Ca Waves Research Articles

The phospholipase C inhibitor U‐73122 inhibits Ca2+ release from the intracellular sarcoplasmic reticulum Ca2+ store by inhibiting Ca2+ pumps in smooth muscle

The sarcoplasmic reticulum (SR) releases Ca(2+) via inositol 1,4,5-trisphosphate receptors (IP(3)R) in response to IP(3)-generating agonists. Ca(2+) release subsequently propagates as Ca(2+) waves. To clarify the role of IP(3) production in wave generation, the contribution of a key enzyme in the production of IP(3) was examined using a phosphoinositide-specific phospholipase C (PI-PLC) inhibitor, U-73122. Single colonic myocytes were voltage-clamped in whole-cell configuration and cytosolic Ca(2+) concentration ([Ca(2+)](cyto)) measured using fluo-3. SR Ca(2+) release was evoked either by activation of IP(3)Rs (by carbachol or photolysis of caged IP(3)) or ryanodine receptors (RyRs; by caffeine). U-73122 inhibited carbachol-evoked [Ca(2+)](cyto) transients. The drug also inhibited [Ca(2+)](cyto) increases, evoked by direct IP(3)R activation (by photolysis of caged IP(3)) and RyR activation (by caffeine), which do not require PI-PLC activation. U-73122 also increased steady-state [Ca(2+)](cyto) and slowed the rate of Ca(2+) removal from the cytoplasm. An inactive analogue of U-73122, U-73343, was without effect on either IP(3)R- or RyR-mediated Ca(2+) release. U-73122 inhibited carbachol-evoked [Ca(2+)](cyto) increases. However, the drug also reduced Ca(2+) release when evoked by direct activation of IP(3)R or RyR, slowed Ca(2+) removal and increased steady-state [Ca(2+)](cyto). These results suggest U-73122 reduces IP(3)-evoked Ca(2+) transients by inhibiting the SR Ca(2+) pump to deplete the SR of Ca(2+) rather than by inhibiting PI-PLC.

Photochemically Initiated Intracellular Astrocytic Calcium Waves in Living Mice Using Two-Photon Uncaging of IP3

We have developed a caged IP(3) analogue for two-photon photolysis in living animals. This probe is a cell permeable version and was coloaded with a fluorescent Ca(2+) dye into astrocytes in layer 1 of the somatosensory cortex of anesthetized mice. Two-photon irradiation of single cells at 720 nm produced rapid and robust increases in intracellular Ca(2+) concentrations monitored using two-photon microscopy at 950 nm. The photoevoked intracellular Ca(2+) waves were similar in magnitude to intrinsic signals in wild type mice. These waves did not propagate to other cells beyond the targeted astrocyte. In contrast, we observed intercellular astrocytic Ca(2+) waves in two mouse models of familial Alzheimer's disease. These data suggest that Alzheimer's might perturb gliotransmission but not IP(3) signaling per se in mouse models of the disease.

Damage-induced cell–cell communication in different cochlear cell types via two distinct ATP-dependent Ca2+ waves

Intercellular Ca(2+) waves can coordinate the action of large numbers of cells over significant distances. Recent work in many different systems has indicated that the release of ATP is fundamental for the propagation of most Ca(2+) waves. In the organ of hearing, the cochlea, ATP release is involved in critical signalling events during tissue maturation. ATP-dependent signalling is also implicated in the normal hearing process and in sensing cochlear damage. Here, we show that two distinct Ca(2+) waves are triggered during damage to cochlear explants. Both Ca(2+) waves are elicited by extracellular ATP acting on P2 receptors, but they differ in their source of Ca(2+), their velocity, their extent of spread and the cell type through which they propagate. A slower Ca(2+) wave (14 mum/s) communicates between Deiters' cells and is mediated by P2Y receptors and Ca(2+) release from IP(3)-sensitive stores. In contrast, a faster Ca(2+) wave (41 mum/s) propagates through sensory hair cells and is mediated by Ca(2+) influx from the external environment. Using inhibitors and selective agonists of P2 receptors, we suggest that the faster Ca(2+) wave is mediated by P2X(4) receptors. Thus, in complex tissues, the expression of different receptors determines the propagation of distinct intercellular communication signals. The online version of this article (doi:10.1007/s11302-010-9193-8) contains supplementary material, which is available to authorized users.

