0021-972X/90/7105-1368$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society
Vol. 71, No. 5 Printed in U.S.A.
Corticotropin-Releasing Hormone Inhibition of Growth Hormone-Releasing Hormone-Induced Growth Hormone Release in Man* ANTONINO BARBARINO, SALVATORE M. CORSELLO, SILVIA DELLA CASA, ANNA TOFANI, ROSA SCIUTO, CARLO ANTONIO ROTA, LUCILLA BOLLANTI, AND ANGELA BARINI Institutes of Endocrinology and Biochemistry (A. Bari), Catholic University School of Medicine, 1-00168 Rome, Italy
ABSTRACT. Recent studies in the rat have shown that intracerebroventricular administration of CRH inhibited spontaneous pulsatile GH secretion and prevented GH-releasing hormone (GHRH)-induced GH release. We have studied the effect of CRH on GHRH-induced GH release in man. In the first study, CRH was injected iv at three different doses (100, 50, or 25 Mg) at 0800 h together with 50 Mg GHRH in six men and six women. In a second study, 100 Mg CRH were given iv at 0800 h, 1 h before the administration of 50 fig GHRH in five men and five women. Each subject demonstrated a normal GH response after the administration of 50 fig GHRH plus saline. All doses of CRH administered simultaneously with GHRH significantly inhibited GHRH-induced GH release in women [peak value ± SE after GHRH plus saline, 28.9 ± 2.9 Mg/L; after GHRH plus 100 ng CRH, 9.9 ± 0.7 Mg/L (P < 0.001); after GHRH plus 50 Hg CRH, 8.7 ± 0.8 Mg/L (P < 0.001); after GHRH plus 25 ng CRH, 9.5 ± 1.6 Mg/L (P < 0.001)]. In contrast, in men, while a
G
H SECRETION appears to be regulated at the pituitary level by a GH-releasing hormone (GHRH) and a GH release-inhibiting hormone, somatostatin (SS). Several neuropeptides can, in turn, modify GH secretion by acting on the central nervous system to modulate the hypothalamic secretion of GHRH and SS. Recent studies in animals have suggested that endogenous CRH can serve as a chemical mediator in the stress-induced inhibition of GH secretion (1, 2); intracerebroventricular injection of CRH inhibited pulsatile GH secretion and also prevented GHRH-stimulated GH release in rats (1, 2). This suppressive action of CRH seems to involve an increased release of SS, since it was abolished by previous treatment with anti-SS serum (3, 4). These data together with the observation that CRH
dose of 100 fig CRH was capable of suppressing GHRH-induced GH secretion (peak value ± SE, 8.1 ± 0.6 vs. 20 ± 2.9 Mg/L; P < 0.001), no inhibition was observed after 50- and 25-Mg doses. When 100 Mg CRH were injected 1 h before the administration of 50 Mg GHRH, it strongly inhibited GHRH-induced GH secretion in both men (peak value ± SE, 6.2 ± 2.8 vs. 24.6 ± 5.9 fig/ L; P < 0.02) and women (peak value ± SE, 14.2 ± 4.5 vs. 37.8 ± 6.7 Mg/L; P < 0.005), and this inhibition lasted up to 2 h postCRH administration. These results demonstrate that CRH is capable of inhibiting GHRH-induced GH release in both men and women. Furthermore, the findings suggest that a sexual dimorphism in the neuroregulation of GH secretion may be present in man. In view of the inhibitory action of CRH on GH secretion, simultaneous administration of CRH and GHRH for testing should be avoided in clinical practice. («/ Clin Endocrinol Metab 7 1 : 1368-1374, 1990)
was capable of inhibiting gonadotropin release in normal nonstressed women (5) prompted us to examine the effect of CRH on GH secretion in humans. Since, unlike in the rat (6, 7), the occurrence of spontaneous GH peaks cannot be easily predicted in the conscious man, we have elected to study the effect of CRH on GHRH-stimulated GH release in normal nonstressed men and normal cycling women. Materials and Methods
Received August 14,1989. Address all correspondence and requests for reprints to: Antonino Barbarino, M.D., via Diano Marina 9/11,1-00168 Rome, Italy. * Presented in part at the 71st Annual Meeting of The Endocrine Society, Seattle, WA, June 21-24, 1989 (Abstract 1760).
Nine normal women, aged 19-26 yr, and nine normal men, aged 18-25 yr, were studied after they had given informed consent. All subjects were nonstressed. All were within 10% of ideal body weight (weight range, 62-77 kg for men and 52-66 kg for women) and were eating their customary diet before and during the interval between the tests. None had evidence of any endocrine disorder or was taking any medication. All women had regular menstrual cycles. After an overnight fast, all subjects were kept in bed from 0700 h until the end of the tests. An iv catheter was inserted
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CRH INHIBITION OF GH RELEASE in one arm 30 min before starting the tests. It was used for drug injection and blood sampling, as specified below. Protocol 1
In 12 subjects (6 women and 6 men) 4 separate GHRH stimulation tests were performed. A 50-/*g bolus dose of GHRH was injected iv at time zero (0800 h) together with either saline (2-cc bolus) or 3 different doses of CRH (100-, 50-, or 25-Mg bolus). Blood samples were collected 30, 15, and 0 min before and 15, 30, 60, and 90 min after GHRH administration.
