In vitro release of growth hormone-releasing factor (GRF) from the hypothalamus: somatostatin inhibits GRF release.

The manner of release of growth hormone-releasing factor (GRF) from the rat hypothalamus was studied in a perifusion system using a highly sensitive radioimmunoassay for rat GRF. The recovery of GRF in this system was 50-60%. The release of GRF from the rat hypothalamic blocks was almost stable for 20-240 min after the start of the perifusion and was stimulated by depolarization induced by high K+ concentration. The release of GRF was inhibited by somatostatin at concentrations of 10(-11) to 10(-8) M with maximum inhibition to 52.5% of the basal release at a concentration of 10(-9) M. These results suggest that this system is useful in studying the regulatory mechanism of GRF release and that, in addition to its action on the pituitary, somatostatin appears to act at the level of the hypothalamus in inhibiting GRF release in the regulation of GH secretion.     

Desensitization of rat pituitary somatotrophs to growth hormone-releasing factor occurs in vitro

Desensitization of rat pituitary somatotrophs to human growth hormone-releasing factor (hGHRF) was investigated using cultured rat anterior pituitary cells. Growth hormone (GH) release decreased but the production of cAMP was still induced in response to subsequently added 10(-9) M hGHRF from cells pretreated with hGHRF at concentrations ranging from 10(-11) to 10(-7) M for 4 h. Desensitization to 10(-9) M hGHRF was also observed in cells pretreated with 10(-9) M hGHRF for 4 h in the presence of 2 mM EGTA, 10 ng/ml nifedipine or 10(-9) M somatostatin-28, which decreased GH release during pretreatment. Forskolin and A23187, at concentrations of 10(-6) M and 10(-4) M, respectively, stimulated GH release from cells pretreated with hGHRF to the same extent as that from the control cells. These results, therefore, suggest that desensitization to GHRF occurs regardless of the presence of releasable GH pool and that some changes such as uncoupling of GHRF receptors with adenylate cyclase and decreased sensitivity to cAMP of cAMP-dependent protein kinase of the secretory mechanism of GH, in addition to the decrease in releasable GH pool and down regulation of GHRF receptors, may be involved in the desensitization mechanism.     

Effect of sodium ion on atrial natriuretic factor release from rat hypothalamic fragments

The effects of Na ion and choline chloride on the release of atrial natriuretic factor (ANF) and growth hormone-releasing factor (GHRF) from rat hypothalamic fragments including the organum vasculosum of the lamina terminalis (OVLT) were examined in vitro. Although the release of ANF was stimulated by Na ion, choline chloride, and glucose in concentration-dependent manners, the release was more sensitive to a change in concentration of Na ion than to those of choline chloride and glucose. On the other hand, the change in Na ion concentration did not affect the release of GHRF. It can be therefore proposed that Na ion is the first candidate controlling ANF release from the brain tissue and that ANF in the hypothalamus and/or OVLT may play some role in the regulation of the Na ion and water balance in the central nervous system.     

Effects of growth hormone-releasing factor and somatostatin on growth hormone secretion in hypophysial stalk-transected beef calves

The effects of growth hormone-releasing factor (GHRF) on growth hormone (GH) secretion were studied in beef calves after hypophysial stalk transection (HST). Peripheral GH concentration during surgery was elevated for 60 min after the initiation of anesthesia to 15 ng/ml, which was greater than plasma levels after HST and during the recovery period (0-30 hr mean, 3 ng/ml; P less than 0.05). Episodic GH secretion normally seen in sham-operated controls (SOC) was abolished after HST. Before HST, calves responded to 80% of the GHRF challenges, whereas after HST calves responded to every challenge of GHRF with an increase in plasma GH. A dose of 0.067 microgram human pancreatic (hp) hpGHRF(1-40)OH/kg body wt 3 days after HST increased plasma GH to 55 ng/ml from a control period mean of 5 ng/ml (P less than 0.04). On Day 8, HST calves received two injections of 0.067 microgram hpGHRF/kg body wt at 3-hr intervals, with feeding 70 min after the first injection. During two preinjection control periods, basal GH averaged less than 4 ng/ml and increased to 17 (P less than 0.02) and 9 (P less than 0.04) ng/ml immediately after the first and second injection of hpGHRF, but the response declined over the 8-day period after surgery. On Days 19 and 20, the HST calves were infused iv with 0.033 and 0.067 microgram somatostatin(SS)-14 (SRIH)/kg body wt, during which a pulse injection of 0.067 microgram hpGHRF/kg body wt was administered. GH increased to 9 and 5 ng/ml during the 0.033- and 0.067-microgram SRIH infusions after GHRF; no somatotropic rebound was observed after the SRIH was discontinued as was seen in the animals while the hypothalamic-hypophysial connections were intact. Five and six months after HST the responses to two analogs of rat hypothalamic GHRF were similar to those in SOC calves. These results indicate that HST calves responded to exogenous GHRF with an abrupt increase in plasma GH, but GH response to GHRF during SRIH infusion was greatly inhibited.     

