Direct kisspeptin-10 stimulation on luteinizing hormone secretion from bovine and porcine anterior pituitary cells

Kisspeptins are peptide hormones encoded by the KiSS-1 gene and act as the principal positive regulator of the reproductive axis by directly stimulating gonadotropin-releasing hormone (GnRH) neuron activity. However, peripheral administration, as well as central administration, of kisspeptin stimulates luteinizing hormone (LH) secretion in some mammalian species. In order to evaluate the direct effects of kisspeptin-10 (the minimal kisspeptin sequence necessary for receptor activation) on LH secretion from bovine and porcine anterior pituitary (AP) cells, LH-releasing effects of kisspeptin-10 on AP cells were compared with GnRH in vitro. The AP cells were prepared from 1-month-old intact male calves, 8-month-old castrated male calves, or 6-month-old barrows, and then the cells were incubated for 2h with the peptides. The 1000 nM and 10,000 nM, but not lower concentrations, of kisspeptin-10 significantly stimulated LH secretion from the bovine AP cells (P<0.05). The 100 nM and 1000 nM, but not lower concentrations, of kisspeptin-10 significantly stimulated LH secretion from porcine AP cells (P<0.05). As 10nM of GnRH strongly stimulated LH secretion from all AP cells tested in this study, the present results suggest that kisspeptin-10 has a direct, but weak, stimulating effect on LH secretion in bovine and porcine AP cells. The present study is the first to examine the direct actions of kisspeptin on the bovine and porcine pituitary gland as far as we know. Kisspeptin might have other actions on the pituitary because the pituitary has multiple roles.     

Differential involvement of protein kinase C in basal versus acetylcholine-regulated prolactin secretion in rat anterior pituitary cells during aging

Although it is well known that plasma concentration of prolactin (PRL) increases during aging in rats, how the anterior pituitary (AP) aging per se affects PRL secretion remains obscure. The objectives of this study were to determine if changes in the pituitary PRL responsiveness to acetylcholine (ACh; a paracrine factor in the AP), as compared with that to other PRL stimulators or inhibitors, contribute to the known age-related increase in PRL secretion, and if protein kinase C (PKC) is involved. We also determined if replenishment with aging-declined hormones such as estrogen/thyroid hormone influences the aging-caused effects on pituitary PRL responses. AP cells were prepared from old (23-24-month-old) as well as young (2-3-month-old) ovariectomized rats. Cells were pretreated for 5 days with diluent or 17beta-estradiol (E(2); 0.6 nM) in combination with or without triiodothyronine (T(3); 10 nM). Then, cells were incubated for 20 min with thyrotropin-releasing hormone (TRH; 100 nM), angiotensin II (AII; 0.2-20 nM), vasoactive intestinal peptide (VIP; 10(-9)-10(-5) M), dopamine (DA; 10(-9)-10(-5) M), or ACh (10(-7)-10(-3) M). Cells were also challenged with ACh, TRH, or phorbol 12-myristate 13-acetate (PMA; 10(-6) M) following PKC depletion by prolonged PMA (10(-6) M for 24 h) pretreatment. We found that estrogen priming of AP cells could reverse the aging-caused effects on pituitary PRL responses to AII and DA. In hormone-replenished cells aging enhanced the stimulation of PRL secretion by TRH and PMA, but not by AII and VIP. Aging also reduced the responsiveness of cells to ACh and DA in suppressing basal PRL secretion, and attenuated ACh inhibition of TRH-induced PRL secretion. Furthermore, ACh suppressed TRH-induced PRL secretion mainly via the PMA-sensitive PKC in the old AP cells, but via additional mechanisms in young AP cells. On the contrary, basal PRL secretion was PKC (PMA-sensitive)-independent in the old AP cells, but dependent in the young AP cells. Taken together, these results suggest differential roles of PMA-sensitive PKC in regulating basal and ACh-regulated PRL responses in old versus young AP cells. The persistent aging-induced differences in AP cell responsiveness to ACh, DA, TRH, and PMA following hormone (E(2)/T(3)) replenishment suggest an intrinsic pituitary change that may contribute, in part, to the elevated in vivo PRL secretion observed in aged rats.     

