The temporal integration of the aldosterone secretory response to angiotensin occurs via two intracellular pathways

Angiotensin II (AII) regulates the secretion of aldosterone from adrenal glomerulosa cells by a calcium-dependent mechanism which involves both the uptake of calcium from the extracellular pool, and the release of calcium from a dantrolene-sensitive intracellular pool. In the present study, it was shown that AII induces the rapid (10 s) hydrolysis of phosphatidylinositol 4-phosphate and -4,5-bisphosphate, leading to the sustained production of inositol bis- and trisphosphate (Ins-P3), and diacylglycerol rich in arachidonic acid. Saponin-permeabilized glomerulosa cells accumulate calcium into a nonmitochondrial pool by an ATP-dependent manner. Ins-P3 (0.5-5 microM) induces a release of Ca2+ from this pool. This release was blocked by dantrolene (10 microM). Adrenal glomerulosa cells were shown to contain the calcium-activated, phospholipid-dependent protein kinase (C-kinase). Perfusion of glomerulosa cells with combined 12-O-tetradecanoyl phorbol 13-acetate and A23187 induced an immediately developing, sustained, maximal secretory response similar to that induced by AII. These data are interpreted in terms of a model in which, after AII addition, there is a flow of information through two separate branches of the calcium messenger system, each with its unique temporal role: a calmodulin branch activated by the transient rise in the [Ca2+] in the cell cytosol, which is largely responsible for the initial transient cellular response; and a C-kinase branch activated by the increase in both cytosolic [Ca2+] and the diacylglycerol content of the plasma membrane, which is largely responsible for the sustained phase of the cellular response. The temporal integration of these two phases underlies the observed pattern of cellular response.     

Stimulatory and inhibitory effects of progesterone on FSH secretion by the anterior pituitary.

The purpose of this study was to investigate whether progesterone exerted progesterone receptor mediated direct effects on the anterior pituitary in the secretion of FSH and whether such effects were mediated through the 5 alpha-reduction of progesterone. Treatment of anterior pituitary dispersed cells for 48 h with 0.5 nM estradiol reduced the ED50 for gonadotropin releasing hormone (GnRH)-stimulated FSH release from 0.58 to 0.36 ng/ml and the ED50 for GnRH-induced LH release from 0.54 to 0.19 ng/ml. When dispersed pituitary cells were treated with 0.5 nM estradiol and exposed to various doses of progesterone for 1 to 6 h, the most consistent rise in basal and GnRH-stimulated FSH release was observed with the 50 nM dose of progesterone with a 3-h exposure period. All three doses of progesterone elevated basal LH and GnRH-stimulated LH was increased by the 50 and 100 nM doses of progesterone during the 3-h period of treatment. Using the 50 nM dose of progesterone, basal and GnRH-stimulated LH was increased after 2, 3 and 6 h of progesterone treatment. When the period of exposure of progesterone was extended to 12, 36 or 48 h, there was a significant inhibition of GnRH-stimulated FSH release. GnRH-stimulated LH release was inhibited at 36 and 48 but not 12 h after progesterone treatment. These studies showed that the effect of progesterone administered for periods of 1 to 6 h enhanced the secretion of LH and FSH whereas progesterone administered for periods beyond 12 h inhibited FSH and LH release by dispersed pituitary cells in culture. These results are similar to those observed in vivo after progesterone treatment. Furthermore estrogen priming of the dispersed pituitary cells was necessary to observe the effects of progesterone. The progesterone antagonist RU486 prevented the progesterone-induced rise in GnRH-stimulated FSH release. Furthermore the 5 alpha-reductase inhibitor N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane- 17 beta-carboxamide also prevented the progesterone-induced rise in GnRH-stimulated FSH release in estrogen-treated dispersed pituitary cells. These results indicate that the anterior pituitary is a major site of action of progesterone in the release of FSH and that 5 alpha-reduction of progesterone plays an important role in FSH release.     

Restoration of responsiveness to gonadotropin releasing hormone (GnRH) in calcium-depleted rat pituitary cells.

