Modulation of intracellular calcium concentrations and T cell activation by prickly pear polyphenols

Opuntia ficus indica(prickly pear) polyphenolic compounds (OFPC) triggered an increase in [Ca²⁺]_i in human Jurkat T-cell lines. Furthermore, OFPC-induced rise in [Ca²⁺]_i was significantly curtailed in calcium-free buffer (0% Ca²⁺) as compared to that in 100% Ca²⁺ medium. Preincubation of cells with tyrphostin A9, an inhibitor of Ca²⁺ release-activated Ca²⁺(CRAC) channels, significantly diminished the OFPC-induced sustained response on the increases in [Ca²⁺]_i. Lanthanum and nifedipine, the respective inhibitors of voltage-dependent and L-type calcium channels, failed to curtail significantly the OFPC-induced calcium response. As OFPC still stimulated increases in [Ca²⁺]_i in 0% Ca²⁺ medium, the role of intracellular calcium was investigated. Hence, addition of thapsigargin (TG), an inhibitor of Ca²⁺-ATPase of the endoplasmic reticulum (ER), during the OFPC-induced peak response exerted an additive effect, indicating that the mechanism of action of these two agents are different. Furthermore, U73122, an inhibitor of IP₃ production, completely abolished increases in [Ca²⁺]_i, induced by OFPC, suggesting that these polyphenols induce the production of IP₃ that recruits calcium from ER pool. Polyphenolic compounds do act extracellularly as addition of fatty acid-free bovine serum albumin (BSA) significantly diminished the rise in [Ca²⁺]_i evoked by the formers. OFPC also induced plasma membrane hyperpolarisation which was reversed by addition of BSA. OFPC were found to curtail the expression of IL-2 mRNA and T-cell blastogenesis. Together these results suggest that OFPC induce increases in [Ca²⁺]_i via ER pool and opening of CRAC channels, and exert immunosuppressive effects in Jurkat T-cells.     

Regulation of calcium signalling by docosahexaenoic acid in human T-cells. Implication of CRAC channels

We elucidated the role of docosahexaenoic acid (DHA) on the increases in free intracellular calcium concentrations, [Ca(2+)]i, in human (Jurkat) T-cell lines. DHA evoked an increase in [Ca(2+)]i in a dose-dependent manner in these cells. Anti-CD3 antibody, known to stimulate increases in Ca(2+) from endoplasmic reticulum (ER) via the production of inositol trisphosphate, also evoked increases in [Ca(2+)]i in Jurkat T-cells. We also used thapsigargin which inhibits Ca(2+)-ATPase of the ER and, therefore, increases Ca(2+) in the cytosol. Interestingly, addition of DHA during the thapsigargin-induced peak response exerted an additive effect on the increases in [Ca(2+)]i in human T-cells, indicating that the mechanisms of action of these two agents are different. However, the DHA-induced calcium response was not observed when this agent was added during the anti-CD3-induced calcium peak, though its addition resulted in a prolonged and sustained calcium response as a function of time, suggesting that DHA recruits calcium, in part, from the ER pool and the prolonged response may be due to Ca(2+) influx. In the medium containing 0% Ca(2+), the DHA-evoked response on the increases in [Ca(2+)]i was significantly curtailed as compared to that in 100% Ca(2+) medium, supporting the notion that the response of the DHA is also due, in part, to the opening of calcium channels. Furthermore, preincubation of cells with tyrphostin A9, an inhibitor of Ca(2+) release-activated Ca(2+) (CRAC) channels also significantly curtailed the DHA-induced sustained response on the increases in [Ca(2+)]i in these cells. These results suggest that DHA induces an increase in [Ca(2+)]i via the ER pool and the opening of CRAC channels in human T-cells.     

Regulation of calcium signalling by 1-O-alkylglycerols in human Jurkat T lymphocytes

We studied the role of natural occurring 1-O-alkylglycerols on the calcium signalling in Jurkat T-cells. Alkylglycerols evoked an increase in free intracellular calcium concentration [Ca2+]i, in a dose-dependent manner. When the experiments were performed in calcium-free buffer, the alkylglycerol response on the rise of [Ca2+]i was wholly abolished compared with the one in calcium-containing buffer, suggesting that these etherlipids induce a calcium influx by the opening of Ca2+ channels. We further employed inhibitors of voltage-gated calcium channels. We observed that omega-conotoxin, a blocker of N-type voltage-activated Ca2+ channels, but not verapamil, a blocker of L-type voltage-activated Ca2+ channels, curtailed significantly the calcium rise evoked by the lipid agents. Alkylglycerols also induced plasma membrane depolarisation, known to be involved in the opening of the voltage-gated calcium channels. Our study shows that alkylglycerols increase [Ca2+]i influx in human Jurkat T-cells possibly by modulating the permeability of calcium channels.     

