Gonadotropin-releasing hormone-stimulated intracellular Ca2+ fluctuations and luteinizing hormone release can be uncoupled from inositol phosphate production.

In order to study the dependence of GnRH-stimulated LH release on inositol phosphate (IP) turnover, this study used an inhibitor of phospholipase C activity, 1-[6-[[17 beta-3- methoxyestra-1,3,5(10)-triene-17-yl]amino]hexyl]-1H-pyrrole-dione (U-73122) and an inactive analog 1-[6[[17 beta-3-methoxyestra-1,3,5(10)- triene-17-yl]amino]hexyl]2,5-pyrrolidine-dione (U-73343). U-73122 (10 microM) decreased GnRH-provoked (1 microM, 45 min) IP accumulation from 873 +/- 61 dpm to 365 +/- 50 dpm (basal accumulation also was decreased from 420 +/- 18 dpm to 207 +/- 16 dpm) while LH release was not inhibited (30.2 +/- 1.4% of cellular LH in control compared to 30.3 +/- 1.1% in U-73122 pretreated cells). GnRH provoked increased IP3 accumulation (123% of basal) after 15 sec of stimulation, IP2 accumulation (131% of basal) after 30 sec, and IP1 (121% of basal) after 1 min. Pretreatment with U-73122 blocked accumulation of IPs at these early timepoints. Sodium fluoride (NaF)-stimulated IP accumulation was also inhibited by U-73122 (from 1539 +/- 132 dpm to 414 +/- 21 dpm) while LH release increased from 22.9 +/- 1.4% total cellular LH to 28.0 +/- 2.2%. In contrast, GnRH- and NaF-stimulated IP accumulation were not significantly decreased in U-73343 pretreated cells (GnRH: 817 +/- 43 dpm compared to 873 +/- 61 dpm in control; NaF: 1133 +/- 74 dpm compared to 1539 +/- 132 dpm in control cells). Results of a perifusion study showed that U-73122 did not block the initial phase of GnRH-stimulated LH release or interfere with the development of desensitization to the releasing hormone. In addition, GnRH-stimulated intracellular Ca2+ fluctuations were similar in magnitude and duration in U-73122-pretreated compared to U-73343-pretreated cells. These results demonstrate that GnRH- as well as NaF-stimulated LH release can be uncoupled from IP production calling to question the role of IP3 as a second messenger for GnRH-stimulated LH release.     

Gonadotropin-releasing hormone stimulates mass changes in phosphoinositides and diacylglycerol accumulation in purified gonadotrope cell cultures

GnRH stimulates rapid and specific hydrolysis of the phosphoinositides and their conversion to diacylglycerols (DAGs) in enriched gonadotrope cultures. In short term labeling studies, we observed stimulation of 32P incorporation into inositol phospholipids; no corresponding change was seen in other major phospholipids. The observation that this response continues in the presence of the Ca2+ channel antagonist D600 suggests that, unlike LH release, stimulation of phosphatidylinositol (PI) production is independent of mobilization of extracellular Ca2+. Agents that substitute for endogenous DAGs (phorbol myristate acetate) or mobilize Ca2+ directly (ionophore A23187) did not stimulate 32P incorporation into PI. Relative mass changes in PIs in response to GnRH were measured in a 32P/33P protocol. These showed a fall (40%) in the level of the inositol phospholipids within 45 sec, with a new steady state achieved at a lower level by 5 min. Phosphatidic acid formation was stimulated by GnRH within 2 min, with a 150% increase above control values at the highest dose of GnRH tested (10(-7) M). In cells prelabeled with [3H]arachidonic acid, GnRH caused a transient increase in DAG formation (twice the control value) within 1 min (with or without D600) which declined thereafter. This effect of GnRH was not seen in cells stimulated to release LH with ionophore A23187 or phorbol myristate acetate, suggesting that these may act distally to inositol phospholipid turnover. The data suggest that GnRH stimulates the hydrolysis of newly synthesized PIs, leading to the formation of DAGs. This pathway appears to be regulated independently of mobilization of extracellular Ca2+.     