GnRH-stimulated, but not basal, luteinizing hormone (LH) release from cultured pituitary cells requires extra-cellular calcium. The present studies were designed to show whether cells which had lost responsiveness to GnRH in the absence of extracellular calcium (“Ca²⁺-depleted cells”) could regain responsiveness by readdition of calcium to the media. The addition of calcium-containing medium to cells which were preincubated (75 min) in calcium-free medium resulted in elevated basal LH release. Addition of GnRH to the media in the presence of calcium did not cause additional stimulation of LH release above the elevated basal level. Incubation of Ca²⁺-depleted cells in calcium-containing media for 2 h before measuring responsiveness depressed the basal level to near that seen in control cells and GnRH was able to stimulate LH release, but not to as high a level as in control cells (which were preincubated in 1 mM Ca²⁺-containing media). After incubation of calcium depleted cells in calcium-containing media for 3 h or 5 h, the basal and stimulated levels of LH response were statistically indistinguishable from those seen in control cells.     

Effect of calcium, glucose and trifluoroperazine on the carboxymethylation of proteins in pancreatic islets of rats

In the isolated pancreatic islets incubated in vitro, carboxylmethylation increases following stimulation with 20 mM glucose. This increase is dependent on extracellular calcium concentration and is suppressed in the presence of trifluoperazine, a calmodulin inhibitor. In conclusion, the increase in carboxylmethylation induced by 20 mM glucose seems to be a calcium-dependent process and it is probably mediated by calmodulin.     

Dependence of secretory responses to gonadotropin-releasing hormone on diacylglycerol metabolism. Studies with a diacylglycerol lipase inhibitor, RHC 80267

The role of diacylglycerol (DG) as a source of arachidonic acid during gonadotropin-releasing hormone (GnRH) stimulation of gonadotropin secretion was analyzed in primary cultures of rat anterior pituitary cells. An inhibitor of DG lipase (RHC 80267, RHC) caused dose-dependent blockade of GnRH-stimulated luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. The DG lipase inhibitor did not alter gonadotropin responses to arachidonic acid, and addition of arachidonic acid reversed its inhibition of GnRH-stimulated LH and FSH release. In [3H]arachidonic acid-prelabeled cells, incubation with RHC increased the accumulation of [3H]DG. These results suggest that DG lipase participates in GnRH action and that arachidonic acid mobilization from DG is involved in the mechanism of gonadotropin release. Gonadotropin responses to tetradecanoyl phorbol acetate and dioctanoyl glycerol were not altered by RHC, and the addition of these activators of protein kinase C (Ca2+- and phospholipid-dependent enzyme) did not prevent the inhibition of GnRH-induced gonadotropin release by RHC. Activation of phospholipase A2 by melittin increased LH and FSH secretion, whereas blockade of this enzyme by quinacrine reduced GnRH-stimulated hormone release. However, RHC did not diminish the gonadotropin response to melittin. The inhibitory actions of RHC and quinacrine were additive and were reversed by concomitant treatment with arachidonic acid. Ionomycin also increased LH and FSH release, and the gonadotropin responses to the ionophore were unaltered by RHC but were reduced by quinacrine. Incubation of cells in Ca2+-depleted (+/- [ethylenebis(oxyethylenenitrilo)]tetraacetic acid) medium reduced but did not abolish the LH and FSH releasing activity of GnRH. Treatment with RHC also reduced the gonadotropin responses to GnRH under Ca2+-depleted conditions. These observations indicate that RHC inhibition of GnRH action is not due to nonspecific actions on Ca2+ entry, protein kinase C activation and actions, nor phospholipase A2 enzyme activity. The results of this study provide further evidence for an extracellular Ca2+-independent mechanism of GnRH action, and suggest that GnRH causes mobilization of arachidonic acid by two distinct lipases, namely, phospholipase A2 and DG lipase, during stimulation of gonadotropin secretion.     

Calcium, Calmodulin and the Control of Respiration in Protoplasts Isolated from Meristematic Tissues8

Owen, J. H., Hetherington, A. M. and Wellburn, A. R. 1987. Calcium,calmodulin and the control of respiration in protoplasts isolatedfrom meristematic tissues by abscisic acid.—J. exp. Bot.38: 1356–1361. A study was made of the possible involvement of calcium channelsand calmodulin during the calcium-dependent inhibition of mitochondrialrespiration by abscisic acid (ABA) in meristematic protoplastsobtained from light-grown barley (Hordeum vulgare L. cv. Patty)seedlings. The calcium channel blockers lanthanum, verapamiland nifedipine were all found to reduce the Ca²⁺-dependent inhibitionof protoplast respiration by ABA. The ionophore A23187 [GenBank] itselfcaused an inhibition of protoplast respiration, possibly becauseit mimicked the action of ABA by increasing plasmalemma permeabilityto extracellular calcium. By contrast, calmodulin antagoniststrifluoperazine and compound 48/80 both caused a partial decreasein the Ca²⁺-dependent inhibition of protoplast respiration byABA. In contrast to the action of ABA, gibberellic acid markedlyincreased the rates of protoplast respiration but this did notappear to require the presence of extracellular calcium ions.These results support the hypothesis that ABA increases plasmalemmapermeability to extracellular calcium which might then directlyor indirectly act as a second messenger, possibly in conjunctionwith calmodulin, to regulate mitochondrial dark respirationwhich is an important part of early meristematic cell development. Key words: Abscisic acid, calcium, calmodulin, meristematic respiration     

