In vitro and in vivo growth hormone responsiveness to growth hormone-releasing factor in male and female Zucker rats

In order to determine whether there is an anomaly in the pituitary responsiveness to growth hormone (GH)-releasing factor in the genetically obese rat, we examined the in vitro and in vivo effects of rGRF(1-29)NH2 (GRF) on GH release in male and female Zucker rats. The effect of increasing GRF concentrations (1.56, 6.25, 12.5 25 and 50 pM) was first tested on GH release from freshly perifused anterior pituitary cells. In both sexes, the GH response per one pituitary equivalent to each GRF concentration tested was reduced in the obese group. However, when GH release was expressed as a percent of initial cell GH content, there was no difference between the lean and the obese groups. Furthermore, under pentobarbital anesthesia, GRF was injected intravenously at two consecutive doses of 0.8 and 4.0 microgram/kg body weight in obese and lean animals. In both sexes, the GH response to each dose of GRF tested was decreased in the obese group. Basal serum GH concentrations were similar in male and female obese rats compared to their respective lean siblings. In conclusion, this study demonstrates a decrease of the in vitro and in vivo pituitary response to GRF in the obese Zucker rat, suggesting a possible secondary defect at the pituitary level.     

Changes in follicle-stimulating hormone secretion in spontaneously hypertensive rats.

FSH and testosterone plasma levels, pituitary FSH content and concentration and the weight of testis, seminal vesicles and ventral prostate have been studied at the ages of 30, 60 and 90 days in spontaneously hypertensive rats (SHR) and normotensive control (WKY) rats. In vitro FSH secretion by pituitaries, and the response to orchidectomy and to exogenous administration of either LHRH (1 microgram) or LHRH agonist (0.05, 0.1, 1, and 5 micrograms/kg) were analyzed in 90-day-old SHR and WKY male rats. Ventral prostate weight and FSH plasma levels were determined in other groups of adult male rats castrated and castrated and implanted for 15 days with silastic capsules containing testosterone, dihydrotestosterone or estradiol. Also FSH plasma levels and pituitary FSH concentration were determined at the ages of 30, 60 and 90 days in SHR and WKY female rats. Male SHR showed increased plasma FSH levels and high testicular weight in all the cases, and enhanced testosterone levels in plasma and pituitary FSH content on days 60 and 90. Weight of seminal vesicles and ventral prostate was normal or reduced, depending on the animal age. Adult SHR had increased FSH secretion in vitro, normal response to orchidectomy and did not exhibit FSH increases after LHRH administration. The efficiency of testosterone to stimulate ventral prostate growth and the ability of estradiol to reduce FSH plasma levels were decreased in SHR. Female SHR showed a significant increase in the pituitary content of FSH on day 30 and on proestrus at the ages of days 60 and 90.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma growth hormone response to human growth hormone releasing factor in rats administered with chlorpromazine and antiserum against somatostatin. Effects of hypo- and hyperthyroidism

The effect of hypo- and hyperthyroidism on the plasma growth hormone (GH) response to synthetic human growth hormone releasing factor (GRF) was determined in conscious, freely moving rats pretreated with chlorpromazine and antiserum against somatostatin. Chlorpromazine plus somatostatin antiserum pretreated rats gave consistent response to GRF which was not observed in untreated rats. Chlorpromazine alone has no effect on GH secretion induced by GRF in rat pituitary monolayer culture. In rats made hypothyroid by thyroidectomy, both basal and peak plasma GH responses to a small (0.25 microgram/kg bw) and a moderate dose of GRF (1 microgram/kg bw) were significantly reduced as compared to controls. In rats made hyperthyroid by the administration of thyroxine, basal and peak plasma GH responses to a small but not to a moderate dose of GRF were significantly reduced as compared to controls. A reduced plasma GH response to a small dose of GRF was observed 8 days after the cessation of thyroxine administration. The pituitary GH reserve was markedly reduced in hypothyroid but not in hyperthyroid rats as compared to their respective controls. These results indicate that plasma GH response to GRF is reduced both in hypo- and hyperthyroidism. The mechanism involved in the phenomenon appears to be different between the two conditions.     