Effect of atrial natriuretic peptide on the release of beta-endorphin from rat hypothalamo-neurohypophysial complex

The aim of this study was to determine whether atrial natriuretic peptide (ANP) alters beta-endorphin (beta-END) secretion from rat intermediate pituitary and whether this effect is a direct action on the intermediate pituitary or an indirect one mediated by hypothalamic factor(s). We studied the release of beta-END from rat neuro-intermediate lobes of the pituitary (NIL) and from the hypothalamo-neurohypophysial complex (HNC), which consists of the hypothalamus, pituitary stalk, intermediate and posterior lobes of the pituitary, by means of an in vitro perifusion system. NIL and HNC were prepared from male Wistar rats and individually perifused for 30 min with perifusion medium followed by 20 min perifusion with medium containing alpha-rat ANP and/or dopamine (DA). Samples of perifusion medium were collected every 5 min and subjected to RIA for beta-END. The basal release of beta-END from NIL was 180% of that from HNC (p less than 0.01), which provides further support for the presence of hypothalamic factors that inhibit beta-END release from the intermediate pituitary. The perifusion of HNC with ANP at 10(-7) and 10(-6) M increased the beta-END concentration by 25 and 50%, respectively (p less than 0.01). In contrast, ANP (10(-8) to 10(-6) M) had no effect on beta-END release from NIL. The inhibitory effect of DA (10(6) M) on beta-END release from NIL and HNC (51% and 50% of the basal release, respectively, p less than 0.01) was confirmed. However, this inhibitory effect was not reversed by ANP.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo and in vitro effects of cholecystokinin octapeptide on the release of growth hormone in rats

The effect of cholecystokinin octapeptide (CCK-8) on the release of growth hormone (GH) in rats was studied in vivo and in vitro. Intravenous injection of 5 micrograms/100 g BW of CCK-8 resulted in significant increase in the plasma GH level after 10 and 20 min. CCK-8 at concentrations of 10(-11)M to 10(-7)M also caused dose-dependent stimulation of GH release from dispersed cells of rat anterior pituitary. On the other hand, somatostatin (SRIF) inhibited GH release from dispersed cells of rat anterior pituitary in a dose-related manner at concentrations of 10(-7)M to 10(-9)M. Release of GH from the cells was increased by addition of K+ at high concentration (50 mM) in a Ca++-dependent manner. Addition of 10(-3)M verapamil to the incubation medium inhibited CCK-8-induced GH release from the cells. Addition of SRIF (10(-7)M) to the incubation medium inhibited GH release from the cells induced by CCK-8 or high K+ (50 mM). These results indicate that CCK-8 acts directly on the anterior pituitary cells to stimulate GH release and that calcium ion is involved in the mechanism of this effect.     

Ghrelin is released from rat hypothalamic explants and stimulates corticotrophin-releasing hormone and arginine-vasopressin.

