0021-972X/90/7101-0157$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society
Vol. 71, No. 1 Printed in U.S.A.
Impaired Inhibitory Effects of Somatostatin on Growth Hormone (GH)-Releasing Hormone Stimulation of GH Secretion after Short Term Infusion* MIRTHA KELIJMAN AND LAWRENCE A. FROHMAN Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
ABSTRACT. We examined the effect of prior exposure to somatostatin (SRIH) on its inhibition of GH and TSH responses to GHRH and TRH stimulation to determine whether SRIH desensitization has physiological significance in man. Six men received GHRH (1 Mg/kg, iv) and TRH (0.3 jig/kg, iv) 20 min after starting a saline or SRIH (5.5 ng/kg/min, iv) infusion and again 6 h later. Hormone responses were quantified by measuring the area under the curve, corrected for GH concentration at injection time. Similar results were obtained when GH responses were quantified by measuring the hormone secretory rate using the program Detect. Plasma GH and TSH responses to the two GHRH and TRH injections during saline were similar. However, the effects of prior exposure to SRIH were hormone specific. SRIH blunted GH responses to GHRH at 20 min (1609 ± 286 jug/L-min us. 451 ± 224), but did not significantly inhibit the responses 6 h later (1422 ± 410 Mg/Lmin us. 1000 ± 302). In contrast, SRIH inhibition of TSH responses to the two TRH injections was similar (first, 946 ± 201 /ig/L-min vs. 700 ± 148; second, 813 ± 175 )ig/L-min us. 562 ± 66). We next used these results to study whether the previously reported attenuation of GH responses to repeated GHRH stimulation at 2-h intervals is
mediated by SRIH. Eight men received GHRH (1 Mg/kg, iv) 380 min after starting a saline or SRIH (5.5 ng/kg/min, iv) infusion or 90 min after starting a primed (5 mg, iv) infusion of propranolol (80 Mg/min, iv) and again 2 h later. As in the first protocol, GH responses to GHRH were not inhibited when preceded by a 6-h SRIH infusion. However, the 6-h SRIH infusion resulted in a partial restoration of plasma GH responses to the second GHRH injection (saline infusion: first, 1429 ± 342 ^g/L-min; second, 254 ± 75; SRIH infusion: first, 1042 ± 247 Mg/L-min; second, 468 ± 105). /3-Blockade by propranolol resulted in enhanced GH responses to GHRH, but did not prevent the attenuation of GH responses to the second GHRH injection (first, 1937 ± 366 Mg/Lmin; second, 614 ± 99). The desensitization to SRIH inhibition of GH responses to GHRH after a 6-h SRIH infusion provides evidence of physiological consequences of SRIH receptor down-regulation. The impaired GH responses to repeated GHRH stimulation are mediated at least in part by enhanced SRIH secretion, which appears independent of a 0adrenergic mechanism. (J CUn Endocrinol Metab 7 1 : 157-163, 1990)
T
physiological significance of SRIH receptor down-regulation has not been reported. In the present study we have investigated whether SRIH receptor desensitization occurs under physiological circumstances in man. Our approach was to determine whether prior exposure to physiological concentrations of SRIH attenuates its suppressive effects on pituitary responsiveness by comparing SRIH-induced inhibition of GH and TSH responses to GHRH and TRH stimulation at the beginning and after a continuous SRIH infusion of sufficient duration to produce receptor down-regulation. It has been previously reported that the GH response to repeated GHRH injections at 2-h intervals is attenuated (6). To test the hypothesis that this reduction is mediated by enhanced SRIH secretion, we determined whether it could be prevented by desensitizing the pituitary to SRIH action by means of prior SRIH exposure. In addition, we explored the possible /3-adrenergic mediation of this effect with the use of propranolol, a purported inhibitor of SRIH secretion (7).
HE SECRETION of GH is regulated by both stimulatory [GH-releasing hormone (GHRH)] and inhibitory [somatostatin (SRIH)] hypothalamic hormones. The pituitary response to these hormones is influenced not only by their concentrations in hypothalamic-pituitary portal blood, but also by the level of their respective receptors on the somatotroph. In experimental animals, GHRH and SRIH receptors have been reported to exhibit down-regulation in vitro (1, 2) and in vivo (3), suggesting a potential role of this process in the control of GH secretion. While several studies have examined the alterations in plasma GH responses to GHRH induced by prior GHRH administration in man (4, 5), the Received August 21,1989. Address all correspondence and requests for reprints to: Lawrence A. Frohman, M.D., Division of Endocrinology and Metabolism, University of Cincinnati Medical Center, 231 Bethesda Avenue, ML 547, Cincinnati, Ohio 45267. * This work was supported in part by USPHS Grants DK-30667 and RR-00068 and a grant from Genentech, Inc. 157
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 18:02 For personal use only. No other uses without permission. . All rights reserved.
KELIJMAN AND FROHMAN
158
Materials and Methods Subjects Thirteen healthy men, aged 19-24 yr (mean ± SE, 22 ± 0.5) and of normal body weight (81.9 ± 1.7 kg), participated in the study after giving informed consent to the protocols, which were approved by the Institutional Review Board. The protocols were performed in the University of Cincinnati General Clinical Research Center, with an interval of at least 7 days between studies. The order of the studies in each protocol was randomly assigned. The subjects fasted for 8 h before and during the studies of the first protocol. Breakfast was kept constant for each subject and was served immediately after starting each study of the second protocol. Heparin lock cannulae were placed in a vein in each forearm, one for infusions and the other for iv injections and blood sampling. Drugs
JCE & M • 1990 Vol 71 • No 1
min intervals throughout the studies. Blood samples for SRIH were collected in heparinized tubes containing aprotinin (Trasylol, Mobay Chemical Corp., New York, NY) during each SRIH infusion at the time of each injection and 5 min before the first injection in the first protocol. Samples for plasma glucose were obtained at 15- to 60-min intervals throughout the studies. All blood samples were immediately chilled, centrifuged at 4 C, and stored at -20 C until assayed. Plasma GH and TSH levels were measured by RIA as previously described (11,12). The intra- and interassay coefficients of variation were 6.1% and 6.8%, and 7.2% and 7.8%, respectively. SRIH concentrations were determined after extraction by RIA in a single assay, as previously described (8, 13), with an intraassay coefficient of variation of 5.4%. We have previously reported plasma SRIH levels in control subjects using this method to be 5 ± 1 ng/L (mean ± SE) (8). Plasma glucose levels were measured by a glucose oxidase method. Statistical analysis
GHRH-(l-44) (Hoffmann-La Roche, Inc., Nutley, NJ) was dissolved in normal saline at a concentration of 100 ^g/mL. TRH (Relefact, Hoechst-Roussel Pharmaceuticals, Inc., Somerville, NJ) was dissolved in 1% human serum albumin (HSA) at a concentration of 20 /ug/mL. Somatostatin (Stilamin, Serono Laboratories, Inc., Randolph, MA) was dissolved in HSA and stored at 4 C. Aliquots of the stock solution were diluted in HSA immediately before administration. SRIH and propranolol (Inderal, Ayerst Laboratories, Inc., New York, NY) were infused, as described below, via a Harvard pump (Harvard Apparatus, Millis, MA). Experimental design Attenuation of SRIH inhibitory effects. On separate days, six subjects received 500-min infusions of saline containing 1% HSA or SRIH (5.5 ng/kg/min, iv), a dose estimated to inhibit plasma GH responsiveness to GHRH by approximately 60% (8). Each study was preceded by a 30-min stabilization period. In each study, GHRH (1 Mg/kg, iv) and TRH (0.3 fig/kg, iv) were injected 20 min after starting the infusions and again 6 h later. The timing of the first injection was chosen to permit plasma SRIH concentrations to reach equilibrium (9). That of the second, 6 h later, was chosen on the basis of 1) the reported lack of attenuation of a repeated GHRH injection at this interval (10), and 2) the demonstration that this interval was sufficient to demonstrate SRIH receptor down-regulation in experimental animals (3). Mediation of GH response attenuation to reaeated GHRH injections. On separate days eight subjects received 620-min infusions of saline or SRIH. In a third study, subjects received a saline infusion for 290 min, followed by a primed (5 mg, iv) infusion of propranolol (80 /ig/min, iv) for the remainder of the 620-min infusion period. GHRH was injected 380 min after the start of each study and again 2 h later. The doses of SRIH and GHRH were the same as in the first protocol. Sample collection and assays. Heparinized blood samples for measurement of GH and TSH were obtained at 15-min intervals for 2 h after GHRH and TRH injections and at 15- to 60-
The 2-h integrated hormonal secretory responses to GHRH and TRH were determined by the trapezoidal method [area under the curve (AUC)] after correcting for preinjection (baseline) values. In the second protocol, the plasma GH concentration had not returned to basal levels by the time of the second GHRH injection. We used two approaches to distinguish the responses to each injection. In the first, the total AUC after the second injection was corrected by subtracting that fraction attributable to the GH response to the first, calculated on the basis of the GH half-life (14). In the second, the instant secretory rates for the 2 h after each injection were determined using the program Detect (15). The results obtained with each method were highly correlated (r = 0.995; P < 0.001). Group differences were determined by repeated measures two-way analysis of variance, after log transformation, when indicated. P < 0.05 was considered significant.
