Evidence that NO/cGMP/PKG signalling cascade mediates endothelium dependent inhibition of IP3R mediated Ca2+ oscillations in myocytes and pericytes of ureteric microvascular network in situ

In ureteric microvessels the antagonistic relationship between Ca²⁺ signalling in endothelium and Ca²⁺ oscillations in myocytes and pericytes of arterioles and venules involves nitric oxide (NO), but the underlying mechanisms are not well understood. In the present study we investigated the effects of carbachol and NO donor SNAP on Ca²⁺ signalling and vasomotor responses of arterioles and venules in intact urteric microvascular network in situ using confocal microscopy. Vasomotor responses of arterioles and venules induced by AVP correlated with the occurrence of Ca²⁺ oscillations in the myocytes and pericytes and were not abolished by the removal of Ca²⁺ from extracellular fluid. Carbachol-induced rise of intracellular Ca²⁺ in endothelium was accompanied by the termination of the Ca²⁺ oscillations in myocytes and pericytes. This carbachol-induced inhibitory effect on Ca²⁺ oscillations in myocytes and pericytes was reversed by ODQ, an inhibitor of soluble guanylyl cyclase (sGC) and by Rp-8-pCPT-cGMPS, an inhibitor of protein kinase G (PKG). Ca²⁺ oscillations in myocytes and pericytes were also effectively blocked by NO donor SNAP. An Inhibitory effect of SNAP was markedly enhanced by zaprinast, a selective inhibitor of cGMP-specific phosphodiesterase-5, and reversed by sGC inhibitor, ODQ and PKG inhibitor, Rp-8-pCPT-cGMPS. The cGMP analogue and selective PKG activator 8pCPT-cGMP also induced inhibition of the AVP-induced Ca²⁺ oscillations in myocytes and pericytes. SNAP had no effects on Ca²⁺ oscillations induced by caffeine in distributing arcade arterioles. Consequently, we conclude that NO- mediated inhibition of Ca²⁺ oscillations in myocytes and pericytes predominantly recruits the cGMP/PKG dependent pathway. The inhibitory effect of NO/cGMP/PKG cascade is associated with suppressed Ca²⁺ release from the SR of myocytes and pericytes selectively via the inositol triphosphate receptor (IP₃R) channels.     

Involvement of mitogen-activated protein kinases and reactive oxygen species in the inotropic action of ouabain on cardiac myocytes. A potential role for mitochondrial KATP channels

Binding of ouabain to Na⁺/K⁺-ATPase activated multiple signal transduction pathways including stimulation of Src, Ras, p42/44 MAPKs and production of reactive oxygen species (ROS) in rat cardiac myocytes. Inhibition of either Src or Ras ablated ouabain-induced increase in both [Ca²⁺]_i and contractility. While PD98059 abolished the effects of ouabain on [Ca²⁺]_i, it only caused a partial inhibition of ouabain-induced increases in contractility. On the other hand, pre-incubation of myocytes with N-acetyl cysteine (NAC) reduced the effects of ouabain on contractility, but not [Ca²⁺]_i. Furthermore, 5-hydroxydecanoate (5-HD) blocked ouabain-induced ROS production and partially inhibited ouabain-induced increases in contractility in cardiac myocytes. Pre-incubation of myocytes with both 5-HD and PD98059 completely blocked ouabain's effect on contractility. Finally, we found that opening of mitochondrial K_ATP channel by diazoxide increased intracellular ROS and significantly raised contractility in cardiac myocytes. These new findings indicate that ouabain regulates cardiac contractility via both [Ca²⁺]_i and ROS. While activation of MAPKs leads to increases in [Ca²⁺]_i, opening of mitochondrial K_ATP channel relays the ouabain signal to increased ROS production in cardiac myocytes.     

