Basal and gonadotrophin-releasing hormone-induced biosynthesis and release of luteinizing hormone: effect of calcium deprivation.

The present study examines the basal and gonadotrophin-releasing hormone (GnRH)-stimulated biosynthesis and release of luteinizing hormone (LH) by pituitary cells in primary culture, and the effect of extracellular calcium deprivation on these events. Pituitaries from ovariectomized adult rats were enzymatically dispersed and cultured for 96 h. The cells were then incubated for 5 h (Expts. 1 and 3) or for different time intervals between 0 and 5 h (Expt. 2), in medium containing [14C]leucine ([14C]leu) and [3H]glucosamine ([3H]gln), with or without GnRH. Total immunoreactive LH (iLH) was measured in the medium and the cell extract by radioimmunoassay. LH translation (as estimated by [14C]leu incorporation into LH; [14C]LH) and LH glycosylation (as estimated by [3H]gln incorporation into LH; [3H]LH) were measured by immunoprecipitation with specific LH beta antiserum in both medium and cell extract. Treating the cells with GnRH caused both time- and dose-dependent increases of iLH in the medium as well as in total (cells plus medium) content, with an approximate ED50 of 0.7 nM. GnRH also stimulated LH biosynthesis by increasing both LH polypeptide chain synthesis and LH glycosylation. The effect of GnRH on LH glycosylation was detected earlier than that on translation, the [3H]LH rates of production and release being higher than those of [14C]LH. These findings suggest that GnRH-induced translation and glycosylation of LH are independently regulated. Removal of extracellular calcium resulted in the loss of cellular responsiveness to GnRH, preventing not only the stimulatory effects of GnRH on total and released iLH but also the GnRH-induced incorporation of both [14C]leu and [3H]gln into newly synthesized LH. These observations suggest that GnRH-stimulated LH glycosylation and LH translation involve calcium-dependent mechanisms. Neither the uptake of radiolabeled precursors nor their incorporation into total protein were affected by GnRH or Ca(2+)-deficient (no added calcium) medium. The results also suggest that the release of newly synthesized LH is regulated differently from previously synthesized stored hormone.     

Regulation of luteinizing hormone subunit biosynthesis in cultured male anterior pituitary cells: effects of gonadotropin-releasing hormone and testosterone

The purpose of this study was to evaluate the direct effects of testosterone (T) on LH subunit apoprotein synthesis, glycosylation, and release by the male pituitary. Cells from 1-week castrate rats were cultured for 48 h in steroid-free medium, followed by 48 h in medium with or without 10 nM T. The cells were then incubated for 2, 4, 6, 8, or 12 h in medium containing [35S]methionine (35S-Met) or [3H]glucosamine (3H-Gln), with or without 1 nM GnRH (Exp 1) or in medium containing precursors with or without 10 nM T and/or 1 nM GnRH (Exp 2). Radiolabeled precursor incorporation into LH subunits was determined by immunoprecipitation, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In Exp 1, precursor incorporation into total protein (TP) and LH subunits increased linearly over time for at least 8 h. GnRH did not affect precursor incorporation into total protein or 35S-Met labeling of LH subunits, but stimulated a linear time-dependent accumulation of 3H-Gln into total (cells plus media) LH subunits and release of radioimmunoassayable LH into the medium. Based on these results, the effects of T on LH subunit biosynthesis (with or without GnRH) were studied during an 8-h incubation. In Exp 2, GnRH enhanced total 3H-Gln (but not 35S-Met) incorporation into both LH subunits. GnRH stimulated the release of 35S-Met LH alpha and 3H-Gln LH subunits and increased the relative glycosylation of secreted LH subunits without altering the relative glycosylation of intracellular LH subunits. T inhibited radioimmunoassayable LH release and incorporation of both precursors into total and secreted LH subunits (with or without GnRH). However, only the relative glycosylation of secreted LH alpha was reduced by T (with or without GnRH). These data indicate that T acts directly at the pituitary to inhibit LH subunit apoprotein synthesis and selectively inhibit LH alpha glycosylation. Further, these data support the hypothesis that changes in LH glycosylation may be one of the ways by which GnRH and T regulate LH release.     

Luteinizing hormone synthesis in cultured fetal human pituitary cells exposed to gonadotropin-releasing hormone.

