present results demonstrate a clear dissociation between radioimmunoassayable p i t u i t a r y LH and PRL release during successive exposures to novel environment stress, and indicate that the frequency of repeated stress exposures is an important determinant of continued p i t u i t a r y responsiveness, p a r t i c u l a r l y with regard to LH release. These findings suggest that separate neuroendocrine mechanisms are involved in the mediation of the LH and the PRL hormonal responses to r e p e t i t i v e novel environment stress. ACKNOWLEDGEMENTS The authors wish to thank Dr. Harold Brody, Department of Anatomical Sciences, SUNY at Buffalo, for his encouragement and support of the present research project. REFERENCES 1. 2. 3. 4. 5. 6. 7.
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