Secretion of LH in spontaneously hypertensive rats

Weights of testes, seminal vesicles, ventral prostate and pituitary, plasma testosterone and LH concentrations, pituitary LH content and concentration, the LH in-vivo response after LHRH administration (1 microgram), and basal and LHRH-stimulated secretion in vitro were analysed in adult male spontaneously hypertensive (SH) and normotensive control (WKY) rats. Spontaneously hypertensive rats showed: testis and pituitary hypertrophy; seminal vesicle and ventral prostate atrophy; increased plasma testosterone and LH concentrations; increased pituitary LH content and concentration; unchanged net increase of plasma concentrations of LH 15 and 45 min after administration of 1 microgram LHRH; and increased basal LH secretion in vitro with a normal response to LHRH stimulation. These results provide evidence that SH rats show increased LH secretion with a normal response to LHRH stimulation. The coexistence of high plasma concentrations of testosterone with seminal vesicle and ventral prostate atrophy suggest a reduction in the effectiveness of testosterone in these structures.     

Mechanisms of impaired growth hormone secretion in genetically obese Zucker rats: roles of growth hormone-releasing factor and somatostatin

GH secretion is markedly blunted in obesity; however, the mechanism(s) mediating this response remains to be elucidated. In the present study we examined the involvement of the two hypothalamic GH-regulatory hormones, GH-releasing factor (GRF) and somatostatin (SRIF), using the genetically obese male Zucker rat. Spontaneous GH, insulin, and glucose secretory profiles obtained from free moving, chronically cannulated rats revealed a marked suppression in amplitude and duration of GH pulses in obese Zucker rats compared to their lean littermates (mean 6-h plasma GH level, 3.9 +/- 0.4 vs. 21.5 +/- 3.8 ng/ml; P less than 0.001). Obese rats also exhibited significant hyperinsulinemia in the presence of normoglycemia. The plasma GH response to an iv bolus of 1 microgram rat GRF-(1-29)NH2, administered during peak and trough periods of the GH rhythm, was significantly attenuated in obese rats at peak (137.4 +/- 26.1 vs. 266.9 +/- 40.7 ng/ml; P less than 0.02), although not at trough, times. Passive immunization of obese rats with a specific antiserum to SRIF failed to restore the amplitude of GH pulses to normal values; the mean 6-h plasma GH level of obese rats given SRIF antiserum was not significantly different from that of obese rats administered normal sheep serum. Both pituitary wet weight and pituitary GH content and concentration were reduced in the obese group. Measurement of hypothalamic GRF immunoreactivity revealed a significant (P less than 0.05) reduction in the mediobasal hypothalamic GRF content in obese rats (503.2 +/- 60.1 pg/fragment) compared to that in lean controls (678.1 +/- 50.2 pg/fragment), although no significant difference was observed in hypothalamic SRIF concentration. Peripheral SRIF immunoreactive levels were significantly (P less than 0.01) elevated in both the pancreas and stomach of obese rats. These results demonstrate that the genetically obese Zucker rat exhibits 1) marked impairment in both spontaneous and GRF-induced GH release, which cannot be reversed by SRIF immunoneutralization, 2) significant reduction in pituitary GH concentration, 3) depressed hypothalamic GRF content, and 4) elevated gastric and pancreatic, but not hypothalamic, SRIF levels. The findings suggest that the defect in pituitary GH secretion observed in the genetically obese Zucker rat is due, at least partially, to insufficient stimulation by hypothalamic GRF, and that SRIF does not play a significant role.     

In vivo studies with growth hormone (GH)-releasing factor and clonidine in rat pups: ontogenetic development of their effect on GH release and synthesis