Atherosclerosis affects calcium signalling in endothelial cells from apolipoprotein E knockout mice before plaque formation

Little is known about how hypercholesterolaemia affects Ca²⁺ signalling in the vasculature of ApoE^−/− mice, a model of atherosclerosis. Our objectives were therefore to determine (i) if hypercholesterolaemia alters Ca²⁺ signalling in aortic endothelial cells before overt atherosclerotic lesions occur, (ii) how Ca²⁺ signals are affected in older plaque-containing mice, and (iii) whether Ca²⁺ signalling changes were translated into contractility differences. Using confocal microscopy we found agonist-specific Ca²⁺ changes in endothelial cells. ATP responses were unchanged in ApoE^−/− cells and methyl-β-cyclodextrin, which lowers cholesterol, was without effect. In contrast, Ca²⁺ signals to carbachol were significantly increased in ApoE^−/− cells, an effect methyl-β-cyclodextrin reversed. Ca²⁺ signals were more oscillatory and store-operated Ca²⁺ entry decreased as mice aged and plaques formed. Despite clearly increased Ca²⁺ signals, aortic rings pre-contracted with phenylephrine had impaired relaxation to carbachol. This functional deficit increased with age, was not related to ROS generation, and could be partially rescued by methyl-β-cyclodextrin. In conclusion, carbachol-induced calcium signalling and handling are significantly altered in endothelial cells of ApoE^−/− mice before plaque development. We speculate that reduction in store-operated Ca²⁺ entry may result in less efficient activation of eNOS and thus explain the reduced relaxatory response to CCh, despite the enhanced Ca²⁺ response.     

Atrial cardiomyocyte calcium signalling

Whereas Ca²⁺ signalling in ventricular cardiomyocytes is well described, much less is known regarding the Ca²⁺ signals within atrial cells. This is surprising given that atrial cardiomyocytes make an important contribution to the refilling of ventricles with blood, which enhances the subsequent ejection of blood from the heart. The dependence of cardiac function on the contribution of atria becomes increasingly important with age and exercise. Disruption of the rhythmic beating of atrial cardiomyocytes can lead to life-threatening conditions such as atrial fibrillation. Atrial and ventricular myocytes have many structural and functional similarities. However, one key structural difference, the lack of transverse tubules (“T-tubules”) in atrial myocytes, make these two cell types display vastly different calcium patterns in response to electrical excitation. The lack of T-tubules in atrial myocytes means that depolarisation provokes calcium signals that originate around the periphery of the cells. Under resting conditions, such Ca²⁺ signals do not propagate towards the centre of the atrial cells and so do not fully engage the contractile machinery. Consequently, contraction of atrial myocytes under resting conditions is modest. However, when atrial myocytes are stimulated with a positive inotropic agonist, such as isoproterenol, the peripheral Ca²⁺ signals trigger a global wave of Ca²⁺ that propagates in a centripetal manner into the cells. Enhanced centripetal movement of Ca²⁺ in atrial myocytes leads to increased contraction and a more substantial contribution to blood pumping. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Calcineurin is part of a negative feedback loop in the InsP3/Ca signalling pathway in blowfly salivary glands

The ubiquitous InsP₃/Ca²⁺ signalling pathway is modulated by diverse mechanisms, i.e. feedback of Ca²⁺ and interactions with other signalling pathways. In the salivary glands of the blowfly Calliphora vicina, the hormone serotonin (5-HT) causes a parallel rise in intracellular [Ca²⁺] and [cAMP] via two types of 5-HT receptors. We have shown recently that cAMP/protein kinase A (PKA) sensitizes InsP₃-induced Ca²⁺ release. We have now identified the protein phosphatase that counteracts the effect of PKA on 5-HT-induced InsP₃/Ca²⁺ signalling. We demonstrate that (1) tautomycin and okadaic acid, inhibitors of protein phosphatases PP1 and PP2A, have no effect on 5-HT-induced Ca²⁺ signals; (2) cyclosporin A and FK506, inhibitors of Ca²⁺/calmodulin-activated protein phosphatase calcineurin, cause an increase in the frequency of 5-HT-induced Ca²⁺ oscillations; (3) the sensitizing effect of cyclosporin A on 5-HT-induced Ca²⁺ responses does not involve Ca²⁺ entry into the cells; (4) cyclosporin A increases InsP₃-dependent Ca²⁺ release; (5) inhibition of PKA abolishes the effect of cyclosporin A on the 5-HT-induced Ca²⁺ responses, indicating that PKA and calcineurin act antagonistically on the InsP₃/Ca²⁺ signalling pathway. These findings suggest that calcineurin provides a negative feedback on InsP₃/Ca²⁺ signalling in blowfly salivary glands, counteracting the effect of PKA and desensitizing the signalling cascade at higher 5-HT concentrations.     

