In vitro regulation of growth hormone (GH) release from ovine pituitary cells during fetal and neonatal development: effects of GH-releasing factor, somatostatin, and insulin-like growth factor I

Using a monolayer approach, we have examined the acute (3 h) effects of GRF, somatostatin (SRIF), and insulin-like growth factor I (IGF-I) on GH release from pituitary cells of male and female 70-, 100-, and 130-day-old fetuses and newborn lambs and of prepubertal male lambs. GRF stimulated basal GH release in a dose-dependent (10(-12)-10(-8) M) manner at each stage in development. There was no linear relationship between maximal response and increasing age of the donor animals. The ED50 values for GRF were similar in all groups, except in the pituitaries from male and female 130-day-old fetuses, where the ED50 values were significantly higher. SRIF elicited a dose-related (10(-10)-10(-6) M) inhibition of basal GH secretion at each stage of fetal life and in the prepubertal period; although the response was lower in the youngest fetal pituitaries, there was no significant change in maximal response during the fetal or prepubertal period. No effect of SRIF on basal GH secretion was observed in newborn lambs. However, SRIF (10(-7) M) was able to block GRF (10(-8) M)-stimulated GH release in 100- and 130-day-old fetal and prepubertal as well as newborn lamb pituitary cells. Plasma IGF-I concentrations increased from 15.0 +/- 0.7 (mean +/- SE) and 13.8 +/- 0.9 ng/ml for male and female animals, respectively, at 70 days gestation to 55.8 +/- 3.2 and 51.8 +/- 11.1 ng/ml at the time of birth. The increase was much more pronounced in prepubertal lambs, especially in male animals, where IGF-I levels reached 300.8 +/- 37.7 ng/ml. IGF-I (100 ng/ml) had no effect on basal GH release in 70- and 100-day-old fetal, newborn, and prepubertal lamb pituitary cultures, but significantly inhibited basal GH secretion from 130-day-old fetal cells. This dose of IGF-I had no effect on GRF (10(-9) M)-stimulated GH release at 70 days gestation. It significantly inhibited this effect at 100 days and in prepubertal lamb cells. In 130-day-old fetal and newborn lamb pituitary cultures, IGF-I completely blocked the GH response to GRF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Influence of gonadal steroids on age- and sex-related secretory patterns of growth hormone in the rat

The influence of sex steroids on the pulsatile GH-secretory pattern was investigated in male and female rats of different ages. Neonatal and prepubertal gonadectomy was performed when the rats were 0-1 and 25 days old, respectively. Determinations of plasma GH levels were made in blood samples obtained from the tip of the tail or from a chronic intracardiac venous cannula. After blood sampling from the tip of the tail, maximum and minimum plasma GH levels were determined in individual rats in order to evaluate pulse height and baseline plasma GH levels. In the chronically cannulated rats the GH pulse height and plasma GH baseline levels in individual rats were determined by means of a pulse analysis computer program. Neonatal gonadectomy of male and female rats did not affect the increase in plasma GH that is observed during late prepubertal life in both sexes. However, the pulsatile, sexually differentiated, secretory pattern of GH seen in sexually mature male and female rats was modulated by gonadectomy. In adult male rats, GH was secreted in regular episodes at 3- to 4-h intervals, baseline levels being low or undetectable. After neonatal or prepubertal gonadectomy, GH baseline levels increased in comparison to the sham-operated controls. Replacement therapy with testosterone completely reversed this effect, indicating that a continuous presence of testosterone is necessary for maintaining the low baseline GH levels in adult male rats. Neonatal, but not prepubertal, gonadectomy decreased GH pulse height in male rats during adult life, suggesting that neonatal androgen secretion is a determinant for the pulse height in adult male rats. In female rats, GH pulse height was lower and baseline levels were higher than in male rats. Neonatal gonadectomy resulted in higher plasma GH pulses during puberty and decreased GH baseline levels postpubertally. There were no apparent differences in the GH-secretory patterns between male and female rats after neonatal gonadectomy. Neonatal gonadectomy of male rats decreased body weight gain as well as longitudinal bone growth, as measured in the proximal tibia by the tetracycline method. In contrast, neonatal gonadectomy of female rats resulted in a stimulation of body weight gain and longitudinal bone growth. The present results demonstrate that gonadal steroids influence GH secretion in the rat. We suggest that the sexual differentiation of the GH secretory pattern may be important for the difference in body growth between male and female rats.     

