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.     

Generation and amplification of the cytosolic calcium signal during secretory responses to gonadotropin-releasing hormone

Gonadotropin-releasing hormone (GnRH) stimulates characteristic biphasic increases in cytosolic calcium concentration ([Ca2+]i) and in luteinizing hormone (LH) release in cultured gonadotrophs, with an early peak followed by a prolonged plateau in both responses. Analysis of [Ca2+]i by dual-wavelength fluorimetric assay and of LH release at 5-sec intervals in perifused pituitary cells revealed increases in both responses within a few seconds of exposure to GnRH. The maximum elevation of [Ca2+]i occurred within 20 sec, and the peak gonadotropin release in 35 sec; the total duration of the spike phase for both [Ca2+]i and LH release was 2.5 min. Under extracellular Ca2(+)-deficient conditions, the GnRH-induced peak in [Ca2+]i was reduced by about 20% and the plateau phase was abolished. Concomitantly, the magnitude of the acute phase of LH release was reduced by 40% and that of the second phase by about 90%. Recovery of the plateau phase of LH release occurred within 25 sec after addition of 1.25 mM Ca2+ to Ca2(+)-deficient medium. In a dose-dependent manner, the non-selective Ca2+ channel blockers Co2+ and Cd2+ reduced the Ca2+ current measured by whole-cell recording in pituitary gonadotrophs and abolished the extracellular Ca2(+)-dependent component of LH release. The selective calcium channel blocker, nifedipine, decreased the magnitude of the Ca2+ current and reduced the plateau phase of LH release by 50%; conversely, the dihydropyridine agonist methyl, 1,4,dihydro-2,6-dimethyl 3-nitro-4-(2-trifluorome) (Bay K 8644) consistently enhanced the amplitudes of both Ca2+ current and GnRH-induced LH release. These data reveal a close temporal correlation between changes in [Ca2+]i and LH release during GnRH action, with Ca2+ mobilization during the spike phase and Ca2+ influx through dihydropyridine-sensitive and insensitive sets of receptor-operated calcium channels during the spike and plateau phases. In addition, analysis of the magnitudes of the [Ca2+]i and LH responses to a wide range of GnRH concentrations in the presence and absence of extracellular Ca2+ is consistent with amplification of the [Ca2+]i signal in agonist-stimulated gonadotrops.     

Mechanisms of secretory responses to gonadotropin-releasing hormone and phorbol esters in cultured pituitary cells. Participation of protein kinase C and extracellular calcium mobilization

The role of protein kinase C in luteinizing hormone (LH) release was analyzed in studies on the actions of gonadotropin releasing hormone (GnRH) and phorbol esters in cultured pituitary cells. During incubation in normal medium, GnRH stimulated LH release with an ED50 of 0.35 nM. Incubation in Ca2+-deficient medium (Ca2+-free, 10 microM) substantially decreased but did not abolish the LH responses to GnRH. The extracellular Ca2+-dependent component of GnRH action could be mimicked by high K+ concentrations, consistent with the presence of voltage-sensitive calcium channels (VSCC) in pituitary gonadotrophs. Ca2+ channel agonist (Bay K 8644) and antagonist (nifedipine) analogs, respectively, enhanced or partially inhibited LH responses to GnRH and also to K+, the latter confirming the participation of two types of VSCC (dihydropyridine-sensitive and -insensitive) in K+-induced secretion. Phorbol esters, including 12-O-tetradecanoylphorbol-13-acetate (TPA), 4 beta-phorbol-12,13-dibenzoate, and 4 beta-phorbol-12,13-diacetate, stimulated LH release with ED50s of 5, 10, and 1000 nM, respectively, and with about 70% of the efficacy of GnRH. Phorbol ester-stimulated LH secretion was decreased but not abolished by progressive reduction of [Ca2+]e in the incubation medium, and the residual LH response was identical with that elicited by GnRH in Ca2+-deficient medium. TPA increased [Ca2+]i to a peak after 20 s in normal medium but not in the absence of extracellular Ca2+, indicating that protein kinase C (Ca2+/phospholipid-dependent enzyme) promotes calcium entry but can also mediate secretory responses without changes in calcium influx and [Ca2+]i. The extracellular Ca2+-dependent action of TPA on LH release was blocked by Co2+. However, nifedipine did not alter TPA action on [Ca2+]i and LH release. These observations indicate that protein kinase C can participate in GnRH-induced LH release that is independent of Ca2+ entry, but also promotes the influx of extracellular Ca2+ through dihydropyridine-insensitive Ca2+-channels.     