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)     

Gonadal steroid modulation of signal transduction and luteinizing hormone release in cultured chicken pituitary cells

The mechanism whereby gonadal steroids modulate GnRH-stimulated LH secretion by primary cultures of chicken pituitary cells was investigated. Estradiol (10(-8) M), testosterone (10(-7) M), and progesterone (10(-7) M) inhibited LH release stimulated by GnRH (10(-7) M) by 56%, 61%, and 53%, respectively, and the inhibitory effects required prolonged preincubation (24-48 h) with the steroids. The steroids inhibited the spike (0-3 min) and plateau (9-30 min) phases of LH release to a similar degree. The ED50 values of estradiol, testosterone, and progesterone for inhibition of GnRH-stimulated LH release were 7 x 10(-11), 2 x 10(-9), and 1 x 10(-9) M, respectively. Estradiol, testosterone, and progesterone inhibited the maximal LH response to GnRH, but the ED50 of GnRH (4 x 10(-9) M) was not altered by steroid pretreatment. Steroid pretreatment did not cause a change in cellular LH content, suggesting that the steroids do not inhibit LH synthesis. Combinations of two or three of the steroids were not additive, suggesting that all three steroids affect GnRH-stimulated LH release via the same mechanism. In experiments investigating their mechanism of action, the steroids inhibited LH release stimulated by GnRH and Ca2+ ionophore A23187, but generally had no effect on the responses to phorbol ester (12-O-tetradecanoylphorbol-13-acetate), forskolin, K+, Bay K8644, or veratridine. Estradiol inhibited GnRH-stimulated 45Ca2+ efflux, but its inhibitory effect on GnRH-induced inositol phosphate production was not significant. Estradiol had no effect on binding of 125I-[His5,D-Tyr6]GnRH to a pituitary cell preparation. These findings suggest that the site of steroid modulation of GnRH action is distal to binding of GnRH to its receptor, and that the inhibitory effects are exerted at two intracellular sites: 1) the coupling events linking receptor activation to mobilization of Ca2+, and 2) a site distal to Ca2+ mobilization.     

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 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.     

Enhanced responsiveness of gonadotropes after protein kinase-C activation: postreceptor regulation of gonadotropin releasing hormone action

GnRH stimulates the release of LH from pituitary gonadotropes in a Ca2+-and calmodulin-dependent manner. Although GnRH also appears to activate protein kinase-C in gonadotropes, the role of this enzyme in GnRH action remains undetermined. In the present work we have assessed the effect of pretreatment of pituitary cell cultures with a protein kinase-C-activating phorbol ester on gonadotrope responsiveness to GnRH. Pretreatment for 6 h with phorbol 12-myristate 13-acetate (PMA) reduced the EC50 for GnRH-stimulated LH release approximately 8-fold without altering the maximum proportion of total cellular LH release. This increase in the potency of GnRH occurred in the absence of any measurable change in receptor affinity. Subsequent studies revealed that PMA pretreatment did not alter the EC50 for GnRH-stimulated [3H]inositol phosphate accumulation (an indicator of phosphoinositide hydrolysis), but did cause a modest reduction (approximately 2-fold) in the EC50 for LH release in response to the Ca2+ ionophore A23187 and the Ca2+ channel-activating compound maitotoxin. These observations demonstrate that the efficiency of coupling of the GnRH receptor to LH release can be regulated at a postreceptor locus by activation of protein kinase-C and that an increased responsiveness of Ca2+-regulated effector systems to mobilized Ca2+ appears to contribute to the observed effect.     

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.     