Results Attenuation of the inhibitory effects of SRIH Plasma GH responses to GHRH and TRH are shown in Fig. 1, and the integrated secretory responses and secretory rates are given in Table 1. Plasma GH responses to the two GHRH injections during saline infusion were similar. During the SRIH infusion, GH responses to the first GHRH injection were significantly blunted (P < 0.05), while those to the second injection were not. As a consequence, the responses to the second injection were greater than those to the first (P < 0.05). Inhibition of plasma TSH responses to the first TRH injection during SRIH infusion (Fig. 2 and Table 1) was less pronounced than that of the plasma GH responses to the first GHRH infusion (AUC, 19 ± 11% vs. 64 ± 15%; P = 0.01). After a 6-h infusion, the inhibitory effects of SRIH on the TSH responses to TRH remained intact. Plasma SRIH concentrations at the time of each GHRH injection (first, 71 ± 17 ng/L; second, 87 ± 19)
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 18:02 For personal use only. No other uses without permission. . All rights reserved.
EFFECT OF SRIH ON GHRH-STIMULATED GH SECRETION
159
SALINE or SRIH, 5.5 ng/kg/min
SALINE or SRIH, 5.5 ng/kg/min
TRH
TRH
20
10
-30
0 20
140
260
380
500 -30
0 20
140
260
380
500
MINUTES
FlG. 1. Effect of a continuous SRIH infusion on the plasma GH responses to repeated GHRH stimulation (1 fig/kg, iv) at a 6-h interval in six normal subjects. Shown are the mean ± SE. TABLE 1. Effects of SRIH and the sequence of stimulation on the GH and TSH responses to GHRH and TRH injections administered at a 6-h interval First injection Plasma GH responses to GHRH Saline (6) AUC (Mg/Lmin) 1609 ± 286 SR (ng/min) 59 ± 1 1 SRIH (6) AUC(Mg/L-min) 451 ± 224° SR (ng/min) 18±8a Plasma TSH responses to TRH Saline (6) AUC(mU/L-min) 946 ± 201 SRIH (6) AUC(mU/L-min) 700 ± 148°
Second injection
1422 ± 410 54 ± 16 1000 ± 302* 37 ± 11*
813 ± 175 562 ± 66°
Values are the mean ± SE. The number of subjects is shown in parentheses. AUC represents the 2-h integrated secretory response corrected for baseline values at the time of injection. SR represents the 15-min secretory rate during the 2-h period after injection. " P < 0.05 us. saline. * P < 0.05 vs. first GHRH injection.
were similar, as were concentrations 5 min before the first injection (86 ± 16 ng/L), indicating that equilibrium had been reached at the time of the first injection. Plasma glucose concentrations at the time of each injection were similar (data not shown).
MINUTES
FlG. 2. Effect of a continuous SRIH infusion on the plasma TSH responses to repeated TRH stimulation (0.3 Mg/kg, iv), at a 6-h interval in six normal subjects. Shown are the mean ± SE.
Effects of SRIH and propranolol on the GH responses to a repeated GHRH injection at 2 h SRIH infusion. Plasma GH responses to GHRH during saline and SRIH infusions are shown in Fig. 3 and Table 2. The first GHRH injection was administered 380 min after the start of the saline or SRIH infusion, a time comparable to that of the second GHRH injection in the previous protocol. SRIH infusion failed to inhibit plasma GH responses to the first GHRH injection, confirming the results of the first protocol. During the saline infusion, plasma GH responses to the second GHRH injection were nearly abolished. However, a SRIH infusion, started 8 h earlier, partially restored the GH responses to the second GHRH injection. Plasma glucose concentrations were slightly lower at the time of the second compared to those at the time of the first GHRH injection during both saline (first, 5.1 ± 0.1 mmol/L; second, 4.7 ± 0.1) and SRIH infusions (first, 5.2 ± 0 . 1 mmol/L; second, 5.0 ± 0.1). Plasma glucose levels at the time of each GHRH injection during saline and SRIH infusions were, however, not statistically different. Propranolol infusion. Plasma GH responses to GHRH during saline and propranolol infusions are shown in Fig. 4 and Table 2. The effects of propranolol and of the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 18:02 For personal use only. No other uses without permission. . All rights reserved.
KELIJMAN AND FROHMAN
JCE & M • 1990 Vol 71 • No 1
30 SALINE or PROPRANOLOL, 5.5 mg + 80 ug/min
SALINE
SALINE or SRIH, 5.5 ng/kg/min
GHRH
GHRH
A
V 20
o CO
I
Q- 10
SAUNE PROPRANOLOL
200 200
260
500
620
MINUTES
FIG. 3. Effect of a continuous SRIH infusion on the plasma GH responses to repeated GHRH stimulation (1 fig/kg, iv) at a 2-h interval in eight normal subjects. Shown are the mean ± SE. TABLE 2. Effects of SRIH and propranolol and the sequence of stimulation on the GH responses to GHRH injections administered at a 2h interval
Saline (8) AUC(Mg/L-min) SR (ng/min) SRIH (8) AUC(Mg/L-min) SR (ng/min) Propranolol (8) AUC (/xg/L-min) SR (ng/min)
First injection
Second injection
1429 ± 342 58 ± 14
254 ± 75° 12 ±3°
1042 ± 247 39 ± 9
468 ± 105 17 ± 4
1937 ± 366 71 ±14
614 ± 99a'6 24 ± Aa'b
Values are the mean ± SE. The number of subjects is shown in parentheses. AUC represents the 2-h integrated secretory response corrected for the plasma GH levels at the time of injection, as described in Materials and Methods. SR represents the 15-min secretory rate during the 2-h period after injection. 0 P < 0.05 us. first injection. 6 P < 0.05 vs. saline.
sequence of stimulation were determined independently of one another, since an analysis of variance revealed no statistical evidence of their interaction (AUC, P = 0.26; SR, P = 0.38). Propranolol infusion enhanced the GH responses to GHRH. However, despite ^-blockade, the GH responses to the second GHRH injection were sig-
260
380
MINUTES
FIG. 4. Effect of propranolol infusion on the plasma GH responses to repeated GHRH stimulation (1 jtg/kg, iv) at a 2-h interval in eight normal subjects. Shown are the mean ± SE.
nificantly lower than those to the first injection. The mean plasma glucose level during propranolol infusion was slightly lower at the time of the second (4.9 ±0.1 mmol/L) compared to that at the time of the first GHRH injection (5.2 ± 0 . 1 mmol/L). Plasma glucose levels at the time of each GHRH injection during saline and propranolol infusions were not statistically different.
