Effect of growth hormone-releasing factor on phosphoinositide hydrolysis in somatotrophs

We studied the role of the phosphatidylinositol system in the action of growth hormone-releasing factor (GRF). We asked whether GRF stimulates the activity of phospholipase C by determining GRF-induced changes in 32P labeling of the individual phosphoinositides and inositol phosphates in purified rat somatotrophs. The somatotrophs were challenged with GRF (10(-7)M) for 0.33, 1, 3, 10, 30, and 90 min. GRF did not significantly or consistently alter 32P incorporation into phosphatidylinositol bisphosphate (PIP2), phosphatidylinositol monophosphate (PIP), or phosphatidylinositol (PI), except for a small reduction in PIP labeling at 90 min. In general the level of 32P incorporation into the inositol phosphates did not increase but instead decreased with GRF. There was a small but significant reduction of labeling of inositol trisphosphate (IP3) at 90 min of GRF incubation. There were also small but significant decreases in 32P incorporation into inositol bisphosphate (IP2) at 0.33, 3, and 30 min. GRF did not significantly alter 32P labeling of inositol monophosphate (IP). These results indicate that GRF does not stimulate phospholipase C activity in somatotrophs. We conclude that the phosphatidylinositol second messenger system does not play an essential role in the action of GRF.     

The inhibition of phosphatidylinositol turnover: a possible postreceptor mechanism for the prolactin secretion-inhibiting effect of dopamine

We studied the association between the inhibition of phosphatidylinositol (PI) turnover and the inhibition of PRL secretion in the presence of dopamine. The incorporation of radiolabeled phosphate into anterior pituitary gland PI as well as serum PRL levels were significantly (P less than 0.01) greater in female than in male rats. No significant sex-related difference was found in the incorporation by pituitary tissue of 32P into phosphatidylcholine (PC) or phosphatidylethanolamine (PE). Dopamine decreased the incorporation of 32P into PI, but not into PC or PE, by female rat pituitary glands; this effect was reversed by two dopamine receptor-blocking agents, haloperidol and pimozide. After dopamine was removed from the incubation medium, basal 32P incorporation into PI was restored within 10 min. The administration of bromocriptine (500 micrograms/kg, ip, 4 h earlier) significantly reduced pituitary PI turnover. Conversely, in vivo injection of alpha-methyl-p-tyrosine (alpha MpT; 200 mg, ip, 2.5 h before death), an inhibitor of catecholamine biosynthesis, dramatically increased serum PRL levels. In vitro incorporation of 32P into PI, but not into PC or PE, increased (+130%) when these glands were incubated for 30 min with radiolabeled phosphate. The in vitro addition of 0.5 microM dopamine to glands from alpha MpT-treated rats counteracted the stimulation of 32P incorporation into PI produced by alph MpT treatment. In rats bearing the transplantable PRL-secreting tumor MtTW15, the hyperprolactinemia produced by the tumor stimulates hypothalamic turnover of dopamine, with a consequent inhibition of pituitary gland PRL secretion. 32P incorporation into PI, but not into PC or PE, was significantly (P less than 0.01) inhibited (-41%) in pituitary glands from these rats. The injection of alpha MpT (200 mg/kg, ip) or haloperidol (2 mg/kg, ip) 12 and 3 h before death into MtTW15 tumor-bearing rats abolished the inhibition of 32P incorporation into pituitary PI. Dopamine also decreased PI turnover in the 7315a PRL-secreting pituitary tumor. Our data indicate that the PI cycle may be an intracellular mechanism controlling PRL release in the rat and that the changes in its cleavage and turnover may be an early postreceptor event responsible for the inhibition of PRL secretion produced by factors such as dopamine.     