Agonist‐evoked Ca2+ wave progression requires Ca2+ and IP3

Smooth muscle responds to IP(3)-generating agonists by producing Ca(2+) waves. Here, the mechanism of wave progression has been investigated in voltage-clamped single smooth muscle cells using localized photolysis of caged IP(3) and the caged Ca(2+) buffer diazo-2. Waves, evoked by the IP(3)-generating agonist carbachol (CCh), initiated as a uniform rise in cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) over a single though substantial length (approximately 30 microm) of the cell. During regenerative propagation, the wave-front was about 1/3 the length (approximately 9 microm) of the initiation site. The wave-front progressed at a relatively constant velocity although amplitude varied through the cell; differences in sensitivity to IP(3) may explain the amplitude changes. Ca(2+) was required for IP(3)-mediated wave progression to occur. Increasing the Ca(2+) buffer capacity in a small (2 microm) region immediately in front of a CCh-evoked Ca(2+) wave halted progression at the site. However, the wave front does not progress by Ca(2+)-dependent positive feedback alone. In support, colliding [Ca(2+)](c) increases from locally released IP(3) did not annihilate but approximately doubled in amplitude. This result suggests that local IP(3)-evoked [Ca(2+)](c) increases diffused passively. Failure of local increases in IP(3) to evoke waves appears to arise from the restricted nature of the IP(3) increase. When IP(3) was elevated throughout the cell, a localized increase in Ca(2+) now propagated as a wave. Together, these results suggest that waves initiate over a surprisingly large length of the cell and that both IP(3) and Ca(2+) are required for active propagation of the wave front to occur.

Calcium channels in photosynthetic eukaryotes: implications for evolution of calcium‐based signalling

Much of our current knowledge on the mechanisms by which Ca(2+) signals are generated in photosynthetic eukaryotes comes from studies of a relatively small number of model species, particularly green plants and algae, revealing some common features and notable differences between 'plant' and 'animal' systems. Physiological studies from a broad range of algal cell types have revealed the occurrence of animal-like signalling properties, including fast action potentials and fast propagating cytosolic Ca(2+) waves. Genomic studies are beginning to reveal the widespread occurrence of conserved channel types likely to be involved in Ca(2+) signalling. However, certain widespread 'ancient' channel types appear to have been lost by certain groups, such as the embryophytes. More recent channel gene loss is also evident from comparisons of more closely related algal species. The underlying processes that have given rise to the current distributions of Ca(2+) channel types include widespread retention of ancient Ca(2+) channel genes, horizontal gene transfer (including symbiotic gene transfer and acquisition of bacterial genes), gene loss and gene expansion within taxa. The assessment of the roles of Ca(2+) channel genes in diverse physiological, developmental and life history processes represents a major challenge for future studies.

Identification of Functionally Segregated Sarcoplasmic Reticulum Calcium Stores in Pulmonary Arterial Smooth Muscle

In pulmonary arterial smooth muscle, Ca2+ release from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) may induce constriction and dilation in a manner that is not mutually exclusive. We show here that the targeting of different sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA) and RyR subtypes to discrete SR regions explains this paradox. Western blots identified protein bands for SERCA2a and SERCA2b, whereas immunofluorescence labeling of isolated pulmonary arterial smooth muscle cells revealed striking differences in the spatial distribution of SERCA2a and SERCA2b and RyR1, RyR2, and RyR3, respectively. Almost all SERCA2a and RyR3 labeling was restricted to a region within 1.5 μm of the nucleus. In marked contrast, SERCA2b labeling was primarily found within 1.5 μm of the plasma membrane, where labeling for RyR1 was maximal. The majority of labeling for RyR2 lay in between these two regions of the cell. Application of the vasoconstrictor endothelin-1 induced global Ca2+ waves in pulmonary arterial smooth muscle cells, which were markedly attenuated upon depletion of SR Ca2+ stores by preincubation of cells with the SERCA inhibitor thapsigargin but remained unaffected after preincubation of cells with a second SERCA antagonist, cyclopiazonic acid. We conclude that functionally segregated SR Ca2+ stores exist within pulmonary arterial smooth muscle cells. One sits proximal to the plasma membrane, receives Ca2+ via SERCA2b, and likely releases Ca2+ via RyR1 to mediate vasodilation. The other is located centrally, receives Ca2+ via SERCA2a, and likely releases Ca2+ via RyR3 and RyR2 to initiate vasoconstriction.

