Ovine Pituitary Cells Research Articles

Direct modification of somatotrope function by long-term leptin treatment of primary cultured ovine pituitary cells.

Leptin is produced primarily in adipocytes and regulates body energy balance. A close link between leptin and pituitary hormones, including GH, has been reported. The mechanisms employed by leptin to influence somatotropes are not clear, however. Here we report a direct action of recombinant ovine leptin on primary cultured ovine somatotropes by analyzing the levels of mRNA encoding for GH or the receptors for GHRH (GHRH-R) and GH-releasing peptides (GHRP). Treatment of ovine somatotropes with leptin (10(-7)-10(-9) M) for 1-3 d reduced the mRNA levels encoding GH and GHRH-R, but increased GHRP receptor mRNA levels in a time- and dose-dependent manner. Three-day treatment of cells with leptin decreased the GH response to GHRH stimulation, but the GH response to GHRP-2 stimulation was increased. The combined effect of GHRH and GHRP-2 on GH secretion was not altered by treatment of the cells with leptin. These results demonstrated a direct action of leptin on ovine pituitary cells, leading to a reduced sensitivity of somatotropes to GHRH. It is also suggested that GHRP may be useful to correct the decrease in GHRH-induced GH secretion by leptin.

Direct Modification of Somatotrope Function by Long-Term Leptin Treatment of Primary Cultured Ovine Pituitary Cells

Leptin is produced primarily in adipocytes and regulates body energy balance. A close link between leptin and pituitary hormones, including GH, has been reported. The mechanisms employed by leptin to influence somatotropes are not clear, however. Here we report a direct action of recombinant ovine leptin on primary cultured ovine somatotropes by analyzing the levels of mRNA encoding for GH or the receptors for GHRH (GHRH-R) and GH-releasing peptides (GHRP). Treatment of ovine somatotropes with leptin (10−7–10−9m) for 1–3 d reduced the mRNA levels encoding GH and GHRH-R, but increased GHRP receptor mRNA levels in a time- and dose-dependent manner. Three-day treatment of cells with leptin decreased the GH response to GHRH stimulation, but the GH response to GHRP-2 stimulation was increased. The combined effect of GHRH and GHRP-2 on GH secretion was not altered by treatment of the cells with leptin. These results demonstrated a direct action of leptin on ovine pituitary cells, leading to a reduced sensitivity of somatotropes to GHRH. It is also suggested that GHRP may be useful to correct the decrease in GHRH-induced GH secretion by leptin.

Inhibin binding sites and proteins in pituitary, gonadal, adrenal and bone cells

Activin signals via complexes of type I (50–55 kDa) and II (70–75 kDa) activin receptors, but the mechanism of inhibin action is unclear. Proposed models range from an anti-activin action at the type II activin receptor to independent actions involving putative inhibin receptors. Two membrane-embedded proteoglycans, betaglycan and p120, have recently been implicated in inhibin binding, but neither appears to be a signalling receptor. The present studies on primary cultures of rat pituitary and adrenal cells, and several murine and human cell lines were undertaken to characterise inhibin binding to its physiological targets. High affinity binding of inhibin to the primary cultures and several of the cell lines, like that previously described for ovine pituitary cells, was saturable and reversible. Scatchard analysis revealed two classes of binding sites ( K d of 40–400 and 500–5000 pM, respectively). Affinity labelling identified [ 125I]inhibin binding proteins with apparent molecular weights of 41, 74, 114 and >170 kDa in all cell types that displayed high affinity, high capacity binding of inhibin. Additional labelling of a 124 kDa species was evident in gonadal TM3 and TM4 cell lines. In several cases, activin (≥20 nM) competed poorly or not at all for binding to these proteins. The 74, 114 and >170 kDa inhibin binding proteins in TM3 and TM4 cells were immunoprecipitated by an anti-betaglycan antiserum. These three proteins correspond in size to the activin receptor type II and the core protein and glycosylated forms of betaglycan, respectively, that have been proposed to mediate anti-activin actions of inhibin, but the identity of the 74 kDa species is yet to be confirmed. Studies of [ 125I]inhibin binding kinetics and competition for affinity labelling of individual binding proteins in several cell lines suggest these three species and the 41 and 124 kDa proteins form a high affinity inhibin binding complex. In summary, common patterns of inhibin binding and affinity labelling were observed in inhibin target cells. Novel inhibin binding proteins of around 41 and 124 kDa were implicated in the high affinity binding of inhibin to cells from several sources.

