Inositol trisphosphate is required for the propagation of calcium waves in Xenopus oocytes.

Stimuli which act through the second messenger inositol 1,4,5-trisphosphate (InsP3) often increase free intracellular Ca2+ concentration ([Ca2+]i) in a localized subcellular area. Actively propagated Ca2+ waves then extend this focal Ca2+ signal to other parts of the cell. To understand how cells may control the spatial distribution of Ca2+, we investigated the mechanism by which Ca2+ waves propagate through the cytoplasm of Xenopus oocytes. Heparin, which inhibits the binding of InsP3 to its receptor, prevented the migration of Ca2+ waves induced by a poorly metabolized InsP3 (InsP3S3). This result suggested that Ca2+ waves move through the cell via the serial release of Ca2+ from InsP3-sensitive stores. Interventions which caused a localized increase in [Ca2+]i without elevations of InsP3 did not trigger Ca2+ waves. In the presence of a Ins-P3S3, however, endogenously released or locally injected Ca2+ elicited Ca2+ waves. A cooperative interaction between Ca2+ and InsP3 may therefore be responsible for the propagation of Ca2+ waves.     

Inositol 1,4,5-trisphosphate and its co-players in the concert of Ca2+ signalling--new faces in the line up

Release of Ca2+ from intracellular stores can occur by different intracellular messengers such as InsP3, cADPR and NAADP. Although in some cells messengers may operate on different stores, there are also Ca2+ stores with sensitivities for all three of these messengers. It is well documented, that InsP3- and cADPR-sensitive Ca2+ stores are involved in the activation of "store-operated Ca2+ channels" (SOCC). It has not yet been unequivocally shown, however, if Ca2+ release from stores, which respond to NAADP but not to InsP3 or cADPR, also generate signals which lead to "store-operated Ca2+ entry". Neither localization nor the mechanism of coupling to the plasma membrane of those InsP3- and cADPR-sensitive Ca2+ stores which activate SOCCs is yet clear. In this review localization and properties of InsP3-, cADPR- and NAADP-sensitive Ca2+ pools and their mutual interactions are discussed. Differential sensitivities of Ca2+ release mechanisms to InsP3, cADPR and NAADP have consequences on Ca2+ release, Ca2+ oscillations, propagation of Ca2+ waves and on activation of SOCC. Possible interaction of InsP3R and cADPR with candidates of SOCCs (TRP channels) and mechanisms involved in the regulation of SOCCs (activation-deactivation) will be elaborated.     

Hormonal regulation of phosphatidylinositol breakdown

Cyclic AMP and Ca2+ are intracellular mediators of hormone action. Catecholamines interact with beta adrenoceptors to activate adenylate cyclase or with alpha 2 adrenoceptors to inhibit adenylate cyclase. Alpha 1 adrenoceptor activation results in elevation of cytosol Ca2+ and an increased breakdown of phosphatidylinositol. In blowfly salivary glands, 5-hydroxytryptamine (5-HT) interacts with beta type receptors resulting in adenylate cyclase activation while alpha type receptors are involved in phosphatidylinositol breakdown and elevation of cytosol Ca2+. The link between Ca2+ mobilization and phosphatidylinositol breakdown remains to be established but breakdown of the receptor-regulated pool of phosphatidylinositol is not secondary to the rise in Ca2+. Direct addition of 5-HT to cell-free homogenates of blowfly salivary glands results in activation of phosphatidylinositol breakdown in the absence of Ca2+. In rat liver plasma membrane preparations, vasopressin increases phosphatidylinositol breakdown in the absence of Ca2+ or cytosol if deoxycholate is present. The data do not indicate whether hormone activation increases the availability of substrate to enzymatic hydrolysis or activates phospholipase C. However, they demonstrate that hormones directly accelerate phosphatidylinositol breakdown.     

Histamine-H1-receptor-mediated phosphoinositide hydrolysis, Ca2+ signalling and membrane-potential oscillations in human HeLa carcinoma cells.

