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Inhibition of rat corticotropin-releasing factor and adrenocorticotropin secretion by an osmotic stimulus
Brain Research, 523 (1990) 1-4 Elsevier BRES 15692
1
Research Reports
Inhibition of rat corticotropin-releasing factor and adrenocorticotropin secretion by an osmotic stimulus D.S. Jessop, H.S. Chowdrey and Stafford L. Lightman Medical Unit, WestminsterHospital, London (U.K.) (Accepted 16 January 1990)
Key words: CRF-41; Corticotropin-releasing hormone; Adrenocorticotropic hormone; Adrenocorticotropin; Hypertonic saline; Adrenalectomy; Hypothalamus
This study demonstrates that a chronic osmotic stimulus can influence the hypothalamo-pituitary axis by inhibiting the secretion of basal and adrenalectomy-elevated adrenocorticotropin (ACTH) from the anterior pituitary. In rats given 340 mM NaC1 instead of tap water to drink for 12 days, plasma concentrations of ACTH decreased to 105 + 27 pM (mean + S.E.M., n = 6) compared with control animals (182 + 13 pM). In adrenalectomised (ADX) rats given 150 mM NaCI to drink for 12 days, plasma ACTH concentrations were greatly increased (783 + 141 pM) but in ADX rats treated with 340 mM NaC1 for the same period, plasma ACTH was similar to controls (237 + 59 pM). The corticotropin-releasing factor (CRF-41) content of the median eminence (ME) was reduced in ADX rats on 150 mM NaC1 (854 + 78 fmol) and further reduced in ADX rats given 340 mM NaC1 (510 + 56 fmol) compared with control animals (1239 + 114 fmol), suggesting that the decrease in plasma ACTH concentrations in saline-treated animals is secondary to a decrease in the secretion of CRF-41 from the ME. These data are the first evidence for a central mechanism, independent of glucocorticoid feedback, through which a chronic osmotic stimulus can inhibit the activity of CRF-41 neurons.
INTRODUCTION Corticotropin-releasing factor (CRF-41) is a 41-residue p e p t i d e synthesised principally in the parvoceUular neurons of the h y p o t h a l a m i c paraventricular nucleus ( P V N ) 2-4's'24'25'33. CRF-41 is released from the m e d i a n e m i n e n c e ( M E ) in association with arginine vasopressin to stimulate the release of a d r e n o c o r t i c o t r o p i n ( A C T H ) from the corticotrophs of the anterior pituitary under basal conditions and also in response to stress 12. Increased release of CRF-41 and A C T H occurs in response to m a n y different stimuli such as a d r e n a l e c t o m y , ether, h e m o r r h a g e and h y p o g l y c a e m i a 1. In contrast, only two conditions have b e e n d e m o n s t r a t e d to inhibit the hypot h a l a m o - p i t u i t a r y stress response; firstly, it has been k n o w n for s o m e time that glucocorticoids decrease circulating concentrations of A C T H l S ; and secondly, it has recently b e e n shown that during the physiological state of lactation, the corticosterone response to stress is a t t e n u a t e d 2°. In addition, increased p l a s m a osmolality o v e r a p e r i o d of 12 days can decrease levels of CRF-41 m R N A in the parvocellular neurons of the P V N 19'36. This suggests that chronic osmotic stress, unlike any o t h e r k n o w n stress, m a y result in d e c r e a s e d p l a s m a A C T H . We
have e m p l o y e d specific r a d i o i m m u n o a s s a y s ( R I A ) to d e t e r m i n e w h e t h e r the decrease in CRF-41 synthesis previously r e p o r t e d following osmotic stress is accompanied by changes in the CRF-41 content of the M E , and in p l a s m a A C T H concentrations. MATERIALS AND METHODS Animal experimentation was undertaken in accordance with the Animals (Scientific Procedures) Act, 1986 (U.K.). Male SpragueDawley rats weighing 200-250 g were given either tap water (control) or 340 mM NaC1 to drink. A separate group of rats was given dexamethasone (2.55 /~M, Sigma Chemical Co., Poole, Dorset, U.K.) dissolved in drinking water. Bilateral adrenalectomies (ADX) were performed in another group of rats anaesthetised with sodium pentobarbitone and the animals were placed on either 150 mM NaC1 (for electrolyte replacement) or 340 mM NaCI. Control animals subjected to sham operation were given 150 mM NaCI. After 12 days of treatment, the animals were decapitated between 09.00 and 11.00 h and the brains were rapidly removed and frozen on dry ice. Separate tissue blocks containing the PVN (1 mm3 either side of the third ventricle) and the ME were carefully dissected away from the rest of the hypothalamus and stored at -80 °C. The methods of CRF-41 tissue extraction and RIA have been fully described9'16. Trunk blood was collected into heparinised tubes and plasma was separated by centrifugation, frozen on dry ice and stored at -20 *C. The method for extracting ACTH from rat plasma by Sep-pak C18 cartridges and subsequent measurement by RIA has been fully described16. Plasma arginine vasopressin (AVP) was measured by RIA 1°.
