Atrial local Ca signaling and inositol 1,4,5-trisphosphate receptors

In atrial myocytes lacking t-tubules, action potential triggers junctional Ca²⁺ releases in the cell periphery, which propagates into the cell interior. The present article describes growing evidence on atrial local Ca²⁺ signaling and on the functions of inositol 1,4,5-trisphosphate receptors (IP₃Rs) in atrial myocytes, and show our new findings on the role of IP₃R subtype in the regulation of spontaneous focal Ca²⁺ releases in the compartmentalized areas of atrial myocytes. The Ca²⁺ sparks, representing focal Ca²⁺ releases from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR) clusters, occur most frequently at the peripheral junctions in isolated resting atrial cells. The Ca²⁺ sparks that were darker and longer lasting than peripheral and non-junctional (central) sparks, were found at peri-nuclear sites in rat atrial myocytes. Peri-nuclear sparks occurred more frequently than central sparks. Atrial cells express larger amounts of IP₃Rs compared with ventricular cells and possess significant levels of type 1 IP₃R (IP₃R1) and type 2 IP₃R (IP₃R2). Over the last decade the roles of atrial IP₃R on the enhancement of Ca²⁺-induced Ca²⁺ release and arrhythmic Ca²⁺ releases under hormonal stimulations have been well documented. Using protein knock-down method and confocal Ca²⁺ imaging in conjunction with immunocytochemistry in the adult atrial cell line HL-1, we could demonstrate a role of IP₃R1 in the maintenance of peri-nuclear and non-junctional Ca²⁺ sparks via stimulating a posttranslational organization of RyR clusters.     

An inositol 1,4,5-trisphosphate receptor-gated intracellular Ca(2+) store is involved in regulating sperm hyperactivated motility.

Hyperactivated motility, a swimming pattern displayed by mammalian sperm in the oviduct around the time of ovulation, is essential to fertilization. Ca(2+) has been shown to be crucial for the initiation and maintenance of hyperactivated motility. Nevertheless, how Ca(2+) reaches the axoneme in the core of the flagellum to switch on hyperactivation is unknown. Ca(2+)-releasing agents were used to determine whether an intracellular store provides Ca(2+) to the axoneme. Hyperactivation was induced immediately in bull sperm by thapsigargin, caffeine, and thimerosal. The responses were dose-dependent and were induced in both capacitated and uncapacitated sperm. When external Ca(2+) was buffered below 50 nM with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, the response to caffeine was significantly reduced; however, the responses to thapsigargin and thimerosal were not affected. This indicates caffeine-induced hyperactivation depends on external Ca(2+) influx, whereas hyperactivation by thapsigargin and thimerosal do not. Acrosome reactions were not induced by these treatments; therefore, an acrosomal store was probably not involved. Indirect immunofluorescence labeling showed type I inositol 1,4,5-trisphosphate receptors (IP(3)R) in the acrosome and neck region, but no ryanodine receptors (RyR) were found using anti-RyR antibodies or BODIPY FL-X ryanodine. These data indicate that there is an IP(3)R-gated Ca(2+) store in the neck region of sperm that regulates hyperactivated motility.     

Stochastic properties of Ca(2+) release of inositol 1,4,5-trisphosphate receptor clusters

Intracellular Ca(2+) release is controlled by inositol 1,4,5-trisphosphate (IP(3)) receptors or ryanodine receptors. These receptors are typically distributed in clusters with several or tens of channels. The random opening and closing of these channels introduces stochasticity into the elementary calcium release mechanism. Stochastic release events have been experimentally observed in a variety of cell types and have been termed sparks and puffs. We put forward a stochastic version of the Li-Rinzel model (the deactivation binding process is described by a Markovian scheme) and a computationally more efficient Langevin approach to model the stochastic Ca(2+) oscillation of single clusters. Statistical properties such as Ca(2+) puff amplitudes, lifetimes, and interpuff intervals are studied with both models and compared with experimental observations. For clusters with tens of channels, a simply decaying amplitude distribution is typically observed at low IP(3) concentration, while a single peak distribution appears at high IP(3) concentration.     