Protocol 2
In 10 subjects (5 women and 5 men, of whom 2 women and 2 men were also included in protocol 1) two separate GHRH stimulation tests were performed. A 50-^g bolus dose of GHRH was injected iv at time zero (0900 h), 1 h after either saline (2cc bolus) or CRH (100-Mg bolus) injection. Blood samples were collected 75, 60, 45, 30,15, and 0 min before and 15, 30, 60, and 90 min after GHRH administration. In individual subjects, the tests were performed at least at 72-h intervals, in a blind fashion. In women they were performed during the luteal phase of the same or a different cycle. The time of the cycle was established by measuring estradiol and progesterone levels. GHRH-(l-44) and human CRH were furnished by Bachem (Bubendorf, Switzerland). The administered doses of GHRH and CRH were diluted in 2 cc saline. Plasma GH levels were measured in duplicate by RIA, using polyethylene glycol to separate free and bound hormone, with reagents obtained by Biodata (Milan, Italy). Normal plasma GH levels ranged from 0.25-10 fig/L.. One nanogram of GH corresponded to 2 fi\J/mL WHO 66/217 standard. The sensitivity of the assay was 0.08 Mg/L- The intra- and interassay coefficients of variation ranged from 2.5-3.9% and from 5.88.5%, respectively. Plasma concentrations of estradiol, progesterone, testosterone, and cortisol levels were measured by RIA, as previously described (8, 9). All samples from a given study were measured in the same assay for each hormone. Plasma glucose levels were measured, using a polarographic method (Beckman Instruments, Palo Alto, CA), in five subjects of protocol 1, when 50 fig GHRH plus 100 ng CRH were administered. The results were evaluated by analysis of variance for repeated measures. The means of each time were compared between control (GHRH plus saline) and experimental groups (GHRH plus CRH at various doses) by Newman-Keuls test. The results are given as the mean ± SE. The significance level was established at P < 0.05.
Results Protocol 1
The effects of different doses of CRH, when simultaneously injected with GHRH, on GHRH-induced GH release and plasma cortisol concentrations in women and
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men are shown in Figs. 1 and 2, respectively. In women, the GH response to GHRH was significantly reduced after all doses of CRH compared to that after saline injection. The mean peak GH value was diminished from 28.9 ± 2.9 fig/L (GHRH plus saline) to 9.9 ± 0.7 Mg/L (GHRH plus 100 fig CRH; P < 0.001), 8.7 ± 0.8 Mg/L (GHRH plus 50 fig CRH; P < 0.001), and 9.5 ± 1.6 fig/L (GHRH plus 25 fig CRH; P < 0.001; Fig. 3). In contrast, in men only a dose of 100 /ug CRH was able to significantly suppress the GH response to GHRH, with a mean peak GH value diminished from 20 ± 2.9 fig/L (GHRH plus saline) to 8.1 ± 0.6 fig/L (P < 0.001). No significant inhibition was observed after 50- and 25Hg doses (GH peaks, 19.8 ± 2.9 and 17.4 ± 4.3 fig/h, respectively; Figs. 2 and 3). Plasma cortisol concentrations increased 111% in women and 95% in men after 100 fig CRH (expressed as peak values vs. the means of three basal values). The first significant increase was apparent 15 min after CRH administration. When 50 fig CRH, 25 fig CRH, or saline were given, plasma cortisol did not increase significantly during the period of sample collection. Protocol 2
The effect of 100 fig CRH, administered 1 h before GHRH, on GHRH-induced GH release in men and women is shown in Fig. 4. After CRH administration, the GH response to GHRH was significantly reduced compared to that after saline injection. The peak GH value was diminished from 37.8 ± 6.7 to 14.2 ± 4.5 Mg/L in women (P < 0.005) and from 24.6 ± 5.9 to 6.2 ± 2.8 fig/L in men (P < 0.02). When CRH was given, the plasma cortisol concentration increased 166% in women and 181% in men (expressed as peak values vs. the means of two basal values). The first significant increase was apparent 15 min after CRH injection. When saline was given, plasma cortisol did not increase during the period of sample collection. CRH produced facial flushing in four of the subjects in whom a 100-fig bolus was injected iv. Besides this, no other side-effects were noted. There was no change in supine blood pressure or heart rate. No significant increase in plasma glucose levels was observed in the five subjects in whom glucose was measured after GHRH plus 100 fig CRH (basal values 4.88 ± 0.23 mmol/L, peak values 5.20 ± 0.32 mmol/L). Estradiol and progesterone plasma concentrations in women were determined during the luteal phase of the menstrual cycle (483.1 ± 36.3 pmol/L and 52.7 ± 13.5 nmol/L, respectively). Plasma testosterone concentrations in men were within the adult normal male range (21.5 ± 3.5 nmol/L).
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JCE & M • 1990 Vol 71 • No 5
BARBARINO ET AL.
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G H R H 50 ug CRH 100 ug or
30L
FIG. 1. Mean (±SE) GH (left panel) and cortisol (rightpanel) responses to administration of saline plus GHRH ( ) and CRH (100, 50, or 25 fig) plus GHRH • — • ) in six normal women. *, P < 0.001, comparing values after GHRH plus CRH with those after GHRH plus saline.
GHRH
Saline
50 H9
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Discussion The observation that iv administration of CRH could significantly decrease plasma gonadotropin levels in normal nonstressed women during the late follicular and luteal phases of the cycle (5) raised the possibility that in humans, CRH could also participate in a variety of hormonal responses and exert an inhibitory role in the
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regulation of GH secretion. While spontaneous GH bursts occur regularly in the conscious rat (6, 7), in man GH peaks do not occur regularly and are greatly influenced by metabolic events (10). Nocturnal GH secretion is characterized in the adult man by one large secretory pulse occurring around sleep onset. The infusion of CRH to normal sleeping subjects somewhat reduced GH release and induced sev-
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CRH INHIBITION OF GH RELEASE
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GHRH 50 yg 3d,
CRH 100 ug or Saline 500.
300
O O 100
500.
FIG. 2. Mean (±SE) GH {left panel) and cortisol (rightpanel) responses to administration of saline plus GHRH ( ) and CRH (100, 50, or 25 ug) plus GHRH (•—•) in six normal men. *, P < 0.001, comparing values after GHRH plus CRH with those after GHRH plus saline.
O
E c 300.
O O 100.
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300.
