0013-7227/91/1282-1198$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 2 Printed in U.S.A.
CORTICOTROPIN-RELEASING HORMONE (CRH) DIRECTLY STIMULATES CORTICOSTERONE SECMTION BY THE RAT ADRENAL GLAND Paola G. Andreis, Giuliano Neri and Gastone G. Nussdorfer Department of Anatomy, University of Padua, 35121 Padua, Italy
ABSTRACT. Corticotropin-releasing hormone (CRH) acute ip administration (10 yg) significantly incre ased the blood concentration of corticosterone (B) in hypophysectomized rats, without inducing any rise in the level of circulating ACTH. CRH (10~6 M) did not affect B production by isolated rat adrenocortical cells, but notably enhanced that by adrenal slices including both cortex and medulla. This last effect of CRH was blocked by corticotropin inhibiting peptide (CIP), at a concentration (10~6 M) which was found to completely annul B response of adrenal slices to ACTH (10~8 M). In light of many findings indicating that adrenal medulla contains and releases CPH and numerous POMC-derived peptides, the hypothesis is advanced that an intra-adrenal CRH/ACTH mechanism may be operative in the control of adrenocortical steroid-hormone secretion.
Evidence has been accumulated indicating that CRH may act on the adrenal cortex independently of pituitary ACTH release (1-3). Moreover, high-affinity binding sites for CRH have been found in the rat adrenal zona medullaris (4,5); this last has been suggested to exert a paracrine control on the cortex (6). To gain insight into this problem, we investigated the direct effects of CRH on the secretory activity of rat inner adreno cortical zones in vivo and in vitro.
MATERIALS AND METHODS In vivo experiments Adult male Wistar rats (300 ± 30 g BW) were purchased from Charles-River (Como, Italy). A group of rats was hypophysectomized by the parapharyngeal approach and, in order to maintain the growth and secretory capacity of their adrenals, was administered daily ip injections of 2 IU/kg ACTH (Sigma, St. Louis, MO) for the first 8 days after the operation (Table 1). The completeness
of hypophysectomy was checked at the time of autopsy. On the 10th day after hypophysectomy, groups of control (sham-operated) and hypophysectomized rats (n=16) were divided into two subgroups (n=8), one of which was given an ip injection of 10 /jg CRH (human and rat, synthetic; Sigma) dissolved in 0.2 ml 0.9% NaCl, 30 min before the sacrifice. The other subgroup was given the saline vehicle (none subgroup). The animals were decapitated between 1000 and 1100 h, and their trunk blood was collected and centrifuged at 2,000 £ for 20 min at 4 C; plasma w.as stored at -70 C until hormonal assay. ACTH was extracted from plasma (7) and the recovery was calculated to be in the range of 65% (8). ACTH plasma concentration was determined by RIA: ACTH-RIA kit (Nichols, Los Angeles, CA; intra- and interassay variations, 6% and 8%). Corticosterone (B) was extra£ ted and purified (9), and its concentration was measu red by RIA: Cortx-RIA kit (Eurogenetix, Milan, Italy; intra- and interassay variations, 7% and 9%). In vitro experiments
TABLE 1. Effect of ACTH on the relative adrenal weight (RAW) and plasma concentration of corti costerone (B) in hypophysectomized (HYPOX) rats.
Controls HYPOX HYPOX/ACTH
RAW (mg/100 g BW)
B (pg/dl)
15.7 * 3.4 8.5 * 1.9« 14.3 * 3.8
11.5 * 3.7 2.6 * 1.5* 5.3 * 2.1».a
Data are means * SD (n > 8 ) . • P«0.01 vs. controls; a P««0.01 vs. HYPOX.
