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.     

Intracellular Ca2+ release through ryanodine receptors contributes to AMPA receptor-mediated mitochondrial dysfunction and ER stress in oligodendrocytes

Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca²⁺ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca²⁺ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP₃Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP₃Rs. Blockade of Ca²⁺-induced Ca²⁺ release by TMB-8 following α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca²⁺ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca²⁺ overload whereas IP₃R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by α subunit of the eukaryotic initiation factor 2α phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca²⁺ release through RyRs contributes to cytosolic Ca²⁺ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes.     

Intracellular calcium release channels mediate their own countercurrent: the ryanodine receptor case study

Intracellular calcium release channels like ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP₃Rs) mediate large Ca²⁺ release events from Ca²⁺ storage organelles lasting >5 ms. To have such long-lasting Ca²⁺ efflux, a countercurrent of other ions is necessary to prevent the membrane potential from becoming the Ca²⁺ Nernst potential in <1 ms. A recent model of ion permeation through a single, open RyR channel is used here to show that the vast majority of this countercurrent is conducted by the RyR itself. Consequently, changes in membrane potential are minimized locally and instantly, assuring maintenance of a Ca²⁺-driving force. This RyR autocountercurrent is possible because of the poor Ca²⁺ selectivity and high conductance for both monovalent and divalent cations of these channels. The model shows that, under physiological conditions, the autocountercurrent clamps the membrane potential near 0 mV within ∼150 μs. Consistent with experiments, the model shows how RyR unit Ca²⁺ current is defined by luminal [Ca²⁺], permeable ion composition and concentration, and pore selectivity and conductance. This very likely is true of the highly homologous pore of the IP₃R channel.     

Fluvoxamine rescues mitochondrial Ca transport and ATP production through σ1-receptor in hypertrophic cardiomyocytes

Aims

We previously reported that fluvoxamine, a selective serotonin reuptake inhibitor with high affinity for the σ₁-receptor (σ₁R), ameliorates cardiac hypertrophy and dysfunction via σ₁R stimulation. Although σ₁R on non-cardiomyocytes interacts with the IP₃ receptor (IP₃R) to promote mitochondrial Ca^2 + transport, little is known about its physiological and pathological relevance in cardiomyocytes.

Main methods

Here we performed Ca^2 + imaging and measured ATP production to define the role of σ₁Rs in regulating sarcoplasmic reticulum (SR)-mitochondrial Ca^2 + transport in neonatal rat ventricular cardiomyocytes treated with angiotensin II to promote hypertrophy.

Key finding

These cardiomyocytes exhibited imbalances in expression levels of σ₁R and IP₃R and impairments in both phenylephrine-induced mitochondrial Ca^2 + mobilization from the SR and ATP production. Interestingly, σ₁R stimulation with fluvoxamine rescued impaired mitochondrial Ca^2 + mobilization and ATP production, an effect abolished by treatment of cells with the σ₁R antagonist, NE-100. Under physiological conditions, fluvoxamine stimulation of σ₁Rs suppressed intracellular Ca^2 + mobilization through IP₃Rs and ryanodine receptors (RyRs). In vivo, chronic administration of fluvoxamine to TAC mice also rescued impaired ATP production.

Significance

These results suggest that σ₁R stimulation with fluvoxamine promotes SR-mitochondrial Ca^2 + transport and mitochondrial ATP production, whereas σ₁R stimulation suppresses intracellular Ca^2 + overload through IP₃Rs and RyRs. These mechanisms likely underlie in part the anti-hypertrophic and cardioprotective action of the σ₁R agonists including fluvoxamine.     

Interaction of 2-APB,dantrolene, and TDMT with IP3R and RyR modulates ER stress-induced programmed cell death I and II in neuron-like PC12 cells: an experimental and computational investigation

Ca²⁺ is an essential second messenger, playing a fundamental role in maintaining cell viability and neuronal activity. Two specific endoplasmic reticulum calcium channels, ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP₃Rs) play an important role in Ca²⁺ regulation. In the present study, we provided a 3D structure of RyR and IP3R by homology modeling, and we predicted their interactions with a known neuroprotective compound, 3-thiomethyl-5,6-(dimethoxyphenyl)-1,2,4-triazine (TDMT), as well as two inhibitors, dantrolene and 2-aminoethoxydiphenyl borate (2-APB). Interestingly, we found that dantrolene and 2-APB can bind to the IP3-binding domain of IP3R and RyR, while TDMT may directly block both channels by interacting with the putative resident domains in the pore. Cell culture experiments showed that these compounds could protect PC12 cells against H₂O₂-induced apoptosis and activate autophagic pathways. Collectively, our computational (in silico) and cell culture studies suggest that RyR and IP3R are novel and promising targets to be used against neurodegenerative diseases.     

