Effects of aging on inositol 1,4,5-triphosphate-induced Ca(2+) release in unfertilized mouse oocytes

We previously demonstrated in the mouse oocyte that in vivo postovulatory aging significantly suppresses activity of the endoplasmic reticulum (ER) Ca(2+)-ATPase (Igarashi et al. 1997. Mol Reprod Dev 48:383-390). We undertook the present study to further examine the effects of oocyte aging on Ca(2+) release from the inositol 1,4,5-triphosphate (InsP(3))-sensitive Ca(2+) channels of the ER membrane, because not only Ca(2+) reuptake, but also Ca(2+) release from the ER, substantially affect Ca(2+) oscillations in fertilized oocytes. A transient increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) was induced by photolysis of caged InsP(3) microinjected into the cytoplasm in both fresh (14 hr post hCG) and aged (20 hr or 24 hr post hCG) oocytes, where the maximum rate of increase in [Ca(2+)](i) significantly decreased in the aged oocytes. Reduced ER Ca(2+) release in the aged oocyte may not be attributable to aging-related desensitization of the InsP(3)-sensitive Ca(2+) channels in the ER because concentrations of caged InsP(3) for half maximal [Ca(2+)](i) increase were identical for fresh and aged oocytes. The peak [Ca(2+)](i) response following administration of 5 microM thapsigargin, a specific ER Ca(2+)-ATPase inhibitor, was significantly reduced in the aged oocyte, suggesting reduction of the ER Ca(2+) stores. We conclude from these results that reduction of Ca(2+) release from the InsP(3)-sensitive Ca(2+) stores in the aged oocyte arises from depletion of the ER Ca(2+) stores with aging. These aging-related changes in Ca(2+) release and reuptake may account for alterations in Ca(2+) oscillations in aged fertilized oocytes.     

A bimodal pattern of InsP(3)-evoked elementary Ca(2+) signals in pancreatic acinar cells

InsP(3)-evoked elementary Ca(2+) release events have been postulated to play a role in providing the building blocks of larger Ca(2+) signals. In pancreatic acinar cells, low concentrations of acetylcholine or the injection of low concentrations of InsP(3) elicit a train of spatially localized Ca(2+) spikes. In this study we have quantified these responses and compared the Ca(2+) signals to the elementary events shown in Xenopus oocytes. The results demonstrate, at the same concentrations of InsP(3), Ca(2+) signals consisting of one population of small transient Ca(2+) release events and a second distinct population of larger Ca(2+) spikes. The signal mass amplitudes of both types of events are within the range of amplitudes for the elementary events in Xenopus oocytes. However, the bimodal Ca(2+) distribution of Ca(2+) responses we observe is not consistent with the continuum of event sizes seen in Xenopus. We conclude that the two types of InsP(3)-dependent events in acinar cells are both elementary Ca(2+) signals, which are independent of one another. Our data indicate a complexity to the organization of the Ca(2+) release apparatus in acinar cells, which might result from the presence of multiple InsP(3) receptor isoforms, and is likely to be important in the physiology of these cells.     

Inositol 1,4,5-trisphosphate receptors are downregulated in mouse oocytes in response to sperm or adenophostin A but not to increases in intracellular Ca(2+) or egg activation

Fertilization in mammals stimulates a series of Ca(2+) oscillations that continue for 3-4 h. Cell-cycle-dependent changes in the ability to release Ca(2+) are one mechanism that leads to the inhibition of Ca(2+) transients after fertilization. The downregulation of InsP(3)Rs at fertilization may be an additional mechanism for inhibiting Ca(2+) transients. In the present study we examine the mechanism of this InsP(3)R downregulation. We find that neither egg activation nor Ca(2+) transients are necessary or sufficient for the stimulation of InsP(3)R downregulation. First, parthenogenetic activation fails to stimulate downregulation. Second, downregulation persists when fertilization-induced Ca(2+) transients and egg activation are inhibited using BAPTA. Third, downregulation can be induced in immature oocytes that do not undergo egg activation. Other than fertilization, the only stimulus that downregulated InsP(3)Rs was microinjection of the potent InsP(3)R agonist adenophostin A. InsP(3)R downregulation was inhibited by the cysteine protease inhibitor ALLN but MG132 and lactacystin were not effective. Finally, we have injected maturing oocytes with adenophostin A and produced MII eggs depleted of InsP(3)Rs. We show that sperm-induced Ca(2+) signaling is inhibited in such InsP(3)R-depleted eggs. These data show that InsP(3)R binding is sufficient for downregulation and that Ca(2+) signaling at fertilization is mediated via the InsP(3)R.     