The glycopeptide moiety of vasopressin-neurophysin precursor is neurohypophysial prolactin releasing factor

All of the classically-described hypothalamic, hypophysiotropic factors that regulate anterior pituitary hormone secretion have now been isolated and identified except for prolactin releasing factor. We report here that the 39-amino acid glycopeptide comprising the carboxyterminus of the neurohypophysial vasopressin-neurophysin precursor stimulates prolactin release from cultured pituitary cells as potently as does thyrotropin releasing hormone but has no effect on the secretion of other pituitary hormones. Furthermore, antisera to the glycopeptide administered to lactating rats attenuated suckling-induced prolactin secretion. Thus, this glycopeptide appears to be the neurohypophysial prolactin releasing factor.     

An acute release of Ca2+ from sequestered intracellular pools is not the primary transduction mechanism causing the initial burst of PRL and TSH secretion induced by TRH in normal rat pituitary cells.

With 1.5 mM [Ca2+]e, 10 nM TRH induced a prompt high-amplitude burst of hormone secretion and an initial high-amplitude [Ca2+]i burst (first phase) followed by a sustained low-amplitude [Ca2+]i increment (second phase) in both tumor-derived GH4C1 and normal adenohypophyseal (AP) cells. With less than 2 microM [Ca2+]e, in both cell types the TRH-induced first phase rise in [Ca2+]i was suppressed 30% while the second phase rise was completely abolished; however, hormone secretion was inhibited only 20-30% in GH4C1 but greater than 80% in AP cells. Thapsigargin induced a first-phase rise in [Ca2+]i in AP cells equal to that induced by 10 nM TRH but only 20% as much first-phase hormone secretion. Blocking Ca2+ channels with nifedipine inhibited TRH-induced secretion in AP cells significantly more than in GH4C1 cells. Our data indicate that the TRH-induced first-phase spike in [Ca2+]i from intracellular Ca2+ stores may play a major transduction role in hormone secretion in GH4C1 cells but not in normal AP cells. Transduction mechanisms coupled to Ca2+ influx through Ca2+ channels in the plasmalemma are apparently a much more important component of TRH-induced secretion in normal than in tumor-derived pituitary cells.     

5,6-Epoxyeicosatrienoic acid mobilizes Ca2+ in anterior pituitary cells

Luteinizing hormone releasing hormone stimulates the concomitant release of luteinizing hormone and 45Ca2+ from prelabeled anterior pituitary cells. Indomethacin (10 microM) and nordihydroguaiaretic acid (10 microM) had no effect on the luteinizing hormone releasing hormone-stimulated release of either luteinizing hormone or 45Ca2+. Eicosatetraynoic acid (10 microM) blocked both luteinizing hormone releasing hormone-stimulated luteinizing hormone secretion and luteinizing hormone releasing hormone-stimulated 45Ca2+ efflux. 5,6-Epoxyeicosatrienoic acid stimulated both luteinizing hormone secretion and 45Ca2+ efflux from anterior pituitary cells. Additionally, 5,6-epoxyeicosatrienoic acid closely mimics the ability of luteinizing hormone releasing hormone to increase intracellular free calcium. These results are consistent with the hypothesis that 5,6-EET alters calcium homeostasis in a manner similar to that observed during luteinizing hormone releasing hormone stimulation of luteinizing hormone release.     

Kisspeptin-10 stimulates the secretion of growth hormone and prolactin directly from cultured bovine anterior pituitary cells