Received in Iowa City November 29, 1990
The left adrenals of control rats were decapsulated and enucleated (in order to eliminate zona glomerulosa and zona medullaris), and isolated inner (zonae fasci culata and reticularis) adrenocortical cells were ob_t ained by collagenase/DNase I disaggregation (10). Viab ility of isolated cells was checked by the trypan-blue exclusion test. The right adrenals were decapsulated and cut into 5 slices (11). Isolated cells and adrenal slices were put in medium 199 (DIFCO, Detroit, MI) and potassium-free Krebs-Ringer bicarbonate buffer with 0.2% glucose (2:1 vol/vol) containing 5 g/1 human serum albumin. Aliquots of cell suspensions (3 x 10 5 / ml) and adrenal fragments (about 1 mg/ml) were incuba ted, in replicates of 6 each) with ACTH 10- 8 M or CRH
1198
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1199
RAPID COMMUNICATIONS lO"^ M or without any peptide. The incubation was car ried out for 90 min in a shaking bath at 37 C in an atmosphere of 95% 0 2 and 5% CC>2Other adrenal slices were incubated, in replicates of 8 each, with CRH 10~^ M in the presence or absence of corticotropin inhibiting peptide (ACTH 7 _ 38 ) 10~ 6 M (CIP; Peninsula, Merseyside, U.K.). The concentration of CIP was that found to almost completely annul the B response of adrenocortical slices to ACTH 10~8 M, without affecting basal B secretion (Table 2 ) . TABLE 2. Effect of increasing concentrations of CIP on basal and ACTH-stimulated B production by adrenal slices. 10 ~ 8 M Basal ACTH
52.4 ±15.6 401.5 ±108.2*
62.5 + 22.4 229.5 ±98.6*
10~ 7 M
10" 6 M
49.5 ±16.2 125.8 ± 38.6*
60.3 ±21.2 70.1 ± 18.7
Data are means ± SD (n = 4 ) . • P^O.01 vs. basal.
The B concentration in the incubation medium measured by RIA, as described above.
was
Statistics Data were expressed as means ± SD, and their statis tical comparison was done by ANOVA, followed by the Multiple Range Test of Duncan.
RESULTS CRH ip administration raised plasma concentrationis of ACTH (4-fold) and B (3-fold) in control rats. Hypophy sectomy provoked notable decreases in the levels of circulating ACTH (-90%) and B (-65%). CRH still caused a significant enhancement in the blood concentration of B in hypophysectomized rats (83%), without inducing any change in that of ACTH (Table 3 ) . TABLE 3. Effect of CRH on ACTH and B plasma concentration!! in control and HYPOX/ACTH-treated rats. Controls ACTH(pg/ml) B (pg/dl)
HYPOX/ACTH
None
CRH
None
CRH
80.3 ±20.4 12.3 ± 4 . 4
316.2 ± 102.8* 38.5 ±13.9*
8.2 ± 5 . 2 a 4.2±1.9a
9.7 ± 4.8 7.7 ± 2.1"
Data are means ± SD (n = 8 ) . • P « 0 . 0 1 v s . the r e s p e c t i v e none;
a
P « 0 . 0 1 v s . c o n t r o l none.
ACTH (10~ 8 M) strikingly elevated B secretion by both isolated inner adrenocortical c e l l s (8-fold) and adrenal s l i c e s (7-fold). CRH (10~ 6 M) did not affect TABLE 4. Effect of ACTH and CRH on B production by isolated inner adrenocortical cells and adrenal slices of control rats. B production Isolated cells (pM/lC-6 cells.h) Adrenal slices (pM/mg.h)
None
ACTH
CRH
120.5 ±37.2
974.5 ± 271.3*
130.2 ±40.1
4B.6±10.7
348.5 ±101.9*
101.4 ±22.4*
Data are means ± SD (n • 6). * P^O.01 vs. none.
B production by isolated cells, but significantly in£ reases that by adrenal slices (Table 4 ) . CIP (10~ 6 M) completely annulled the stimulating effect of CRH (10~ 6 M) on B production by adrenal sli. ces (Table 5 ) . TABLE 5. Effect of CIP on the B response of adrenal slices to CRH.
B (pM/mg.h)
None
CRH
CRH/CIP
57.2 ± 11.6
126.1 ± 36.2*
50.7 ± 15.9
Data are means ± SD (n = 8 ) . * P^O.01 vs. none.