Activin Modulates Differential Effects of Estradiol on Synthesis and Secretion of Follicle-Stimulating Hormone in Ovine Pituitary Cells1

In several physiological paradigms, secretion of FSH and LH are not coordinately regulated. Because these hormones appear to be produced by a single cell type in the anterior pituitary gland, their discordant regulation must be related to differential intracellular responses to various stimuli. Estradiol-17beta (estradiol) has been shown to influence secretion of both FSH and LH and some of its effects are mediated directly on the gonadotrope. Changes in expression of intrapituitary factors such as activin and follistatin may mediate effects of estradiol and account for discordant patterns of FSH and LH. The aims of this study were 1) to determine if estradiol alters expression of genes encoding activin, follistatin, or both in ovine pituitary cells; and 2) to observe the effects of immunoneutralizing activin B in vitro on gonadotropin secretion. Pituitary cells from five ewes in the anestrous season were cultured for 24 h with estradiol (0.01 or 1.0 nM). Estradiol reduced basal secretion of FSH in a dose-dependent manner (P: < 0.001) and simultaneously increased basal secretion of LH (P: < 0.001). Decreased secretion of FSH in estradiol-treated cultures was accompanied by suppressed levels of FSHbeta subunit mRNA (P: < 0.001). Amounts of mRNA for activin beta(B) were reduced in a dose-dependent manner by estradiol (27% +/- 4.9% at 0.01 nM, P: < 0.02; and 46% +/- 3.9% at 1.0 nM, P: < 0.002). In contrast, mRNA for follistatin was not affected by treatment with estradiol. Treatment of pituitary cells with an antibody to activin B reduced secretion of FSH by 50% (P: < 0.01) without influencing secretion of LH. These data lead us to conclude that discordant secretion of gonadotropins can be induced by estradiol acting directly at the pituitary level. The inhibitory effect of estradiol on FSH secretion may be mediated indirectly through decreased pituitary expression of the activin gene.

The in vitro effect of leptin on growth hormone secretion from primary cultured ovine somatotrophs.

Although existing data suggest an influence of leptin on circulating levels of growth hormone (GH), the action site and properties of leptin are still controversial. Using primary cultured ovine pituitary cells, we studied the direct effect of leptin on the secretion of GH. Pituitary cells were dissociated by collagenase and subjected to Percoll gradient centrifugation to enrich the somatotroph population to 60-80% of cells. Treatment of primary cultured ovine somatotrophs with leptin (10(-9)-10(-7) M) for 30 min did not affect basal, GH-releasing hormone (GHRH) (10(-7) M)- or GH-releasing peptide-2 (GHRP-2)(10(-7) M)-stimulated GH secretion. Following treatment of cells for 1-3 d with leptin, GHRH-stimulated GH secretion was reduced and GHRP-2-stimulated GH secretion increased. The combined effect of GHRH and GHRP-2 on GH secretion was not altered by the treatment of cells with leptin for 3 d. GHRH receptor mRNA levels in cultured somatotrophs were decreased but GHRP receptor mRNA levels were increased by 3-d leptin treatment. These results suggest that leptin has a long-term effect on somatotrophs to reduce GHRH receptor synthesis leading to a decrease in GHRH-stimulated GH secretion. Leptin appears, however, to have an opposite effect on GHRP receptor synthesis leading to an increase in GHRP-stimulated GH secretion.

Adrenoceptor subtype involvement in suppression of prolactin secretion by noradrenaline.

In sheep, injection of noradrenaline suppresses prolactin secretion by a direct effect at the pituitary gland. The aims of this study were to use primary cultures of ovine pituitary cells to examine the receptor subtypes that mediate the inhibitory effect of noradrenaline on prolactin secretion and, by using receptor antagonists in vivo, determine whether noradrenaline acts as a prolactin release-inhibiting factor (PIF). Noradrenaline and dopamine suppressed prolactin secretion from ovine pituitary cells with ED50s of 60.9+/-46.6 and 1.5+/-1.0x10-9 mol/l, respectively (P<0.05). The in-vitro prolactin release-inhibiting effect of noradrenaline (10-7 mol/l) was not blocked by the dopamine antagonists pimozide (D2) or SCH23390 (D1) but was blocked by each of the adrenoceptor antagonists (alpha1-adrenoceptor antagonists prazosin and WB4101, the alpha2-adrenoceptor antagonist yohimbine and the beta-adrenoceptor antagonist propranolol). The response to adrenoceptor agonists was also tested in vitro. The alpha1-adrenoceptor agonists phenylephrine and cirazoline significantly suppressed prolactin. Of the alpha2-agonists, clonidine had no effect whereas oxymetazoline and p-aminoclonidine both suppressed prolactin. The beta-adrenoceptor agonist isoproterenol also suppressed prolactin while the specific beta3-antagonist BRL37344 had no effect. When the adrenoceptor antagonists were tested in vivo in ewes manipulated to be in the luteal phase, only WB4101 significantly (P<0.05) increased plasma prolactin concentrations but this response was small and only observed in one of two experiments. In summary, these experiments suggest that adrenoceptors and not dopamine receptors are responsible for the inhibitory effect of noradrenaline on prolactin secretion in vitro but do not implicate a particular adrenoceptor subtype. The in-vivo experiments do not provide convincing evidence for a role for noradrenaline as a physiologically important PIF.