Ghrelin and synthetic growth hormone secretagogues have diverse effects on the hypothalamus including effects on appetite and the growth hormone axis as well as on the hypothalamus-pituitary-adrenal (HPA) axis. We previously studied the effect of synthetic growth hormone secretagogues on CRH and AVP release from rat hypothalami in vitro, and now report on the effects of ghrelin on CRH and AVP release. The ghrelin protein content and ghrelin output from rat hypothalamic explants was measured using a specific novel ghrelin enzyme immunoassay. The effect of 10(-8) M to 10(-6) M ghrelin on CRH and AVP release was studied in the rat hypothalamic explants, where stimulation with des-octanoyl ghrelin was used as control. The presence of both ghrelin mRNA and protein could be shown in the rat hypothalamus. Ghrelin output was detected in the incubation fluid of rat hypothalamic explants and could be stimulated with high potassium concentrations. Our data also demonstrated a dose-dependent effect of ghrelin on both CRH and AVP release, while des-octanoylated ghrelin showed no effect on either peptide. In summary, the current data suggest that ghrelin is expressed in the hypothalamus both at RNA and the protein levels. Ghrelin stimulates the HPA axis in the rat via stimulation of both CRH, and particularly, AVP release from the hypothalamus. The local autocrine/paracrine and endocrine effects of ghrelin in the hypothalamus could influence all the hormonal systems involved in ghrelin effects, including growth hormone release, the HPA axis and appetite.     

Effect of dopamine and dopamine-receptor blockade on in vitro renin release, tissue renin content and tissue cyclic AMP content in the rat

This study evaluated the in vitro renin release, tissue cyclic AMP content (TcAMPc), and tissue renin content (TRC) changes with time, in response to administration of dopamine (DOP) and of the dopamine-receptor blocking agent pimozide (PIM) to renal cortical slices from sodium deficient (SD) rats. Addition of 10(-3)M DOP to the slice preparation resulted in a gradual stimulation of RR with time, which was significantly different from that seen in control samples after 60 min of incubation. In contrast, TcAMPc of the DOP-treated samples was significantly greater than that of controls after 5 min of incubation. At 60 min, mean TRC of DOP-treated samples was greater than that of controls but not significantly. Two PIM doses (10(-8)M and 10(-6)M, whether added alone or together with 10(-3)M DOP to the cortical slice system, significantly increased RR in each instance while simultaneously depressing TcAMP content markedly below that of unstimulated controls at all incubation times examined. Mean TRC of pimozide-treated samples was also lower than that of controls by 60 min. These in vitro data in the SD rat suggest that: 1) stimulation of renin release by DOP is time-dependent and is mediated by a TcAMP-generating mechanism, and 2) the increase in renin release by PIM administration appears to involve pharmacological inactivation of TcAMP-generating pathways and disruption of membrane permeability, leading to uncontrolled RR.     

Growth hormone and prolactin secretion in hypophysial stalk-transected pigs as affected by growth hormone and prolactin-releasing and inhibiting factors.

Control of growth hormone (GH) and prolactin (PRL) release was investigated in hypophysial stalk-transected (HST) and stalk-intact pigs by determining the effects of analogs of GH-releasing factors (GHRF), somatostatin (SRIF), arginine, thyrotropin-releasing hormone, alpha-methyl-rho-tyrosine, and haloperidol. HST and control gilts were challenged with intravenous injections of human pancreatic GHRF(1-40)OH, thyrotropin-releasing hormone, and analogs of rat hypothalamic GHRF. HST animals remained acutely responsive to GHRF by releasing 2-fold greater quantities of GH than seen in controls. This occurred in spite of a 38% reduction in pituitary gland weight and a 32 and 55% decrease in GH concentration and total content. During SRIF infusion, GH remained at similar basal concentrations in HST and control gilts, but increased immediately after stopping SRIF infusion only in the controls. Releasable pituitary GH appears to accumulate during SRIF infusion. GHRF given during SRIF infusion caused a 2-fold greater release of GH than seen in animals receiving only GHRF. Arginine increased (P less than 0.05) GH release in controls, but not in HST gilts, which suggests that it acts through the central nervous system. Basal PRL concentrations were greater (P less than 0.05) in HST gilts than in control gilts. TRH acutely elevated circulating PRL (P less than 0.001) in HST gilts, suggesting that it acts directly on the pituitary gland. Haloperidol, a dopamine receptor antagonist, increased circulating PRL in controls but not in HST animals. alpha-Methyl-rho-tyrosine did not consistently increase circulating PRL, however, suggesting that it did not sufficiently alter turnover rate of the tyrosine hydroxylase pool. The results indicate that the isolated pituitary after HST remains acutely responsive to hypothalamic releasing and inhibiting factors for both GH and PRL release in the pig.     