DISCUSSION Our in vivo experiment clearly shows that the acute administration of a high dose of CRH is able to stimu late the secretion of B, the main glucocorticoid pro duced by adrenal glands of rodents (for review, see Ref. 12), independently of any increase in the ACTH release by the hypophysis. This effect of CRH is not conceivably exerted directly on adrenocortical cells, since this peptide does not elevate B production by isolated inner adrenocortical cells. CRH direct secre tory effect seems to require the integrity of adrenal tissue, and is probably mediated by local ACTH produc tion. In fact, B response of adrenal slices to CRH is completely blunted by CIP, a well-known competitive inhibitor of ACTH (13). Our findings do not allow us to settle the locus of the supposed intra-adrenal CRH-dependent production of ACTH. According to Jones and Edwards (14), the main candidate appears to be adrenal medulla, whose cells possess high-affinity binding sites for CRH (4,5) and are found to co-release with catecholamines, many POMCderived peptides, including ACTH (6,15). The modality whereby intra-adrenal secreted ACTH may reach the cor tex and so stimulate steroidogenesis could be very simple in the rat, although cortex and medulla are supplied with separate capillary beds (16). In fact, many rays of medullary tissue are commonly found to project into the cortex, sometimes reaching the capsule (17): thus ACTH produced by chromaffin cells may be released near steroidogenic ones. This strict interla cement between cortical and medullary tissues could also explain why CRH exerts its secretory effect also on tissue fragments obtained from enucleated adrenals (data not shown). Investigations are in progress aimed at ascertaining whether CRH is able to evoke any seer etory response by slices of ACTH-responsive capsular adrenocortical autotransplants, which always lack chro maffin tissue (11). Another possibility, not necessa rily conflicting with the previous one, is that CRH may also act on local leukocytes, which are known to secrete ACTH/endorphin in response, to CRH (for review, see Ref. 18). CRH is contained in adrenal chromaffin cells of various species (15,19-21), and is released in response to hemorrhage (22) and splanchnic nerve stimulation (23). Parenthetically, this last procedure was also found to provoke a small output of ACTH from the calf adrenal glands (14). In light of the literature data
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RAPID COMMUNICATIONS
1200
now reviewed and of our present findings, it appears conceivable to hypothesize the existence of an intraadrenal CRH/ACTH mechanism replicating that operating at the hypothalamo-hypophyseal level. It remains to be investigated whether this intra-adrenal control mecha nism pleys any physiological routinary role or may be activated only under extremely stressful conditions capable of stimulating the splanchnic sympathetic system.
Acknowledgements. The authors wish to thank Dr. G. P. De Tos and Prof. M. Boscaro for their help with RIA assays.
REFERENCES 1. Fehm HL, Holl R, Spatschwalbe E, Born J, Voight KH 1988 Ability of corticotropin releasing hormone to stimulate cortisol secretion independent from pi£ uitary adrenocorticotropin. Life Sci 42:679-686 2. Mazzocchi G, Rebuffat P, Meneghelli V, Nussdorfer GG 1989 Effects of the infusion with ACTH or CRH on the secretory activity of rat adrenal cortex. J Steroid Biochem 32:841-843 3. Bornstein SR, Ehrhart M, Scherbaum WA, Pfeiffer EF 1990 Adrenocortical atrophy of hypophysectomized rats can be reduced by corticotropin-releasing hormone (CRH). Cell Tissue Res 260:161-166 4. Dave JR, Eiden