Calcium ions as a mediator in GnRH action on gonadotropin release in the common carp (Cyprinus carpio L)

Collagenase-dispersed carp pituitary cells in a perifusion system were used to study the role of calcium ions in the mechanism of GnRH action on the release of maturational gonadotropin (GtH) in fish. The specific calcium chelator EGTA and the calcium antagonist manganese (Mn2+) caused a 40% inhibition in the basal GtH release and completely blocked GnRH-stimulated GtH release. Short-term application of graded doses of calcium ionophore A23187 caused a dose-dependent increase in GtH secretion. A23187 failed to stimulate GtH secretion in the presence of EGTA. Depolarization of the membrane by K+ caused a strong stimulation of GtH release similar to the action of GnRH. Stimulatory action of K+ was inhibited by EGTA. These data suggest a role for extracellular calcium as an intracellular mediator in GnRH-stimulated, as well as in basal, GtH release in carp. The stimulation of GtH release by K+ also indicates that voltage-dependent processes could be involved in this phenomenon.     

Phorbol esters reduce gonadotrope responsiveness to protein kinase C activators but not to Ca2+-mobilizing secretagogues. Does protein kinase C mediate gonadotropin-releasing hormone action?

The demonstration that activators of the Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C), such as phorbol esters and diacylglycerols, can provoke luteinizing hormone (LH) release from pituitary gonadotropes, suggests a possible role for protein kinase C in stimulus-release coupling. We now report that administration of phorbol myristate acetate (PMA) to pituitary cell cultures causes a sustained reduction in Triton X-100-extracted protein kinase C activity. Further, phorbol ester- and diacylglycerol-stimulated LH release, as well as inhibition by PMA of gonadotropin-releasing hormone (GnRH)-stimulated inositol phosphate production, were reduced by pretreatment with PMA. The effects of phorbol ester pretreatment on PMA-stimulated LH release and protein kinase C activity were dose-dependent, sustained (greater than or equal to 24 h) and specific (no measurable effect with 4 alpha-phorbol didecanoate). The effect on PMA-stimulated LH release was apparently Ca2+-independent. In pituitary cell cultures with reduced protein kinase C activity, the gonadotropes have reduced responsiveness to PMA but release a similar proportion of cellular LH in response to Ca2+-mobilizing secretagogues (GnRH and A23187) as do control cells. The normal responsiveness to GnRH of cells with reduced responsiveness to protein kinase C activators calls into question the requirement for this enzyme for GnRH-stimulated LH release.     

Phosphatidic acid and the calcium-dependent actions of gonadotropin-releasing hormone in pituitary gonadotrophs

The stimulation of luteinizing hormone (LH) release and cyclic GMP (cGMP) production in rat anterior pituitary cells by gonadotropin-releasing hormone (GnRH) are receptor mediated and calcium dependent, and have been shown to be accompanied by increased phospholipid turnover and arachidonic acid release. The incorporation of 32Pi into the total phospholipid fraction of pituitary gonadotrophs was significantly elevated by 10(-8) M GnRH, with specific increases in the labeling of phosphatidylinositol and phosphatidic acid (PA). Since PA acts as a calcium ionophore in several cell types, its effects upon calcium-mediated gonadotroph responses were compared with those elicited by GnRH. In rat pituitary gonadotrophs prepared by centrifugal elutriation, PA stimulated LH release and cGMP production by 9-fold and 5-fold, respectively. The stimulation of LH release by 30 microM PA was biphasic in its dependence on extracellular calcium concentration, rising from zero in the absence of calcium to a maximum of 10-fold at 0.5 mM Ca2+ and declining at higher calcium concentrations. In dose-response experiments, PA was 3-fold more potent at 0.5 mM Ca2+ than at 1.2 mM Ca2+. The cGMP response to PA in cultured gonadotrophs was also calcium dependent, and was progressively enhanced by increasing Ca2+ concentrations up to 1.5 mM. The ability of PA to stimulate both LH release and cGMP formation in a calcium-dependent manner suggests that endogenous PA formed in response to GnRH receptor activation could function as a Ca2+ ionophore in pituitary gonadotrophs, and may participate in the stimulation of gonadotroph responses by GnRH and its agonist analogs.     