In human HeLa carcinoma cells, histamine causes a dose-dependent formation of inositol phosphates, production of diacylglycerol and a transient rise in intracellular [Ca2+]. These responses are completely blocked by the H1-receptor antagonist pyrilamine. In streptolysin-O-permeabilized cells, formation of inositol phosphates by histamine is strongly potentiated by guanosine 5'-[gamma-thio]triphosphate and inhibited by guanosine 5'-[beta-thio]diphosphate, suggesting the involvement of a GTP-binding protein. Histamine stimulates the rapid but transient formation of Ins(1,4,5)P3, Ins(1,3,4)P3 and InsP4. InsP accumulates in a much more persistent manner, lasting for at least 30 min. Studies with streptolysin-O-permeabilized cells indicate that InsP accumulation results from dephosphorylation of Ins(1,4,5)P3, rather than direct hydrolysis of PtdIns. The rise in intracellular [Ca2+] is biphasic, with a very fast release of Ca2+ from intracellular stores, that parallels the Ins(1,4,5)P3 time course, followed by a more prolonged phase of Ca2+ influx. In individual cells, histamine causes a rapid initial hyperpolarization of the plasma membrane, which can be mimicked by microinjected Ins(1,4,5)P3. Histamine-induced hyperpolarization is followed by long-lasting oscillations in membrane potential, apparently owing to periodic activation of Ca2+-dependent K+ channels. These membrane-potential oscillations can be mimicked by microinjection of guanosine 5'-[gamma-thio]triphosphate, but are not observed after microinjection of Ins(1,4,5)P3. We conclude that H1-receptors in HeLa cells activate a PtdInsP2-specific phospholipase C through participation of a specific G-protein, resulting in long-lasting oscillations of cytoplasmic free Ca2+.     

Calcium release and subsequent development induced by modification of sulfhydryl groups in porcine oocytes.

The mechanism of Ca2+ release induced by modification of sulfhydryl groups and the subsequent activation of porcine oocytes were investigated. Thimerosal, a sulfhydryl-oxidizing compound, induced Ca2+ oscillation in matured oocytes. In thimerosal-preincubated oocytes, the amount of Ca2+ released after microinjection of inositol 1,4,5-trisphosphate (InsP3) or ryanodine increased strikingly, indicating that thimerosal potentiated both InsP3- and ryanodine-sensitive Ca2+ release pathways. Thimerosal also enhanced the sensitivity of oocytes to microinjected Ca2+ so that in pretreated oocytes a Ca2+ injection triggered a larger transient. Heparin at concentrations that normally block the InsP3-induced Ca2+ release were without effect; higher doses significantly increased the time leading up to the first spike. The thimerosal-induced Ca2+ release could not be blocked by procaine, and it did not require the formation of InsP3 since preinjection with neomycin did not prevent the oscillation. Immunocytochemistry revealed that thimerosal treatment destroyed the meiotic spindle, preventing further development, an effect that could be reversed by dithiothreitol. The combined thimerosal/dithiothreitol treatment triggered second polar body extrusion in 50% of the oocytes, and as a result of this activation scheme approximately 15% of the in vitro- and approximately 60% of the in vivo-matured oocytes developed to blastocyst during a 7-day culture in vitro.     

