Inhibitory effect of pure 31-kilodalton bovine inhibin on gonadotropin-releasing hormone (GnRH)-induced up-regulation of GnRH binding sites in cultured rat anterior pituitary cells

Primary cultures of enzymatically dispersed rat anterior pituitary cells were used to examine the effect of pure 31 kilodalton bovine inhibin on GnRH-induced up-regulation of GnRH binding sites. After 2 days in culture, the cells were exposed to stimuli with or without test substances for 10 h, followed by evaluation of GnRH binding sites using iodinated GnRH-A (Buserelin) as tracer. Inhibin suppressed GnRH-induced up-regulation of GnRH binding sites in a dose-dependent manner with an IC50 of 0.13 U/ml (5.5 pM). The inhibin-related peptides transforming growth factor-beta, and Müllerian inhibitory substance had no detectable effect (stimulatory or inhibitory), suggesting that the action is specific to inhibin. In addition, inhibin inhibited the calcium ionophore A23187-induced up-regulation of GnRH binding sites, indicating that this effect of inhibin can occur, at least in part, at a stage subsequent to Ca2+ mobilization. Inhibin did not compete with iodinated GnRH-A for GnRH binding sites. In conclusion, pure 31 kilodalton bovine inhibin suppressed GnRH-induced up-regulation of GnRH binding sites in cultured rat anterior pituitary cells, providing direct evidence that inhibin modulates delayed actions of GnRH.     

Desensitization to gonadotropin-releasing hormone in perifused chicken anterior pituitary cells

A perifusion method consisting of dispersed chicken anterior pituitary cells suspended in columns of Bio-Gel was developed to monitor the dynamics of LH release. The perifused cells responded to chicken I GnRH (Gln8-GnRH) in a dose-dependent manner. The ED50 was 3 X 10(-10) M, and maximal LH release occurred in response to 4 X 10(-9) M Gln8-GnRH. Continuous administration of 10(-7) M Gln8-GnRH and agonist stimulated an initial 8- to 10-fold increase in LH release within minutes. LH release then declined rapidly, reaching basal levels within 100 min. A biphasic response was noted. Calcium ionophore A23187 was effective in releasing additional LH from cells desensitized to 10(-7) Gln8-GnRH and agonist, indicating that total cellular LH was not depleted. In contrast, delivery of 2-min pulses of 10(-7) M and 10(-9) M Gln8-GnRH at a frequency of one pulse every 30 or 60 min for 3-5 h maintained pituitary responsiveness. Exposure to 10(-7) M Gln8-GnRH for 20 min was sufficient to desensitize pituitary cells to subsequent Gln8-GnRH stimulation. However, 20-min exposure to 10(-7) M GnRH antagonist neither evoked LH release nor had a desensitizing effect on subsequent stimulation by 10(-7) M Gln8-GnRH, indicating that receptor activation, not merely receptor binding, is necessary for Gln8-GnRH-mediated homologous desensitization. Pituitary cells desensitized by 20-min exposure to 10(-8) M Gln8-GnRH maintained responsiveness to a higher dose (10(-6 M) of Gln8-GnRH, suggesting that down-regulation of pituitary GnRH receptors might play a part in desensitization. Calcium ionophore A23187 partially desensitized pituitary cells to subsequent stimulation with Gln8-GnRH, probably due to depletion of releasable LH or desensitization of calcium-coupled secretory mechanisms. In calcium-free medium, 10(-7) M Gln8-GnRH did not evoke LH release, but nevertheless partially desensitized cells to subsequent 10(-7) M Gln8-GnRH stimulation. Thus desensitization is partially calcium-dependent. These findings demonstrate that the GnRH-mediated desensitization of gonadotrophs is a characteristic of chicken pituitary cells as in the mammal. However, chicken pituitary cells differ from mammalian cells in that desensitization is more rapid and partially dependent on extracellular calcium.     