O
O 100
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eral changes in sleep architecture, indicating a possible inhibitory action of CRH on spontaneous GH secretion (11). Previous studies on the effect of exogenous GHRH on GH secretion have shown that GHRH, in appropriate doses, selectively induces GH release in both men and women (12). A considerable variability of the GH responses to GHRH among individual subjects has been
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previously reported (12). In addition, the nutritional status, body weight, and time of experiment can modify the GH response to GHRH (10). In an effort to eliminate endogenous as well as exogenous factors that may influence the GH response to exogenously administered GHRH, we have studied subjects of similar age, nutritional status, and body weight and compared in each subject the magnitude of the GH
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BARBARINOETAL.
1372 30-i
20-
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GHRH SALINE
GHRH CRH toot
GHRH CRH 50(10
G HRH CRHlOOpg
CRH
GHRH CRH
30 -i
20-
10-
SALINE
G HRH
G HRH
CRH 25\XQ
FlG. 3. Mean (±SE) basal and peak values of plasma GH after the simultaneous injection of GHRH (50 ng) plus saline and GHRH (50 ixg) plus CRH (100, 50, and 25 fig) in six normal men (upper panel) and six normal women (lower panel). The mean of three basal values has been considered the basal level. *, P < 0.001, comparing peak values after GHRH plus CRH with those after GHRH plus saline.
response to GHRH alone and GHRH plus CRH administration, so that each individual served as his/her own control. Normal nonstressed men and women studied at 0900 h after an overnight fast exhibited rapid GH release after iv administration of 50 ng GHRH. This response was quite variable among the subjects. Nevertheless, graded doses (100, 50, and 25 fig) of CRH had a differential inhibitory effect on GH secretion in the two sexes when the same subject was studied repeatedly. In women, GHRH-induced GH secretion was significantly inhibited after all doses of CRH, whereas in men CRH induced GH inhibition only after a 100-jig dose. In addition, a dose of 100 ng CRH injected 1 h before GHRH administration was also effective in inhibiting GHRH-induced GH secretion in both normal men and women. These results are in agreement with experiments showing that in rats the icv injection of CRH prevented spontaneous as well as GHRH-induced GH secretion (1, 2). Our
JCE & M • 1990 Vol 71 • No 5
results also indicate that CRH exerts an inhibitory influence on pituitary hormone secretion for up to 2 h after its administration and are in agreement with our previous data demonstrating that after CRH injection, basal LH secretion significantly decreased during the 4 h following CRH injection and returned to normal levels 5 h after injection (5). The mechanism by which CRH exerts its inhibitory action on GHRH-induced GH release is not evident from our experiments. However, previous animal studies have shown that this effect is possibly mediated by SS, since SS antiserum blocked the inhibitory action of CRH in conscious rats (3, 4). Studies on brain localization of SS have shown high concentration of this hormone in various areas of the central nervous system, with the highest concentration found in the median eminence (13). It is, therefore, possible that large doses of peripherally injected CRH can reach the central nervous system and induce somatostatin release from brain sites outside the blood-brain barrier, especially in the region of the median eminence, and this, in turn, will inhibit GHRH action at the pituitary level. Of some interest is our observation that in female subjects CRH was effective at doses much lower than those required in males, even if we consider the difference in body weight range between sexes. These results could be due to gender differences in the neuroregulation of GH secretion. A sexual dimorphism in the secretory pattern of GH has been shown in the mature rat, and it has been attributed to a higher somatostatinergic tone in male animals (14,15). It is possible that, analogously, men have a higher somatostatinergic tone than women, and thus, small doses of peripherally injected CRH are unable to induce a variation in GH secretion in male subjects. On the contrary, women may show an inhibition of GH secretion after small doses of CRH by virtue of a lower somatostatinergic tone. Another possible explanation for our results is that CRH modified the somatotroph responsiveness to GHRH. However, it has been documented in the rat that CRH is devoid of direct effects on GH release at the pituitary level (16). CRH inhibition of GHRH-induced GH release could be mediated by a neurohormonal action of cortisol, which is released after CRH administration. However, reports on the effect of glucocorticoids on GH secretion are conflicting. In vitro studies have shown that glucocorticoids potentiate basal GH secretion in a variety of pituitary culture systems, including human pituitary explants (17). A similar stimulatory action of corticosteroids on GHRH-stimulated GH secretion has been demonstrated in vivo in the rat after administration of 40 Mg/day dexamethasone for 7 days (18). On the other hand,
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CRH INHIBITION OF GH RELEASE
GHRM so pg
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GHRH soug
N=5
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-75 -6O
-45
-3O -15
-75 -6O - 4 5
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FlG. 4. Mean (±SE) GH responses to administration of saline plus GHRH (•—•) and CRH plus GHRH (O—O) in five normal women (left panel) and five normal men (right panel). CRH or saline was given 1 h before GHRH. *, P < 0.02; **, P < 0.005 (comparing values after GHRH plus CRH with those after GHRH plus saline).
administration of 60 mg/day prednisone for 4 days to normal men decreased their GH response to GHRH (19). Thus, administration of glucocorticoids for several days (4-7) seems to elicit differential effects on GH secretion in the rat and man. Very recently, Casaneuva et al. (20) have reassessed the effects of very short term administration of glucocorticoids on GHRH-induced GH secretion in normal men. These researchers have demonstrated that 4 mg dexamethasone given 3 h before GHRH significantly potentiated GHRH-induced GH secretion, while 8 mg dexamethasone given 12 h before GHRH administration completely blocked GH secretion (20). These last results seem to exclude the possibility that the suppressive effect of CRH observed in the present paper is due to hypercortisolism, which occurs after CRH administration. Rather, a potentiating effect of cortisol on GH secretion should have occurred, especially when 100 ng CRH were administered together with GHRH. Our results strongly suggest that CRH per se inhibits GH secretion, and this counteracts the stimulatory action of the concomitant hypercortisolism that is present after injection of large doses of CRH. Additional support for lack of mediation by cortisol comes from present data in females, in which CRH at 50 and 25 fig inhibited GHRH-induced GH release despite no significant increase in plasma cortisol. Although it has been reported that the icv administration of CRH produces hyperglycemia in rats (21), the lack of any increase in plasma glucose levels after iv CRH injection in our subjects excludes a hyperglycemiamediated attenuation of the GH response to GHRH.