Calcium-dependent release of NO from intracellular S-nitrosothiols

The paper describes a novel cellular mechanism for rapid calcium-dependent nitric oxide (NO) release. This release occurs due to NO liberation from S-nitrosothiols. We have analysed the changes of NO concentration in acutely isolated pancreatic acinar cells. Supramaximal acetylcholine (ACh) stimulation induced a Ca(2+)-dependent increase in the fluorescence in the majority of cells loaded with the NO probe DAF-FM via a patch pipette. The ACh-induced NO signals were insensitive to inhibitors of calmodulin and protein kinase C but were inhibited by calpain antagonists. The initial part of the NO signals induced by 10 muM ACh showed little sensitivity to inhibition of NO synthase (NOS); however, cell pretreatment with NO donors (increasing cellular S-nitrosothiol contents) substantially enhanced the initial component of NO responses. Pancreatic acinar cells were able to generate fast calcium-dependent NO responses when stimulated with physiological or supramaximal doses of secretagogues. Importantly, the source of this NO is the already available S-nitrosothiol store rather than de novo synthesis by NOS. A similar mechanism of NO release was found in dorsal root ganglia neurons.     

The endothelium-derived vasoconstrictor peptide endothelin inhibits renin release in vitro

The effect of endothelin, a newly identified endothelium-derived vasoconstrictor peptide, on renin release from rat kidney cortical slices was examined. Endothelin produced a concentration-dependent inhibition of renin release and this inhibitory effect was dependent on extracellular calcium. The dihydropyridine calcium channel blockers nifedipine and nicardipine did not antagonize the inhibitory effect induced by endothelin. On the other hand, nifedipine completely antagonized the extracellular high potassium- or Bay K 8644-induced inhibition of renin release. The endothelin-induced inhibition of the release was markedly blocked by the addition of Co2+. Similar blocking effects of Co2+ were also observed with extracellular high potassium or Bay K 8644. Thus, endothelin exerts an inhibitory action on renin release in vitro, in a calcium-dependent manner. This inhibition may be mediated by the increased calcium influx through dihydropyridine-insensitive calcium channels.     

Gonadotropin-releasing hormone stimulation of pituitary gonadotrope cells produces an increase in intracellular calcium

Gonadotropin-releasing hormone (GnRH) stimulates pituitary gonadotrope cells to release luteinizing hormone (LH). Previous studies have indicated a role for Ca+2 in this process; however, the present study provides the first measurements of an increased intracellular Ca+2 concentration. Pituitary cell cultures enriched for gonadotropes were loaded with quin 2, a fluorescent Ca+2-sensitive molecule. Subsequent addition of GnRH to these cells produced a rapid (within 10 sec) increase in fluorescence (indicating an increase in intracellular free Ca+2). In contrast, two GnRH analogs, des1 GnRH (a very low-affinity binder to the GnRH receptor) and Ac[D-pCl-Phe1,2] DTrp3 DLys6 DAla10-GnRH (a pure GnRH antagonist) produced no such Ca+2 change, thus showing a correlation between increased intracellular Ca+2 and LH release. A functional relationship between increased Ca+2 and LH release was suggested by experiments in which LH release was inhibited from cells loaded with high levels of intracellular quin 2 (in order to chelate intracellular Ca+2). Since this inhibition was completely reversed by addition of the Ca+2 ionophore A23187, quin 2 was not toxic at the concentrations used and apparently inhibited LH release by buffering intracellular Ca+2. The results presented here are consistent with the hypothesis that GnRH-stimulated LH release is mediated by increased intracellular Ca+2 and support the notion that the rate-limiting step in GnRH-stimulated LH release is distal to Ca+2 mobilization.     