Intercellular Ca2+ waves in mechanically stimulated articular chondrocytes

Articular cartilage is a tissue designed to withstand compression during joint movement and, in vivo, is subjected to a wide range of mechanical loading forces. Mechanosensitivity has been demonstrated to influence chondrocyte metabolism and cartilage homeostasis, but the mechanisms underlying mechanotransduction in these cells are poorly understood. In many cell types mechanical stimulation induces increases of the cytosolic Ca2+ concentration that propagates from cell to cell as an intercellular Ca2+ wave. Cell-to-cell communication through gap junctions underlies tissue co-ordination of metabolism and sensitivity to extracellular stimuli: gap junctional permeability to intracellular second messengers allows signal transduction pathways to be shared among several cells, ultimately resulting in co-ordinated tissue responses. Mechanically-induced Ca2+ signalling was investigated with digital fluorescence video imaging in primary cultures of rabbit articular chondrocytes. Mechanical stimulation of a single cell, obtained by briefly distorting the plasmamembrane with a micropipette, induced a wave of increased Ca2+ that was communicated to surrounding cells. Intercellular Ca2+ spreading was inhibited by 18 alpha-glycyrrhetinic acid, suggesting the involvement of gap junctions in signal propagation. The functional expression of gap junctions was assessed, in confluent chondrocyte cultures, by the intercellular transfer of Lucifer yellow dye in microinjection experiments while the expression of connexin 43 could be detected in Western blots. A series of pharmacological tools known to interfere with the cell calcium handling capacity were employed to investigate the mechanism of mechanically-induced Ca2+ signalling. In the absence of extracellular Ca2+ mechanical stimulation induced communicated Ca2+ waves similar to controls. Mechanical stress induced Ca2+ influx both in the stimulated chondrocyte but not in the adjacent cells, as assessed by the Mn2+ quenching technique. Cells treatment with thapsigargin and with the phospholipase C inhibitor U73122 blocked mechanically-induced signal propagation. These results provide evidence that in chondrocytes mechanical stimulation activates phospholipase C, thus leading to an increase of intracellular inositol 1,4,5-trisphosphate. The second messenger, by permeating gap junctions, stimulates intracellular Ca2+ release in neighbouring cells. Intercellular Ca2+ waves may provide a mechanism to co-ordinate tissue responses in cartilage physiology.     

Receptor-oriented intercellular calcium waves evoked by vasopressin in rat hepatocytes.

Agonist-induced intracellular calcium signals may propagate as intercellular Ca2+ waves in multicellular systems as well as in intact organs. The mechanisms initiating intercellular Ca2+ waves in one cell and determining their direction are unknown. We investigated these mechanisms directly on fura2-loaded multicellular systems of rat hepatocytes and on cell populations issued from peripheral (periportal) and central (perivenous) parts of the hepatic lobule. There was a gradient in vasopressin sensitivity along connected cells as demonstrated by low vasopressin concentration challenge. Interestingly, the intercellular sensitivity gradient was abolished either when D-myo-inositol 1,4, 5-trisphosphate (InsP3) receptor was directly stimulated after flash photolysis of caged InsP3 or when G proteins were directly stimulated with AlF4-. The gradient in vasopressin sensitivity in multiplets was correlated with a heterogeneity of vasopressin sensitivity in the hepatic lobule. There were more vasopressin-binding sites, vasopressin-induced InsP3 production and V1a vasopressin receptor mRNAs in perivenous than in periportal cells. Therefore, we propose that hormone receptor density determines the cellular sensitivity gradient from the peripheral to the central zones of the liver cell plate, thus the starting cell and the direction of intercellular Ca2+ waves, leading to directional activation of Ca2+-dependent processes.     

Regulatory and spatial aspects of inositol trisphosphate-mediated calcium signals

Hormones that act to release Ca2+ from intracellular stores initiate a signaling cascade that culminates in the production of inositol 1,4,5-trisphosphate (InsP3). The Ca2+ response mediated by InsP3 is not a sustained increase in the cytosolic Ca2+ concentration, but rather a series of periodic spikes that manifest as waves in larger cells. In vitro studies have determined that the key positive feedback parameter driving spikes and waves is a highly localized direct Ca(2+)-activation of InsP3-gated Ca2+ channels. Advances in fluorescent Ca2+ imaging have facilitated the resolution of individual positive feedback units. These studies have revealed that there are several modes of channel coupling underlying global Ca2+ signals; single channel openings or Ca2+ "blips," synchronized clusters of channels or Ca2+ "puffs," and cell wide calcium waves. It appears that the channel clusters that produce Ca2+ puffs are synchronized by the highly localized positive feedback that was predicted by the in vitro studies of channel regulation. Localization of InsP3-induced Ca2+ signals has been shown to be important for activation of several cellular processes including uni-directional salt flow and mitochondrial activation.     