Dissociation of Ca2+ entry and Ca2+ mobilization responses to angiotensin II in bovine adrenal chromaffin cells

In fura-2-loaded bovine adrenal chromaffin cells, 0.5 microM angiotensin II (AII) stimulated a 185 +/- 19 nM increase of intracellular-free calcium [( Ca2+]i) approximately 3 s after addition. The time from the onset of the response until achieving 50% recovery (t 1/2) was 67 +/- 10 s. Concomitantly, AII stimulated both the release of 45Ca2+ from prelabeled cells, and a 4-5-fold increase of [3H]inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) levels. In the presence of 50 microM LaCl3, or when extracellular-free Ca2+ [( Ca2+]o) was less than 100 nM, AII still rapidly increased [Ca2+]i by 95-135 nM, but the t 1/2 for recovery was then only 23-27 s. In medium with 1 mM MnCl2 present, AII also stimulated a small amount of Mn2+ influx, as judged by quenching of the fura-2 signal. When [Ca2+]o was normal (1.1 mM) or low (less than 60 nM), 1-2 microM ionomycin caused [Ca2+]i to increase 204 +/- 26 nM, while also releasing 45-55% of bound 45Ca2+. With low [Ca2+]o, ionomycin pretreatment abolished both the [Ca2+]i increase and 45Ca2+ release stimulated by AII. However, after ionomycin pretreatment in normal medium, AII produced a La3+-inhibitable increase of [Ca2+]i (103 +/- 13 nM) with a t 1/2 of 89 +/- 8 s, but no 45Ca2+ release. No pretreatment condition altered AII-induced formation of [3H]Ins(1,4,5)P3. We conclude that AII increased [Ca2+]i via rapid and transient Ca2+ mobilization from Ins(1,4,5)P3- and ionomycin-sensitive stores, accompanied (and/or followed) by Ca2+ entry through a La3+-inhibitable divalent cation pathway. Furthermore, the ability of AII to activate Ca2+ entry in the absence of Ca2+ mobilization (i.e. after ionomycin pretreatment) suggests a receptor-linked stimulus other than Ca2+ mobilization initiates Ca2+ entry.     

Relationship of thyrotropin-releasing hormone-induced spike and plateau phases in cytosolic free Ca2+ concentrations to hormone secretion. Selective blockade using ionomycin and nifedipine

In clonal rat pituitary cells (GH cells), thyrotropin-releasing hormone (TRH) induced a pattern of changes in cytosolic free calcium concentrations [( Ca2+]i) composed of two phases: an acute spike phase to micromolar levels which decayed (t1/2 = 8 s) to a near-basal concentration and then rose to a prolonged plateau phase of elevated [Ca2+]i (as measured using Quin 2). Closely following these changes in [Ca2+]i, TRH stimulated a rapid "spike phase" of pronounced, but brief, enhancement of the rate of prolactin and growth-hormone secretion and then a "plateau phase" of prolonged enhancement. These two phases were dissociated using two classes of pharmacologic agents: the ionophore ionomycin, and a calcium channel antagonist nifedipine. Ionomycin (100 nM) specifically blocked (less than 90%) the spike phase of TRH action by rapidly emptying the TRH-regulated reservoir of cellular Ca2+ to generate a TRH-like spike in [Ca2+]i; nifedipine inhibited (less than 50%) the plateau phase of TRH-induced changes in [Ca2+]i and hormone secretion by preventing Ca2+ influx through voltage-dependent Ca2+ channels. These agents demonstrated that the TRH-induced spike in [Ca2+]i in GH cells is caused by release of an ionomycin-sensitive pool of cellular Ca2+ with a small component (10%) due to influx of extracellular Ca2+. The TRH-induced plateau in [Ca2+]i is due to influx of extracellular Ca2+, about half of which enters through voltage-dependent calcium channels and half of which enters via nifedipine/verapamil-insensitive influx. The TRH-induced spike in [Ca2+]i led to a burst in hormone secretion, and the plateau in [Ca2+]i produced a prolonged enhancement of secretion; the spike and plateau phases were generated independently by TRH. A spike in [Ca2+]i is necessary, but not sufficient, to induce burst release of hormone, while the prolonged rate of hormone secretion is intimately related to the steady-state [Ca2+]i.     