Identification of high affinity binding sites for inhibin on ovine pituitary cells in culture.

The aim of this study was to identify and characterize binding sites for inhibin in primary cultures of ovine anterior pituitary cells. Recombinant human 31-kDa inhibin A was iodinated by an optimized lactoperoxidase procedure. Fractionation of the labeled protein by gel filtration chromatography on Sephadex G-100 in 0.1 M HCl yielded two immunoactive peak regions, the second of which was bioactive as assessed by in vitro bioassay, with a ratio of bioactivity/immunoactivity of 0.62-0.77 and an iodine incorporation ratio of 1.7-2.0 mol 125I/mol inhibin. The specific binding of purified [125I]inhibin to cultured ovine pituitary cells varied with time, temperature, and cell number. Displacement of the tracer by unlabeled inhibin, as assessed by Scatchard analysis, revealed two binding sites with average Kd values of 0.28 and 3.9 nM and with approximately 250 and 3100 binding sites/anterior pituitary cell, respectively. There was little cross-reaction between inhibin and activin A (<2%), transforming growth factor-beta (<0.2%), or follistatin (<<0.1%). Examination of cell lines that were not expected to have inhibin receptors showed that there was no specific binding of inhibin to human leukemia (Jurkat) cells, whereas the binding to human embryonic kidney (293) cells was displaced by both inhibin and activin with a similar degree of cross-reaction, which suggests binding to an activin receptor. It is concluded that inhibin-binding sites with high affinity and specificity have been identified on ovine pituitary cells, consistent with both inhibin action on the pituitary and the presence of the putative inhibin receptor.

Identification of High Affinity Binding Sites for Inhibin on Ovine Pituitary Cells in Culture

Identification of High Affinity Binding Sites for Inhibin on Ovine Pituitary Cells in Culture

The in vitro Effect of Leptin onBasal and Growth Hormone-Releasing Hormone-StimulatedGrowth Hormone Secretion from theOvine Pituitary Gland

We have studied the direct effect of leptin on the secretion of GH from the anterior pituitary gland of the sheep. Using primary cultures of ovine pituitary cells, leptin (10^–9–10^–7 M) treatment for 30 min did not affect basal or GHRH (10^–7 M)-stimulated GH secretion. Following treatment for 24 h, a dose of 10^–7 M leptin stimulated basal GH secretion. In contrast, doses of 10^–7 and 10^–8 M leptin inhibited GHRH-stimulated GH secretion after 24 h of treatment. These results suggest that leptin can have long-term effects on the somatotropes, but no acute effect. Furthermore, leptin appears to have opposite effects on basal and GHRH-stimulated GH secretion.

Effects of interleukins on secretion of luteinizing hormone from ovine pituitary cells

Abstract Objective To determine whether cytokines of homologous species might mediate the stimulatory effects of endotoxin on release of luteinizing hormone (LH) from pituitary cells. Sample Population Cells from pituitary glands collected from 8- to 14-month-old wethers. Procedure Cells from the anterior pituitary gland were cultured in the presence of recombinant ovine or bovine cytokines (interleukin [IL]-1α, IL-1β, and IL-2), tumor necrosis factor-α (TNF), and interferon-γ (IFN-γ). Luteinizing hormone that was released into the medium was measured. Cells were also cultured with modulators of signal transduction pathways to evaluate the second messenger system used by IL-1α and IL-1β. Results Similar to effects of endotoxin, IL-1α and IL-1β stimulated release of LH. Interleukin 2, TNF, and IFN-γ did not have a detectable effect on release of LH. Stimulation of LH release by IL-1αand IL-1β required activation of voltage-dependent Ca2+ channels and appeared to involve protein kinase C. Conclusions IL-1αand IL-1β may mediate the direct stimulatory effect of endotoxin on release of LH in vitro. Interleukin 2, TNF, and IFN-γ do not have a direct effect on release of LH; therefore, they do not mediate this effect of endotoxin. Clinical Relevance Stressors, including infection, are often associated with reduced fertility. Infection resulting in endotoxin release, production of interleukins, or both, can lead to direct stimulation of LH release from the pituitary gland. Inopportune release of LH via cytokines may interfere with normal pulsatile release of LH, thereby suppressing gonadal function. (Am J Vet Res 1998;59:1488–1493)