Spontaneous Ca waves in ventricular myocytes from failing hearts depend on Ca2+-calmodulin-dependent protein kinase II

Increased cardiac ryanodine receptor (RyR)-dependent diastolic SR Ca leak is present in heart failure and in conditions when adrenergic tone is high. Increasing Ca leak from the SR could result in spontaneous Ca wave (SCaW) formation. SCaWs activate the inward Na/Ca exchanger (NCX) current causing a delayed afterdepolarization (DAD), potentially leading to arrhythmia. Here we examine SCaWs in ventricular myocytes isolated from failing and healthy rabbit hearts. Myocytes from healthy hearts did not exhibit SCaWs under baseline conditions versus 43% of those exposed to isoproterenol (ISO). This ISO-induced increase in activity was reversed by inhibition of Ca-calmodulin-dependent protein kinase II (CaMKII) by KN93. Inhibition of cAMP-dependent protein kinase (PKA) by H89 had no observed effect. Of myocytes treated with forskolin 50% showed SCaW activity, attributable to a large increase in SR Ca load ([Ca]SRT) versus control. At similar [Ca]SRT (121μM) myocytes treated with ISO plus KN93 had significantly fewer SCaWs versus those treated with ISO or ISO plus H89 (0.2±0.28 vs. 1.1±0.28 and 1.29±0.39 SCaWs cell−1, respectively). In myocytes isolated from failing hearts ISO induced an increase in the percentage of cells generating SCaWs vs. baseline (74% vs. 11%) with no increase in [Ca]SRT. Inhibiting CaMKII reversed this effect (14%). At similar [Ca]SRT (71µM) myocytes treated with ISO or ISO plus H89 had significantly more SCaWs per cell vs. untreated (2.5±0.5; 1.6±0.7 vs. 0.36±0.3, respectively). Treatment with ISO plus KN93 completely abolished this effect. The evidence suggests the ISO-dependent increase in SCaW activity in both healthy and failing myocytes is CaMKII-dependent, implicating CaMKII in arrhythmogenesis.

Generation and propagation of gastric slow waves

1. Mechanisms underlying the generation and propagation of gastrointestinal slow wave depolarizations have long been controversial. The present review aims to collate present knowledge on this subject with specific reference to slow waves in gastric smooth muscle. 2. At present, there is strong agreement that interstitial cells of Cajal (ICC) are the pacemaker cells that generate slow waves. What has been less clear is the relative role of primary types of ICC, including the network in the myenteric plexus (ICC-MY) and the intramuscular network (ICC-IM). It is concluded that both ICC-MY and ICC-IM are likely to serve a major role in slow wave generation and propagation. 3. There has been long-standing controversy as to how slow waves 'propagate' circumferentially and down the gastrointestinal tract. Two mechanisms have been proposed, one being action potential (AP)-like conduction and the other phase wave-based 'propagation' resulting from an interaction of coupled oscillators. Studies made on single bundle gastric strips indicate that both mechanisms apply with relative dominance depending on conditions; the phase wave mechanism is dominant under circumstances of rhythmically generating slow waves and the AP-like propagation is dominant when the system is perturbed. 4. The phase wave mechanism (termed Ca(2+) phase wave) uses cyclical Ca(2+) release as the oscillator, with coupling between oscillators mediated by several factors, including: (i) store-induced depolarization; (ii) resultant electrical current flow/depolarization through the pacemaker cell network; and (iii) depolarization-induced increase in excitability of downstream Ca(2+) stores. An analogy is provided by pendulums in an array coupled together by a network of springs. These, when randomly activated, entrain to swing at the same frequency but with a relative delay along the row giving the impression of a propagating wave. 5. The AP-like mechanism (termed voltage-accelerated Ca(2+) wave) propagates sequentially like a conducting AP. However, it is different in that it depends on regenerative store Ca(2+) release and resultant depolarization rather than regenerative activation of voltage-dependent channels in the cell membrane. 6. The applicability of these mechanisms to describing propagation in large intact gastrointestinal tissues, where voltage-dependent Ca(2+) entry is also likely to be functional, is discussed.

Intercellular calcium signaling between astrocytes and oligodendrocytes via gap junctions in culture

To understand further how oligodendrocytes regulate brain function, the mechanism of communication between oligodendrocytes and other cell types needs to be explored. An important mode of communication between various cell types in the nervous system involves gap junctions. Astroglial cells are extensively connected through gap junctions forming the glial syncytium. Although the presence of gap junctions between oligodendrocytes and astrocytes have been well documented, evidence for gap junction-mediated calcium transfer between these two glial populations is still missing. To measure functional coupling between astrocytes and oligodendrocytes and to test whether this coupling is mediated by gap junctions we used laser photostimulation and monitored Ca2+ propagation in cultures from transgenic animals in which oligodendrocytes express enhanced green fluorescent protein (eGFP). We show that waves of Ca2+ spread from astrocytes to oligodendrocytes and that these waves are blocked by the broad-spectrum gap junction blocker carbenoxolone, but not the neuron-specific gap junction blocker quinine. We also show that the spread of Ca2+ waves between astrocytes and oligodendrocytes is bi-directional. Thus, increase of Ca2+ concentration in astrocytes triggered by surrounding neuronal activity may feed back onto different neuronal populations through oligodendrocytes.