Discussion Down-regulation of pituitary SRIH receptors has been demonstrated in vitro by direct exposure of somatotrophs to SRIH. The physiological significance of this phenomenon is, however, unclear, since exposure of cultured rat pituitary cells to SRIH concentrations 2 orders of magnitude higher than the IC50 for inhibiting GH release was needed to induce significant down-regulation (2). In experimental animals injection of GH, a potent stimulus for SRIH release, results in decreased binding capacity of pituitary SRIH receptors (3). In humans no attenuation of the inhibition of GH secretion has been observed after long term treatment of acromegaly with SMS 210995, a SRIH analog (16). However, there are no reports of dose-response studies of SRIH sensitivity before and during treatment with this agent. Our first protocol was designed to investigate whether SRIH desensitization occurs in man under physiological
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 18:02 For personal use only. No other uses without permission. . All rights reserved.
EFFECT OF SRIH ON GHRH-STIMULATED GH SECRETION
conditions. During saline infusion, plasma GH responses to GHRH injections administered at a 6-h interval were similar, indicating the absence of interaction between the two GHRH injections. SRIH infusion resulted in a 64% inhibition of GH responsiveness to GHRH administered shortly after starting the infusion. In contrast, there was no significant inhibition of plasma GH responses to GHRH after the 6-h SRIH infusion, suggesting that somatotroph responsiveness to SRIH was diminished by previous exposure to the hormone. Plasma SRIH concentrations in the human hypothalamic-pituitary portal system are unknown. However, the incomplete somatotroph inhibition observed and the reports of SRIH levels in the portal system of rats (17) and sheep (18) similar to those in the present study suggest that the plasma SRIH levels achieved during our infusions were within the physiological range. The relative increase in the GH response to the second injection of GHRH during SRIH infusion could alternatively be explained by an up-regulation of GHRH receptors or the accumulation of a readily releasible pool of GH. Although SRIH can suppress GHRH secretion (19), which, in turn, might be expected to up-regulate GHRH receptors, there is no evidence indicating that this can occur in response to systemic, as opposed to central, SRIH administration. Continuous infusion of SRIH does lead to the accumulation of a readily releasible pool of GH, demonstrable upon elimination of the SRIH influence (20). However, it is uncertain whether this pool is either accessible to GHRH stimulation (21) or releasible during the continued presence of SRIH. Thus, our results are most consistent with SRIH desensitization, although the level at which this occurs {e.g. receptor down-regulation or a postreceptor event, such as second messenger alteration) cannot be inferred from indirect studies as in the present report. Decreased responsiveness to SRIH after a relatively brief exposure to physiological concentrations of the hormone is consistent with a participatory role of SRIH receptor down-regulation in the control of GH secretion. Down-regulation of SRIH receptors may contribute to the ultradian rhythm of GH secretion. Bursts of GH secretion occur upon interruption of a SRIH infusion, and a pattern of intermittent SRIH administration in rats results in pulsatile GH secretion with increased body weight (22). In addition, episodic pulses of GH secretion in experimental animals are associated with asynchronous changes in hypothalamic GHRH and SRIH secretion (17, 18). Thus, SRIH receptor down-regulation that occurs as a result of SRIH pulses might potentiate the effects of trough SRIH concentrations both on the somatotroph (23), resulting in increased GH secretion, and on the hypothalamic arcuate nucleus, resulting in enhanced GHRH secretion (24).
161
The present study confirms our previous observation that SRIH inhibition of the TSH response to TRH is considerably less potent than that of the GH response to GHRH (8). SRIH inhibition of the TSH response to TRH, unlike that of the GH response to GHRH, was not impaired after a 6-h SRIH infusion. The discordant effects of a previous SRIH infusion on SRIH inhibition of GH and TSH responses to direct stimulation suggest that somatotroph and thyrotroph SRIH receptors have distinct binding characteristics. In an in vitro study by Smith et al. (2), much higher SRIH concentrations inhibited TSH responses to TRH and induced down-regulation of thyrotroph SRIH receptors, suggesting an association between the biological effects of SRIH and its ability to induce desensitization. The mediation of the GH response attenuation to repeated GHRH stimulation has been the subject of considerable debate. GHRH receptor down-regulation has been observed in in vitro studies (1). However, GHRH concentrations similar to or greater than those in plasma as early as 10 min after the iv injection of 1 Mg/kg GHRH (~ 25 pg/mL) (25) fail to produce GHRH receptor down-regulation in vitro (1). Depletion of a readily releasable storage pool of GH is unlikely in view of preserved and even enhanced plasma GH responses to hypoglycemia or arginine or clonidine stimulation after GHRH administration (26-28). However, depletion of a GH pool specifically responsive to GHRH (29) cannot be excluded. Finally, increased hypothalamic SRIH secretion induced by exogenous GHRH (30), GH (31), or locally produced insulin-like growth factor-I (32) might mediate the inhibition of GH responses to repeated GHRH stimulation. To investigate this hypothesis, we attempted to reverse the inhibition by reducing 1) the SRIH effect, based on the results of our first protocol, and 2) SRIH secretion, using propranolol, a purported inhibitor of SRIH release. Infusion of SRIH, started 6 h earlier, failed to significantly inhibit GH responses to the first GHRH injection (confirming the results of the first protocol). Plasma GH responses to the second GHRH injection, which were almost completely abolished during saline infusion, were partially restored during SRIH infusion. The lack of an enhanced GH response to the second GHRH injection in the SRIH-infused group compared to that in the saline-infused group is probably attributable to a residual inhibitory effect of SRIH on GH secretion. The actual restoration of the GH reponse to the second GHRH injection by SRIH pretreatment, therefore, suggests that the impaired GH responses to repeated GHRH stimulation are mediated at least in part by increased hypothalamic SRIH secretion. It is, therefore, possible that the gradual recovery of GH responses to GHRH stimulation repeated every 2 h for 12 h (5) as well as the normal GH responses to
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 18:02 For personal use only. No other uses without permission. . All rights reserved.
KELIJMAN AND FROHMAN
162
GHRH observed after 14 days (33), but not to shorter durations of GHRH infusions or injections, are a manifestation of SRIH receptor desensitization. SRIH inhibits GH responses to all known stimuli, and thus, responses to any stimulus administered after a GHRH injection should seemingly be suppressed by the enhanced SRIH secretion. However, enhanced hypothalamic SRIH secretion induced by previous GHRH stimulation might not impair GH responses to stimuli that are believed to suppress SRIH release (34-36). Propranolol potentiates GH responses to a maximally stimulatory dose of GHRH, suggesting /?-adrenergic stimulation of hypothalamic SRIH secretion (7). In the present study infusion of propranolol enhanced GH responses to each GHRH injection, administered at a 2-h
8.
9.
10.
11. 12.
interval, to a similar degree. However, it did not prevent
the attenuation of GH responses to the second, compared to the first, GHRH injection, indicating that previous GHRH stimulation and ^-blockade modify hypothalamic SRIH secretion through separate mechanisms. In summary, the present study indicates that relatively brief exposure to SRIH at a physiological concentration impairs its inhibition of GH secretion. We suggest that this effect has physiological significance and contributes to the pulsatile pattern of GH secretion. Our results also suggest that the inhibition of GH responses to repeated GHRH stimulation is mediated in part by enhanced hypothalamic SRIH secretion, which appears independent of a /3-adrenergic pathway.
Acknowledgments We are indebted to the staff of the General Clinical Research Center, to Gladys Perisutti and Amy Steinkamp for technical assistance, and to Dr. Peter Laskarzewski and Ismail Kaya for statistical advice. Materials for the GH and TSH RIAs were provided by the National Hormone and Pituitary Program, NIH.
13. 14.
15.
16.
17. 18.
19. 20.
