Calcium antagonists and hormone release. II. Effects of verapamil on basal, gonadotropin-releasing hormone- and thyrotropin-releasing hormone-induced pituitary hormone release in normal subjects

Although it has been well established that Ca2+ plays an essential role in the release of several hormones, very little is known of the interactions between Ca2+ and secretagogues in the process of pituitary hormone release. One possible way of studying the mechanism of action of hypothalamic releasing hormones is to study how organic calcium antagonists affect their action. Consequently, we infused the commonly used calcium antagonist, verapamil, into 20 normal subjects (10 men and 10 women; aged 19-37 yr) and studied its effects on both basal pituitary hormone levels and augmented hormonal release induced by gonadotropin-releasing hormone (GnRH) and TRH. Verapamil, infused at a rate of 5 mg/h for 3 h, induced a significant and marked suppression of circulating LH and FSH levels in both men and women. By the end of the infusion, the suppression of release was greater for LH (60%) than for FSH (54%). After the termination of the infusion, plasma gonadotropin concentrations returned progressively to basal levels within 2 h. Verapamil was also capable of blunting the peak incremental gonadotropin response to GnRH. Although the basal TSH concentration was apparently unaffected by verapamil, the incremental TSH response to TRH was significantly inhibited in both men and women. Verapamil infusion did not affect either the basal PRL concentration or the PRL response to TRH. Our data provide evidence that verapamil exerts different effects on the release of pituitary hormones in normal subjects. It inhibits the centrally mediated as well as the peripherally mediated gonadotropin release and blunts the TSH response to TRH. On the contrary, verapamil does not seem to affect basal or TRH-mediated PRl release. The use of organic calcium antagonists in experimental models in vitro as well as in vivo appears to offer a promising tool for further studies on the mechanism of action of secretagogues in the process of hormone release.     

The role of calcium in gonadotropin-releasing hormone induction of follicle-stimulating hormone release by the pituitary gonadotrope

Binding of gonadotropin-releasing hormone (GnRH) to the pituitary gonadotrope induces activation of a membrane associated calcium channel, resulting ultimately in luteinizing hormone release. The role of calcium mobilization in GnRH-induced follicle-stimulating hormone (FSH) release was explored using anterior pituitary glands from female rats in a perifusion tissue culture system. While perifusion with GnRH (10 ng/ml) induced a constant level of gonadotropin release, the calcium channel blocker verapamil (10(-4)M) depressed FSH release, as did dantrolene (10(-4)M), an antagonist of intracellular calcium mobilization. When the calcium ionophore A23187 (10(-5) M) was substituted for GnRH, FSH release was not only maintained but increased. Antagonism of the activity of calmodulin (CAM) with trifluoperazine (10(-4)M), however, did not depress FSH release. Cellular content of cAMP and cGMP increased in response to GnRH. When FSH secretion was ionophoretically induced by A23187, however, little cAMP was detected. These results support a role for calcium mobilization in the second messenger cascade underlying GnRH-induced FSH release. The role for calcium in the disparate release of FSH and LH were further discussed in the context of these data.     

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.     

Pituitary gonadotrophin-releasing hormone receptor up-regulation in vitro: dependence on calcium and microtubule function