Alterations in the Function of Endoplasmic Reticulum and Neuronal Signalling

For many years, the endoplasmic reticulum (ER) was considered to be involved in rapid signalling events due to its ability to serve as a dynamic calcium store capable of accumulating large amounts of Ca²⁺ ions and of releasing them in response to physiological stimulation. Recent data significantly increased the importance of the ER as a signalling organelle, by demonstrating that the ER is associated with specific pathways regulating long-lasting adaptive processes and controlling cell survival. The ER lumen is enriched by enzymatic systems involved in protein synthesis and correcting post-translational folding of these proteins. The processes of post-translational protein processing are controlled by a class of specific enzymes known as chaperones, which in turn are regulated by the free Ca²⁺ concentration within the ER lumen ([Ca²⁺]_L). At the same time, a high [Ca²⁺]_L determines the ability of the ER to generate cytosolic Ca²⁺ signals. Thus, the ER is able to produce signals interacting within different temporal domains. Fast ER signals result from Ca²⁺ release via specific Ca²⁺-release channels and from rapid movements of Ca²⁺ ions within the ER lumen (calcium tunneling). Long-lasting signals involve Ca²⁺-dependent regulation of chaperones with subsequent changes in protein processing and synthesis. Any malfunctions in the ER Ca²⁺ homeostasis result in accumulation of unfolded proteins, which in turn activates several signalling systems aimed at appropriate compensatory responses or (in the case of severe ER dysregulation) in cellular pathology and death (ER stress responses). Thus, the Ca²⁺ ion emerges as a messenger molecule, which integrates various signals within the ER: fluctuations of the [Ca²⁺]_L induced by signals originating at the level of the plasmalemma (i.e., Ca²⁺ entry or activation of the metabotropic receptors) regulate in turn protein synthesis and processing via generating secondary signalling events between the ER and the nucleus.     

Mitochondrial permeability transition in Ca(2+)-dependent apoptosis and necrosis

A variety of stimuli utilize an increase of cytosolic free Ca²⁺ concentration as a second messenger to transmit signals, through Ca²⁺ release from the endoplasmic reticulum or opening of plasma membrane Ca²⁺ channels. Mitochondria contribute to the tight spatiotemporal control of this process by accumulating Ca²⁺, thus shaping the return of cytosolic Ca²⁺ to resting levels. The rise of mitochondrial matrix free Ca²⁺ concentration stimulates oxidative metabolism; yet, in the presence of a variety of sensitizing factors of pathophysiological relevance, the matrix Ca²⁺ increase can also lead to opening of the permeability transition pore (PTP), a high conductance inner membrane channel. While transient openings may serve the purpose of providing a fast Ca²⁺ release mechanism, persistent PTP opening is followed by deregulated release of matrix Ca²⁺, termination of oxidative phosphorylation, matrix swelling with inner membrane unfolding and eventually outer membrane rupture with release of apoptogenic proteins and cell death. Thus, a rise in mitochondrial Ca²⁺ can convey both apoptotic and necrotic death signals by inducing opening of the PTP. Understanding the signalling networks that govern changes in mitochondrial free Ca²⁺ concentration, their interplay with Ca²⁺ signalling in other subcellular compartments, and regulation of PTP has important implications in the fine comprehension of the main biological routines of the cell and in disease pathogenesis.     

Fluctuations in the Concentration of Extracellular ATP Synchronized with Intracellular Ca2+ Oscillatory Rhythm in Cultured Cardiac Myocytes

Isolated and cultured neonatal cardiac myocytes contract spontaneously and cyclically. The intracellular concentration of free Ca²⁺ also changes rhythmically in association with the rhythmic contraction of myocytes (Ca²⁺ oscillation). Both the contraction and Ca²⁺ oscillatory rhythms are synchronized among myocytes, and intercellular communication via gap junctions has been considered primarily responsible for the synchronization. However, a recent study has demonstrated that intercellular communication via extracellular ATP‐purinoceptor signaling is also involved in the intercellular synchronization of intracellular Ca²⁺ oscillation. In this study, we aim to elucidate whether the concentration of extracellular ATP changes cyclically and contributes to the intercellular synchronization of Ca²⁺ oscillation among myocytes. In almost all the cultured cardiac myocytes at four days in vitro (4 DIV), intracellular Ca²⁺ oscillations were synchronized with each other. The simultaneous measurement of the concentration of extracellular ATP and intracellular Ca²⁺ revealed the extracellular concentration of ATP actually oscillated concurrently with the intracellular Ca²⁺ oscillation. In addition, power spectrum and cross‐correlation analyses suggested that the treatment of cultured cardiac myocytes with suramin, a blocker of P2 purinoceptors, resulted in the asynchronization of Ca²⁺ oscillatory rhythms among cardiac myocytes. Treatment with suramin also resulted in a significant decrease in the amplitudes of the cyclic changes in both intracellular Ca²⁺ and extracellular ATP. Taken together, the present study demonstrated the possibility that the concentration of extracellular ATP changes cyclically in association with intracellular Ca²⁺, contributing to the intercellular synchronization of Ca²⁺ oscillation among cultured cardiac myocytes.     