Kisspeptins are peptide hormones encoded by the KiSS-1 gene, and act as the principal positive regulator of the reproductive axis by directly stimulating gonadotropin-releasing hormone (GnRH) neuron activity. We recently observed that kisspeptin-10 (the minimal kisspeptin sequence necessary for receptor activation) also has a direct stimulating effect on luteinizing hormone (LH) secretion in bovine anterior pituitary (AP) cells. In the present study, we evaluated the direct effect of kisspeptin-10 on the secretion of other pituitary hormones, growth hormone (GH) and prolactin (PRL), from bovine AP cells. The AP cells, which were prepared from 1- or 8-month-old male calves, were incubated for 2h with the peptides. Kisspeptin-10 at 100 nM (P<0.05), 1000 nM (P<0.01) and 10,000 nM (P<0.01), but not at 10 nM, significantly stimulated GH secretion from the AP cells of 1-month-old calves, while in 8-month-old calves it was significantly (P<0.05) stimulated at 1000 nM (P<0.01) and 10,000 nM (P<0.01), but not at 10nM and 100 nM. The response of GH to 100 nM (P<0.01), 1000 nM (P<0.05) and 10,000 nM (P<0.01) kisspeptin-10 in the AP cells of 1-month-old calves was significantly greater than in those of 8-month-old calves. All tested doses of kisspeptin-10 had no effect on PRL secretion from AP cells of 1-month-old calves. However, 1000 nM (P<0.05) and 10,000 nM (P<0.01), but not lower concentrations, of kisspeptin-10 significantly stimulated PRL secretion from the AP cells of 8-month-old calves. The present study is, as far as we know, the first to examine the direct actions of kisspeptin on the secretion of GH and PRL from the bovine pituitary gland. Further studies are necessary to evaluate the importance of multiple actions of kisspeptin on the pituitary of various animals in vivo.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates cyclic AMP formation as well as peptide output of cultured pituitary melanotrophs and AtT-20 corticotrophs.

The present study was aimed at investigating whether PACAP stimulates accumulation of cAMP, as well as hormonal secretion of homogeneous populations of pituitary proopiomelanocortin (POMC) cells, namely melanotrophs and AtT-20 corticotrophs. PACAP was shown to enhance cAMP accumulation in a dose-dependent fashion in both cell types (with EC50 values of approx. 10(-10) M) and elicited additive increases of cAMP production with CRF in melanotrophs, but not in corticotrophs. PACAP also stimulated dose-dependently the secretion of alpha-MSH and ACTH, with EC50 concentrations of about 10(-9) M. In melanotrophs, bromocriptine significantly depressed PACAP-induced cAMP formation and blunted by more than 90% stimulated alpha-MSH release. This study shows that (1) pituitary POMC cells did respond to PACAP by enhancing cAMP accumulation and elevating hormone secretion as well; (2) the effect of PACAP was additive with CRF on cAMP production in melanotrophs, but not in corticotrophs, while there was no additivity on peptide output from both cell types; (3) activation of dopamine receptors in melanotrophs dampened both cAMP formation and peptide secretion. These findings are consistent with PACAP playing a possible hypophysiotropic role in the regulation of pituitary POMC cell activity.     

Effect of guanine nucleotides on phospholipase C activity in permeabilized pituitary cells: possible involvement of an inhibitory GTP-binding protein

Cultured pituitary cells prelabeled with myo-[2-3H] inositol were permeabilized by ATP4-, exposed to guanine nucleotides and resealed by Mg2+. Addition of guanosine 5'-0-(3-thio triphosphate) (GTP gamma S) to permeabilized cells, or gonadotropin releasing hormone (GnRH) to resealed cells, resulted in enhanced phospholipase C activity as determined by [3H] inositol phosphate (Ins-P) production. The effect was not additive, but the combined effect was partially inhibited by guanosine 5'-0-(2-thiodiphosphate) (GDP beta S) or by neomycin. Surprisingly, addition of GDP beta S (100-600 microM) on its own resulted in a dose-related increase in [3H]Ins-P accumulation. Several nucleoside triphosphates stimulated phospholipase C activity in permeabilized pituitary cells with the following order: UTP greater than GTP gamma S greater than ATP greater than CTP. The stimulatory effect of UTP, ATP and CTP, but not GTP gamma S or GDP beta S, could also be demonstrated in normal pituitary cells suggesting a receptor-activated mechanism. GTP and GTP gamma S decreased the affinity of GnRH binding to pituitary membranes and stimulated LH secretion in permeabilized cells. These results suggest the existence of at least two G-proteins (stimulatory and inhibitory) which are involved in phospholipase C activation and GnRH action in pituitary cells.     