In conclusion, our present findings demonstrate that CRH is capable of inhibiting GHRH-induced GH release in both men and women, and suggest that this action of CRH is not dependent on its ability to stimulate cortisol secretion. Moreover, our results suggest that CRH modulation of GH secretion is different in men and women. Simultaneous administration of CRH and GHRH for testing should be avoided in clinical practice in view of the inhibitory effect of CRH on GH secretion demonstrated in this study. References 1. Rivier C, Vale W. Corticotropin-releasing factor (CRF) acts centrally to inhibit growth hormone secretion in the rat. Endocrinology. 1984;114:2409-ll. 2. Ono N, Lumpkin MD, Samson WK, McDonald JK, McCann SM. Intrahypothalamic action of corticotropin-releasing factor (CRF) to inhibit growth hormone and LH release in the rat. Life Sci. 1984;35:1117-23. 3. Katakami H, Arimura A, Frohman LA. Involvement of hypothalamic somatostatin in the suppression of growth hormone secretion by central corticotropin-releasing factor in conscious male rats. Neuroendocrinology. 1985;41:390-3. 4. Rivier C, Vale V. Involvement of corticotropin-releasing factor and somatostatin in stress-induced inhibition of growth hormone secretion in the rat. Endocrinology. 1985;117:2478-82. 5. Barbarino A, De Marinis L, Folli G, et al. Corticotropin-releasing hormone inhibition of gonadotropin secretion during the menstrual cycle. Metabolism. 1989;38:504-6. 6. Tannenbaum GS, Martin JB. Evidence for an endogenous ultradian rhythm governing growth hormone secretion in the rat. Endocrinology. 1976;98:562-70. 7. Katakami H, Kato Y, Matsushita N, Hiroto S, Shimatsu A, Imura
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9.
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12. 13.
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H. Involvement of alpha-adrenergic mechanisms in growth hormone release induced by opioid peptides in conscious rats. Neuroendocrinology. 1981;33:129-35. Barbarino A, De Marinis L, Anile C, Menini E, Merlini G, Maira G. Dopaminergic mechanisms regulating prolactin secretion in patients with prolactin secreting pituitary adenoma. Long term studies after selective transsphenoidal surgery. Metabolism. 1982;31:1100-4. De Marinis L, Mancini A, Calabro' F, Massari M, Torlontano M, Barbarino A. Differential effects of a dopaminergic drug (piribedil) on pituitary hormone release in normal men and women. Acta Endocrinol (Copenh). 1983;104:385-9. De Marinis L, Folli G, D'Amico C, et al. Differential effects of feeding on the ultradian variation of the GH response to GHRH in normal subjects and patients with obesity and anorexia nervosa. J Clin Endocrinol Metab. 1988;66:598-604. Holsboer F, von Bardeleben U, Steiger A. Effect of intravenous corticotropin-releasing hormone upon sleep-related growth hormone surge and sleep EEG in man. Neuroendocrinology. 1988;48:32-8. Thorner MO, Vance ML, Evans S. Physiological and clinical studies of GRF and GH. Recent Prog Horm Res. 1986;42:589-640. Brownstein MJ, Palkovits M, Saavedra JM, Kizer JS. Distribution of hypothalamic hormones and neurotransmitters within the diencephalon. In: Ganong WF, Martini L, eds. Frontiers in neuroendocrinology, chapt. 1. New York: Oxford University Press; 1976; 1:23.
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14. Jansson JO, Eden S, Isaksson O. Sexual dimorphism in the control of growth hormone secretion. Endocr Rev. 1985;6:128-50. 15. Chowen-Breed JA, Steiner RA, Clifton DK. Sexual dimorphism and testosterone-dependent regulation of somatostatin gene expression in the periventricular nucleus of the rat brain. Endocrinology. 1989;125:357-62. 16. Vale W, Vaughan J, Smith M, Yamamoto G, Rivier J, Rivier C. Effects of synthetic ovine corticotropin-releasing factor, glucocorticoids, catecholamines, neurohypophysial peptides and other substances on cultured corticotropic cells. Endocrinology. 1983;113:1121-31. 17. Bridson WE, Kohler PO. Cortisol stimulation of growth hormone production by human pituitary tissue in culture. J Clin Endocrinol Metab. 1970;30:538-40. 18. Wehrenberg WB, Baird A, Ling N. Potent interaction between glucocorticoids and growth hormone-releasing factor in vivo. Science. 1983;221:556-8. 19. Kaufmann S, Jones KL, Wehrenberg WB, Culler FL. Inhibition by prednisone of growth hormone (GH) response to GH-releasing hormone in normal men. J Clin Endocrinol Metab. 1988;67:125861. 20. Casanueva FF, Burguera B, Tome MA, et al. Depending on the time of administration, dexamethasone potentiates or blocks growth hormone-releasing hormone-induced growth hormone release in man. Neuroendocrinology. 1988;47:46-9. 21. Brown MR, Fisher LA, Spiess J, Rivier C, Rivier J, Vale W. Corticotropin-releasing factor: actions on the sympathetic nervous system and metabolism. Endocrinology. 1982;111:928-31.
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Vol. 71, No. 5 Printed in U.S.A.