Effect of antiserum to rat growth hormone (GH)-releasing factor on physiological GH secretion in the female rat

To further study the physiological role of GH-releasing factor (GRF), we examined the effect of antiserum to rat hypothalamic GRF on spontaneous GH secretion in the normal female rat. Two groups of six conscious female rats were passively immunized with either nonimmune rabbit serum (NRS) or antirat GRF serum via a chronic indwelling atrial catheter. The secretory profiles of GH were observed by collecting blood samples at 15-min intervals for 1 h before and 4 h after administration. The NRS-treated rats showed a characteristic female pattern of spontaneous GH secretion. GH pulses were of low amplitude (mean +/- SEM, 26.8 +/- 2.4 ng/ml) and occurred irregularly at a frequency of 4.2 +/- 0.2/5 h, while interpeak through levels of GH were relatively high, with nadir values of 8.6 +/- 0.7 ng/ml. Synthetic rat GRF, given iv at a dose of 1 microgram/kg BW after the last blood sampling, stimulated GH release to a peak level of 153 +/- 37 ng/ml in the control rats. Administration of GRF antiserum caused a profound suppression of both pulse and trough components of GH secretion. This effect occurred rapidly, within 15 min after injection of antiserum, and GH secretion decreased uniformly to very low levels (3.4 +/- 0.1 ng/ml), with little or no fluctuation throughout the observation period. GRF antiserum also abolished the synthetic rat GRF-induced GH release, indicating sufficient potency of immunoneutralization. These results demonstrate that both GH pulses and troughs are dependent upon hypothalamic GRF in normal female rats, thereby substantiating earlier observations in male rats which demonstrated the physiological role of GRF in GH secretion.     

Sexual dimorphism in the control of growth hormone secretion

The secretory pattern of GH in the mature rat is sexually differentiated. In male rats GH is secreted in pulses occurring at regular 3- to 4-h intervals. In females the pulses are lower and plasma GH levels between the pulses are higher than in males. The continuous presence of testosterone appears to be necessary to maintain low basal GH levels in adult male rats. Neonatal, but not prepubertal, gonadectomy decreases GH pulse height in adult male rats to female levels. Administration of testosterone neonatally to castrated animals returns GH pulse height to normal suggesting that neonatal testicular androgen secretion is one determinant for GH pulse height in adult male rats. Administration of testosterone neonatally or during adult life to neonatally ovariectomized rats also produces higher GH pulses. In contrast to testosterone, estrogens elevate basal plasma GH levels and suppress the GH pulses under some conditions. Estrogens may stimulate basal GH secretion by acting directly on the pituitary. The physiological significance of the secretory pattern of GH has been investigated in hypophysectomized rats by simulating different plasma patterns of GH. The results suggest that high, infrequent GH pulses with low plasma GH levels in between (i.e. a masculine plasma GH pattern) promotes growth more effectively than an intermediate, rather constant level of plasma GH (i.e. a feminine plasma GH pattern). Since male sex steroids masculinize the secretory pattern of GH and have only minor growth-promoting effects in hypophysectomized animals it appears that the growth promoting effect of androgens is indirect and is due to an altered secretory pattern of GH. Presumably, neonatal androgen secretion stimulates body growth during adult life by irreversibly masculinizing the secretory pattern of GH. In contrast, estrogens appear to influence body growth by mechanisms that are mainly independent of the secretory pattern of GH. Evidence is accumulating that the secretory pattern of GH in the rat also affects various sexually differentiated hepatic characteristics such as steroid metabolism and prolactin receptor concentration. Thus, a feminization of the liver develops after continuous, but not intermittent, administration of GH to hypophysectomized rats. GH secretion is predominantly regulated by two hypothalamic peptides; GRF, and the GH-release-inhibiting factor, somatostatin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of testosterone on growth hormone secretion in female rats during a continuous infusion of growth hormone releasing factor