Effect of phorbol ester on stimulus-secretion coupling mechanisms in gonadotropin releasing hormone-stimulated pituitary gonadotrophs

The feedback regulatory control mechanism exerted by activated Ca2+/phospholipid-dependent protein C kinase upon gonadotropin releasing hormone (GnRH) binding, stimulation of phosphoinositide turnover and gonadotropin secretion was investigated in cultured pituitary cells. Addition of the tumor promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), at concentrations which activate pituitary protein C kinase, to cultured pituitary cells resulted in up-regulation of GnRH receptors (155% at 4 h). The stimulatory effect of GnRH on [3H]inositol phosphates (Ins-P) production in myo-[2-3H]inositol prelabeled pituitary cells was not inhibited by prior treatment of the cells with TPA (10(-9)-10(-7) M). Higher concentrations of TPA (10(-6)-10(-5) M) inhibited the effect of GnRH on [3H]Ins-P production. Increasing concentrations of TPA or the permeable analog of diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) stimulated luteinizing hormone (LH) release from cultured pituitary cells with ED50 values of 5 x 10(-9) M and 10 micrograms/ml, respectively. No consistent inhibition or additivity of LH release was observed when increasing doses of TPA or OAG were added with a submaximal dose of GnRH. These results suggest that protein C kinase might mediate the known homologous up-regulation of GnRH receptors during the reproductive cycle. Protein C kinase is positively involved in mediating the process of gonadotropin secretion. Unlike many other systems, activation of protein C kinase in pituitary gonadotrophs is not involved in negative feed-back regulation of stimulus-secretion-coupling mechanisms in GnRH-stimulated gonadotrophs.     

The effect of stimulators and blockers of adrenergic receptors on LH secretion and cyclic nucleotide (cAMP and cGMP) production by porcine pituitary cells in vitro.

The direct effects of alpha- and beta-adrenergic agents on luteinizing hormone (LH) secretion in vitro by porcine pituitary cells and the participation of secondary messengers, adenosine 3'5'-monophosphate (cAMP) and guanosine 3'5'-monophospate (cGMP), in transduction of signals induced by adrenergic agents and gonadotropin-releasing hormone (GnRH) in these cells have been investigated. Pituitary glands were obtained from mature gilts, which were ovariectomized (OVX) 1 month before slaughter. OVX gilts, assigned to four groups, were primed with: (1) vehicle (OVX); (2 and 3) estradiol benzoate (EB; 2.5mg/100kg b.w.) at 30-36h (OVX+EB I) or 60-66h (OVX+EB II) before slaughter, respectively; (4) progesterone (P(4); 120mg/100kg b.w.) for 5 consecutive days before slaughter (OVX+P(4)). Anterior pituitaries were dispersed with trypsin and then pituitary cells were cultured (10(6) per well) in McCoy's 5a medium containing horse serum (10%) and fetal calf serum (2.5%) for 3 days, at 37 degrees C and under the atmosphere of 95% air and 5% CO(2). On day 4 of the culture, the cells were submitted to 3.5h incubation in the presence of GnRH (a positive control), alpha- and beta-adrenergic agonists (phenylephrine (PHEN) and isoproterenol (ISOP), respectively), and alpha- and beta-adrenergic blockers (phentolamine (PHENT) and propranolol (PROP), respectively). The culture media were assayed for LH (experiment I) and cyclic nucleotides (experiment II).In experiment I, addition of GnRH (100ng/ml) increased LH secretion by pituitary cells taken from gilts of all experimental groups. The effects of alpha- and beta-adrenergic agents on LH secretion by the cells depended on hormonal status of gilts. The LH secretion by pituitary cells of OVX gilts was potentiated in the presence of PHEN (10, 100nM, and 1microM) and PHENT (1microM), alone or in combination with PHEN (100nM) and by the cells derived from OVX+EB I and OVX+P(4) animals in response to PHEN (100nM) and ISOP (1microM). ISOP (1microM) also stimulated LH secretion by the cells taken from OVX+EB II gilts. In experiment II, GnRH (100ng/ml) increased cGMP production by pituitary cells obtained from all groups of gilts and cAMP secretion by the cells taken from OVX and OVX+P(4) animals. PHEN (100nM) decreased and PROP (1microM) enhanced cAMP production by pituitary cells derived from OVX+EB I and OVX gilts, respectively. Moreover, PHEN (100nM) reduced, while PHENT (1microM) stimulated the release of cGMP by pituitary cells taken from OVX+EB II animals. In turn, ISOP (100nM) decreased and increased cGMP production by the cells derived from OVX+EB II and OVX+P(4) gilts, respectively. PROP (1microM) potentiated cGMP accumulation by pituitary cells taken from OVX+EB I and OVX+P(4) animals.In conclusion, our results suggest that adrenergic agents can modulate LH release by porcine pituitary cells acting through guanyl and adenylyl cyclase and in a manner dependent on hormonal status of gilts.     