Age-dependent changes in the mechanisms controlling prolactin secretion and phosphatidylinositol turnover in male rats: effect of phosphatidylserine

The present study was undertaken in order to better characterize the functional state of anterior pituitary gland in young and old rats by using prolactin secretion and incorporation of radioactive phosphate into phosphatidylinositol (PI) as markers. The in vitro incorporation of radiolabeled phosphate into anterior pituitary PI was significantly (p less than 0.01) greater in young (3-5 months) than in aged (24-25 months) male Sprague-Dawley rats. No significant difference was found in the incorporation by pituitary tissue of 32P into phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Also, the extent of prolactin secretion from isolated pituitary was significantly greater in young than in aged rats, while the prolactin pituitary content was significantly higher in aged animals. In vitro dopamine (DA) decreased the incorporation of 32P into PI, both in young and old pituitary glands, and inhibited prolactin secretion into the incubation medium. Brain cortex-phosphatidylserine (BC-PS), a pharmacologically active purified phospholipid, capable of stimulating the dopaminergic system in the hypothalamus and of decreasing prolactin secretion both in humans and rats in vitro and in vivo, inhibited the incorporation of labeled phosphate into PI of pituitary glands from either young or old rats, but did not alter the prolactin secretion from the glands incubated in vitro. Baseline prolactin plasma levels did not differ significantly between young and old rats either when blood was collected from the trunk after decapitation or underwent sampling from chronically cannulated rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Galanin secretion from anterior pituitary cells in vitro is regulated by dopamine, somatostatin, and thyrotropin-releasing hormone.

Lactotrophs, somatotrophs, and thyrotrophs have been shown to contain immunoreactive galanin. Furthermore, estrogen stimulates galanin mRNA and peptide levels in the rat anterior pituitary, particularly within lactotrophs. To determine whether galanin is released from the anterior pituitary in a regulated manner, we used cultured pituitary cells from male and ovariectomized Fischer 344 rats implanted with estrogen-containing capsules. Anterior pituitary cells (5 x 10(5) cells/well) were challenged (0.5-3 h) with hypothalamic factors known to regulate anterior pituitary hormone secretion, and medium galanin levels were measured by RIA. In female pituitary cells, galanin secretion was inhibited by dopamine (10 and 100 nM) and stimulated by TRH (20 and 100 nM). Although galanin release was significantly lower in male pituitary cells, dopamine and TRH inhibited and stimulated galanin secretion, respectively. Medium galanin levels were also significantly reduced by somatostatin (5 nM) in both female and male cells. The pattern of PRL release in response to dopamine, TRH, and somatostatin was similar to that observed for galanin, regardless of the sex of the pituitary donor. Although galanin has been localized in somatotrophs, 5 nM GH-releasing hormone (GRF) failed to alter galanin release in male as well as female pituitary cells; GH secretion was significantly increased by GRF. LHRH (5 nM) and CRF (5 nM) failed to alter galanin release in vitro. We conclude that in estrogen-exposed pituitary cells obtained from male and ovariectomized Fischer 344 rats: 1) galanin secretion is inhibited by dopamine and somatostatin, and stimulated by TRH; 2) GRF, LHRH, and CRF do not regulate galanin release in these cells; and 3) the profile of the regulated pathway for galanin release suggests that the primary location of galanin is the lactotroph, probably within secretory granules.     

Stimulation of phosphatidic acid and phosphatidylinositol labeling in luteal cells by luteinizing hormone releasing hormone

Luteinizing hormone-releasing hormone (LHRH) causes a rapid and marked increase of [32P]orthophosphate incorporation into phosphatidylinositol (PI) and phosphatidic acid (PA) in rat luteal cells in culture. The neurohormone exerts its stimulatory effect at an ED50 value of approximately 15 nM. Human chorionic gonadotropin (hCG) has no effect alone and does not interfere with the LHRH-induced PA-PI labeling. The rapidity and the specificity of the effect of LHRH suggest that the stimulation of the PA-PI cycle may well serve as a potent transducing mechanism responsible for the direct action of LHRH and its agonists at the ovarian level.     

Gonadotropin-releasing hormone (GnRH) stimulates phosphatidylinositol metabolism in rat granulosa cells: mechanism of action of GnRH.