Involvement of plasma membrane Ca2+ channels,IP3 receptors,and ryanodine receptors in the generation of spontaneous rhythmic contractions of the cricket lateral oviduct

In the present study, the isolated cricket (Gryllus bimaculatus) lateral oviduct exhibited spontaneous rhythmic contractions (SRCs) with a frequency of 0.29 ± 0.009 Hz (n = 43) and an amplitude of 14.6 ± 1.25 mg (n = 29). SRCs completely disappeared following removal of extracellular Ca²⁺ using a solution containing 5 mM EGTA. Application of the non-specific Ca²⁺ channel blockers Co²⁺, Ni²⁺, and Cd²⁺ also decreased both the frequency and amplitude of SRCs in dose-dependent manners, suggesting that Ca²⁺ entry through plasma membrane Ca²⁺ channels is essential for the generation of SRCs. Application of ryanodine (30 μM), which depletes intracellular Ca²⁺ by locking ryanodine receptor (RyR)-Ca²⁺ channels in an open state, gradually reduced the frequency and amplitude of SRCs. A RyR antagonist, tetracaine, reduced both the frequency and amplitude of SRCs, whereas a RyR activator, caffeine, increased the frequency of SRCs with a subsequent increase in basal tonus, indicating that RyRs are essential for generating SRCs. To further investigate the involvement of phospholipase C (PLC) and inositol 1,4,5-trisphosphate receptors (IP₃Rs) in SRCs, we examined the effect of a PLC inhibitor, U73122, and an IP₃R antagonist, 2-aminoethoxydiphenyl borate (2-APB), on SRCs. Separately, U73122 (10 μM) and 2-APB (30–50 μM) both significantly reduced the amplitude of SRCs with little effect on their frequency, further indicating that the PLC/IP₃R signaling pathway is fundamental to the modulation of the amplitude of SRCs. A hypotonic-induced increase in the frequency and amplitude of SRCs and a hypertonic-induced decrease in the frequency and amplitude of SRCs indicated that mechanical stretch of the lateral oviduct is involved in the generation of SRCs. The sarcoplasmic reticulum Ca²⁺-pump ATPase inhibitors thapsigargin and cyclopiazonic acid impaired or suppressed the relaxation phase of SRCs. Taken together, the present results indicate that Ca²⁺ influx through plasma membrane Ca²⁺ channels and Ca²⁺ release from RyRs play an essential role in pacing SRCs and that Ca²⁺ release from IP₃Rs may play a role in modulating the amplitude of SRCs, probably via activation of PLC.     

Haloperidol Affects Plasticity of Differentiated NG-108 Cells Through σ1R/IP<Subscript>3</Subscript>R1 Complex

Haloperidol is an antipsychotic agent that primarily acts as an antagonist of D2 dopamine receptors. Besides other receptor systems, it targets sigma 1 receptors (σ1Rs) and inositol 1,4,5-trisphosphate receptors (IP₃Rs). Aim of this work was to investigate possible changes in IP₃Rs and σ1Rs resulting from haloperidol treatment and to propose physiological consequences in differentiated NG-108 cells, i.e., effect on cellular plasticity. Haloperidol treatment resulted in up-regulation of both type 1 IP₃Rs (IP₃R1s) and σ1Rs at mRNA and protein levels. Haloperidol treatment did not alter expression of other types of IP₃Rs. Calcium release from endoplasmic reticulum (ER) mediated by increased amount of IP₃R1s elevated cytosolic calcium and generated ER stress. IP₃R1s were bound to σ1Rs, and translocation of this complex from ER to nucleus occurred in the group of cells treated with haloperidol, which was followed by increased nuclear calcium levels. Haloperidol-induced changes in cytosolic, reticular, and nuclear calcium levels were similar when specific σ1 blocker -BD 1047- was used. Changes in calcium levels in nucleus, ER, and cytoplasm might be responsible for alterations in cellular plasticity, because length of neurites increased and number of neurites decreased in haloperidol-treated differentiated NG-108 cells.     