Mitochondrial Ca(2+) signals in autophagy

Macroautophagy (autophagy) is a lysosomal degradation pathway that is conserved from yeast to humans that plays an important role in recycling cellular constituents in all cells. A number of protein complexes and signaling pathways impinge on the regulation of autophagy, with the mammalian target of rapamycin (mTOR) as the central player in the canonical pathway. Cytoplasmic Ca(2+) signaling also regulates autophagy, with both activating and inhibitory effects, mediated by the canonical as well as non-canonical pathways. Here we review this regulation, with a focus on the role of an mTOR-independent pathway that involves the inositol trisphosphate receptor (InsP(3)R) Ca(2+) release channel and Ca(2+) signaling to mitochondria. Constitutive InsP(3)R Ca(2+) transfer to mitochondria is required for autophagy suppression in cells in nutrient-replete media. In its absence, cells become metabolically compromised due to insufficient production of reducing equivalents to support oxidative phosphorylation. Absence of this Ca(2+) transfer to mitochondria results in activation of AMPK, which activates mTOR-independent pro-survival autophagy. Constitutive InsP(3)R Ca(2+) release to mitochondria is an essential cellular process that is required for efficient mitochondrial respiration, maintenance of normal cell bioenergetics and suppression of autophagy.     

Unitary Ca(2+) current through recombinant type 3 InsP(3) receptor channels under physiological ionic conditions

The ubiquitous inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) channel, localized primarily in the endoplasmic reticulum (ER) membrane, releases Ca(2+) into the cytoplasm upon binding InsP(3), generating and modulating intracellular Ca(2+) signals that regulate numerous physiological processes. Together with the number of channels activated and the open probability of the active channels, the size of the unitary Ca(2+) current (i(Ca)) passing through an open InsP(3)R channel determines the amount of Ca(2+) released from the ER store, and thus the amplitude and the spatial and temporal nature of Ca(2+) signals generated in response to extracellular stimuli. Despite its significance, i(Ca) for InsP(3)R channels in physiological ionic conditions has not been directly measured. Here, we report the first measurement of i(Ca) through an InsP(3)R channel in its native membrane environment under physiological ionic conditions. Nuclear patch clamp electrophysiology with rapid perfusion solution exchanges was used to study the conductance properties of recombinant homotetrameric rat type 3 InsP(3)R channels. Within physiological ranges of free Ca(2+) concentrations in the ER lumen ([Ca(2+)](ER)), free cytoplasmic [Ca(2+)] ([Ca(2+)](i)), and symmetric free [Mg(2+)] ([Mg(2+)](f)), the i(Ca)-[Ca(2+)](ER) relation was linear, with no detectable dependence on [Mg(2+)](f). i(Ca) was 0.15 +/- 0.01 pA for a filled ER store with 500 microM [Ca(2+)](ER). The i(Ca)-[Ca(2+)](ER) relation suggests that Ca(2+) released by an InsP(3)R channel raises [Ca(2+)](i) near the open channel to approximately 13-70 microM, depending on [Ca(2+)](ER). These measurements have implications for the activities of nearby InsP(3)-liganded InsP(3)R channels, and they confirm that Ca(2+) released by an open InsP(3)R channel is sufficient to activate neighboring channels at appropriate distances away, promoting Ca(2+)-induced Ca(2+) release.     