This report describes the effects of gonadotropin-releasing hormone (GnRH; gonadoliberin) and an agonist, [D-Ala6, des-Gly10]GnRH ethyl amide (GnRHa), on phospholipid metabolism in rat granulosa cells isolated from mature Graafian follicles. As indicated by the incorporation of 32PO4, GnRHa rapidly (less than 2 min) stimulated the labeling of phosphatidic acid and phosphatidylinositol but had no effect on the labeling of other phospholipids. Increased phosphatidylinositol labeling was also observed when myo-[2-3H]inositol was incubated with granulosa cells in the presence of GnRHa. Increases in labeling were dependent on the dose of GnRH and time of incubation. Thyrotropin-releasing hormone and a specific GnRH antagonist had no effect on labeling, but a GnRH antagonist prevented the stimulatory action of GnRH. In addition, treatment with GnRHa slightly increased the levels of phosphatidylinositol (15%) in 60-min incubations but had no effect on the levels of other phospholipids. Significant increases in progesterone accumulation were observed after 30 min of incubation with GnRHa, and further increases were correlated with the time of incubation. The stimulatory action of GnRH on phospholipid metabolism and progesterone accumulation was not related to increases in cyclic nucleotide accumulation. In incubations lasting up to 30 min, GnRHa had no effect on cAMP accumulation. However, a transient decrease in cGMP levels was observed in response to GnRHa. These studies suggest that the rapid and specific effects of GnRH on phospholipid metabolism in rat granulosa cells represent early events in the action of GnRH.     

Effect of LHRH on incorporation of [32P]-orthophosphate into phosphatidylinositol by dispersed anterior pituitary cells

Stimulated release of luteinizing hormone (LH) from the anterior pituitary in response to luteinizing hormone releasing hormone (LHRH), is apparently controlled by Ca2+-mediated events. In many tissues, when an involvement of Ca2+ in secretion has been found, there is an associated increased metabolic turnover of phosphatidylinositol. The objective of this investigation was to determine the effect of LHRH on the incorporation of [32P]orthophosphate into phosphatidylinositol by anterior pituitary cells maintained in vitro. When anterior pituitary cells were incubated for 5-40 min in the presence of [32P]orthophosphate, phosphatidylinositol and phosphatidylcholine were the most rapidly radiolabelled phospholipids. The addition of LHRH to the cell culture medium at concentrations previously demonstrated to release LH, increased the incorporation of 32P]orthophosphate into phosphatidylinositol in a dose-dependent manner. Incorporation of [32P]orthophosphate into other phospholipids was unaffected by LHRH at all concentrations employed. These results are consistent with the hypothesis that LHRH causes a receptor-mediated increase in turnover of phosphatidylinositol and this may be among the early metabolic events in the mechanism of LHRH-stimulated LH secretion from the anterior pituitary gland.     

Specific binding of a potent LHRH agonist in rat testis

High affinity binding sites for the potent LHRH agonist [¹²⁵I][d-Ser(TBU)⁶, des-GlyNH₂¹⁰]LHRH ethylamide are present in dissociated rat testicular interstitial cells, a preparation rich in Leydig cells. The iodinated LHRH agonist binds to a single class of high affinity sites at a K_D value of 0.12 nM and the number of binding sites is approx. 2500 per interstitial cell. A close correlation is observed between the potency of representative LHRH agonists to stimulate LH release in anterior pituitary cells in culture and their affinity for the testicular binding sites. The presence of specific LHRH receptors in an enriched population of Leydig cells suggests that these receptors could play a role, not only in the antifertility effects of LHRH agonists, but also in the physiological control of testicular functions.     

Ca2+, but not membrane lipid hydrolysis, mediates human chorionic gonadotropin production by luteinizing hormone-releasing hormone in human term placenta

We postulated a role for lipid metabolism and Ca2+ in the LHRH-induced release of hCG by human placentas. Term placental cells in suspension prelabeled with [3H]myoinositol were stimulated without or with increasing concentrations of LHRH in the presence of 10 mM LiCl, and total inositol phosphate (IP) was measured by ion exchange chromatography; a nonsignificant 0.9 +/- 0.08-fold increase over the control value was observed. In contrast, placental cells stimulated with equimolar concentrations of angiotensin II (AII) induced a 4.6 +/- 0.9-fold increase in total IP (P less than 0.01), while rat pituitary cells showed 1.9 +/- 0.2- and a 2.4 +/- 0.07-fold increases in total IP production after stimulation with LHRH or AII, respectively (P less than 0.05). These increases were blocked by coincubation with specific LHRH and AII antagonists. When 1 x 10(6) placental cells were incubated with 45Ca2+ without and with increasing doses of LHRH for 0-75 s and then filtered under negative pressure, we observed significant incorporation of 45Ca2+. This influx was linear with incubation time, significantly more pronounced in cells exposed to LHRH than in control cells, and showed a dose-response curve to LHRH that reached maximal influx rates of 3.6 +/- 0.3 nM/min.1 x 10(6) cells with 10(-5) M LHRH. This response was completely blocked by coincubation with 10(-5) M LHRH antagonists; cobalt chloride and verapamil reduced it by 60% and 80%, respectively. Compared to placental cells stimulated with LHRH alone, those coincubated with LHRH and specific LHRH or Ca2+ antagonists released from 10-100% less hCG. We conclude that Ca2+ participates in the LHRH action in human placentas, but uncoupled to PI turnover.     