The demonstration that GH-releasing factor (GRF) stimulates GH synthesis and release in rat pups prompted studies to evaluate the effects on the same indices of clonidine (CLO), an alpha 2-adrenoceptor and potent GH secretagogue, purported to act in adult rats via GRF release. Our first aim was to ascertain whether CLO elicits GH release in rat pups via GRF, and if this is the case, to evaluate the ontogenetic development in 1- to 10-day-old pups of the GH response to acute CLO or GRF administration and, finally, the effects of short term CLO or GRF treatment on plasma and pituitary GH concentrations and on the GH response to an acute challenge with the homologous secretagogue. CLO (15 micrograms/100 g BW, sc) induced a clearcut GH rise in 10-day-old rats but not in pups pretreated with a specific anti-GRF serum. Moreover, unlike GRF (10(-8) M), CLO (10(-6) to 10(-5) M) did not stimulate GH release in vitro from anterior pituitaries of 10-day-old rats. In 1-day-old rats, neither CLO (15 micrograms/100 g BW, sc) nor GRF (20 ng/100 g BW, sc) stimulated GH release, whereas significant GH stimulation was elicited by GRF, but not CLO, in 5-day-old rats and by both secretagogues in 10-day-old rats. Short term treatment with CLO (15 micrograms/100 g BW, sc, twice daily) or GRF (20 ng/100 g BW, sc, twice daily) on postnatal days 1 through 5 did not modify either plasma or pituitary GH concentrations 14 h after the last drug administration, but did so when either secretagogue was administered on postnatal days 5 through 9. Finally, an acute challenge with GRF, but not with CLO, induced a further rise in the already elevated plasma GH levels of pups pretreated from postnatal day 5 through 9, but neither secretagogue did so in pups pretreated from postnatal days 1 to 5. Viewed together, these data indicate that in infant rats CLO releases GH via GRF release and that the somatotropes respond earlier to GRF (5 days) than the GRF-secreting structures do to alpha 2-adrenergic stimulation (10 days). Both GRF and CLO stimulate GH synthesis when administered repeatedly. However, whereas repeated GRF treatment has a priming effect on the somatotropes, CLO does not, probably because of down-regulation of hypothalamic alpha 2-adrenoceptors.     

Down-regulation of growth hormone releasing factor receptors following continuous infusion of growth hormone releasing factor in vivo

The in vivo chronic infusion of growth hormone releasing factor (GRF) results in a loss of the pituitary growth hormone (GH) response to GRF as well as in a substantial depletion of pituitary GH content. To evaluate if the loss in response is due to the down-regulation of GRF receptors the specific GRF binding capacity of pituitary homogenates prepared from rats infused with saline or GRF (1 or 15 micrograms/h for 24 h) was determined. The pituitary binding capacity of animals infused with GRF was significantly reduced as compared to animals infused with saline.     

Thyroidectomy abolishes pulsatile growth hormone secretion without affecting hypothalamic somatostatin

The effects of thyroid hormone deprivation and of subsequent replacement therapy on growth hormone (GH) secretion were investigated in unrestrained unanesthetized rats. Male rats were thyroparathyroidectomized (TPTX) 5 weeks prior to plasma sampling for GH assay, or to decapitation for evaluation of hypothalamic somatostatin (SRIF) content and in vitro SRIF and GH release. Thyroid hormone deprivation suppressed pulsatile GH secretion as well as GH release induced by clonidine (150 micrograms/kg). Treatment of TPTX rats with small doses of triiodothyronine (T3) restored an episodic pattern of GH secretion, but with lower peak values than controls, as well as the GH response to clonidine. Thyroid deprivation induced a 92-fold decrease in GH release from the pituitary; however, the ratio between GH release and GH content was similar in TPTX and normal rats, and human pancreatic growth hormone-releasing factor (GRF) (3 X 10(-8) M) was still able to stimulate residual GH release by hemipituitaries from TPTX rats in a manner similar to that in euthyroid controls (295 and 254% stimulation, respectively). Thyroid deprivation or T3 replacement did not modify SRIF content in the hypothalamus or other brain structures tested. The capacity of K+ depolarization to release SRIF in vitro from the hypothalamus was not modified by TPTX. These findings indicate that thyroid hormones are necessary to maintain both pulsatile and induced GH secretion in unanesthetized rats. In addition they suggest that impairment of GH secretion in thyroidectomized rats does not depend upon changes in the hypothalamic SRIF regulation of the hormone but could be dependent on a defect in GRF release and/or, most probably, GH synthesis directly at the pituitary level.     

Effect of Fetal Alcohol Exposure on Postnatal Pituitary Adenosine 3',5' -Cyclic Phosphate Content and Growth Hormone Release