Melatonin inhibits pituitary adenylyl cyclase-activating polypeptide-induced increase of cyclic AMP accumulation and [Ca2+]i in cultured cells of neonatal rat pituitary

The effects of melatonin on pituitary adenylyl cyclase-activating polypeptide-induced increase of cyclic AMP and [Ca2+]i were studied in neonatal rat pituitary cells. The polypeptide increased cyclic AMP accumulation. In the presence of melatonin the increase of cyclic AMP was inhibited in a dose-dependent manner, the maximal inhibition was achieved with 1-10 nM melatonin. Pituitary adenylyl cyclase-activating polypeptide also increased [Ca2+]i in 30% of the pituitary cells and melatonin inhibited the effect. Most of the cells sensitive to adenylyl cyclase-activating polypeptide (77%) were also sensitive to GnRH, suggesting they are gonadotrophs. The remaining cells were not identified. The polypeptide-induced [Ca2+]i increase was inhibited in Ca2+-free medium in 2/3 of the cells indicating that Ca2+ influx was involved. To examine causal relationship between cyclic AMP and [Ca2+]i increase, we have studied the effect of adenylyl cyclase activation by forskolin on intracellular Ca2+ concentration. Forskolin had similar effects as adenylyl cyclase-activating polypeptide: it increased [Ca2+]i in the pituitary cells and the increase was dependent on presence of Ca2+ in the medium. Melatonin inhibited the forskolin induced [Ca2+]i increase. Our observations indicate that increase of cyclic AMP stimulates Ca2+ influx in the pituitary cells of neonatal rat and that this mechanism is involved in [Ca2+]i increase induced by the pituitary adenylyl cyclase-activating polypeptide. Because melatonin inhibits increase of cyclic AMP induced by pituitary adenylyl cyclase-activating polypeptide or forskolin, the inhibitory effect of melatonin on Ca2+-influx may be mediated by the decrease of cyclic AMP concentration. This mechanism of melatonin action has not been described previously. Because melatonin inhibits the polypeptide- or forskolin-induced [Ca2+]i also in the cells not sensitive to GnRH, melatonin receptors seem to be present on both gonadotrophs and non-gonadotrophic pituitary cells.     

Feedback inhibition of Ca2+ release by Ca2+ is the underlying mechanism of agonist-evoked intracellular Ca2+ oscillations in pancreatic acinar cells.

Oscillations of free intracellular Ca2+ concentration ([Ca2+]i) are known to occur in many cell types during physiological cell signaling. To identify the basis for the oscillations, we measured both [Ca2+]i and extracellular Ca2+ concentration ([Ca2+]o) to follow the fate of Ca2+ during stimulation of [Ca2+]i oscillations in pancreatic acinar cells. [Ca2+]i oscillations were initiated by either t-butyloxycarbonyl-Tyr(SO3)-Nle-Gly-Tyr-Nle-Asp-2-phenylethyl ester (CCK-J), which mobilized Ca2+ from the inositol 1,4,5-trisphosphate (IP3)-insensitive pool, or low concentration of cholecystokinin octapeptide (CCK-OP), which mobilized Ca2+ from the IP3-sensitive internal pool. Little Ca2+ efflux occurred during the oscillations triggered by CCK-J or CCK-OP in spite of a large average increase in [Ca2+]i. When internal store Ca2+ pumps were inhibited with thapsigargin (Tg) during [Ca2+]i oscillations, a rapid Ca2+ efflux occurred similar to that measured in intensely stimulated, nonoscillatory cells. Tg also stimulated 45Ca efflux from internal pools of cells stimulated with CCK-J or a low concentration of CCK-OP. Hence, a large fraction of the Ca2+ released during each spike is reincorporated by the internal store Ca2+ pumps. Surprisingly, when the increase in [Ca2+]i during stimulation of oscillations was prevented by loading the cells with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid, a persistent activation of Ca2+ release and Ca2+ efflux occurred. This was reflected as a persistent increase in [Ca2+]o in cells suspended at low [Ca2+]o or persistent efflux of 45Ca from internal stores of cells maintained at high [Ca2+]o. Since agonist-stimulated Ca2+ release evidently remains activated when [Ca2+]i is highly buffered, the primary mechanism determining Ca2+ oscillations must include an inhibition of Ca2+ release by [Ca2+]i. Loading the cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid had no apparent effect on the levels or kinetics of IP3 formation in agonist-stimulated cells. This suggests that [Ca2+]i regulated the oscillation by inhibition of Ca2+ release independent of its possible effects on cellular levels of IP3.     