Gonadotropin-releasing hormone (GnRH) rapidly stimulates the formation of inositol phosphates and diacyglycerol in rat granulosa cells: further evidence for the involvement of Ca2+ and protein kinase C in the action of GnRH

GnRH provokes a phospholipid response in rat granulosa cells that has been characterized by increased incorporation of radioactive precursors into phosphatidic acid and phosphatidylinositol, and by depletion of 32P-prelabeled polyphosphoinositides. In this report, rat granulosa cells from mature Graafian follicles were incubated with GnRH under various conditions to follow the hydrolysis of phosphoinositides and the generation of the metabolic byproducts of phospholipase C action. Granulosa cells were prelabeled for 3 h with myo[2-3H]inositol. GnRH provoked rapid (10 sec) and sustained (up to 60 min) increases in the levels of inositol monophosphates, inositol bisphosphates, and inositol trisphosphates (IP3). Time-course studies revealed that IP3 was formed more rapidly than inositol bisphosphate and inositol monophosphate after GnRH treatment. The response to GnRH was concentration dependent (maximal at 10 ng/ml) and was prevented by a specific GnRH antagonist. Lithium chloride (1-10 mM) greatly enhanced the GnRH-provoked accumulation of all [3H]inositol phosphates, presumably by inhibiting the action of inositol phosphate phosphatases. No changes were observed in the levels of free [3H] inositol and [3H]phosphatidylinositol in GnRH-treated cells. However, treatment with both lithium and GnRH for 30 min significantly reduced the levels of free [3H]inositol and [3H] phosphatidylinositol. In the presence of lithium, the rate of hormone-stimulated inositol phosphate formation was not altered by 30 min of prior treatment with GnRH, indicating that phospholipase C activity is not readily desensitized. GnRH also increased the formation of diacylglycerol (DAG), another product of phospholipase C action. In cells prelabeled with [3H] arachidonic acid, GnRH significantly increased levels of DAG in incubations lasting 2-5 min. Concomitant increases in [3H] phosphatidic acid were also observed in GnRH-treated cells. In conjunction with these studies, intracellular free Ca2+ levels were measured by Quin 2 fluorescence. GnRH and its agonistic analog rapidly increased (5 sec) cytosolic free Ca2+ levels (approximately double). The results demonstrate that an early event in the action of GnRH is the hydrolysis of phosphoinositides by a phospholipase C-dependent mechanism. The products resulting from this action of GnRH, i.e. IP3 and DAG, may serve as intracellular mediators for the mobilization of intracellular calcium, or the activation of protein kinase C and arachidonic acid release.     

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.     

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.     

Regulation of phospholipid hydrolysis in streptolysin-O-permeabilized rat pancreatic acini.

To investigate the mechanism of phospholipid hydrolysis in pancreatic acinar cells, the effects of Ca2+, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and cholecystokinin (CCK) on both polyphosphoinositide (PI) and phosphatidylcholine (PC) hydrolysis were studied in rat pancreatic acini permeabilized with the bacterial toxin, streptolysin-O. When acini were prelabeled with myo-[3H]inositol, permeabilized, and then incubated with various concentrations of free Ca2+ for 15 min, Ca2+ stimulated [3H]inositol phosphate release at a concentration of 100 nM and was maximally effective at 100 microM. Both GTP gamma S and CCK enhanced Ca(2+)-induced [3H]inositol phosphate release, although these agents had no effect in the absence of Ca2+. At a physiological concentration of Ca2+ (100 nM), CCK stimulated [3H]inositol phosphate release which was further enhanced by GTP gamma S. When acini were similarly prelabeled with [3H]choline before permeabilization, [3H]choline phosphate release was also stimulated by free Ca2+ over the concentration range from 100 nM to 10 microM. In contrast to PI hydrolysis, however, neither GTP gamma S, CCK, or GTP gamma S plus CCK had an additional effect on [3H]choline phosphate release stimulated by 100 nM-100 microM free Ca2+. Furthermore, Ca(2+)-induced [3H]choline phosphate release appeared to be due to the redistribution from cell to the medium rather than to an increase in choline phosphate production. Therefore, choline phosphate release following prelabeling with [3H]choline is not useful as an indicator of PC hydrolysis in permeabilized acini.     

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.     

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.     

Na+ dependence of gonadotropin-releasing hormone action: characterization of the Na+/H+ antiport in pituitary gonadotropes.