LE, Eskay RL 1985 Corticotropinreleasing factor binding to peripheral tissue and activation of the adenylate cyclase-adenosine 3',5'monophosphate system. Endocrinology 116:2152-2159 5. Aguilera G, Millan MA, Hauger RL, Catt KJ 1987 Corticotropin-releasing factor receptors: distri^ bution and regulation in brain, pituitary and peri pherial tissues. Ann NY Acad Sci 512:48-66 6. Hinson JP 1990 Paracrine control of adrenocortical function: a new role for the medulla? J Endocrinol 124:7-9 7. Rees LH, Cook DM, Kendall JW, Allen CF, Kramer RM, Ratcliffe JG, Knight RA 1971 A radioimmunoassay for rat plasma ACTH. Endocrinology 89:254-261 8. Nussdorfer GG, Mazzocchi G 1987 Vasoactive intest inal peptide (VIP) stimulates aldosterone secretion by rat adrenal glands in vivo. J Steroid Biochem 26:203-205 9. Sippell WG, Bidlingmaier F, Becker H, Brilnig T, Dorr H, Hahn H, Golder W, Hollmann G, Knorr D 1978 Simultaneous radioimmunoassay of plasma aldosterone, corticosterone, 11-deoxycorticosterdne, progesterone, 17-hydroxyprogesterone, 11-deoxycortisol, cortisol, and cortisone. J Steroid Biochem 9:63-74
Endo'1991 Voll28»No2
10. Szalay KZs 1981 Effect of pituitary intermediate lobe extract on uteroid production by the isolated zona glomerulose. and fasciculata cells. Acta Phy_ siol Acad Sci Hung 57:225-231 11. Belloni AS, Neri G, Musajo FG, Andreis PG, Boscaro M, D'Agostino D, Rebuffat P, Boshier DP, Gottardo G, Mazzocchi G, Nussdorfer GG 1990 Investigations on the morphology and function of adrenocortical tissue regenerated from gland capsular fragments autotransplanted in the musculus gracilis of the rat. Endocrinology 126:3251-3262 12. Nussdorfer GG 1986 Cytophysiology of the adrenal cortex. Int Rev Cytol 98:1-405 13. Li CH, Chung D, Yamashiro D, Lee CY 1978 Isolation, characterization and synthesis of a corticotropininhibiting peptide from human pituitary glands. Proc Nat Acad Sci USA 75:4306-4309 14. Jones CT, Edwards AV 1990 Release of adrenocorti cotrophin from the adrenal gland in the conscious calf. J Physiol (London) 426:397-407 15. Suda T, Tomori M, Yajima F, Odagiri E, Demura H, Shizume K 1986 Characterization of immunoreactive corticotropin eind corticotropin-releasing factor in human adrenal and ovarian tumors. Acta Endocr inol (Copenh) 111:546-552 16. Sparrow RA, Coupland RE 1987 Blood flow to the adrenal gland of the rat. J Anat 155:51-61 17. Gallo-Payet N, Pothier P, Isler H 1987 On the pre sence of chromaffin cells in the adrenal cortex: their possible role in adrenocortical function. Biochem Cell Biol 65:588-592 18. Buckingham JC 1987 A role for leukocytes in the control of adrenal steroidogenesis? J Endocrinol 114:1-2 19. Hashimoto K, Murakami K, Hattori T, Niimi M, Fujimo K, Ota Z 1984 Corticotropin-releasing factor (CRF)like immunoreactivity in the adrenal medulla. Pe£ tides 5:707-712 20. Suda T, Tomori N, TozawaF, Mouri T, Demura H, Shiz ume K 1984 Distribution and characterization of immunoreactive oorticotropin-releasing factor in human tissues. J Clin Endocrinol Metab 59:861-867 21. Minamino N, Uehara A, Arimura A 1988 Biological and immunological characterization of corticotropinreleasing activity in the bovine adrenal medulla. Peptides 9:37-45 22. Bruhn TO, Engeland WC, Anthony ELP, Gann DS, Jackson IDM 1987 Corticotropin-releasing factor in the dog adrenal medulla is secreted in response to hemor rhage. Endocrinology 120:25-33 23. Edwards AV, Jones CT 1988 Secretion of corticotro phin releasing factor from the adrenal during splanchnic nerve stimulation in conscious calves. J Physiol (London) 400:89-100
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Vol. 128, No. 2 Printed in U.S.A.