References 1. Bilezikjian LM, Seifert H, Vale W. Desensitization to growth hormone-releasing factor (GRF) is associated with down-regulation of GRF-binding sites. Endocrinology. 1986;118:2045-52. 2. Smith MA, Yamamoto G, Vale WW. Somatostatin desensitization in rat anterior pituitary cells. Mol Cell Endocrinol. 1984;37:311-8. 3. Katakami H, Berelowitz M, Marbach M, Frohman LA. Modulation of somatostatin binding to rat pituitary membranes by exogenously administered growth hormone. Endocrinology. 1985;117:557-60. 4. Vance ML, Kaiser DL, Evans WS, et al. Evidence for a limited growth hormone (GH)-releasing hormone (GHRH)-releasable quantity of GH: effects of 6-hour infusions of GHRH on GH secretion in normal man. J Clin Endocrinol Metab. 1985;60:370-5. 5. Hulse JA, Rosenthal SM, Cuttler L, Kaplan SL, Grumbach MM. The effect of pulsatile administration, continuous infusion, and diurnal variation on the growth hormone (GH) response to GHreleasing hormone in normal men. J Clin Endocrinol Metab. 1986;63:872-8. 6. Shibasaki T, Hotta M, Masuda A, et al. Plasma GH responses to GHRH and insulin-induced hypoglycemia in man. J Clin Endocrinol Metab. 1985;60:1265-7. 7. Chihara K, Kodama H, Kaji H, et al. Augmentation by propranolol
21.
22. 23. 24. 25.
26.
JCE & M • 1990 Vol 71 • No 1
of growth hormone-releasing hormone-(l-44)-NH2-induced growth hormone release in normal short and normal children. J Clin Endocrinol Metab. 1985;61:229-33. Williams TC, Kelijman M, Crelin WC, Downs TR, Frohman LA. Differential effects of somatostatin (SRIH) and a SRIH analog, SMS 201-995, on the secretion of growth hormone and thyroidstimulating hormone in man. J Clin Endocrinol Metab. 1988;66:3945. Skamene A, Patel YC. Infusion of graded concentrations of somatostatin in man: pharmacokinetics and differential inhibitory effects on pituitary and islet hormones. Clin Endocrinol (Oxf). 1984;20:555-64. Losa M, Schopohl J, Muller OA, von Werder K. Stimulation of growth hormone secretion with human growth hormone releasing factors ( G R F ^ , GRF^o, GRF,-29) in normal subjects. Klin Wochenschr. 1984;62:1140-3. Frohman LA, Horton ES, Lebovitz HE. Growth hormone releasing effects of a pseudomonas endotoxin (Piromen). Metabolism. 1967;16:57-67. Pekary AE, Hershman JM, Parlow AF. A sensitive and precise radioimmunoassay for human thyroid stimulating hormone. J Clin Endocrinol Metab. 1975;41:676-84. Kronheim S, Berelowitz M, Pimstone BL. The characterization of somatostatin-like immunoreactivity in human serum. Diabetes. 1978;27:523-9. Henry DP, Nyhart EH, Bowsher RR. Pharmacokinetics of Humatrope (biosynthetic human growth hormone) in man: I.V..I.M., subcutaneous administration. Proc of the 69th Annual Meet of The Endocrine Soc. 1987;924. Oerter KE, Guardabasso V, Rodbard D. Detection and characterization of peaks and estimation of instantaneous secretory rate for episodic pulsatile hormone secretion. Com Biomed Res. 1986;19:170-91. Lamberts SWJ, Uitterlinden P, Del Pozo E. SMS 201-995 induces a continuous decline in circulating growth hormone and somatomedin-C levels during therapy of acromegalic patients for over two years. J Clin Endocrinol Metab. 1987;65:703-10. Plotsky PM, Vale W. Patterns of growth hormone-releasing factor and somatostatin secretion into the hypophysial-portal circulation of the rat. Science. 1985;230:461-3. Frohman LA, Downs TR, Clarke IJ, Thomas GB. Measurement of growth hormone-releasing hormone and somatostatin in hypothalamic-portal plasma of unanesthetized sheep: spontaneous secretion and response to insulin-induced hypoglycemia. J Clin Invest. 1990; In Press. Katakami H, Downs TR, Frohman LA. Inhibitory effect of hypothalamic medial preoptic area somatostatin on growth hormonereleasing factor in the rat. Endocrinology. 1988;123:1103-9. Stachura ME. Influence of synthetic somatostatin upon growth hormone release from perifused rat pituitaries. Endocrinology. 1976;99:678-83. Rittmaster RS, Loriaux DL, Merriam GR. Effect of continuous somatostatin and growth hormone-releasing hormone (GHRH) infusions on the subsequent growth hormone (GH) response to GHRH. Evidence for somatotroph desensitization independent of GH pool depletion. Neuroendocrinology. 1987;45:118-22. Clark RG, Robinson ICA. Paradoxical growth-promoting effects induced by patterned infusions of somatostatin in female rats. Endocrinology. 1988;122:2675-82. Soya H, Suzuki M. Somatostatin rapidly restores rat growth hormone (GH) release response attenuated by prior exposure to human GH-releasing factor in vitro. Endocrinology. 1988;122:2492-8. Miki N, Ono M, Shizume K. Withdrawal of endogenous somatostatin induces secretion of growth hormone-releasing factor in rats. J Endocrinol. 1988;117:245-52. Frohman LA, Downs TR, Williams TC, Heimer EP, Yu-Ching EP, Felix AM. Rapid enzymatic degradation of growth hormone-releasing hormone by plasma in vitro and in vivo to a biologically inactive product cleaved at the NH2 terminus. J Clin Invest. 1986;78:906-13. Vance ML, Kaiser DL, Rivier J, Vale W, Thorner MO. Dual effects of growth hormone (GH)-releasing hormone infusion in normal
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 18:02 For personal use only. No other uses without permission. . All rights reserved.
EFFECT OF SRIH ON GHRH-STIMULATED GH SECRETION
27. 28. 29. 30. 31.
32.
men: somatotroph desensitization and increase in releasable GH. J Clin Endocrinol Metab. 1986;62:591-4. Page MD, Dieguez C, Valcavi R, Edwards C, Hall R, Scanlon MF. Growth hormone (GH) responses to arginine and L-dopa alone and after GHRH pretreatment. Clin Endocrinol (Oxf). 1988;28:551-8. Valcavi R, Dieguez C, Page MD, et al. Alpha-2-adrenergic pathways release growth hormone via a non-GRF-dependent mechanism in normal human subjects. Clin Endocrinol (Oxf). 1988;29:309-16. Richardson SB, Twente S. Evidence that diminished pituitary responsitivity to GHRF is secondary to intracellular GH pool depletion. Am J Physiol. 1988;254:E358-64. Aguila MC, McCann SM. Stimulation of somatostatin release in vitro by synthetic human growth hormone-releasing factor by a nondopaminergic mechanism. Endocrinology. 1985;117:762-5. Berelowitz M, Firestone SL, Frohman LA. Effects of growth hormone excess and deficiency on hypothalamic somatostatin content and release and on tissue somatostatin distribution. Endocrinology. 1981;109:714-9. Berelowitz M, Szabo M, Frohman LA, Firestone S, Chu L, Hintz
33.
34.
35.
36.
163
RL. Somatomedin-C mediates growth hormone negative feedback by effects on both the hypothalamus and the pituitary. Science. 1981;212:1279-81. Vance ML, Kaiser DL, Martha PM, et al. Lack of in vivo somatotroph desensitization or depletion after 14 days of continuous growth hormone (GH)-releasing hormone administration in normal men and a GH-deficient boy. J Clin Endocrinol Metab. 1989;68:22-8. Kelijman M, Frohman LA. Discordant effects of insulin-hypoglycemia on growth hormone-stimulated GH and thyrotropin (TSH)releasing hormone-stimulated TSH secretion. J Clin Endocrinol Metab. 1988;66:872-5. Page MD, Koppeschaar HPF, Edwards CA, Dieguez C, Scanlon MF. Additive effects of growth hormone releasing factor and insulin hypoglycemia on growth hormone release in man. Clin Endocrinol (Oxf). 1987;26:589-95. Alba-Roth J, Muller OA, Schopohl J, Von Werder K. Arginine stimulates growth hormone secretion by suppressing endogenous somatostatin secretion. J Clin Endocrinol Metab. 1988;67:1186-9.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 18:02 For personal use only. No other uses without permission. . All rights reserved.