Lateral inhibition of inositol 1,4,5-trisphosphate receptors by cytosolic Ca(2+).

Ryanodine and inositol 1,4,5-trisphosphate (IP(3)) receptors - two related families of Ca(2+) channels responsible for release of Ca(2+) from intracellular stores [1] - are biphasically regulated by cytosolic Ca(2+) [2] [3] [4]. It is thought that the resulting positive feedback allows localised Ca(2+)-release events to propagate regeneratively, and that the negative feedback limits the amplitude of individual events [5] [6]. Stimulation of IP(3) receptors by Ca(2+) occurs through a Ca(2+)-binding site that becomes exposed only after IP(3) has bound to its receptor [7] [8]. Here, we report that rapid inhibition of IP(3) receptors by Ca(2+) occurs only if the receptor has not bound IP(3). The IP(3) therefore switches its receptor from a state in which only an inhibitory Ca(2+)-binding site is accessible to one in which only a stimulatory site is available. This regulation ensures that Ca(2+) released by an active IP(3) receptor may rapidly inhibit its unliganded neighbours, but it cannot terminate the activity of a receptor with IP(3) bound. Such lateral inhibition, which is a universal feature of sensory systems where it improves contrast and dynamic range, may fulfil similar roles in intracellular Ca(2+) signalling by providing increased sensitivity to IP(3) and allowing rapid graded recruitment of IP(3) receptors.     

Bcl-2 interaction with the inositol 1,4,5-trisphosphate receptor: role in Ca(2+) signaling and disease

The Bcl-2 protein, best known for its ability to inhibit apoptosis, interacts with the inositol 1,4,5-trisphosphate receptor (IP₃R) Ca²⁺ channel to regulate IP₃-mediated Ca²⁺ release from the endoplasmic reticulum. This review summarizes the current state of knowledge regarding the interaction of Bcl-2, and also its homologue Bcl-xl, with the IP₃R and how these interactions regulate Ca²⁺ signaling. The dual role of these interactions in promoting prosurvival Ca²⁺ signals, while at the same time inhibiting proapoptotic Ca²⁺ signals, is discussed. Moreover, this review will elucidate the recently recognized importance of the Bcl-2-IP₃R interaction in human disease.     

Stereospecific mobilization of intracellular Ca2+ by inositol 1,4,5-triphosphate. Comparison with inositol 1,4,5-trisphosphorothioate and inositol 1,3,4-trisphosphate.

The isolated activation segment of pig procarboxypeptidase A binds two Tb3+ ions in a strong and specific way. In contrast, the binding of Ca2+, Cd2+ and Mg2+ is weak. The binding of Tb3+ increases the resistance of the isolated activation segment against proteolysis and competes for the binding of the carbocyanine dye Stains-All. This dye forms complexes with the activation segment showing spectral properties similar to those observed with EF-hand structures. The presented results support a previous hypothesis on the existence of two regions in the activation segment of pancreatic procarboxypeptidases structurally related to Ca2+-binding domains of the EF-hand protein family.     

Ca(2+)-induced Ca(2+) release via inositol 1,4,5-trisphosphate receptors is amplified by protein kinase A and triggers exocytosis in pancreatic beta-cells