Oxytocin induces intracellular Ca2+ release in cardiac fibroblasts from neonatal rats

Pituitary neuropeptide oxytocin is increasingly recognised as a cardiovascular hormone, in addition to its many regulatory roles in other organ systems. Studies in atrial and ventricular myocytes from the neonatal and adult rats have identified synthesis of oxytocin and the expression of oxytocin receptors in these cells. In cardiac fibroblasts, the most populous non-myocyte cell type in mammalian heart, the oxytocin receptors have not been described before. In the present study, we have investigated the direct effects of oxytocin on intracellular Ca²⁺ dynamics in ventricular myocytes and fibroblasts from new born rats. In myocytes, oxytocin increased the frequency of spontaneous Ca²⁺ transients and decreased their amplitude. Our data suggest that oxytocin receptors are also present and functional in the majority of cardiac fibroblasts. We used selective oxytocin receptor inhibitor L-371,257 and a number of intracellular Ca ²⁺ release blockers to investigate the mechanism of oxytocin induced Ca²⁺ signalling in cardiac fibroblasts. Our findings suggest that oxytocin induces Ca²⁺ signals in cardiac fibroblasts by triggering endoplasmic reticulum Ca²⁺ release via inositol trisphosphate activated receptors. The functional significance of the oxytocin induced Ca²⁺ signalling in cardiac fibroblasts, especially for their activation into secretory active myofibroblasts, remains to be investigated.     

Purinergic stimulation of K+-dependent Na+/Ca2+ exchanger isoform 4 requires dual activation by PKC and CaMKII

K⁺-dependent Na⁺/Ca²⁺-exchanger isoform 4 (NCXK4) is one of the most broadly expressed members of the NCKX (K⁺-dependent Na⁺/Ca²⁺-exchanger) family. Recent data indicate that NCKX4 plays a critical role in controlling normal Ca²⁺ signal dynamics in olfactory and other neurons. Synaptic Ca²⁺ dynamics are modulated by purinergic regulation, mediated by ATP released from synaptic vesicles or from neighbouring glial cells. Previous studies have focused on modulation of Ca²⁺ entry pathways that initiate signalling. Here we have investigated purinergic regulation of NCKX4, a powerful extrusion pathway that assists in terminating Ca²⁺ signals. NCKX4 activity was stimulated by ATP through activation of the P2Y receptor signalling pathway. Stimulation required dual activation of PKC (protein kinase C) and CaMKII (Ca²⁺/calmodulin-dependent protein kinase II). Mutating T312, a putative PKC phosphorylation site on NCKX4, partially prevented purinergic stimulation. These data illustrate how purinergic regulation can shape the dynamics of Ca²⁺ signalling by activating a signal damping and termination pathway.     

Potassium Currents in Freshly Dissociated Uterine Myocytes from Nonpregnant and Late-Pregnant Rats

In freshly dissociated uterine myocytes, the outward current is carried by K⁺ through channels highly selective for K⁺. Typically, nonpregnant myocytes have rather noisy K⁺ currents; half of them also have a fast-inactivating transient outward current (I_TO). In contrast, the current records are not noisy in late pregnant myocytes, and I_TO densities are low. The whole-cell I_K of nonpregnant myocytes respond strongly to changes in [Ca²⁺]_o or changes in [Ca²⁺]_i caused by photolysis of caged Ca²⁺ compounds, nitr 5 or DM-nitrophene, but that of late-pregnant myocytes respond weakly or not at all. The Ca²⁺ insensitivity of the latter is present before any exposure to dissociating enzymes. By holding at −80, −40, or 0 mV and digital subtractions, the whole-cell I_K of each type of myocyte can be separated into one noninactivating and two inactivating components with half-inactivation at approximately −61 and −22 mV. The noninactivating components, which consist mainly of iberiotoxin-susceptible large-conductance Ca²⁺-activated K⁺ currents, are half-activated at 39 mV in nonpregnant myocytes, but at 63 mV in late-pregnant myocytes. In detached membrane patches from the latter, identified 139 pS, Ca²⁺-sensitive K⁺ channels also have a half-open probability at 68 mV, and are less sensitive to Ca²⁺ than similar channels in taenia coli myocytes. Ca²⁺-activated K⁺ currents, susceptible to tetraethylammonium, charybdotoxin, and iberiotoxin contribute 30–35% of the total I_K in nonpregnant myocytes, but <20% in late-pregnant myocytes. Dendrotoxin-susceptible, small-conductance delayed rectifier currents are not seen in nonpregnant myocytes, but contribute ∼20% of total I_K in late-pregnant myocytes. Thus, in late-pregnancy, myometrial excitability is increased by changes in K⁺ currents that include a suppression of the I_TO, a redistribution of I_K expression from large-conductance Ca²⁺-activated channels to smaller-conductance delayed rectifier channels, a lowered Ca²⁺ sensitivity, and a positive shift of the activation of some large-conductance Ca²⁺-activated channels.     