This study examines the influence of fetal ethanol (ETOH) exposure and pair-feeding dams on postnatal, releasing factor-induced pituitary growth hormone (GH) release and adenosine 3′,5′-cyclic phosphate (CAMP) accumulation. Fetuses were exposed to ETOH in utero by feeding dams a 36% (calories derived from ETOH: 6.6% v/v) ETOH liquid diet. Postnatal body weights were measured at sacrifice to evaluate the influence of ETOH on growth. Pituitary weight and protein content were measured to determine if changes in GH secretion or cAMP are proportional to the overall effect of ETOH on the pituitary. Pituitaries from 1-, 10-, and 60-day-old pups were explanted and incubated without hormones or with either somatostatin [somatotropin-release inhibiting factor (SRIF); 10^?9 M], or GH-releasing factor (GRF; 5 × 10^?9 M). Radioimmunoassays were used to determine tissue cAMP content, after extraction, and media GH concentration. Results indicate that fetal ETOH exposure specifically reduces the weight of both male and female pups. However, by 60 days of age, this reduction is not different from that found in pups of pair-fed controls, and both groups weighed less than pups of ad libitum controls. Furthermore, both pituitary weight and protein content were proportionately reduced in ETOH-exposed pups. In regard to releasing factor sensitivity, compared with pituitaries from ad libitum controls, the capacity of GRF to simulate GH release was diminished in 10-day-old males (p < 0.00s) exposed to ETOH. On the other hand, the capacity of GRF to stimulate cAMP accumulation was generally enhanced by prenatal ETOH exposure. The capacity of SRIF to depress GH release was diminished in ETOH pups, compared with both pair-fed and ad libitum-fed controls (p < 0.0001). This difference in GH release was more apparent in pituitaries from females than males (p < 0.001). However, the depressed SRIF response was not associated with altered cAMP accumulation. These data suggest that fetal ETOH exposure has a sexually dimorphic effect on pituitary sensitivity to GH-releasing factors that may be related to altered regulation of GH release and susceptibility to growth retardation.     

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)     

Human pancreatic growth hormone (GH)-releasing hormone stimulates GH synthesis and release in infant rats. An in vivo study

Administration of human pancreatic GH-releasing factor 1-40 (hpGRF-40) at doses of 1, 10, 20, 100, and 500 ng/100 g BW sc induced in 10-day-old rats a clear-cut rise in plasma GH 15-min post-injection, although the effect was not dose-related and peak GH levels were already present after the lowest GRF dose. In 25-day-old rats, hpGRF induced only a slight rise in plasma GH at the dose of 500 ng/100 g BW sc, whereas it was completely ineffective at the lower doses. In 5-day-old rats, hpGRF (20 ng/100 g BW sc twice daily), administered for 5 days, induced a marked rise in pituitary GH content and plasma GH levels determined 14 h after the last hpGRF injection. In these rats, at the end of treatment, a challenge hpGRF dose (20 ng/100 g BW) induced a rise in plasma GH significantly higher than in infant rats receiving only the challenge hpGRF dose. These data show that: 1) pituitary responsiveness to hpGRF is strikingly higher in infant than in post-weaning rats; 2) in infant rats, subacute administration of hpGRF stimulates GH synthesis and release.     

Growth hormone releasing hormone induced release of growth hormone in aging male rats: dependence on pharmacological manipulation and endogenous somatostatin release

These studies were designed to assess whether a decline in pituitary response to growth hormone-releasing factor (GRF) contributes to the decline in amplitude of growth hormone (GH) pulses with age. Atrial cannulated young (3-4 months), middle-aged (12-14 months) and old (20-22 months) Fischer 344 male rats were anesthetized with pentobarbital to suppress pulsatile release of GH. One hour later, animals were injected with hpGRF(1-44) (10 micrograms/kg) and blood samples were removed at 0, 5, 10, 20, 40 and 80 min. Animals were then passively immunized with somatostatin antiserum and hpGRF(1-44) was reinjected. Samples were removed at the time intervals previously described. In response to 10 micrograms/kg GRF, plasma GH in young and middle-aged animals increased to 921 +/- 105 and 866 +/- 126 ng/ml, respectively, at 10 min and declined. In old animals, GH concentrations only increased to 654 +/- 80 ng/ml (p less than 0.05 compared to young rats). This represents a 28% reduction in the peak GH response to GRF. Despite passive immunization with somatostatin antiserum, the peak GH response to a second injection of GRF was diminished in all age-groups. However, integration of the area under the response curve indicated that GH secretion was similar in young and old animals during the 80-min sampling period after somatostatin antiserum. In another study, diethyldithiocarbamate (DDC; 250 mg/kg, i.v.), a dopamine-beta-hydroxylase inhibitor, was used to inhibit pulsatile GH release. One hour later, animals were injected with hpGRF(1-44) (50 micrograms/kg, i.v.) and blood samples were removed as previously described.(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.     