Calcium signaling in liver

In hepatocytes, hormones linked to the formation of the second messenger inositol 1,4,5-trisphosphate (InsP3) evoke transient increases or spikes in cytosolic free calcium ([Ca2+]i), that increase in frequency with the agonist concentration. These oscillatory Ca2+ signals are thought to transmit the information encoded in the extracellular stimulus to down-stream Ca2+-sensitive metabolic processes. We have utilized both confocal and wide field fluorescence microscopy techniques to study the InsP3-dependent signaling pathway at the cellular and subcellular levels in the intact perfused liver. Typically InsP3-dependent [Ca2+]i spikes manifest as Ca2+ waves that propagate throughout the entire cytoplasm and nucleus, and in the intact liver these [Ca2+]i increases are conveyed through gap junctions to encompass entire lobular units. The translobular movement of Ca2+ provides a means to coordinate the function of metabolic zones of the lobule and thus, liver function. In this article, we describe the characteristics of agonist-evoked [Ca2+]i signals in the liver and discuss possible mechanisms to explain the propagation of intercellular Ca2+ waves in the intact organ.     

Subcellular organization of agonist-evoked Ca(2+) waves in the blowfly salivary gland

We have studied the subcellular organization of intra- and intercellular Ca(2+)waves elicited by the neurohormone 5-hydroxytryptamine (5-HT) in intact blowfly salivary glands by using Ca(2+)-sensitive fluorescent probes and confocal microscopy. 5-HT (3 nM) elicited repetitive Ca(2+)waves (1) that were initiated at Ca(2+)-release sites close to the basal plasma membrane, (2) that sequentially spread to the cell apex and (3) that, after a delay of 0.7 +/- 0.20 s at the cell boundaries, spread into adjacent cells. [Ca(2+)](i)increases in the adjacent cells were first detectable at those portions of the lateral plasma membrane that faced a previously activated cell. Electron microscopy revealed that the sites of Ca(2+)wave transmission between the cells are correlated with the distribution of gap junctions that cluster in the basal cell portions. The ensuing intracellular Ca(2+)wave propagated at constant velocity (27 +/- 7.3 microm/s) in the lateral cell plane. Moreover, a basally to apically propagating wavefront was detectable at the cell membrane that bordered on the neighbor that provided the excitatory signal, whereas [Ca(2+)](i)increased simultaneously both apically and basally at the opposite lateral cell border. Overall, the subcellular patterns of Ca(2+)wave propagation differed from the patterns observed in mammalian secretory epithelial cells. The findings impose some constraints on the functional significance of intra- and intercellular Ca(2+)waves and potential mechanisms underlying 5-HT-evoked fluid secretion.     

Inositol 1,4,5-triphosphate microinjection triggers activation, but not meiotic maturation in amphibian and starfish oocytes

Inositol 3,4,5-triphosphate (InsP3) brought about cortical granule exocytosis and elevation of a fertilization membrane, due to a rapid increase of free calcium in cytoplasm, when injected into oocytes of the amphibian Xenopus laevis arrested at second meiotic metaphase. The same result was observed when injection was performed into oocytes of the starfish Marthasterias glacialis arrested either at the first meiotic prophase or after completion of meiosis. Although meiotic maturation was induced in both animals by specific hormones which have been previously shown to release Ca2+ within cytoplasm, InsP3 microinjection into prophase-arrested oocytes did not release them from prophase block.     