灰斑古毒蛾口腔反吐物诱导沙冬青细胞Ca2+内流及H2O2积累

植物对昆虫取食活动进行成功防御的关键,取决于对昆虫口腔反吐物的激发子的快速识别。实验利用无损伤微测系统及激光共聚焦显微镜,研究了沙冬青细胞经灰斑古毒蛾口腔反吐物诱导后Ca2+流及H2O2的变化。结果发现:灰斑古毒蛾口腔反吐物诱导沙冬青细胞Ca2+内流及H2O2的积累,表明Ca2+内流及H2O2的积累是沙冬青细胞对口腔反吐物产生应答的早期响应事件;Ca2+钙通道阻断剂仅部分抑制Ca2+内流,说明Ca2+内流除经过质膜上的Ca2+通道进入细胞外,尚存在其他的内流途径;灰斑古毒蛾口腔反吐物中的某些成分与沙冬青细胞的质膜结合后,诱导质膜上形成允许Ca2+通过的孔道,而GdCl3不能抑制这类孔道的活性。胞外Ca2+螯合剂EGTA完全抑制H2O2的积累,GdCl3预处理仅部分抑制了H2O2的积累,说明灰斑古毒蛾诱导的沙冬青细胞内H2O2的积累依赖于Ca2+内流;抑制剂实验表明,H2O2的积累主要来源于质膜上NADPH氧化酶的作用。     

Importance of Ca2+ influx by Na+/Ca2+ exchange under normal and sodium-loaded conditions in mammalian ventricles

Na⁺/Ca²⁺ exchange (NCX) is a major Ca²⁺ extrusion system in cardiac myocytes, but can also mediate Ca²⁺ influx and trigger sarcoplasmic reticulum Ca²⁺ release. Under conditions such as digitalis toxicity or ischemia/reperfusion, increased [Na⁺]_i may lead to a rise in [Ca²⁺]_i through NCX, causing Ca²⁺ overload and triggered arrhythmias. Here we used an agent which selectively blocks Ca²⁺ influx by NCX, KB-R7943 (KBR), and assessed twitch contractions and Ca²⁺ transients in rat and guinea pig ventricular myocytes loaded with indo-1. KBR (5 M) did not alter control steady-state twitch contractions or Ca²⁺ transients at 0.5 Hz in rat, but significantly decreased them in guinea pig myocytes. When cells were Na⁺-loaded by perfusion of strophanthidin (50 M), the addition of KBR reduced diastolic [Ca²⁺]_i and abolished spontaneous Ca²⁺ oscillations. In guinea pig papillary muscles exposed to substrate-free hypoxic medium for 60 min, KBR (10 M applied 10 min before and during reoxygenation) reduced both the incidence and duration of reoxygenation-induced arrhythmias. KBR also enhanced the recovery of developed tension after reoxygenation. It is concluded that (1) the importance of Ca²⁺ influx via NCX for normal excitation-contraction coupling is species-dependent, and (2) Ca²⁺ influx via NCX may be critical in causing myocardial Ca²⁺ overload and triggered activities induced by cardiac glycoside or reoxygenation.     