Paradoxical enhancement of pituitary growth hormone (GH) responsiveness to GH-releasing factor in the face of high somatostatin tone

Pituitary GH secretion is regulated by a delicate interplay between stimulatory (GRF) and inhibitory [somatostatin (SRIF)] hypothalamic hormones, although the nature of the GRF/SRIF interaction remains to be elucidated. In the present study, we documented a significant elevation of plasma SRIF-like immunoreactivity in 72-h fasted rats compared to that in fed controls (129.0 +/- 17.9 vs. 38.2 +/- 5.8 pg/ml; P less than 0.01) and used this model of high SRIF tone to further delineate the interrelation between GRF and SRIF in physiological regulation of pulsatile GH secretion. We examined pituitary GH responsiveness to GRF, both in vivo and in vitro, after 72-h exposure to nutritional deprivation and high SRIF secretion. In vivo, GRF-induced GH release was markedly enhanced in the face of high circulating SRIF; freely moving, starved rats released 4- to 8-fold more GH than fed controls in response to rat GRF iv. In vitro, both basal and human GRF-induced GH release were augmented 2- to 4-fold in perifused dispersed anterior pituitary cells of starved rats compared to those in fed controls, and this enhanced responsiveness persisted in the presence of 10(-9) M SRIF. These results demonstrate that SRIF not only inhibits GH secretion stimulated by GRF, but that under different temporal conditions SRIF may act in a paradoxically positive manner to sensitize pituitary GH responsiveness to GRF. Such a cooperative interaction of the two peptides may be necessary to optimize pulsatile GH release. Our findings provide support for the hypothesis that the temporal patterning of hypothalamic GRF/SRIF signals to pituitary somatotrophs may be the major determinant for pulsatile GH secretion and, ultimately, body growth.     

Altered pituitary growth hormone (GH) regulation in streptozotocin-diabetic rats: a combined defect of hypothalamic somatostatin and GH-releasing factor

Diabetes mellitus in the rat is associated with loss of pulsatile GH secretion. An interplay between hypothalamic GH-releasing factor (GRF) and inhibitory factor [somatostatin (SRIF)] secretion is thought to account for episodic pituitary GH release. An increase in SRIF tone/action or a decrease in GRF release/response in diabetic rats could account for the suppressed GH levels. Pituitaries from streptozotocin-diabetic rats contained less GH than controls (15.9 +/- 2.5 vs. 29.5 +/- 4.6 micrograms/mg; P less than 0.05) despite normal somatotrope representation, as demonstrated using immunofluorescence studies. Basal GH secretion from monolayer culture of dispersed anterior pituitary (AP) cells from diabetic rats was proportionately decreased (150 +/- 10 vs. 103 +/- 10 ng/10(5) cells; P less than 0.005). GRF (10(-11)-10(-8) M)-induced release of GH from AP cells was decreased in diabetic rats (maximum response to 10(-8) M GRF, 401 +/- 60 vs. 618 +/- 41 ng/10(5) cells; P less than 0.01); however, sensitivity to GRF was unchanged (EC50, 79 +/- 41 vs. 128 +/- 67 pM). By contrast, SRIF (10(-7)-10(-10)-induced inhibition of GRF (10(-8) M)-mediated GH release was impaired in AP cells of diabetic rats compared to that in controls (IC50, 112 +/- 33 vs. 55 +/- 31 pM; P less than 0.05) associated with a decrease in AP plasma membrane SRIF receptor concentration (63.4 +/- 15.6 vs. 160.3 +/- 13.7 fmol/mg protein; P less than 0.05), with no change in affinity. These findings are consistent with chronic exposure to increased hypothalamic SRIF influence. GH synthesis has been shown to be independent of SRIF regulation; however, insulin-like growth factor-I and GRF inhibit and stimulate GH synthesis, respectively. In diabetic rats insulin-like growth factor-I levels were decreased, appropriate to low GH status, in serum (290 +/- 66 vs. 1662 +/- 92 ng/ml; P less than 0.001) and hypothalamus (6.8 +/- 1.0 vs. 13.0 +/- 0.4 pg/mg wet wt; P less than 0.001) and, thus, did not seem to account for the low AP GH content. Hypothalamic GRF content in diabetic rats (1.11 +/- 0.10 ng/hypothalamus) did not differ from that in controls (1.16 +/- 0.17 ng/hypothalamus). GRF mRNA levels, however, were reduced by 80% in diabetic rats compared to controls. Taken together these data support a combined role for decreased hypothalamic GRF and increased SRIF in mediating alterations of GH synthesis and secretion in streptozotocin-induced diabetes.     

