Induced Growth Hormone Release Research Articles

Involvement of Rabphilin-3A in Ca2+-Dependent Exocytosis from PC12 Cells

Rabphilin-3A, a putative target molecule of Rab3A small GTP-binding protein implicated in Ca2+-dependent exocytosis, consists of two functionally different domains: the N-terminal Rab3A-binding domain and the C-terminal two C2-like domains (C2A and C2B domains) interacting with Ca2+and phospholipid. Here, we used the growth hormone (GH) co-expression assay system of PC12 cells in which expressed GH is released in response to high K+. Reduction of endogenous rabphilin-3A inhibited the Ca2+-dependent, high K+-induced GH release. Various rabphilin-3A mutants expressing an N-terminal, C-terminal, or C2B fragment, but not the rabphilin-3A mutant expressing a C2A fragment, inhibited the high K+-induced GH release. These results indicate that rabphilin-3A is involved at least in Ca2+-dependent exocytosis from PC12 cells and that the C2A and C2B domains have different functions.

Inverse control of growth hormone and prolactin secretion in clonidine-stimulated dairy cattle.

Clonidine is a specific alpha-2-adrenoreceptor agonist that stimulates growth hormone (GH) release in animals and humans. This drug was used to study the GH and prolactin (PRL) secretory response in dairy cows and heifers. An i.v. infusion of 10 micrograms/kg body weight induced GH release to a peak concentration after 30-60 min, while 2 micrograms/kg had no effect on GH secretory patterns. Plasma PRL decreased significantly (P < 0.01) starting 15-60 min after both doses of clonidine, this effect lasting up to 6 h. Clonidine significantly lowered plasma insulin (P < 0.01) and raised plasma glucose (P < 0.01). The changes in plasma GH, PRL, insulin and glucose differed significantly between doses, the 10 micrograms/kg dose being more effective (P < 0.01). The results of our investigation in dairy cattle provide evidence of (i) an increase in GH release after 10 micrograms/kg clonidine; (ii) a concomitant decrease in PRL secretion, hence GH and PRL secretion in cattle appear inversely controlled; (iii) a significant difference between the effects of the 2 and 10 micrograms/kg doses and (iv) no relationship between the changes in plasma GH and PRL after clonidine and plasma hormone levels before treatment.

The interactive effects of VIP, PHI, GHRH, and SRIF on the release of growth hormone from cultured adenohypophysial cells in cattle.

The effects of hypothalamic peptides [vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), growth hormone (GH)-releasing hormone (GHRH) and somatostatin (SRIF) on GH release from cultured bovine adenohypophysial cells were studied. The cells were incubated for 2 h with the peptides after preincubation for 3.5 days. At doses from 10(-9) to 10(-7)M VIP, the amount of GH released was significantly greater than in the controls (P < 0.05 to P < 0.001). PHI (10(-10 to 10(-7)M did not alter the bovine GH concentration in the media. Incubation with the media containing 10(-7)M GHRH, 10(-7)M VIP, and combined treatment with the VIP plus GHRH increased GH by 186, 40 and 182%, respectively (P < 0.001). Furthermore, although VIP-induced GH release was significantly decreased by SRIF compared with the treatment with VIP alone (P<0.001), the VIP significantly blunted the inhibitory effect of the SRIF on GH release by 24% when compared with that of the SRIF plus GHRH without the VIP (P < 0.05). GH release in combined treatments with VIP, GHRH and SRIF was significantly less than that of the VIP plus GHRH (P < 0.001), but it was significant 29% increase compared with the SRIF plus GHRH (P < 0.05). The combined effects of the VIP (10(-7)M) with GHRH (10(-7), 10(-8) and 10(-10)M significantly induced GH release compared with the controls (P < 0.001), but no additive effect was not observed when compared with the GHRH alone. The results indicate that VIP, but not PHI, acts directly on cultured adenohypophysial cells to induce GH release in cattle.

Somatotropic dysregulation in old mammals.