Correspondence: D.S. Jessop, Medical Unit, Westminster Hospital, 17 Page St., London SWlP 2AP, U.K. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
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CONTROL
DEX
SHAM 150raM NaCI
340mM NaCI
ADX 150mM NaCl
ADX 340raM NaCl
Fig. 1. Plasma concentrations of ACTH in rats given either tap water to drink (control), 340 mM NaCI in drinking water or dexamethasone (DEX). Values are means + S.E.M. (n = group number). *P < 0.05 compared with control group (Duncan's one-way ANOVA). The decrease in plasma ACTH following 340 mM NaCI has been observed in 4 separate experiments.
Fig. 3. Total corticotropin-releasing factor (CRF-41) content in rat median eminence (ME) tissue extracts from adrenaleetomised (ADX) rats on either 150 or 340 mM NaCI, and from sham-operated rats on 150 mM NaCI. Values are means + S.E,M. (n = group number). *P < 0.05 compared with control group; **P < 0.05 compared with ADX/150 mM NaCI group (Duncan's one-way ANOVA).
RESULTS
increase in p l a s m a A V P concentrations from 3.6 + 0.4 to 22.7 + 3.7 p M ( P < 0.05, m e a n + S . E . M . , n = 6). Saline t r e a t m e n t decreased circulating A C T H concentrations from 182 + 13 to 105 + 27 p M , a decrease of similar magnitude to that following d e x a m e t h a s o n e (Fig. 1). Plasma A C T H concentrations in A D X rats given 150 m M NaCI for 12 days were greatly elevated, but t r e a t m e n t with 340 m M NaCI for 12 days following A D X r e d u c e d plasma A C T H to levels found in s h a m - o p e r a t e d rats (Fig.
R e p l a c e m e n t of drinking water with 340 m M NaCI for 12 days resulted in increased p l a s m a osmolality and an
*
1000
2).
800
N & [..., o<
The total a m o u n t of CRF-41 in the M E was reduced after A D X and was further r e d u c e d following t r e a t m e n t of A D X rats with 340 m M NaCI (Fig. 3). In extracts of tissue containing the PVN, CRF-41 content was unchanged in A D X rats on 150 m M NaCI (154 + 12 fmol, m e a n + S . E . M . ) or 340 m M NaC! (197 + 14 fmol) c o m p a r e d with s h a m - o p e r a t e d rats on 150 m M NaCI (152 + 14 fmol).
600
400 -
200 !
DISCUSSION
~,",,~j,)7~P'/////~ SHAM 150mM NaCI
ADX 150raM NaCI
ADX 340mM NaCI
Fig. 2. Plasma concentrations of ACTH in rats 12 days after bilateral adrenalectomy (ADX) or sham operation. ADX rats were placed on either 150 or 340 mM NaCI in drinking water while sham controls were given 150 mM NaCI. Values are means + S.E.M. (n = group number which represents the pooled data from 2 separate experiments). *P < 0.05 compared with control group (Duncan's one-way ANOVA).