Action kinetics of inhibin in superfused pituitary cells depend on gonadotropin-releasing hormone treatment

We examined the effects of partly purified inhibin from porcine follicular fluid on FSH and LH release in superfused rat pituitary cell cultures exposed to different GnRH stimuli. Pituitary cells from immature male rats were cultured in chemically defined medium. After 4 days of static culture in the absence of inhibin preparation and GnRH, the cell monolayers were superfused for approximately 10 h at a constant speed (0.15 or 0.25 ml/min) with medium with or without inhibin preparation (1 micrograms/ml). During the superfusion, some cultures were stimulated with GnRH (10 nM) continuously or intermittently (1 min/0.5 h or 6 min/1 h). In the basal condition (no GnRH), inhibin suppressed FSH release after 5 h of exposure (P less than 0.01), whereas LH secretion was not affected. In cultures treated with GnRH pulses (of either frequency), the inhibitory effects on the GnRH-stimulated FSH and LH release were statistically significant (P less than 0.01) after 2 h of exposure, became more pronounced in the next several hours, then remained stable until the end of the experiment. In cultures exposed to GnRH continuously, the suppressing effects of inhibin preparation became significant (P less than 0.01) after 3 h of exposure and were maximal at 4 h (52% and 61% of control values for FSH and LH, respectively). Later, the suppressing effect became less pronounce due to the decreasing rate of gonadotropin secretion in control (no inhibin) cultures exposed continuously to GnRH. The magnitude of FSH and LH suppression after 9 h of exposure to the inhibin preparation was statistically different (P less than 0.05) for different GnRH treatments and was more pronounced with GnRH pulses (24-27% and 54-57% of control values for FSH and LH, respectively) than with cultures exposed to GnRH continuously (77% and 89% of control values for FSH and LH, respectively) or in the absence of GnRH (50% and 92% of control values for FSH and LH, respectively). We conclude that both the kinetics and magnitude of action of the inhibin preparation on FSH and LH release can differ significantly depending on the presence or absence of GnRH as well as on the mode of GnRH stimulation. Of particular importance is the observation that suppressive effects of inhibin preparation decline in cultures that have been desensitized to GnRH after prolonged continuous GnRH exposures. These differences stress the role of GnRH-inhibin interactions in the regulation of gonadotropin secretion and emphasize the importance of the mode of GnRH stimulation in studies concerning inhibin action on pituitary cells in vitro.     

Effects of 31 kilodalton bovine inhibin on follicle-stimulating hormone and luteinizing hormone in rat pituitary cells in vitro: actions under basal conditions

The effects of purified 31 kilodalton (kDa) bovine inhibin on the basal release and cell contents of FSH and LH in pituitary cells from adult male Sprague-Dawley rats have been investigated for periods up to 28 days in tissue culture. A constant rate of basal FSH release (1.90 +/- 0.24 ng/10(6) cells.h) was observed during the first 10 days of culture and this release was blocked by inhibin in a concentration-dependent manner (IC50 = 0.16 U/ml, IC100 greater than 2.5 U/ml). The constant basal LH release between days 2 and 12 of culture (0.15 +/- 0.02 ng/10(6) cells.h) was also blocked by inhibin in a concentration-dependent manner to a minimum of 25% of control (IC50 = 0.66 U/ml, ICmax greater than 6 U/ml). FSH and LH cell contents decreased exponentially in the presence and absence of inhibin over the first 20 days of culture permitting the calculation of decay half-times (t1/2). Inhibin reduced the FSH cell content t1/2 from 5.0 to 1.8 days and the LH cell content t1/2 from 5.8 to 3.6 days (IC50 = 0.81-0.83 U/ml, ICmax greater than 6 U/ml for both FSH and LH). The sum of the FSH released and FSH cell content (i.e. total FSH) increased with time in culture and this increase was blocked by inhibin. In contrast, total LH decreased with time in all cultures. The data suggest that in pituitary cells separated from hypothalamic and gonadal inputs 1) inhibin primarily suppresses tonic FSH synthesis coupled to its basal release; 2) at higher concentrations inhibin partially suppresses basal LH release and promotes intracellular degradation of FSH and LH. Thus, based on differences in sensitivity, inhibin acts at two or more sites, one associated with FSH tonic synthesis-basal release and the other(s) with FSH/LH mobilization and/or degradation.     