Hormones, such as glucagon and glucagon-like peptide-1, potently amplify nutrient stimulated insulin secretion by raising cAMP. We have studied how cAMP affects Ca(2+)-induced Ca(2+) release (CICR) in pancreatic beta-cells from mice and rats and the role of CICR in secretion. CICR was observed as pronounced Ca(2+) spikes on top of glucose- or depolarization-dependent rise of the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)). cAMP-elevating agents strongly promoted CICR. This effect involved sensitization of the receptors underlying CICR, because many cells exhibited the characteristic Ca(2+) spiking at low or even in the absence of depolarization-dependent elevation of [Ca(2+)](i). The cAMP effect was mimicked by a specific activator of protein kinase A in cells unresponsive to activators of cAMP-regulated guanine nucleotide exchange factor. Ryanodine pretreatment, which abolishes CICR mediated by ryanodine receptors, did not prevent CICR. Moreover, a high concentration of caffeine, known to activate ryanodine receptors independently of Ca(2+), failed to mobilize intracellular Ca(2+). On the contrary, a high caffeine concentration abolished CICR by interfering with inositol 1,4,5-trisphosphate receptors (IP(3)Rs). Therefore, the cell-permeable IP(3)R antagonist 2-aminoethoxydiphenyl borate blocked the cAMP-promoted CICR. Individual CICR events in pancreatic beta-cells were followed by [Ca(2+)](i) spikes in neighboring human erythroleukemia cells, used to report secretory events in the beta-cells. The results indicate that protein kinase A-mediated promotion of CICR via IP(3)Rs is part of the mechanism by which cAMP amplifies insulin release.     

Counting functional inositol 1,4,5-trisphosphate receptors into the plasma membrane

Inositol 1,4,5-trisphosphate receptors (IP(3)R) within the endoplasmic reticulum mediate release of Ca(2+) from intracellular stores. Different channels usually mediate Ca(2+) entry across the plasma membrane. In B lymphocytes and a cell line derived from them (DT40 cells), very few functional IP(3)R (approximately 2/cell) are invariably expressed in the plasma membrane, where they mediate about half the Ca(2+) entry evoked by activation of the B-cell receptor. We show that cells reliably count approximately 2 functional IP(3)R into the plasma membrane even when their conductance and ability to bind IP(3) are massively attenuated. We conclude that very small numbers of functional IP(3)R can be reliably counted into a specific membrane compartment in the absence of feedback signals from the active protein.     

Prolonged exposure to inositol 1,4,5-trisphosphate does not cause intrinsic desensitization of the intracellular Ca(2+)-mobilizing receptor.

The rapid release of Ca2+ from intracellular stores stimulated with inositol 1,4,5-trisphosphate (InsP3) has required superfusion or stopped-flow techniques to resolve the kinetics of Ca2+ mobilization and made it difficult to determine whether the InsP3 receptor desensitizes during prolonged stimulation. Here we have overloaded the intracellular Ca2+ stores of permeabilized rat hepatocytes by incubating them with ATP and 45Ca2+ in the presence of pyrophosphate, which precipitates Ca2+ within the lumen of the stores. Subsequent ATP removal initiated slow 45Ca2+ efflux that followed zero-order kinetics, allowing us to examine the effects of InsP3 over a prolonged time course. InsP3 produced a concentration-dependent increase in the 45Ca2+ efflux rate that was sustained for several min. The rate rapidly returned to the unstimulated level after the addition of decavanadate, a competitive antagonist of InsP3 at its receptor. Prior incubation with a submaximal concentration of InsP3 (1 microM) did not affect the subsequent enhanced rate of 45Ca2+ efflux stimulated by a higher, but still submaximal, concentration of InsP3 (3 microM). We conclude that prolonged exposure to InsP3 does not desensitize the InsP3 receptor and that intrinsic receptor desensitization cannot provide an explanation for the quantal responses to InsP3 observed in several cell types.     