Corticotropin-Releasing Hormone Inhibition of Growth Hormone-Releasing Hormone-Induced Growth Hormone Release in Man* ANTONINO BARBARINO, SALVATORE M. CORSELLO, SILVIA DELLA CASA, ANNA TOFANI, ROSA SCIUTO, CARLO ANTONIO ROTA, LUCILLA BOLLANTI, AND ANGELA BARINI Institutes of Endocrinology and Biochemistry (A. Bari), Catholic University School of Medicine, 1-00168 Rome, Italy
ABSTRACT. Recent studies in the rat have shown that intracerebroventricular administration of CRH inhibited spontaneous pulsatile GH secretion and prevented GH-releasing hormone (GHRH)-induced GH release. We have studied the effect of CRH on GHRH-induced GH release in man. In the first study, CRH was injected iv at three different doses (100, 50, or 25 Mg) at 0800 h together with 50 Mg GHRH in six men and six women. In a second study, 100 Mg CRH were given iv at 0800 h, 1 h before the administration of 50 fig GHRH in five men and five women. Each subject demonstrated a normal GH response after the administration of 50 fig GHRH plus saline. All doses of CRH administered simultaneously with GHRH significantly inhibited GHRH-induced GH release in women [peak value ± SE after GHRH plus saline, 28.9 ± 2.9 Mg/L; after GHRH plus 100 ng CRH, 9.9 ± 0.7 Mg/L (P < 0.001); after GHRH plus 50 Hg CRH, 8.7 ± 0.8 Mg/L (P < 0.001); after GHRH plus 25 ng CRH, 9.5 ± 1.6 Mg/L (P < 0.001)]. In contrast, in men, while a
G
H SECRETION appears to be regulated at the pituitary level by a GH-releasing hormone (GHRH) and a GH release-inhibiting hormone, somatostatin (SS). Several neuropeptides can, in turn, modify GH secretion by acting on the central nervous system to modulate the hypothalamic secretion of GHRH and SS. Recent studies in animals have suggested that endogenous CRH can serve as a chemical mediator in the stress-induced inhibition of GH secretion (1, 2); intracerebroventricular injection of CRH inhibited pulsatile GH secretion and also prevented GHRH-stimulated GH release in rats (1, 2). This suppressive action of CRH seems to involve an increased release of SS, since it was abolished by previous treatment with anti-SS serum (3, 4). These data together with the observation that CRH
dose of 100 fig CRH was capable of suppressing GHRH-induced GH secretion (peak value ± SE, 8.1 ± 0.6 vs. 20 ± 2.9 Mg/L; P < 0.001), no inhibition was observed after 50- and 25-Mg doses. When 100 Mg CRH were injected 1 h before the administration of 50 Mg GHRH, it strongly inhibited GHRH-induced GH secretion in both men (peak value ± SE, 6.2 ± 2.8 vs. 24.6 ± 5.9 fig/ L; P < 0.02) and women (peak value ± SE, 14.2 ± 4.5 vs. 37.8 ± 6.7 Mg/L; P < 0.005), and this inhibition lasted up to 2 h postCRH administration. These results demonstrate that CRH is capable of inhibiting GHRH-induced GH release in both men and women. Furthermore, the findings suggest that a sexual dimorphism in the neuroregulation of GH secretion may be present in man. In view of the inhibitory action of CRH on GH secretion, simultaneous administration of CRH and GHRH for testing should be avoided in clinical practice. («/ Clin Endocrinol Metab 7 1 : 1368-1374, 1990)
was capable of inhibiting gonadotropin release in normal nonstressed women (5) prompted us to examine the effect of CRH on GH secretion in humans. Since, unlike in the rat (6, 7), the occurrence of spontaneous GH peaks cannot be easily predicted in the conscious man, we have elected to study the effect of CRH on GHRH-stimulated GH release in normal nonstressed men and normal cycling women. Materials and Methods
Received August 14,1989. Address all correspondence and requests for reprints to: Antonino Barbarino, M.D., via Diano Marina 9/11,1-00168 Rome, Italy. * Presented in part at the 71st Annual Meeting of The Endocrine Society, Seattle, WA, June 21-24, 1989 (Abstract 1760).
Nine normal women, aged 19-26 yr, and nine normal men, aged 18-25 yr, were studied after they had given informed consent. All subjects were nonstressed. All were within 10% of ideal body weight (weight range, 62-77 kg for men and 52-66 kg for women) and were eating their customary diet before and during the interval between the tests. None had evidence of any endocrine disorder or was taking any medication. All women had regular menstrual cycles. After an overnight fast, all subjects were kept in bed from 0700 h until the end of the tests. An iv catheter was inserted
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CRH INHIBITION OF GH RELEASE in one arm 30 min before starting the tests. It was used for drug injection and blood sampling, as specified below. Protocol 1
In 12 subjects (6 women and 6 men) 4 separate GHRH stimulation tests were performed. A 50-/*g bolus dose of GHRH was injected iv at time zero (0800 h) together with either saline (2-cc bolus) or 3 different doses of CRH (100-, 50-, or 25-Mg bolus). Blood samples were collected 30, 15, and 0 min before and 15, 30, 60, and 90 min after GHRH administration.