GnRH stimulates LH release from gonadotropes in a Ca2(+)-dependent manner. Because of the apparent relationship between cellular Ca2+ metabolism and Na(+)-driven antiports, we investigated their influence on GnRH action. We also assessed the influence of bicarbonate, because its transport may alter effects of Na+/H+ exchange on intracellular pH. In pituitary cell cultures without bicarbonate, GnRH-stimulated LH release was reduced by Na+ omission, by amiloride, and by amiloride analogs that selectively block Na+/H+ exchange. The Na+ dependence of amiloride action (EC50, 14 and 100 microM in medium with 20 and 135 mM NaCl, respectively, and no effect in Na(+)-free medium) and the order of potency of these analogs, indicated specific inhibition of Na+/H+ exchange. 5-(N,N-Di-methyl)amiloride (DMA; a potent Na+/H+ exchange inhibitor) reduced GnRH-stimulated LH release but not GnRH receptor binding or Ca2+ ionophore (A23187)-stimulated LH release, suggesting inhibition at a locus beyond receptor occupancy but before exocytosis. Amiloride analogs that selectively inhibit Na+/Ca2+ exchange also modestly reduced GnRH-stimulated LH release. Bicarbonate (10 mM) reduced the inhibitory effects of DMA and Na+ omission (but not the effects of the Na+/Ca2+ exchange inhibitors or of a Ca2+ channel antagonist), and the effect of bicarbonate was inhibited by a blocker of bicarbonate-dependent antiports. These observations reveal the Na+ dependence of GnRH action and that gonadotropes possess a Na+/H+ exchanger. The Na+ dependence of GnRH-stimulated LH release appears to reflect at least in part dependence upon this antiport. Prevention of the Na+/H+ exchange inhibitor effects by bicarbonate supports the specificity of their action, but suggests regulation of this antiport as an unlikely means of controlling LH release in vivo.     

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.     

Divergent roles of protein kinase C in luteinizing hormone biosynthesis versus release in rat anterior pituitary cells.

We previously demonstrated that protein kinase C (PKC) activators, i.e. L-alpha-1,2-dioctanoyl glycerol (C8) and phorbol 12-myristate 13-acetate (PMA), mimic the stimulatory effects of GnRH on both LH glycosylation and release. To further evaluate the roles of PKC, we determined: 1) the interaction between PKC activator and GnRH; and 2) the effects of depleting cellular PKC with a high dose of PMA on LH glycosylation vs. release. Anterior pituitaries excised from ovariectomized rats were enzymatically dispersed and cultured. In series 1 experiments, day 3 monolayer cells were incubated in the presence of radiolabeled precursors and GnRH (0, 1, or 100 nM), with or without C8 (200 microM). In series 2 experiments, day 2 cells were pretreated with either PMA (1 microM) or vehicle (0.08% dimethyl sulfoxide) for 24 h and then incubated with diluent, GnRH (1 nM), or PMA (20 nM), and radiolabeled precursors for 4 h. LH translation and glycosylation were monitored by measuring incorporation of [14C]alanine ([14C]A) and [3H]glucosamine ([3H]GA), respectively, into LH. Immunoreactive LH (IRLH) was measured by RIA. In series 1 experiments, C8 increased basal release of IRLH, potentiated IRLH release stimulated by 1 nM GnRH, but not by 100 nM GnRH. C8 elevated total [3H]GA-LH but had no additive effects with GnRH. In series 2 experiments, PMA pretreatment inhibited subsequent PMA-stimulated IRLH release. However, PMA pretreatment did not affect GnRH-induced IRLH release even though PMA pretreatment decreased cellular IRLH content. In comparison, PMA pretreatment reduced both GnRH- and PMA-stimulated total [3H]GA-LH. PMA pretreatment had no effects on total [14C]A-LH in the presence of GnRH or PMA, but reduced the basal level. In summary, PKC activators had no additive effects on either IRLH release or LH glycosylation stimulated by a maximal dose of GnRH. However, PMA pretreatment decreased GnRH-induced LH glycosylation without depressing LH release. These results suggest differential roles of PKC in the actions of GnRH on LH glycosylation vs. LH release.     