Role of three isoforms of phospholipase A2 in capacitative calcium influx in human T-cells.

The present study was conducted on human Jurkat T-cell lines in order to elucidate the role of phospholipase A2 in capacitative calcium entry. We have employed thapsigargin (TG) that induces increases in [Ca2+]i by emptying the calcium pool of endoplasmic reticulum, followed by capacitative calcium entry. We designed a Ca2+ free/Ca2+ reintroduction (CFCR) protocol for the experiments, conducted in Ca2+-free medium. By employing CFCR protocol, we observed that addition of exogenous arachidonic acid (AA) stimulated TG-induced capacitative calcium influx. The liberation of endogenous AA and its autocrine action seems to be implicated during TG-induced capacitative calcium influx: TG potentiates the induction of constitutively expressed mRNA of four PLA2 isoforms (type 1B, IV, V, VI), the inhibitors of the three PLA2 isotypes (type 1B, V, VI) inhibit TG-induced release of [3H]AA into the extracellular medium, and finally, these PLA2 inhibitors do curtail TG-stimulated capacitative calcium entry in these cells. These results suggest that stimulation of three isoforms of PLA2 by thapsigargin liberates free AA that, in turn, induces capacitative calcium influx in human T-cells.     

Quasi-synaptic calcium signal transmission between endoplasmic reticulum and mitochondria.

Transmission of cytosolic [Ca2+] ([Ca2+]c) oscillations into the mitochondrial matrix is thought to be supported by local calcium control between IP3 receptor Ca2+ channels (IP3R) and mitochondria, but study of the coupling mechanisms has been difficult. We established a permeabilized cell model in which the Ca2+ coupling between endoplasmic reticulum (ER) and mitochondria is retained, and mitochondrial [Ca2+] ([Ca2+]m) can be monitored by fluorescence imaging. We demonstrate that maximal activation of mitochondrial Ca2+ uptake is evoked by IP3-induced perimitochondrial [Ca2+] elevations, which appear to reach values >20-fold higher than the global increases of [Ca2+]c. Incremental doses of IP3 elicited [Ca2+]m elevations that followed the quantal pattern of Ca2+ mobilization, even at the level of individual mitochondria. In contrast, gradual increases of IP3 evoked relatively small [Ca2+]m responses despite eliciting similar [Ca2+]c increases. We conclude that each mitochondrial Ca2+ uptake site faces multiple IP3R, a concurrent activation of which is required for optimal activation of mitochondrial Ca2+ uptake. This architecture explains why calcium oscillations evoked by synchronized periodic activation of IP3R are particularly effective in establishing dynamic control over mitochondrial metabolism. Furthermore, our data reveal fundamental functional similarities between ER-mitochondrial Ca2+ coupling and synaptic transmission.     

The T-cell antigen receptor regulates sustained increases in cytoplasmic free Ca2+ through extracellular Ca2+ influx and ongoing intracellular Ca2+ mobilization.

Signal transduction by the T-cell antigen receptor involves the turnover of polyphosphoinositides and an increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). This increase in [Ca2+]i is due initially to the release of Ca2+ from intracellular stores, but is sustained by the influx of extracellular Ca2+. To examine the regulation of sustained antigen-receptor-mediated increases in [Ca2+]i, we studied the relationships between extracellular Ca2+ influx, the mobilization of Ca2+ from intracellular stores, and the contents of inositol polyphosphates after stimulation of the antigen receptor on a human T-cell line, Jurkat. We demonstrate that sustained antigen-receptor-mediated increases in [Ca2+]i are associated with ongoing depletion of intracellular Ca2+ stores. When antigen-receptor-ligand interactions are disrupted, [Ca2+]i and inositol 1,4,5-trisphosphate return to basal values over 3 min. Under these conditions, intracellular Ca2+ stores are repleted if extracellular Ca2+ is present. There is a tight temporal relationship between the fall in [Ca2+]i, the return of inositol 1,4,5-trisphosphate to basal values, and the repletion of intracellular Ca2+ stores. Reversal of the increase in [Ca2+]i preceeds any fall in inositol tetrakisphosphate by 2 min. These studies suggest that sustained antigen-receptor-induced increases in [Ca2+]i, although dependent on extracellular Ca2+ influx, are also regulated by ongoing inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ mobilization. In addition, an elevated concentration of inositol tetrakisphosphate in itself is insufficient to sustain an increase in [Ca2+]i within Jurkat cells.     