The Bcl-2 Protein Family Member Bok Binds to the Coupling Domain of Inositol 1,4,5-Trisphosphate Receptors and Protects Them from Proteolytic Cleavage

Bok is a member of the Bcl-2 protein family that controls intrinsic apoptosis. Bok is most closely related to the pro-apoptotic proteins Bak and Bax, but in contrast to Bak and Bax, very little is known about its cellular role. Here we report that Bok binds strongly and constitutively to inositol 1,4,5-trisphosphate receptors (IP₃Rs), proteins that form tetrameric calcium channels in the endoplasmic reticulum (ER) membrane and govern the release of ER calcium stores. Bok binds most strongly to IP₃R1 and IP₃R2, and barely to IP₃R3, and essentially all cellular Bok is IP₃R bound in cells that express substantial amounts of IP₃Rs. Binding to IP₃Rs appears to be mediated by the putative BH4 domain of Bok and the docking site localizes to a small region within the coupling domain of IP₃Rs (amino acids 1895–1903 of IP₃R1) that is adjacent to numerous regulatory sites, including sites for proteolysis. With regard to the possible role of Bok-IP₃R binding, the following was observed: (i) Bok does not appear to control the ability of IP₃Rs to release ER calcium stores, (ii) Bok regulates IP₃R expression, (iii) persistent activation of inositol 1,4,5-trisphosphate-dependent cell signaling causes Bok degradation by the ubiquitin-proteasome pathway, in a manner that parallels IP₃R degradation, and (iv) Bok protects IP₃Rs from proteolysis, either by chymotrypsin in vitro or by caspase-3 in vivo during apoptosis. Overall, these data show that Bok binds strongly and constitutively to IP₃Rs and that the most significant consequence of this binding appears to be protection of IP₃Rs from proteolysis. Thus, Bok may govern IP₃R cleavage and activity during apoptosis.     

Expression and reconstitution of the bioluminescent Ca2+ reporter aequorin in human embryonic stem cells,and exploration of the presence of functional IP3 and ryanodine receptors during the early stages of their differentiation into cardiomyocytes

In order to develop a novel method of visualizing possible Ca~(2+) signaling during the early differentiation of h ESCs into cardiomyocytes and avoid some of the inherent problems associated with using fluorescent reporters, we expressed the bioluminescent Ca~(2+) reporter, apo-aequorin, in HES2 cells and then reconstituted active holo-aequorin by incubation with f-coelenterazine. The temporal nature of the Ca~(2+) signals generated by the holo-f-aequorin-expressing HES2 cells during the earliest stages of differentiation into cardiomyocytes was then investigated. Our data show that no endogenous Ca~(2+) transients(generated by release from intracellular stores) were detected in 1–12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KCl or Ca Cl_2, indicating that holo-f-aequorin was functional in these cells. Furthermore, following the addition of exogenous ATP, an inositol trisphosphate receptor(IP_3R) agonist, small Ca~(2+) transients were generated from day 1 onward. That ATP was inducing Ca~(2+) release from functional IP_3 Rs was demonstrated by treatment with 2-APB, a known IP_3 R antagonist. In contrast, following treatment with caffeine, a ryanodine receptor(Ry R) agonist, a minimal Ca~(2+) response was observed at day 8 of differentiation only. Thus, our data indicate that unlike Ry Rs, IP_3 Rs are present and continually functional at these early stages of cardiomyocyte differentiation.     