Elevated InsP3R expression underlies enhanced calcium fluxes and spontaneous extra-systolic calcium release events in hypertrophic cardiac myocytes

Cardiac hypertrophy is associated with profound remodelling of Ca²⁺ signalling pathways. During the early, compensated stages of hypertrophy, Ca²⁺ fluxes may be enhanced to facilitate greater contraction, whereas as the hypertrophic heart decompensates, Ca²⁺ homeostatic mechanisms are dysregulated leading to decreased contractility, arrhythmia and death. Although ryanodine receptor Ca²⁺ release channels (RyR) on the sarcoplasmic reticulum (SR) intracellular Ca²⁺ store are primarily responsible for the Ca²⁺ flux that induces myocyte contraction, a role for Ca²⁺ release via the inositol 1,4,5-trisphosphate receptor (InsP₃R) in cardiac physiology has also emerged. Specifically, InsP₃-induced Ca²⁺ signals generated following myocyte stimulation with an InsP₃-generating agonist (e.g. endothelin, ET-1), lead to modulation of Ca²⁺ signals associated with excitation-contraction coupling (ECC) and the induction of spontaneous Ca²⁺ release events that cause cellular arrhythmia. Using myocytes from spontaneously hypertensive rats (SHR), we recently reported that expression of the type 2 InsP₃R (InsP₃R2) is significantly increased during hypertrophy. Notably, this increased expression was restricted to the junctional SR in close proximity to RyRs. There, enhanced Ca²⁺ release via InsP₃Rs serves to sensitise neighbouring RyRs causing an augmentation of Ca²⁺ fluxes during ECC as well as an increase in non-triggered Ca²⁺ release events. Although the sensitization of RyRs may be a beneficial consequence of elevated InsP₃R expression during hypertrophy, the spontaneous Ca²⁺ release events are potentially of pathological significance giving rise to cardiac arrhythmia. InsP₃R2 expression was also increased in hypertrophic hearts from patients with ischemic dilated cardiomyopathy and aortically-banded mice demonstrating that increased InsP₃R expression may be a general phenomenon that underlies Ca²⁺ changes during hypertrophy.     

Modulation of calcium signalling by mitochondria

In this review we will attempt to summarise the complex and sometimes contradictory effects that mitochondria have on different forms of calcium signalling. Mitochondria can influence Ca²⁺ signalling indirectly by changing the concentration of ATP, NAD(P)H, pyruvate and reactive oxygen species — which in turn modulate components of the Ca²⁺ signalling machinery i.e. buffering, release from internal stores, influx from the extracellular solution, uptake into cellular organelles and extrusion by plasma membrane Ca²⁺ pumps. Mitochondria can directly influence the calcium concentration in the cytosol of the cell by importing Ca²⁺ via the mitochondrial Ca²⁺ uniporter or transporting Ca²⁺ from the interior of the organelle into the cytosol by means of Na⁺/Ca²⁺ or H⁺/Ca²⁺ exchangers. Considerable progress in understanding the relationship between Ca²⁺ signalling cascades and mitochondrial physiology has been accumulated over the last few years due to the development of more advanced optical techniques and electrophysiological approaches.     

Mechanisms by which cytoplasmic calcium wave propagation and alternans are generated in cardiac atrial myocytes lacking T-tubules-insights from a simulation study

This study investigated the mechanisms underlying the propagation of cytoplasmic calcium waves and the genesis of systolic Ca²⁺ alternans in cardiac myocytes lacking transverse tubules (t-tubules). These correspond to atrial cells of either small mammals or large mammals that have lost their t-tubules due to disease-induced structural remodeling (e.g., atrial fibrillation). A mathematical model was developed for a cluster of ryanodine receptors distributed on the cross section of a cell that was divided into 13 elements with a spatial resolution of 2 μm. Due to the absence of t-tubules, L-type Ca²⁺ channels were only located in the peripheral elements close to the cell-membrane surface and produced Ca²⁺ signals that propagated toward central elements by triggering successive Ca²⁺-induced Ca²⁺ release (CICR) via Ca²⁺ diffusion between adjacent elements. Under control conditions, the Ca²⁺ signals did not fully propagate to the central region of the cell. However, with modulation of several factors responsible for Ca²⁺ handling, such as the L-type Ca²⁺ channels (Ca²⁺ influx), SERCA pumps (sarcoplasmic reticulum (SR) Ca²⁺ uptake), and ryanodine receptors (SR Ca²⁺ release), Ca²⁺ wave propagation to the center of the cell could occur. These simulation results are consistent with previous experimental data from atrial cells of small mammals. The model further reveals that spatially functional heterogeneity in Ca²⁺ diffusion within the cell produced a steep relationship between the SR Ca²⁺ content and the cytoplasmic Ca²⁺ concentration. This played an important role in the genesis of Ca²⁺ alternans that were more obvious in central than in peripheral elements. Possible association between the occurrence of Ca²⁺ alternans and the model parameters of Ca²⁺ handling was comprehensively explored in a wide range of one- and two-parameter spaces. In addition, the model revealed a spontaneous second Ca²⁺ release in response to a single voltage stimulus pulse with SR Ca²⁺ overloading and augmented Ca²⁺ influx. This study provides what to our knowledge are new insights into the genesis of Ca²⁺ alternans and spontaneous second Ca²⁺ release in cardiac myocytes that lack t-tubules.     