Components of the Neuronal Exocytotic Machinery in the Anterior Pituitary of the Ovariectomised Ewe and the Effects of Oestrogen in Gonadotropes as Studied with Confocal Microscopy

We have investigated exocytotic proteins in ovine pituitary cells and sought to identify changes in expression of these proteins related to the effects of estrogen on luteinising hormone (LH) secretion in the ovariectomised ewe. Sheep were treated with either oestradiol benzoate, or oil (i.m.) and blood samples collected for LH assay. Pituitaries were perfusion-fixed and dual-label immunohistochemistry was performed to identify hormone-secreting cells, and colocalise synaptic proteins within different cell types. Synaptophysin, SNAP-25, VAMP-2, rab3A, Munc-18-1, α/β-SNAP, csp, and secretogranin II were detected in gonadotropes and somatotropes. Lactotropes were positive for SNAP-25 and synaptophysin (other synaptic proteins not investigated). Synaptotagmin I was detected in gonadotropes and lactotropes, but not somatotropes. Synaptophysin, SNAP-25, synaptotagmins I, II and III, VAMP-2, rab3A, Munc-18-1, α/β-SNAP, csp, and secretogranin II were detected in nerve fibres of the posterior lobe. Membrane staining for SNAP-25 and weak cytoplasmic labelling for both synaptotagmin I and secretogranin II were detected in the intermediate lobe. Syntaxin and complexin II antibodies did not label any region of the ovine pituitary. Oestrogen treatment, to induce a pre-ovulatory-like LH surge, caused migration of LH-containing secretory granules toward the plasma membrane of gonadotropes, but did not alter the percentage of gonadotropes expressing each exocytotic protein. Oestrogen treatment caused a similar redistribution of csp and secretogranin II staining in gonadotropes. We conclude that synaptic protein expression is not altered in the anterior pituitary at the time when LH secretion is maximal. The ubiquitous distribution of many exocytotic proteins suggests that all hormone-secreting cells of the pituitary gland contain the same, or similar exocytotic machinery, but distinct ‘activating factors’ are required to selectively trigger the secretion of individual hormones.

In vivo effects of the GH-releasing heptapeptide GHRP-1 in lambs.

The novel synthetic growth hormone-releasing heptapeptide GHRP-1 is reported to be more potent than growth hormone-releasing hormone (GHRH) in eliciting GH release in vivo in rats and man. However, in ovine pituitary cells in primary culture in a perifusion system, GHRP-1 was 10-fold less active than GHRH. The purpose of this work was to study the effect of GHRP-1 in sheep in vivo. Ovine GH release stimulated by either GHRP-1 or GHRH(1-29)NH2, in eight pre-ruminant lambs, was determined. GHRP-1 was administered at doses of 1.2, 2.4 and 6 nmole/kg by i.v. bolus, and GHRH(1-29)NH2 at 0.3 nmole/kg. Mean+/-s.e.m. peak GH levels in the plasma after injection of saline, 1.2, 2.4, 6 nmole/kg GHRP-1 and 0.3 nmole/kg GHRH were 2.2+/-0.9, 9.3+/-2.5, 8.8+/-2.4, 35.1+/-5.8 and 51.6+/-10.5 ng/mL, respectively. As spontaneous 20 ng/mL peaks were observed, only peaks above this level can be considered as significant. The highest dose of GHRP-1 (6 nmole/kg) elicited oGH release, but its action was surpassed by GHRH 0.3 nmole/kg. Furthermore GHRP-1 and GHRH appear to behave inversely when response amplitudes are considered. Animals exhibiting a strong reaction to GHRH-1, show a correspondingly weak reaction to GHRH and vice-versa. This may reflect differences in intracellular mechanisms at the pituitary level. Our data support the results in vitro that in sheep GHRP-1 is a weaker stimulant of GH secretion than GHRH.

Effects of bovine follicular fluid and passive immunization against gonadotropin-releasing hormone (GnRH) on messenger ribonucleic acid for GnRH receptor and gonadotropin subunits in ovariectomized ewes.