In old mammals, including humans, the spontaneous growth hormone (GH) secretory pattern is markedly reduced resulting in lower amounts of GH released over 24 h, and the GH response to administration of GH-releasing hormone (GHRH) is reduced. In agreement with these in vivo findings, an impaired responsiveness to GHRH is evident in the pituitary of old male and female rats in vitro, and this is linked with a diminished stimulation of adenylate cyclase by GHRH. The poor GH responsiveness to GHRH in old mammals, which in the rat is coupled to a defective number of GHRH receptors in the somatotrophs, is likely due to a primary deficiency of GHRH availability, as implied by the diminished GHRH immunoreactivity and gene expression in and GHRH release from the hypothalamus of old rats. Attempts have been made to stimulate the sluggish somatotrophic function in elderly humans and dogs using GHRH; in either species positive results were obtained though, overall, it would seem that the GHRH hypofunction does not entirely account for the GH hyposecretory state during ageing. Concerning somatostatin, although the expression of this peptide decreases with age in the rat hypothalamus, secretion and activity of this hormone is increased, resulting in an altered relationship between GHRH and somatostatin gene expression and secretion. It is likely that defects, especially in catecholaminergic and cholinergic neurons, are instrumental in altering specific peptidergic neurons. Reportedly, catecholamines induce GH release by stimulating GHRH neurons and inhibiting somatostatin-releasing neurons; acetylcholine stimulates GH release via muscarinic receptors, in this way inhibiting the action of somatostatin neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Entry of Extracellular Calcium Mediates Dopamine D1-Stimulated Growth Hormone Release from Goldfish Pituitary Cells

Unlike mammals, the goldfish is unique in having dopamine (DA) D1 receptors in the anterior pituitary. In this species, DA stimulates growth hormone (GH) release via D1 receptors coupled to the cAMP-dependent pathway. To further examine the postreceptor mechanisms of this novel pituitary DA D1 system, the role of extracellular Ca2+ ([Ca2+]c] in mediating DA D1-stimulated GH release was studied using dispersed goldfish pituitary cells. The GH responses to DA (1 nM-10 μM), the D1 agonist SKF38393 (1 μM), and the Ca2+ ionophore A23187 (10 μM) were abolished by incubation with Ca2+-deficient medium. Incubation with depolarizing doses of KCl (10-25 mM), which activate voltage-sensitive Ca2+ channels (VSCC), induced GH release in a dose-dependent manner. In contrast, the VSCC blockers nifedipine (10 μM), nicardipine (10 μM), and verapamil (10 μM) and the inorganic competitor of Ca2+ entry CoCl2 (5 mM) blocked the GH responses to DA (1 μM) as well as SKF38393 (1 μM). These results strongly indicate that the entry of [Ca2+]c via VSCC is an essential part of the signal transduction mechanisms mediating DA D1-stimulated GH release in the goldfish. In this study, the possible interactions between the Ca2+- and cAMP-dependent pathways in DA-induced GH secretion were also investigated. The membrane-permeant cAMP analogue 8Br.cAMP (I mM) and the adenylate cyclase activator forskolin (10 μM) stimulated GH release from goldfish pituitary cells. These GH responses were suppressed by incubation with Ca2+-deficient medium or with the VSCC blocker nifedipine (10 μM). Furthermore, the GH responses to forskolin (10 μM) and the nonselective DA agonist apomorphine (1 μM) were not additive to that of the Ca2+ ionophore A23187 (10 μM). These results suggest that [Ca2+]c entry induced by DA D1 stimulation occur at steps after activation of the cAMP-dependent pathway.

Effect of growth hormone (GH)-releasing peptide (GHRP) on the release of GH from cultured anterior pituitary cells in cattle.