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.     

Hydrogen sulphide triggers VEGF-induced intracellular Ca signals in human endothelial cells but not in their immature progenitors

Hydrogen sulphide (H₂S) is a newly discovered gasotransmitter that regulates multiple steps in VEGF-induced angiogenesis. An increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) is central to endothelial proliferation and may be triggered by both VEGF and H₂S. Albeit VEGFR-2 might serve as H₂S receptor, the mechanistic relationship between VEGF- and H₂S-induced Ca²⁺ signals in endothelial cells is unclear. The present study aimed at assessing whether and how NaHS, a widely employed H₂S donor, stimulates pro-angiogenic Ca²⁺ signals in Ea.hy926 cells, a suitable surrogate for mature endothelial cells, and human endothelial progenitor cells (EPCs). We found that NaHS induced a dose-dependent increase in [Ca²⁺]_i in Ea.hy926 cells. NaHS-induced Ca²⁺ signals in Ea.hy926 cells did not require extracellular Ca²⁺ entry, while they were inhibited upon pharmacological blockade of the phospholipase C/inositol-1,4,5-trisphosphate (InsP₃) signalling pathway. Moreover, the Ca²⁺ response to NaHS was prevented by genistein, but not by SU5416, which selectively inhibits VEGFR-2. However, VEGF-induced Ca²⁺ signals were suppressed by dl-propargylglycine (PAG), which blocks the H₂S-producing enzyme, cystathionine γ-lyase. Consistent with these data, VEGF-induced proliferation and migration were inhibited by PAG in Ea.hy926 cells, albeit NaHS alone did not influence these processes. Conversely, NaHS elevated [Ca²⁺]_i only in a modest fraction of circulating EPCs, whereas neither VEGF-induced Ca²⁺ oscillations nor VEGF-dependent proliferation were affected by PAG. Therefore, H₂S-evoked elevation in [Ca²⁺]_i is essential to trigger the pro-angiogenic Ca²⁺ response to VEGF in mature endothelial cells, but not in their immature progenitors.     

Physiologic gating properties of unitary cardiac L-type Ca channels

The contraction of adult mammalian ventricular cardiomyocytes is triggered by the influx of Ca²⁺ ions through sarcolemmal L-type Ca²⁺ channels (LCCs). However, the gating properties of unitary LCCs under physiologic conditions have remained elusive. Towards this end, we investigated the voltage-dependence of the gating kinetics of unitary LCCs, with a physiologic concentration of Ca²⁺ ions permeating the channel. Unitary LCC currents were recorded with 2 mM external Ca²⁺ ions (in the absence of LCC agonists), using cell-attached patches on K-depolarized adult rat ventricular myocytes. The voltage-dependence of the peak probability of channel opening (Po vs. Vm) displayed a maximum value of 0.3, a midpoint of −12 mV, and a slope factor of 8.5. The maximum value for Po of the unitary LCC was significantly higher than previously assumed, under physiologic conditions. We also found that the mean open dwell time of the unitary LCC increased twofold with depolarization, ranging from 0.53 ± 0.02 ms at −30 mV to 1.08 ± 0.03 ms at 0 mV. The increase in mean LCC open time with depolarization counterbalanced the decrease in the single LCC current amplitude; the latter due to the decrease in driving force for Ca²⁺ ion entry. Thus, the average amount of Ca²⁺ ions entering through an individual LCC opening (∼300-400 ions) remained relatively constant over this range of potentials. These novel results establish the voltage-dependence of unitary LCC gating kinetics using a physiologic Ca²⁺ ion concentration. Moreover, they provide insight into local Ca²⁺-induced Ca²⁺ release and a more accurate basis for mathematical modeling of excitation-contraction coupling in cardiac myocytes.