Hg(2+) affects the intracellular free Ca(2+) oscillatory pattern and the correlated membrane conductance changes in Mg(2+)-stimulated oocytes of the prawn Palaemon serratus

The impact of mercuric ions (Hg(2+)) on prawn oocytes was examined. Prawn oocytes constitute an unusual system in that they are activated at spawning by seawater Mg(2+), which mediates correlated dynamic changes in intracellular free calcium concentration [(Ca(2+))(i)] and membrane conductance associated with the meiosis resumption. Using a voltage clamp technique and intracellular calcium measurements, we observed that treatment with Hg(2+) (5, 10, and 20 microM) resulted in simultaneous impairments of both (Ca(2+))(i) and membrane current responses to external Mg(2+). Treatment with Hg(2+) also resulted in a gradual dose-dependent slow increase in the baseline level of both (Ca(2+))(i) and membrane conductance, independent of stimulation with external Mg(2+). The effect of Hg(2+) on (Ca(2+))(i) and membrane conductance changes resulted from a block of the signal transduction pathway at some point before the InsP(3) receptor channel involved in Ca(2+) release from the endoplasmic reticulum (ER) stocks. The Hg(2+)-dependent gradual increase in both (Ca(2+))(i) and membrane conductance baseline levels may potentially result from a slow permeabilization of the ER membrane, resulting in Ca(2+) leaking into the cytosol. Indeed, this effect could be blocked with the cell permeable Hg(2+) competitor dithiothreitol, which was able to displace Hg(2+) from its intracellular target regardless of whether external Ca(2+) was present or not.     

alpha(1)-adrenergic stimulation mediates Ca(2+)-dependent inositol phosphate formation through the alpha(1B)-like adrenoceptor subtype in adult rat cardiac myocytes.

We studied the effects of increased Ca(2+) influx on alpha(1)-adrenoceptor-stimulated InsP formation in adult rat cardiac myocytes. We further examined if such effects could be mediated through a specific alpha(1)-adrenoceptor subtype. [(3)H]InsP responses to adrenaline were dependent on extracellular Ca(2+) concentration, from 0.1 microM to 2 mM, and were completely blocked by Ca(2+) removal. However, in cardiac myocytes preloaded with BAPTA, a highly selective calcium chelating agent, Ca(2+) concentrations higher than 1 microM had no effect on adrenaline-stimulated [(3)H]InsP formation. Taken together these results suggest that [(3)H]InsP formation induced by alpha(1)-adrenergic stimulation is in part mediated by increased Ca(2+) influx. Consistent with this, ionomycin, a calcium ionophore, stimulated [(3)H]InsP formation. This response was additive with the response to adrenaline stimulation implying that different signaling mechanisms may be involved. In cardiac myocytes treated with the alpha(1B)-adrenoceptor alkylating agent, CEC, [(3)H]InsP formation remained unaffected by increased Ca(2+) concentrations, a pattern similar to that observed when intracellular Ca(2+) was chelated with BAPTA. In contrast, addition of the alpha(1A)-subtype antagonist, 5'-methyl urapidil, did not affect the Ca(2+) dependence of [(3)H]InsP formation. Neither nifedipine, a voltage-dependent Ca(2+) channel blocker nor the inorganic Ca(2+) channel blockers, Ni(2+) and Co(2+), had any effect on adrenaline stimulated [(3)H]InsP, at concentrations that inhibit Ca(2+) channels. The results suggest that in adult rat cardiac myocytes, in addition to G protein-mediated response, alpha(1)-adrenergic-stimulated [(3)H]InsP formation is activated by increased Ca(2+) influx mediated by the alpha(1B)-subtype.     

ATP regulation of type 1 inositol 1,4,5-trisphosphate receptor channel gating by allosteric tuning of Ca(2+) activation.