The effect of His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP) on growth hormone (GH) release from cultured bovine anterior pituitary (AP) cells was studied in vitro with the interactive effects of GH-releasing factor (GRF: hpGRF (1-29)-NH2) and somatostatin (SRIF). The AP cells (5 x 10(4) cells per well) were incubated with media, and the media were changed 3 days after plating. After 3.5 days in culture, cells were incubated for 2 h with the peptides. GHRP stimulated GH release from cultured cells in a dose-related manner. At doses from 10(-11) to 10(-7) M GHRP, the amount of GH released was significantly greater than the controls (P < 0.05 to P < 0.001). The amounts of GH released at lower doses of GHRP (10(-14) to 10(-12) M) were not significantly different from the controls. GH concentrations after treatment with 10(-11) and 10(-7) M GHRP were 3.98 +/- 0.27 and 4.81 +/- 0.16 ng/ml, respectively. In experiments performed similarly, the 10(-7) M GHRH, GHRP, and combined treatment with GHRP plus GHRH increased GH 126, 57, and 139%, respectively (P < 0.001). The GH releasing effects of either GHRH alone or GHRP plus GHRH were significantly more potent than that of GHRP alone (P < 0.001). The additive effect was not significant when compared with GHRH alone. GH release induced by either GHRH or GHRP was significantly inhibited by SRIF (P < 0.01) compared with the untreated control. The inhibitory effect of SRIF in combined treatment with GHRP plus GHRH was significantly less than that of SRIF with GHRH or with GHRP (P < 0.01). The present study suggests that GH-releasing peptide (GHRP) induced GH release in cattle via a direct action on anterior pituitary cells in vitro.

Cyclic 3',5'-adenosine monophosphate mediates dopamine D1-stimulated growth hormone release from goldfish pituitary cells.

Previously, we have demonstrated that dopamine (DA) stimulates growth hormone (GH) release from the goldfish pituitary through DA D1 receptors. In the present study, the role of cAMP in DA D1-stimulated GH release was investigated using static incubation of goldfish pituitary cells. The D1 agonist SKF38393 (1 nM-10 microM) induced GH release and cAMP accumulation in a dose-dependent manner with ED50s of 73 +/- 32 and 109 +/- 53 nM, respectively. In contrast, the D2 agonist LY171555 (1 nM-10 microM) was not effective in these regards. The GH-releasing action of SKF38393 was mimicked by the adenylate cyclase activator forskolin (0.1-40 microM) as well as the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.1 microM-1 mM). Dideoxyforskolin (0.1-40 microM), a derivative of forskolin inactive in stimulating adenylate cyclase, did not affect basal GH secretion. Similar stimulatory effects on GH release were also observed using the membrane-permeant cAMP analogs (10 microM-2 mM), dibutyryl cAMP and 8-bromo cAMP (8Br.cAMP). In the presence of a high dose (1 mM) of Br.cAMP, the ability of SKF38393 (1 nM-10 microM) to stimulate GH release was abolished, suggesting that the GH-releasing actions of cAMP and DA D1 stimulation are mediated through a common signal transduction mechanism. In the present study, the possible involvement of the cAMP-dependent enzyme protein kinase A (PKA) in DA D1-stimulated GH release was also examined. The GH responses to 8Br.cAMP (1 mM) and SKF38393 (1 microM) were blocked by simultaneous treatment with the PKA inhibitor H89 (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Pyridostigmine induced growth hormone release in mania: focus on the cholinergic/somatostatin system

The release of growth hormone (GH) from the anterior pituitary is partly under cholinergic control. Pyridostigmine, the acetylcholinesterase inhibitor, releases GH by this mechanism. Pyridostigmine/GH responses have been reported as enhanced in depression. The aim of the current study was to examine such responses during a manic episode. A between subjects design was employed. Seven male manic patients and seven male healthy controls were studied. They were matched in terms of age and body mass index. GH response to pyridostigmine (120 mg) challenge was measured as the net increase above baseline. Cortisol levels were also measured. Release of GH in the manic patients was significantly enhanced and their baseline cortisol levels were elevated. These results demonstrate enhanced pyridostigmine/GH responsiveness in mania which may be due to enhanced somatostatin tone or increased cholinergic receptor responsivity.