To investigate whether GnRH regulates LH synthesis during human development, pituitary cells from second trimester fetuses were incubated with [35S]methionine ([35S]met) and [3H]glucosamine ([3H]gln) for 48 h with 0, 10(-9), and 10(-7) mol/L GnRH. Immunoassayable (i) LH was measured in media and cellular lysates, and dual label scintillation analysis was used to quantitate incorporation of radiolabeled precursors into cells, trichloroacetic acid-precipitable proteins, and immunoprecipitated LH subjected to electrophoresis. Exposure of cells to GnRH did not affect cellular uptake or incorporation of precursors into proteins, but specifically increased total (secreted plus cellular) LH synthesis. Both GnRH concentrations significantly increased iLH release and enhanced secreted and cellular [3H]gln-LH. The secretion of [35S] met-LH was stimulated only by 10(-7) mol/L GnRH. The proportion of newly synthesized LH that was secreted and the 3H/35S ratio of secreted and cellular LH were uninfluenced by GnRH. Although basal LH synthesis was not sex dependent, total iLH content and GnRH-stimulated LH translation were greater in cells from females than in those from males. Therefore, GnRH regulates LH synthesis by second trimester fetal human gonadotrophs without influencing the proportion of total radiolabeled LH that is secreted. The existence of a sex difference in total iLH content and GnRH-stimulated LH translation is consistent with the sexual dimorphism in pituitary LH content occurring during human development.     

Testosterone suppresses 8-bromo-adenosine 3',5'-monophosphate and gonadotropin-releasing hormone-stimulated luteinizing hormone subunit synthesis.

The major objective of this study was to determine the effects of testosterone (T) on 8-bromo-cAMP (8-br-cAMP)- and GnRH-stimulated LH subunit polypeptide synthesis and glycosylation in cultured male anterior pituitary cells. The anterior pituitaries from 1-week castrate male rats were enzymatically dispersed and incubated for 48 h in steroid-free medium, followed by a 48-h incubation with or without 10 nM T. The cells were then incubated for 12 h in medium containing [35S]methionine ([35S]Met) and [3H]glucosamine ([3H]Gln) with or without 1 mM 8-br-cAMP or 1 nM GnRH, with or without 10 nM T. Incorporation of radiolabeled precursors into LH subunits was determined by specific immunoprecipitation of the LH dimer with subsequent analysis of the individual LH alpha- and beta-subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. LH content was quantified by RIA (iLH). Both 8-br-cAMP and GnRH stimulated iLH release. T suppressed basal and GnRH-induced iLH secretion, whereas it enhanced iLH release stimulated by 8-br-cAMP. Both 8-br-cAMP and GnRH stimulated total (cell plus medium) [35S]Met and [3H]Gln incorporation into LH alpha and LH beta, and these responses were suppressed by T. Basal [35S]Met incorporation into the LH subunits was inhibited by T, whereas T had no effect on basal levels of [3H]Gln incorporation. Neither T nor GnRH altered [3H]Gln cell uptake or incorporation into total proteins, whereas 8-br-cAMP increased these responses. There were no treatment effects on [35S]Met cell uptake or incorporation into total proteins. These results suggest that 8-br-cAMP, similar to GnRH, stimulates both polypeptide synthesis and glycosylation of the LH alpha- and beta-subunits and that T suppresses these responses to 8-br-cAMP and GnRH in a similar fashion. These data indicate that cAMP is involved in mediating the actions of GnRH on LH subunit biosynthesis and that the inhibition of LH subunit polypeptide synthesis and glycosylation by T involves postreceptor events that are regulated by cAMP-dependent mechanisms.     

Synthesis and release of luteinizing hormone by rat anterior pituitary cells: effects of cytochalasins B and D

We determined the role of microfilaments in regulating LH synthesis (translation or glycosylation) and release from cultured rat anterior pituitary cells under basal and GnRH-stimulated conditions. Cells were pretreated for 2 h with microfilament-disrupting drugs, cytochalasin B (CB; 2 and 20 microM) or cytochalasin D (CD; 1 and 10 microM). LH synthesis and release were measured after 4 h of incubation with or without 1 nM GnRH and drugs. LH translation and glycosylation were monitored by measuring the incorporation of [14C]alanine and [3H]glucosamine, respectively, into total (cell and medium) immunoprecipitable LH. Immunoreactive LH (IRLH) in medium and cells was measured by RIA. GnRH at 1 nM significantly (P less than 0.01) increased the release of IRLH and total [3H]LH (glycosylation), but had no effect on total [14C]LH (translation), uptake, or incorporation of precursors into total protein. Neither CB (2 and 20 microM) nor CD (10 microM) altered basal or GnRH-stimulated IRHL release. Neither drug altered basal medium concentrations of [3H]LH or [14C]LH. In contrast, both CB and CD reduced (P less than 0.01) GnRH-stimulated [3H]LH in the medium and total system (LH glycosylation). CB reduced (P less than 0.01) [3H]glucosamine uptake, total [3H]protein synthesis, and basal level of total [3H]LH, while CD had no effects on these parameters. Thus, CD exerted a more specific inhibitory effect on GnRH-stimulated LH glycosylation than CB. CB (2 and 20 microM) increased (P less than 0.01), while CD (10 microM) decreased (P less than 0.01) [14C]alanine uptake, total [14C]LH, and [14C]protein under both basal and GnRH-stimulated conditions. These results demonstrated that while the cytochalasins did not inhibit either basal or GnRH-stimulated IRLH release, they did inhibit GnRH-stimulated LH glycosylation, although the effect of CB was due partially to reduced [3H]glucosamine uptake. Integrity of microfilaments appears to be important for GnRH-enhanced LH glycosylation, but not for GnRH-enhanced LH release.     