Effects of porcine diazepam-binding inhibitor on insulin and glucagon secretion in vitro from the rat endocrine pancreas.

Porcine diazepam-binding inhibitor (pDBI) is a novel peptide that has been isolated from the small bowel of the pig, and that occurs also in the islet D-cells. We have studied its effects on hormone release in vitro from the endocrine pancreas of the rat. In isolated islets, pDBI (10(-9)-10(-6)M) did not affect basal insulin release at 3.3 mM glucose, whereas stimulated release at 8.3 mM glucose was dose-dependently suppressed by 32-69% (P less than 0.01). Furthermore, insulin secretion stimulated by either 16.7 mM glucose or 1 mM IBMX (3-isobutyl-1-methylxanthine) or 1 micrograms/ml glibenclamide was suppressed by pDBI at 10(-8) M (by 28-30%, P less than 0.05) and 10(-7) M (by 43-47%, P less than 0.01). In contrast, islet insulin secretion induced by 20 mM arginine was unaffected by these concentrations of pDBI. In the perfused rat pancreas, pDBI (10(-8) M) enhanced by 30% (P less than 0.05) the first phase (0-5 min) of arginine-stimulated insulin release, whereas the second phase (5-20 min) was unchanged. Moreover, pDBI suppressed by 28% (P less than 0.05) the second phase of arginine-induced glucagon release. Arginine-induced somatostatin release was not significantly affected by the peptide. Since pDBI immunoreactivity has been localized also to islet D-cells, the present results suggest that pDBI may act as a local modulator of islet hormone release.     

Functional interaction between transforming growth factor alpha and capsaicin-sensitive sensory neurons in the rat stomach

BACKGROUND AND AIMS: Transforming growth alpha (TGFalpha) and sensory neurons have been shown to promote gastric mucosal protection and healing. Aims were to examine in vitro interactions between gastric sensory neurons, the sensory neuropeptide calcitonin gene-related peptide (CGRP), and TGFalpha. METHODS: Gastric mucosal/submucosal tissue fragments from Sprague-Dawley (SD) rats were incubated in short-term (30 min) culture. Peptide release into media and TGFalpha tissue content were measured by radioimmunoassay. RESULTS: TGFalpha (1 x 10(-8) to 1 x 10(-6) M) caused dose-dependent stimulation of CGRP release. Maximal CGRP release (+87%) was observed with 1 x 10(-6) M TGFalpha: 28.6+/-3.8 vs. control of 15.5+/-2.7 pg/g tissue; P<0.05. Both CGRP (1 x 10(-7) to 1 x 10(-5) M) and capsaicin (1 x 10-(8) to 1 x 10(-6)M) significantly inhibited basal TGFalpha release in a dose-dependent fashion that ranged from -20% to -39%. In contrast, capsaicin-induced sensory denervation caused significant increases in both basal TGFalpha release and TGFalpha tissue content. CONCLUSION: Function interactions between TGFalpha and gastric sensory neurons are suggested by the observations that (1) TGFalpha stimulated CGRP release from gastric sensory neurons; (2) CGRP and acute capsaicin treatment inhibited TGFalpha release and; (3) capsaicin-induced sensory denervation caused significant increases in both gastric TGFalpha basal release and tissue content.     

Insulin-like growth factor-1 stimulates growth of mouse preimplantation embryos in vitro.