Exogenous cyclic adenosine nucleotides increase gonadotrophin-releasing hormone (GnRH) receptors in intact cultured rat pituitary cells in a similar manner to that observed with GnRH itself. In this study the calcium and microtubule dependency of GnRH receptor up-regulation was examined in vitro. Treatment of pituitary cells in Ca2+ and serum-containing media with either GnRH (1 nmol/l), K+ (58 mmol/l) or dibutyryl cyclic AMP (dbcAMP; 1 mmol/l) for 7-10 h routinely resulted in a 50-100% increase in GnRH receptors. Incubation of pituitary cells with the calcium channel blocker verapamil, for 7 h, or the calcium chelator EGTA, for 10 h, had no effect on basal receptor levels but prevented the increase in GnRH receptors stimulated by either GnRH, K+ or dbcAMP. Luteinizing hormone release measured with the same stimulators over a 3-h period was prevented by both verapamil and EGTA. Calcium ionophore (A23187) increased GnRH receptors by 40-60% at low concentrations (10 and 100 nmol/l) while higher concentrations (10 and 100 mumol/l) reduced receptor levels. Luteinizing hormone release was not increased by receptor-stimulating concentrations of A23187, but was by higher concentrations (10 mumol/l). None of these pretreatments, for up to 10 h, impaired the subsequent LH response of the cells to increasing doses of GnRH. Vinblastine (1 mumol/l did not affect basal receptor levels but markedly reduced the increase in GnRH receptors stimulated by GnRH, K+ and dbcAMP. This concentration of vinblastine had no effect on LH release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Divergent influences of the structurally dissimilar calcium entry blockers, diltiazem and verapamil, on thyrotropin- and gonadotropin-releasing hormone-stimulated anterior pituitary hormone secretion in man

We have tested the influence of a new calcium ion channel antagonist, diltiazem, on hypothalamic releasing hormone-stimulated secretion of LH and other anterior pituitary hormones in man. To this end, six normal men received a continuous infusion of GnRH (1 microgram/min) and TRH (2 micrograms/min) for 3 h under three different experimental conditions: 1) saline (control) infusion; 2) iv diltiazem (0.3 mg/kg bolus dose, and 0.002 mg/kg . min) infusion for 4 h beginning 1 h before releasing hormone injection; and 3) oral diltiazem (60 mg, every 6 h) administration for 1 week before pituitary stimulation. Blood was sampled at 10-min intervals for the subsequent immunoassay of LH, FSH, TSH, PRL, and GH concentrations and at hourly intervals for the assay of plasma diltiazem concentrations by high performance liquid chromatography. Despite sustained plasma diltiazem concentrations of 80-120 ng/ml during either iv or oral drug administration, the GnRH/TRH-stimulated release of LH, FSH, TSH, and PRL or the basal secretion of GH did not differ significantly from that during saline infusion. In contrast, when these subjects underwent the same infusion schedule using a structurally dissimilar calcium influx blocker, verapamil (5-mg bolus dose and 15 mg/h, continuous infusion), there was significant suppression of the delayed component of GnRH/TRH-stimulated LH release, with simultaneous enhancement of PRL secretion. We conclude that exogenously stimulated anterior pituitary hormone secretion in man exhibits differential susceptibility to the structurally discrete calcium entry blockers diltiazem and verapamil. Moreover, the differential influence of these two calcium ion channel antagonists on gonadotropes is distinct from that described in cardiac and smooth muscle cells.     

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.     

Effects of cupric acetate on hypothalamic gonadotropin-releasing hormone release in intact and ovariectomized rabbits

The effects of intravenous injection of cupric acetate (CuAc) on release of hypothalamic gonadotropin-releasing hormone (GnRH) and pituitary luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin (PRL) were explored in conscious, ovarian intact does and in ovariectomized (OVEX) does that received subcutaneous implants, either blank Silastic capsules or Silastic capsules containing crystalline estradiol-17 beta (E2). Animals were subjected to push-pull perfusion of the posterior median eminence for 6 h, and CuAc was intravenously injected (2.5 mg/kg body weight) at the end of the 2nd h of push-pull perfusion. Perfusate samples were collected continuously and pooled for assay at 10-min intervals. Peripheral blood samples were obtained at 10- to 30-min intervals. Levels of GnRH in push-pull perfusate and LH, FSH, and PRL in plasma were measured by specific radioimmunoassays. In intact does (n = 4), intravenous injection of CuAc stimulated the release of hypothalamic GnRH and pituitary LH, FSH, and PRL. The initial increase and subsequent decrease in hypothalamic GnRH after CuAc injection preceded those in plasma LH, FSH, and PRL. Injection of saline into intact does (n = 4) had no effect on any of these hormone levels. In OVEX does that received blank Silastic capsules (n = 4), CuAc failed to stimulate either hypothalamic GnRH release or pituitary LH, FSH, and PRL release. In contrast, CuAc stimulated the release of all four hormones in OVEX does that received E2 containing Silastic capsules (n = 3).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of hypothalamic gonadotropin-releasing hormone release by endogenous opioid peptides in the female rabbit