The response of A V P to increased p l a s m a osmolality is w e l l - d o c u m e n t e d 17'31'35 hut t h e r e are few reports of plasma A C T H m e a s u r e m e n t s following osmotic stimulus. Increases have been r e p o r t e d in the horse 14 and in m a n 3° while no changes have been o b s e r v e d in sheep 29 or dogs 28. All these studies were u n d e r t a k e n within hours of an osmotic stimulus. W e have d e m o n s t r a t e d that, in response to chronically elevated p l a s m a osmolality, cir-
culating concentrations of A C T H are decreased through a mechanism which can operate either in the presence or absence of glucocorticoids. Decreased CRF-41 m R N A in the parvocellular neurons of the PVN following the osmotic stimulus 19'36 indicates that osmotic control over the release of A C T H is effected at the hypothalamic level and not directly at the anterior pituitary. Decreased synthesis of CRF-41 would also explain why the ME content of CRF-41 in A D X rats on 340 mM NaCI was lower than that observed after isotonic saline. A decrease in ME content of CRF-41 in A D X rats given isotonic saline has been previously reported 13'15, which is consistent with increased secretion of CRF-41 into hypophysial portal blood 11'26. With the single exception of lactation during which the response to stress is attenuated 2°, increased plasma osmolality is the only physiological condition which has been found to inhibit the hypothalamo-pituitary secretion of CRF-41 and ACTH. It is also the only known condition which exerts opposite effects upon the secretion of A C T H and AVP from the adenohypophysis and neurohypophysis respectively. It is clearly of great interest to identify the mechanism whereby increased plasma osmolality results in decreased plasma ACTH. Diminished parvocellular CRF-41 m R N A 19'36 and ME content of CRF-41 following saline treatment strongly suggest action at the hypothalamic level. However, the factor(s) integrating the hypothalamic responses to an osmotic stimulus remain unidentified. Expression of AVP m R N A in the magnocellular neurons of the PVN, in contrast REFERENCES 1 Antoni, F.A., Hypothalamic control of adrenocorticotropin secretion: advances since the discovery of 41-residue corticotropin-releasing factor, Endocr. Rev., 7 (1986) 351-379. 2 Antoni, EA., Palkovits, M., Makara, G.B., Linton, E.A., Lowry, P.J. and Kiss, J.Z., Immunoreactive corticotropin releasing hormone (CRF) in the hypothalamo-infundibular tract, Neuroendocrinology, 36 (1983) 415-423. 3 Bloom, EE., Battenberg, E.L.E, Rivier, J. and Vale, W., Corticotropin releasing factor (CRF): immunoreactive neurones and fibers in rat hypothalamus, Regul. Pept., 4 (1982) 43-48. 4 Bugnon, C., Fellman, D., Gouget, A., Bresson, J., Clavequin, M.C., Hadjiyiassemis, M. and Cardot, J., Corticoliberin neurons; cytophysiology, phylogeny and ontogeny, J. Steroid Biochem., 20 (1984) 183-188. 5 Burbach, J.P.H., De Hoop, M.J., Schmale, H., Richter, D., De Kloet, E.R., ten Haaf, J.A. and De Wied, D., Differential responses to osmotic stress of vasopressin-neurophysin mRNA in hypothalamic nuclei, Neuroendocrinology, 39 (1984) 582-584. 6 Burbach, J.P.H., Van Tol, H.H.M., Bakkus, M.H.C., Schmale, H. and Ivell, R., Quantitation of vasopressin mRNA and oxytocin mRNA in hypothalamic nuclei by solution hybridization assays, J. Neurochem., 47 (1986) 1814-1821. 7 Chowdrey, H.S., Jessop, D.S. and Lightman, S.L., Substance P stimulates arginine vasopressin and inhibits adrenocorticotropin release in vivo, Neuroendocrinology, in press. 8 Cummings, S.R., EIde, R., Ells, J. and Lindall, A., Corticotropin-releasing immunoreactivity is widely distributed within
with that of CRF-41 in the parvoceUular neurons, increases in response to osmotic stimulus 5'6'19'22'23'32'34'37. Therefore, increased plasma osmolality has opposite effects upon the synthesis as well as the secretion of CRF-41 and AVE Since both AVP and CRF-41 are synthesised in adjacent and overlapping divisions of the PVN, it is possible that the same neurotransmitter may be involved in both responses. With regard to this, we would like to draw attention to our recent observation that intracerebroventricular injection of the peptide substance P (SP) in rats resulted in an increase in plasma AVP and a decrease in plasma A C T H 7, the same combination of responses observed in saline-treated rats. Since SP is present in large amounts in the hypothalamus 21'27, this peptide has both the neuroanatomical and pharmacological potential to centrally mediate changes in circulating AVP and A C T H . We are currently investigating whether SP is involved in the responses of the hypothalamo-pituitary axis to osmotic stimulus. In conclusion, our results reveal a hitherto unknown mechanism controlling plasma A C T H concentrations in response to changes in plasma osmolality. Decreased levels of CRF-41 in the ME, and decreased CRF-41 m R N A in the parvocellular neurons of the hypothalamus following saline treatment, indicate a central site of control. Acknowledgements. We thank Mr. H. Patel for the AVP RIA data. We are also grateful for financial support from the Joint Research Committee of the Westminster Hospital group. ACTH antiserum was kindly provided by Dr. G. Court (Wellcome plc).