The effect of testosterone on growth hormone (GH) secretory pattern during a 6-hour continuous infusion of human GH-releasing factor (GRF) (1-44) NH2 was observed in unrestrained adult female Wistar rats. Rats had been ovariectomized or sham operated 6 weeks previously. Three weeks after the ovariectomy, the rats received sesame oil or testosterone propionate at a dose of 1 or 2 mg s.c. daily for 21 days. All rats were provided with two indwelling cannulae: one in the right atrium for undisturbed blood collection and the other in the inferior vena cava for vehicle or GRF infusion. Vehicle or GRF was administered by an infusion pump at a dose of 50 ng/kg/min for 6 h. Serial blood specimens were obtained every 20 min. Sham-operated adult female Wistar rats exhibited a high-frequency, low-amplitude pulsatile GH secretion during a 6-hour vehicle infusion. When they received a 6-hour continuous infusion of GRF, the amplitudes of GH pulses and baseline GH values were markedly augmented, but the pulse frequency remained unaltered. The GH secretory pattern during a 6-hour vehicle infusion among ovariectomized rats was similar to that of sham-operated female rats, whereas the magnitude of elevation of GH pulse and baseline level in ovariectomized rats were significantly lower than in sham-operated rats. The ovariectomized female rats that had received 2 mg testosterone for 21 days showed a low-frequency, regularly timed, high-amplitude pulsatile GH secretion, and GH values during the intervening period were low. This GH secretory pattern was indistinguishable from that in adult male rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth hormone (GH) secretion in the conscious rat: negative feedback of GH on its own release

The negative-feedback effects of GH on its own secretion were studied in conscious male and female rats bearing indwelling double-bore venous cannulae. Intravenous infusions of human GH (hGH; 20-60 micrograms/h) or somatostatin (SS; 10 micrograms/h) were given while frequent serial microsamples of blood were withdrawn using an automatic blood-sampling system. In both sexes, i.v. infusions of hGH for 6 h inhibited endogenous GH secretory pulses, with a slow onset of the inhibition. There was no rebound GH secretion immediately following the removal of the hGH infusion, but spontaneous GH secretion gradually returned to normal. Infusions of hGH did not inhibit the pituitary GH response to repeated GH-releasing factor (GRF) injections (1 microgram) given i.v. every 40 min to female rats. By contrast, infusions of SS, which also blocked spontaneous GH release, dramatically reduced the GH responses to serial GRF injections. When SS Infusions were stopped, the subsequent GRF-induced GH secretory responses were enhanced. These results show that GH can inhibit its own release when given by i.v. infusion to conscious male and female rats. Since GH responses to GRF are maintained during a GH infusion, the feedback effect of GH is unlikely to be exerted directly on the pituitary or by increasing SS release. Our results are consistent with the idea that GH feedback in the conscious rat involves an inhibition of GRF release.     

Imprinting of growth hormone secretion, body growth, and hepatic steroid metabolism by neonatal testosterone

The influence of endogenous sex steroids and exogenous testosterone treatment on pulsatile GH secretion, body weight, longitudinal bone growth, and hepatic steroid metabolism was studied in male and female adult rats. Blood samples were obtained from the tip of the tail, and maximum and minimum GH levels were determined in individual rats to evaluate pulse heights and baseline levels. Longitudinal bone growth was measured using the intravital marker tetracycline, and hepatic steroid metabolism was evaluated by determining the enzyme activities of 16 alpha-hydroxylase and 5 alpha-reductase. Neonatal, but not prepubertal, gonadectomy of male rats suppressed maximum and mean plasma GH levels during adult life. The body weight and the rate of longitudinal bone growth were also decreased. Testosterone treatment neonatally reversed all of these effects. Neonatal gonadectomy of male rats also caused an elevation of minimum plasma GH levels, an effect, however, which was not reversed by testosterone replacement during neonatal life. Neonatally gonadectomized females treated with testosterone neonatally or during adult life increased their maximum and decreased their minimum GH levels. Their longitudinal bone growth was increased. The body weight of these rats was increased by neonatal, but not adult, testosterone treatment. There was no effect of neonatal ovariectomy on plasma GH levels in 3- to 4-month-old female rats. However, neonatal ovariectomy did increase the maximum and mean plasma GH levels immediately postpubertally, suggesting that the effect of the ovaries on GH secretion differs among mature female rats of different ages. Prepubertal gonadectomy of male rats feminized their hepatic steroid metabolism by decreasing 16 alpha-hydroxylase and increasing 5 alpha-reductase activities. Neonatal gonadectomy caused an even more pronounced feminization, which was partly reversed in rats given testosterone replacement therapy neonatally. In neonatally gonadectomized female rats, treatment with testosterone during adult life increased 16 alpha-hydroxylase and decreased 5 alpha-reductase to levels seen in intact male rats. The present results indicate that neonatally secreted testicular androgens imprint the high amplitude pulses characteristic of GH secretion in adult male rats. Neonatal androgens also stimulate somatic growth and partially account for the masculinized hepatic steroid metabolism in the adult animal. It is proposed that imprinting of the GH secretory pattern contributes to the influence of neonatal testicular androgens on body growth and hepatic steroid-metabolizing enzymes.     