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)     

Plasma growth hormone (GH) response to intravenous GH-releasing factor (GRF) in adult rats: evidence for transient pituitary desensitization after GRF stimulation

The ability of human (h)GRF-(1-29)NH2 to stimulate GH secretion was studied in cannulated adult rats. In order to suppress endogenous GRF secretion and the inhibitory action of hypothalamic somatostatin (SRIF), rats were anesthetized with sodium pentobarbital. Intravenous administration of hGRF-(1-29)NH2 elicited a dose-dependent response of plasma GH, with 250 ng/kg being the smallest effective dose in male rats. In female rats, for each dose tested (250 to 70,000 ng/kg), the GH response represented only about 60% that of male rats. Repeated iv stimulations with hGRF-(1-29)NH2 at short time intervals (45 min) produced transient desensitization of pituitary responsiveness to GRF: a blunted GH response to the second and third stimulations was observed both in male and in female rats and for each dose tested. Similar blunted responses were also obtained with repeated injections of native hGRF-(1-44)NH2. The possibility that these blunted responses could be due to incomplete suppression of hypothalamic SRIF secretion by sodium pentobarbital was excluded by the use of rats that were passively immunized against SRIF; in these rats, it was shown that at least 65% of the inhibition of the GH response after the second GRF stimulation was unrelated to SRIF action. Similar transient desensitization to repeated hGRF-(1-29)NH2 stimulations was also observed in conscious rats that were passively immunized against SRIF. This occurrence of blunted responses was shown to be related to the length of the time interval between GRF stimulations, with longer intervals resulting in less or no desensitization. It appears thus that modulation of pituitary responsiveness to the action of GRF is mediated by at least two independent mechanisms in the rat: in addition to the inhibitory action imposed by hypothalamic SRIF, which induces periods of refractoriness to the action of GRF, it was shown in this study that in the pituitary level each GRF stimulation also induces a transient desensitization of somatotrophs for about 1 h. This period of refractoriness might not be due to excessive stimulation with GRF, since it was also observed with the lowest dose of hGRF-(1-29)NH2 that gave a significant release of GH. Finally, a sex difference was confirmed for the response of anesthetized adult rats to stimulation with hGRF-(1-29)NH2, reflecting a sex steroid-induced modification of pituitary responsiveness to GRF stimulation.     

Diminished pituitary responsiveness to growth hormone-releasing factor in aging male rats

The pattern of GH secretion undergoes substantial changes in the aging rat, resulting in decreased daily secretion of GH. In this study, the pituitary responsiveness to GH-releasing factor (GRF) was examined in young (2- to 5-month old) and aging (14- to 18-month old) male rats. In vivo studies were performed under sodium pentobarbital anesthesia. After injection of 250 ng GRF/100 g BW, young rats experienced more GH secretion [peak level, 544.5 +/- 209.5 (+/- SEM) ng/ml] than did 18-month-old rats (89.3 +/- 13.7 ng/ml). To investigate the locus of this insensitivity to GRF, anterior pituitary cells from young and aging rats were dispersed and placed in primary culture. While basal GH secretion from the cultured pituitary cells was similar in the two groups (49.7 +/- 3.5 vs. 47.8 +/- 2.7 ng/ml X 4 h for the 2- and 18-month old rats, respectively), the GH-releasing ability of GRF was partially but significantly impaired in cells derived from both 14- and 18-month old rats; 100 nM GRF stimulated the release of 96.7 +/- 1.8 ng/ml X 4 h in the 18-month old rats as opposed to 115.0 +/- 6.0 (P less than 0.05) ng/ml X 4 h in the 2-month-old rats. Since GRF stimulates GH release through the activation of adenylate cyclase, intracellular cAMP levels were measured in the cultured pituitary cells. GRF stimulated 65% less intracellular cAMP accumulation in the 18-month-old rats. In 14-month-old rats, the ability of forskolin and (Bu)2 cAMP to release GH was impaired, while phorbol ester-elicited GH secretion was unchanged. In conclusion, the GH response to GRF is blunted in aging rats. While much of the insensitivity to GRF may be mediated by the increased somatostatin tone reported in aging rats, a diminished pituitary cAMP response to GRF may also be an important etiological factor in the hyposomatotropinemia of the aging male rat.     