Inositol 1,4,5-trisphosphate (InsP(3)) mobilizes intracellular Ca(2+) by binding to its receptor (InsP(3)R), an endoplasmic reticulum-localized Ca(2+) release channel. Patch clamp electrophysiology of Xenopus oocyte nuclei was used to study the effects of cytoplasmic ATP concentration on the cytoplasmic Ca(2+) ([Ca(2+)](i)) dependence of single type 1 InsP(3)R channels in native endoplasmic reticulum membrane. Cytoplasmic ATP free-acid ([ATP](i)), but not the MgATP complex, activated gating of the InsP(3)-liganded InsP(3)R, by stabilizing open channel state(s) and destabilizing the closed state(s). Activation was associated with a reduction of the half-maximal activating [Ca(2+)](i) from 500 +/- 50 nM in 0 [ATP](i) to 29 +/- 4 nM in 9.5 mM [ATP](i), with apparent ATP affinity = 0.27 +/- 0.04 mM, similar to in vivo concentrations. In contrast, ATP was without effect on maximum open probability or the Hill coefficient for Ca(2+) activation. Thus, ATP enhances gating of the InsP(3)R by allosteric regulation of the Ca(2+) sensitivity of the Ca(2+) activation sites of the channel. By regulating the Ca(2+)-induced Ca(2+) release properties of the InsP(3)R, ATP may play an important role in shaping cytoplasmic Ca(2+) signals, possibly linking cell metabolic state to important Ca(2+)-dependent processes.     

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.     

Calpain-cleaved type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1) has InsP(3)-independent gating and disrupts intracellular Ca(2+) homeostasis

The type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1) is a ubiquitous intracellular Ca(2+) release channel that is vital to intracellular Ca(2+) signaling. InsP(3)R1 is a proteolytic target of calpain, which cleaves the channel to form a 95-kDa carboxyl-terminal fragment that includes the transmembrane domains, which contain the ion pore. However, the functional consequences of calpain proteolysis on channel behavior and Ca(2+) homeostasis are unknown. In the present study we have identified a unique calpain cleavage site in InsP(3)R1 and utilized a recombinant truncated form of the channel (capn-InsP(3)R1) corresponding to the stable, carboxyl-terminal fragment to examine the functional consequences of channel proteolysis. Single-channel recordings of capn-InsP(3)R1 revealed InsP(3)-independent gating and high open probability (P(o)) under optimal cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) conditions. However, some [Ca(2+)](i) regulation of the cleaved channel remained, with a lower P(o) in suboptimal and inhibitory [Ca(2+)](i). Expression of capn-InsP(3)R1 in N2a cells reduced the Ca(2+) content of ionomycin-releasable intracellular stores and decreased endoplasmic reticulum Ca(2+) loading compared with control cells expressing full-length InsP(3)R1. Using a cleavage-specific antibody, we identified calpain-cleaved InsP(3)R1 in selectively vulnerable cerebellar Purkinje neurons after in vivo cardiac arrest. These findings indicate that calpain proteolysis of InsP(3)R1 generates a dysregulated channel that disrupts cellular Ca(2+) homeostasis. Furthermore, our results demonstrate that calpain cleaves InsP(3)R1 in a clinically relevant injury model, suggesting that Ca(2+) leak through the proteolyzed channel may act as a feed-forward mechanism to enhance cell death.     

Signal-induced Ca2+ oscillations: properties of a model based on Ca(2+)-induced Ca2+ release.

We consider a simple, minimal model for signal-induced Ca2+ oscillations based on Ca(2+)-induced Ca2+ release. The model takes into account the existence of two pools of intracellular Ca2+, namely, one sensitive to inositol 1,4,5 trisphosphate (InsP3) whose synthesis is elicited by the stimulus, and one insensitive to InsP3. The discharge of the latter pool into the cytosol is activated by cytosolic Ca2+. Oscillations in cytosolic Ca2+ arise in this model either spontaneously or in an appropriate range of external stimulation; these oscillations do not require the concomitant, periodic variation of InsP3. The following properties of the model are reviewed and compared with experimental observations: (a) Control of the frequency of Ca2+ oscillations by the external stimulus or extracellular Ca2+; (b) correlation of latency with period of Ca2+ oscillations obtained at different levels of stimulation; (c) effect of a transient increase in InsP3; (d) phase shift and transient suppression of Ca2+ oscillations by Ca2+ pulses, and (e) propagation of Ca2+ waves. It is shown that on all these counts the model provides a simple, unified explanation for a number of experimental observations in a variety of cell types. The model based on Ca(2+)-induced Ca2+ release can be extended to incorporate variations in the level of InsP3 as well as desensitization of the InsP3 receptor; besides accounting for the phenomena described by the minimal model, the extended model might also account for the occurrence of complex Ca2+ oscillations.     