Somatostatin rapidly restores rat growth hormone (GH) release response attenuated by prior exposure to human GH-releasing factor in vitro

To clarify the role of somatostatin (SRIF) in pulsatile GH secretion, profiles of the hormone release from intact anterior pituitaries of male rats were examined in an in vitro perifusion system. Infusion of human (h) GRF (0.1 microM) into the perifusion system for 10 min stimulated GH release, which peaked within 30-40 min. Two hours after the first stimulation, when basal GH release had not yet fallen to the original levels, the response to a second hGRF stimulation was attenuated to as low as 47.7 +/- 10.0% (+/- SE) of the first response. However, when GH release after the first stimulation had returned to the basal level after the first stimulation had returned to the basal level after perifusion with the medium for 3 h, the second response to hGRF was restored to a level similar to that of the first response. In contrast, when SRIF (0.1 microM) was infused for 50 min 1 h after the first stimulation to lower the GH baseline, the second response to hGRF was also restored to the level of the first response. Neither SRIF infusion after the first hGRF stimulation nor infusion of SRIF without hGRF caused any rebound increase in GH release after cessation of the perifusion. To determine whether SRIF exerts a direct action on the GH response, a prestimulatory perifusion with SRIF (0.1 microM) for 50 min was performed. The treatment tended to facilitate the pituitary response to hGRF. When 50-min pretreatment with SRIF at a lower concentration (0.05 microM) was given, a significantly facilitated response to the first hGRF stimulation (0.05 microM) was observed. These results suggest that 1) SRIF perifusion rapidly restores the attenuated response to a second hGRF challenge by lowering GH release to basal levels; and 2) SRIF pretreatment facilitates the GH response to the first hGRF challenge.     

Effects of sex steroids on the response of cultured rat pituitary cells to growth hormone-releasing hormone and somatostatin

In the male rat, GH secretion is characterized by high amplitude pulses that appear at regular intervals of 3-4 h, with low basal levels between such pulses. In the female, the pulses are irregular and more frequent, with lower amplitudes, while basal secretion is higher. The present study was designed to exclude the indirect effects of sex steroids on the pituitary, enabling investigation of the direct effects of sex steroids on the pituitary. Rats were gonadectomized at 22 days of age, and 12 days later their anterior pituitaries were trypsinized for cell dispersion. Testosterone (T) or 17 beta-estradiol (E2), 5 nM, was added to the medium for 6 days, and subsequently, GRF or somatostatin was added for 4 h. In a perifusion system, the male-derived cell response to GRF was augmented after pretreatment with T, but not with E2. The female-derived cell response to GRF was augmented by E2, but not by T. T increased the sensitivity of the cells to GRF from 3.0-0.03 nM and increased the maximal potency of GH secretion 3-fold. E2 had no significant effect on the sensitivity, but lowered the potency. Somatostatin (1 nM) inhibited GH secretion by 44% in T-treated cells. In E2-treated cells, somatostatin was ineffective. GRF increased the total amount of GH (medium plus cells) in both T- and E2-treated cells, but not in control pituitary cells. It is suggested that T has direct effects on the male somatotroph. By increasing the pituitary cell responses to GRF and somatostatin, T contributes to the high amplitude peak/low baseline pattern of the male. By decreasing the pituitary cell responses to GRF and somatostatin, E2 contributes to the low amplitude peak/high baseline pattern of the female.     

Different regulation of growth hormone-releasing factor-sensitive adenylate cyclase in the anterior pituitary of young and aged rats