In cultured ovine pituitary cells, inhibin increases concentrations of mRNA encoding GnRH receptor and numbers of GnRH receptors. The objective of this study was to test the hypothesis that inhibin increases concentrations of ovine GnRH receptor mRNA in vivo. Ovariectomized ewes were used to eliminate effects of endogenous ovarian hormones, and passive immunization against GnRH was employed to avoid possible confounding influences of GnRH on GnRH receptor gene expression. Two groups of ewes (n = 5/group) were treated with 50 ml GnRH antiserum on Days 0 and 3 of the experiment. One group of immunized ewes received 10 ml charcoal-extracted bovine follicular fluid (bFF) as a source of inhibin every 8 h for 48 h on Days 4-6 of the experiment. A third group of ewes was not passively immunized and was treated only with bFF, and control ewes received no treatments. Anterior pituitary glands were collected from all ewes on Day 6. Passive immunization against GnRH, alone or in combination with treatment with bFF, decreased mean concentrations of LH (p < 0.01) and LH pulse amplitude (p < 0.001). In ewes treated only with GnRH antiserum, number of LH pulses was also reduced (p < 0.03). Circulating concentrations of FSH tended to be lower (p = 0.06) in passively immunized ewes compared to controls. Treatment with bFF, alone or in combination with GnRH antiserum, reduced circulating concentrations of FSH (p < 0.02) and amounts of FSHbeta subunit mRNA (p < 0.001) to less than 30% and 10% of control values, respectively. Despite effects of bFF on concentrations of FSHbeta mRNA and secretion of FSH, concentrations of GnRH receptor mRNA were similar among controls, ewes treated with bFF alone, and passively immunized ewes treated with bFF. Passive immunization against GnRH did not affect concentrations of GnRH receptor mRNA but resulted in a reduction (p < 0.05) in amount of LHbeta mRNA. Treatment with bFF did not affect amounts of either alpha subunit or LHbeta subunit mRNA except when combined with treatment with antiserum, when amounts of both alpha and LHbeta subunit mRNA were reduced (p < 0.05). These results do not support the hypothesis that inhibin increases concentrations of GnRH receptor mRNA in the ewe, and they provide evidence that inhibin is not an acute regulator of ovine GnRH receptor gene expression in vivo.

A novel approach to assess changes in endocrine secretion: analysis of GnRH antagonist (Nal-Glu) suppression of gonadotropin release in ovariectomized ewes.

Circulating hormone levels reflect the outcome of multiple feedback systems. A method to accurately assess the dynamics of hormonal changes in samples collected at infrequent intervals and compare these dynamic processes among treatment groups is presented. In this approach, a smooth curve is fitted to each time series of concentrations produced in an experiment, the curves are summarized by numerical measurements, and the measurements are subjected to statistical analysis. The method is demonstrated on data from an experiment that explores the differential effects of a competitive GnRH receptor antagonist (Nal-Glu) on circulating levels of LH and FSH. In this experiment, six adult ovariectomized Suffolk ewes were treated with one of three doses of Nal-Glu using a crossover design. LH and FSH concentrations were determined in hourly samples of jugular blood for 24 h after treatment. Applying the analytical approach, we observed differential effects of increasing concentrations of Nal-Glu on circulating LH and FSH concentrations. The magnitude of LH suppression was similar from dose to dose, while the duration of LH suppression was dose-dependent. In contrast, all doses of Nal-Glu elicited similar effects on the amplitude, duration and time to recovery of FSH suppression. Studies conducted in vitro utilizing dispersed ovine pituitary cells in culture demonstrated that the differential effects of Nal-Glu on FSH and LH secretion were not the outcome of differential sensitivity of FSH and LH to GnRH. The differential effects of Nal-Glu on circulating LH and FSH concentrations may be due to a number of factors, including other releasing or release-inhibiting hormones, paracrine modulators involved in selective regulation of FSH, and/or differences in clearances.

The biological and immunological characterization of inhibin A and B forms in human follicular fluid and plasma.