Contraceptive progestins and gonadotropin secretion in vitro

In an in vitro bioassay using rat pituitary cell cultures the effect of contraceptive progestins was tested on basal and gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion in vitro. Progestins diminished gonadotropin release in pituitary cells stimulated with GnRH, but did not alter basal values. This inhibitory effect was dose dependent in a range of 10(-10)-10(-5) M tested and the inhibitory action of most of the progestins examined was more potent than that of progesterone. The maximal reduction of LH and FSH values was by 60% of GnRH-induced control levels. Progestins also caused a shift in sensitivity of cells to GnRh (10(-12)-10(-6) M). When time dependence was investigated, some progestins potentiated GnRH effect on gonadotropins in pituitary cell cultures pre-incubated for a short time (4 h) with steroids. More prolonged pre-incubations from 23 to 71 h resulted in a progressive suppression of LH and FSH response to GnRH (10(-7) M). In order to examine intracellular effects, cells were pretreated with progestins and inositol phosphate metabolism was investigated. The data obtained in pituitary cells give evidence that polyphosphoinositide breakdown is potentially an early step in the action of GnRH on gonadotropin secretion by providing diacylglycerol and inositol phosphates. Addition of gonadotropin-releasing hormone to myo-2[3H]inositol-prelabeled rat pituitary cells in primary culture evoked a dose-dependent increase of the accumulation of [3H]inositol phosphates with a rise of inositol triphosphate, inositol diphosphate and inositol monophosphate within 1 min. Using one contraceptive progestin, gestoden, inositol phosphate production was inhibited by 80% compared to controls of GnRH-treated cells without the addition of steroids. The data obtained in this study suggest that this in vitro bioassay using rat pituitary cells is a useful tool in testing progestational compounds regarding their potency on gonadotropin release. In addition, these results show that one possible site of interference of progestins with GnRH-induced gonadotropin secretion may involve polyphosphoinositide breakdown.     

Relation of endogenous opioid peptides and morphine to neuroendocrine functions.

Morphine and the endogenous opioid peptides (EOP) exert similar effects on the neuroendocrine system. When adminstered acutely, they stimulate growth hormone (GH), prolactin (PRL), and adrenocorticotropin (ACTH) release, and inhibit release of luteinizing hormone (LH), follicle stimulating hormone (FSH),and thyrotropin (TSH). Recent studies indicate that the EOP probably have a physiological role in regulating pituitary hormone secretion. Thus injection of naloxone (opiate antagonist) alone in rats resulted in a rapid fall in serum concentrations of GH and PRL, and a rise in serum LH and FSH, suggesting that the EOP help maintain basal secretion of these hormones. Prior administration of naloxone or naltrexon inhibited stress-induced PRL release, and elevated serum LH in castrated male rats to greater than normal castrate levels. Studies on the mechanisms of action of the EOP and morphine on hormone secretion indicate that they have no direct effect on the pituitary, but act via the hypothalamus. There is no evidence that the EOP or morphine alter the action of the hypothalamic hypophysiotropic hormones on pituitary hormone secretion; they probably act via hypothalamic neurotransmitters to influence release of the hypothalamic hormones into the pituitary portal vessels. Preliminary observations indicate that they may increase serotonin and decrease dopamine metabolism in the hypothalamus, which could account for practically all of their effects on pituitary hormone secretion.     