the central nervous system of the rat: an immunohistochemical study, J. Neurosci., 3 (1983) 1355-1368. 9 Cunnah, D., Jessop, D.S., Besser, G.M. and Rees, L.H., Measurement of circulating corticotrophin-releasing factor in man, J. Endocrinol., 113 (1987) 123-131. 10 Eckland, D.J.A., Todd, K. and Lightman, S.L., Immunoreactive vasopressin and oxytocin in hypothalamo-hypophysial portal blood of the Brattleboro and Long-Evans rat: effect of adrenalectomy and dexamethasone, J. Endocrinol., 117 (1988) 27-34. 11 Fink, G., Robinson, I.C.A.E and Tannahill, L.A., Effects of adrenalectomy and glucocorticoids on the peptides CRF-41, AVP and oxytocin in rat hypophysial portal blood, J. Physiol., 401 (1988) 329-345. 12 Gillies, G.E. and Lowry, P.J., Adrenal function. In S.L. Lightman and B.J. Everitt (Eds.), Neuroendocrinology, Blackwell Scientific Publications, London, 1986, pp. 360-388. 13 Hisano, S., Tsuruo, Y., Katoh, S., Daikoku, S., Yanaihara, N. and Shibasaki, T., Intragranular colocalization of arginine vasopressin and methionine-enkephalin-octapeptide in CRFaxons in the rat median eminence, Cell Tissue Res., 249 (1987) 497-507. 14 Irvine, C.H.G., Alexander, S.L. and Donald, R.A., Effect of an osmotic stimulus on the secretion of arginine vasopressin and adrenocorticotropin in the horse, Endocrinology, 124 (1989) 3102-3108. 15 Jeandel, L., Van Dorsselaer, A., Lutz-Bucher, B. and Koch, B., Characterization and modulation of corticotropin-releasing factor in the neurointermediate pituitary gland, Neuroendocrinology, 45 (1987) 146-151.
4 16 Jessop, D.S., Eckland, D.J.A., Todd, K. and Lightman, S.L., Osmotic regulation of hypothalamo-neurointermediate lobe corticotrophin releasing factor-41 in the rat, J. Endocrinol., 120 (1989) 119-124. 17 Jones, C.W. and Pickering, B.T., Comparison of the effects of water deprivation and sodium chloride imbibation on the hormone content of the neurohypophysis of the rat, J. Physiol., 203 (1969) 449-458. 18 Keller-Wood, M.E. and Dallman, M.F., Corticosteroid inhibition of ACTH secretion, Endocr. Rev., 5 (1984) 1-24. 19 Lightman, S.L. and Young III, W.S., Vasopressin, oxytocin, dynorphin, enkephalin and corticotrophin-releasing factor mRNA stimulation in the rat, J. Physiol., 394 (1987) 23-39. 20 Lightman, S.L. and Young III, W.S., Lactation inhibits stressmediated secretion of corticosterone and oxytocin and hypothalamic accumulation of corticotropin-releasing factor and enkephalin messenger ribonucleic acids, Endocrinology, 124 (1989) 2358-2364. 21 Ljungdahl, A., Hokfelt, T. and Nilsson, G., Distribution of substance P-like immunoreactivity in the central nervous system of the rat. 1. Cell bodies and nerve terminals, Neuroscience, 3 (1978) 861-943. 22 McCabe, J.T., Morrell, J.L., Richter, D. and Pfaff, D.W., Localisation of neuroendocrinologically relevant RNA in brain by in situ hybridisation. In W.F. Ganong and L. Martini (Eds.), Frontiers in Neuroendocrinology, Vol. 9, Raven, New York, 1986, pp. 149-167. 