Protocol 2
In 10 subjects (5 women and 5 men, of whom 2 women and 2 men were also included in protocol 1) two separate GHRH stimulation tests were performed. A 50-^g bolus dose of GHRH was injected iv at time zero (0900 h), 1 h after either saline (2cc bolus) or CRH (100-Mg bolus) injection. Blood samples were collected 75, 60, 45, 30,15, and 0 min before and 15, 30, 60, and 90 min after GHRH administration. In individual subjects, the tests were performed at least at 72-h intervals, in a blind fashion. In women they were performed during the luteal phase of the same or a different cycle. The time of the cycle was established by measuring estradiol and progesterone levels. GHRH-(l-44) and human CRH were furnished by Bachem (Bubendorf, Switzerland). The administered doses of GHRH and CRH were diluted in 2 cc saline. Plasma GH levels were measured in duplicate by RIA, using polyethylene glycol to separate free and bound hormone, with reagents obtained by Biodata (Milan, Italy). Normal plasma GH levels ranged from 0.25-10 fig/L.. One nanogram of GH corresponded to 2 fi\J/mL WHO 66/217 standard. The sensitivity of the assay was 0.08 Mg/L- The intra- and interassay coefficients of variation ranged from 2.5-3.9% and from 5.88.5%, respectively. Plasma concentrations of estradiol, progesterone, testosterone, and cortisol levels were measured by RIA, as previously described (8, 9). All samples from a given study were measured in the same assay for each hormone. Plasma glucose levels were measured, using a polarographic method (Beckman Instruments, Palo Alto, CA), in five subjects of protocol 1, when 50 fig GHRH plus 100 ng CRH were administered. The results were evaluated by analysis of variance for repeated measures. The means of each time were compared between control (GHRH plus saline) and experimental groups (GHRH plus CRH at various doses) by Newman-Keuls test. The results are given as the mean ± SE. The significance level was established at P < 0.05.
Results Protocol 1
The effects of different doses of CRH, when simultaneously injected with GHRH, on GHRH-induced GH release and plasma cortisol concentrations in women and
1369
men are shown in Figs. 1 and 2, respectively. In women, the GH response to GHRH was significantly reduced after all doses of CRH compared to that after saline injection. The mean peak GH value was diminished from 28.9 ± 2.9 fig/L (GHRH plus saline) to 9.9 ± 0.7 Mg/L (GHRH plus 100 fig CRH; P < 0.001), 8.7 ± 0.8 Mg/L (GHRH plus 50 fig CRH; P < 0.001), and 9.5 ± 1.6 fig/L (GHRH plus 25 fig CRH; P < 0.001; Fig. 3). In contrast, in men only a dose of 100 /ug CRH was able to significantly suppress the GH response to GHRH, with a mean peak GH value diminished from 20 ± 2.9 fig/L (GHRH plus saline) to 8.1 ± 0.6 fig/L (P < 0.001). No significant inhibition was observed after 50- and 25Hg doses (GH peaks, 19.8 ± 2.9 and 17.4 ± 4.3 fig/h, respectively; Figs. 2 and 3). Plasma cortisol concentrations increased 111% in women and 95% in men after 100 fig CRH (expressed as peak values vs. the means of three basal values). The first significant increase was apparent 15 min after CRH administration. When 50 fig CRH, 25 fig CRH, or saline were given, plasma cortisol did not increase significantly during the period of sample collection. Protocol 2
The effect of 100 fig CRH, administered 1 h before GHRH, on GHRH-induced GH release in men and women is shown in Fig. 4. After CRH administration, the GH response to GHRH was significantly reduced compared to that after saline injection. The peak GH value was diminished from 37.8 ± 6.7 to 14.2 ± 4.5 Mg/L in women (P < 0.005) and from 24.6 ± 5.9 to 6.2 ± 2.8 fig/L in men (P < 0.02). When CRH was given, the plasma cortisol concentration increased 166% in women and 181% in men (expressed as peak values vs. the means of two basal values). The first significant increase was apparent 15 min after CRH injection. When saline was given, plasma cortisol did not increase during the period of sample collection. CRH produced facial flushing in four of the subjects in whom a 100-fig bolus was injected iv. Besides this, no other side-effects were noted. There was no change in supine blood pressure or heart rate. No significant increase in plasma glucose levels was observed in the five subjects in whom glucose was measured after GHRH plus 100 fig CRH (basal values 4.88 ± 0.23 mmol/L, peak values 5.20 ± 0.32 mmol/L). Estradiol and progesterone plasma concentrations in women were determined during the luteal phase of the menstrual cycle (483.1 ± 36.3 pmol/L and 52.7 ± 13.5 nmol/L, respectively). Plasma testosterone concentrations in men were within the adult normal male range (21.5 ± 3.5 nmol/L).
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JCE & M • 1990 Vol 71 • No 5
BARBARINO ET AL.
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G H R H 50 ug CRH 100 ug or
30L
FIG. 1. Mean (±SE) GH (left panel) and cortisol (rightpanel) responses to administration of saline plus GHRH ( ) and CRH (100, 50, or 25 fig) plus GHRH • — • ) in six normal women. *, P < 0.001, comparing values after GHRH plus CRH with those after GHRH plus saline.
GHRH
Saline
50 H9
C R H 50 >ig
500. 20.
300.
or I O O
O
30 H
GHRH
100.
50 M9
+ CRH
25 Mg
500. 20.
O
E
x o
C
300
o
100
10.
30
15
0
15
30
M INUTES
Discussion The observation that iv administration of CRH could significantly decrease plasma gonadotropin levels in normal nonstressed women during the late follicular and luteal phases of the cycle (5) raised the possibility that in humans, CRH could also participate in a variety of hormonal responses and exert an inhibitory role in the
60
90
30
15
0
15
30
60
90
MINUTES
regulation of GH secretion. While spontaneous GH bursts occur regularly in the conscious rat (6, 7), in man GH peaks do not occur regularly and are greatly influenced by metabolic events (10). Nocturnal GH secretion is characterized in the adult man by one large secretory pulse occurring around sleep onset. The infusion of CRH to normal sleeping subjects somewhat reduced GH release and induced sev-
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CRH INHIBITION OF GH RELEASE
1371
GHRH 50 yg 3d,
CRH 100 ug or Saline 500.
300
O O 100
500.
FIG. 2. Mean (±SE) GH {left panel) and cortisol (rightpanel) responses to administration of saline plus GHRH ( ) and CRH (100, 50, or 25 ug) plus GHRH (•—•) in six normal men. *, P < 0.001, comparing values after GHRH plus CRH with those after GHRH plus saline.