Glucocorticoid induction of growth hormone synthesis in a strain of rat pituitary cells

Conditions were determined for measuring growth hormone synthesis by a clonal strain of rat pituitary cells grown in suspension culture. Incubation of the cells with [3H]leucine in either continuous labeling or pulse-chase experiments showed that secretion of newly synthesized growth hormone commences only after a lag of about 15 min. The pulse-chase experiments also demonstrated that there is no detectable degradation by the cells of growth hormone. Thus growth hormone synthesis could be measured, in the absence of complications arising either from secretion or degradation of growth hormone, by incubating the cells with [3H]leucine for 10 min. Exposure of cells grown under the usual culture conditions to dexamethasone (a synthetic glucocorticoid) led to an average stimulation of specific growth hormone synthesis (growth hormone synthesis/total cytoplasmic protein synthesis) of only 2.6-fold. However, two other growth conditions were found in which dexamethasone routinely yielded a 5- to 15-fold stimulation of specific growth hormone synthesis. One of these conditions, involving substitution of 10% fetal calf serum for the normal serum supplement, was employed in subsequent experiments. A stimulation of specific growth hormone synthesis could be observed at 10(-9) M dexamethasone, and the maximum stimulation was observed at dexamethasone concentrations of about 10(-8) to 10(-7) M. There was a lag of about 6 h before a stimulation by dexamethasone of specific growth hormone synthesis was detected. Thereafter, the stimulation increased in a nearly linear fashion until maximum stimulation was reached at about 48 h.     

Dexamethasone increases inhibin and estradiol secretion mediated by endogenous FSH in equine chorionic gonadotropin (eCG)-primed immature female rats.

In the present study, we have examined whether the effects of dexamethasone on follicle stimulating hormone (FSH) secretion were mediated by hypophysiotropic factors, and whether the increased levels of FSH induced by dexamethasone can stimulate ovarian functions in equine chorionic gonadotropin (eCG)-primed immature female rats. Dexamethasone (500 microg) significantly increased serum concentrations of FSH in hypophysectomized rats implanted with pituitary under the kidney capsule, as well as in intact rats. Serum concentrations of inhibin and estradiol in eCG (2.5, 5 i.u.)-primed rats were significantly increased by simultaneous treatment with dexamethasone (500 microg) and eCG. These simultaneous effects were not confirmed in hypophysectomized rats. The results had shown that hypophysiotropic factors do not mediate the selective increase of FSH secretion caused by dexamethasone. Dexamethasone induces the excess amount of FSH secretion from anterior pituitary and this FSH can stimulate inhibin and estradiol secretion in eCG-primed immature female rat.     

Mosquito larvae proteins modulate luteinizing hormone and prolactin release in pituitary cells of normal and estrogenized rats

Whilst looking for vertebrate growth factor homologues in insects, we found that a soluble fraction of a 12-80 kDa molecular weight band peaking at 25 kDa, isolated from mosquito larvae extracts by gel permeation chromatography, had a modulatory effect on mouse hepatocytes and adult human mononuclear cell proliferation. The effect disappeared after heating the extract at 90 degrees C for 30 min, suggesting that the active factor may be a protein. In order to determine the activity of the extract on cell function, we assessed the effect of the extract on pituitary hormone secretion in vitro. We assayed a dialyzed fraction (MW greater than 12 kDa) of mosquito larvae for its effect on the release of luteinizing hormone (LH) and prolactin (PRL) from dispersed rat pituitary cells. In normal anterior pituitary (AP) cells we found that the extract had a stimulatory effect on LH release but an inhibitory action on prolactin secretion. In AP cells obtained from estrogen-induced hyperplasia, the extract had an inhibitory effect on prolactin secretion. In all cases the effects were time- and dose-dependent. Interference of the mosquito proteins with the radioimmunoassay was checked and found to be negligible. After a 60 min incubation, cell viability was comparable in control and treated cells. Furthermore, the biological effect of the extract was thermally unstable. Our results suggest that mosquito larvae may share common factors with mammals, probably peptidic in nature, which are able to modulate cell function.     