Single-Molecule Tracking of Inositol Trisphosphate Receptors Reveals Different Motilities and Distributions

Puffs are local Ca²⁺ signals that arise by Ca²⁺ liberation from the endoplasmic reticulum through the concerted opening of tightly clustered inositol trisphosphate receptors/channels (IP₃Rs). The locations of puff sites observed by Ca²⁺ imaging remain static over several minutes, whereas fluorescence recovery after photobleaching (FRAP) experiments employing overexpression of fluorescently tagged IP₃Rs have shown that the majority of IP₃Rs are freely motile. To address this discrepancy, we applied single-molecule imaging to locate and track type 1 IP₃Rs tagged with a photoswitchable fluorescent protein and expressed in COS-7 cells. We found that ∼70% of the IP₃R1 molecules were freely motile, undergoing random walk motility with an apparent diffusion coefficient of ∼0.095 μm s⁻¹, whereas the remaining molecules were essentially immotile. A fraction of the immotile IP₃Rs were organized in clusters, with dimensions (a few hundred nanometers across) comparable to those previously estimated for the IP₃R clusters underlying functional puff sites. No short-term (seconds) changes in overall motility or in clustering of immotile IP₃Rs were apparent following activation of IP₃/Ca²⁺ signaling. We conclude that stable clusters of small numbers of immotile IP₃Rs may underlie local Ca²⁺ release sites, whereas the more numerous motile IP₃Rs appear to be functionally silent.     

Endoplasmic reticulum Ca release through ryanodine and IP3 receptors contributes to neuronal excitotoxicity

Overactivation of ionotropic glutamate receptors induces a Ca²⁺ overload into the cytoplasm that leads neurons to excitotoxic death, a process that has been linked to several neurodegenerative disorders. While the role of mitochondria and its involvement in excitotoxicity have been widely studied, the contribution of endoplasmic reticulum (ER), another crucial intracellular store in maintaining Ca²⁺ homeostasis, is not fully understood. In this study, we analyzed the contribution of ER-Ca²⁺ release through ryanodine (RyR) and IP₃ (IP₃R) receptors to a neuronal in vitro model of excitotoxicity. NMDA induced a dose-dependent neuronal death, which was significantly decreased by ER-Ca²⁺ release inhibitors in cortical neurons as well as in organotypic slices. Furthermore, ryanodine and 2APB, RyR and IP₃R inhibitors respectively, attenuated NMDA-triggered intracellular Ca²⁺ increase and oxidative stress, whereas 2APB reduced mitochondrial membrane depolarization and caspase-3 cleavage. Consistent with ER-Ca²⁺ homeostasis disruption, we observed that NMDA-induced ER stress, characterized here by eIF2α phosphorylation and over-expression of GRP chaperones which were regulated by ER-Ca²⁺ release inhibitors. These results demonstrate that Ca²⁺ release from ER contributes to neuronal death by both promoting mitochondrial dysfunction and inducing specific stress and apoptosis pathways during excitotoxicity.     

IP3-induced cytosolic and nuclear Ca2+ signals in HL-1 atrial myocytes: Possible role of IP3 receptor subtypes

HL-1 cells are the adult cardiac cell lines available that continuously divide while maintaining an atrial phenotype. Here we examined the expression and localization of inositol 1,4,5-trisphosphate receptor (IP₃R) subtypes, and investigated how pattern of IP₃-induced subcellular local Ca²⁺ signaling is encoded by multiple IP₃R subtypes in HL-1 cells. The type 1 IP₃R (IP₃R1) was expressed in the perinucleus with a diffuse pattern and the type 2 IP₃R (IP₃R2) was expressed in the cytosol with a punctate distribution. Extracellular ATP (1 mM) elicited transient intracellular Ca²⁺ releases accompanied by a Ca²⁺ oscillation, which was eliminated by the blocker of IP₃Rs, 2-APB, and attenuated by ryanodine. Direct introduction of IP₃ into the permeabilized cells induced Ca²⁺ transients with Ca²⁺ oscillations at ⩾ 20 μM of IP₃, which was removed by the inhibition of IP₃Rs using 2-APB and heparin. IP₃-induced local Ca²⁺ transients contained two distinct time courses: a rapid oscillation and a monophasic Ca²⁺ transient. The magnitude of Ca²⁺ oscillation was significantly larger in the cytosol than in the nucleus, while the monophasic Ca²⁺ transient was more pronounced in the nucleus. These results provide evidence for the molecular and functional expression of IP₃R1 and IP₃R2 in HL-1 cells, and suggest that such distinct local Ca²⁺ signaling may be correlated with the punctate distribution of IP₃R2s in the cytosol and the diffuse localization of IP₃R1 in the peri-nucleus.     