Development of gonadotrope desensitization to gonadotropin-releasing hormone (GnRH) and recovery are not coupled to inositol phosphate production or GnRH receptor number.

After initial GnRH pretreatment (10 nM, 5 h), subsequent GnRH-stimulated LH release from the gonadotrope was diminished (1 microM GnRH stimulated release of 36.4 +/- 1.4% total cellular LH over 3 h in cells initially pretreated with medium alone compared to 27.4 +/- 1.2% in GnRH-pretreated cells); however, inositol phosphate (IP) production in response to the releasing hormone remained unaffected (1 microM GnRH provoked IP accumulation of 161 +/- 9% above basal levels after 45 min in control cells and 162 +/- 11% in GnRH-pretreated cells). Pretreatment of pituitary cell cultures with NaF (a guanyl nucleotide binding protein activator, 10 mM, 3 h) also decreased subsequent GnRH-stimulated LH release, and in addition, provoked a decrease in GnRH receptor number, an increase in GnRH receptor affinity, reduction of GnRH-stimulated IP production to basal levels, and an increase in the amount of LH released in response to stimulation with the calcium ionophore A23187. In order to determine if the changes in LH release were a result of decreased IP production and/or decreased GnRH receptor binding, the time course of recovery to control levels of these processes was assessed. GnRH receptor binding continued to decrease after NaF pretreatment, reaching a nadir (62% of control) at 6 h after the pretreatment period and recovering at 48 h (90% of control). In contrast, GnRH-provoked IP accumulation did not return to control levels even after 48 h of recovery after NaF pretreatment (1 microM GnRH-stimulated IP accumulation in NaF-pretreated cells was 57% compared to control cells after 48 h of recovery). GnRH-stimulated LH release was inhibited immediately after NaF pretreatment (1 microM GnRH-stimulated LH release in NaF-pretreated cells was 65% of control levels). Cells began to recover within 3 h (80% of control) and were almost completely recovered by 6 h (90% of control). A23187-provoked LH release was enhanced immediately after NaF pretreatment (30 microM A23187-stimulated LH release in NaF-pretreated cells was 170% of control levels). Responsiveness to ionophore was 133% of control by 0.5 h, and complete recovery was measured within 1 h (100% of control). Furthermore, both NaF and GnRH pretreatment still provoked a decrease in gonadotrope responsiveness when IP production was inhibited by the phospholipase C inhibitor U-73122. The results suggest that the development of gonadotrope desensitization (by either NaF or GnRH pretreatment) can be uncoupled from changes in IP production.(ABSTRACT TRUNCATED AT 400 WORDS)     

GnRH action in rat anterior pituitary gland: regulation of protein, glycoprotein and LH synthesis.

The effect of synthetic GnRH on the synthesis of proteins and glycoproteins in the anterior pituitary and in vitro release of LH into the medium was studied. A maximal dose (25 ng/ml) of of synthetic GnRH caused optimum release of radioimmunoassayable LH into the medium after 2 h of incubation. A concomitant increase in cyclic AMP accumulation in the tissue and LH in the incubation medium was also observed under the influence of GnRH during different periods of incubation time. Incubation of the rat anterior pituitary with GnRH stimulated the incorporation of [3H] proline into acid precipitable proteins in a time- and dose-dependent manner, similar to radioimmunoassayable LH released into the medium. Similar results were obtained when pituitary was incubated with dibutyryl cyclic AMP. LH, in addition, enhanced the incorporation of [3H] glucosamine and [3H] amino acids mixture into acid-precipitable proteins suggesting that proteins including glycoproteins are synthesized by the rat anterior pituitary under the influence of GnRH. Approximately 10% of the radioactivity associated with proteins comigrated with radioimmunoassayable LH on the gels. GnRH also enhanced the incorporation of [3H] glucosamine and [3H] amino acid mixture into immunoprecipitable LH. The GnRH-induced incorporation of [3H] proline into anterior pituitary proteins was abolished by specific translation inhibitors.     