Because recent studies have particularly implicated the insulin growth factor family in early development, the effects of insulin-like growth factor (IGF-1) on the development of mouse embryos in vitro were investigated in detail. When added to the medium for culture of two-cell embryos, IGF-1 stimulated the number of cells in the resultant blastocysts after 54 hr, entirely by increasing the number of cells in the inner cell mass (ICM) (16.0 +/- 0.5 vs. 12.6 +/- 0.5 cells/ICM). This stimulation was also achieved when ICMs were isolated from blastocysts prior to culture for 24 hr with IGF-1 (22.3 +/- 1.0 vs. 17.5 +/- 0.8 cells/ICM). There was no effect on IGF-1 on trophectoderm (TE) cell proliferation. In morphology studies, IGF-1 also increased the proportion of blastocysts (62% +/- 3% vs. 49% +/- 4%) while decreasing the number of embryos remaining as morulae (32% +/- 3% vs. 38% +/- 2%) or in the early cleavage stages (7% +/- 3% vs. 13% +/- 3%) after 54 hr culture from the two-cell stage. All these effects were achieved with EC50s of approximately 60 pM IGF-1, which is in the range for IGF-1 receptor mediation; however, cross reaction with insulin, IGF-2, or other unknown receptors is not excluded. Nonetheless, the results show that physiological concentrations of IGF-1 (17-170 pM, 0.1-1 ng/ml), which have been observed in the reproductive tract, affect the early embryo, suggesting a normal role for this factor in the regulation of growth of the developing conceptus before implantation.     

Immunocytochemical localization of growth hormone releasing factor (GHRF)-containing structures in the rat brain using anti-rat GHRF serum

The distribution of growth hormone releasing factor (GHRF) immunoreactive structures in the rat hypothalmus was studied after colchicine treatment with PAP immunocytochemistry in vibratome sections using an antiserum directed to rat hypothalamic GHRF. The majority of the GHRF-immunoreactive cell bodies were found in the arcuate nucleus, the medial perifornical region, and the ventral premammillary nuclei of the hypothalamus. Scattered cells were seen in the lateral basal hypothalamus, the medial and lateral portions of the ventromedial nucleus, and the dorsomedial and paraventricular nuclei. Immunoreactive fibers were observed in all the regions mentioned above. GHRF terminals were located in the central region of the median eminence. In addition, GHRF-immunoreactive neuronal processes were seen in the ventral region of the dorsomedial nucleus, the medial preoptic and suprachiasmatic regions, dorsal portion of the suprachiasmatic nucleus, bed nucleus of the stria terminals and the hypothalamic portion of the stria terminals. The localization of GHRF-immunoreactive terminals in the median eminence reinforces the view that GHRF plays a physiological role in the regulation of pituitary function. In addition, the localization of GHRF-immunoreactive structures in areas not usually considered to project to the median eminence suggest that GHRF may act as a neuromodulator or neurotransmitter.     

Growth hormone releasing factor: comparison of two analogues and demonstration of hypothalamic defect in growth hormone release after radiotherapy.

Human pancreatic growth hormone releasing factor (hpGHRF(1-40] stimulates the release of growth hormone in normal subjects and some patients with growth hormone deficiency. A study comparing the shorter chain amidated analogue hpGHRF(1-29) with an equivalent dose of hpGHRF(1-40) in seven normal subjects showed no significant difference in growth hormone response between the two preparations. Six patients with prolactinomas were also tested; these patients had received megavoltage radiotherapy previously but had developed growth hormone deficiency as shown by insulin induced hypoglycaemia. In all six patients 200 micrograms hpGHRF(1-40) or hpGHRF(1-29)NH2 produced an increase in the serum growth hormone concentration. These data suggest that hpGHRF(1-29)NH2 may be useful for testing the readily releasable pool of growth hormone in the pituitary and that cases of hypothalamo-pituitary irradiation resulting in growth hormone deficiency may be due to failure of synthesis or delivery of endogenous GHRF from the hypothalamus to pituitary cells.     