Dopamine receptor-mediated Ca(2+) signaling in striatal medium spiny neurons

Inositol 1,4,5-trisphosphate (InsP(3)) and cAMP are the two second messengers that play an important role in neuronal signaling. Here, we investigated the interactions of InsP(3)- and cAMP-mediated signaling pathways activated by dopamine in striatal medium spiny neurons (MSN). We found that in approximately 40% of the MSN, application of dopamine elicited robust repetitive Ca(2+) transients (oscillations). In pharmacological experiments with specific agonists and antagonists, we found that the observed Ca(2+) oscillations were triggered by activation of D1 class dopamine receptors (DARs). We further demonstrated that activation of phospholipase C was required for induction of dopamine-induced Ca(2+) oscillations and that maintenance of dopamine-evoked Ca(2+) oscillations required both Ca(2+) influx and Ca(2+) mobilization from internal Ca(2+) stores. In "priming" experiments with a type 2 5-hydroxytryptamine receptor agonist, we have shown a likely role for calcyon in coupling D1 class DARs with Ca(2+) oscillations in MSN. In experiments with the DAR-specific agonist SKF83959, we discovered that phospholipase C activation alone could not account for dopamine-induced Ca(2+) oscillations. We further demonstrated that direct activation of protein kinase A by 8-bromo-cAMP or inhibition of protein phosphatase-1 (PP1) or calcineurin (PP2B) resulted in elevation of basal Ca(2+) levels in MSN, but not in Ca(2+) oscillations. In experiments with competitive peptides, we have shown an importance of type 1 InsP(3) receptor association with PP1alpha and with AKAP9.protein kinase A for dopamine-induced Ca(2+) oscillations. In experiments with MSN from DARPP-32 knock-out mice, we demonstrated a regulatory role of DARPP-32 in dopamine-induced Ca(2+) oscillations. Our results indicate that, following D1 class DAR activation, InsP(3) and cAMP signaling pathways converge on the type 1 InsP(3) receptor, resulting in Ca(2+) oscillations in MSN.     

Analysis of Mn(2+)/Ca(2+) influx and release during Ca(2+) oscillations in mouse eggs injected with sperm extract

Repetitive Ca(2+) release from the endoplasmic reticulum (ER) is necessary for activation of mammalian eggs. Influx and release of Mn(2+) and Ca(2+) during Ca(2+) oscillations induced by injection of sperm extract (SE) into mouse eggs were investigated by Mn(2+)-quenching of intracellular Fura-2 after adding Mn(2+) to external medium. Mn(2+)/Ca(2+) influx was detected at the resting state. A marked Mn(2+)/Ca(2+) influx occurred during the first Ca(2+) release upon SE injection, and persistently facilitated Mn(2+)/Ca(2+) influx was observed during steady Ca(2+) oscillations. As intracellular Mn(2+) concentration ([Mn(2+)](i)) increased progressively, periodic [Mn(2+)](i) rises appeared, corresponding to each Ca(2+)transient but taking a slower time course. A numerical simulation based on continuous Mn(2+)/Ca(2+) influx-extrusion across the plasma membrane and release-uptake across the ER membrane in a competitive manner mimicked well the Mn(2+) oscillations calculated from experimental data, strongly suggesting that repetitive Mn(2+) release develops after Mn(2+) entry and uptake into the ER. In other experiments, a marked Mn(2+) influx occurred upon Mn(2+) addition to Ca(2+)-free medium after depletion of the ER using an ER Ca(2+) pump inhibitor plus repeated injection of inositol 1,4,5-trisphosphate (InsP(3)). No significant increase in Mn(2+) influx was induced by injection of SE, InsP(3), or Ca(2+), when Ca(2+) release was prevented by pre-injection of an antibody against the InsP(3) receptor. We concluded that Ca(2+) influx is activated during the initial large Ca(2+)release possibly by a capacitative mechanism and kept facilitated during steady Ca(2+) oscillations. The finding that repetitive Mn(2+) release is caused by continuous Mn(2+) entry suggests that continuous Ca(2+) influx may play a critical role in refilling the ER and, thereby, maintaining Ca(2+)oscillations in mammalian fertilization.     