Histamine release from human basophils by synthetic block co-polymers composed of polyoxyethylene and polyoxypropylene and synergy with immunologic and non-immunologic stimuli

Co-polymers composed of polyoxyethylene and polyoxypropylene have been shown previously to trigger histamine release from mouse peritoneal mast cells; this property quantitatively is directly related to the ionophorous ability of these compounds to cause a functional exchange of intracellular K+ for extracellular Na+ across the cell membrane. We investigated the effect of an inflammatory copolymer, T130R2, on human basophils. The data demonstrate that T130R2 can cause calcium-dependent histamine release from human basophils in vitro. Further, at concentrations that do not cause histamine release, this co-polymer markedly augments release by suboptimal concentrations of the lectin Con A or anti-IgE antibody and the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate but not the calcium ionophore A23187. Thus, these co-polymers induce mediator release from cells of both rodents and humans. In both instances it is likely that calcium-dependent cell triggering is the result of an influx of sodium ions with concomitant depolarization of the transmembrane potential. In common with the calcium ionophore A23187, the co-polymer T130R2 has the ability to synergize with stimuli which trigger the IgE receptor as well as those which directly activate the cellular calcium- and phospholipid-dependent protein kinase.     

The influence of gabaergic drugs upon LH secretion from the pituitary of the common carp: an in vitro study

In the present study, the perifusions of whole pituitary glands of spermiating male common carp were performed in the presence of several GABAergic drugs. Muscimol (agonist of GABA(A) receptors) and bicuculline (the antagonist of the same type of GABA receptors) did not modify basal LH release. LH basal secretion was not modified when pituitaries were perifused with baclofen--an agonist of GABAB receptors. On the other hand, baclofen at doses of 10(-8) and 10(-4) M significantly decreased GnRH-A-induced LH release to about 86% and 88% of LH levels in control group, respectively. In our previous study we have shown that GABA decreased basal and GnRH-A-stimulated in vivo and in vitro LH release. In conclusion, it can be suggested that in the mature male carp GABA exerts an inhibitory influence on GnRH-stimulated LH release, probably through the inhibition of the GnRH action on gonadotropes. This inhibition seems to be mediated by the B type of GABA receptors.     

Modulatory actions of estradiol and progesterone on phorbol ester-stimulated LH secretion from cultured rat pituitary cells.

We compared the ability of estradiol and progesterone to modulate gonadotropin-releasing hormone (GnRH) and protein kinase C (PKC)-mediated luteinizing hormone (LH) secretion. Long-term (48 h) treatment of rat pituitary cells with 1 nM estradiol enhanced GnRH and phorbol ester (TPA)-stimulated LH secretion. This positive effect was facilitated by additional short-term (4 h) treatment with progesterone (100 nM). However, long-term progesterone treatment, which inhibited GnRH-stimulated LH secretion, did not influence TPA-stimulated gonadotropin release. These steroid actions occurred without an effect on the total amount of LH in the cell cultures (total LH = LH secreted + LH remaining in the cell) and neither the secretagogues nor the steroids altered total LH. Since GnRH or TPA-induced LH secretion depends on Ca2+ influx into the gonadotroph, we also analyzed the effects of estradiol and progesterone under physiological extracellular Ca2+ concentrations and in the absence of extracellular Ca2+. The steroids were able to influence GnRH or TPA-induced LH secretion under both conditions. However, when TPA was used as stimulus in Ca(2+)-deficient medium the relative changes induced by estradiol and progesterone were more pronounced, possibly indicating that the extracellular Ca(2+)-independent component of PKC-mediated LH secretion is more important for the regulation of the steroid effects. It is concluded that estradiol and progesterone might mediate their modulatory actions on GnRH-stimulated LH secretion via an influence on PKC. This effect can occur independently from de novo synthesis of LH and Ca2+ influx into gonadotrophs.     

Calcium mobilization and influx during the biphasic cytosolic calcium and secretory responses in agonist-stimulated pituitary gonadotrophs