Vol. 71, No. 1 Printed in U.S.A.
Impaired Inhibitory Effects of Somatostatin on Growth Hormone (GH)-Releasing Hormone Stimulation of GH Secretion after Short Term Infusion* MIRTHA KELIJMAN AND LAWRENCE A. FROHMAN Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
ABSTRACT. We examined the effect of prior exposure to somatostatin (SRIH) on its inhibition of GH and TSH responses to GHRH and TRH stimulation to determine whether SRIH desensitization has physiological significance in man. Six men received GHRH (1 Mg/kg, iv) and TRH (0.3 jig/kg, iv) 20 min after starting a saline or SRIH (5.5 ng/kg/min, iv) infusion and again 6 h later. Hormone responses were quantified by measuring the area under the curve, corrected for GH concentration at injection time. Similar results were obtained when GH responses were quantified by measuring the hormone secretory rate using the program Detect. Plasma GH and TSH responses to the two GHRH and TRH injections during saline were similar. However, the effects of prior exposure to SRIH were hormone specific. SRIH blunted GH responses to GHRH at 20 min (1609 ± 286 jug/L-min us. 451 ± 224), but did not significantly inhibit the responses 6 h later (1422 ± 410 Mg/Lmin us. 1000 ± 302). In contrast, SRIH inhibition of TSH responses to the two TRH injections was similar (first, 946 ± 201 /ig/L-min vs. 700 ± 148; second, 813 ± 175 )ig/L-min us. 562 ± 66). We next used these results to study whether the previously reported attenuation of GH responses to repeated GHRH stimulation at 2-h intervals is
mediated by SRIH. Eight men received GHRH (1 Mg/kg, iv) 380 min after starting a saline or SRIH (5.5 ng/kg/min, iv) infusion or 90 min after starting a primed (5 mg, iv) infusion of propranolol (80 Mg/min, iv) and again 2 h later. As in the first protocol, GH responses to GHRH were not inhibited when preceded by a 6-h SRIH infusion. However, the 6-h SRIH infusion resulted in a partial restoration of plasma GH responses to the second GHRH injection (saline infusion: first, 1429 ± 342 ^g/L-min; second, 254 ± 75; SRIH infusion: first, 1042 ± 247 Mg/L-min; second, 468 ± 105). /3-Blockade by propranolol resulted in enhanced GH responses to GHRH, but did not prevent the attenuation of GH responses to the second GHRH injection (first, 1937 ± 366 Mg/Lmin; second, 614 ± 99). The desensitization to SRIH inhibition of GH responses to GHRH after a 6-h SRIH infusion provides evidence of physiological consequences of SRIH receptor down-regulation. The impaired GH responses to repeated GHRH stimulation are mediated at least in part by enhanced SRIH secretion, which appears independent of a 0adrenergic mechanism. (J CUn Endocrinol Metab 7 1 : 157-163, 1990)
T
physiological significance of SRIH receptor down-regulation has not been reported. In the present study we have investigated whether SRIH receptor desensitization occurs under physiological circumstances in man. Our approach was to determine whether prior exposure to physiological concentrations of SRIH attenuates its suppressive effects on pituitary responsiveness by comparing SRIH-induced inhibition of GH and TSH responses to GHRH and TRH stimulation at the beginning and after a continuous SRIH infusion of sufficient duration to produce receptor down-regulation. It has been previously reported that the GH response to repeated GHRH injections at 2-h intervals is attenuated (6). To test the hypothesis that this reduction is mediated by enhanced SRIH secretion, we determined whether it could be prevented by desensitizing the pituitary to SRIH action by means of prior SRIH exposure. In addition, we explored the possible /3-adrenergic mediation of this effect with the use of propranolol, a purported inhibitor of SRIH secretion (7).
HE SECRETION of GH is regulated by both stimulatory [GH-releasing hormone (GHRH)] and inhibitory [somatostatin (SRIH)] hypothalamic hormones. The pituitary response to these hormones is influenced not only by their concentrations in hypothalamic-pituitary portal blood, but also by the level of their respective receptors on the somatotroph. In experimental animals, GHRH and SRIH receptors have been reported to exhibit down-regulation in vitro (1, 2) and in vivo (3), suggesting a potential role of this process in the control of GH secretion. While several studies have examined the alterations in plasma GH responses to GHRH induced by prior GHRH administration in man (4, 5), the Received August 21,1989. Address all correspondence and requests for reprints to: Lawrence A. Frohman, M.D., Division of Endocrinology and Metabolism, University of Cincinnati Medical Center, 231 Bethesda Avenue, ML 547, Cincinnati, Ohio 45267. * This work was supported in part by USPHS Grants DK-30667 and RR-00068 and a grant from Genentech, Inc. 157
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 18:02 For personal use only. No other uses without permission. . All rights reserved.
KELIJMAN AND FROHMAN
158
Materials and Methods Subjects Thirteen healthy men, aged 19-24 yr (mean ± SE, 22 ± 0.5) and of normal body weight (81.9 ± 1.7 kg), participated in the study after giving informed consent to the protocols, which were approved by the Institutional Review Board. The protocols were performed in the University of Cincinnati General Clinical Research Center, with an interval of at least 7 days between studies. The order of the studies in each protocol was randomly assigned. The subjects fasted for 8 h before and during the studies of the first protocol. Breakfast was kept constant for each subject and was served immediately after starting each study of the second protocol. Heparin lock cannulae were placed in a vein in each forearm, one for infusions and the other for iv injections and blood sampling. Drugs
JCE & M • 1990 Vol 71 • No 1
min intervals throughout the studies. Blood samples for SRIH were collected in heparinized tubes containing aprotinin (Trasylol, Mobay Chemical Corp., New York, NY) during each SRIH infusion at the time of each injection and 5 min before the first injection in the first protocol. Samples for plasma glucose were obtained at 15- to 60-min intervals throughout the studies. All blood samples were immediately chilled, centrifuged at 4 C, and stored at -20 C until assayed. Plasma GH and TSH levels were measured by RIA as previously described (11,12). The intra- and interassay coefficients of variation were 6.1% and 6.8%, and 7.2% and 7.8%, respectively. SRIH concentrations were determined after extraction by RIA in a single assay, as previously described (8, 13), with an intraassay coefficient of variation of 5.4%. We have previously reported plasma SRIH levels in control subjects using this method to be 5 ± 1 ng/L (mean ± SE) (8). Plasma glucose levels were measured by a glucose oxidase method. Statistical analysis
GHRH-(l-44) (Hoffmann-La Roche, Inc., Nutley, NJ) was dissolved in normal saline at a concentration of 100 ^g/mL. TRH (Relefact, Hoechst-Roussel Pharmaceuticals, Inc., Somerville, NJ) was dissolved in 1% human serum albumin (HSA) at a concentration of 20 /ug/mL. Somatostatin (Stilamin, Serono Laboratories, Inc., Randolph, MA) was dissolved in HSA and stored at 4 C. Aliquots of the stock solution were diluted in HSA immediately before administration. SRIH and propranolol (Inderal, Ayerst Laboratories, Inc., New York, NY) were infused, as described below, via a Harvard pump (Harvard Apparatus, Millis, MA). Experimental design Attenuation of SRIH inhibitory effects. On separate days, six subjects received 500-min infusions of saline containing 1% HSA or SRIH (5.5 ng/kg/min, iv), a dose estimated to inhibit plasma GH responsiveness to GHRH by approximately 60% (8). Each study was preceded by a 30-min stabilization period. In each study, GHRH (1 Mg/kg, iv) and TRH (0.3 fig/kg, iv) were injected 20 min after starting the infusions and again 6 h later. The timing of the first injection was chosen to permit plasma SRIH concentrations to reach equilibrium (9). That of the second, 6 h later, was chosen on the basis of 1) the reported lack of attenuation of a repeated GHRH injection at this interval (10), and 2) the demonstration that this interval was sufficient to demonstrate SRIH receptor down-regulation in experimental animals (3). Mediation of GH response attenuation to reaeated GHRH injections. On separate days eight subjects received 620-min infusions of saline or SRIH. In a third study, subjects received a saline infusion for 290 min, followed by a primed (5 mg, iv) infusion of propranolol (80 /ig/min, iv) for the remainder of the 620-min infusion period. GHRH was injected 380 min after the start of each study and again 2 h later. The doses of SRIH and GHRH were the same as in the first protocol. Sample collection and assays. Heparinized blood samples for measurement of GH and TSH were obtained at 15-min intervals for 2 h after GHRH and TRH injections and at 15- to 60-
The 2-h integrated hormonal secretory responses to GHRH and TRH were determined by the trapezoidal method [area under the curve (AUC)] after correcting for preinjection (baseline) values. In the second protocol, the plasma GH concentration had not returned to basal levels by the time of the second GHRH injection. We used two approaches to distinguish the responses to each injection. In the first, the total AUC after the second injection was corrected by subtracting that fraction attributable to the GH response to the first, calculated on the basis of the GH half-life (14). In the second, the instant secretory rates for the 2 h after each injection were determined using the program Detect (15). The results obtained with each method were highly correlated (r = 0.995; P < 0.001). Group differences were determined by repeated measures two-way analysis of variance, after log transformation, when indicated. P < 0.05 was considered significant.