The involvement of dihydropyridine-sensitive calcium channels in phorbol ester-induced luteinizing hormone and growth hormone release

We examined the role of voltage-activated, L-type, Ca 2+ channels in phorbol ester-induced luteinizing hormone (LH) and growth hormone (GH) release from rat anterior pituitary tissue. The L-type Ca 2+ channel inhibitor, nimodipine (NMD), inhibited phorbol 12,13-dibutyrate (PDBu)-induced GH release but had no significant effect on LH release. The L-type Ca 2+ channel activator BAY K 8644 had no effect on PDBu-induced GH release but potentiated PDBu-induced LH release. In contrast, 60 mM K +-induced LH and GH release were inhibited by NMD, whereas BAY K 8644 had no effect. When PDBu and either K + or BAY K 8644 were used together, they acted synergistically to evoke levels of LH release greater than addition of release caused by each secretagogue alone. However, the release of GH was additive with PDBu and either K +, BAY K 8644. The protein kinase C (PKC) inhibitor staurosporine inhibited both PDBu-induced LH release and GH release. A structurally different PKC inhibitor, H7, significantly inhibited PDBu-induced LH release but had no effect on PDBu-induced GH release. Both staurosporine and H7 inhibited LH release induced by PDBu and BAY K 8644 together. In contrast, although staurosporine inhibited GH release induced by PDBu and BAY K 8644, H7 significantly potentiated this response. A difference in the action of these two inhibitors was also apparent on K +-induced hormone release where staurosporine partially blocked K +-induced LH and GH release but H7 had no effect on the release of either hormone. Data obtained in 45Ca 2+ influx experiments further suggested that a staurosporine-sensitive, but H7-resistant, PKC-like kinase may tonically maintain L-channels in a voltage-sensitive state, as down-regulation of PKC in dispersed anterior pituitary cells by long term PDBu treatment caused a significant reduction in K +-induced 45Ca 2+ influx. We conclude that phorbol ester-induced GH release, but not LH release, is a result of L-type Ca 2+ channel activation which may occur by means of alterations in the channel itself to increase its responsiveness to a given depolarisation.

Demonstration of biological activity of a growth hormone-releasing hormone-like substance produced by a pheochromocytoma

The biological characteristics of a growth hormone-releasing hormone (GHRH)-like substance produced by a pheochromocytoma were studied. Analysis by gel filtration chromatography combined with the use of two distinct GHRH antisera that recognize the N- and C-termini of authentic GHRH(1-44)NH2 indicated molecular heterogeneity of the immunoreactive GHRH in the tumor extract, but a component corresponding to GHRH(1-44)NH2 was the predominant form. The biological activity of this immunoreactive component was assessed in vitro by measuring its ability to induce growth hormone release from dispersed rat anterior pituitary cells. At concentrations of 0.125-2.0 nmol/l, the test materials induced a dose-related increase in growth hormone release from the cells into the incubation medium (range 992 +/- 68-1872 +/- 32 ng.1.7 x 10(5) cells-1 x 3 h-1), similar to that observed with synthetic GHRH(1-44)NH2. (control value 640 +/- 30 ng.1.7 x 10(5) cells-1 x 3 h-1). This suggests that immunoreactive GHRH in the tumor has almost the same biological activity as the synthetic product and that a combination of pheochromocytoma and acromegaly is not always fortuitous because both diseases may be caused by a single neoplasm.

Pyridostigmine potentiates growth hormone (GH)-releasing hormone- induced GH release in both men and women

Pyridostigmine potentiates growth hormone (GH)-releasing hormone- induced GH release in both men and women