Dependence of gonadotropin-releasing hormone-stimulated luteinizing hormone release upon intracellular Ca2+ pools is revealed by desensitization and thapsigargin blockade.

Sustained GnRH-stimulated LH release requires extracellular Ca2+, but GnRH transiently increases LH release in Ca(2+)-free medium. Here we have tested the dependence of the transient effect on intracellular Ca2+ pools. In superfused pituitary cells three Ca(2+)-mobilizing stimuli (GnRH, A23187, and endothelin-1) all caused sustained increases in LH release in normal medium (plateau responses), but only transient increases in Ca(2+)-free medium (spike responses). In Ca(2+)-free medium, GnRH (10(-10) or 10(-9) M) increased LH release transiently and desensitized the cells to the LH-releasing effect of subsequent stimulation with 10(-7) M GnRH. This desensitization was reversed by brief exposure to Ca(2+)-containing medium between the two GnRH stimulation periods. Heterologous desensitization between GnRH and A23187 and between GnRH and endothelin-1 also occurred in Ca(2+)-free medium. Thapsigargin, which inhibits the endoplasmic reticulum Ca(2+)-ATPase and thereby elevates cytosolic Ca2+, stimulated LH release (EC50, approximately 20 microM) in static culture, an effect which, unlike those of GnRH and A23187, was not markedly reduced in Ca(2+)-free medium. Low doses of thapsigargin, which had no effect on LH release alone, inhibited both sustained GnRH-stimulated LH release from static cultures in normal medium and transient GnRH-stimulated LH release from cells superfused in Ca(2+)-free medium. These data suggest that the spike phase of GnRH-stimulated LH release is not only associated with but is also dependent upon the mobilization of a GnRH- and thapsigargin-sensitive intracellular Ca2+ pool and that the Ca2+ pool mediating this GnRH effect is identical to or substantially interchangeable with A23187- and endothelin-1-mobilizable intracellular Ca2+ pools. Inhibition of sustained GnRH-stimulated LH release by thapsigargin also suggests the involvement of an intracellular Ca2+ pool in this phase of GnRH action.     

The relationship between gonadotropin-releasing hormone-stimulated luteinizing hormone release and inositol phosphate production: studies with calcium antagonists and protein kinase C activators

The coupling between GnRH-stimulated phosphoinositide (PI) turnover and LH release has been investigated in rat pituitary cell cultures. Accumulation of [3H]inositol phosphates ([3H]IPs) formed by hydrolysis of PIs was measured in cells that had been preloaded with [3H]myo-inositol. GnRH stimulated both LH release and incorporation of [3H]inositol into total [3H]IPs with similar dose and time dependencies. [3H] IP production in response to GnRH could be blocked by a GnRH antagonist, but was stimulated by a compound that provokes receptor microaggregation. GnRH-stimulated IP production persisted in the presence of either the Ca2+ channel blocker D600 or the calmodulin antagonist pimozide at concentrations that reduced LH release to 60% and 20% of control, respectively. Stimulated [3H]IP production was inhibited at higher concentrations of D600. In 1-h incubations, GnRH-stimulated [3H]IP production, but not LH release, was markedly inhibited by the protein kinase C activators phorbol myristate acetate and 1,2-dioctanoylglycerol. These findings indicate that in the gonadotrope, GnRH-stimulated LH release and [3H]IP production are closely coupled to receptor activation by an agonist; Ca2+ antagonists uncouple stimulated LH release from [3H]IP production; and protein kinase C activators uncouple stimulated PI turnover from LH release. Thus, GnRH-stimulated production of PI metabolites, as measured by [3H]IP accumulation, is apparently not sufficient to support LH release in the absence of Ca2+. In addition, GnRH-stimulated LH release is apparently not dependent on full expression of the PI response.     

Ca+2 dependence of gonadotropin-releasing hormone-stimulated luteinizing hormone secretion: in vitro studies using continuously perifused dispersed rat anterior pituitary cells