Ca(2+) oscillations induced by a cytosolic sperm protein factor are mediated by a maternal machinery that functions only once in mammalian eggs

At fertilization in mammals, the sperm activates the egg by inducing a series of oscillations in the intracellular free Ca(2+) concentration. There is evidence showing that this oscillatory event is triggered by a sperm-derived protein factor which diffuses into egg cytoplasm after gamete membrane fusion. At present the identity of this factor and its precise mechanism of action is unknown. Here, we studied the specificity of action of the sperm factor in triggering Ca(2+) oscillations in mammalian eggs. In doing so, we examined the patterns of Ca(2+) signaling in mouse eggs, zygotes, parthenogenetic eggs and maturing oocytes following the stimulation of bovine sperm extracts which contain the sperm factor. It is observed that the sperm factor could induce Ca(2+) oscillations in metaphase eggs, maturing oocytes and parthenogenetically activated eggs but not in the zygotes. We present evidence that Ca(2+) oscillations induced by the sperm factor require a maternal machinery. This machinery functions only once in mammalian oocytes and eggs, and is inactivated by sperm-derived components but not by parthenogenetic activation. In addition, it is found that neither InsP(3) receptor sensitivity to InsP(3) nor Ca(2+) pool size are the determinants that cause the fertilized egg to lose its ability to generate sperm-factor-induced Ca(2+) oscillations at metaphase. In conclusion, our study suggests that the orderly sequence of Ca(2+) oscillations in mammalian eggs at fertilization is critically dependent upon the presence of a functional maternal machinery that determines whether the sperm-factor-induced Ca(2+) oscillations can persist.     

Calpain-cleaved type 1 inositol 1,4,5-trisphosphate receptor impairs ER Ca(2+) buffering and causes neurodegeneration in primary cortical neurons

Disruption of neuronal Ca(2+) homeostasis plays a well-established role in cell death in a number of neurodegenerative disorders. Recent evidence suggests that proteolysis of the type 1 inositol 1,4,5-trisphosphate receptor (InsP(3) R1), a Ca(2+) release channel on the endoplasmic reticulum, generates a dysregulated channel, which may contribute to aberrant Ca(2+) signaling and neurodegeneration in disease states. However, the specific effects of InsP(3) R1 proteolysis on neuronal Ca(2+) homeostasis are unknown, as are the functional contributions of this pathway to neuronal death. This study evaluates the consequences of calpain-mediated InsP(3) R1 proteolysis on neuronal Ca(2+) signaling and survival using adeno-associated viruses to express a recombinant cleaved form of the channel (capn-InsP(3) R1) in rat primary cortical neurons. Here, we demonstrate that expression of capn-InsP(3) R1 in cortical cultures reduced cellular viability. This effect was associated with increased resting cytoplasmic Ca(2+) concentration ([Ca(2+) ](i) ), increased [Ca(2+) ](i) response to glutamate, and enhanced sensitivity to excitotoxic stimuli. Together, our results demonstrate that InsP(3) R1 proteolysis disrupts neuronal Ca(2+) homeostasis, and potentially acts as a feed-forward pathway to initiate or execute neuronal death.     