Rapid inactivation of depletion-activated calcium current (ICRAC) due to local calcium feedback

Rapid inactivation of Ca2+ release-activated Ca2+ (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole-cell patch clamp recording and [Ca2+]i measurement techniques. In the presence of 22 mM extracellular Ca2+, the Ca2+ current declined with a biexponential time course (time constants of 8-30 ms and 50-150 ms) during hyperpolarizing pulses to potentials more negative than -40 mV. Several lines of evidence suggest that the fast inactivation process is Ca2+ but not voltage dependent. First, the speed and extent of inactivation are enhanced by conditions that increase the rate of Ca2+ entry through open channels. Second, inactivation is substantially reduced when Ba2+ is present as the charge carrier. Third, inactivation is slowed by intracellular dialysis with BAPTA (12 mM), a rapid Ca2+ buffer, but not by raising the cytoplasmic concentration of EGTA, a slower chelator, from 1.2 to 12 mM. Recovery from fast inactivation is complete within 200 ms after repolarization to -12 mV. Rapid inactivation is unaffected by changes in the number of open CRAC channels or global [Ca2+]i. These results demonstrate that rapid inactivation of ICRAC results from the action of Ca2+ in close proximity to the intracellular mouths of individual channels, and that Ca2+ entry through one CRAC channel does not affect neighboring channels. A simple model for Ca2+ diffusion in the presence of a mobile buffer predicts multiple Ca2+ inactivation sites situated 3-4 nm from the intracellular mouth of the pore, consistent with a location on the CRAC channel itself.     

Mechanisms of calcium elevation in the micromeres of sea urchin embryos

The micromeres, the first cells to be specified in sea urchin embryos, are generated by unequal cleavage at the fourth cell division. The micromeres differentiate autonomously to form spicules and dispatch signals to induce endomesoderm in the neighbouring macromeres cells in the embryo. Using a calcium indicator Fura-2/AM and a mixture of dextran conjugated Oregon green-BAPTA 488 and Rhodamine red, the intracellular calcium ion concentration ([Ca2+]i) was studied in embryos at the 16-cell stage. [Ca2+]i was characteristically elevated in the micromeres during furrowing at the 4th cleavage. Subsequently, Ca2+ oscillated for about 10 min in the micromeres, resulting in episodic high levels of [Ca2+]i. High [Ca2+]i regions were associated with regional localizations of the endoplasmic reticulum (ER), though not with ER accumulated at the vegetal pole of the micromeres during the 4th division. Pharmacological studies, using a blocker of IP3-mediated Ca2+ release (Xestospongin), a store-operated Ca2+ entry inhibitor (2 aminoethoxydiphenyl borate (2-APB)) and an inhibitor of stretch-dependent ion channels (gadolinium), suggest that the high [Ca2+]i and oscillations in the micromeres are triggered by calcium influx caused by the activation of stretch-dependent calcium channels, followed by the release of calcium ions from the endoplasmic reticulum. On the basis of these new findings, a possible mechanism for autonomous formation of the micromeres is discussed.     

Ceramide increase cytoplasmic Ca2+ concentration in Jurkat T cells by liberation of calcium from intracellular stores and activation of a store-operated calcium channel

The effect of ceramide on the cytoplasmic Ca2+ concentration ([Ca2+]i) varies depending on the cell type. We have found that in Jurkat human T cells ceramide increases the [Ca2+]i from a thapsigargin-sensitive calcium pool and the subsequent activation of a capacitative Ca2+ entry. This effect occurs both in the presence and in the absence of extracellular calcium. Addition of ceramine, a non-hydrolysable analogue of ceramide, reproduced its effect on the [Ca2+]i ruling out that this is due to the conversion of ceramide to sphingosine. The effect of ceramide was additive to that obtained by sphingosine, but not to the Jurkat T cells specific antibody OKT3. However, different to the latter, ceramide do not induced an elevation of InsP3. The opening of a store operated Ca2+ channel by ceramide was corroborated by experiments of Fura-2 quenching, using Mn2+ as a surrogate for Ca2+ and confirmed by whole-cell recording patch clamp techniques.     