Growth hormone-releasing factor does not stimulate phosphoinositides breakdown in primary cultures of rat and human pituitary cells

It has been reported that rat growth hormone releasing factor (rat GRF-43), similarly to the two human GRFs (GRF-40 and 44) stimulates adenylate cyclase activity in pituitary cells. Controversial findings have been presented by two different groups on the action of GRF on phosphoinositides (PI) metabolism, a phenomenon linked to Ca-- mediated intracellular mechanisms. In the work to be reported, we evaluated the accumulation of inositol phosphates induced by GRF exposure in primary cultures of rat and human pituitary cells. Addition of rat GRF-43 to rat pituitary cells at doses up to 1 microM had no effect on inositol phosphates accumulation, while already at a dose as low as 0.05 nM it increased growth hormone secretion in the incubation medium significantly. In the same cell system, TRH, a known activator of PI breakdown, significantly increased [3H]inositol phosphates. In primary cultures of human somatotrophs from acromegalic subjects as in rats, addition of hpGRF-40 and also of TRH did not elicit any modification in the accumulation of [3H]inositol phosphates. Consistent with in vivo findings, both peptides induced a significant release of GH in the medium. Our results show that the GH releasing effect of GRF does not involve the hydrolysis of phosphatidylinositol in normal rat as well as in tumoral human somatotrophs. In addition it appears that the anomalous response of TRH on adenomatous cells from acromegalic patients is differently mediated in respect to the action of the tripeptide on normal lactotrophs and thyrotrophs.     

Effects of prostaglandin F2 alpha and a gonadotropin-releasing hormone agonist on inositol phospholipid metabolism in isolated rat corpora lutea of various ages

The sensitivity of rat corpora lutea to luteolytic agents increases with luteal age. We examined the effect of prostaglandin F2 alpha (PGF2 alpha) and [D-Ala6,Des-Gly10]GnRH ethylamide (GnRHa) on inositol phospholipid metabolism in day 2 and day 7 corpora lutea from PMSG-treated rats. Isolated corpora lutea were incubated with 32PO4 or [3H]inositol and were treated with LH, PGF2 alpha, or GnRHa. Phospholipids were purified by TLC, and the water-soluble products of phospholipase-C activity (inositol phosphates) were isolated by ion exchange chromatography. In day 2 corpora lutea, PGF2 alpha, (10 microM) and GnRHa (100 ng/ml) significantly increased 32PO4 incorporation into phosphatidic acid (PA) and phosphatidylinositol (PI), but not into other fractions. LH provoked slight increases in PA. Results were similar with 30 min of prelabeling or simultaneous addition of 32PO4 and stimulants. In other experiments, PGF2 alpha and GnRHa provoked rapid increases (1-5 min) in the accumulation of inositol mono-, bis-, and trisphosphates. LH did not significantly increase inositol phosphate accumulation, but stimulated cAMP accumulation in 2-day-old corpora lutea. Inositol phospholipid metabolism was increased in day 7 corpora lutea compared to that in day 2 corpora lutea. This increase was associated with increased incorporation of 32PO4 into PA and PI and increased accumulation of [3H]inositol phosphates. In day 7 corpora lutea, which are very sensitive to the luteolytic effect of PGF2 alpha, the PG-induced increase in PA labeling was small and inconsistent, whereas PI labeling was unaffected in 30-min incubations. GnRHa was without effect in such corpora lutea. LH, PGF2 alpha, or GnRHa did not increase inositol phosphate accumulation in 7-day-old corpora lutea. These studies demonstrate that the transformation of young (day 2) to mature (day 7) corpora lutea is associated with an increase in luteal inositol phospholipid metabolism. The results also show that PGF2 alpha and GnRHa stimulate phospholipase-C activity in young corpora lutea, but are ineffective in mature corpora lutea, and suggest that an increase in inositol phospholipid metabolism by itself is not sufficient to explain the acute luteolytic action of PGF2 alpha and GnRH in vitro. However, phospholipase-C-derived second messengers may be involved in the action of hormones that control luteal function.     