Regulation of phosphoinositide breakdown by guanine nucleotides

Phosphoinositide hydrolysis is coupled to receptor systems involved in the elevation of cytosolic Ca2+ and activation of protein kinase C. In cell-free systems, guanine nucleotides are required to transduce the effects of receptor activation to phosphoinositide breakdown. Non-hydrolyzable guanine nucleotides stimulate phosphoinositide breakdown in permeabilized cells as well as membranes prepared from salivary glands, GH3 cells, neutrophils, hepatocytes and cerebral cortical tissue. In blowfly salivary gland membranes, 5-hydroxytryptamine stimulates a guanine-nucleotide dependent breakdown of both endogenous and exogenous phosphoinositide substrate through activation of phospholipase C. These data suggest that a GTP-binding protein modulates phospholipase C activity. The identity of this GTP-binding protein has not been established but may resemble other regulatory GTP-binding proteins which have been identified as transducing proteins in a variety of receptor systems.     

Mechanism of Ca2+ inhibition of inositol 1,4,5-trisphosphate (InsP3) binding to the cerebellar InsP3 receptor.

Ca2+ efficiently inhibits binding of inositol 1,4,5-trisphosphate (InsP3) to the InsP3 receptor in cerebellar membranes but not to the purified receptor. We have now investigated the mechanism of action by which Ca2+ inhibits InsP3 binding. Our results suggest that Ca2+ does not cause the stable association of a Ca(2+)-binding protein with the receptor. Instead, Ca2+ leads to the production of a soluble, heat-stable, low molecular weight substance from cerebellar membranes that competes with InsP3 for binding. This inhibitory substance probably represents endogenously generated InsP3 as judged by the fact that it co-purifies with InsP3 on anion-exchange chromatography, competes with [3H]InsP3 binding in a pattern similar to unlabeled InsP3, and is in itself capable of releasing 45Ca2+ from permeabilized cells. A potent Ca(2+)-activated phospholipase C activity producing InsP3 was found in cerebellar microsomes that exhibited a Ca2+ dependence identical to the Ca(2+)-dependent inhibition of InsP3 binding. Together these results suggest that the Ca(2+)-dependent inhibition of InsP3 binding to the cerebellar receptor is due to activation of a Ca(2+)-sensitive phospholipase C enriched in cerebellum. Nevertheless, Ca2+ probably also modulates the InsP3 receptor function by a direct interaction with the receptor that does not affect InsP3 binding but regulates InsP3-dependent channel gating.     

NMDA-receptor regulation of muscarinic-receptor stimulated inositol 1,4,5-trisphosphate production and protein kinase C activation in single cerebellar granule neurons

Inositol 1,4,5-trisphosphate (InsP(3)) production in single cerebellar granule neurons (CGNs) grown in culture was measured using the PH domain of phospholipase C delta1 tagged with enhanced green fluorescent protein (eGFP-PH(PLCdelta1)). These measurements were correlated with changes in intracellular free Ca2+ determined by single cell imaging. In control CGNs, intracellular Ca2+ stores appeared replete. However, the refilling state of these stores appeared dependent on the fluorophore used to measure Ca2+-release. Thus, methacholine (MCH), acting via muscarinic acetylcholine-receptors (mAchRs), mobilised intracellular Ca2+ in cells loaded with fluo-3 and fura-4f, but not fura-2. Confocal measurements of single CGNs expressing eGFP-PH(PLCdelta1) demonstrated that MCH stimulated a robust peak increase in InsP(3), which was followed by a sustained plateau phase of InsP(3) production. In contrast, glutamate-induced InsP(3) signals were weak or not detectable. MCH-stimulated InsP(3) production was reduced by chelation of intracellular Ca2+ with BAPTA, and emptying of intracellular stores with thapsigargin, indicated a positive feedback effect of Ca2+ mobilisation onto PLC activity. In CGNs, NMDA- and KCl-mediated Ca2+-entry significantly enhanced MCH-induced InsP(3) production. Furthermore, mAchR-mediated PLC activation appeared sensitive to the full dynamic range of intracellular Ca2+ increases stimulated by 100 microm NMDA. This dynamic regulation was also observed at the level of PKC activation indicated by an enhanced translocation of eGFP-tagged myristoylated alanine-rich C kinase substrate (MARCKS) protein in cells stimulated with MCH. Thus, NMDA-mediated Ca2+ influx and PLC activation may represent a coincident-detection system whereby ionotropic and metabotropic signals combine to stimulate InsP(3) production and PKC-mediated phosphorylation events in CGNs.     