Prenatal development of gonadotropin-releasing hormone receptors in the rat anterior pituitary

The development of pituitary GnRH receptors was studied in the rat with in vitro and in vivo autoradiography. GnRH receptors were first seen in pituitary primordia of 13-day-old fetuses. The binding was specific and saturable and was abolished in the presence of 10 microM synthetic GnRH. To examine whether GnRH was available to the fetus, amnionic fluid was collected on days E 12-18. RIA analyses showed that GnRH levels in the amnionic fluid were low on days 12 and 13 (0-20 pM/ml) and rose to 225 pM/ml on day E 16 before they declined to 110 pM/ml on fetal day E 18. The highest levels of GnRH in the amnionic fluid on day E 16 coincided with the first appearance of immunoreactive LH cells, as determined by immunohistochemistry. Intravenous injection of 500 microliters amnionic fluid into pentobarbital-anesthetized adult rats caused a transient 40-60% increase in circulating serum LH in the recipient animal. To show that GnRH from the amnionic fluid has access to the developing pituitary, the 125I-labeled GnRH agonist Buserelin was injected into the amnionic fluid of 13-, 14-, and 15-day-old fetuses in the presence or absence of 10 microM unlabeled GnRH. Autoradiographic analysis of the fetal tissue indicated that the labeled GnRH agonist bound to specific receptors in the primordial pituitaries. The results suggest that the pituitary gonadotropes are differentiated before day E 13 because the expression of GnRH receptors is already an indication of cell determination. Since GnRH is present in the amnionic fluid in a biologically active form and can reach the fetal pituitary, it is concluded that GnRH may be an important factor determining the onset LH synthesis, but not the differentiation, of primordial pituitary cells.     

Growth hormone-releasing hormone and gonadotropin-releasing hormone stimulate nitric oxide production in 17beta-estradiol-primed rat anterior pituitary cells

It was reported that neuronal nitric oxide synthase (nNOS) was expressed only in gonadotrophs and folliculo-stellate cells in the anterior lobe of the pituitary gland. However, recent studies have demonstrated the occurrence of nNOS in the somatotrophs and lactotrophs. In the present study, we investigated effects of growth hormone-releasing hormone (GHRH), gonadotropin-releasing hormone (GnRH), and 17β-estradiol on nitric oxide (NO) release in cultured rat anterior pituitary cells in vitro. The NO ₂ ⁻ level in the incubation medium of the rat anterior pituitary cells was dependent on the cell density. Pretreatment with 10 μM 17β-estradiol resulted in an increase in medium NO ₂ ⁻ level. GHRH and GnRH failed to change medium NO ₂ ⁻ levels, but they elicited increases in medium NO ₂ ⁻ levels in estrogen-treated cells. The GHRH-induced increase in NO ₂ ⁻ level was inhibited by Nχ-nitro-l-arginine methyl ester, a NOS inhibitor. These findings suggest that GnRH and GHRH could activate nNOS in the gonadotrophs and the somatotrophs, respectively.     

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.     

Regulation of the synthetic rate of gonadotropin-releasing hormone receptors in rat pituitary cell cultures by inhibin