Ca2+ homeostasis in permeabilized human neutrophils. Characterization of Ca2+-sequestering pools and the action of inositol 1,4,5-triphosphate

The regulation of Ca2+ transport by intracellular compartments was studied in digitonin-permeabilized human neutrophils, using a Ca2+-selective electrode. When incubated in a medium containing ATP and respiratory substrates, the cells lowered within 6 min the ambient [Ca2+] to a steady state of around 0.2 microM. A vesicular ATP-dependent and vanadate-sensitive non-mitochondrial pool maintained this low [Ca2+] level. In the absence of ATP, a higher Ca2+ steady state of 0.6 microM was seen, exhibiting the characteristics of a mitochondrial Ca2+ "set point." Both pools were shown to act in concert to restore the previous ambient [Ca2+] following its elevation. Thus, the mitochondria participate with the other pool(s) in decreasing [Ca2+] to the submicromolar range whereas only the nonmitochondrial pool(s) lowers [Ca2+] to the basal level. The action of inositol 1,4,5-triphosphate (IP3) which has been inferred to mediate Ca2+ mobilization in a few cell types was studied. IP3 released (detectable within 2 s) Ca2+ accumulated in the ATP-dependent pool(s) but had no effect on the mitochondria. The response was transient and resulted in desensitization toward subsequent IP3 additions. Under experimental conditions in which the ATP-dependent Ca2+ influx was blocked, the addition of IP3 resulted in a very large Ca2+ release from nonmitochondrial pool. The results strongly suggest that IP3 is a second messenger mediating intracellular Ca2+ mobilization in human neutrophils. Furthermore, the nonmitochondrial pool appears to have independent influx and efflux pathways for Ca2+ transport, a Ca2+ ATPase (the influx component) and an IP3-sensitive efflux component activated during Ca2+ mobilization.     

Ca2+ entry in T cells is activated by emptying the inositol 1,4,5-triphosphate sensitive Ca2+ pool

Using alpha-linolenic acid (ALA), one of several polyunsaturated fatty acids (PUFAs) that have previously been shown to both mobilize intracellular Ca2+ from the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pool independently of IP3 production and inhibit Ca2+ influx, the relationship between Ca2+ mobilization from intracellular stores and Ca2+ influx in T cells (JURKAT) was studied. JURKAT cells were treated with 30 microM ALA to deplete the IP3-sensitive Ca2+ pool. When the intracellular free Ca2+ concentration [( Ca2+]i) returned to basal level, fatty acid free bovine serum albumin (BSA) was added to remove extracellular and membrane bound ALA. This resulted in a sustained increase in [Ca2+]i in the absence of inositol phosphates' formation. This sustained increase in [Ca2+]i was insensitive to protein kinase C activation but was inhibited by Ni2+ ions. The extent of Ca2+ influx was found to be correlated to the amount of Ca2+ initially discharged from the IP3-sensitive Ca2+ pool by sub-optimal concentrations of ALA. Ligation of the CD3 complex of the T cell antigen receptor with an anti-CD3 antibody (OKT3) during the sustained [Ca2+]i increased (induced by a sub-optimal concentration of ALA), produced a greater response. No increase in the sustained response was observed when the CD3 complex was activated in cells pretreated with an optimal concentration of ALA. In summary, Ca2+ entry in T cells is activated by emptying of the IP3-sensitive Ca2+ pool which can be dissociated from inositol phosphate production. The rate of Ca2+ influx appears to be closely correlated to the initial discharge of Ca2+ from the IP3-sensitive Ca2+ pool, suggesting that Ca2+ may first enter the depleted pool and then is released into the cytosol.     

Interactions between calcium and protein kinase C in the control of signaling and secretion in pituitary gonadotrophs.

Single pituitary gonadotrophs exhibit episodes of spontaneous fluctuations in cytoplasmic calcium concentration [( Ca2+]i) due to entry through voltage-sensitive calcium channels (VSCC) and show prominent agonist-induced oscillations in [Ca2+]i that are generated by periodic release of intracellular Ca2+. Gonadotropin releasing hormone (GnRH) elicited three types of Ca2+ responses: at low doses, subthreshold, with an increase in basal [Ca2+]i; at intermediate doses, oscillatory, with dose-dependent modulation of spiking frequency; and at high doses, biphasic, without oscillations. Elevation of [Ca2+]i or activation of protein kinase C (PKC) did not influence the frequency of agonist-induced [Ca2+]i spikes but caused dose-dependent reductions in amplitude for all types of Ca2+ response. Stimulation of transient Ca2+ spikes by GnRH was followed by inhibition of the spontaneous fluctuations. GnRH also reduced the ability of high extracellular K+ to promote Ca2+ influx through VSCC. Activation of PKC by phorbol esters stimulated Ca2+ influx in quiescent cells but inhibited influx when VSCC were already activated, either spontaneously or by high K+. In contrast to their biphasic actions on [Ca2+]i, phorbol esters exerted only stimulatory actions on gonadotropin release, even when Ca2+ influx was concomitantly reduced. However, pituitary cells had to be primed with an appropriate [Ca2+]i level before exocytosis could be amplified by PKC. In PKC-depleted cells, all actions of phorbol esters on Ca2+ entry and amplitude modulation, and on LH release, were abolished. GnRH-induced LH secretion was also significantly reduced, especially the plateau phase of the response. These data indicate that Ca2+ and PKC serve as interacting signals during the cascade of cellular events triggered by agonist stimulation, in which Ca2+ turns cell responses on or off, and PKC amplifies the positive and negative effects of Ca2+.     