Calcium wave propagation in pancreatic acinar cells: functional interaction of inositol 1,4,5-trisphosphate receptors, ryanodine receptors, and mitochondria

In pancreatic acinar cells, inositol 1,4,5-trisphosphate (InsP(3))-dependent cytosolic calcium ([Ca(2+)](i)) increases resulting from agonist stimulation are initiated in an apical "trigger zone," where the vast majority of InsP(3) receptors (InsP(3)R) are localized. At threshold stimulation, [Ca(2+)](i) signals are confined to this region, whereas at concentrations of agonists that optimally evoke secretion, a global Ca(2+) wave results. Simple diffusion of Ca(2+) from the trigger zone is unlikely to account for a global [Ca(2+)](i) elevation. Furthermore, mitochondrial import has been reported to limit Ca(2+) diffusion from the trigger zone. As such, there is no consensus as to how local [Ca(2+)](i) signals become global responses. This study therefore investigated the mechanism responsible for these events. Agonist-evoked [Ca(2+)](i) oscillations were converted to sustained [Ca(2+)](i) increases after inhibition of mitochondrial Ca(2+) import. These [Ca(2+)](i) increases were dependent on Ca(2+) release from the endoplasmic reticulum and were blocked by 100 microM ryanodine. Similarly, "uncaging" of physiological [Ca(2+)](i) levels in whole-cell patch-clamped cells resulted in rapid activation of a Ca(2+)-activated current, the recovery of which was prolonged by inhibition of mitochondrial import. This effect was also abolished by ryanodine receptor (RyR) blockade. Photolysis of d-myo InsP(3) P(4(5))-1-(2-nitrophenyl)-ethyl ester (caged InsP(3)) produced either apically localized or global [Ca(2+)](i) increases in a dose-dependent manner, as visualized by digital imaging. Mitochondrial inhibition permitted apically localized increases to propagate throughout the cell as a wave, but this propagation was inhibited by ryanodine and was not seen for minimal control responses resembling [Ca(2+)](i) puffs. Global [Ca(2+)](i) rises initiated by InsP(3) were also reduced by ryanodine, limiting the increase to a region slightly larger than the trigger zone. These data suggest that, while Ca(2+) release is initially triggered through InsP(3)R, release by RyRs is the dominant mechanism for propagating global waves. In addition, mitochondrial Ca(2+) import controls the spread of Ca(2+) throughout acinar cells by modulating RyR activation.     

Regulation of inositol trisphosphate receptors by luminal Ca2+ contributes to quantal Ca2+ mobilization.

The quantal behaviour of inositol trisphosphate (InsP3) receptors allows rapid graded release of Ca2+ from intracellular stores, but the mechanisms are unknown. In Ca2+-depleted stores loaded with Fura 2, InsP3 caused concentration dependent increases in the rates of fluorescence quench by Mn2+ that were unaffected by prior incubation with InsP3, indicating that InsP3 binding did not cause desensitization. When Fura 2 was used to report the luminal free [Ca2+] after inhibition of further Ca2+ uptake, submaximal concentrations of InsP3 caused rapid, partial decreases in fluorescence ratios. Subsequent addition of a maximal InsP3 concentration caused the fluorescence to fall to within 5% of that recorded after ionomycin. Addition of all but the lowest concentrations of InsP3 to stores loaded with the lower affinity indicator, Calcium Green-5N, caused almost complete emptying of the stores at rates that increased with InsP3 concentration. The lowest concentration of InsP3 (10 nM) slowly emptied approximately 80% of the stores, but within 3 min the rate of Ca2+ release slowed leaving approximately 7 microM Ca2+ within the stores, which was then rapidly released by a maximal InsP3 concentration. In stores co-loaded with both indicators, InsP3-evoked Ca2+ release appeared quantal with Fura 2 and largely non-quantal with Calcium Green-5N; the discrepancy is not, therefore, a direct effect of the indicators. The fall in luminal [Ca2+] after activation of InsP3 receptors may, therefore, cause their inactivation, but only after the Ca2+ content of the stores has fallen by approximately 95% to < or = 10 microM.     