Regulation of steady state levels of plasma membrane receptors for GnRH is the arithmetic result of processes that contribute to the appearance of receptors (synthesis, recycling, and unmasking) less those that contribute to the loss of receptors (degradation, internalization, and inactivation). We have adapted the density shift technique to evaluate specifically the rate of synthesis of GnRH receptors in rat pituitary cell cultures. Recently, it has been shown that inhibin can decrease the steady state levels of GnRH receptors in rat pituitary cell cultures and can block homologous up-regulation of GnRH receptors. In the present study we have evaluated the ability of purified inhibin to affect the synthesis rate of GnRH receptors under basal conditions and after exposure of cultured gonadotropes (from female weanling rats) to GnRH. Cells were exposed to inhibin alone (4 or 12 ng/ml) or to GnRH (10(-10) M) plus inhibin (0.4, 4, or 12 ng/ml) in the presence of densely labeled amino acids. GnRH was administered as a 20-min pulse, but inhibin treatment was continued for up to 2 days. After these treatments, GnRH receptors were covalently linked to a radio-labeled photoaffinity probe (125I- Tyr5-[azido-benzoyl-D-Lys6] GnRH) and solubilized with 1% sodium dodecyl sulfate. Newly synthesized GnRH receptors (those that had incorporated the dense amino acids) were separated from previously synthesized receptors (those containing normal amino acids) by velocity sedimentation through sucrose gradients (O-20% sucrose, 1% sodium dodecyl sulfate, and 10 mM Tris-HCl, pH 7.0; centrifuged at 156,000 x g for 24 h). After velocity sedimentation, gradients were fractionated, and the radioactivity in each fraction was quantified. Treatment with inhibin alone had no effect on the synthesis rate of GnRH receptors compared to that of control cultures (t1/2, 23.5 +/- 0.3 vs. 23.3 +/- 0.3 vs. 22.9 +/- 0.9 h for control, 4 ng/ml inhibin, and 12 ng/ml inhibin, respectively). In contrast, inhibin blocked the stimulation of homologous receptor synthesis by GnRH in a dose-dependent manner (t1/2, 12.2 +/- 0.7 vs. 14.0 +/- 0.7 vs. 19.2 +/- 1.5 vs. 20.0 +/- 2.9 h for GnRH alone and GnRH plus 0.4, 4, or 12 ng/ml inhibin, respectively). These data indicate that in rat pituitary cell cultures, inhibin does not decrease basal levels of GnRH receptors by affecting the synthesis rate of receptors, but prevents up-regulation of GnRH receptors by blocking stimulation of GnRH receptor synthesis by homologous hormone.     

Characterization of gonadotropin-releasing hormone receptors in cultured rat pituitary cells

The properties of gonadotropin-releasing hormone (GnRH) receptors were analyzed in isolated pituitary cells prepared by enzymatic dispersion with trypsin or collagenase-hyaluronidase. The initial impairment of LH responses to GnRH in isolated cells prepared by both methods was reversed during culture for 2 days in multiwell vessels. However, specific binding sites for GnRH, assayed by equilibration with [125I]iodi0[D-Ser(t-BU)6]des-Gly10-GnRH N-ethylamide (GnRH-A) were demonstrable in collagenase-dispersed cells, both initially and after 2 days in culture. Cellular uptake of GnRH-A was temperature dependent, with rapid and saturable binding to a limited number of specific receptor sites with high affinity for the labeled analog (Ka = 4.0 +/- 0.8 X 10(9) M-1). These sites showed common binding specificity for GnRH-A and the native GnRH peptide, with significantly lower affinity for the natural peptide (Ka = 2.3 X 10(7) M-1). Other protein and peptide hormones, including ovine LH, ovine PRL, hCG, TRH, somatostatin, and angiotensin II (up to 10(-6) M) did not inhibit binding of GnRH-A to its receptors. Cellular binding of GnRH-A was followed by increased cGMP production and LH release within 10 min. The analog was 50 times more potent than native GnRH in stimulating LH release. The Kact values derived for GnRH and GnRH-A were 0.5 and 0.01 nM, respectively, considerably lower than the Kd values of 50 and 0.25 nM derived from receptor binding analysis. These results indicate that GnRH receptors can be identified in isolated pituitary cells, in which peptide binding is followed by increased cGMP production and LH release. The impaired LH responses in acutely dispersed pituitary cells are not due to the loss of receptor sites but to a reversible postreceptor defect. Occupancy of about 20% of the GnRH-binding sites elicits a near-maximal LH response, indicating the nonlinearity of GnRH-receptor coupling to secretory responses in the cultured gonadotroph.     