CORTICOTROPIN-RELEASING HORMONE (CRH) DIRECTLY STIMULATES CORTICOSTERONE SECMTION BY THE RAT ADRENAL GLAND Paola G. Andreis, Giuliano Neri and Gastone G. Nussdorfer Department of Anatomy, University of Padua, 35121 Padua, Italy
ABSTRACT. Corticotropin-releasing hormone (CRH) acute ip administration (10 yg) significantly incre ased the blood concentration of corticosterone (B) in hypophysectomized rats, without inducing any rise in the level of circulating ACTH. CRH (10~6 M) did not affect B production by isolated rat adrenocortical cells, but notably enhanced that by adrenal slices including both cortex and medulla. This last effect of CRH was blocked by corticotropin inhibiting peptide (CIP), at a concentration (10~6 M) which was found to completely annul B response of adrenal slices to ACTH (10~8 M). In light of many findings indicating that adrenal medulla contains and releases CPH and numerous POMC-derived peptides, the hypothesis is advanced that an intra-adrenal CRH/ACTH mechanism may be operative in the control of adrenocortical steroid-hormone secretion.
Evidence has been accumulated indicating that CRH may act on the adrenal cortex independently of pituitary ACTH release (1-3). Moreover, high-affinity binding sites for CRH have been found in the rat adrenal zona medullaris (4,5); this last has been suggested to exert a paracrine control on the cortex (6). To gain insight into this problem, we investigated the direct effects of CRH on the secretory activity of rat inner adreno cortical zones in vivo and in vitro.
MATERIALS AND METHODS In vivo experiments Adult male Wistar rats (300 ± 30 g BW) were purchased from Charles-River (Como, Italy). A group of rats was hypophysectomized by the parapharyngeal approach and, in order to maintain the growth and secretory capacity of their adrenals, was administered daily ip injections of 2 IU/kg ACTH (Sigma, St. Louis, MO) for the first 8 days after the operation (Table 1). The completeness
of hypophysectomy was checked at the time of autopsy. On the 10th day after hypophysectomy, groups of control (sham-operated) and hypophysectomized rats (n=16) were divided into two subgroups (n=8), one of which was given an ip injection of 10 /jg CRH (human and rat, synthetic; Sigma) dissolved in 0.2 ml 0.9% NaCl, 30 min before the sacrifice. The other subgroup was given the saline vehicle (none subgroup). The animals were decapitated between 1000 and 1100 h, and their trunk blood was collected and centrifuged at 2,000 £ for 20 min at 4 C; plasma w.as stored at -70 C until hormonal assay. ACTH was extracted from plasma (7) and the recovery was calculated to be in the range of 65% (8). ACTH plasma concentration was determined by RIA: ACTH-RIA kit (Nichols, Los Angeles, CA; intra- and interassay variations, 6% and 8%). Corticosterone (B) was extra£ ted and purified (9), and its concentration was measu red by RIA: Cortx-RIA kit (Eurogenetix, Milan, Italy; intra- and interassay variations, 7% and 9%). In vitro experiments
TABLE 1. Effect of ACTH on the relative adrenal weight (RAW) and plasma concentration of corti costerone (B) in hypophysectomized (HYPOX) rats.
Controls HYPOX HYPOX/ACTH
RAW (mg/100 g BW)
B (pg/dl)
15.7 * 3.4 8.5 * 1.9« 14.3 * 3.8
11.5 * 3.7 2.6 * 1.5* 5.3 * 2.1».a
Data are means * SD (n > 8 ) . • P«0.01 vs. controls; a P««0.01 vs. HYPOX.
Received in Iowa City November 29, 1990
The left adrenals of control rats were decapsulated and enucleated (in order to eliminate zona glomerulosa and zona medullaris), and isolated inner (zonae fasci culata and reticularis) adrenocortical cells were ob_t ained by collagenase/DNase I disaggregation (10). Viab ility of isolated cells was checked by the trypan-blue exclusion test. The right adrenals were decapsulated and cut into 5 slices (11). Isolated cells and adrenal slices were put in medium 199 (DIFCO, Detroit, MI) and potassium-free Krebs-Ringer bicarbonate buffer with 0.2% glucose (2:1 vol/vol) containing 5 g/1 human serum albumin. Aliquots of cell suspensions (3 x 10 5 / ml) and adrenal fragments (about 1 mg/ml) were incuba ted, in replicates of 6 each) with ACTH 10- 8 M or CRH
1198
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1199
RAPID COMMUNICATIONS lO"^ M or without any peptide. The incubation was car ried out for 90 min in a shaking bath at 37 C in an atmosphere of 95% 0 2 and 5% CC>2Other adrenal slices were incubated, in replicates of 8 each, with CRH 10~^ M in the presence or absence of corticotropin inhibiting peptide (ACTH 7 _ 38 ) 10~ 6 M (CIP; Peninsula, Merseyside, U.K.). The concentration of CIP was that found to almost completely annul the B response of adrenocortical slices to ACTH 10~8 M, without affecting basal B secretion (Table 2 ) . TABLE 2. Effect of increasing concentrations of CIP on basal and ACTH-stimulated B production by adrenal slices. 10 ~ 8 M Basal ACTH
52.4 ±15.6 401.5 ±108.2*
62.5 + 22.4 229.5 ±98.6*
10~ 7 M
10" 6 M
49.5 ±16.2 125.8 ± 38.6*
60.3 ±21.2 70.1 ± 18.7
Data are means ± SD (n = 4 ) . • P^O.01 vs. basal.