Stimulus-dependent control of inositol 1,4,5-trisphosphate-induced Ca(2+) oscillation frequency by the endoplasmic reticulum Ca(2+)-ATPase

In many cell types, receptor stimulation evokes cytosolic calcium oscillations with a frequency that depends on agonist dose. Previous studies demonstrated controversial effects of changing the activity of the endoplasmic reticulum Ca(2+)-ATPase upon the frequency of oscillations. By numerical simulations, we found that the model of De Young and Keizer (J. Keizer and G.W. De Young, 1994, J. Theor. Biol. 166: 431-442), unlike other models, can explain the observed discrepancies, assuming that the different experiments were performed at different stimulus levels. According to model predictions, partial inhibition of internal calcium pumps is expected to increase frequency at low stimulus strength and should have an opposite effect at strong stimuli. Similar results were obtained using an analytical estimation of oscillation period, based on calcium-dependent channel activation and inactivation. In experiments on HeLa cells, 4 nM thapsigargin increased the frequency of calcium oscillations induced by 1 and 2.5 microM histamine but had no effect on supramaximally stimulated cells. In HEp-2 cells, 2 nM thapsigargin slowed down the rapid, ATP-induced oscillations. Our results suggest that in the investigated cell types, the De Young-Keizer model based on inositol 1,4,5-trisphosphate-dependent calcium-induced calcium release can properly describe intracellular calcium oscillations.     

Bcl-2 functionally interacts with inositol 1,4,5-trisphosphate receptors to regulate calcium release from the ER in response to inositol 1,4,5-trisphosphate

Inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) are channels responsible for calcium release from the endoplasmic reticulum (ER). We show that the anti-apoptotic protein Bcl-2 (either wild type or selectively localized to the ER) significantly inhibited InsP3-mediated calcium release and elevation of cytosolic calcium in WEHI7.2 T cells. This inhibition was due to an effect of Bcl-2 at the level of InsP3Rs because responses to both anti-CD3 antibody and a cell-permeant InsP3 ester were decreased. Bcl-2 inhibited the extent of calcium release from the ER of permeabilized WEHI7.2 cells, even at saturating concentrations of InsP3, without decreasing luminal calcium concentration. Furthermore, Bcl-2 reduced the open probability of purified InsP3Rs reconstituted into lipid bilayers. Bcl-2 and InsP3Rs were detected together in macromolecular complexes by coimmunoprecipitation and blue native gel electrophoresis. We suggest that this functional interaction of Bcl-2 with InsP3Rs inhibits InsP3R activation and thereby regulates InsP3-induced calcium release from the ER.     

Polycystin 2 interacts with type I inositol 1,4,5-trisphosphate receptor to modulate intracellular Ca2+ signaling

Autosomal dominant polycystic kidney disease, a common cause of renal failure, arises from mutations in either the PKD1 or the PKD2 gene. The precise function of both PKD gene products polycystins (PCs) 1 and 2 remain controversial. PC2 has been localized to numerous cellular compartments, including the endoplasmic reticulum, plasma membrane, and cilia. It is unclear what pools are the most relevant to its physiological function as a putative Ca2+ channel. We employed a Xenopus oocyte Ca2+ imaging system to directly investigate the role of PC2 in inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ signaling. Cytosolic Ca2+ signals were recorded following UV photolysis of caged IP3 in the absence of extracellular Ca2+. We demonstrated that overexpression of PC2, as well as type I IP3 receptor (IP3R), significantly prolonged the half-decay time (t1/2) of IP3-induced Ca2+ transients. However, overexpressing the disease-associated PC2 mutants, the point mutation D511V, and the C-terminally truncated mutation R742X did not alter the t1/2. In addition, we found that D511V overexpression significantly reduced the amplitude of IP3-induced Ca2+ transients. Interestingly, overexpression of the C terminus of PC2 not only significantly reduced the amplitude but also prolonged the t1/2. Co-immunoprecipitation assays indicated that PC2 physically interacts with IP3R through its C terminus. Taken together, our data suggest that PC2 and IP3R functionally interact and modulate intracellular Ca2+ signaling. Therefore, mutations in either PC1 or PC2 could result in the misregulation of intracellular Ca2+ signaling, which in turn could contribute to the pathology of autosomal dominant polycystic kidney disease.     