Effects of a low calcium environment on luteinizing hormone biosynthesis in cultured rat anterior pituitary cells

The purpose of this study was to investigate the effects of lowering the extracellular calcium concentration on GnRH-stimulated LH glycosylation and LH translation, as measured by the incorporation of [3H]glucosamine (3H-Gln) and [35S]methionine (35S-Met) into immunoprecipitable LH. Cultured anterior pituitary cells, previously exposed to estradiol (5 X 10(-10) M) to maximize precursor incorporation were incubated for 4 h in normal calcium (2.5 mM) or low calcium medium (less than 15 microM) containing radiolabeled precursors with or without 1 nM GnRH. In the presence of normal calcium, GnRH significantly increased 3H-Gln-labeled LH in the medium (278%) and cells (290%), as well as total (cells plus medium) 3H- Gln LH (280%) compared to the control value (no GnRH). GnRH also significantly increased the 35S-Met LH released into the medium (164%) and total 35S-Met LH (186%) over control values. Depletion of extracellular calcium completely inhibited GnRH-stimulated 3H-Gln LH and 35S-Met LH production. Total immunoreactive LH (iLH), as measured by RIA, was also increased significantly by GnRH treatment in the presence of calcium, but this response was prevented by removal of calcium from the medium. Lowering extracellular calcium had no effect on cellular uptake or incorporation of 3H-Gln or 35S-Met into total trichloroacetic acid-precipitable protein. Approximately 80% of newly synthesized LH was released into the medium in all treatment groups independent of whether calcium or GnRH was present. The specific activity (disintegrations per min/microgram iLH) of radiolabeled LH released into the medium was significantly reduced by treatment with GnRH due to the large amount of unlabeled iLH released into the medium. However, when the cells were incubated in low calcium, the SA of 3H-Gln LH and 35S-Met LH in the medium was unaltered by GnRH, whereas GnRH-stimulated iLH release was inhibited. We conclude that GnRH stimulation of LH glycosylation and LH apoprotein synthesis involves extracellular calcium-dependent events, and the release of newly synthesized LH is closely coupled to LH biosynthesis and is less dependent on extracellular calcium, whereas the GnRH-stimulated release of previously synthesized, stored LH is dependent on extracellular calcium.     

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.     

Stimulation by phorbol ester and diacylglycerol of luteinizing hormone glycosylation and release by rat anterior pituitary cells

We studied the effects of protein kinase C (PKC) activators on LH glycosylation and release and the effect of 17 beta-estradiol on PKC activator-induced LH release. Rat anterior pituitary cells were incubated for 4 h with diluent, GnRH, and the PKC activators, phorbol 12-myristate 13-acetate (PMA), L-alpha-1,2-dioctanoyl glycerol (C8), and 1-oleoyl-2-acetyl-glycerol. LH translation and glycosylation were monitored by measuring incorporation of [14C]alanine ([14C]A) and [3H]glucosamine ([3H]GA), respectively, into total (medium + cell) immunoprecipitable LH. Immunoreactive LH (IRLH) was measured by RIA. PMA (10(-9) M) and 1-oleoyl-2-acetyl-glycerol (50-200 microM) had no significant effects. PMA at 10(-7) M elevated (P less than 0.01) medium IRLH, medium and total [3H]GA-LH, and medium but not total [14C]A-LH. PMA at 10(-7) M increased (P less than 0.01) uptake and incorporation of [3H]GA, but not [14C]A, into total pituitary protein. C8 increased both medium IRLH and total [3H]GA-LH (P less than 0.01) without altering total [14C]A-LH. Two hundred micromolar C8 increased medium concentrations of [3H]GA-LH (P less than 0.01) and [14C]A-LH (P less than 0.05). C8 (50-200 microM) had no detectable effects on uptake and incorporation of precursors into protein. GnRH (1 nM) enhanced (P less than 0.01) both medium IRLH and total [3H]GA-LH, but had no effect on total [14C]A-LH. Pretreatment of pituitary cells with 17 beta-estradiol (6 X 10(-10) M) greatly enhanced LH release induced by C8. In conclusion, PMA and C8, like GnRH, stimulated both LH glycosylation and release. These results suggest that PKC may regulate both LH release and glycosylation and may be important in estrogen modulation of LH release.