Sensing and refilling calcium stores in an excitable cell.

Inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ mobilization leads to depletion of the endoplasmic reticulum (ER) and an increase in Ca2+ entry. We show here for the gonadotroph, an excitable endocrine cell, that sensing of ER Ca2+ content can occur without the Ca2+ release-activated Ca2+ current (Icrac), but rather through the coupling of IP3-induced Ca2+ oscillations to plasma membrane voltage spikes that gate Ca2+ entry. Thus we demonstrate that capacitative Ca2+ entry is accomplished through Ca(2+)-controlled Ca2+ entry. We develop a comprehensive model, with parameter values constrained by available experimental data, to simulate the spatiotemporal behavior of agonist-induced Ca2+ signals in both the cytosol and ER lumen of gonadotrophs. The model combines two previously developed models, one for ER-mediated Ca2+ oscillations and another for plasma membrane potential-driven Ca2+ oscillations. Simulations show agreement with existing experimental records of store content, cytosolic Ca2+ concentration ([Ca2+]i), and electrical activity, and make a variety of new, experimentally testable predictions. In particular, computations with the model suggest that [Ca2+]i in the vicinity of the plasma membrane acts as a messenger for ER content via Ca(2+)-activated K+ channels and Ca2+ pumps in the plasma membrane. We conclude that, in excitable cells that do not express Icrac, [Ca2+]i profiles provide a sensitive mechanism for regulating net calcium flux through the plasma membrane during both store depletion and refilling.     

Interplay between ER Ca2+ uptake and release fluxes in neurons and its impact on [Ca2+] dynamics

In neurons, depolarizing stimuli open voltage-gated Ca2+ channels, leading to Ca2+ entry and a rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i). While such [Ca2+]i elevations are initiated by Ca2+ entry, they are also influenced by Ca2+ transporting organelles such as mitochondria and the endoplasmic reticulum (ER). This review summarizes contributions from the ER to depolarization-evoked [Ca2+]i responses in sympathetic neurons. As in other neurons, ER Ca2+ uptake depends on SERCAs, while passive Ca2+ release depends on ryanodine receptors (RyRs). RyRs are Ca2+ permeable channels that open in response to increases in [Ca2+]i, thereby permitting [Ca2+]i elevations to trigger Ca2+ release through Ca(2+)-induced Ca2+ release (CICR). However, whether this leads to net Ca2+ release from the ER critically depends upon the relative rates of Ca2+ uptake and release. We found that when RyRs are sensitized with caffeine, small evoked [Ca2+]i elevations do trigger net Ca2+ release, but in the absence of caffeine, net Ca2+ uptake occurs, indicating that Ca2+ uptake is stronger than Ca2+ release under these conditions. Nevertheless, by increasing ER Ca2+ permeability, RyRs reduce the strength of Ca2+ buffering by the ER in a [Ca2+](I)-dependent manner, providing a novel mechanism for [Ca2+]i response acceleration. Analysis of the underlying Ca2+ fluxes provides an explanation of this and two other modes of CICR that are revealed as [Ca2+]i elevations become progressively larger.     

Gonadotropin-releasing hormone-induced rise in cytosolic free Ca2+ levels: mobilization of cellular and extracellular Ca2+ pools and relationship to gonadotropin secretion

Addition of GnRH to pituitary gonadotrophs preloaded with Quin 2 resulted in a rapid (approximately 8 s) mobilization of an ionomycin-sensitive intracellular Ca2+ pool. A second component of Ca2+ entry via voltage dependent channels contributed about 45% of the peak cytosolic free Ca2+ concentration ([Ca2+]i). Thereafter, influx of Ca2+ via voltage-sensitive and -insensitive channels is responsible for maintenance of elevated [Ca2+]i during the second phase of GnRH action. Addition of inositol 1,4,5-trisphosphate (IP3) to permeabilized pituitary cells resulted in a Ca2+ transient, released from a nonmitochondrial pool, which maintained ambient free Ca2+ concentration around 170 nM in an ATP-dependent mechanism. Successive stimulations of the cells with IP3 produced an attenuated response. Elevation of the gonadotroph [Ca2+]i by ionomycin, to levels equivalent to that induced by GnRH, resulted in LH release amounting to only 45% of the response to the neurohormone. Activation of the voltage-dependent Ca2+ channels by the dihydropyridine Ca2+-agonist [methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine- 5-carboxylate (BAYK8644)] stimulated LH release, 36% of the GnRH (100 nM) response being reached by 10(-8) M of the drug, both [Ca2+]i elevation and GnRH-induced LH release were inhibited similarly (40-50%) by the dihydropyridine Ca2+-antagonist nifedipine. The results indicate that peak [Ca2+]i induced by GnRH in pituitary gonadotrophs is derived mainly from ionomycin-sensitive cellular stores most likely via IP3 formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphatidic acid increases inositol-1,4,5,-trisphosphate and [Ca2+]i levels in neonatal rat cardiomyocytes.