O
E c 300.
O O 100.
500
300.
O
O 100
-30
15
0
15
30
MINUTES
eral changes in sleep architecture, indicating a possible inhibitory action of CRH on spontaneous GH secretion (11). Previous studies on the effect of exogenous GHRH on GH secretion have shown that GHRH, in appropriate doses, selectively induces GH release in both men and women (12). A considerable variability of the GH responses to GHRH among individual subjects has been
30
15
0
15
30
60
90
MINUTES
previously reported (12). In addition, the nutritional status, body weight, and time of experiment can modify the GH response to GHRH (10). In an effort to eliminate endogenous as well as exogenous factors that may influence the GH response to exogenously administered GHRH, we have studied subjects of similar age, nutritional status, and body weight and compared in each subject the magnitude of the GH
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BARBARINOETAL.
1372 30-i
20-
10-
GHRH SALINE
GHRH CRH toot
GHRH CRH 50(10
G HRH CRHlOOpg
CRH
GHRH CRH
30 -i
20-
10-
SALINE
G HRH
G HRH
CRH 25\XQ
FlG. 3. Mean (±SE) basal and peak values of plasma GH after the simultaneous injection of GHRH (50 ng) plus saline and GHRH (50 ixg) plus CRH (100, 50, and 25 fig) in six normal men (upper panel) and six normal women (lower panel). The mean of three basal values has been considered the basal level. *, P < 0.001, comparing peak values after GHRH plus CRH with those after GHRH plus saline.
response to GHRH alone and GHRH plus CRH administration, so that each individual served as his/her own control. Normal nonstressed men and women studied at 0900 h after an overnight fast exhibited rapid GH release after iv administration of 50 ng GHRH. This response was quite variable among the subjects. Nevertheless, graded doses (100, 50, and 25 fig) of CRH had a differential inhibitory effect on GH secretion in the two sexes when the same subject was studied repeatedly. In women, GHRH-induced GH secretion was significantly inhibited after all doses of CRH, whereas in men CRH induced GH inhibition only after a 100-jig dose. In addition, a dose of 100 ng CRH injected 1 h before GHRH administration was also effective in inhibiting GHRH-induced GH secretion in both normal men and women. These results are in agreement with experiments showing that in rats the icv injection of CRH prevented spontaneous as well as GHRH-induced GH secretion (1, 2). Our
JCE & M • 1990 Vol 71 • No 5
results also indicate that CRH exerts an inhibitory influence on pituitary hormone secretion for up to 2 h after its administration and are in agreement with our previous data demonstrating that after CRH injection, basal LH secretion significantly decreased during the 4 h following CRH injection and returned to normal levels 5 h after injection (5). The mechanism by which CRH exerts its inhibitory action on GHRH-induced GH release is not evident from our experiments. However, previous animal studies have shown that this effect is possibly mediated by SS, since SS antiserum blocked the inhibitory action of CRH in conscious rats (3, 4). Studies on brain localization of SS have shown high concentration of this hormone in various areas of the central nervous system, with the highest concentration found in the median eminence (13). It is, therefore, possible that large doses of peripherally injected CRH can reach the central nervous system and induce somatostatin release from brain sites outside the blood-brain barrier, especially in the region of the median eminence, and this, in turn, will inhibit GHRH action at the pituitary level. Of some interest is our observation that in female subjects CRH was effective at doses much lower than those required in males, even if we consider the difference in body weight range between sexes. These results could be due to gender differences in the neuroregulation of GH secretion. A sexual dimorphism in the secretory pattern of GH has been shown in the mature rat, and it has been attributed to a higher somatostatinergic tone in male animals (14,15). It is possible that, analogously, men have a higher somatostatinergic tone than women, and thus, small doses of peripherally injected CRH are unable to induce a variation in GH secretion in male subjects. On the contrary, women may show an inhibition of GH secretion after small doses of CRH by virtue of a lower somatostatinergic tone. Another possible explanation for our results is that CRH modified the somatotroph responsiveness to GHRH. However, it has been documented in the rat that CRH is devoid of direct effects on GH release at the pituitary level (16). CRH inhibition of GHRH-induced GH release could be mediated by a neurohormonal action of cortisol, which is released after CRH administration. However, reports on the effect of glucocorticoids on GH secretion are conflicting. In vitro studies have shown that glucocorticoids potentiate basal GH secretion in a variety of pituitary culture systems, including human pituitary explants (17). A similar stimulatory action of corticosteroids on GHRH-stimulated GH secretion has been demonstrated in vivo in the rat after administration of 40 Mg/day dexamethasone for 7 days (18). On the other hand,
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CRH INHIBITION OF GH RELEASE
GHRM so pg
N-5
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C R H IOOyg or saline iv
GHRH soug
N=5
20
10
-75 -6O
-45
-3O -15
-75 -6O - 4 5
-30
FlG. 4. Mean (±SE) GH responses to administration of saline plus GHRH (•—•) and CRH plus GHRH (O—O) in five normal women (left panel) and five normal men (right panel). CRH or saline was given 1 h before GHRH. *, P < 0.02; **, P < 0.005 (comparing values after GHRH plus CRH with those after GHRH plus saline).
administration of 60 mg/day prednisone for 4 days to normal men decreased their GH response to GHRH (19). Thus, administration of glucocorticoids for several days (4-7) seems to elicit differential effects on GH secretion in the rat and man. Very recently, Casaneuva et al. (20) have reassessed the effects of very short term administration of glucocorticoids on GHRH-induced GH secretion in normal men. These researchers have demonstrated that 4 mg dexamethasone given 3 h before GHRH significantly potentiated GHRH-induced GH secretion, while 8 mg dexamethasone given 12 h before GHRH administration completely blocked GH secretion (20). These last results seem to exclude the possibility that the suppressive effect of CRH observed in the present paper is due to hypercortisolism, which occurs after CRH administration. Rather, a potentiating effect of cortisol on GH secretion should have occurred, especially when 100 ng CRH were administered together with GHRH. Our results strongly suggest that CRH per se inhibits GH secretion, and this counteracts the stimulatory action of the concomitant hypercortisolism that is present after injection of large doses of CRH. Additional support for lack of mediation by cortisol comes from present data in females, in which CRH at 50 and 25 fig inhibited GHRH-induced GH release despite no significant increase in plasma cortisol. Although it has been reported that the icv administration of CRH produces hyperglycemia in rats (21), the lack of any increase in plasma glucose levels after iv CRH injection in our subjects excludes a hyperglycemiamediated attenuation of the GH response to GHRH.