Results Attenuation of the inhibitory effects of SRIH Plasma GH responses to GHRH and TRH are shown in Fig. 1, and the integrated secretory responses and secretory rates are given in Table 1. Plasma GH responses to the two GHRH injections during saline infusion were similar. During the SRIH infusion, GH responses to the first GHRH injection were significantly blunted (P < 0.05), while those to the second injection were not. As a consequence, the responses to the second injection were greater than those to the first (P < 0.05). Inhibition of plasma TSH responses to the first TRH injection during SRIH infusion (Fig. 2 and Table 1) was less pronounced than that of the plasma GH responses to the first GHRH infusion (AUC, 19 ± 11% vs. 64 ± 15%; P = 0.01). After a 6-h infusion, the inhibitory effects of SRIH on the TSH responses to TRH remained intact. Plasma SRIH concentrations at the time of each GHRH injection (first, 71 ± 17 ng/L; second, 87 ± 19)
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 18:02 For personal use only. No other uses without permission. . All rights reserved.
EFFECT OF SRIH ON GHRH-STIMULATED GH SECRETION
159
SALINE or SRIH, 5.5 ng/kg/min
SALINE or SRIH, 5.5 ng/kg/min
TRH
TRH
20
10
-30
0 20
140
260
380
500 -30
0 20
140
260
380
500
MINUTES
FlG. 1. Effect of a continuous SRIH infusion on the plasma GH responses to repeated GHRH stimulation (1 fig/kg, iv) at a 6-h interval in six normal subjects. Shown are the mean ± SE. TABLE 1. Effects of SRIH and the sequence of stimulation on the GH and TSH responses to GHRH and TRH injections administered at a 6-h interval First injection Plasma GH responses to GHRH Saline (6) AUC (Mg/Lmin) 1609 ± 286 SR (ng/min) 59 ± 1 1 SRIH (6) AUC(Mg/L-min) 451 ± 224° SR (ng/min) 18±8a Plasma TSH responses to TRH Saline (6) AUC(mU/L-min) 946 ± 201 SRIH (6) AUC(mU/L-min) 700 ± 148°
Second injection
1422 ± 410 54 ± 16 1000 ± 302* 37 ± 11*
813 ± 175 562 ± 66°
Values are the mean ± SE. The number of subjects is shown in parentheses. AUC represents the 2-h integrated secretory response corrected for baseline values at the time of injection. SR represents the 15-min secretory rate during the 2-h period after injection. " P < 0.05 us. saline. * P < 0.05 vs. first GHRH injection.
were similar, as were concentrations 5 min before the first injection (86 ± 16 ng/L), indicating that equilibrium had been reached at the time of the first injection. Plasma glucose concentrations at the time of each injection were similar (data not shown).
MINUTES
FlG. 2. Effect of a continuous SRIH infusion on the plasma TSH responses to repeated TRH stimulation (0.3 Mg/kg, iv), at a 6-h interval in six normal subjects. Shown are the mean ± SE.
Effects of SRIH and propranolol on the GH responses to a repeated GHRH injection at 2 h SRIH infusion. Plasma GH responses to GHRH during saline and SRIH infusions are shown in Fig. 3 and Table 2. The first GHRH injection was administered 380 min after the start of the saline or SRIH infusion, a time comparable to that of the second GHRH injection in the previous protocol. SRIH infusion failed to inhibit plasma GH responses to the first GHRH injection, confirming the results of the first protocol. During the saline infusion, plasma GH responses to the second GHRH injection were nearly abolished. However, a SRIH infusion, started 8 h earlier, partially restored the GH responses to the second GHRH injection. Plasma glucose concentrations were slightly lower at the time of the second compared to those at the time of the first GHRH injection during both saline (first, 5.1 ± 0.1 mmol/L; second, 4.7 ± 0.1) and SRIH infusions (first, 5.2 ± 0 . 1 mmol/L; second, 5.0 ± 0.1). Plasma glucose levels at the time of each GHRH injection during saline and SRIH infusions were, however, not statistically different. Propranolol infusion. Plasma GH responses to GHRH during saline and propranolol infusions are shown in Fig. 4 and Table 2. The effects of propranolol and of the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 18:02 For personal use only. No other uses without permission. . All rights reserved.
KELIJMAN AND FROHMAN
JCE & M • 1990 Vol 71 • No 1
30 SALINE or PROPRANOLOL, 5.5 mg + 80 ug/min
SALINE
SALINE or SRIH, 5.5 ng/kg/min
GHRH
GHRH
A
V 20
o CO
I
Q- 10
SAUNE PROPRANOLOL
200 200
260
500
620
MINUTES
FIG. 3. Effect of a continuous SRIH infusion on the plasma GH responses to repeated GHRH stimulation (1 fig/kg, iv) at a 2-h interval in eight normal subjects. Shown are the mean ± SE. TABLE 2. Effects of SRIH and propranolol and the sequence of stimulation on the GH responses to GHRH injections administered at a 2h interval
Saline (8) AUC(Mg/L-min) SR (ng/min) SRIH (8) AUC(Mg/L-min) SR (ng/min) Propranolol (8) AUC (/xg/L-min) SR (ng/min)
First injection
Second injection
1429 ± 342 58 ± 14
254 ± 75° 12 ±3°
1042 ± 247 39 ± 9
468 ± 105 17 ± 4
1937 ± 366 71 ±14
614 ± 99a'6 24 ± Aa'b
Values are the mean ± SE. The number of subjects is shown in parentheses. AUC represents the 2-h integrated secretory response corrected for the plasma GH levels at the time of injection, as described in Materials and Methods. SR represents the 15-min secretory rate during the 2-h period after injection. 0 P < 0.05 us. first injection. 6 P < 0.05 vs. saline.
sequence of stimulation were determined independently of one another, since an analysis of variance revealed no statistical evidence of their interaction (AUC, P = 0.26; SR, P = 0.38). Propranolol infusion enhanced the GH responses to GHRH. However, despite ^-blockade, the GH responses to the second GHRH injection were sig-
260
380
MINUTES
FIG. 4. Effect of propranolol infusion on the plasma GH responses to repeated GHRH stimulation (1 jtg/kg, iv) at a 2-h interval in eight normal subjects. Shown are the mean ± SE.
nificantly lower than those to the first injection. The mean plasma glucose level during propranolol infusion was slightly lower at the time of the second (4.9 ±0.1 mmol/L) compared to that at the time of the first GHRH injection (5.2 ± 0 . 1 mmol/L). Plasma glucose levels at the time of each GHRH injection during saline and propranolol infusions were not statistically different.