P2Y(2) receptor-stimulated phosphoinositide hydrolysis and Ca(2+) mobilization in tracheal epithelial cells

Extracellular nucleotides have been implicated in the regulation of secretory function through the activation of P2 receptors in the epithelial tissues, including tracheal epithelial cells (TECs). In this study, experiments were conducted to characterize the P2 receptor subtype on canine TECs responsible for stimulating inositol phosphate (InsP(x)) accumulation and Ca(2+) mobilization using a range of nucleotides. The nucleotides ATP and UTP caused a concentration-dependent increase in [(3)H]InsP(x) accumulation and Ca(2+) mobilization with comparable kinetics and similar potency. The selective agonists for P1, P2X, and P2Y(1) receptors, N(6)-cyclopentyladenosine and AMP, alpha,beta-methylene-ATP and beta, gamma-methylene-ATP, and 2-methylthio-ATP, respectively, had little effect on these responses. Stimulation of TECs with maximally effective concentrations of ATP and UTP showed no additive effect on [(3)H]InsP(x) accumulation. The response of a maximally effective concentration of either ATP or UTP was additive to the response evoked by bradykinin. Furthermore, ATP and UTP induced a cross-desensitization in [(3)H]InsP(x) accumulation and Ca(2+) mobilization. These results suggest that ATP and UTP directly stimulate phospholipase C-mediated [(3)H]InsP(x) accumulation and Ca(2+) mobilization in canine TECs. P2Y(2) receptors may be predominantly mediating [(3)H]InsP(x) accumulation, and, subsequently, inositol 1,4,5-trisphosphate-induced Ca(2+) mobilization may function as the transducing mechanism for ATP-modulated secretory function of tracheal epithelium.     

Ca(2+) response to cADPr during maturation and fertilization of starfish oocytes.

During the reinitiation of the meiotic cycle (maturation) induced by the hormone 1-methyladenine (1-MA), starfish oocytes undergo structural and biochemical changes in preparation for successful fertilization. Previous work has shown that the sensitivity of internal Ca(2+) stores to InsP(3) increases during maturation of the oocytes. Since Astropecten auranciacus oocytes also respond to cADPr, we have studied whether the response to cADPr also changes during maturation. We have found that the photoactivation of injected cADPr in immature oocytes immediately induces multiple patches of Ca(2+) release in the cortical region. The Ca(2+) signal then spreads from these initial points of increase to the entire cell. In mature oocytes, the uncaging of cADPr induces instead a single (or at most a dual) initial point of Ca(2+) release, which is immediately followed by the formation of a cortical Ca(2+) flash and then by the globalization of the wave and by the elevation of the fertilization envelope. External Ca(2+) plays a role in the Ca(2+) responses. Inhibition of L-type Ca(2+) channels does not affect the initial Ca(2+) release, but abolishes the cortical flash and impairs the elevation of the fertilization envelope. External Ca(2+) has other effects, as shown by the irregular appearance of the surface of oocytes incubated in Ca(2+)-free sea water. The sequence of Ca(2+) responses induced by cADPr in mature oocytes mimics those seen at fertilization, i.e., a first localized Ca(2+) increase followed by a cortical flash and by the globalization of the Ca(2+) signal. As in the case of maturation, L-type Ca(2+) channel blockers abolish the sperm induced cortical flash.     

InsP(3)-induced Ca(2+) release in permeabilized invertebrate photoreceptors: a link between phototransduction and Ca(2+) stores