Studying brain receptor function: a neuroendocrine approach

AbstractA significant component of psychiatric practice relates to the management of patients with behavioural disturbance whose aetiology lies in the subtle alteration of brain biochemistry. The major handicap in assessing such patients, both from a clinical and a research point of view has been a lack of suitably sophisticated technology for studying brain function. Despite significant improvements in imaging technique and the development of positron emission tomography we are still lacking tools which assess brain receptor functioning. The neuroendocrine axis provides us with the means of assessing specific neurotransmitters in a safe and relatively inexpensive way. Such an approach is now widely used in research and has considerable potential within a clinical setting.The fact that classic monoamine neurotransmitters are implicated both in affective disorders and schizophrenia and at the same time control hypothalmic-anterior pituitary function provides the basis for many psychoneuroendocrine investigations. The stimulation of certain central neurotransmitter systems results in the elevation of anterior pituitary hormones. If a pharmacologically selective drug is used, the rise in the anterior pituitary hormone gives some index of the integrity of the neurotransmitter pathway and the sensitivity of its receptor system. This approach is heavily dependent on the development of selective drugs for challenging specific receptor systems. As there are a myriad of potential confounding variables it is essential that there be rigorous control over such factors as gender, age, psychotropic drug exposure, weight loss etc.The release of growth hormone (GH) from the anterior pituitary is under the control of 2 peptides, namely growth hormone releasing hormone (GHRH) and somatostatin (SS). Noradrenaline acting via the GHRH containing neurones stimulates the release of GH (1,2). We now know that the stimulated release of GH through this mechanism is significantly blunted in patients with major depression (3,4,5). When sone suppression and noradrenergic mediated GH release are both investigated in depressed patients, those subjects who show dexamethasone non-suppression are more likely to demonstrate blunted GH release than those with normal dexamethasone responses (5).Acetylcholine (ACh) stimulates growth hormone release via the SS method (6). It is now clear that depressed patients show enhanced release when their cholinergic system is challenged with pyridostigmine (7). Overall therefore, depressed patients seem to have a downregulation or under activity of their NA receptors and an up-regulation or over-activity of their ACh receptors.GH release is also under GABAergic control. In a study of patients with major depression baclofen the GABA-B agonist was used to induce GH release. Baclofen (20 mg) significantly elevated GH levels in all healthy subjects but a blunting of response was seen in those patients with major depressive illness. The finding indicates diminished responsivity of the GABA-B receptor system in depression (7a).The release of prolactin from the anterior pituitary is under the inhibitory control of dopamine (DA) which acts directly on the lactotrophs of the pituitary. The release of prolactin is stimulated by serotonin (8,9). There is now unequivocal evidence to indicate that 5-HT mediated prolactin release is blunted in major depression (10,11). Such blunting has been demonstrated with a wide variety of probe drugs including 1-tryptophan and fenfluramine. The abnormality is however not entirely specific to depression as patients with obsessive compulsive disorder and sociopathic personality disorder also demonstrate such blunting even in the absence of mood disturbance (12,13).The first evidence to emerge that central DA receptors might in some way control GH release was demonstrated by the administration of 1-dopa which led to an increase in GH levels (14). GH responses to the DA agonist apomorphine have been reported to be greater in patients with first rank symptoms of schizophrenia, but to be blunted in those patients with significant egative symptoms such as emotional flattening and social withdrawal (15,16).The very complex neurotransmitter control of such hormones as GH and prolactin provides a window to the biology of neuro-behavioural disturbance. As a tool psychoneuroendocrinology is of relevance not just to the researcher but to the practicing psychiatrist as well.

Evidence for a role of protein kinase-C in His-D-Trp-Ala-Trp-D-Phe-Lys-NH2-induced growth hormone release from rat primary pituitary cells.

We have recently reported that His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP-6) synergizes with GH-releasing factor (GRF) to increase GH release and cAMP accumulation in rat pituitary cells in vitro. This study was undertaken to further investigate the mechanism of action of GHRP-6 on GH release, particularly the involvement of protein kinase-C. Forskolin (10(-5) M), A23187 (10(-6) M), and phorbol 12-myristate 13-acetate (PMA; 10(-7) M) all stimulated GH release. However, only PMA can mimic the synergistic effects of GHRP-6 on GRF-stimulated GH release and intracellular cAMP accumulation. 4 alpha-Phorbol 12,13-didecanoate, an inactive phorbol ester, was unable to stimulate GH release or potentiate the effect of GRF. Extracellularly added phospholipase-C not only stimulated GH release in a dose-dependent manner, but also potentiated GRF-induced GH release. Phloretin, a protein kinase-C inhibitor, in a concentration range of 10-250 microM had very little or no effect on basal and GRF-stimulated GH release, but markedly inhibited the stimulatory effects induced by either PMA or GHRP-6. Incubation of rat pituitary cells with 10(-6) M PMA for 24 h completely down-regulated protein kinase-C, since such PMA-pretreated cells did not release GH in response to a second dose of PMA. The protein kinase-C-depleted cells had an attenuated GHRP-6 response, but they responded normally to GRF. Moreover, the synergistic effects of GHRP-6 and GRF on GH release and cAMP accumulation were also greatly inhibited by protein kinase-C down-regulation. These data suggest that the effects of GHRP-6 on GH release, either alone or together with GRF, are at least partially mediated via the activation of protein kinase-C.