Ghrelin-induced growth hormone release from goldfish pituitary cells involves voltage-sensitive calcium channels

Ghrelin (GRL) is a stimulator of growth hormone (GH) release in many organisms, including goldfish. As a first study to examine the signalling mechanisms mediating GRL action on GH release in goldfish, we tested the hypothesis that GLR induces GH release from goldfish pituitary cells by enhancing Ca²⁺ entry through L-type voltage-sensitive Ca²⁺ channels (LVSCCs) using perifusion GH release and fura-2/AM Ca²⁺-imaging experiments. Goldfish (g)GRL₁₉ at 1 nM elicited reversible and repeatable GH responses from dispersed goldfish mixed pituitary cultures. However, the lack of a dose-response relationship in sequential treatments with decreasing concentrations of gGRL₁₉ (ranging from 10 to 0.01 nM) implicated rapid desensitization of the GH response. Sequential applications of gGRL₁₉ (1 nM) and salmon GnRH (100 nM), a known Ca²⁺-dependent stimulator of GH release, increased intracellular free Ca²⁺ levels ([Ca²⁺]_i) from the same identified somatotropes, suggesting co-expression of GRL and GnRH receptors on single cells. In contrast, 1 nM gGRL₁₉ failed to elicit GH release and elevation in [Ca²⁺]_i when the cells are incubated with nominally Ca²⁺-free media. When GH release and [Ca²⁺]_i increases were already stimulated by the LVSCC agonist Bay K8644 (10 μM), addition of 1 nM gGRL₁₉ did not further elevate these responses. Finally, the LVSCC inhibitors nifedipine (1 μM) and verapamil (1 μM) abolished 1 nM gGRL₁₉-induced GH release responses while nifedipine eliminated gGRL₁₉-induced [Ca²⁺]_i increase. Taken together, the results of this study provide evidence that entry of extracellular Ca²⁺ through LVSCCs is a key component of the GRL signalling pathway leading to GH release in the goldfish pituitary.     

Luteinizing hormone-releasing hormone and neuropeptide Y influence deoxyribonucleic acid replication in three anterior pituitary cell types. Evidence for mediation by growth factors released from gonadotrophs.

Treatment of anterior pituitary reaggregate cell cultures from 14-day-old female rats with physiological doses of the gonadotropin-releasing hormone LHRH or neuropeptide Y (NPY) for 40 h dose-dependently increased [3H]thymidine ([3H]T) incorporation into DNA of cells expressing PRL immunoreactivity (PRL-ir) and of those expressing ACTH-ir, whereas these peptides decreased the number of [3H]T-labeled cells expressing GH-ir. The effects of NPY were of the same magnitude as those of LHRH. The effects of LHRH were not seen in a gonadotroph-deprived cell population obtained by sequential velocity and buoyant density gradient sedimentation. When the latter cell population was coaggregated with purified gonadotrophs from 14-day-old rats, LHRH did enhance [3H]T labeling of lactotrophs and decreased that of somatotrophs. Gonadotroph-conditioned medium obtained by continuous perifusion of gonadotroph-rich reaggregates contained four different high molecular weight substances mimicking the effects of LHRH and NPY on [3H]T incorporation in the respective pituitary cell types. These substances were partially purified and separated from each other by concentration on a Bond-elut C18-reversed phase cartridge, ultrafiltration, and C18-reversed phase HPLC. One factor stimulated [3H]T labeling of lactotrophs, another that of corticotrophs, and two others inhibited [3H]T labeling of somatotrophs. The present data suggest that the development of PRL-, GH-, and ACTH-containing cells in the pituitary is modulated by LHRH and/or NPY and that the action of LHRH and probably also of NPY is mediated by specific paracrine growth factors released from gonadotrophs.     