Sexual differentiation of growth hormone feedback effects on hypothalamic growth hormone-releasing hormone and somatostatin

To investigate possible sex differences in the feedback regulation of growth hormone (GH) secretion, concentrations of immunoreactive GH-releasing hormone (GRF) and somatostatin (SS) were measured in the median eminence (ME) and the hypothalamus of male and female rats bearing the MtTW15 tumor, which secretes high amounts of GH and prolactin (PRL). Four weeks after tumor implantation in male rats, the GRF concentration in the whole hypothalamus, including the ME, was decreased by 37% (0.29 +/- 0.02 vs. 0.46 +/- 0.02 ng/mg protein in intact male controls; p less than 0.001) and the concentration of SS was increased by 40% (11.5 +/- 0.7 vs. 8.1 +/- 0.3 ng/mg protein in male controls; p less than 0.01). In female rats, the presence of tumor for 4 weeks caused a smaller (18%) reduction in GRF concentrations (0.27 +/- 0.02 vs. 0.33 +/- 0.03 ng/mg protein in intact female controls; p less than 0.05) and no significant change in SS concentrations (10.2 +/- 0.08 vs. 9.7 +/- 0.8 ng/mg protein in female controls). Tumor-related changes in GRF and SS concentrations were also more pronounced in male rats than in females, when determined separately in the microdissected ME and in the remaining hypothalamus. These differences occurred despite similar increases in serum GH, PRL and insulin-like growth factor I concentrations in male and female tumor-bearing rats. To assess which hormone (GH or PRL) was responsible for these changes, intact male rats were treated for 10 days with 2 daily s.c. injections of rat GH (rGH; 100 and 250 micrograms/day), rat PRL (100 and 250 micrograms/day) or vehicle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulatory role of galanin in control of hypothalamic-anterior pituitary function.

The role of the neuropeptide galanin in the regulation of anterior pituitary function was studied in vivo in conscious male rats and in vitro with cultured anterior pituitary cells. Galanin (50-200 ng; 15-60 pmol) injected into the third cerebral ventricle of rats produced highly significant, dose-related increases of plasma growth hormone (GH) concentrations, whereas galanin increased prolactin (PRL) and decreased thyroid-stimulating hormone (TSH) levels only at the highest dose (60 pmol) tested. Intravenous galanin failed to alter PRL and TSH levels in these rats. In contrast with the results with intraventricular injection of the peptide, intravenous injection of 30 or 300 pmol of galanin produced small, brief, dose-related increases in plasma GH. The response to the 300-pmol dose was less than that induced by a factor-of-20-lower intraventricular dose, which establishes a central action of galanin. Galanin in concentrations ranging from 1 nM to 1 microM failed to alter significantly GH, PRL, or TSH release from dispersed anterior pituitary cells. It also failed to alter GH secretion in response to 100 nM GH-releasing hormone; however, at this dose galanin did potentiate the effect of 100 nM TSH-releasing hormone on TSH and PRL release. Thus, the effects of third-ventricular injection of the peptide are mediated by the hypothalamus. To determine the physiological significance of galanin in control of pituitary hormone release, highly specific antiserum against galanin was injected intraventricularly. Third-ventricular injection of 3 microliter of galanin antiserum resulted in a dramatic decrease in plasma GH values as compared with those of normal rabbit serum-injected controls within 15 min, which persisted until the end of the experiment (5 hr postinjection). Galanin antiserum did not decrease plasma PRL or TSH levels at any time period after its third-ventricular injection; however, a transient increase of plasma TSH levels occurred after 30 and 60 min in comparison with TSH levels in normal rabbit serum-injected controls. Since there was no effect of the antiserum on plasma PRL and only a transient elevation in TSH, galanin may not be physiologically significant enough during resting conditions to alter PRL and TSH release in the male rat. The results of the experiments with galanin antiserum indicate that endogenous galanin has a tonic action within the hypothalamus to stimulate GH release. The rapidity of onset of the effects of galanin and the antiserum directed against it suggest that it acts to stimulate release of GH-releasing hormone from periventricular structures, which then stimulates the release of GH.