Low basal GH secretion and reduced GH responsiveness to different GH secretagogues, including GHRF, have been reported in aged animals and humans. Parallel to the in vivo findings, an impaired GH responsiveness to GHRF is evident in somatotropes from old rats of either sex. We report here that in anterior pituitaries (APs) from aged male and female rats GHRF-induced stimulation of adenylate cyclase (AC) activity was strikingly reduced (male rats, change from baseline 700% in young and 100% in old rats) or lacking (female rats, change from baseline 430% in young and 13% in old rats) when compared to that evoked by GHRF in the APs from young counterparts. Pretreatment with GHRH (5 micrograms/rat iv for 3 days) decreased the high basal AC activity of old male rats [from 33.38 +/- 3.60 to 15.99 +/- 5.75 (SEM) pmol cAMP/min.mg protein], did not alter the GHRF-stimulated rise in AC activity in old male rats, and induced a small but unequivocal rise in AC activity in old female rats (change from baseline 35% vs. 13%, respectively). Pretreatment with GHRF markedly reduced the acute effect of GHRF in the APs from young rats of both sexes (male rats, change from baseline 360% and 700%; female rats, change from baseline 230% and 430% in GHRF-pretreated and control rats, respectively). In parallel studies performed in female rats, it was shown that in vivo pretreatment with GHRF at the same schedule markedly reduced the effect of acute GHRF stimulation on GH secretion from cultured pituitary cells of young rats but left unchanged GHRF-induced stimulation of GH secretion from pituitary cells of old rats. In all, these data suggest that deficiency of endogenous GHRF synthesis and/or release may underlie defective GH secretion in old rats and that a GHRF replacement regimen that reduces the sensitivity of the young somatotrope cells does not alter the sensitivity of (male rats) or exerts a priming effect (female rats) on the old somatotrope cell.     

Sex difference in growth hormone feedback in the rat

Growth hormone inhibits its own secretion in animals and man but the mechanism for this inhibition is unclear: both stimulation of somatostatin release and inhibition of GH-releasing factor (GRF) release have been implicated. We have now studied the GRF responsiveness of conscious male and female rats under conditions of GH feedback induced by constant infusion of exogenous human GH (hGH). Intravenous infusions of hGH (60 micrograms/h) were maintained for 3 to 6 h whilst serial injections of GRF(1-29)NH2 (0.2-1 microgram) were given at 45-min intervals. The GH responses were studied by assaying blood samples withdrawn at frequent intervals using an automatic blood sampling system. We have confirmed that male and female rats differ in their ability to respond to a series of GRF injections; female rats produced consistent GH responses for up to 13 consecutive GRF injections, whereas male rats showed a 3-hourly pattern of intermittent responsiveness. In female rats, multiple injections of GRF continued to elicit uniform GH responses during hGH infusions, whereas hGH infusions in male rats disturbed their intermittent pattern of responsiveness to GRF, and their regular 3-hourly cycle of refractoriness was prolonged. We suggest that this sex difference in GH feedback may be due to GH altering the pattern of endogenous somatostatin release differentially in male and female rats. Such a mechanism of GH autofeedback could be involved in the physiological control of the sexually differentiated pattern of GH secretion in the rat.     

Inhibitory effect of the ovaries on neonatal androgen imprinting of growth hormone secretion in female rats

The effect of neonatal androgen treatment on the GH secretory pattern was examined in intact and ovariectomized adult female rats. Neonatal ovariectomy or sham operation was performed at 1-2 days of age; thereafter, the animals were immediately given testosterone propionate (250 micrograms) or vehicle. Other rats, also treated neonatally with testosterone, were ovariectomized 15-22 days before blood sampling. Plasma GH was measured in blood samples obtained from indwelling intraatrial cannulae every 20 min for 8 h when the animals were 100-140 days old. Plasma GH secretory patterns were analyzed by a pulse analysis computer program (PULSAR). Neonatal testosterone treatment did not affect the GH secretory pattern of female rats with intact ovaries. In contrast, neonatal androgen treatment enhanced GH pulse height as well as mean GH concentration in neonatally ovariectomized female rats to levels comparable to those in intact male rats. Neonatal testosterone administration also significantly increased GH pulse height and mean plasma GH concentration in female rats that were ovariectomized during adulthood. However, the GH secretory pattern of ovariectomized female rats given testosterone neonatally still differed markedly from that of normal males, in that GH pulses occurred less regularly and baseline levels were higher. Pituitary GH content and concentration in neonatally ovariectomized female rats were increased to levels indistinguishable from those in male rats by neonatal testosterone treatment. No significant effect of neonatal testosterone was observed in sham-operated females. Neonatal ovariectomy decreased basal plasma GH levels, but did not affect plasma GH pulse height or pituitary GH levels. The serum estradiol concentration was markedly decreased in ovariectomized female rats, but was unchanged in sham-operated rats given neonatal testosterone, raising the possibility that serum estradiol secretion mediated the antagonistic effect of the ovaries on neonatal androgen imprinting. These results indicate that the presence of ovaries can prevent the stimulatory effect of neonatal androgen exposure on GH storage and secretion in adult female rats.     