Type 1 inositol 1,4,5-trisphosphate receptors mediate UTP-induced cation currents, Ca2+ signals, and vasoconstriction in cerebral arteries

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) regulate diverse physiological functions, including contraction and proliferation. There are three IP(3)R isoforms, but their functional significance in arterial smooth muscle cells is unclear. Here, we investigated relative expression and physiological functions of IP(3)R isoforms in cerebral artery smooth muscle cells. We show that 2-aminoethoxydiphenyl borate and xestospongin C, membrane-permeant IP(3)R blockers, reduced Ca(2+) wave activation and global intracellular Ca(2+) ([Ca(2+)](i)) elevation stimulated by UTP, a phospholipase C-coupled purinergic receptor agonist. Quantitative PCR, Western blotting, and immunofluorescence indicated that all three IP(3)R isoforms were expressed in acutely isolated cerebral artery smooth muscle cells, with IP(3)R1 being the most abundant isoform at 82% of total IP(3)R message. IP(3)R1 knockdown with short hairpin RNA (shRNA) did not alter baseline Ca(2+) wave frequency and global [Ca(2+)](i) but abolished UTP-induced Ca(2+) wave activation and reduced the UTP-induced global [Ca(2+)](i) elevation by approximately 61%. Antibodies targeting IP(3)R1 and IP(3)R1 knockdown reduced UTP-induced nonselective cation current (I(cat)) activation. IP(3)R1 knockdown also reduced UTP-induced vasoconstriction in pressurized arteries with both intact and depleted sarcoplasmic reticulum (SR) Ca(2+) by approximately 45%. These data indicate that IP(3)R1 is the predominant IP(3)R isoform expressed in rat cerebral artery smooth muscle cells. IP(3)R1 stimulation contributes to UTP-induced I(cat) activation, Ca(2+) wave generation, global [Ca(2+)](i) elevation, and vasoconstriction. In addition, IP(3)R1 activation constricts cerebral arteries in the absence of SR Ca(2+) release by stimulating plasma membrane I(cat).     

Increase in Ca2+ permeability of intracellular Ca2+ store membrane of saponin-treated guinea pig peritoneal macrophages by inositol 1,4,5-trisphosphate

Inositol 1,4,5-trisphosphate (InsP3) releases Ca2+ from the non-mitochondrial Ca2+ store site of various types of cells. To study the mechanisms of the Ca2+ release from the store site, the effect of InsP3 on the passive Ca2+ release and influx, and the active Ca2+ uptake in the presence of oxalate, was examined using saponin-treated guinea pig peritoneal macrophages. InsP3 stimulated the passive Ca2+ release and influx. Although InsP3 slightly inhibited the active Ca2+ uptake in the presence of oxalate, it seems unlikely that the Ca2+ release by this agent is caused by the inhibition of the Ca2+ uptake, because the addition of apyrase or hexokinase (which removes ATP within 30 s, so that no more Ca2+ can be accumulated) or vanadate (which inhibits the Ca2+ uptake) resulted in very slow release of Ca2+. These results suggest that the Ca2+ permeability of the Ca2+ store membrane is increased by InsP3. InsP3 did not cause an increase in the Ca2+ permeability of phospholipid vesicles (liposomes), indicating that this agent may bring about Ca2+ release by a specific effect on the physiologically relevant Ca2+ channels or carriers in the non-mitochondrial Ca2+ store site. The passive Ca2+ release by InsP3 was enhanced by ATP and an unhydrolyzable ATP analogue, 5'-adenylyimidodiphosphate, but not by ADP or AMP. The passive Ca2+ release by InsP3 was observed even at 0 degree C.     