Recent evidence suggests that the endogenous opioid peptides (EOPs) inhibit luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by suppression of hypothalamic gonadotropin-releasing hormone (GnRH) release, and that the feedback inhibition by EOPs is influenced by ovarian steroids. In the present studies, intact (INT) and ovariectomized (OVX) adult female rabbits were fitted with femoral vein catheters and mediobasal hypothalamic (MBH) push-pull perfusion (PPP) cannulae. One week after brain cannulation, does were subjected to 6 h of PPP and sequential blood sampling. In experiment I, INT (n = 6) and OVX (n = 5) does were infused intravenously with saline for 4 h followed by 2 h of infusion of the opiate antagonist naloxone (NAL; 10 micrograms/min/kg) while the MBH was simultaneously perfused with media. In experiment II, INT (n = 5) and OVX (n = 5) does were perfused with media for 4 h followed by 2 h of intrahypothalamic (IHP) NAL perfusion (0.2 microgram/min). The GnRH in push-pull perfusates and LH and FSH in plasma samples collected at 10-min intervals were measured by specific radioimmunoassays. In INT does, neither intravenous infusion nor IHP perfusion of NAL altered pulsatile parameters of GnRH or LH release. In contrast, both intravenous and IHP NAL administration stimulated GnRH and LH release within 30-50 min in OVX does by marked increases in both GnRH and LH pulse amplitudes. Neither route of NAL administration affected FSH secretion in any of the treatment groups. We conclude that EOPs are involved in the inhibition of hypothalamic GnRH secretion in OVX does; the feedback inhibition by ovarian steroids on the hypothalamic-pituitary axis in the rabbit is sufficient to compromise the effects of EOPs, and under these experimental conditions, the hypothalamic mechanisms which regulate the secretion of pituitary LH and FSH may be independent.     

Sustained intermittent release of gonadotropin-releasing hormone in the prepubertal male rhesus monkey induced by N-methyl-DL-aspartic acid

The purpose of the present study was to determine whether gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus of the prepubertal monkey may be prematurely provoked into producing a sustained train of intermittent GnRH release N-methyl-DL-aspartic acid (NMA), an analog of the putative excitatory neurotransmitter aspartate, was used to stimulate the hypothalamus. In order to utilize pituitary luteinizing hormone (LH) secretion as a bioassay of hypothalamic GnRH release, juvenile males were castrated and the responsiveness of their gonadotrophs to GnRH was enhanced prior to the study with a chronic intermittent intravenous infusion of the synthetic decapeptide (0.1 microgram/min for 3 min every hour). Treatment with this regimen of GnRH, which appears to provide the pituitary gonadotrophs with a hypophysiotropic stimulus similar to that produced by the hypothalamus of castrated adults, elicited a pattern of pulsatile LH secretion in prepubertal animals similar to that observed in the open-loop situation in adults. This episodic pattern of LH release was sustained without decrement following termination of GnRH priming and initiation of an intermittent intravenous infusion of NMA (4.5-6.5 mg NMA/kg body weight/pulse, administered over 1 min) delivered at a frequency of 1 pulse/1 h for 50 h. In contrast, an intermittent infusion of the vehicle employed to administer NMA (saline) failed to maintain LH secretion. Administration of the same dose of NMA at a slower frequency of 1 pulse/2 h for 52 h, while also sustaining LH secretion without decrement, resulted in an exaggeration in the LH response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of leukotrienes in gonadotropin-releasing hormone-stimulated pituitary cells: potential role in luteinizing hormone release.