Differential effects of T4 and T3 on TRHand GRF-induced GH secretion in the domestic fowl

The in vivo growth hormone (GH) response of immature domestic fowl to thyrotrophin-releasing hormone (TRH) and GH-releasing factor (GRF) was suppressed in birds fed diets supplemented (1 ppm) with triiodothyronine (T3) or given bolus intraperitoneal (ip) injections (100 micrograms/kg for 10 d) of T3. Supplementation (ppm) of the diet with T4 had no effect on secretagogue- induced GH release. Exogenous T3 or T4 suppressed basal, TRH- and GRF-induced GH release 2 h after daily ip administration (100 micrograms/kg for 10 d). 24 h after the last injection, only T3 was effective in inhibiting basal and stimulated GH secretion in vivo. The systemic administration of T3 was followed 2 h and 24 h later by a downregulation of pituitary TRH binding sites. T4 administration had no effect on pituitary TRH binding. When chicken pituitary glands were incubated in vitro, basal GH release was unaffected by the addition of 10(-9)-10(-5) M T3 or T4 to the incubation media. The in vitro GH response to TRH (10(-6) M) or GRF (10(-6) M) challenge was, however, suppressed in a dose-related manner by T3 but was unaffected by the coincubation of T4. These results demonstrate inhibitory effects of T3 and T4 on basal and secretagogue-induced GH secretion in fowl. T4 is less active than T3 and probably exerts some of its effects via T3-independent mechanisms.

Pharmacological Testing of Growth Hormone Secretion

The laboratory confirmation of growth hormone (GH) deficiency (GHD) has been extensively studied. Multiple stimuli induce GH release, but insulin-induced hypoglycemia usually is considered the 'gold standard'. Seventy-five to 90% of normal children have significant increments of hGH to any single test. Complete and partial syndromes of GHD have been defined, but some patients with a clinical appearance of GHD release hGH during provocative testing. Discordant results on varied tests may occur in the same child. Sequential and simultaneous tests have been attempted with diverse time patterns; testing sequence may significantly affect data interpretation. Persistent problems with GH provocative tests remain: normal data not strictly defined throughout childhood, multiple tests with discordant results, and substantial discrepancies of immunopotency estimates with different radioimmunoassays. Some children with 'normal' hGH increments during provocative tests, despite clinical GHD, may require short-term treatment with hGH to finally establish the diagnosis.