23 Majzoub, J.A., Rich, A., van Boom, J. and Habener, J.E, Vasopressin and oxytocin mRNA regulation in the rat assessed by hybridisation with synthetic oligonucleotides, J. Biol. Chem., 258 (1983) 14061-14064. 24 Merchenthaler, I., Vigh, S., Petrusz, P. and Schally, A.V., The paraventricular-infundibular corticotropin releasing factor (CRF)-pathway as revealed by immunocytochemistry in longterm hypophysectomised or adrenalectomised rats, Regul. Pept., 5 (1983) 295-305. 25 Olschowka, J.A., O'Donohue, T.L., Mueller, G.P. and Jacobowitz, D.M., The distribution of corticotropin releasing factorlike immunoreactive neurons in rat brain, Peptides, 3 (1982) 995-1015. 26 Plotsky, P.M. and Sawchencko, P.E., Hypophyseal portal plasma levels, median eminence content, and immunohistochemical staining of corticotropin releasing factor, arginine vasopressin, and oxytocin after pharmacological adrenalectomy,
Endocrinology, 120 (1987) 1361-1369. 27 Powell, D., Leeman, S., Tregear, G.W., Niall, H.D. and Potts, J.T., Radioimmunoassay for substance P, Nature New Biol., 241 (1973) 252-254. 28 Raft, H., Skelton, M.M., Merrill, D.C. and Cowley Jr., A.W., Vasopressin responses to corticotropin releasing factor and hyperosmolahty in conscious dogs, Am. J. Physiol., 251 (1986) R1235-1239. 29 Redekopp, C., Livesey, J.H., Sadler, W. and Donald, R.A., The physiological significance of arginine vasopressin in potentiating the response to corticotropin-releasing factor in sheep, J. Endocrinol., 108 (1986) 309-312. 30 Rittmaster, R.S., Cutler Jr., J.B., Gold, P.W., Brandon, D.D., Tomai, T., Loriaux, D.L. and Chrousos, G.P., The relationship of saline-induced changes in vasopressin secretion to basal and corticotropin-releasing hormone-stimulated adrenocorticotropin and cortisol secretion in man, J. Clin. EndocrinoL Metab., 64 (1987) 371-376. 31 Robertson, G.L. and Athar, S., The interaction of blood osmolality and blood volume in regulating plasma vasopressin in man, J. Clin. Endocrinol. Metab., 42 (1976) 613-620. 32 Sherman, T.G., McKelvy, J.E and Watson, S.J., Vasopressin mRNA regulation in individual hypothalamic nuclei: a Northern and in situ hybridisation analysis, J. Neurosci., 6 (1986) 1685-1694. 33 Swanson, L.W., Sawchencko, P.E., Rivier, J. and Vale, W.W., Organization of ovine corticotropin-releasing immunoreactive cells and fibers in the rat brain: an immunohistochemical study, Neuroendocrinology, 58 (1983) 165-186. 34 Uhl, G.R., Zingg, H.H. and Habener, J.F., Vasopressin mRNA in situ hybridisation: localisation and regulation studied with oligonucleotide cDNA probes in normal and Brattleboro rat hypothalamus, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 55555559. 35 Verney, E.B., The antidiuretic hormone and the factors which determine its release, Proc. Roy. Soc., B135 (1947) 25-106. 36 Young III, W.S., Corticotropin-releasing factor mRNA in the hypothalamus is affected differentially by drinking saline and by dehydration, FEBS Len., 208 (1986) 158-162. 37 Zingg, H.H., Lefebre, D. and Almazan, G., Regulation of vasopressin gene expression in rat hypothalamus neurons. Response to osmotic stimulation, J. Biol. Chem., 261 (1986) 12956-12959.