Somatostatin is targeted to the regulated secretory pathway of gonadotrophs in transgenic mice expressing a metallothionein-somatostatin gene

The pituitaries of transgenic mice that express a metallothionein-somatostatin fusion gene contain high concentrations of somatostatin-14 exclusively in the gonadotrophic cells. The purpose of this study was to determine whether somatostatin expressed from the foreign fusion gene enters the normal secretory pathway within these cells. Immuno-gold labeling of serial thin sections localized somatostatin to the secretory granules of gonadotropin-producing cells. The gonadotroph-specific hypophysiotropic factor, luteinizing hormone-releasing hormone caused a dose-dependent secretion of somatostatin when applied to primary pituitary cultures from these mice. Growth hormone-releasing hormone, thyrotropin-releasing hormone, corticotropin releasing factor, and dopamine did not affect somatostatin secretion. These experiments demonstrate that a neurosecretory peptide encoded by a foreign gene can enter the regulated secretory pathway of pituitary cells from transgenic mice.     

Effects of growth hormone-releasing factor, somatostatin and dopamine on growth hormone and prolactin secretion from cultured ovine pituitary cells

A synthetic form of human pancreatic growth hormone releasing factor (GRF-44-NH2) was shown to be a potent stimulator of growth hormone (GH) secretion and cellular cyclic AMP levels in cultured sheep pituitary cells. A small dose-dependent stimulation of prolactin secretion was also observed. Somatostatin (0.5 microM) completely blocked the maximal GRF (1 nM)-stimulated secretion without a significant effect on cyclic AMP levels. Dopamine (0.1 microM) inhibited the GRF-elevated GH secretion by 50% and lowered cyclic AMP levels by 30%. Dopamine (0.1 microM) inhibition of basal prolactin secretion was not affected by GRF (1 nM). The data support the hypothesis that cyclic AMP is involved in the action of GRF but suggest that somatostatin can inhibit GRF-induced secretion of GH independently of cyclic AMP.     

Effect of [D-Trp6]LHRH infusion on prolactin secretion by perifused rat pituitary cells

The effect of a superactive agonistic analog of luteinizing hormone-releasing hormone (LHRH), [D-Trp6]LHRH on prolactin (PRL) secretion by perifused rat pituitary cells was investigated. Constant infusion of [D-Trp6]LHRH (0.5 ng/min) for 2-3 h elicited a significant decrease in PRL secretion by these cells. This decrease in PRL release started ca. 30 min after the beginning of the infusion with the LHRH analog and lasted up to 1.5-2 h. [D-Trp6]LHRH significantly stimulated luteinizing hormone (LH) secretion during the first 30 min of peptide infusion; thereafter, LH levels began to return to control values. In animals pretreated in vivo with 50 micrograms of [D-Trp6]LHRH (s.c.) 1 h before sacrifice, PRL secretion by the rat pituitary cell perifusion system was significantly lower than vehicle-injected controls throughout the entire [D-Trp6]LHRH infusion period. On the other hand, thyrotropin-releasing hormone (TRH)-stimulated PRL secretion was slightly, but significantly imparied by [D-Trp6]LHRH infusion, while dopamine (DA) inhibition of PRL release was unaffected by this same treatment. These results reinforce previous observations of a modulatory effect of [D-Trp6]LHRH, probably mediated by pituitary gonadotrophs, on PRL secretion by the anterior pituitary. In addition, our findings suggest that basal PRL secretion by the lactotroph may be dependent on a normal function of the gonadotroph. The collected data from this and previous reports support the existence of a functional link between gonadotrophs and lactotrophs in the rat pituitary gland.     

Parathyroid hormone secretion in the absence of extracellular free Ca2+ and transmembrane Ca2+ influx

The involvement of extracellular Ca2+ and Ca2+ influx across the plasma membrane in parathyroid hormone (PTH) secretion was investigated in vitro using a new preparation of bovine parathyroid cells. Incubation of these cells in the presence of 25 microM or 2.5 microM free ambient Ca2+ induced a maximal rate of PTH secretion. Low free Ca2+ secretion is not associated with changes in membrane permeability, requires metabolic energy, and is reversible. The Ca2+ channel blocker D600 had no effect on either 45Ca-influx or PTH secretion in these cells. These results, showing that extracellular Ca2+ and Ca2+ influx across the plasma membrane are not required for PTH secretion by parathyroid cells, emphasize the differences in the cellular mechanisms underlying the secretion of PTH vs that of other secretory cells.