A continuously perifused dispersed rat anterior pituitary cell system was used to determine the importance of calcium (Ca+2) on the release of LH by GnRH. In response to continuous exposure to 10 nM GnRH, LH was released in a biphasic fashion; arbitrarily, phase I was defined as being the LH released during the initial 40 min and phase II as the subsequent release. Withdrawal of Ca+2 from the perifusion medium during phases I or II of LH release led to a rapid inhibition of the LH secretion. Cells were exposed to GnRH for 2.5 min, washed with medium for 30 min, and then reexposed to GnRH for 30 min. This sequence was repeated 1 h later under identical conditions in the presence of a Ca+2 blocking agent; D600 (20 or 100 microM). D600 inhibited both the 2.5- and the 30-min GnRH-stimulated LH release. The results were expressed as the ratio obtained by dividing the total LH released during the second GnRH exposure (either 2.5 or 30 min) by the total LH released during the respective initial GnRH exposure of same duration. For the cells perifused with 20 microM D600 the ratios +/- SE (D600 vs. control) were 0.48 +/- 0.06 vs. 1.28 +/- 0.13 (P = 0.0001) and 0.29 +/- 0.05 vs. 1.01 +/- 0.08 (P less than or equal to 0.0001) for the 2.5- and 30-min exposures, respectively. For the cells perifused with 100 microM D600 the ratios +/- SE (D600 vs. control) were 0.18 +/- 0.05 vs. 1.28 +/- 0.13 (P less than or equal to 0.00001) and 0.12 +/- 0.03 vs. 1.01 +/- 0.08 (P = 0.002) for the 2.5- and 30-min exposures, respectively, revealing an even more profound inhibitory effect of D600 on GnRH stimulated LH secretion. Our data both confirm previous reports that Ca+2 is involved in LH release and demonstrate that Ca+2 is an essential requirement during both phases of GnRH-stimulated LH release in perifused dispersed rat anterior pituitary cells.     

Modifications of luteinizing hormone biosynthesis and release by gonadotropin-releasing hormone, cycloheximide, and actinomycin D.

In the first experiment, the influence of synthetic gonadotropin-releasing hormone (GnRH) on the time course of [3H]glucosamine ([3H]GA) and [14C]alanine ([14C]A) incorporation into LH by quartered rat anterior pituitary glands and the sequence of release of radiolabeled and total immunoreactive LH (IR-LH) was investigated. Radiolabeled LH was measured by immunoprecipitation and total IR-LH was measured by RIA. After lag periods of 5 and 20 min, respectively, incorporation of [14C]A and [3H]GA into tissue LH increased linearly for 4 h. GnRH stimulated incorporation of [3H]GA only. The ratio of [14C]A-LH to [3H]GA-LH (14C:3H ratio) in the tissue decreased significantly with time and with GnRH treatment. In the second experiment, replenishment of GnRH in the medium every 0.5 h elevated the release rate (release during each sequential 0.5 h) of both [3H]GA and [14C]A-labeled LH within 1.5 h. The release rate of radiolabeled LH increased linearly until 3.5 h. The 14C:3H ratio in LH released during each time interval was reduced by GnRH. The release rate of IR-LH increased linearly with time, plateaued by 1.5-3 h, and started to decline. In other experiments, cycloheximide blocked synthesis of [14C]A-LH and greatly reduced the GnRH-induced synthesis and release of [3H]GA-LH, but reduced release of IR-LH by only 25%. Actinomycin D had no effect on GnRH-induced synthesis and release of LH at 2 h, but significantly reduced both at 4 h. These data suggest that 1) the time course for the release of preexisting IR-LH differs from that for newly synthesized LH, 2) the newly synthesized LH released in response to high levels of GnRH has more sugar residues than that released under basal conditions, 3) the GnRH-induced LH release can occur under conditions in which LH synthesis has been blocked, and 4) synthesis of messenger RNA is not required for GnRH-induced LH release or short term LH synthesis but seems to be required for continued synthesis and subsequent release of LH.     

Maintenance of gonadotropin-releasing hormone (GnRH)-stimulated luteinizing hormone release despite desensitization of GnRH-stimulated cytosolic calcium responses.

Involvement of ionized cytosolic calcium ([Ca2+]i) and protein kinase-C (PKC) in GnRH-stimulated LH release was assessed by correlating measurable changes in [Ca2+]i and LH release in PKC-depleted and nondepleted gonadotropes. Primary cultures of anterior pituitary cells were loaded with the calcium-sensitive fluorescent dye fura-2 and placed in a perifusion chamber. GnRH pulses were delivered to the cells, and changes in fura-2 fluorescence and LH release were determined. The level of [Ca2+]i (assessed by fura-2) increased rapidly to a maximum within 20-40 sec, followed by a slower decline over the next minute (spike phase) to a sustained intermediate value (plateau phase). GnRH-stimulated LH release was unaffected by loading cells with fura-2. Both LH release and changes in [Ca2+]i were directly dependent on GnRH concentration. Pretreatment with the GnRH antagonist Antide (50 nM; [NAcD2Nal1-DpClPhe2-D3Pal3-Ser4-NicLys5-++ +DNicLys6-Leu7-ILys8-Pro9-DAla10]NH2 ) had no effect on basal [Ca2+]i or basal LH release, but did block both GnRH-stimulated calcium mobilization and GnRH-stimulated LH release. GnRH pretreatment (3.5 nM; 10 min) blocked the calcium spike phase, but not the plateau phase occurring in response to a GnRH pulse (10 nM; 5 min) delivered immediately after pretreatment. Inhibition of the calcium spike phase was transient (recovery within 15 min) and was dependent on pretreatment concentrations of GnRH. Calcium spike phase inhibition by GnRH pretreatment prevented increased LH release from PKC-depleted cells in response to a subsequent pulse of GnRH, but not from gonadotropes with normal levels of PKC. This suggests that initial LH release is dependent on changes in [Ca2+]i, but enhancement of LH release after periods of elevated GnRH concentrations may be dependent on PKC.     