Granulosa cells express three inositol 1,4,5-trisphosphate receptor isoforms: cytoplasmic and nuclear Ca<Superscript>2+</Superscript> mobilization

Background  

Granulosa cells play an important endocrine role in folliculogenesis. They mobilize Ca2+ from intracellular stores by a coordinated action between 1,4,5 inositol trisphosphate and ryanodine receptors (IP3R and RyR). The aim of this study was to explore the isoforms of IP₃Rs expressed in mouse C57BL/6 NHsd granulosa cells, characterizing their intranuclear localization and the relation with other Ca2+-handling proteins.     

Regulation of transient receptor potential melastatin 4 channel by sarcoplasmic reticulum inositol trisphosphate receptors: Role in human detrusor smooth muscle function

We recently reported key physiologic roles for Ca²⁺-activated transient receptor potential melastatin 4 (TRPM4) channels in detrusor smooth muscle (DSM). However, the Ca²⁺-signaling mechanisms governing TRPM4 channel activity in human DSM cells are unexplored. As the TRPM4 channels are activated by Ca²⁺, inositol 1,4,5-trisphosphate receptor (IP₃R)-mediated Ca²⁺ release from the sarcoplasmic reticulum represents a potential Ca²⁺ source for TRPM4 channel activation. We used clinically-characterized human DSM tissues to investigate the molecular and functional interactions of the IP₃Rs and TRPM4 channels. With in situ proximity ligation assay (PLA) and perforated patch-clamp electrophysiology, we tested the hypothesis that TRPM4 channels are tightly associated with the IP₃Rs and are activated by IP₃R-mediated Ca²⁺ release in human DSM. With in situ PLA, we demonstrated co-localization of the TRPM4 channels and IP₃Rs in human DSM cells. As the TRPM4 channels and IP₃Rs must be located within close apposition to functionally interact, these findings support the concept of a potential Ca²⁺-mediated TRPM4-IP₃R regulatory mechanism. To investigate IP₃R regulation of TRPM4 channel activity, we sought to determine the consequences of IP₃R pharmacological inhibition on TRPM4 channel-mediated transient inward cation currents (TICCs). In freshly-isolated human DSM cells, blocking the IP₃Rs with the selective IP₃R inhibitor xestospongin-C significantly decreased TICCs. The data suggest that IP₃Rs have a key role in mediating the Ca²⁺-dependent activation of TRPM4 channels in human DSM. The study provides novel insight into the molecular and cellular mechanisms regulating TRPM4 channels by revealing that TRPM4 channels and IP₃Rs are spatially and functionally coupled in human DSM.     

Dynamic regulation of IP3 receptor clustering and activity by IP3

Inositol 1,4,5-trisphosphate receptors (IP₃Rs) are intracellular Ca²⁺ channels. Their regulation by both IP₃ and Ca²⁺ allows interactions between IP₃Rs to generate a hierarchy of intracellular Ca²⁺ release events. These can progress from openings of single IP₃R, through near-synchronous opening of a few IP₃Rs within a cluster to much larger signals that give rise to regenerative Ca²⁺ waves that can invade the entire cell. We have used patch-clamp recording from excised nuclear membranes of DT40 cells expressing only IP₃R3 and shown that low concentrations of IP₃ rapidly and reversibly cause IP₃Rs to assemble into small clusters. In addition to bringing IP₃Rs close enough to allow Ca²⁺ released by one IP₃R to regulate the activity of its neighbors, clustering also retunes the regulation of IP₃Rs by IP₃ and Ca²⁺. At resting cytosolic [Ca²⁺], lone IP₃R are more sensitive to IP₃ and the mean channel open time (~10ms) is twice as long as for clustered IP₃R. When the cytosolic free [Ca²⁺] is increased to 1µM, to mimic the conditions that might prevail when an IP₃R within a cluster opens, clustered IP₃R are no longer inhibited and their gating becomes coupled. IP₃, by dynamically regulating IP₃R clustering, both positions IP₃R for optimal interactions between them and it serves to exaggerate the effects of Ca²⁺ within a cluster. During the course of these studies, we have observed that nuclear IP₃R stably express one of two single channel K ⁺ conductances (γ_K ~120 or 200pS). Here we demonstrate that for both states of the IP₃R, the effects of IP₃ on clustering are indistinguishable. These observations reinforce our conclusion that IP₃ dynamically regulates assembly of IP₃Rs into clusters that underlie the hierarchical recruitment of elementary Ca²⁺ release events.     