In a previous study (see Ref.7), the molecular weight distribution of inhibin activity in fractionated human follicular fluid (hFF) and human male and female plasma/serum was determined by in vitro bioassay using ovine pituitary cells in culture and various specific inhibin A and inhibin alpha-subunit-directed immunoassays. It was shown, however, that the ovine in vitro bioassay detected inhibin B poorly. These findings are extended in the present study by the determination of the molecular weight profile of in vitro bioactivity using rat pituitary cells, which detects both inhibin A and B, a specific inhibin B enzyme-linked immunosorbent assay (ELISA), an RIA detecting the alpha N region of the alpha-subunit, an alpha-subunit ELISA (Pro-alpha C) directed to the inhibin forms containing the Pro sequence, and an alpha C subunit immunofluorometric assay that detects all inhibin forms. The profile in hFF of inhibin in vitro bioactivity, using rat pituitary cells in culture, significantly (P < 0.001) correlated with in vitro bioactivity using ovine pituitary cells (r = 0.85), inhibin A immunoactivity (r = 0.70), inhibin B immunoactivity (r = 0.89), and the combination of inhibin A + B immunoactivities (r = 0.93), with peaks of activity identified at 66K, 55K, 36K and 33K, consistent with presumed known mol wt forms of inhibin. Inhibin B profiles in fractionated serum from women stimulated with gonadotropins and male plasma consisted of two forms (66K and 36K), whereas inhibin A in female serum included, in addition, the 55K form. These findings indicated that higher molecular weight forms of inhibin B are present in biological samples, and their distribution differs from that of inhibin A, suggesting a differential processing of the precursor forms in the circulation. Pro-alpha C immunoactivity was identified in serum samples with prominent peaks at 36K and 29K (known Pro-alpha C subunit forms) and not with any high mol wt dimeric forms of inhibin. If this observation applies to a wider range of serum samples, the Pro-alpha C ELISA may provide an appropriate and specific assay for the measurement of free alpha-subunit. To compare immunoactivity levels between assays, the inhibins A, B, and Pro-alpha C standards were calibrated in terms of their alpha C subunit content, as determined by an alpha C subunit immunoassay, with the inhibin B standard containing 60% of the alpha C subunit content compared with either the inhibin A or Pro-alpha C standard. After adjustments of the various standards for this difference in alpha C subunit content, a comparison was undertaken of the combined levels of inhibins A, B, and Pro-alpha C immunoactivity across the hFF and serum chromatograms and compared with levels determined by the alpha-subunit-directed immunoassays. A high correlation (r = 0.59-0.96) was observed, indicating that the alpha-subunit immunoactivity in serum consists largely of a composite of presumed known molecular weight forms of inhibins A, B, and Pro-alpha C. It is concluded that: 1) inhibin in vitro bioactivity in hFF is largely attributed to the presence of 33-36K and 50-66K forms of inhibins A and B; and 2) inhibin alpha-subunit immunoactivity in hFF and serum is a composite of presumed known forms of inhibin A, inhibin B, and the alpha-subunit.

Use of newly designed microperifusion system with amperometric sensors for near-continuous on-line monitoring of hormone secretion. I. correlation with the luteinizing hormone secretory response to gonadotropin-releasing hormone.

LH is rapidly secreted from the anterior pituitary gland in response to episodic release of GnRH. To understand better the detailed dynamics of this secretory process and related time- and dose-dependent effects, we developed a microperifusion system able to preserve resolution of dynamic secretory responses by minimizing dispersion and with capabilities for automation and continuous on-line monitoring. To monitor secretion at a frequency greater than that feasible with discrete samples, we determined whether LH or another molecule cosecreted by anterior pituitary cells could be monitored on a near-continuous basis using amperometric electrochemical sensors operated under conditions of cyclic voltammetry. The developed culture system incorporated a 32-mu l cell chamber in a controllable constant environment. Miniature sensors were positioned immediately adjacent to the cells to permit differential measurements of the input and effluent streams of medium. With the system, square wave pulses of electrochemically active, biologically inert molecules, e.g. ascorbate and phenol red, showed similar redox profiles before and after the cells, with minimal dispersion. Enzymatically dispersed ovine anterior pituitary cells were cultured on SoloHill glass beads for 4 days, loaded into the chamber, and perifused with DMEM containing 10% FCS for 2 h. After stabilization, the medium was switched to protein-free, HEPES-buffered HBSS with L-glutamine and allowed to flow for a 1-h washout period, followed by GnRH challenges of varying concentrations in a random order. Perifusate was collected in six-drop fractions at approximately 30-sec intervals. Although LH, its individual subunits, and FSH could not be detected amperometrically, GnRH stimulation of the cells simultaneously induced a several log order dose-dependent secretion of both an electrochemically detectable molecule and LH, as measured by RIA. The secretory profiles of the amperometrically detected signal and immunoactive LH were very similar. Dose-response relationships of the amperometric signal and LH to a wide range of GnRH were similar. The responses of both secreted LH and the amperometric signaling molecule(s) to GnRH were triphasic; an initial peak of activity was observed within 20-40 sec, a lower plateau level was observed for the duration of the 4-min GnRH stimulation, and a gradual return to baseline followed. The cells then maintained a constant level of GnRH-independent basal secretion. These results indicate that it is feasible to monitor the complex dynamics of endocrine and cellular responsiveness to secretagogues from endocrine cells/tissues continuously in real-time.