Biosynthesis of gonadotropins by rat pituitary cells in culture and in pituitary homogenates: effect of gonadotropin-releasing hormone.

The incorporation of labeled amino acids and glucosamine into LH and FSH by cultured rat anterior pituitary cells and anterior pituitary homogenates is reported. There was a significant augmentation in this incorporation by cells after 6 days of culture in the presence of GnRH. Tritiated LH and FSH were found in the cell extracts as well as in the media by the method of immunoprecipitation. An increase of approximately 7--13-fold in the release of LH and FSH into the cell incubation medium was observed in the presence of GnRH (3 ng/ml). The rate of incorporation of [3H]proline was higher than that of [3H]glucosamine into LH and FSH. At the same time a higher incorporation of labeled amino acids was observed in the case of FSH than with LH. Cycloheximide inhibited completely the incorporation of labeled proline but the inhibition was partial for the incorporation of labeled glucosamine. Freshly dispersed cells, short-time cultures maintained for 20 h and pituitary homogenates also incorporated labeled amino acids into LH and FSH, but GnRH had no effect on this incorporation. Pituitary homogenates also incorporated [3H]glucosamine into LH and FSH with an optimal incorporation after 30 min of incubation. Three different concentrations of GnRH had no effect on the incorporation of [3H]proline by homogenates. Cycloheximide and puromycin inhibited this incorporation completely.     

Inhibitory action of ethanol on luteinizing hormone secretion by rat anterior pituitary cells in culture

Ethanol (EtOH) inhibits LH secretion in humans and animals. In these studies we examined the gonadotroph as a possible site of action of EtOH by treating cultured pituitary cells with several concentrations of EtOH (200, 400, or 800 mg/100 ml) for 4 days. Cells were incubated for 2 h with LHRH on the last day of the experiment. Whereas basal LH release was unaffected over the 4 days of EtOH treatment, the LH response to LHRH was inhibited by EtOH in a dose-related fashion. Inhibitory responses at the lowest concentration of EtOH (200 mg/100 ml) occurred inconsistently. At the highest concentration (800 mg/100 ml), the curve of log LHRH dose vs. LH release had a lower maximum and was shifted to the right of that for untreated cells. Total LH content and total number of cells attached to the culture wells were unchanged after EtOH treatment. These data suggest that EtOH at high concentrations can lower the responsiveness of the gonadotroph to LHRH.     

The effects of inhibin purified from bovine follicular fluid in several in vitro pituitary cell culture systems

The effects of inhibin purified from bovine follicular fluid (bFF) and a charcoal-treated bFF preparation were investigated in several inhibin in vitro systems based on the use of pituitary cells in culture. FSH, LH, TSH and PRL were determined in the medium and cell extracts before and after a 4 h LHRH stimulation test. Both pure inhibin and bFF preparations markedly inhibited the basal release, cell content and LHRH-stimulated release of FSH in a parallel dose-dependent manner with minor or negligible effects on LH, TSH and PRL. Using parallel line bioassay statistics the inhibin activities of the purified inhibin preparations in the various in vitro systems were calculated with the charcoal-treated bFF as standard. Significantly higher inhibin values were obtained using the basal release procedure than with the cell content or LHRH-stimulated release procedures. This difference was influenced by the length of time the inhibin preparations were in culture. The highly purified preparations showed no signs of cytotoxicity in culture as assessed by a 51Cr release test. It is concluded that purified bFF inhibin is specific in suppressing pituitary FSH and not LH, TSH or PRL. The observation that purified bFF inhibin is more active in the basal release procedure is attributed to a loss of activity of the bFF standard in this system in contrast to that observed in either the cell content or LHRH-stimulated release procedure.     