Hypothyroidism blunts the growth hormone (GH) releasing effect of human pancreatic GH releasing factor in the adult male rat in vivo and in vitro

The effect of the 44-amino-acid peptide human pancreatic GH releasing factor (hpGRF-44) upon the secretion of GH was studied in control and hypothyroid adult male rats. In animals rendered hypothyroid by ingestion of propylthiouracil (PTU), basal and hpGRF-44-stimulated secretion of GH was depressed in vivo. Administration of T4 together with PTU prevented the decline in basal and hpGRF-provoked GH secretion in vivo. HpGRF-44-stimulated release of rat GH was also impaired in vitro, an effect partially reversed by administration of low doses of T4 in vivo. The depressed in vitro secretion of GH could not be restored in hypothyroidism pituitaries by incubation of the glands with T3. Thus, hypothyroidism blunts hpGRF-44-stimulated secretion of GH in vivo and in vitro in the hypothyroid adult male rat.     

Time-dependent reduction and potentiation of growth hormone (GH) responsiveness to GH-releasing factor induced by exogenous GH: role for somatostatin.

Exogenous GH is known to exert a negative feedback effect on its own responsiveness to GH-releasing factor (GRF); however, the mechanism is not known. In the present study we examined the time course of effects of a single sc administration of recombinant human (rh) GH on GH responsiveness to GRF and investigated the possible involvement of somatostatin (SRIF) in this response. Free-moving adult male rats were administered 200 micrograms rhGH, sc, at 0800 h and subsequently challenged with 1 microgram GRF-(1-29)NH2, iv, at times of spontaneous peaks (1100 and 1500 h) and troughs (1300 h) in GH secretion during a 6-h (1000-1600 h) sampling period. H2O-injected control rats exhibited the typical cyclic responsiveness to GRF stimulation, with GRF-induced GH release significantly greater during peak compared to trough periods of the GH rhythm. Pretreatment with rhGH 3 h before GRF injection markedly inhibited the GH response to GRF at a peak time [integrated GH release over 30 min, 1135 +/- 271 vs. 6372 +/- 1185 ng/ml.30 min in H2O-injected controls (mean +/- SE); P less than 0.01]. In striking contrast, 5 h after rhGH administration, there was a 6-fold augmentation of GH responsiveness to GRF compared to that in H2O-injected controls at a trough time (7032 +/- 1622 vs. 1128 +/- 216 ng/ml.30 min; P less than 0.01). High GH responsiveness to GRF was preserved 7 h after rhGH injection. Passive immunization of rhGH-treated rats with SRIF antiserum reversed the rhGH-induced blunted GH response at 3 h (7985 +/- 366 vs. 1705 +/- 431 ng/ml.30 min in rhGH-treated control rats given normal sheep serum; P less than 0.01) and completely restored GH responsiveness to levels as high as those in H2O-injected controls. These results demonstrate that 1) a single sc injection of rhGH markedly attenuates GH responsiveness to GRF acutely for about 3 h, but subsequently enhances somatotroph sensitivity to the stimulatory actions of GRF; and 2) the short term blunting of GRF-induced GH release by rhGH is due at least in part to increased release of endogenous SRIF. The subsequent potentiation of GH responsiveness to GRF is probably due to a SRIF-mediated build-up of pituitary GH stores in a readily releasable pool. Such a mechanism of GH autofeedback may play a physiological role in the genesis of pulsatile GH secretion.     

Growth hormone (GH)-releasing factor stimulates hypothalamic somatostatin release: an inhibitory feedback effect on GH secretion