Interaction of cyclic AMP- and calcium-dependent mechanisms in the regulation of growth hormone-releasing hormone-stimulated growth hormone release from ovine pituitary cells

Growth hormone-releasing hormone (GHRH)-stimulated growth hormone (GH) release from the sheep pituitary is mediated through Ca 2+- and cyclic AMP-dependent mechanisms. The initial Ca 2+ influx is suggested to result from depolarization, whereas a secondary Ca 2+ influx is thought to result from second messengers. This study sought to determine whether there was an interaction between these two signal transduction pathways. Sheep pituitary cells were placed in culture for 4 d and were then washed and incubated for 1 hr in serum-free medium before the application of specific antagonists and/or agonists. Both KCl and forskolin stimulated GH release (P < 0.05), but neither produced an effect similar to that of GHRH. The combination of both stimuli, however, mimicked GH release, as seen with a maximal dose of GHRH. Pretreatment with H89 (protein kinase A [PKA] inhibitor) inhibited GHRH, forskolin- and KCl-stimulated GH release (P < 0.001) but had no effect on phorbol myristate acetate (PMA)-stimulated GH release. Verapamil (voltage-dependent Ca 2+ channel blocker) inhibited the GHRH effects on GH release (P < 0.0002) but did not influence forskolin or PMA actions. These data suggest that Ca 2+-dependent pathways converge with cyclic AMP-dependent pathways before or with the activation of PKA. The data also suggest that PKA activation by cyclic AMP alone is insufficient to reproduce either the effects of GHRH stimulation or the combined effects of Ca 2+, influx plus PKA activation on GH release. A calmodulin blocker, W7, reduced GHRH-stimulated GH release, a reduction equivalent to the Ca 2+ effect on GH release. This suggests that Ca 2+ activates calmodulin, which in turn enhances adenylyl cyclase and/or PKA activity to release GH from the sheep pituitary.

The effects of GH-releasing peptide-6 (GHRP-6) and GHRP-2 on intracellular adenosine 3′,5′-monophosphate (cAMP) levels and GH secretion in ovine and rat somatotrophs

The mechanism of action of GH-releasing peptide-6 (GHRP-6) and GHRP-2 on GH release was investigated in ovine and rat pituitary cells in vitro. In partially purified sheep somatotrophs, GHRP-2 and GH-releasing factor (GRF) increased intracellular cyclic AMP (cAMP) concentrations and caused GH release in a dose-dependent manner; GHRP-6 did not increase cAMP levels. An additive effect of maximal doses of GRF and GHRP-2 was observed in both cAMP and GH levels whereas combined GHRP-6 and GHRP-2 at maximal doses produced an additive effect on GH release only. Pretreatment of the cells with MDL 12,330A, an adenylyl cyclase inhibitor, prevented cAMP accumulation and the subsequent release of GH that was caused by either GHRP-2 or GRF. The cAMP antagonist, Rp-cAMP also blocked GH release in response to GHRP-2 and GRF. The cAMP antagonist did not prevent the effect of GHRP-6 on GH secretion whereas MDL 12,330A partially reduced the effect. An antagonist for the GRF receptor, [Ac-Tyr1,D-Arg2]-GRF 1-29, significantly diminished the effect of GHRP-2 and GRF on cAMP accumulation and GH release, but did not affect GH release induced by GHRP-6. Somatostatin prevented cAMP accumulation and GH release responses to GHRP-2, GRF and GHRP-6. Ca2+ channel blockade did not affect the cAMP increase in response to GHRP-2 or GRF but totally prevented GH release in response to GHRP-2, GRF and GHRP-6. These results indicated that GHRP-2 acts on ovine pituitary somatotrophs to increase cAMP concentration in a manner similar to that of GRF; this occurs even during the blockade of Ca2+ influx. GHRP-6 caused GH release without an increase in intracellular cAMP levels. GH release in response to all three secretagogues was reduced by somatostatin and was dependent upon the influx of extracellular Ca2+. The additive effect of GHRP-2 and GRF or GHRP-6 suggested that the three peptides may act on different receptors. In rat pituitary cell cultures, GHRP-6 had no effect on cAMP levels, but potentiated the effect of GRF on cAMP accumulation. The synergistic effect of GRF and GHRP-6 on cAMP accumulation did not occur in sheep somatotrophs. Whereas GHRP-2 caused cAMP accumulation in sheep somatotrophs, it did not do so in rat pituitary cells. These data indicate species differences in the response of pituitary somatotrophs to the GHRPs and this is probably due to different subtypes of GHRP receptor in rat or sheep.