1
Research Reports
Inhibition of rat corticotropin-releasing factor and adrenocorticotropin secretion by an osmotic stimulus D.S. Jessop, H.S. Chowdrey and Stafford L. Lightman Medical Unit, WestminsterHospital, London (U.K.) (Accepted 16 January 1990)
Key words: CRF-41; Corticotropin-releasing hormone; Adrenocorticotropic hormone; Adrenocorticotropin; Hypertonic saline; Adrenalectomy; Hypothalamus
This study demonstrates that a chronic osmotic stimulus can influence the hypothalamo-pituitary axis by inhibiting the secretion of basal and adrenalectomy-elevated adrenocorticotropin (ACTH) from the anterior pituitary. In rats given 340 mM NaC1 instead of tap water to drink for 12 days, plasma concentrations of ACTH decreased to 105 + 27 pM (mean + S.E.M., n = 6) compared with control animals (182 + 13 pM). In adrenalectomised (ADX) rats given 150 mM NaCI to drink for 12 days, plasma ACTH concentrations were greatly increased (783 + 141 pM) but in ADX rats treated with 340 mM NaC1 for the same period, plasma ACTH was similar to controls (237 + 59 pM). The corticotropin-releasing factor (CRF-41) content of the median eminence (ME) was reduced in ADX rats on 150 mM NaC1 (854 + 78 fmol) and further reduced in ADX rats given 340 mM NaC1 (510 + 56 fmol) compared with control animals (1239 + 114 fmol), suggesting that the decrease in plasma ACTH concentrations in saline-treated animals is secondary to a decrease in the secretion of CRF-41 from the ME. These data are the first evidence for a central mechanism, independent of glucocorticoid feedback, through which a chronic osmotic stimulus can inhibit the activity of CRF-41 neurons.
INTRODUCTION Corticotropin-releasing factor (CRF-41) is a 41-residue p e p t i d e synthesised principally in the parvoceUular neurons of the h y p o t h a l a m i c paraventricular nucleus ( P V N ) 2-4's'24'25'33. CRF-41 is released from the m e d i a n e m i n e n c e ( M E ) in association with arginine vasopressin to stimulate the release of a d r e n o c o r t i c o t r o p i n ( A C T H ) from the corticotrophs of the anterior pituitary under basal conditions and also in response to stress 12. Increased release of CRF-41 and A C T H occurs in response to m a n y different stimuli such as a d r e n a l e c t o m y , ether, h e m o r r h a g e and h y p o g l y c a e m i a 1. In contrast, only two conditions have b e e n d e m o n s t r a t e d to inhibit the hypot h a l a m o - p i t u i t a r y stress response; firstly, it has been k n o w n for s o m e time that glucocorticoids decrease circulating concentrations of A C T H l S ; and secondly, it has recently b e e n shown that during the physiological state of lactation, the corticosterone response to stress is a t t e n u a t e d 2°. In addition, increased p l a s m a osmolality o v e r a p e r i o d of 12 days can decrease levels of CRF-41 m R N A in the parvocellular neurons of the P V N 19'36. This suggests that chronic osmotic stress, unlike any o t h e r k n o w n stress, m a y result in d e c r e a s e d p l a s m a A C T H . We
have e m p l o y e d specific r a d i o i m m u n o a s s a y s ( R I A ) to d e t e r m i n e w h e t h e r the decrease in CRF-41 synthesis previously r e p o r t e d following osmotic stress is accompanied by changes in the CRF-41 content of the M E , and in p l a s m a A C T H concentrations. MATERIALS AND METHODS Animal experimentation was undertaken in accordance with the Animals (Scientific Procedures) Act, 1986 (U.K.). Male SpragueDawley rats weighing 200-250 g were given either tap water (control) or 340 mM NaC1 to drink. A separate group of rats was given dexamethasone (2.55 /~M, Sigma Chemical Co., Poole, Dorset, U.K.) dissolved in drinking water. Bilateral adrenalectomies (ADX) were performed in another group of rats anaesthetised with sodium pentobarbitone and the animals were placed on either 150 mM NaC1 (for electrolyte replacement) or 340 mM NaCI. Control animals subjected to sham operation were given 150 mM NaCI. After 12 days of treatment, the animals were decapitated between 09.00 and 11.00 h and the brains were rapidly removed and frozen on dry ice. Separate tissue blocks containing the PVN (1 mm3 either side of the third ventricle) and the ME were carefully dissected away from the rest of the hypothalamus and stored at -80 °C. The methods of CRF-41 tissue extraction and RIA have been fully described9'16. Trunk blood was collected into heparinised tubes and plasma was separated by centrifugation, frozen on dry ice and stored at -20 *C. The method for extracting ACTH from rat plasma by Sep-pak C18 cartridges and subsequent measurement by RIA has been fully described16. Plasma arginine vasopressin (AVP) was measured by RIA 1°.