Effects of phorbol ester on GH, TSH and PRL release by superfused rat adenohypophysis

The present study was undertaken to examine the effects of 12-0-tetradecanoyl-phorbol-13-acetate (TPA), one of the potent tumor promoting agents, on GH, TSH and PRL release by rat adenohypophyseal dispersed cells and fragments, using a superfusion technique. TPA (10(-6) to 10(-5) M) stimulated GH release from acutely dispersed rat adenohypophyseal cells. Neither TSH nor PRL was affected, but both were increased by TRH in a dose-dependent fashion (10(-9) to 10(-7) M). In fragments, TPA (10(-8) to 10(-6) M) elicited a dose-related release of GH. Exposure of the fragments to 10(-6) M TPA for 5 min promptly caused a 5-fold increase in GH release which continued for at least 40 min after stopping the stimulation. The addition of somatostatin (SRIF) (10(-7) M) decreased basal GH release and abolished GH release induced by 10(-6) M TPA. In contrast to GH, neither TSH nor PRL release was affected by TPA, but both were stimulated by TRH. These results indicate 1) that GH release is more sensitive to stimulation with TPA in normal rat anterior pituitaries in vitro than the release of TSH and PRL, and 2) that SRIF abolishes TPA-induced GH release.     

Endothelin-1 and endothelin-3 stimulate prostaglandin E2 secretion from the rat median eminence.

The in vitro effects of endothelin-1 (ET-1) and endothelin-3 (ET-3) on the release of prostaglandin (PG)E2 from the rat median eminence were investigated. The addition of ET-1 from 10(-9) M to 10(-6) M stimulated PGE2 release in a dose-dependent manner (from 10.5 +/- 2.1 to 54.4 +/- 5.6 pg/ME fragment/30 min; mean +/- SEM, p less than 0.001). ET-3 also stimulated the release of PGE2 from 10(-7) M to 10(-5) M dose dependently (from 18.1 +/- 0.7 to 60.9 +/- 17.4 pg/ME fragment/30 min p less than 0.05). The time course effect of ET-3 (10(-6) M) showed that PGE2 release was stimulated within five minutes (control, 1.5 +/- 0.5; ET-3, 15.8 +/- 3.0 pg/ME fragment/5 min, p less than 0.01). These results suggest that ET-1 and ET-3 have some physiological effects on the rat median eminence.     

In vitro, release of cholecystokinin from hypothalamus and frontal cortex of Sprague-Dawley, Zucker lean (Fa/-) and obese (fa/fa) rats

Cholecystokinin (CCK) has been suggested as a putative satiety factor, whose site of action is in the hypothalamus. The genetically obese (fa/fa) Zucker rat has been proposed as a model of human obesity. Though hypothalamic tissue levels of CCK did not vary between the fa/fa rat and age-matched lean littermates (25.5 +/- 5.7 vs. 27.6 +/- 5.2 pmoles/g tissue) we sought to determine if the releasability of hypothalamic and cortical CCK was the same in lean and obese rats. The in vitro superfusion paradigm was used to study the release of CCK and substance P (sP) from hypothalamus, and CCK and vasoactive intestinal polypeptide (VIP) from frontal cortex. The potassium stimulated release of CCK from obese rat hypothalamic tissue was significantly higher than from lean rat hypothalamus (3.62 +/- 0.3 vs. 1.91 +/- 0.3 fmole equivalents CCK-8/mg tissue/10 min). Similarly, sP release was exaggerated in obese rats in a parallel fashion (5.56 +/- 0.44 vs. 2.761 +/- 0.46 fmoles/mg tissue/10 min). However, the potassium stimulated release of CCK and VIP from cortical tissue was the same in all three groups of rats. The obese Zucker rat thus, may have an anomalous release of CCK and sP from the hypothalamus, but not from the frontal cortex, an area not presumably associated with satiety.     

Effect of dopamine, bombesin and cysteamine hydrochloride on plasma growth hormone response to synthetic growth hormone-releasing factor in rats

In urethane anesthetized rats, an intracerebroventricular (icv) injection of 2 micrograms bombesin 5 min prior to the administration of synthetic human growth hormone-releasing factor (GRF) (1 microgram/kg, iv) inhibited plasma growth hormone (GH) response, while cysteamine hydrochloride (90 mg/kg, sc) administered 150 min beforehand depleted immunoreactive somatostatin content in the pituitary-stalk median eminence and consequently potentiated the response to GRF. Under the same experimental conditions, central injection of 1.89 micrograms (10(-8)M) dopamine hydrochloride or iv administration of L-DOPA (10 mg/kg) did not influence the subsequent plasma GH response to GRF. Results suggest indirectly that bombesin and cysteamine, but not dopamine, predominantly modulate somatostatin release from the hypothalamus.     