Synthesis and release of luteinizing hormone in vitro by rat anterior pituitary cells: effects of gallopamil hydrochloride (D600) and pimozide

We compared the role of Ca2+ in regulating GnRH-induced LH synthesis and release from cultured rat pituitary cells. LH synthesis and release were measured after a 4-h treatment of cells with gallopamil hydrochloride (D600; 1 and 100 microM), a Ca2+ channel blocker, or pimozide (0.5 and 5.0 microM), a calmodulin inhibitor, with or without 1 nM GnRH. LH translation and glycosylation were monitored by measuring incorporation of [14C]alanine and [3H]glucosamine, respectively, into total (cell and medium) immunoprecipitable LH. GnRH significantly (P less than 0.01) increased total [3H]LH (glycosylation), but had no effect on total [14C]LH (translation). D600 significantly (P less than 0.01) depressed (1 microM) and completely blocked (100 microM) GnRH-induced LH glycosylation and release of [3H]LH, [14C]LH, and immunoreactive LH. D600 (100 microM) also reduced (P less than 0.05) total basal synthesis of [14C]LH. Neither dose of D600 altered uptake of [3H]glucosamine, but 100 microM D600 significantly (P less than 0.01) depressed its incorporation into total protein. D600 (100 microM) significantly (P less than 0.01) depressed [14C]alanine uptake and incorporation into total protein. Pimozide significantly (P less than 0.01) reduced, in a dose-related manner, GnRH-induced LH glycosylation, and release of immunoreactive LH, [3H]LH, and [14C]LH. Pimozide did not alter LH translation or uptake of radiolabeled precursors or their incorporation into total protein. These results demonstrate that D600 and pimozide inhibit both GnRH-induced LH glycosylation and release. Thus, the actions of GnRH on LH glycosylation and release are both mediated by similar Ca2+-dependent pathways.     

Tunicamycin and neuraminidase effects on luteinizing hormone (LH)-releasing hormone binding and LH release from rat pituitary cells in culture

We have studied the effects of tunicamycin (TM) and neuraminidase on the binding of 125I-labeled Buserelin, a GnRH agonist, and on GnRH-stimulated LH release in cultured rat pituitary cells. Treatment with TM, an antibiotic which inhibits protein glycosylation, abolished the development of elongated cell processes without any effect on cell viability. Concomitantly, TM caused a time- and dose-dependent inhibition of specific binding of Buserelin and of GnRH-stimulated LH release. The inhibition of binding was due to a decrease in the number of GnRH receptors without any significant effect on binding affinity. Protein synthesis was not affected under these experimental conditions, suggesting that the aglycosylated GnRH receptors are probably intracellularly accumulated and are not expressed on the cell surface. Treatment with neuraminidase inhibited only 50% of GnRH agonist binding and did not affect GnRH-stimulated LH release. These results indicate that the oligosaccharide portion is essential for the functional properties of the GnRH receptor.     

Differential actions of arachidonic acid and melittin on luteinizing hormone release and synthesis

We studied the actions of arachidonic acid (AA) on luteinizing hormone (LH) release versus synthesis (translation or glycosylation) by cultured rat anterior pituitary cells. Monolayer cells were incubated for 3-4 h with secretagogues (AA, melittin, gonadotropin-releasing hormone; GnRH) which either increase endogenous AA levels or release AA. LH translation and glycosylation were monitored by measuring the incorporation of [14C]alanine and [3H]glucosamine, respectively, into total (cell plus medium) immunoprecipitable LH. Immunoreactive (IR) LH was measured by radioimmunoassay. Nonlytic doses of AA increased (p less than 0.01) IR-LH release without increasing total [3H]glucosamine-LH, [14C]alanine-LH, or [3H]glucosamine-protein. AA either had no effect or slightly increased (p less than 0.05) [3H]glucosamine uptake, but decreased (p less than 0.01) [14C]alanine uptake and incorporation into total [14C]alanine protein. AA at 250 microM lysed cells, thus increasing medium IR-LH and decreasing (p less than 0.01) [3H]glucosamine and [14C]alanine uptake and incorporation into total LH and protein. Melittin, which releases AA by activating phospholipase A2 (245 and 490 nM), increased medium IR-LH (p less than 0.01) without affecting any other parameter measured. GnRH at 1 nM enhanced (p less than 0.01) both LH (IR-LH, [3H]glucosamine-LH and [14C]alanine-LH) release and total [3H]glucosamine-LH, but had no effect on total [14C]alanine-LH. In summary, AA and melittin at doses which stimulated LH release did not stimulate either LH glycosylation or translation. This suggests that increased LH release by nonspecific secretagogues is not necessarily accompanied by increased LH glycosylation or translation.     