Using the low-affinity fluorescent Ca(2+) indicators, Mag-Fura-2 and Mag-Fura Red, we studied light- and InsP(3)-induced Ca(2+) release in permeabilized microvillar photoreceptors of the medicinal leech, Hirudo medicinalis. Two major components of the phosphoinositide signaling pathway, phospholipase-C and the InsP(3) receptor, were characterized immunologically and appropriately localized in photoreceptors. Whereas phospholipase-C was abudantly expressed in photoreceptive microvilli, InsP(3) receptors were found mostly in submicrovillar endoplasmic reticulum (SER). Permeabilization of the peripheral plasma membrane with saponin allowed direct measurements of luminal free Ca(2+) concentration (Ca(L)) changes. Confocal Ca(2+) imaging using Mag-Fura Red demonstrated that Ins(1,4,5)P(3) mobilizes Ca(2+) from SER. As detected with Mag-Fura-2, a brief 50ms light flash activated rapid Ca(2+) depletion of SER, followed by an effective refilling within 1min of dark adaptation after the light flash. Sensitivity to Ins(1,4,5)P(3) of the Ca(2+) release from SER in leech photoreceptors was accompanied by irreversible uncoupling of phototransduction from Ca(2+) release. Depletion of Ca(2+) stores was induced by Ins(1,4,5)P(3)(EC(50)= 4.75 microM) and the hyper-potent agonist adenophostin A (EC(50)/40nM) while the stereoisomer L-myo Ins(1,4,5)P(3) was totally inactive. Ins(1,4,5)P(3)- or adenophostin A-induced Ca(2+) release was inhibited by 0.1-1mg/ml heparin. The Ca(2+) pump inhibitors, cyclopiazonic acid and thapsigargin, in the presence of Ins(1,4,5)P(3), completely depleted Ca(2+) stores in leech photoreceptors.     

Single-channel properties in endoplasmic reticulum membrane of recombinant type 3 inositol trisphosphate receptor

The inositol 1,4,5-trisphosphate receptor (InsP(3)R) is an intracellular Ca(2+)-release channel localized in endoplasmic reticulum (ER) with a central role in complex Ca(2+) signaling in most cell types. A family of InsP(3)Rs encoded by several genes has been identified with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. This diversity suggests that cells require distinct InsP(3)Rs, but the functional correlates of this diversity are largely unknown. Lacking are single-channel recordings of the recombinant type 3 receptor (InsP(3)R-3), a widely expressed isoform also implicated in plasma membrane Ca(2+) influx and apoptosis. Here, we describe functional expression and single-channel recording of recombinant rat InsP(3)R-3 in its native membrane environment. The approach we describe suggests a novel strategy for expression and recording of recombinant ER-localized ion channels in the ER membrane. Ion permeation and channel gating properties of the rat InsP(3)R-3 are strikingly similar to those of Xenopus type 1 InsP(3)R in the same membrane. Using two different two-electrode voltage clamp protocols to examine calcium store-operated calcium influx, no difference in the magnitude of calcium influx was observed in oocytes injected with rat InsP(3)R-3 cRNA compared with control oocytes. Our results suggest that if cellular expression of multiple InsP(3)R isoforms is a mechanism to modify the temporal and spatial features of [Ca(2+)](i) signals, then it must be achieved by isoform-specific regulation or localization of various types of InsP(3)Rs that have relatively similar Ca(2+) permeation properties.     

Reciprocal inhibition of Cd(2+) and Ca(2+) uptake in human intestinal crypt cells for voltage-independent Zn-activated pathways

Cadmium-Ca-Zn interactions for uptake have been studied in human intestinal crypt cells HIEC. Our results failed to demonstrate any significant cross-inhibition between Cd and Ca uptake under single metal exposure conditions. However, they revealed a strong reciprocal inhibition for a Zn-stimulated mechanism of transport. Optimal stimulation was observed under exposure conditions that favor an inward-directed Zn gradient, suggesting activation by extracellular rather than intracellular Zn. The effect of Zn on the uptake of Ca was concentration-dependent, and zinc-induced stimulation of Cd uptake resulted in a 3- and 5.8-fold increase in the K(m) and V(max) values, respectively. Neither basal nor Zn-stimulated Ca uptakes were sensitive to membrane depolarization. However, the stimulated component of uptake was inhibited by the trivalent cations Gd(3+), and La(3+) and to a lesser extent by Mg(2+) and Ba(2+). RT-PCR analysis as well as uptake measurement performed with extracellular ATP and/or suramin do not support the involvement of purinergic P2X receptor channels. Uptake and fluorescence data led to the conclusion that Zn is unlikely to trigger Ca influx in response to Ca release from thapsigargin-sensitive intracellular pools. Our data show that Zn may potentiate Cd accumulation in intestinal crypt cells through mechanism that still needs to be clarified.