The type I inositol 1,4,5-trisphosphate receptor interacts with protein 4.1N to mediate neurite formation through intracellular Ca waves

Ca(2+) waves are an important mechanism for encoding Ca(2+) signaling information, but the molecular basis for wave formation and how this regulates neuronal function is not entirely understood. Using nerve growth factor-differentiated PC12 cells as a model system, we investigated the interaction between the type I inositol 1,4,5-trisphosphate receptor (IP3R1) and the cytoskeletal linker, protein 4.1N, to examine the relationship between Ca(2+) wave formation and neurite development. This was examined using RNAi and overexpressed dominant negative binding regions of each protein. Confocal microscopy was used to monitor neurite formation and Ca(2+) waves. Knockdown of IP3R1 or 4.1N attenuated neurite formation, as did binding regions of IP3R1 and 4.1N, which colocalized with endogenous 4.1N and IP3R1, respectively. Upon stimulation with the IP3-producing agonist carbachol, both RNAi and dominant negative molecules shifted signaling events from waves to homogeneous patterns of Ca(2+) release. These findings provide evidence that IP3R1 localization, via protein 4.1N, is necessary for Ca(2+) wave formation, which in turn mediates neurite formation.     

Ca2+-induced Ca2+ release by activation of inositol 1,4,5-trisphosphate receptors in primary pancreatic beta-cells

The effect of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibition on the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) was studied in primary insulin-releasing pancreatic beta-cells isolated from mice, rats and human subjects as well as in clonal rat insulinoma INS-1 cells. In Ca(2+)-deficient medium the individual primary beta-cells reacted to the SERCA inhibitor cyclopiazonic acid (CPA) with a slow rise of [Ca(2+)](i) followed by an explosive transient elevation. The [Ca(2+)](i) transients were preferentially observed at low intracellular concentrations of the Ca(2+) indicator fura-2 and were unaffected by pre-treatment with 100 microM ryanodine. Whereas 20mM caffeine had no effect on basal [Ca(2+)](i) or the slow rise in response to CPA, it completely prevented the CPA-induced [Ca(2+)](i) transients as well as inositol 1,4,5-trisphosphate-mediated [Ca(2+)](i) transients in response to carbachol. In striking contrast to the primary beta-cells, caffeine readily mobilized intracellular Ca(2+) in INS-1 cells under identical conditions, and such mobilization was prevented by ryanodine pre-treatment. The results indicate that leakage of Ca(2+) from the endoplasmic reticulum after SERCA inhibition is feedback-accelerated by Ca(2+)-induced Ca(2+) release (CICR). In primary pancreatic beta-cells this CICR is due to activation of inositol 1,4,5-trisphosphate receptors. CICR by ryanodine receptor activation may be restricted to clonal beta-cells.     

Inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate formation in Ca2+-mobilizing-hormone-activated cells.

The inositol trisphosphate liberated on stimulation of guinea-pig hepatocytes, pancreatic acinar cells and dimethyl sulphoxide-differentiated human myelomonocytic HL-60 leukaemia cells is composed of two isomers, the 1,4,5-trisphosphate and the 1,3,4-trisphosphate. Inositol 1,4,5-trisphosphate was released rapidly, with no measurable latency on hormone stimulation, and, consistent with its proposed role as an intracellular messenger for Ca2+ mobilization, there was good temporal correlation between its formation and Ca2+-mediated events in these tissues. There was a definite latency before an increase in the formation of inositol 1,3,4-trisphosphate could be detected. In all of these tissues, however, it formed a substantial proportion of the total inositol trisphosphate by 1 min of stimulation. In guinea-pig hepatocytes, where inositol trisphosphate increases for at least 30 min after hormone application, inositol 1,3,4-trisphosphate made up about 90% of the total inositol trisphosphate by 5-10 min. In pancreatic acinar cells, pretreatment with 20 mM-Li+ caused an increase in hormone-induced inositol trisphosphate accumulation. This increase was accounted for by a rise in inositol 1,3,4-trisphosphate; inositol 1,4,5-trisphosphate was unaffected. This finding is consistent with the observation that Li+ has no effect on Ca2+-mediated responses in these cells. The role, if any, of inositol 1,3,4-trisphosphate in cellular function is unknown.