Gonadotropin-releasing hormone (GnRH) stimulated the formation of two major metabolites of the 5-lipoxygenase pathway, leukotriene (LT) B4 and LTC4, as well as luteinizing hormone (LH) release in primary cultures of rat anterior pituitary cells. Several lines of evidence suggested the presence of a GnRH-dependent pituitary endocrine system in which LTs act as second messengers for LH release: (i) GnRH-dependent LT formation was observed within 1 min and immediately preceded GnRH-induced LH release, whereas exogenous LTs stimulated LH release at low concentrations; (ii) the dose responses of GnRH-induced LT production and LH release were similar and both effects required the presence of extracellular Ca2+ ions; (iii) GnRH-induced LH release was blocked by up to 45% following the administration of several LT receptor antagonists; (iv) LTE4 action on LH secretion was entirely abolished by LT receptor antagonists; and (v) an activator of protein kinase C acted synergistically with LTE4 to induce LH release. The major source of LT formation in the pituitary cell cultures appeared to be the gonadotrophs, as shown by GnRH receptor desensitization experiments. The results demonstrate the presence of a GnRH-activatable 5-lipoxygenase pathway in anterior pituitary cells and provide strong support for the hypothesis that LTs play a role in LH release in the GnRH signaling pathway.     

Progesterone modulation of gonadotropin secretion by dispersed rat pituitary cells in culture. III. A23187, cAMP, phorbol ester and DiC8-stimulated luteinizing hormone release

Dispersed estradiol-treated rat pituitary cells were used to characterize progesterone (P) modulation of luteinizing hormone (LH) secretion in response to a variety of pharmacologic secretagogues which influence cell biochemistry. Acute (less than 3 h) and chronic (24 h) exposures to P prior to secretagogue challenge respectively enhanced and inhibited Ca2+ ionophore (A23187)-stimulated and gonadotropin-releasing hormone (GnRH)-stimulated LH release in similar quantitative fashion without any effect on concurrent prolactin release. Similar responses were also noted with cholera toxin-stimulated secretion. However, when protein kinase C activators such as phorbol esters and dioctanoylglycerol were used to trigger LH release, chronic exposure to P did not inhibit, but rather enhanced, LH release. Again, P had no effect on prolactin release. 'Washout' studies indicated that chronic treatments with P would suppress LH secretion stimulated by these compounds, but only when the steroid was cleared from the cells 4 h beforehand. These studies provide further evidence that P specifically modulates gonadotroph secretory function via mechanisms which bypass GnRH receptors. Moreover, they suggest that P exerts many different actions within the gonadotroph and question the role of protein kinase C in GnRH action.     

Effects of somatostatin and verapamil on growth hormone release and 45Ca fluxes

Increasing the extracellular K+ concentration to 71 mM causes a phasic release of growth hormone and efflux of 45Ca from perifused bovine pituitary cells. Verapamil (20 micron) partially inhibits the initial phase of growth hormone release and 45Ca efflux and completely inhibits the second phase. Somatostatin (1 microgram/ml) partially inhibits both phases of growth hormone release but does not modify 5+-induced 45Ca efflux. Incubation of pituitary cells in 71 mM K+ increases 45Ca incorporation; verapamil (20 micron) completely prevents, and somatostatin (1 microgram/ml) partially inhibits, the K+-induced increase in 45Ca incorporation. The results suggest that 71 mM K+ increases both calcium entry into the cells and calcium redistribution within them, and that verapamil only inhibits the K+-induced calcium entry. Somatostatin may inhibit calcium entry into tissue stores.     

Annexin 5 messenger ribonucleic acid expression in pituitary gonadotropes is induced by gonadotropin-releasing hormone (GnRH) and modulates GnRH stimulation of gonadotropin release.