Phosphatidic acid (PA), which can be synthesized de novo, or as a product of phosphatidylcholine hydrolysis and/or phosphorylation of 1,2-diacylglycerol (DAG), mediates diverse cellular functions in various cell types, including cardiomyocytes. We set out to characterize the effect of PA on intracellular free calcium ([Ca2+]i) and inositol-1,4,5-trisphosphate (IP(3)) levels in primary cultures of neonatal rat cardiomyocytes. Addition of PA led to rapid, concentration and time dependent increases in both IP(3) and [Ca2+]i levels in adherent cells. There was strong correlation in the concentration-response relationships between IP(3) and [Ca2+]i increases evoked by PA. Incubation with the sarcoplasmic reticulum (SR) Ca2+ pump inhibitor, cyclopiazonic acid (CPA), significantly attenuated the PA evoked [Ca2+]i increase but had no significant effect on IP(3) accumulation. The phospholipase C (PLC) inhibitor, D-609, attenuated both IP(3) and [Ca2+]i elevations evoked by PA whereas staurosporine (STS), a potent and non-selective PKC inhibitor, had no significant effect on either. Another PLC inhibitor, U73122, but not its inactive analog, U73343, also inhibited PA evoked increases in [Ca2+]i. Depletion of extracellular calcium attenuated both basal and PA evoked increases in [Ca2+]i. The PLA(2) inhibitors, bromophenylacyl-bromide (BPB) and CDP-choline, had no effect on PA evoked [Ca2+]i responses. Neither the DAG analog, dioctanoylglycerol, nor the DAG kinase inhibitor, R59949, affected PA evoked changes in [Ca2+]i. Taken together, these data indicate that PA, in a manner independent of PKC, DAG, or PLA(2), may enhance Ca2+ release from IP(3) sensitive SR Ca(2+) stores via activation of PLC in neonatal rat cardiomyocytes.     

Epileptogenesis induces long-term alterations in intracellular calcium release and sequestration mechanisms in the hippocampal neuronal culture model of epilepsy

Calcium and calcium-dependent processes have been hypothesized to be involved in the induction of epilepsy. It has been shown that epileptic neurons have altered calcium homeostatic mechanisms following epileptogenesis in the hippocampal neuronal culture (HNC) and pilocarpine models of epilepsy. To investigate the mechanisms causing these alterations in [Ca2+]i homeostatic processes following epileptogenesis, we utilized the HNC model of in vitro 'epilepsy' which produces spontaneous recurrent epileptiform discharges (SREDs). Using [Ca2+]i imaging, studies were initiated to evaluate the mechanisms mediating these changes in [Ca2+]i homeostasis. 'Epileptic' neurons required much longer to restore a glutamate induced [Ca2+]i load to baseline levels than control neurons. Inhibition of Ca2+ entry through voltage and receptor gated Ca2+ channels and stretch activated Ca2+ channels had no effect on the prolonged glutamate induced increase in [Ca2+]i in epileptic neurons. Employing thapsigargin, an inhibitor of the sarco/endoplasmic reticulum calcium ATPase (SERCA), it was shown that thapsigargin inhibited sequestration of [Ca2+]i by SERCA was significantly decreased in 'epileptic' neurons. Using Ca2+ induced Ca2+ release (CICR) cell permeable inhibitors for the ryanodine receptor (dantrolene) and the IP3 receptor (2-amino-ethoxydiphenylborate, 2APB) mediated CICR, we demonstrated that CICR was significantly augmented in the 'epileptic' neurons, and determined that the IP3 receptor mediated CICR was the major release mechanism altered in epileptogenesis. These data indicate that both inhibition of SERCA and augmentation of CICR activity contribute to the alterations accounting for the impaired calcium homeostatic processes observed in 'epileptic' neurons. The results suggest that persistent changes in [Ca2+]i levels following epileptogenesis may contribute to the long-term plasticity changes manifested in epilepsy and that understanding the basic mechanisms mediating these changes may provide an insight into the development of novel therapeutic approaches to treat epilepsy and prevent or reverse epileptogenesis.     