Effect of aging on GHRF-induced growth hormone release from anterior pituitary cells in primary culture

Five criteria were developed to validate the primary cell culture model for comparison of GRF-induced release of growth hormone in pituitary tissue from aging animals. Pituitaries from young (5-mo), middle-aged (14-mo), and old (24-mo) male Fischer 344 rats were dispersed using either trypsin/trypsin inhibitor or dispase and compared with respect to the number of pituitary cells recovered, cell viability, 3H-leucine incorporation into total protein, time course for recovery of optimal response to GRF, and the dose-relationship for GRF-induced release of growth hormone 2, 4, and 6 days after dispersal. Results indicated that direct comparison of cellular responses between tissues from young, middle-aged, and old rats in primary cell culture is confounded by variations in time for recovery of optimal responses, the effects of the enzymes used for dispersal, and the methods used to express the data.     

Effects of bovine 35 kDa FSH-suppressing protein on FSH and LH in rat pituitary cells in vitro: comparison with bovine 31 kDa inhibin

The mode of action of a recently isolated gonadal protein, termed FSH-suppressing protein (FSP) or follistatin, on basal and gonadotrophin-releasing hormone (GnRH)-stimulated release of FSH and LH and on pituitary cell content of FSH and LH was examined in rat pituitary cell cultures and compared with the previously reported effects of inhibin. Pituitary cells were cultured for 3-9 days in the presence of graded doses of FSP and the basal release rates and changes in cell contents of FSH and LH determined during this period. FSP suppressed both the basal release rate and the cell content of FSH with median inhibitory concentrations (IC50) of 135 and 161 pmol/l respectively. The corresponding effects of FSP on LH basal release rate and LH cell content (IC50 = 200 pmol/l) were limited compared with the effects on FSH. The effect of FSP on GnRH-stimulated release of FSH and LH during 4 h was determined in cells which had been preincubated with FSP for 3 days, and the GnRH-stimulated release of FSH and LH analysed as a percentage of the respective gonadotrophin available for release. FSP antagonized GnRH action with dose-related increases in the GnRH median effective (stimulatory) concentrations for FSH and LH release (EC50 values = 56 and 400 pmol/l respectively) and a suppression in the maximum release of FSH and LH by excess GnRH (IC50 values = 142 and 150 pmol/l respectively). The effect of FSP on FSH cell content after 3 days in culture was insensitive to the neutralizing effects of an inhibin antiserum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Androgen metabolism in rat anterior pituitary cells in culture

The metabolism in vitro of 4 androgens, namely testosterone, androstenedione, 5α-dihydrotestosterone and 3α-androstanediol has been studied in male and female rat anterior pituitary cells in primary culture. When testosterone was used as precursor, androstenedione, 5a-dihydrotestosterone and 3α-androstanediol were the main metabolites whereas androstenedione was mainly converted into testosterone, 5α-androstanedione, 5α-dihydrotestosterone and androsterone. Studies on the metabolism of 5α-dihydrotestosterone and 3α-androstanediol showed that these compounds were easily interconverted and were also significantly metabolized to 5α-androstanedione and androsterone. No aromatized compounds could be detected suggesting that androgen action in the pituitary cell occurs directly via the androgen receptor rather than through prior conversion into estrogens.     

Stimulation of growth hormone release and synthesis by estrogens in rat anterior pituitary cells in culture

We have investigated the potential effect of estrogens in the control of GH secretion in rat anterior pituitary cells in primary culture. We have found that a 72-h preincubation with 17 beta-estradiol (E2) caused an approximately 2- to 3-fold stimulation of basal and GH-releasing factor (GRF)-induced GH release as well as cellular GH content at EC50 values of 44, 35, and 15 pM, respectively. Estrone and estriol also increased GH release at respective EC50 values of 100 and 250 pM. The stimulatory effects of these steroids on GH release and cellular GH content were competitively blocked by simultaneous incubation with the antiestrogen LY156758. In contrast to thyroid and glucocorticoid hormones, a 72-h pretreatment with E2 failed to potentiate GRF-induced cAMP accumulation or enhance the sensitivity of the GH response to GRF. However, E2 increased the stimulatory effect of submaximal concentrations of dexamethasone on spontaneous and GRF-induced GH release as well as on total GH, but did not further increase the effect of maximal dexamethasone concentrations. As determined by a 60-min pulse labeling with [35S]methionine performed after a 72-h preincubation with E2, GH and PRL synthesis were increased by about 50% above control values (P less than 0.005). The present data clearly indicate for the first time that E2, at physiological concentrations, exerts a stimulatory effect on spontaneous and GRF-induced GH release as well as on cellular GH content, probably resulting, at least in part, from stimulation of GH synthesis.     