Animal-vegetal axis patterning mechanisms in the early sea urchin embryo

During mouse fertilization the spermatozoon induces a series of low-frequency long-lasting Ca(2+) oscillations. It is generally accepted that these oscillations are due to Ca(2+) release through the inositol 1,4,5-trisphosphate (InsP(3)) receptor. However, InsP(3) microinjection does not mimic sperm-induced Ca(2+) oscillations, leading to the suggestion that the spermatozoon causes Ca(2+) release by sensitizing the InsP(3) receptor to basal levels of InsP(3). This contradicts recent evidence that the spermatozoon triggers Ca(2+) oscillations by introducing a phospholipase C or else an activator of phospholipase C. Here we show for the first time that sperm-induced Ca(2+) oscillations may be mimicked by the photolysis of caged InsP(3) in both mouse metaphase II eggs and germinal vesicle stage oocytes. Eggs, and also oocytes that had displayed spontaneous Ca(2+) oscillations, gave long-lasting Ca(2+) oscillations when fertilized or when caged InsP(3) was photolyzed. In contrast, oocytes that had shown no spontaneous Ca(2+) oscillations did not generate many oscillations when fertilized or following photolysis of caged InsP(3). Fertilization in eggs was most closely mimicked when InsP(3) was uncaged at relatively low amounts for extended periods. Here we observed an initial Ca(2+) transient with superimposed spikes, followed by a series of single transients with a low frequency; all characteristics of the Ca(2+) changes at fertilization. We therefore show that InsP(3) can mimic the distinctive pattern of Ca(2+) release in mammalian eggs at fertilization. It is proposed that a sperm Ca(2+)-releasing factor operates by generating a continuous small amount of InsP(3) over an extended period of time, consistent with the evidence for the involvement of a phospholipase C.     

Mechanism of Ca2+ release at fertilization in mammals.

At fertilization in mammals the sperm triggers a series of oscillations in intracellular Ca2+ within the egg. These Ca2+ oscillations activate the development of the egg into an embryo. It is not known how the sperm triggers these Ca2+ oscillations. There are currently three different theories for Ca2+ signaling in eggs at fertilization. One idea is that the sperm acts as a conduit for Ca2+ entry into the egg after membrane fusion. Another idea is that the sperm acts upon plasma membrane receptors to stimulate a phospholipase C (PLC) within the egg which generates inositol 1,4, 5-trisphosphate (InsP(3)). We present a third idea that the sperm causes Ca2+ release by introducing a soluble protein factor into the egg after gamete membrane fusion. In mammals this sperm factor is also referred to as an oscillogen because, after microinjection, the factor causes sustained Ca2+ oscillations in eggs. Our recent data in sea urchin egg homogenates and intact eggs suggests that this sperm factor has phospholipase C activity that leads to the generation of InsP(3). We then present a new version of the soluble sperm factor theory of signaling at fertilization. J. Exp. Zool. (Mol. Dev. Evol.) 285:267-275, 1999.     

A mammalian capacitative calcium entry channel homologous to Drosophila TRP and TRPL.

Intracellular Ca2+ signalling evoked by Ca2+ mobilizing agonists, like angiotensin II in the adrenal gland, involves the activation of inositol(1,4,5)trisphosphate(InsP3)-mediated Ca2+ release from internal stores followed by activation of a Ca2+ influx termed capacitative calcium entry. Here we report the amino acid sequence of a functional capacitative Ca2+ entry (CCE) channel that supports inward Ca2+ currents in the range of the cell resting potential. The expressed CCE channel opens upon depletion of Ca2+ stores by InsP3 or thapsigargin, suggesting that the newly identified channel supports the CCE coupled to InsP3 signalling.