Effects of chronic GRF treatment on lambs having low or normal birth weight

The effects of a long term treatment with human GRF(1–29)NH2 on plasma growth hormone (GH), somatomedin C (Sm-C), histomorphometric parameters of bone growth and body composition were investigated in normal and low birthweight male lambs. The animals were divided into two groups according to their birthweight: 24 normal birthweight (NBW) lambs weighing more than 4 kg and 22 low birthweight (LBW) lambs weighing less than 2.5 kg at birth. Half of the animals in each group received two daily subcutaneous injections (8 μg/kg body weight) of hGRF(1–29)NH2 (GRF) from birth to slaughter at 45 or 90 days of age. The other animals received the solvant only. At the beginning and at the end of the treatment, plasma GH and serum Sm-C concentrations were measured in all groups. After slaughter, a histomorphometric study was performed on undecalcified sections of metacarpal growth plates, and the remaining of the carcass was pulverized to study the chemical body composition. GRF induced GH release in both GRF-treated groups. However, plasma GH reached higher (P<.001) concentrations and the GRF-induced GH peak lasted longer in LBW than in NBW lambs. At day 45, the GRF treatment increased (P<.05) serum Sm-C concentrations in LBW. Most of histomorphometric parameters reflecting the metacarpal growth in length, were not statistically modified under GRF treatment. However, the size of degenerative cells was smaller (P<.05) in LBW treated lambs as compared to controls. Consequently, the cell production in the growth plate was increased (P<.05) under GRF treatment. In both NBW and LBW groups at 45 days of treatment, GRF treatment reduced the amounts of lipids (P<.025) and energy (P<.05), while increased (P<.01) phosphorus deposition in the body. In contrast, there was no effect of GRF treatment on protein content. We conclude from this experiment that the induction of GH secretion by a chronic treatment with GRF is able to modify some patterns of growth. However, most of the effects of GRF were observed in LBW lambs and after 45 days of treatment only. This suggests that treatment with GRF may serve to compensate for growth in growth-retarded animals. Further studies with different mode of GRF administration should indicate whether it is as much effective in normal animals.

Full oestrogenic activity of C 19- Δ5 adrenal steroids in rat pituitary lactotrophs and somatotrophs

Oestrogens are known to exert specific stimulatory effects on basal and dopamine (DA)-inhibited prolactin (PRL) release as well as on PRL cell content in rat adenohypophysial cells in primary culture. Recently, we have demonstrated that classical oestrogens increase growth hormone (GH) release and cellular GH content in rat pituitary somatotrophs. Since there is evidence that 5-androstene-3/?,17/J-diol ( Δ 5-diol), a metabolite of dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S) can induce typical oestrogenic responses in target tissues, we have investigated the effect of C 19 adrenal steroids and compared them to that of 17β-oestradiol (E 2) on the above-indicated parameters. Following a 72-h incubation, maximal concentrations of E 2, Δ 5-diol and DHEA caused a similar increase in PRL cell content at respective EC 50 values of 0.020, 14 and 115 nM. The sensitivity of lactotrophs to DA action was decreased by approximately 4-fold after a 48-h exposure to maximal concentrations of Δ 5-diol or DHEA. The time course of the antidopaminergic effect of Δ 5-diol and DHEA was almost superimposable to that of E 2. A 72-h incubation with 5 μM DHEA-S, a concentration within the range of blood levels found in women, doubled ( P ≤ 0.001) cellular PRL accumulation. On the other hand, androstenedione ( Δ 4-dione) was without effect on any of the parameters measured. All stimulatory effects induced by maximal effective concentrations of Δ 5-diol, DHEA or DHEA-S were competitively inhibited by simultaneous incubation with the antioestrogen LY156758. The amplitude of the stimulatory effects of Δ 5-diol and DHEA on GH cell content as well as on spontaneous GH-releasing factor (GRF)-induced GH release was superimposable to that observed with E 2. The effect of the steroids on GH cell content was exerted at 0.016 nM (E 2), 12 nM ( Δ 5-diol) and 250 nM (DHEA). Simultaneous incubation with LY156758 completely blocked the effect of maximally effective concentrations of E 2, Δ 5-diol and DHEA in somatotrophs. Furthermore, a physiological concentration of DHEA-S (5 μM) enhanced spontaneous as well as GRF-induced GH release. On the other hand, Δ 4-dione as well as testosterone and dihydrotestosterone did not alter GH release. The present data demonstrate that Δ 5-diol, DHEA and DHEA-S can exert full oestrogenic effects in lactotrophs and somatotrophs, thus supporting their potential oestrogenic role under both physiological and pathological conditions.