Effects of glucocorticoids on responsiveness of luteinizing hormone and follicle-stimulating hormone to gonadotropin-releasing hormone by male rat pituitary cells in vitro

To determine if the inhibitory effects of glucocorticoids on GnRH-stimulated secretion of LH observed in male rats in vivo are exerted directly on the pituitary, dispersed pituitary cells from adult male rats were treated with 60 or 600 ng/ml cortisol (F) or corticosterone (B) during one or two 48-h incubations. Control cells received no glucocorticoids. During the second 48 h, some cells from each group were treated with GnRH (2.4 X 10(-11)-6.2 X 10(-8) M). Concentrations of LH and FSH in media and cells were measured by RIA. Treatment with steroids had no effect on basal secretion or maximal GnRH-stimulated secretion of LH, or on maximal secretion of FSH. Treatment with 600 ng/ml B for 96 h increased basal secretion of FSH relative to controls. All treatments with glucocorticoids increased the slopes of the GnRH dose-response curves for both LH and FSH, cell content of LH, total (cells + medium) LH, and total FSH. Incubation with 6 micrograms/ml F or B or 60 ng/ml dexamethasone gave similar results. Decreasing the time period of the second incubation to 6 h results in no significant differences between control cells and cells treated with B or F. These results show that glucocorticoids have different effects in vivo and in vitro, suggesting that inhibitory effects of glucocorticoids on secretion of LH in vivo may not be exerted directly on the pituitary but are exerted elsewhere, perhaps by altered hypothalamic secretion of GnRH. Also, these results show that male and female pituitaries in vitro respond differently to glucocorticoids.     

Interaction between purified ovine inhibin and steroids on the release of gonadotropins from cultured rat pituitary cells

Primary pituitary cell cultures derived from adult male rats were used to explore the interaction between purified 32K ovine inhibin and gonadal steroids on the modulation of basal and GnRH-stimulated secretion of FSH and LH. Purified o-inhibin was a potent inhibitor of basal FSH secretion (IC50 = 3.4 pM) and 10 nM GnRH-stimulated FSH and LH secretion (IC50 = 8.8 and 18.4 pM, respectively). Purified o-inhibin had no effect on basal LH secretion. In this system, the androgens testosterone, 5 alpha-dihydrotestosterone, and androstenedione slightly stimulated basal FSH secretion, and o-inhibin could reverse this stimulatory effect. By contrast, androgens suppress GnRH-stimulated FSH and LH secretion. In combination, androgens and o-inhibin provided greater inhibition of GnRH-mediated gonadotropin secretion than either did alone.     

Solubilization of the gonadotropin-releasing hormone receptor from bovine pituitary plasma membranes

The plasma membrane receptor for GnRH in bovine anterior pituitaries has been solubilized with the cholic acid derivative detergent 3([3-cholamidopropyl)dimethylammonio]propane sulfonate. The soluble receptor in the supernatant from a 100,000 x g x 90 min centrifugation displays high affinity, saturability and specific binding to the agonist, [DA1a6,N alpha MeLeu7, Pro9-NEt]-GnRH. The dissociation constant, KD, for the soluble receptor is 0.56 +/- 0.07 nM (mean +/- SEM) and the number of sites, Ro, is 85 +/- 17 fmols/mg protein. For the membrane-bound receptor the KD is 0.50 +/- 0.04 nM and Ro is 300 +/- 18 fmols/mg protein. The relative potencies of GnRH and the potent antagonist, [Ac-delta 3Pro1,pFDPhe2, DTrp3,6]-GnRH are similar for both the soluble and membrane-bound receptor.