Discussion Down-regulation of pituitary SRIH receptors has been demonstrated in vitro by direct exposure of somatotrophs to SRIH. The physiological significance of this phenomenon is, however, unclear, since exposure of cultured rat pituitary cells to SRIH concentrations 2 orders of magnitude higher than the IC50 for inhibiting GH release was needed to induce significant down-regulation (2). In experimental animals injection of GH, a potent stimulus for SRIH release, results in decreased binding capacity of pituitary SRIH receptors (3). In humans no attenuation of the inhibition of GH secretion has been observed after long term treatment of acromegaly with SMS 210995, a SRIH analog (16). However, there are no reports of dose-response studies of SRIH sensitivity before and during treatment with this agent. Our first protocol was designed to investigate whether SRIH desensitization occurs in man under physiological
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 18:02 For personal use only. No other uses without permission. . All rights reserved.
EFFECT OF SRIH ON GHRH-STIMULATED GH SECRETION
conditions. During saline infusion, plasma GH responses to GHRH injections administered at a 6-h interval were similar, indicating the absence of interaction between the two GHRH injections. SRIH infusion resulted in a 64% inhibition of GH responsiveness to GHRH administered shortly after starting the infusion. In contrast, there was no significant inhibition of plasma GH responses to GHRH after the 6-h SRIH infusion, suggesting that somatotroph responsiveness to SRIH was diminished by previous exposure to the hormone. Plasma SRIH concentrations in the human hypothalamic-pituitary portal system are unknown. However, the incomplete somatotroph inhibition observed and the reports of SRIH levels in the portal system of rats (17) and sheep (18) similar to those in the present study suggest that the plasma SRIH levels achieved during our infusions were within the physiological range. The relative increase in the GH response to the second injection of GHRH during SRIH infusion could alternatively be explained by an up-regulation of GHRH receptors or the accumulation of a readily releasible pool of GH. Although SRIH can suppress GHRH secretion (19), which, in turn, might be expected to up-regulate GHRH receptors, there is no evidence indicating that this can occur in response to systemic, as opposed to central, SRIH administration. Continuous infusion of SRIH does lead to the accumulation of a readily releasible pool of GH, demonstrable upon elimination of the SRIH influence (20). However, it is uncertain whether this pool is either accessible to GHRH stimulation (21) or releasible during the continued presence of SRIH. Thus, our results are most consistent with SRIH desensitization, although the level at which this occurs {e.g. receptor down-regulation or a postreceptor event, such as second messenger alteration) cannot be inferred from indirect studies as in the present report. Decreased responsiveness to SRIH after a relatively brief exposure to physiological concentrations of the hormone is consistent with a participatory role of SRIH receptor down-regulation in the control of GH secretion. Down-regulation of SRIH receptors may contribute to the ultradian rhythm of GH secretion. Bursts of GH secretion occur upon interruption of a SRIH infusion, and a pattern of intermittent SRIH administration in rats results in pulsatile GH secretion with increased body weight (22). In addition, episodic pulses of GH secretion in experimental animals are associated with asynchronous changes in hypothalamic GHRH and SRIH secretion (17, 18). Thus, SRIH receptor down-regulation that occurs as a result of SRIH pulses might potentiate the effects of trough SRIH concentrations both on the somatotroph (23), resulting in increased GH secretion, and on the hypothalamic arcuate nucleus, resulting in enhanced GHRH secretion (24).
161
The present study confirms our previous observation that SRIH inhibition of the TSH response to TRH is considerably less potent than that of the GH response to GHRH (8). SRIH inhibition of the TSH response to TRH, unlike that of the GH response to GHRH, was not impaired after a 6-h SRIH infusion. The discordant effects of a previous SRIH infusion on SRIH inhibition of GH and TSH responses to direct stimulation suggest that somatotroph and thyrotroph SRIH receptors have distinct binding characteristics. In an in vitro study by Smith et al. (2), much higher SRIH concentrations inhibited TSH responses to TRH and induced down-regulation of thyrotroph SRIH receptors, suggesting an association between the biological effects of SRIH and its ability to induce desensitization. The mediation of the GH response attenuation to repeated GHRH stimulation has been the subject of considerable debate. GHRH receptor down-regulation has been observed in in vitro studies (1). However, GHRH concentrations similar to or greater than those in plasma as early as 10 min after the iv injection of 1 Mg/kg GHRH (~ 25 pg/mL) (25) fail to produce GHRH receptor down-regulation in vitro (1). Depletion of a readily releasable storage pool of GH is unlikely in view of preserved and even enhanced plasma GH responses to hypoglycemia or arginine or clonidine stimulation after GHRH administration (26-28). However, depletion of a GH pool specifically responsive to GHRH (29) cannot be excluded. Finally, increased hypothalamic SRIH secretion induced by exogenous GHRH (30), GH (31), or locally produced insulin-like growth factor-I (32) might mediate the inhibition of GH responses to repeated GHRH stimulation. To investigate this hypothesis, we attempted to reverse the inhibition by reducing 1) the SRIH effect, based on the results of our first protocol, and 2) SRIH secretion, using propranolol, a purported inhibitor of SRIH release. Infusion of SRIH, started 6 h earlier, failed to significantly inhibit GH responses to the first GHRH injection (confirming the results of the first protocol). Plasma GH responses to the second GHRH injection, which were almost completely abolished during saline infusion, were partially restored during SRIH infusion. The lack of an enhanced GH response to the second GHRH injection in the SRIH-infused group compared to that in the saline-infused group is probably attributable to a residual inhibitory effect of SRIH on GH secretion. The actual restoration of the GH reponse to the second GHRH injection by SRIH pretreatment, therefore, suggests that the impaired GH responses to repeated GHRH stimulation are mediated at least in part by increased hypothalamic SRIH secretion. It is, therefore, possible that the gradual recovery of GH responses to GHRH stimulation repeated every 2 h for 12 h (5) as well as the normal GH responses to
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 18:02 For personal use only. No other uses without permission. . All rights reserved.
KELIJMAN AND FROHMAN
162
GHRH observed after 14 days (33), but not to shorter durations of GHRH infusions or injections, are a manifestation of SRIH receptor desensitization. SRIH inhibits GH responses to all known stimuli, and thus, responses to any stimulus administered after a GHRH injection should seemingly be suppressed by the enhanced SRIH secretion. However, enhanced hypothalamic SRIH secretion induced by previous GHRH stimulation might not impair GH responses to stimuli that are believed to suppress SRIH release (34-36). Propranolol potentiates GH responses to a maximally stimulatory dose of GHRH, suggesting /?-adrenergic stimulation of hypothalamic SRIH secretion (7). In the present study infusion of propranolol enhanced GH responses to each GHRH injection, administered at a 2-h
8.
9.
10.
11. 12.
interval, to a similar degree. However, it did not prevent
the attenuation of GH responses to the second, compared to the first, GHRH injection, indicating that previous GHRH stimulation and ^-blockade modify hypothalamic SRIH secretion through separate mechanisms. In summary, the present study indicates that relatively brief exposure to SRIH at a physiological concentration impairs its inhibition of GH secretion. We suggest that this effect has physiological significance and contributes to the pulsatile pattern of GH secretion. Our results also suggest that the inhibition of GH responses to repeated GHRH stimulation is mediated in part by enhanced hypothalamic SRIH secretion, which appears independent of a /3-adrenergic pathway.
Acknowledgments We are indebted to the staff of the General Clinical Research Center, to Gladys Perisutti and Amy Steinkamp for technical assistance, and to Dr. Peter Laskarzewski and Ismail Kaya for statistical advice. Materials for the GH and TSH RIAs were provided by the National Hormone and Pituitary Program, NIH.
13. 14.
15.
16.
17. 18.
19. 20.