Receptor-mediated internalization of LHRH antagonists by pituitary cells

A fluorescently labeled antagonist of luteinizing hormone releasing hormone (LHRH), d-pGlu-d-Phe-d-Trp-Ser-Tyr-d-Lys⁶-(tetramethylrhodamine)-Leu-Arg-Pro-Gly-NH₂, was prepared. This peptide retained high-affinity binding to the LHRH receptor of pituitary plasma membrane preparations. The analog was able to block LHRH-stimulated LH release from pituitaries incubated in vitro, and exhibited minor agonistic activity. This rhodamine-labeled antagonist was utilized for the microscopic visualization and localization of LHRH receptors in dispersed rat pituitary cells. The fluorescently labeled receptors were initially distributed uniformly on the cell surface. The hormone-receptor complexes were redistributed after incubation at 23 °C and formed clusters which subsequently became internalized (at 37°C) into endocytic vesicles. Addition of LHRH (10^-6 M) abolished these processes, indicating specific binding sites for the rhodamine-labeled peptide to the gonadotrope cells, A quantitative comparison of temperature-dependent internalization by iodinated LHRH agonist and antagonist revealed that both analogs were internalized to a similar extent. These findings suggest that LHRH-receptor complex internalization is related to LHRH receptor regulation.     

Thyrotropin-releasing hormone (TRH) selectively and rapidly stimulates phosphatidylinositol turnover in GH pituitary cells: a possible second step of TRH action

TRH was found to rapidly influence 32PO4 incorporation into phospholipids of PRL-secreting GH pituitary cells. Analogs of TRH were found to exert similar effects, with potencies related to receptor-binding affinity. Additional PRL-releasing agents were also tested. Bombesin exerted a similar effect, whereas vasoactive intestinal polypeptide, 8-bromo cAMP, phosphodiesterase inhibitors, 50 mM K+, and scorpion venom toxin had no influence. Cationophore A23187 stimulated phospholipid labeling in a manner distinguishable from that of TRH. Chromatographic analysis showed the action of TRH to be restricted to the labeling of phosphatidylinositol and phosphatidic acid. Kinetic studies indicated a rapid influence of TRH on phosphatidylinositol breakdown, with subsequent accelerated 32PO4 incorporation into phosphatidylinositol and phosphatidic acid. These studied identified a rapid, receptor-mediated, cAMP-independent action of TRH on phospholipid metabolism. Similar effects of other hormones are believed to be involved in promoting cellular Ca2+ translocation. The rapid onset of the response reported here suggests that this event may play a role in mediating the PRL-releasing effects of TRH and bombesin in GH cells.     

Regulation of Phosphatidylinositol 4-phosphate 5-kinase by Protein Kinase C in Human Platelet Membranes

Phorbol myristate acetate (PM A) increased the formation of |³²P | PI 4,5-P, in ³²P-prelabeled human platelet. In saponin-permeabilized platelets, in which ³²P from exogenous |γ-³²P| ATP was incorporated into PI 4-P and PI 4,5-P₂, addition of 10 nM PMA resulted in increased formation of |³²P|PI 4,5-P₂ and |³²P|PI 4-P. In order to distinguish whether increased [³²P]PI 4,5-P₂ formation by PMA reflected merely an increase of [³²P]PI 4-P, the substrate for PI 4-P 5-kinase, or activation of PI 4-P Skinase, we examined the membrane fraction in which most of the kinase activity was located. Although PMA itself did not affect the PI 4-P 5-kinase activity in the control membranes, the kinase activity was increased nearly 2-fold in membranes pretreated with 10 nM PMA but not 4α-phorbol didecanoate which does not activate protein kinase C (PKC). These results suggested that membrane PI 4-P 5-kinase activity was stimulated by the activation of PKC.

However, 100 nM PMA did not stimulate [³²P]PI 4,s-P₂ formation in saponin-permeabilized platelets, and the PI 4-P 5-kinase activity in membranes from platelets pretreated with 100 nM PMA was almost the same as that in control membranes. This can be explained by product inhibition, since PI 4,5-P₂ inhibited concentration-dependently the membrane PI 4-P 5-kinase activity. The Ca²⁺ -dependent PKC fraction partially purified from the platelet cytosol stimulated the membrane PI 4-P 5-kinase activity, whereas the Ca²'-independent PKC fraction inhibited the kinase activity. Taken together, the present results suggest that the platelet membrane PI 4-P 5-kinase activity is stimulated by Ca²⁺ -dependent PKC (cPKC) and is negatively regulated by PI 4,s-p₂ and Ca²⁺ -independent PKC(nPKC).     