Correspondence: D.S. Jessop, Medical Unit, Westminster Hospital, 17 Page St., London SWlP 2AP, U.K. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
200
2000
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1500
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100 -
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o <
(_)
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500"
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CONTROL
DEX
SHAM 150raM NaCI
340mM NaCI
ADX 150mM NaCl
ADX 340raM NaCl
Fig. 1. Plasma concentrations of ACTH in rats given either tap water to drink (control), 340 mM NaCI in drinking water or dexamethasone (DEX). Values are means + S.E.M. (n = group number). *P < 0.05 compared with control group (Duncan's one-way ANOVA). The decrease in plasma ACTH following 340 mM NaCI has been observed in 4 separate experiments.
Fig. 3. Total corticotropin-releasing factor (CRF-41) content in rat median eminence (ME) tissue extracts from adrenaleetomised (ADX) rats on either 150 or 340 mM NaCI, and from sham-operated rats on 150 mM NaCI. Values are means + S.E,M. (n = group number). *P < 0.05 compared with control group; **P < 0.05 compared with ADX/150 mM NaCI group (Duncan's one-way ANOVA).
RESULTS
increase in p l a s m a A V P concentrations from 3.6 + 0.4 to 22.7 + 3.7 p M ( P < 0.05, m e a n + S . E . M . , n = 6). Saline t r e a t m e n t decreased circulating A C T H concentrations from 182 + 13 to 105 + 27 p M , a decrease of similar magnitude to that following d e x a m e t h a s o n e (Fig. 1). Plasma A C T H concentrations in A D X rats given 150 m M NaCI for 12 days were greatly elevated, but t r e a t m e n t with 340 m M NaCI for 12 days following A D X r e d u c e d plasma A C T H to levels found in s h a m - o p e r a t e d rats (Fig.
R e p l a c e m e n t of drinking water with 340 m M NaCI for 12 days resulted in increased p l a s m a osmolality and an
*
1000
2).
800
N & [..., o<
The total a m o u n t of CRF-41 in the M E was reduced after A D X and was further r e d u c e d following t r e a t m e n t of A D X rats with 340 m M NaCI (Fig. 3). In extracts of tissue containing the PVN, CRF-41 content was unchanged in A D X rats on 150 m M NaCI (154 + 12 fmol, m e a n + S . E . M . ) or 340 m M NaC! (197 + 14 fmol) c o m p a r e d with s h a m - o p e r a t e d rats on 150 m M NaCI (152 + 14 fmol).
600
400 -
200 !
DISCUSSION
~,",,~j,)7~P'/////~ SHAM 150mM NaCI
ADX 150raM NaCI
ADX 340mM NaCI
Fig. 2. Plasma concentrations of ACTH in rats 12 days after bilateral adrenalectomy (ADX) or sham operation. ADX rats were placed on either 150 or 340 mM NaCI in drinking water while sham controls were given 150 mM NaCI. Values are means + S.E.M. (n = group number which represents the pooled data from 2 separate experiments). *P < 0.05 compared with control group (Duncan's one-way ANOVA).