Gonadotropin-releasing hormone modulation of protein kinase-C activity in perifused anterior pituitary cell cultures

The ability of GnRH to modulate protein kinase-C (PKC) activity was examined in perifused rat pituitary cell cultures. Under these conditions, LH release and GnRH receptor number remained unchanged after repeated pulses of 1 nM GnRM, whereas PKC (measured both enzymatically and by radioligand assay) showed an initial increase in kinase activity after the first pulse of GnRH (approximately 2-fold), followed by down-regulation of PKC activity with subsequent pulses of the releasing hormone. It was also observed that the GnRH-stimulated down-regulation of PKC was dependent on the presence of extracellular calcium, which was not the case for the initial up-regulation of PKC. These findings are consistent with a modulating role of the GnRH receptor on PKC activity through a Ca2(+)-dependent process. This study also provides further evidence that GnRH-stimulated LH release and PKC activity can be uncoupled.     

The role of calcium in LH release from the pituitary by phorbol ester

GnRH stimulates LH release from pituitary cells, and this process is calcium dependent. On the other hand, phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA), a potent activator of calcium- and phospholipid-dependent protein kinase (protein kinase C), stimulates luteinizing hormone (LH) release from rat pituitary cells. To investigate the involvement of the calcium dependent process in LH release by TPA, the effects of calcium channel antagonists, verapamil and nifedipine, on TPA-mediated LH release were compared with those of a GnRH superagonist, [D-Ala6] des-Gly10-GnRH N-ethylamide (GnRHa) in cultured pituitary cells. Furthermore, pituitary cells saturated with 45Ca2+ were stimulated by GnRHa or TPA and calcium mobilization after the stimuli were monitored. The pituitary cells from adult male rats were dispersed by trypsin and cultured for 3 days. Cultured pituitary cells were incubated with GnRHa or TPA in the presence of increasing concentrations of verapamil or nifedipine for 3hrs, and LH released into medium was measured by RIA for rat LH. For 45Ca2+ experiment, 3 day-cultured pituitary cells were saturated with 45Ca2+ (10(6) cells/1 microCi/100 microliters) and incubated with secretagogues for the indicated times. Incubations were terminated by filtration, and the radioactivity on the filter was measured by a beta-counter. LH release was stimulated by 0.1 nM TPA, and the maximum response at 10 nM TPA was 50% of the LH response to GnRHa. A23187 also stimulated LH release in relatively high concentrations (10(-5)-10(-4) M), and no additive stimulatory effect was observed when a half-maximal dose of TPA (10(-9) M) was added with increasing concentrations of A23187. Verapamil partially inhibited both GnRHa- and TPA-stimulated LH release, and a similar inhibitory effect on LH release was observed when nifedipine was incubated with GnRHa or TPA, although high concentrations (10(-5)-10(-4) M) of nifedipine stimulated LH release induced by GnRHa and TPA. GnRHa and TPA stimulated 45Ca2+ influx into the cells, and its peak was observed 15 and 30 seconds after stimulation, respectively, while GnRH antagonist did not mobilize 45Ca2+ until 120 seconds after stimulation. These results suggest that TPA-stimulated LH release from pituitary cells involves a calcium dependent process as does GnRH-stimulated LH release.     

Requirement of extracellular calcium in fish pituitary gonadotropin release by gonadotropin hormone-releasing hormone