Our previous studies on annexin 5, a member of the annexin family of proteins, have shown its expression in the anterior pituitary gland, its preferential distribution in gonadotropes, and its increase after ovariectomy. In the present study, we examined (1) whether annexin 5 is synthesized in gonadotropes, (2) whether its expression is under the control of gonadotropin-releasing hormone (GnRH), and (3) the effect of annexin 5 on gonadotropin release. Large cells, also called castration cells, appeared in anterior pituitary tissue 3 weeks after ovariectomy. These cells have been confirmed to be hyperfunctioning gonadotropes and are easily discriminated from other pituitary cells without immunostaining. Using in situ hybridization with a digoxigenin-labeled ribonucleic acid probe, enhanced expression of annexin 5 messenger ribonucleic acid (mRNA) in these gonadotropes was clearly demonstrated. Northern blot analysis showed an increase in the level of annexin 5 mRNA expression 3 weeks after ovariectomy. It was lessened 3 h after the injection of Cetrorelix (GnRH antagonist, 10 microg i.v.). Administration of a GnRH analog [GnRHa; Des-Gly 10 (Pro9) GnRH ethylamide, 0.2 ml of 2.5 microg/ml saline ten times intraperitoneally at 30-min intervals] significantly increased pituitary annexin 5 mRNA. In primary cultures of anterior pituitary cells, recombinant rat annexin 5 stimulated luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release in a dose-dependent manner. Concomitant administration of annexin 5 (1 microg/ml) and GnRHa augmented the LH and FSH release induced by GnRHa. After a 1-hour incubation, cycloheximide (10 microg/ml) apparently inhibited the LH response to GnRHa, while annexin 5 (2 microg/ml) moderated this inhibition. Further, the antisense oligodeoxynucleotide to annexin 5 mRNA blunted the LH response to GnRHa. It is thus concluded that annexin 5 is synthesized in the gonadotropes under the effect of GnRH, and it is suggested that annexin 5 synthesis mediates at least partly GnRH receptor signaling to stimulate gonadotropin secretion.     

Changes in plasma luteinizing hormone-releasing hormone and gonadotropin concentrations during constant rate intravenous infusion of luteinizing hormone-releasing hormone in cyclic rats.

Further analysis has been made of the response of the rat pituitary gland to LHRH during the 4-day estrous cycle. LHRH was infused iv at a constant rate (50 ng/h) into phenobarbital-treated rats at different times during the estrous cycle. Infusion at this rate in proestrous rats simulates the rising and plateau phases of the spontaneous proestrous surges of LH and FSH in plasma. Plasma LH rose to similar heights during the "initial phase" of LH release (during the first 40 min of infusion) on the afternoons of estrus, diestrous day one, and proestrus and during the morning of proestrus. The increase during the afternoon of diestrous day two was significantly less than that in all the other groups. A similar response was seen in the case of FSH release. A "rapid rising" or "augmented" phase of LH release (during 40-120 min of infusion) was present in all groups and the magnitude of the response was greatest during the afternoon of proestrus. In the case of FSH, an augmented phase of release started 60 min after the start of infusion, and the response during the afternoon of proestrus was slightly greater than the responses measured at the other times tested. The responses on diestrous day one were not altered when phenobarbital was omitted or when rats were ovariectomized shortly before LHRH infusion. Other differences in the LH and FSH responses during both initial and augmented phases of release were seen in rats tested at different times during the estrous cycle with an LHRH infusion rate which caused a supraphysiological response on proestrus. The results suggest that 1) the initial rising phases in plasma LH and FSH during the spontaneous surges during proestrus are not the result of an increase in pituitary responsiveness to LHRH during the estrous cycle, 2) augmented phases of LH and FSH release can be elicited on all days of the estrous cycle, and 3) the increases in magnitude of the augmented phases of LH and FSH release on proestrus, as compared to those on other days of the cycle, are the result of an increase in pituitary responsiveness to LHRH during the estrous cycle.     

Gonadotropin, estrogen and progesterone response to long term gonadotropin-releasing hormone infusion at various stages of the menstrual cycle.