GH-releasing factor (GRF) is a hypothalamic peptide that stimulates the secretion of pituitary GH. The possibility of feedback effects of GRF within the central nervous system was studied in conscious freely moving male rats with indwelling iv and intracerebroventricular (icv) cannulae. Animals were injected icv or iv with 10 ng-10 micrograms human (h) GRF(1-40)-OH (hGRF-40) or GRF(1-44)-NH2 (hGRF-44), and blood samples were obtained every 10-20 min from 1000-1400 h. GH secretion was pulsatile, with major secretory peaks at around 1200 h in most control animals. When 10 ng hGRF-40 were injected icv at 1100 h, immediately before the expected onset of the spontaneous GH secretory burst, GH secretion was suppressed during the following 2-h period. An iv injection of 10 ng hGRF-40 was without effect. In contrast, when 1 microgram hGRF-40 was injected icv or iv, plasma GH levels peaked at 20 and 10 min, respectively, and returned toward baseline shortly thereafter. The spontaneous GH secretory pulse after 1 microgram hGRF-40 (icv or iv) was suppressed in proportion to the magnitude of the GH secretory response to GRF (r = 0.78, p less than 0.01), and the prolongation of the interval between the injection of GRF and the subsequent spontaneous GH surge was directly related to the GH response to GRF (r = 0.85, p less than 0.001). The icv or iv injection of a larger dose of either hGRF-40 or hGRF-44 (10 micrograms) at 1100 h also resulted in marked and comparable increases in plasma GH levels, with peaks at 20 min (icv) and 10 min (iv) after injection. No changes in behavior or plasma glucose were observed up to 3 h after icv injection of any of the doses of hGRF-40 or of hGRF-44. The suppressive effect of centrally administered hGRF-40 (10 ng) on GH secretion was blocked by the iv administration of a specific antisomatostatin serum immediately before the injection of hGRF. These results demonstrate a dual action of GRF on spontaneous GH secretion and indicate the presence of an inhibitory feedback system within the central nervous system for the regulation of GH secretion which is mediated by hypothalamic somatostatin.     

Inhibitory effect of hypothalamic medial preoptic area somatostatin on growth hormone-releasing factor in the rat

Possible inhibitory effects of somatostatin (SRIF) on GRF were studied by assessing spontaneous GH secretion and GRF content and release in adult male rats depleted of hypothalamic SRIF by anterolateral hypothalamic deafferentation (AHD) or electrolytic lesions in the medial preoptic area (MPO). Plasma GH levels were measured 7 days postoperatively every 20 min in conscious animals with indwelling iv cannulae. Median eminence SRIF was markedly reduced 8 days postoperatively in both AHD and MPO rats, as determined by immunohistochemistry and RIA (P less than 0.01). Although GRF immunoreactivity in the median eminence of AHD and MPO animals appeared well preserved immunocytochemically, hypothalamic GRF content by RIA was significantly decreased at 8 days (P less than 0.01). Spontaneous GH secretion was pulsatile in sham-operated animals. In contrast, basal GH levels in AHD and MPO animals were markedly elevated (P less than 0.01), and secretory pulses were absent. Intravenous injection of specific anti-GRF serum into MPO animals decreased the elevated plasma GH levels (P less than 0.01), indicating increased hypothalamic GRF secretion. GRF release from hypothalamic median eminence-arcuate nucleus complexes in vitro was significantly greater in AHD and MPO animals than in control animals 4 and 8 days postoperatively in response to 30 mM K+ (P less than 0.01), but not under basal conditions. These results suggest that hypothalamic medial preoptic area somatostatinergic neurons play a tonic inhibitory role in the regulation of GRF release and that GH hypersecretion observed after MPO and AHD is attributable to changes in both SRIF and GRF.     

Roles of somatostatin and growth hormone-releasing factor in ether stress inhibition of growth hormone release.

In order to evaluate the release of somatostatin (SRIF) and growth hormone-releasing factor (GRF) into the pituitary gland in response to ether stress, a push-pull perifusion (PPP) technique has been used in freely moving rats. Push-pull cannulae (PPC) were implanted into the anterior pituitary (AP) glands of male rats. After a 7-day recovery period the rats were fitted with indwelling jugular catheters. The next day the animals were subjected to PPP of the AP during 1 h followed by ether stress (2 min) and another hour of perifusion. The perifusion flow was 20 microliters/min and 10-min fractions were collected and assayed for SRIF and GRF by RIA. Plasma growth hormone (GH) levels were assayed every 10 min. At the end of the experiments, the accuracy of PPC tip placements was ascertained with a dissecting microscope. Under basal conditions there were 2.9 pulses/h of SRIF with an amplitude of 12.76 +/- 0.46 pg. The output of SRIF and GRF in the 10-min period beginning with application of ether was increased 2-fold (p less than 0.005 and p less than 0.01, respectively). Interestingly, the increased release of SRIF continued for an additional 10 min, whereas GRF output decreased and was almost undetectable. The release of both GRF and SRIF had returned to basal values 20-30 min after stress. Mean plasma GH levels were significantly lowered 10 min after stress. Each of the 9 animals showed a restoration of pulsatile GH release to basal levels within 20-30 min after stress. Our findings provide compelling evidence that SRIF plays a prominent role in stress-induced inhibition of GH release in the rat by blocking the response to the transient elevation of GRF and continuing to suppress GH release for 20 min.     