Ontogeny of the response to growth hormone-releasing factor

Primary cell cultures were prepared from fetal, neonatal and adult rat pituitaries and evaluated for their ability to secrete growth hormone (GH) in response to growth hormone-releasing factor (GRF). Pituitary cells prepared from fetuses at days 19 and 21 of gestation, neonatal animals at the day of birth (day 0) or the following day (day 1) and peripubertal male rats showed full dose response curves to GRF with maximal GH release when stimulated with 1 X 10(-10) M rat GRF. At this concentration of GRF, the amount of GH released was not different from that elicited by activation of adenylate cyclase with 1 X 10(-5) M forskolin. In contradistinction, a preparation of cells from fetuses at day 18 of gestation did not show the same release of GH when challenged with 1 X 10(-10) M GRF and forskolin (0.057 +/- 0.001, compared to 0.076 +/- 0.003 micrograms/10(5) cells per 4.5 h), although the cells clearly responded to both secretagogues (basal levels of GH, 0.029 +/- 0.002 micrograms/10(5) cells per 4.5 h). While cells prepared from fetuses at day 21 of gestation or from animals after birth released 5-10% of their total cellular GH content, those prepared from 18- and 19-day fetuses released as much as 40% of their total GH suggesting there is a maturation of intracellular GH processing that occurs late in gestation. The results show that, in late pregnancy, the rat fetal pituitary is highly responsive to growth hormone-releasing factor and suggest that this peptide participates in regulating GH levels during the perinatal period.     

Binding and action of insulin-like growth factors and insulin in bovine luteal tissue during the oestrous cycle.

The effect of insulin-like growth factors (IGFs) and insulin on the release of progesterone and oxytocin from bovine corpus luteum was investigated at early (days 5-7), mid- (days 8-12) and late (days 15-18) luteal phases of the oestrous cycle in an in vitro microdialysis system. The expression of specific receptors was evaluated in bovine corpora lutea of the respective luteal stages. A 30 min infusion of IGF-1, IGF-2 (1.3, 13 and 130 nmol l-1) or insulin (13, 130 and 1300 nmol l-1) caused a stimulation of the release of progesterone (P < 0.05). IGF-1 was most effective in releasing progesterone. Oxytocin release from corpora lutea was stimulated by insulin at all doses tested (13-1300 nmol l-1), whereas the IGFs were only effective at the highest dose (130 nmol l-1) applied. The high doses of IGFs (130 nmol l-1) and insulin (1300 nmol l-1) stimulated the release of progesterone and oxytocin throughout the luteal phase (P < 0.05). For all three peptides, greatest stimulation was seen during the late luteal phase (days 15-18 of the oestrous cycle) with the peak of progesterone release directly related to peptide infusion (P < 0.05). In addition, IGF-1 stimulated total release of progesterone (units in 4 h) after the beginning of the stimulation during this phase (P < 0.05). IGF-1 caused a gradual increase of progesterone even beyond the time of peptide perfusion, whereas IGF-2 and insulin stimulated progesterone release only during the peptide perfusion. Distinct receptors for IGF-1 and IGF-2 were present in corpora lutea membrane preparations at all stages investigated. Specific binding for insulin was also seen in all stages of the cycle without any cycle-dependent changes in the amount of binding. The displacement of labelled insulin by unlabelled IGF-1 and IGF-2 did not show the rank of order that has been described as typical for insulin receptors (i.e. insulin > IGF-1 > IGF-2), but comparable binding affinities were observed for the three unlabelled ligands. Specific binding of IGF-2 was markedly higher than that of IGF-1 or insulin throughout the cycle (1.9- and 4.9-fold higher compared with IGF-1 and insulin, respectively). Receptor specificity did not change during luteal development. Binding affinity and capacity of IGF-1 receptor was constant throughout the oestrous cycle. Specific IGF-2 binding increased and showed a positive co-operativity towards the end of the cycle. Specific binding of insulin was not significantly different in the three luteal stages examined.