Properties of different Ca2+ pools in permeabilized rat thymocytes

The regulation of free Ca2+ concentration by intracellular pools and their participation in the mitogen-induced changes of the cytosolic free Ca2+ concentration, [Ca2+]i, was studied in digitonin-permeabilized and intact rat thymocytes using a Ca2+-selective electrode, chlortetracycline fluorescence and the Ca2+ indicator quin-2. It is shown that in permeabilized thymocytes Ca2+ can be accumulated by two intracellular compartments, mitochondrial and non-mitochondrial. Ca2+ uptake by the non-mitochondrial compartment, presumably the endoplasmic reticulum, is observed only in the presence of MgATP, is increased by oxalate and inhibited by vanadate. The mitochondria do not accumulate calcium at a free Ca2+ concentration below 1 microM. The non-mitochondrial compartment has a greater affinity for calcium and is capable of sequestering Ca2+ at a free Ca2+ concentration less than 1 microM. At free Ca2+ concentration close to the cytoplasmic (0.1 microM) the main calcium pool in permeabilized thymocytes is localized in the non-mitochondrial compartment. Ca2+ accumulated in the non-mitochondrial pool can be released by inositol 1,4,5-triphosphate (IP3) which has been inferred to mediate Ca2+ mobilization in a number of cell types. Under experimental conditions in which ATP-dependent Ca2+ influx is blocked, the addition of IP3 results in a large Ca2+ release from the non-mitochondrial pool; thus IP3 acts by activation of a specific efflux pathway rather than by inhibiting Ca2+ influx. SH reagents do not prevent IP3-induced Ca2+ mobilization. Addition of the mitochondrial uncouplers, FCCP or ClCCP, to intact thymocytes results in no increase in [Ca2+]i measured with quin-2 tetraoxymethyl ester whereas the Ca2+ ionophore A23187 induces a Ca2+ release from the non-mitochondrial store(s). Thus, the data obtained on intact cells agree with those obtained in permeabilized thymocytes. The mitogen concanavalin A increases [Ca2+]i in intact thymocytes suspended in both Ca2+-containing an Ca2+-free medium. This indicates a mitogen-induced mobilization of an intracellular Ca2+ pool, probably via the IP3 pathway.     

Role of voltage-sensitive calcium channels in [Ca2+]i and secretory responses to activators of protein kinase C in pituitary gonadotrophs

The gonadotropin secretory response of anterior pituitary cells to phorbol esters includes both extracellular Ca2(+)-dependent and -independent components (Stojilkovi? et al, 1988; J. Biol. Chem. 263, 17301-17306, 1988). In cultured pituitary cells, measurements of [Ca2+]i using Fura-2 and of LH release during cell perifusion studies revealed that the initial effects of phorbols and permeant diacylglycerols on these responses are extracellular Ca2(+)-dependent and are mediated through activation of voltage- and dihydropyridine-sensitive calcium channels. On the other hand, pretreatment with phorbol esters for 30 to 60 min inhibited subsequent [Ca2+]i responses to diacylglycerols and phorbols and significantly reduced agonist-induced biphasic [Ca2+]i responses, with no change in the number of GnRH receptors. These findings demonstrate that protein kinase C exerts both positive and negative control of [Ca2+]i, and indicate that the calcium, phospholipid dependent enzyme participates in the activation of voltage-sensitive calcium channels and hormone secretion in pituitary gonadotrophs.     