Influence of extracellular calcium on gonadotropin hormone-releasing hormone (GnRH)-stimulated gonadotropin hormone (GtH) release from a teleostean fish (Channa punctatus) pituitary was examined in vitro by preparing enzymatically dispersed pituitary cell incubation. Effect of Ca2+ on GnRH-augmented GtH release was evaluated with partially purified C. punctatus GnRH (cGnRH) and synthetic mammalian GnRH (mGnRH). Cells were dispersed by 0.3% collagenase plus 0.05% trypsin in culture medium and a high yield of viable cells were obtained. Addition of cGnRH (10 micrograms/ml) to pituitary cells in Ca2+-free medium resulted in a significant increase in GtH release, but the addition of Ca2+ (2 mM) enhanced it to about four- and threefold over cGnRH and mGnRH, respectively. Increasing concentrations of Ca2+ (0.1-2.0 mM/well) with fixed concentrations of GnRH (10 micrograms/ml) or increasing doses of GnRH (2.5 to 20 micrograms/ml) with fixed amount of Ca2+ (2 mM/well) resulted in a dose dependent increase in GtH release. EDTA or EGTA (2 mM/well) completely suppressed the Ca2+-augmenting effect of GnRH-stimulated GtH release. Addition of lanthanum (La3+, 4 mM/well), a competitive inhibitor of Ca2+, reduced 60% of the Ca2+ (2 mM/well) stimulation. Verapamil, a specific Ca2+ channel blocker, when added in increasing concentrations (1-100 microM/well) to pituitary cell incubations containing GnRH-stimulated GtH release in Ca2+-free medium could be waived by EGTA (2 mM/well), indicating availability of extracellular calcium from tissue sources. The uptake of radioactive Ca2+ by pituitary cells was greatly enhanced by GnRH while the addition of verapamil (10 microM/well) not only inhibited the GnRH-stimulated uptake, but also reduced it below the control level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gonadotropin-releasing hormone stimulates luteinizing hormone secretion by extracellular calcium-dependent and -independent mechanisms

The dependence of LH responses to GnRH on extracellular calcium was investigated in cultured rat pituitary cells exposed to GnRH for 3 h in static culture or for 2 min during column perifusion. During static culture in normal medium, LH release was stimulated by GnRH with an ED50 of 0.3 nM and by K+ with an ED50 of 32 mM. Incubation in Ca2+-deficient (no added Ca2+) or Ca2+-free medium (containing 100 microM EGTA) substantially decreased, but did not abolish, the LH responses to 10 and 100 nM GnRH, whereas K+-induced LH release was almost completely abolished in Ca2+-deficient medium. The Ca2+ channel agonist (BK 8644) and antagonists (nifedipine, nicardipine, verapamil, and Co2+) respectively enhanced or reduced the LH responses to both GnRH and K+. However, the calcium antagonists completely abolished the LH response to depolarization by K+, but only partially inhibited the LH response to GnRH, confirming the existence of a significant component of GnRH action that is not dependent on extracellular Ca2+. In perifused pituitary cells, exposure to Ca2+-deficient medium or normal medium containing 5 mM EGTA or 5 mM EDTA, reduced the initial rapid LH response to 2-min pulses of 10 nM GnRH and abolished the second phase of LH release. Reintroduction of Ca2+-containing medium at the end of the GnRH pulse caused recovery of the second phase of LH secretion, demonstrating that influx of extracellular Ca2+ is not required for the early phase of the LH response to GnRH but, rather, appears to be essential for its prolongation. The release of LH in response to arachidonic acid, which has been implicated in the mechanism of the secretory action of GnRH, was completely independent of extracellular Ca2+ and unaffected by addition of 10 nM BK 8644. These observations indicate that the initiation of the secretory response to GnRH is largely independent of calcium entry, whereas the prolongation of gonadotropin secretion is maintained by calcium influx, in part through voltage-sensitive calcium channels. The role of arachidonic acid metabolites in GnRH action is probably related to the calcium-independent component of GnRH-induced LH secretion. Since GnRH is secreted episodically and for short periods, much of its physiological action on pulsatile gonadotropin release could be independent of calcium influx from the extracellular fluid.     

Increase in luteinizing hormone content occurs in cultured human fetal pituitary cells exposed to gonadotropin-releasing hormone

To investigate the mechanisms by which GnRH regulates LH production during intrauterine life, dispersed pituitary cells from second trimester human fetuses were cultured on extracellular matrix-coated plates for 48 h. Exposure of cells to 3 x 10(-10) mol/L GnRH for 1-48 h significantly increased cumulative LH secretion compared to that in respective controls (P less than 0.01). The rate of GnRH-stimulated LH release was accelerated during the first 6 h, after which it declined to a level similar to that of basal release. This phenomenon was associated with a decrease in the GnRH concentration of the medium. Exposure of cells to GnRH (3 x 10(-10) to 10(-6) mol/L) for 48 h induced a dose-dependent elevation of total LH which correlated with an increase in releasable, but not cellular, LH. Desensitization to GnRH (10(-7) mol/L) occurred when cells were cultured with pharmacological amounts of GnRH for 48 h. These results indicate that GnRH induces the increase in total and releasable LH in human fetal pituitary cells. These cells also appear to inactivate GnRH. Thus, GnRH may increase LH production in the human fetal pituitary and the pituitary receptor mechanism may be involved in GnRH action on LH release during intrauterine life.