Termination of calcium puffs and coupled closings of inositol trisphosphate receptor channels

Calcium puffs are localized Ca²⁺ signals mediated by Ca²⁺ release from the endoplasmic reticulum (ER) through clusters of inositol trisphosphate receptor (IP₃R) channels. The recruitment of IP₃R channels during puffs depends on Ca²⁺-induced Ca²⁺ release, a regenerative process that must be terminated to maintain control of cell signaling and prevent Ca²⁺ cytotoxicity. Here, we studied puff termination using total internal reflection microscopy to resolve the gating of individual IP₃R channels during puffs in intact SH-SY5Y neuroblastoma cells. We find that the kinetics of IP₃R channel closing differ from that expected for independent, stochastic gating, in that multiple channels tend to remain open together longer than predicted from their individual open lifetimes and then close in near-synchrony. This behavior cannot readily be explained by previously proposed termination mechanisms, including Ca²⁺-inhibition of IP₃Rs and local depletion of Ca²⁺ in the ER lumen. Instead, we postulate that the gating of closely adjacent IP₃Rs is coupled, possibly via allosteric interactions, suggesting an important mechanism to ensure robust puff termination in addition to Ca²⁺-inactivation.     

Inositol 1,4,5-trisphosphate receptor-isoform diversity in cell death and survival

Cell-death and -survival decisions are critically controlled by intracellular Ca^2 + homeostasis and dynamics at the level of the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) play a pivotal role in these processes by mediating Ca^2 + flux from the ER into the cytosol and mitochondria. Hence, it is clear that many pro-survival and pro-death signaling pathways and proteins affect Ca^2 + signaling by directly targeting IP₃R channels, which can happen in an IP₃R-isoform-dependent manner. In this review, we will focus on how the different IP₃R isoforms (IP₃R1, IP₃R2 and IP₃R3) control cell death and survival. First, we will present an overview of the isoform-specific regulation of IP₃Rs by cellular factors like IP₃, Ca^2 +, Ca^2 +-binding proteins, adenosine triphosphate (ATP), thiol modification, phosphorylation and interacting proteins, and of IP₃R-isoform specific expression patterns. Second, we will discuss the role of the ER as a Ca^2 + store in cell death and survival and how IP₃Rs and pro-survival/pro-death proteins can modulate the basal ER Ca^2 + leak. Third, we will review the regulation of the Ca^2 +-flux properties of the IP₃R isoforms by the ER-resident and by the cytoplasmic proteins involved in cell death and survival as well as by redox regulation. Hence, we aim to highlight the specific roles of the various IP₃R isoforms in cell-death and -survival signaling. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

The role of cAMP dependent protein kinase in modulating spontaneous intracellular Ca waves in interstitial cells of Cajal from the rabbit urethra

Interstitial cells of Cajal (ICC) serve as electrical pacemakers in the rabbit urethra. Pacemaking activity in ICC results from spontaneous intracellular Ca²⁺ waves that rely on Ca²⁺ release from endoplasmic reticulum (ER) stores. The purpose of this study was to investigate if the action of protein kinase A (PKA) affected the generation of Ca²⁺ waves in ICC. Intracellular [Ca²⁺] was measured in fluo-4 loaded ICC, freshly isolated from the rabbit urethra using a Nipkow spinning disc confocal microscope. Application of the PKA inhibitor H-89 (10 μM) significantly inhibited the generation of spontaneous Ca²⁺ waves in ICC and this was associated with a significant decrease in the ER Ca²⁺ load, measured with 10 mM caffeine responses. Ca²⁺ waves could be rescued in the presence of H-89 by stimulating ryanodine receptors (RyRs) with 1 mM caffeine but not by activation of inositol 1,4,5 tri-phosphate receptors (IP₃Rs) with 10 μM phenylephrine. Increasing intracellular PKA with the cAMP agonists forskolin and 8-bromo-cAMP failed to yield an increase in Ca²⁺ wave activity. We conclude that PKA may be maximally active under basal conditions in ICC and that inhibition of PKA with H-89 leads to a decreased ER Ca²⁺ load sufficient to inactivate IP₃Rs but not RyRs.