Gonadotropin-releasing hormone activates a rapid Ca2+-independent phosphodiester hydrolysis of polyphosphoinositides in pituitary gonadotrophs

Addition of gonadotropin releasing hormone (GnRH) to pituitary cells prelabeled with [32P]Pi or with myo-[2-3H]inositol, resulted in a rapid decrease in the level of [32P]phosphatidylinositol 4,5-bisphosphate (approximately 10 s), and in [32P]phosphatidylinositol 4-phosphate (approximately 1 min), followed by increased labeling of [32P]phosphatidylinositol and [32P]phosphatidic acid (1 min). GnRH stimulated the appearance of [3H]myo-inositol 1,4,5-trisphosphate (10 s), [3H]myo-inositol 1,4-bisphosphate (15 s), and [3H]myo-inositol 1-phosphate (1 min) in the presence of Li+ (10 mM). Li+ alone stimulated the accumulation of [3H]myo-inositol 1-phosphate and [3H]myo-inositol 1,4-bisphosphate but not [3H]myo-inositol 1,4,5-trisphosphate, but had no effect on luteinizing hormone release. The effect of GnRH on inositol phosphates (Ins-P) production was dose-related (ED50 = 1-5 nM), and was blocked by a potent antagonist [D-pGlu,pClPhe,D-Trp]GnRH. Elevation of cytosolic free Ca2+ levels ([Ca2+]i), by ionomycin and A23187 from intracellular or extracellular Ca2+ pools, respectively, had no significant effect on [3H]Ins-P production. GnRH-induced [3H]Ins-P production was not dependent on extracellular Ca2+ and was noticed also after extracellular or intracellular Ca2+ mobilization by A23187 or ionomycin, respectively. The effect of GnRH on [3H]Ins-P accumulation was not affected by prior treatment of the cells with the tumor promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate or with islet-activating protein pertussis toxin. These results indicate that GnRH stimulates a rapid phosphodiester hydrolysis of polyphosphoinositides. The stimulatory effect is not mediated via an islet-activating protein-substrate, is not dependent on elevation of [Ca2+]i, neither is it negatively regulated by 12-O-tetradecanoylphorbol-13-acetate which activates Ca2+/phospholipid-dependent protein C kinase. The results are consistent with the hypothesis that GnRH-induced phosphoinositide turnover is responsible for Ca2+ mobilization followed by gonadotropin release.     

The role of Ca2+ in steroidogenesis in Leydig cells. Stimulation of intracellular free Ca2+ by lutropin (LH), luliberin (LHRH) agonist and cyclic AMP.

The requirements of purified rat Leydig cells for intra- and extra-cellular Ca2+ during steroidogenesis stimulated by LH (lutropin), cyclic AMP analogues and LHRH (luliberin) agonist were investigated. The intracellular Ca2+ concentrations ([Ca2+]i) were measured by using the fluorescent Ca2+ chelator quin-2. The basal [Ca2+]i was found to be 89.4 +/- 16.6 nM (mean +/- S.D., n = 25). LH, 8-bromo cyclic AMP and dibutyryl cyclic AMP increased [Ca2+]i, by 300-500 nM at the highest concentrations of each stimulator, whereas LHRH agonist only increased [Ca2+]i by a maximum of approx. 60 nM. Low concentrations of LH (less than 1 pg/ml) and all concentrations of LHRH agonist increased testosterone without detectable changes in cyclic AMP. With amounts of LH greater than 1 pg/ml, parallel increases in cyclic AMP and [Ca2+]i occurred. The steroidogenic effect of the LHRH agonist was highly dependent on extracellular Ca2+ concentration ([Ca2+]e), whereas LH effects were only decreased by 35% when [Ca2+]e was lowered from 2.5 nM to 1.1 microM. No increase in [Ca2+]i occurred with the LHRH agonist in the low-[Ca2+]e medium, whereas LH (100 ng/ml) gave an increase of 52 nM. It is concluded that [Ca2+]i can be modulated in rat Leydig cells by LH via mechanisms that are both independent of and dependent on cyclic AMP, whereas LHRH-agonist action on [Ca2+]i is independent of cyclic AMP. The evidence obtained suggests that, at sub-maximal rates of testosterone production, Ca2+, rather than cyclic AMP, is the second messenger, whereas for maximum steroidogenesis both Ca2+- and cyclic-AMP-dependent pathways may be involved.     