References 1. Bilezikjian LM, Seifert H, Vale W. Desensitization to growth hormone-releasing factor (GRF) is associated with down-regulation of GRF-binding sites. Endocrinology. 1986;118:2045-52. 2. Smith MA, Yamamoto G, Vale WW. Somatostatin desensitization in rat anterior pituitary cells. Mol Cell Endocrinol. 1984;37:311-8. 3. Katakami H, Berelowitz M, Marbach M, Frohman LA. Modulation of somatostatin binding to rat pituitary membranes by exogenously administered growth hormone. Endocrinology. 1985;117:557-60. 4. Vance ML, Kaiser DL, Evans WS, et al. Evidence for a limited growth hormone (GH)-releasing hormone (GHRH)-releasable quantity of GH: effects of 6-hour infusions of GHRH on GH secretion in normal man. J Clin Endocrinol Metab. 1985;60:370-5. 5. Hulse JA, Rosenthal SM, Cuttler L, Kaplan SL, Grumbach MM. The effect of pulsatile administration, continuous infusion, and diurnal variation on the growth hormone (GH) response to GHreleasing hormone in normal men. J Clin Endocrinol Metab. 1986;63:872-8. 6. Shibasaki T, Hotta M, Masuda A, et al. Plasma GH responses to GHRH and insulin-induced hypoglycemia in man. J Clin Endocrinol Metab. 1985;60:1265-7. 7. Chihara K, Kodama H, Kaji H, et al. Augmentation by propranolol
21.
22. 23. 24. 25.
26.
JCE & M • 1990 Vol 71 • No 1
of growth hormone-releasing hormone-(l-44)-NH2-induced growth hormone release in normal short and normal children. J Clin Endocrinol Metab. 1985;61:229-33. Williams TC, Kelijman M, Crelin WC, Downs TR, Frohman LA. Differential effects of somatostatin (SRIH) and a SRIH analog, SMS 201-995, on the secretion of growth hormone and thyroidstimulating hormone in man. J Clin Endocrinol Metab. 1988;66:3945. Skamene A, Patel YC. Infusion of graded concentrations of somatostatin in man: pharmacokinetics and differential inhibitory effects on pituitary and islet hormones. Clin Endocrinol (Oxf). 1984;20:555-64. Losa M, Schopohl J, Muller OA, von Werder K. Stimulation of growth hormone secretion with human growth hormone releasing factors ( G R F ^ , GRF^o, GRF,-29) in normal subjects. Klin Wochenschr. 1984;62:1140-3. Frohman LA, Horton ES, Lebovitz HE. Growth hormone releasing effects of a pseudomonas endotoxin (Piromen). Metabolism. 1967;16:57-67. Pekary AE, Hershman JM, Parlow AF. A sensitive and precise radioimmunoassay for human thyroid stimulating hormone. J Clin Endocrinol Metab. 1975;41:676-84. Kronheim S, Berelowitz M, Pimstone BL. The characterization of somatostatin-like immunoreactivity in human serum. Diabetes. 1978;27:523-9. Henry DP, Nyhart EH, Bowsher RR. Pharmacokinetics of Humatrope (biosynthetic human growth hormone) in man: I.V..I.M., subcutaneous administration. Proc of the 69th Annual Meet of The Endocrine Soc. 1987;924. Oerter KE, Guardabasso V, Rodbard D. Detection and characterization of peaks and estimation of instantaneous secretory rate for episodic pulsatile hormone secretion. Com Biomed Res. 1986;19:170-91. Lamberts SWJ, Uitterlinden P, Del Pozo E. SMS 201-995 induces a continuous decline in circulating growth hormone and somatomedin-C levels during therapy of acromegalic patients for over two years. J Clin Endocrinol Metab. 1987;65:703-10. Plotsky PM, Vale W. Patterns of growth hormone-releasing factor and somatostatin secretion into the hypophysial-portal circulation of the rat. Science. 1985;230:461-3. Frohman LA, Downs TR, Clarke IJ, Thomas GB. Measurement of growth hormone-releasing hormone and somatostatin in hypothalamic-portal plasma of unanesthetized sheep: spontaneous secretion and response to insulin-induced hypoglycemia. J Clin Invest. 1990; In Press. Katakami H, Downs TR, Frohman LA. Inhibitory effect of hypothalamic medial preoptic area somatostatin on growth hormonereleasing factor in the rat. Endocrinology. 1988;123:1103-9. Stachura ME. Influence of synthetic somatostatin upon growth hormone release from perifused rat pituitaries. Endocrinology. 1976;99:678-83. Rittmaster RS, Loriaux DL, Merriam GR. Effect of continuous somatostatin and growth hormone-releasing hormone (GHRH) infusions on the subsequent growth hormone (GH) response to GHRH. Evidence for somatotroph desensitization independent of GH pool depletion. Neuroendocrinology. 1987;45:118-22. Clark RG, Robinson ICA. Paradoxical growth-promoting effects induced by patterned infusions of somatostatin in female rats. Endocrinology. 1988;122:2675-82. Soya H, Suzuki M. Somatostatin rapidly restores rat growth hormone (GH) release response attenuated by prior exposure to human GH-releasing factor in vitro. Endocrinology. 1988;122:2492-8. Miki N, Ono M, Shizume K. Withdrawal of endogenous somatostatin induces secretion of growth hormone-releasing factor in rats. J Endocrinol. 1988;117:245-52. Frohman LA, Downs TR, Williams TC, Heimer EP, Yu-Ching EP, Felix AM. Rapid enzymatic degradation of growth hormone-releasing hormone by plasma in vitro and in vivo to a biologically inactive product cleaved at the NH2 terminus. J Clin Invest. 1986;78:906-13. Vance ML, Kaiser DL, Rivier J, Vale W, Thorner MO. Dual effects of growth hormone (GH)-releasing hormone infusion in normal
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 18:02 For personal use only. No other uses without permission. . All rights reserved.
EFFECT OF SRIH ON GHRH-STIMULATED GH SECRETION
27. 28. 29. 30. 31.
32.
men: somatotroph desensitization and increase in releasable GH. J Clin Endocrinol Metab. 1986;62:591-4. Page MD, Dieguez C, Valcavi R, Edwards C, Hall R, Scanlon MF. Growth hormone (GH) responses to arginine and L-dopa alone and after GHRH pretreatment. Clin Endocrinol (Oxf). 1988;28:551-8. Valcavi R, Dieguez C, Page MD, et al. Alpha-2-adrenergic pathways release growth hormone via a non-GRF-dependent mechanism in normal human subjects. Clin Endocrinol (Oxf). 1988;29:309-16. Richardson SB, Twente S. Evidence that diminished pituitary responsitivity to GHRF is secondary to intracellular GH pool depletion. Am J Physiol. 1988;254:E358-64. Aguila MC, McCann SM. Stimulation of somatostatin release in vitro by synthetic human growth hormone-releasing factor by a nondopaminergic mechanism. Endocrinology. 1985;117:762-5. Berelowitz M, Firestone SL, Frohman LA. Effects of growth hormone excess and deficiency on hypothalamic somatostatin content and release and on tissue somatostatin distribution. Endocrinology. 1981;109:714-9. Berelowitz M, Szabo M, Frohman LA, Firestone S, Chu L, Hintz
33.
34.
35.
36.
163
RL. Somatomedin-C mediates growth hormone negative feedback by effects on both the hypothalamus and the pituitary. Science. 1981;212:1279-81. Vance ML, Kaiser DL, Martha PM, et al. Lack of in vivo somatotroph desensitization or depletion after 14 days of continuous growth hormone (GH)-releasing hormone administration in normal men and a GH-deficient boy. J Clin Endocrinol Metab. 1989;68:22-8. Kelijman M, Frohman LA. Discordant effects of insulin-hypoglycemia on growth hormone-stimulated GH and thyrotropin (TSH)releasing hormone-stimulated TSH secretion. J Clin Endocrinol Metab. 1988;66:872-5. Page MD, Koppeschaar HPF, Edwards CA, Dieguez C, Scanlon MF. Additive effects of growth hormone releasing factor and insulin hypoglycemia on growth hormone release in man. Clin Endocrinol (Oxf). 1987;26:589-95. Alba-Roth J, Muller OA, Schopohl J, Von Werder K. Arginine stimulates growth hormone secretion by suppressing endogenous somatostatin secretion. J Clin Endocrinol Metab. 1988;67:1186-9.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 18:02 For personal use only. No other uses without permission. . All rights reserved.