Activation of calcium oscillations by thapsigargin in parotid acinar cells.

The tumor promoter thapsigargin releases Ca2+ from intracellular stores by specific inhibition of microsomal Ca-ATPase activity without inositol phosphate formation. Recent studies of the actions of thapsigargin support the concept that the level of Ca2+ within the inositol (1,4,5)-trisphosphate (IP3)-sensitive intracellular pool regulates the Ca2+ permeability of the plasma membrane. We examined the effects of thapsigargin on intracellular Ca2+ concentration ([Ca2+]i) in single rat parotid cells using digital fluorescence microscopy. In the absence of extracellular Ca2+ (Ca2+o), thapsigargin transiently increased [Ca2+]i. Following the thapsigargin-induced [Ca2+]i transient, carbachol in the continued absence of Ca2+o was unable to raise [Ca2+]i, indicating that thapsigargin mobilizes Ca2+ from the IP3-sensitive store. In the converse experiment, carbachol prevented a rise of [Ca2+]i by thapsigargin, suggesting that the IP3- and thapsigargin-sensitive Ca2+ pools are the same. Depletion of Ca2+ from the IP3-sensitive pool by thapsigargin enhanced plasma membrane Ca2+ permeability. Thapsigargin triggered sustained Ca2+ oscillations in Ca2(+)-containing medium which are highly reminiscent of agonist-induced oscillations in these cells. Carbachol addition rapidly raised IP3 levels during oscillations triggered by thapsigargin but did not elevate [Ca2+]i, indicating that the IP3-sensitive pool remains continuously depleted during [Ca2+]i fluctuations. The results from this study rule out the involvement of the IP3-sensitive pool in the mechanisms involved in thapsigargin-induced (and by analogy, agonist-induced) oscillations in parotid cells.     

Synergistic release of Ca2+ from IP3-sensitive stores evoked by synaptic activation of mGluRs paired with backpropagating action potentials

Increases in postsynaptic [Ca2+]i can result from Ca2+ entry through ligand-gated channels or voltage-gated Ca2+ channels, or through release from intracellular stores. Most attention has focused on entry through the N-methyl-D-aspartate (NMDA) receptor in causing [Ca2+]i increases since this pathway requires both presynaptic stimulation and postsynaptic depolarization, making it a central component in models of synaptic plasticity. Here, we report that repetitive synaptic activation of metabotropic glutamate receptors (mGluRs), paired with backpropagating action potentials, causes large, wave-like increases in [Ca2+]i predominantly in restricted regions of the proximal apical dendrites and soma of hippocampal CA1 pyramidal neurons. [Ca2+]i changes of several micromolars can be reached by regenerative release caused by the synergistic effect of mGluR-generated inositol 1,4,5-trisphosphate (IP3) and spike-evoked Ca2+ entry acting on the IP3 receptor.     

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.     

Roles of Thapsigargin-Sensitive Ca2+ Stores in the Survival of Developing Cultured Neurons

The roles of the intracellular calcium pool involved in regulating the Ca2+ profile and the neuronal survival rate during development were studied by using thapsigargin (TG), a specific inhibitor of endoplasmic reticulum (ER) Ca2+-ATPase in cultured cerebellar granule neurons. Measuring the neuronal [Ca2+]i directly in the culture medium, we found a bell-shaped curve for [Ca2+]i versus cultured days in cerebellar granule neurons maintained in medium containing serum and 25 mM K+. The progressive increase in [Ca2+]i of the immature granule neurons (1-4 days in vitro) was abolished by TG, which resulted in massive neuronal apoptosis. When the [K+] was lowered from 25 to 5 mM, neither the progressively increasing [Ca2+]i nor the survival of immature granule neurons was significantly changed over 24-h incubation. Similarly, TG caused a dramatic decrease in the [Ca2+]i and survival rate of these immature neurons when switched to 5 mM K+ medium. Following maturation, the granule neurons became less sensitive to TG for both [Ca2+]i and neuronal survival. However, TG can protect mature granule neurons from the detrimental effect of switching to a 5 mM K+ serum-free medium by decreasing [Ca2+]i to an even lower level than in the respective TG-free group. Based on these findings, we propose that during the immature stage, TG-sensitive ER Ca2+-ATPase plays a pivotal role in the progressive increase of [Ca2+]i, which is essential for the growth and maturation of cultured granule neurons.