GnRH stimulates LH release directly via inositol phosphate and indirectly via cAMP in African catfish

In African catfish, two gonadotropin-releasing hormone (GnRH) peptides have been identified: chicken GnRH (cGnRH)-II and catfish GnRH (cfGnRH). The GnRH receptors on pituitary cells producing gonadotropic hormone signal through inositol phosphate (IP) elevation followed by increases in intracellular calcium concentration (?Ca(2+)(i)). In primary pituitary cell cultures of male African catfish, both cGnRH-II and cfGnRH dose dependently elevated IP accumulation, ?Ca(2+)(i), and the release of the luteinizing hormone (LH)-like gonadotropin. In all cases, cGnRH-II was more potent than cfGnRH. The GnRH-stimulated LH release was not associated with elevated cAMP levels, and forskolin-induced cAMP elevation had no effect on LH release. With the use of pituitary tissue fragments, however, cAMP was elevated by GnRH, and forskolin was able to stimulate LH secretion. Incubating these fragments with antibodies against cfGnRH abolished the forskolin-induced LH release but did not compromise the forskolin-induced cAMP elevation. This suggests that cfGnRH-containing nerve terminals are present in pituitary tissue fragments and release cfGnRH via cAMP signaling on GnRH stimulation, whereas the GnRH receptors on gonadotrophs use IP/?Ca(2+)(i) to stimulate the release of LH.     

Thyrotropin-releasing hormone-induced spike and plateau in cytosolic free Ca2+ concentrations in pituitary cells. Relation to prolactin release

Using the acetoxymethyl ester of "Quin 2," a fluorescent Ca2+-indicator, we have loaded prolactin (PRL)-producing rat pituitary cells with non-toxic concentrations of Quin 2 and quantitated changes in cytosolic free calcium concentration ( [Ca2+]i) during stimulation of PRL release by thyrotropin-releasing hormone (TRH) and 40 mM K+. TRH induced a biphasic response, with an immediate (less than 1 s) spike in [Ca2+]i from basal levels (350 +/- 80 nM) to a peak of 1-3 microM, which decayed rapidly (t 1/2 = 8 s) to a near basal nadir, then rising to a plateau in [Ca2+]i of 500-800 nM. The TRH-induced spike phase was attenuated but not abolished by prior addition of EGTA, while the plateau phase was eliminated by EGTA. Addition of 40 mM K+ caused an immediate spike in [Ca2+]i to 1-3 microM which equilibrated slowly (t 1/2 = 1 min) directly to a plateau of 600-800 nM. The K+-induced spike and plateau phases were both abolished by prior addition of EGTA. The biphasic nature of TRH action on [Ca2+]i parallels the biphasic actions of TRH on 45Ca2+ fluxes and the biphasic release of PRL by GH cells in suspension. These findings provide evidence that Ca2+-dependent agonist-mediated increases in [Ca2+]i and hormone release are linked, and may generally have two modes: an acute "spike" mode, dependent primarily on redistribution of intracellular Ca2+ stores; and a sustained "plateau" mode, dependent on influx of extracellular Ca2+.     

Production of NAADP and its role in Ca2+ mobilization associated with lysosomes in coronary arterial myocytes

The present study was designed to determine the production of nicotinic acid adenine dinucleotide phosphate (NAADP) and its role associated with lysosomes in mediating endothelin-1 (ET-1)-induced vasoconstriction in coronary arteries. HPLC assay showed that NAADP was produced in coronary arterial smooth muscle cells (CASMCs) via endogenous ADP-ribosyl cyclase. Fluorescence microscopic analysis of intracellular Ca2+ concentration ([Ca2+]i) in CASMCs revealed that exogenous 100 nM NAADP increased [Ca2+]i by 711 +/- 47 nM. Lipid bilayer experiments, however, demonstrated that NAADP did not directly activate ryanodine (Rya) receptor Ca2+ release channels on the sarcoplasmic reticulum. In CASMCs pretreated with 100 nM bafilomycin A1 (Baf), an inhibitor of lysosomal Ca2+ release and vacuolar proton pump function, NAADP-induced [Ca2+]i increase was significantly abolished. Moreover, ET-1 significantly increased NAADP formation in CASMCs and resulted in the rise of [Ca2+]i in these cells with a large increase in global Ca2+ level of 1,815 +/- 84 nM. Interestingly, before this large Ca2+ increase, a small Ca2+ spike with an increase in [Ca2+]i of 529 +/- 32 nM was observed. In the presence of Baf (100 nM), this ET-1-induced two-phase [Ca2+]i response was completely abolished, whereas Rya (50 microM) only markedly blocked the ET-1-induced large global Ca2+ increase. Functional studies showed that 100 nM Baf significantly attenuated ET-1-induced maximal constriction from 82.26 +/- 4.42% to 51.80 +/- 4.36%. Our results suggest that a lysosome-mediated Ca2+ regulatory mechanism via NAADP contributes to ET-1-induced Ca2+ mobilization in CASMCs and consequent vasoconstriction of coronary arteries.     