Stimulation of enriched pituitary gonadotrophs by gonadotropin-releasing hormone (GnRH) elicits dose-dependent biphasic elevations of cytosolic calcium ([Ca2+]i) and luteinizing hormone (LH) release, with rapid initial peaks followed by sustained plateaus during continued exposure to the agonist. A potent GnRH-antagonist, [N-acetyl-D-p-Cl-Phe1,2,D-Trp3,D-Lys6,D-Ala10]GnRH, prevented the biphasic [Ca2+]i and LH responses when added before GnRH, and rapidly abolished both responses to GnRH when added during the plateau phase. In low Ca2+ medium the LH peak responses to GnRH were reduced and the subsequent sustained responses were almost completely abolished; reduction of extracellular Ca2+ during exposure to GnRH caused a prompt decline of LH release. The initial [Ca2+]i peak is derived largely from intracellular calcium mobilization with a partial contribution from calcium influx, while the sustained phase is dependent on the entry of extracellular Ca2+ through both L-type and dihydropyridine-insensitive channels. The presence of L-type voltage-sensitive calcium channels (VSCC) in pituitary gonadotrophs was indicated by the ability of elevated extracellular [K+] to stimulate calcium influx and LH release, and the sensitivity of these responses to dihydropyridine agonist and antagonist analogs. In cells pretreated with high [K+], the peak [Ca2+]i response to GnRH was enhanced but the subsequent plateau phase was markedly attenuated. This divergent effect of sustained membrane depolarization on the biphasic [Ca2+]i response suggests that calcium entry through VSCC initially potentiates agonist-induced mobilization of Ca2+ from intracellular storage sites. However, established Ca2+ entry through depolarization-activated VSCC cannot be further increased by agonist stimulation because both processes operate through the same channels, probably by changes in their activation-inactivation kinetics. Finally, the reciprocal potentiation by the dihydropyridine agonist, BK 8644, and GnRH of [Ca2+]i and LH responses confirms that both compounds act on the same type of channels, i.e., L-type VSCC, that participate in agonist-mediated calcium influx and gonadotropin secretion.     

Calcium signalling in wound-responsive normal human urothelial cell monolayers

Epithelial tissue repair requires coordination of migratory and proliferative activity both adjacent to and remote from the wound edge. Although calcium signalling is implicated, the specific mechanisms are poorly understood. This study characterises the calcium signal invoked in response to scratch wounding of normal human urothelial (NHU) cells and relates it to the localised cellular response. Immediately after wounding of confluent NHU cell monolayers, cells adjacent to the wound edge showed a sustained (>30 min) rise in [Ca(2+)](i), while there was an independent, but simultaneous calcium wave that propagated out from the wound edge. The transient signal involved release of calcium from intracellular stores and was not mediated via gap junctions, but by diffusion of extracellular agonists. We demonstrated that ATP was partially responsible for the initiation and propagation of the calcium wave and showed that the calcium release mechanism was mediated in part via activation of inositol-1,4,5-triphosphate (IP(3)) receptors. By contrast, the sustained calcium signal originated from the extracellular milieu and correlated with an increased rate of migration by these cells. The work presented here provides supportive evidence that the calcium signature, defined by its temporal and amplitude characteristics, is important in co-ordinating the response of cells within an epithelial cell monolayer after wounding.     

Coordinate actions of arachidonic acid and protein kinase C in gonadotropin-releasing hormone-stimulated secretion of luteinizing hormone

The relative contributions of arachidonic acid and protein kinase C during GnRH-stimulated LH release were investigated in cultured rat anterior pituitary cells. Maximal or near-maximal concentrations of arachidonic acid or the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate, were less effective than a maximal dose of GnRH in stimulating LH release. However, the effect of a combination of arachidonic acid and phorbol ester was equivalent with that of GnRH. The protein kinase C inhibitor, retinal, significantly reduced GnRH- and phorbol-induced, but not arachidonic acid-stimulated, LH release. The lipoxygenase inhibitors, 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid, partially inhibited GnRH- and arachidonic acid-stimulated, but not phorbol-induced, LH secretion. Simultaneous addition of retinal and either lipoxygenase inhibitor completely abolished LH responses elicited by GnRH, as well as by combined treatment with arachidonic acid and the phorbol ester. These results suggest that hormone release is mediated by phospholipid-dependent mechanisms that are coordinated during the stimulation of LH secretion by GnRH.     

Biphasic activation of cytosolic free calcium and LH responses by gonadotropin-releasing hormone

Gonadotropin-releasing hormone (GnRH) stimulates rapid peak increases in [Ca2+]i and LH release, followed by lower but sustained elevations of both [Ca2+]i and hormone secretion. Omission of extracellular Ca2+ only slightly decreased the peak of [Ca2+]i, but reduced the peak LH response by 40% and prevented the prolonged increases in [Ca2+]i and LH release. Dihydropyridine calcium antagonists did not affect the peak [Ca2+]i and LH responses, but reduced the sustained increases by up to 50%. Whereas GnRH-induced mobilization of intracellular calcium initiates the LH peak, and Ca2+ entry through dihydropyridine-insensitive channels contributes to the peak and plateau phases of LH release, dihydropyridine-sensitive L-type Ca2+ channels participate only in the sustained phase of gonadotropin secretion.