The response of A V P to increased p l a s m a osmolality is w e l l - d o c u m e n t e d 17'31'35 hut t h e r e are few reports of plasma A C T H m e a s u r e m e n t s following osmotic stimulus. Increases have been r e p o r t e d in the horse 14 and in m a n 3° while no changes have been o b s e r v e d in sheep 29 or dogs 28. All these studies were u n d e r t a k e n within hours of an osmotic stimulus. W e have d e m o n s t r a t e d that, in response to chronically elevated p l a s m a osmolality, cir-
culating concentrations of A C T H are decreased through a mechanism which can operate either in the presence or absence of glucocorticoids. Decreased CRF-41 m R N A in the parvocellular neurons of the PVN following the osmotic stimulus 19'36 indicates that osmotic control over the release of A C T H is effected at the hypothalamic level and not directly at the anterior pituitary. Decreased synthesis of CRF-41 would also explain why the ME content of CRF-41 in A D X rats on 340 mM NaCI was lower than that observed after isotonic saline. A decrease in ME content of CRF-41 in A D X rats given isotonic saline has been previously reported 13'15, which is consistent with increased secretion of CRF-41 into hypophysial portal blood 11'26. With the single exception of lactation during which the response to stress is attenuated 2°, increased plasma osmolality is the only physiological condition which has been found to inhibit the hypothalamo-pituitary secretion of CRF-41 and ACTH. It is also the only known condition which exerts opposite effects upon the secretion of A C T H and AVP from the adenohypophysis and neurohypophysis respectively. It is clearly of great interest to identify the mechanism whereby increased plasma osmolality results in decreased plasma ACTH. Diminished parvocellular CRF-41 m R N A 19'36 and ME content of CRF-41 following saline treatment strongly suggest action at the hypothalamic level. However, the factor(s) integrating the hypothalamic responses to an osmotic stimulus remain unidentified. Expression of AVP m R N A in the magnocellular neurons of the PVN, in contrast REFERENCES 1 Antoni, F.A., Hypothalamic control of adrenocorticotropin secretion: advances since the discovery of 41-residue corticotropin-releasing factor, Endocr. Rev., 7 (1986) 351-379. 2 Antoni, EA., Palkovits, M., Makara, G.B., Linton, E.A., Lowry, P.J. and Kiss, J.Z., Immunoreactive corticotropin releasing hormone (CRF) in the hypothalamo-infundibular tract, Neuroendocrinology, 36 (1983) 415-423. 3 Bloom, EE., Battenberg, E.L.E, Rivier, J. and Vale, W., Corticotropin releasing factor (CRF): immunoreactive neurones and fibers in rat hypothalamus, Regul. Pept., 4 (1982) 43-48. 4 Bugnon, C., Fellman, D., Gouget, A., Bresson, J., Clavequin, M.C., Hadjiyiassemis, M. and Cardot, J., Corticoliberin neurons; cytophysiology, phylogeny and ontogeny, J. Steroid Biochem., 20 (1984) 183-188. 5 Burbach, J.P.H., De Hoop, M.J., Schmale, H., Richter, D., De Kloet, E.R., ten Haaf, J.A. and De Wied, D., Differential responses to osmotic stress of vasopressin-neurophysin mRNA in hypothalamic nuclei, Neuroendocrinology, 39 (1984) 582-584. 6 Burbach, J.P.H., Van Tol, H.H.M., Bakkus, M.H.C., Schmale, H. and Ivell, R., Quantitation of vasopressin mRNA and oxytocin mRNA in hypothalamic nuclei by solution hybridization assays, J. Neurochem., 47 (1986) 1814-1821. 7 Chowdrey, H.S., Jessop, D.S. and Lightman, S.L., Substance P stimulates arginine vasopressin and inhibits adrenocorticotropin release in vivo, Neuroendocrinology, in press. 8 Cummings, S.R., EIde, R., Ells, J. and Lindall, A., Corticotropin-releasing immunoreactivity is widely distributed within
with that of CRF-41 in the parvoceUular neurons, increases in response to osmotic stimulus 5'6'19'22'23'32'34'37. Therefore, increased plasma osmolality has opposite effects upon the synthesis as well as the secretion of CRF-41 and AVE Since both AVP and CRF-41 are synthesised in adjacent and overlapping divisions of the PVN, it is possible that the same neurotransmitter may be involved in both responses. With regard to this, we would like to draw attention to our recent observation that intracerebroventricular injection of the peptide substance P (SP) in rats resulted in an increase in plasma AVP and a decrease in plasma A C T H 7, the same combination of responses observed in saline-treated rats. Since SP is present in large amounts in the hypothalamus 21'27, this peptide has both the neuroanatomical and pharmacological potential to centrally mediate changes in circulating AVP and A C T H . We are currently investigating whether SP is involved in the responses of the hypothalamo-pituitary axis to osmotic stimulus. In conclusion, our results reveal a hitherto unknown mechanism controlling plasma A C T H concentrations in response to changes in plasma osmolality. Decreased levels of CRF-41 in the ME, and decreased CRF-41 m R N A in the parvocellular neurons of the hypothalamus following saline treatment, indicate a central site of control. Acknowledgements. We thank Mr. H. Patel for the AVP RIA data. We are also grateful for financial support from the Joint Research Committee of the Westminster Hospital group. ACTH antiserum was kindly provided by Dr. G. Court (Wellcome plc).
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