Contribution of external and internal Ca2+ to changes in intracellular free Ca2+ produced by mitogens in Swiss 3T3 fibroblasts: the role of dihydropyridine sensitive Ca2+ channels

Changes in intracellular free Ca2+ concentration [( Ca2+]i) produced by growth factors and mitogens have been studied using aequorin-loaded Swiss 3T3 cells. Decreasing free Ca2+ in the external medium by using EGTA had no significant effect on the increase in [Ca2+]i produced by vasopressin, bradykinin, bombesin or prostaglandin E2, but reduced the increase in [Ca2+]i produced by platelet derived growth factor (PDGF) by 58%, by prostaglandin E1 44% and by prostaglandin F2 alpha 47%. The dihydropyridine Ca2+-channel antagonist nifedipine at 10 microM inhibited the [Ca2+]i response to PDGF by 41% in both the presence of and in the absence of external Ca2+. Methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl) pyridine-5-carboxylate (BAY K8644), a Ca2+-channel agonist, at 10 microM produced an increase in [Ca2+]i and decreased the [Ca2+]i response to PDGF by 39%. Nifedipine did not block 45Ca2+ uptake or release by inositol 1,4,5-trisphosphate in saponin-permeabilized Swiss 3T3 fibroblasts but BAY K8644 inhibited 45Ca2+ release by inositol 1,4,5-trisphosphate. The results suggest that the increase in [Ca2+]i caused by PDGF in Swiss 3T3 fibroblasts is due to the influx of external Ca2+ through dihydropyridine sensitive Ca2+ channels, as well as release of internal Ca2+.     

Ca2+ dependence of Ca2+ release from intracellular stores of intact and permeabilized Ehrlich ascites tumour cells

Effects of Ca2+ ions on the mobilization of Ca2+ from intracellular stores of intact and permeabilized (15 microM digitonin) Ehrlich ascites tumour cells (EATC) have been compared. For permeabilized cells, the dependences of the initial rate and amplitude of Ca2+ mobilization evoked by the addition of 100 nM inositol 1,4,5-trisphosphate (IP3) on preexisting [Ca2+] were bell-shaped within a [Ca2+] range 10(-7)-10(-6) M with the maxima at [Ca2+] = 166 nM. In intact cells, different concentrations of free cytosolic Ca2+ ([Ca2+]i) were produced using low (up to 0.005%) concentrations of digitonin which selectively increased the permeability of the plasma membrane. Stimulation of cells by exogenous ATP at [Ca2+]i = 10(-8)-10(-6) M resulted in Ca2+ mobilization the rate and amplitude of which were maximal at 102-115 nM Ca2+. The experimental Ca2+ dependences were fit by a model which includes channel opening upon Ca2+ binding and transition to the inactive states upon Ca2+ binding to the closed and open channel forms. Three inactivation types (including two particular cases) demonstrate a slight priority of inhibitory binding of Ca2+ only to the open channel, but predict markedly different parameter values. We conclude that an increase in [Ca2+] can stimulate IP3-induced mobilization, but in intact EATC, deviations of [Ca2+]i from the resting level (about 100 nM) attenuate responses to the agonist stimulation.     

Ba2+ stimulation of luteinizing-hormone release demonstrates two mechanisms of Ca2+ entry in gonadotrope cells.

Kinetic studies on gonadotropin-releasing-hormone (gonadoliberin, GnRH)-stimulated luteinizing-hormone (lutropin, LH) release in the cultured rat gonadotrope demonstrated a biphasic pattern of LH release. The first rapid phase of release was unaffected by the voltage-gated Ca2+-channel blockers methoxyverapamil (D600) and nifedipine [a dihydropyridine (DHP)], whereas the later second phase was partially inhibited by both drugs. These results suggested that the initial phase of LH release is independent of Ca2+ entry through dihydropyridine (DHP)-sensitive Ca2+ channels and might depend on entry of extracellular Ca2+ by another mechanism. These mechanisms were further studied by utilizing Ba2+ as a Ca2+ substitute. Ba2+, which freely permeates DHP-sensitive Ca2+ channels in the absence of GnRH, induced LH release which was sensitive to blockade by D600 and nifedipine. However, in the presence of the channel blockers, Ba2+-induced LH release could be elicited when GnRH was added to the system. This indicates that GnRH stimulates LH release by initially activating a DHP-insensitive Ca2+-entry mechanism and then a DHP-sensitive mechanism. The DHP-sensitive mechanism freely allows Ba2+ entry in the absence of GnRH-receptor occupancy, whereas the DHP-insensitive mechanism requires GnRH-receptor activation for Ba2+ entry.