Iso stimulation of GH and cAMP: comparison of beta-adrenergic- to GRF-stimulated GH release and cAMP accumulation in monolayer cultures of anterior pituitary cells in vitro

Growth hormone (GH) release and cAMP content were measured in monolayer cultures of anterior pituitary cells after beta-adrenergic and GH-releasing factor (GRF) receptor activation. Isoproterenol (Iso, ED50-20 nM) was less potent than GRF (ED50-20 pM) in stimulating GH release. Iso caused a rapid stimulation of GH release that was maximal after 15 min and declined thereafter, while GRF caused a more gradual increase in GH secretion that was maximal after 30 min and remained elevated after 3 h. Both Iso- and GRF-stimulated GH release were preceded by an increase in cAMP content in the pituitary cells. Further, the addition of 3-isobutyl-1-methylxanthine (IBMX) to the medium enhanced the GH-stimulatory and cAMP-accumulating effects of both secretagogues. Experiments performed with native catecholamines and synthetic catecholamine agonists and antagonists indicated that the GH-stimulatory effect of Iso was mediated by a mixed population of beta 1-adrenergic and beta 2-adrenergic receptors. Additionally, experiments performed with cultured GH3 tumor cells, found that incubation with GRF, Iso, vasoactive intestinal polypeptide, forskolin, or cholera toxin caused an increase in cAMP content in the cells. However, compared to the responses observed in primary pituitary cultures the GH secretory response to these agents was comparatively small. Together, these studies suggest that a mixed population of beta 1-adrenergic and beta 2-adrenergic receptors may act, at least in part, on somatotrophs in the anterior pituitary to stimulate GH release. Although both GRF and beta 2-adrenergic receptor agents affect GH release through a common second messenger system, their differing pharmacokinetic properties suggest distinct intracellular mechanisms.     

Impairments in hepatocyte phosphoinositide metabolism in endotoxemia

The status of phospholipid metabolism and inositol lipids-mediated transmembrane signaling in rat hepatocytes was analyzed during chronic, nonlethal endotoxemia. Rats were infused intravenously (IV) with Escherichia coli endotoxin (ET) via subcutaneously implanted osmotic pumps at a rate of 0.1 mg/100 g bw/day. The experiments were performed after 30 hours of ET or sterile saline (NaCl) infusion, in hepatocytes prelabelled "in vitro" with 32P (15 microCi/mL) and further stimulated with vasopressin (VP, 0.23 mumol/L). Similar experiments were done with food-restricted animals, whose food intake was matched with the voluntary intake of ET-infused rats. Uptake of 32P label into phosphatidic acid (PA), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2) occurs rapidly in cells from pair-fed, saline and ET-infused animals, and reaches a plateau between 60 and 80 minutes of incubation. Labeling of phosphatidylinositol (PI), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) proceeds linearly after a ten-minute lag period for PI and 20 minutes for the two other lipids. The nutritional state greatly affects the distribution of 32P uptake into lipids, resulting in very low labeling of PA and PI and a high labeling of poly-PI as compared with control (taken from untreated rats) cells. In ET-v saline-infused rats, the labeling of PI and PE was depressed concomitantly with a proportional increase in the labeling of PIP and PC. The ability of VP to induce polyphosphoinositide (poly-PI) degradation in hepatocytes from saline-infused animals was similar to that observed in control cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Androgens decrease LHRH binding sites in rat anterior pituitary cells in culture

[125I]-[D-Ser(TBU)6]LHRH-EA binds to a single class of high affinity sites in rat anterior pituitary cells in culture at an apparent dissociation constant of 0.25 nM at 0-4C. The order of potency of a representative group of LHRH agonists and antagonists to displace the labeled ligand is similar to their LH-releasing activity. Treatment of pituitary cells for 48 h with 100 nM 5 alpha-dihydrotestosterone leads to a 40% decrease of the number of LHRH receptors with no change of binding affinity. This loss of LHRH receptors is accompanied by a similar decrease of the LH responsiveness to LHRH, thus providing the first evidence for a direct effect of sex steroids on pituitary LHRH receptors as a possible mechanism of feedback action.