The B concentration in the incubation medium measured by RIA, as described above.
was
Statistics Data were expressed as means ± SD, and their statis tical comparison was done by ANOVA, followed by the Multiple Range Test of Duncan.
RESULTS CRH ip administration raised plasma concentrationis of ACTH (4-fold) and B (3-fold) in control rats. Hypophy sectomy provoked notable decreases in the levels of circulating ACTH (-90%) and B (-65%). CRH still caused a significant enhancement in the blood concentration of B in hypophysectomized rats (83%), without inducing any change in that of ACTH (Table 3 ) . TABLE 3. Effect of CRH on ACTH and B plasma concentration!! in control and HYPOX/ACTH-treated rats. Controls ACTH(pg/ml) B (pg/dl)
HYPOX/ACTH
None
CRH
None
CRH
80.3 ±20.4 12.3 ± 4 . 4
316.2 ± 102.8* 38.5 ±13.9*
8.2 ± 5 . 2 a 4.2±1.9a
9.7 ± 4.8 7.7 ± 2.1"
Data are means ± SD (n = 8 ) . • P « 0 . 0 1 v s . the r e s p e c t i v e none;
a
P « 0 . 0 1 v s . c o n t r o l none.
ACTH (10~ 8 M) strikingly elevated B secretion by both isolated inner adrenocortical c e l l s (8-fold) and adrenal s l i c e s (7-fold). CRH (10~ 6 M) did not affect TABLE 4. Effect of ACTH and CRH on B production by isolated inner adrenocortical cells and adrenal slices of control rats. B production Isolated cells (pM/lC-6 cells.h) Adrenal slices (pM/mg.h)
None
ACTH
CRH
120.5 ±37.2
974.5 ± 271.3*
130.2 ±40.1
4B.6±10.7
348.5 ±101.9*
101.4 ±22.4*
Data are means ± SD (n • 6). * P^O.01 vs. none.
B production by isolated cells, but significantly in£ reases that by adrenal slices (Table 4 ) . CIP (10~ 6 M) completely annulled the stimulating effect of CRH (10~ 6 M) on B production by adrenal sli. ces (Table 5 ) . TABLE 5. Effect of CIP on the B response of adrenal slices to CRH.
B (pM/mg.h)
None
CRH
CRH/CIP
57.2 ± 11.6
126.1 ± 36.2*
50.7 ± 15.9
Data are means ± SD (n = 8 ) . * P^O.01 vs. none.