Effect of Human Growth Hormone-releasing Factor (1–29)NH2 on Growth Hormone Release and Milk Production in Dairy Cows

Twelve cows (209 d in lactation, 642kg BW) were used in an experiment conducted over four 10-d periods (one preinjection, one injection, and two postinjection). Gelatine (n = 6) or 10mg of growth hormone-releasing factor in gelatine (n = 6) was injected subcutaneously at 1000h every day on d 11 to 20. Data were averaged for the last 5 d of each period. During the injection period, milk, fat, and protein yields increased by 3 kg·d−1 (14.3%), .14 kg·d−1 (16.7%), and .12 kg·d−1 (15.4%), respectively. Moreover, milk, fat, and protein yields for the treated cows remained higher than for the control cows until the last postinjection period. Growth hormone response was evaluated from blood samples withdrawn from 2h prior to 8h postinjection on d 11, 15, and 20 and on d 40. After growth hormone-releasing factor injection, peaks and area under the curve were 24.5, 30.8, and 47.0 ng·ml−1 and 2475, 3979, and 3741 ng·ml−1 · min−1 on d 11, 15, and 20, respectively. On d 40, there was no difference in growth hormone concentrations in blood between control and treated cows. These results demonstrate that 10 d of daily injection of a growth hormone-releasing factor increases milk production by 14.3% (3 kg·d−1) and still induces growth hormone release at the end of the injection period without any sign of refractoriness.

Influence of age and sex on basal secretion of growth hormone (GH) and on GH-induced release by porcine GH-releasing factor pGRF(1–29NH 2) in growing pigs

The aim of this study was to determine the effect of age and sex on basal secretory patterns of growth hormone (GH) and growth hormone-releasing factor (GRF) induced GH release. Eighteen pigs (9 castrated males and 9 females) were stimulated with pGRF(1–29)NH 2 at 7,11,15,19 and 23 weeks of age. Blood samples were taken from each animal via jugular vein cannulate every 20 min, from 6 hr before to 5 hr after iv GRF administration at a dose of 4 μg/kg. GH baseline levels, amplitude of the GH peaks, area under the GH peaks and the overall mean of GH serum levels decreased (P<.001) with age in both sexes. Age also had a marked effect on GRF-induced GH release: the amplitude of GH peaks and area under the GH peaks decreased (P<.001) with age. The GH response to pGRF(1–29)NH 2 varied considerably, depending on the timing of the episodic endogenous secretion of GH. An immediate response (<30 min) was observed when GRF was injected at the end of a trough period or at the beginning of a peak, but there was no immediate response when GRF was injected at the end of a peak or at the beginning of a trough period. Our results show that both endogenous GH secretion and pGRF(1–29)NH 2-induced GH release declines with age, suggesting a decreased sensitivity of the somatotroph cells to GRF with age; and that the high variability of the GH response to pGRF(1–29)NH 2 stimulation depends greatly on the timing of the episodic endogenous GH release, thus implying a possible episodic endogenous somatostatin secretion by the hypothalamus.

Influence of cell cycle phases on the electrical activity and hormone release in a transformed line of anterior pituitary cells

Electrophysiological experiments have shown that about 50% of cultured GH3 cells (tumoral cell line, from the anterior pituitary gland) are inexcitable i.e. they do not display action potentials either spontaneously or when depolarized by a current pulse. We report here this inexcitability may be related to cellular kinetics. Thus we have studied the relationship between the various phases of the cell cycle, the electrophysiological properties of GH3/B6 cells and spontaneous or induced Prolactin and Growth Hormone (GH) release rates. Asynchronous populatinos of viable cells were stained with Hoechst 33 342 DNA fluorescent dye, and sorted using a flow cytometer into G1 and S phases. After selection intracellular potentials were recorded using a single glass micro-electrode; the basal or TRH stimulated rates of PRL and GH secretions were determined by RIA. Electrical properties of the cells i.e. resting potentials, input membrane resistance and excitability, reached a maximum for cells in G2+M phases. Only cells in G2+M displayed action potentials and TRH increased their secretion by 5 times for GH and by 6 times for PRL. In G1 and S phases the cells were electrically inactive and secretion rates remained at their basal levels. These findings demonstrate that the mechanism of stimulus secretion coupling is dependent upon the phases of the cell cycle.