Serotonin stimulates GH secretion through a direct pituitary action: studies in hypophysectomized autografted animals and in perifused pituitaries

To analyze a possible direct action of serotonin (5-hydroxytryptamine) at pituitary level in GH secretion, two experimental models were used: hypophysectomized autografted rats and perifused pituitaries. Adult male rats were hypophysectomized and their own pituitaries placed under the right kidney capsule. Ten days later an intra-atrial cannula was inserted. The next day, blood samples were obtained before and every 10 min during a 2 h period after the injection of saline or 5-hydroxytryptamine (1 or 2 mg/kg iv). Plasma volume was replaced with saline. Both doses of 5-hydroxytryptamine elicit a strong release of GH, the effect being dose-dependent. In pituitaries perifused with 5-hydroxytryptamine (100 microM during 115 min or 1, 10 and 100 microM during 15 min), a significant release of GH was also observed. These results suggested that 5-hydroxytryptamine may stimulate GH secretion through a direct pituitary action.     

Growth hormone (GH) secretion in the dwarf rat: release, clearance and responsiveness to GH-releasing factor and somatostatin

The new mutant GH-deficient dwarf (Dw) rat was used to study the effects of GH-releasing factor (GRF) or somatostatin (SRIF) on GH release. In anaesthetized adult Dw female rats, i.v. injections of GRF (0.031-2.0 micrograms) elicited a dose-dependent release of GH. Although the peak plasma GH responses to maximal GRF doses were much lower in adult Dw rats compared with normal rats of this strain (AS), the responses largely reflected their relative pituitary GH contents (140 +/- 17 micrograms vs 2.9 +/- 0.4 micrograms, AS vs Dw (means +/- S.E.M.), P less than 0.001). Except at 20 days of age, normal AS rats were more sensitive to GRF than Dw rats despite their larger body weight. Peak GH responses to injection of 31.25 ng GRF increased nine-fold in normal rats between 20 and 40 days, whereas the GH responses to this GRF dose diminished in Dw rats over this age range, and their pituitary GH content was only 2-5% of that of age-matched AS rats. Treatment with human GH (200 micrograms/day for 7 days) stimulated growth in 40-day-old Dw rats and slightly increased the GH response to a low dose of GRF. Basal GH levels in adult Dw animals were sevenfold lower than in AS rats (2.4 +/- 0.3 vs 17.6 +/- 3.3 micrograms/l P less than 0.001) and were further suppressed by i.v. infusion of SRIF (25 micrograms/h). As in normal rats, a rebound GH secretion occurred in Dw rats after stopping SRIF, which was blocked by injection of anti-GRF serum.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of gastrin-releasing peptide on growth hormone secretion in the rat

Several investigators have reported gastrin-releasing peptide (GRP)-like immunostaining in several regions of the rat brain. The objective of this study was to determine the possible effects of this peptide on GH release. Porcine GRP was injected intraventricularly (third ventricular) in a volume of 2 microliters into ovariectomized female rats. A significant decrease in basal GH release, as evidenced by decreased plasma GH levels, was observed within 10 min which lasted for 90 min after the injection of 2 micrograms (0.7 nmol) GRP (P less than 0.001). In addition, all GH pulses were abolished during this time. In subsequent experiments, varying doses of GRP were administered, and human pancreatic GH-releasing factor (GRF) was injected iv at a dose of 0.1 microgram/kg 20 min later to determine the responsiveness of the pituitary. The minimal effective dose of GRP to lower plasma GH was approximately 10 ng (3.6 pmol); however, the GH-releasing action of GRF was blocked by even the lowest dose of the peptide tested (5 ng; 1.8 pmol). To determine if GRP had any direct action on the pituitary, overnight-cultured pituitary cells from ovariectomized animals were incubated for 1 h with GRP in various concentrations. There was a slight dose-dependent stimulation of GH release with concentrations of GRP ranging from 10(-9)-10(-6) M; however, the GH-releasing action was much less than that of GRF. To confirm the direct stimulatory effect of GRP on GH release, dispersed pituitary cells were perifused with medium containing 2 X 10(-6) M GRP. An immediate increase in GH release was observed in the perfusate. Since GRP has a direct stimulatory action to release GH in the pituitary, but ivt injection of the peptide inhibits GH release and blocks the response to GRF, we suggest that GRP may act on periventricular structures to release somatostatin, which reduces GH release and blocks the response to GRF.