Isolation of Nine Different Biologically and Immunologically Active Molecular Variants of Bovine Follicular Inhibin1

A combination of immunoaffinity chromatography, SDS-PAGE, and electroelution was used to simultaneously isolate 0.36-4.65 mg of nine different molecular forms of inhibin (pro alpha C-29 kDa; fully processed 34 kDa; and large inhibin forms 49, 53, 58, 77, 88, 110, and > 160 kDa) from 0.675 L of bovine follicular fluid (bFF). Each inhibin form, except pro alpha C, cross-reacted with inhibin alpha C 1-26-and beta A 82-114-subunit-directed antibodies during immunoblot analysis. Pro alpha C cross-reacted only with alpha-subunit antibodies. The inhibin forms consisted of 22-, 29-, 49-, or 58-kDa alpha subunits and 17- or 58-kDa beta subunits. During cultures of ovine pituitary cells, a 5-ng/ml dose of each inhibin form (except pro alpha C) suppressed basal accumulation of FSH 30% to 50% but increased GnRH-induced LH release 40% to 248%. The various inhibin forms cross-reacted in parallel fashion with standard curves generated during homologous and heterologous RIAs but with markedly different relative immunopotencies. In the RIAs, pro alpha cross-reacted 3- to 18-fold more than the fully processed inhibin form. The fully processed and the seven different large forms of inhibin cross-reacted with different relative immunopotencies in a two-site dimer-specific ELISA. We concluded that 1) a combination of immunoaffinity extraction, SDS-PAGE, and electroelution simultaneously isolated relatively large amounts of highly enriched preparations of nine different molecular forms of immunologically and biologically active inhibin from bFF; 2) eight different dimeric forms of bovine inhibin may regulate both basal FSH and GnRH-induced LH secretion by the pituitary gland, and 3) eight or nine different molecular forms of inhibin cross-react with different relative immunopotencies in the two-site dimer-specific assay or RIAs.

Ovine anterior pituitary production of follistatin in vitro.

Rat pituitary cells express messenger RNA for the activin-binding protein, follistatin (FS), and rat and bovine pituitary cell cultures secrete FS into the medium. In the present study, a previously validated, heterologous RIA for ovine FS was employed to investigate FS synthesis, secretion, and regulation in cultures of ovine anterior pituitary cells. The validity of the RIA was confirmed by the finding that FS immunoreactivity in ovine pituitary cell culture-conditioned medium diluted in parallel with purified bovine FS, and fractionation of the conditioned medium resulted in the coelution of activin-binding activity with the FS immunoactivity. The concentration of endogenous ovine FS achieved in the culture medium (0.08-0.6 nM) was in the range over which bovine FS suppresses FSH secretion in these cultures (IC50 = 0.5 nM). To characterize the relationship between endogenous FS and FSH secretion, dispersed ovine pituitary cells were preincubated with 10% fetal bovine serum for 2 days, then cultured between days 2-5 in the presence of a chemically defined serum substitute. Under these conditions, FS was continuously secreted at a rate of 12.1 +/- 1.8 ng/10(6) cells.day (mean +/- SEM; n = 18), whereas FSH was secreted at 64 +/- 13 ng/10(6) cells.day (n = 7). The secretion of FS and FSH changed in a reciprocal way as culture conditions were altered either by maintaining exposure of the cells to fetal bovine serum or by plating the cells at a 6- to 10-fold higher seeding density. Under the latter circumstance, for instance, FS secretion during the 3-day test period decreased to 47 +/- 14% (n = 10) and FSH secretion increased to 137 +/- 6% (n = 6) of the respective values in cultures of dispersed cells. FS secretion was increased nearly 3-fold (P < 0.05) in a dose-dependent manner by continuous exposure of ovine pituitary cells between days 2-5 to recombinant human activin A (1-10 nM), which concomitantly increased FSH secretion. Recombinant human inhibin A (0.003-10 nM); the synthetic glucocorticoids, RU28362 and dexamethasone (each 1-100 nM); the sex steroids, testosterone (1-100 nM), 17 beta-estradiol (0.001-5 nM), and progesterone (4-2500 nM); and the vitamin A derivative, retinoic acid (0.3-32 microM), each inhibited FSH secretion from these cultures, but only the last agent significantly (P < 0.05) increased FS secretion. Inhibin prevented the stimulation of FSH secretion by activin A without affecting its stimulation of FS secretion.(ABSTRACT TRUNCATED AT 400 WORDS)