In conclusion, our present findings demonstrate that CRH is capable of inhibiting GHRH-induced GH release in both men and women, and suggest that this action of CRH is not dependent on its ability to stimulate cortisol secretion. Moreover, our results suggest that CRH modulation of GH secretion is different in men and women. Simultaneous administration of CRH and GHRH for testing should be avoided in clinical practice in view of the inhibitory effect of CRH on GH secretion demonstrated in this study. References 1. Rivier C, Vale W. Corticotropin-releasing factor (CRF) acts centrally to inhibit growth hormone secretion in the rat. Endocrinology. 1984;114:2409-ll. 2. Ono N, Lumpkin MD, Samson WK, McDonald JK, McCann SM. Intrahypothalamic action of corticotropin-releasing factor (CRF) to inhibit growth hormone and LH release in the rat. Life Sci. 1984;35:1117-23. 3. Katakami H, Arimura A, Frohman LA. Involvement of hypothalamic somatostatin in the suppression of growth hormone secretion by central corticotropin-releasing factor in conscious male rats. Neuroendocrinology. 1985;41:390-3. 4. Rivier C, Vale V. Involvement of corticotropin-releasing factor and somatostatin in stress-induced inhibition of growth hormone secretion in the rat. Endocrinology. 1985;117:2478-82. 5. Barbarino A, De Marinis L, Folli G, et al. Corticotropin-releasing hormone inhibition of gonadotropin secretion during the menstrual cycle. Metabolism. 1989;38:504-6. 6. Tannenbaum GS, Martin JB. Evidence for an endogenous ultradian rhythm governing growth hormone secretion in the rat. Endocrinology. 1976;98:562-70. 7. Katakami H, Kato Y, Matsushita N, Hiroto S, Shimatsu A, Imura
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9.
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12. 13.
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H. Involvement of alpha-adrenergic mechanisms in growth hormone release induced by opioid peptides in conscious rats. Neuroendocrinology. 1981;33:129-35. Barbarino A, De Marinis L, Anile C, Menini E, Merlini G, Maira G. Dopaminergic mechanisms regulating prolactin secretion in patients with prolactin secreting pituitary adenoma. Long term studies after selective transsphenoidal surgery. Metabolism. 1982;31:1100-4. De Marinis L, Mancini A, Calabro' F, Massari M, Torlontano M, Barbarino A. Differential effects of a dopaminergic drug (piribedil) on pituitary hormone release in normal men and women. Acta Endocrinol (Copenh). 1983;104:385-9. De Marinis L, Folli G, D'Amico C, et al. Differential effects of feeding on the ultradian variation of the GH response to GHRH in normal subjects and patients with obesity and anorexia nervosa. J Clin Endocrinol Metab. 1988;66:598-604. Holsboer F, von Bardeleben U, Steiger A. Effect of intravenous corticotropin-releasing hormone upon sleep-related growth hormone surge and sleep EEG in man. Neuroendocrinology. 1988;48:32-8. Thorner MO, Vance ML, Evans S. Physiological and clinical studies of GRF and GH. Recent Prog Horm Res. 1986;42:589-640. Brownstein MJ, Palkovits M, Saavedra JM, Kizer JS. Distribution of hypothalamic hormones and neurotransmitters within the diencephalon. In: Ganong WF, Martini L, eds. Frontiers in neuroendocrinology, chapt. 1. New York: Oxford University Press; 1976; 1:23.
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14. Jansson JO, Eden S, Isaksson O. Sexual dimorphism in the control of growth hormone secretion. Endocr Rev. 1985;6:128-50. 15. Chowen-Breed JA, Steiner RA, Clifton DK. Sexual dimorphism and testosterone-dependent regulation of somatostatin gene expression in the periventricular nucleus of the rat brain. Endocrinology. 1989;125:357-62. 16. Vale W, Vaughan J, Smith M, Yamamoto G, Rivier J, Rivier C. Effects of synthetic ovine corticotropin-releasing factor, glucocorticoids, catecholamines, neurohypophysial peptides and other substances on cultured corticotropic cells. Endocrinology. 1983;113:1121-31. 17. Bridson WE, Kohler PO. Cortisol stimulation of growth hormone production by human pituitary tissue in culture. J Clin Endocrinol Metab. 1970;30:538-40. 18. Wehrenberg WB, Baird A, Ling N. Potent interaction between glucocorticoids and growth hormone-releasing factor in vivo. Science. 1983;221:556-8. 19. Kaufmann S, Jones KL, Wehrenberg WB, Culler FL. Inhibition by prednisone of growth hormone (GH) response to GH-releasing hormone in normal men. J Clin Endocrinol Metab. 1988;67:125861. 20. Casanueva FF, Burguera B, Tome MA, et al. Depending on the time of administration, dexamethasone potentiates or blocks growth hormone-releasing hormone-induced growth hormone release in man. Neuroendocrinology. 1988;47:46-9. 21. Brown MR, Fisher LA, Spiess J, Rivier C, Rivier J, Vale W. Corticotropin-releasing factor: actions on the sympathetic nervous system and metabolism. Endocrinology. 1982;111:928-31.
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