6 normally menstruating women, aged 22-27, were given constant infusions of 12.5-25 mcg/hour gonadotropin releasing hormone (GnRH) for 24 hours during 10 cycles. 4 were infused in the early follicular, 3 in the late follicular, and 3 in the luteal phase. Frequent blood samples were assayed for luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol, progesterone, and GnRH. The increase in gonadotropin and patterns of response varied in the different stages of the cycle. Quantitatively the response was minimal in the early follicular phase, maximal at midcycle, and moderate in midluteal phase. In the latter 2 phases most of the gonadotropins were released during the first 8 hours of infusion. The ratio of the LH-FSH areas under the curves favored FSH in the early follicular phase and LH at midcycle and luteal phase. In all the cycles there was an initial increase in both gonadotropins which lasted 6-8 hours after which the levels declined but nevertheless remained above baseline as long as the infusion was continued. Plasma GnRH measured during 6 infusions was undetectable prior to the starting and after discontinuation of the infusion, but during infusion fluctuations were considerable ranging from 150 to 500 pg/ml. These studies bring additional evidence to the possible existence of 2 gonadotropin pools in the human pituitary and point to the complexity of the response mechanism to GnRH stimulation and its relation to ovarian secretion.     

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)     

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.     

Calcium-dependent actions of gonadotropin-releasing hormone on pituitary guanosine 3',5'-monophosphate production and gonadotropin release

The effects of gonadotropin-releasing hormone (GnRH) on cGMP production and LH release in cultured rat pituitary cells are markedly dependent upon the extracellular calcium concentration. The absence of calcium from incubation media caused almost complete loss of the GnRH effects on cGMP production and LH release but did not change the stimulation of cAMP accumulation by GnRH in the pituitary of the adult male rat. In female rat pituitary cells, reduction of the extracellular calcium concentration increased the concentration of GnRH required to produce half-maximal LH release and decreased the maximal gonadotropin output but had no significant effect on basal LH release. The divalent cation ionophore A23187 stimulated LH release, and this action was dependent on extracellular calcium. Both GnRH and A23187 were found to have maximal effects when the calcium concentration was 0.6 mM, and their actions were not additive. The calcium antagonists, verapamil and lanthanum, caused concentration-dependent inhibition of the actions of GnRH, with half-maximal blockade values of 10(-5) and 3 X 10(-6) M, respectively, and had no effect on basal LH release. The binding of a radioiodinated GnRH analog, [D-Ser(t-Bu)6]des-Gly10-GnRH-N-ethylamide, to pituitary GnRH receptors was unchanged in the absence of extracellular calcium. These observations demonstrate that stimulation of pituitary cGMP production and LH release by GnRH is dependent on extracellular calcium. The site at which calcium is required during GnRH action is at a postreceptor locus before cGMP formation.     

Kisspeptin-1 directly stimulates LH and GH secretion from goldfish pituitary cells in a Ca(2+)-dependent manner

It has been established that kisspeptin regulates reproduction via stimulation of hypothalamic gonadotropin-releasing hormone (GnRH) secretion, which then induces pituitary luteinizing hormone (LH) release. Kisspeptin also directly stimulates pituitary hormone release in some mammals. However, in goldfish, whether kisspeptin directly affects pituitary hormone release is controversial. In this study, synthetic goldfish kisspeptin-1((1-10)) (gKiss1) enhances LH and growth hormone (GH) release from primary cultures of goldfish pituitary cells in column perifusion. gKiss1 stimulation of LH and GH secretion were still manifested in the presence of the two native goldfish GnRHs, salmon (s)GnRH (goldfish GnRH-3) and chicken (c)GnRH-II (goldfish GnRH-2), but were attenuated by two voltage-sensitive calcium channel blockers, verapamil and nifedipine. gKiss-induced increases in intracellular Ca(2+) in Fura-2AM pre-loaded goldfish pars distalis cells were also inhibited by nifedipine. These results indicate that, in goldfish, (1) direct gKiss1 actions on pituitary LH and GH secretion exist, (2) these actions are independent of GnRH and (3) they involve Ca(2+) signalling.     

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.