DISCUSSION Our in vivo experiment clearly shows that the acute administration of a high dose of CRH is able to stimu late the secretion of B, the main glucocorticoid pro duced by adrenal glands of rodents (for review, see Ref. 12), independently of any increase in the ACTH release by the hypophysis. This effect of CRH is not conceivably exerted directly on adrenocortical cells, since this peptide does not elevate B production by isolated inner adrenocortical cells. CRH direct secre tory effect seems to require the integrity of adrenal tissue, and is probably mediated by local ACTH produc tion. In fact, B response of adrenal slices to CRH is completely blunted by CIP, a well-known competitive inhibitor of ACTH (13). Our findings do not allow us to settle the locus of the supposed intra-adrenal CRH-dependent production of ACTH. According to Jones and Edwards (14), the main candidate appears to be adrenal medulla, whose cells possess high-affinity binding sites for CRH (4,5) and are found to co-release with catecholamines, many POMCderived peptides, including ACTH (6,15). The modality whereby intra-adrenal secreted ACTH may reach the cor tex and so stimulate steroidogenesis could be very simple in the rat, although cortex and medulla are supplied with separate capillary beds (16). In fact, many rays of medullary tissue are commonly found to project into the cortex, sometimes reaching the capsule (17): thus ACTH produced by chromaffin cells may be released near steroidogenic ones. This strict interla cement between cortical and medullary tissues could also explain why CRH exerts its secretory effect also on tissue fragments obtained from enucleated adrenals (data not shown). Investigations are in progress aimed at ascertaining whether CRH is able to evoke any seer etory response by slices of ACTH-responsive capsular adrenocortical autotransplants, which always lack chro maffin tissue (11). Another possibility, not necessa rily conflicting with the previous one, is that CRH may also act on local leukocytes, which are known to secrete ACTH/endorphin in response, to CRH (for review, see Ref. 18). CRH is contained in adrenal chromaffin cells of various species (15,19-21), and is released in response to hemorrhage (22) and splanchnic nerve stimulation (23). Parenthetically, this last procedure was also found to provoke a small output of ACTH from the calf adrenal glands (14). In light of the literature data
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RAPID COMMUNICATIONS
1200
now reviewed and of our present findings, it appears conceivable to hypothesize the existence of an intraadrenal CRH/ACTH mechanism replicating that operating at the hypothalamo-hypophyseal level. It remains to be investigated whether this intra-adrenal control mecha nism pleys any physiological routinary role or may be activated only under extremely stressful conditions capable of stimulating the splanchnic sympathetic system.
Acknowledgements. The authors wish to thank Dr. G. P. De Tos and Prof. M. Boscaro for their help with RIA assays.
REFERENCES 1. Fehm HL, Holl R, Spatschwalbe E, Born J, Voight KH 1988 Ability of corticotropin releasing hormone to stimulate cortisol secretion independent from pi£ uitary adrenocorticotropin. Life Sci 42:679-686 2. Mazzocchi G, Rebuffat P, Meneghelli V, Nussdorfer GG 1989 Effects of the infusion with ACTH or CRH on the secretory activity of rat adrenal cortex. J Steroid Biochem 32:841-843 3. Bornstein SR, Ehrhart M, Scherbaum WA, Pfeiffer EF 1990 Adrenocortical atrophy of hypophysectomized rats can be reduced by corticotropin-releasing hormone (CRH). Cell Tissue Res 260:161-166 4. Dave JR, Eiden LE, Eskay RL 1985 Corticotropinreleasing factor binding to peripheral tissue and activation of the adenylate cyclase-adenosine 3',5'monophosphate system. Endocrinology 116:2152-2159 5. Aguilera G, Millan MA, Hauger RL, Catt KJ 1987 Corticotropin-releasing factor receptors: distri^ bution and regulation in brain, pituitary and peri pherial tissues. Ann NY Acad Sci 512:48-66 6. Hinson JP 1990 Paracrine control of adrenocortical function: a new role for the medulla? J Endocrinol 124:7-9 7. Rees LH, Cook DM, Kendall JW, Allen CF, Kramer RM, Ratcliffe JG, Knight RA 1971 A radioimmunoassay for rat plasma ACTH. Endocrinology 89:254-261 8. Nussdorfer GG, Mazzocchi G 1987 Vasoactive intest inal peptide (VIP) stimulates aldosterone secretion by rat adrenal glands in vivo. J Steroid Biochem 26:203-205 9. Sippell WG, Bidlingmaier F, Becker H, Brilnig T, Dorr H, Hahn H, Golder W, Hollmann G, Knorr D 1978 Simultaneous radioimmunoassay of plasma aldosterone, corticosterone, 11-deoxycorticosterdne, progesterone, 17-hydroxyprogesterone, 11-deoxycortisol, cortisol, and cortisone. J Steroid Biochem 9:63-74
Endo'1991 Voll28»No2
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