The Connexin43 C-Terminal Region Mediates Neuroprotection During Stroke

Connexin43 plays an important role in neuroprotection in experimental stroke models; reducing the expression of this gap junction protein in astrocytes enhances injury upon middle cerebral artery occlusion (MCAO). Because the C-terminal region of connexin43 isimportant for channel activity, we carried out MCAO stroke experiments in mice expressing a truncated form of connexin43 (Cx43DeltaCT mice). Brain sections were analyzed for infarct volume, astrogliosis, and inflammatory cell invasion 4 days after MCAO. Adult cortices and astrocyte cultures were examined for connexin43 (Cx43) expression by immunohistochemistry and Western blot. Cultured astrocytes were also examined for dye coupling, channel conductance, hemichannel activity, and Ca wave propagation. The Cx43DeltaCT mice exhibit enhanced cerebral injury after stroke. Astrogliosis was reduced and inflammatory cell invasion was increased inthe peri-infarct region in these mice compared with controls; Cx43 expression was also altered. Lastly, cultured astrocytes from Cx43DeltaCT mice were less coupled and displayed alterations in channel gating, hemichannel activity, and Ca wave properties. These results suggest that astrocytic Cx43 contributed to the regulation of cell death after stroke and support the view that the Cx43 C-terminal region is important in protection in cerebral ischemia.

Na+-dependent SR Ca2+ overload induces arrhythmogenic events in mouse cardiomyocytes with a human CPVT mutation

Mutations in the cardiac ryanodine receptor Ca(2+) release channel, RyR2, underlie catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited life-threatening arrhythmia. CPVT is triggered by spontaneous RyR2-mediated sarcoplasmic reticulum (SR) Ca(2+) release in response to SR Ca(2+) overload during beta-adrenergic stimulation. However, whether elevated SR Ca(2+) content--in the absence of protein kinase A activation--affects RyR2 function and arrhythmogenesis in CPVT remains elusive. Isolated murine ventricular myocytes harbouring a human RyR2 mutation (RyR2(R4496C+/-)) associated with CPVT were investigated in the absence and presence of 1 micromol/L JTV-519 (RyR2 stabilizer) followed by 100 micromol/L ouabain intervention to increase cytosolic [Na(+)] and SR Ca(2+) load. Changes in membrane potential and intracellular [Ca(2+)] were monitored with whole-cell patch-clamping and confocal Ca(2+) imaging, respectively. At baseline, action potentials (APs), Ca(2+) transients, fractional SR Ca(2+) release, and SR Ca(2+) load were comparable in wild-type (WT) and RyR2(R4496C+/-) myocytes. Ouabain evoked significant increases in diastolic [Ca(2+)], peak systolic [Ca(2+)], fractional SR Ca(2+) release, and SR Ca(2+) content that were quantitatively similar in WT and RyR2(R4496C+/-) myocytes. Ouabain also induced arrhythmogenic events, i.e. spontaneous Ca(2+) waves, delayed afterdepolarizations and spontaneous APs, in both groups. However, the ouabain-induced increase in the frequency of arrhythmogenic events was dramatically larger in RyR2(R4496C+/-) when compared with WT myocytes. JTV-519 greatly reduced the frequency of ouabain-induced arrhythmogenic events. The elevation of SR Ca(2+) load--in the absence of beta-adrenergic stimulation--is sufficient to increase the propensity for triggered arrhythmias in RyR2(R4496C+/-) cardiomyocytes. Stabilization of RyR2 by JTV-519 effectively reduces these triggered arrhythmias.

Abnormal Intra-Store Calcium Handling and Arrhythmogenesis in Heart Failure

Heart failure (HF) patients are known to have increased susceptibility to ventricular arrhythmias. Although abnormal intracellular calcium (Ca) cycling is recognized as an important contributor to the pathogenesis of ventricular arrhythmias, the specific cellular and molecular mechanisms of these arrhythmias remain to be defined. The objective of present study was to investigate the sub-cellular mechanisms of Ca-dependent arrhythmia using time-resolved Ca imaging in the cytosolic and sarcoplasmic reticulum (SR) luminal compartments and the patch-clamp technique in a canine model of tachypacing-induced HF. When rhythmically paced in the presence of the β-adrenergic agonist, isoproterenol, HF myocytes displayed a higher frequency of diastolic Ca waves than control myocytes. In both HF and control myocytes, diastolic Ca waves occurred when [Ca]SR rose above a certain threshold level, which was significantly lower in HF than in control myocytes. Ca signaling refractoriness determined as the time delay between systolic SR Ca depletion and Ca wave initiation was significantly reduced in HF myocytes. Electrical and Ca signaling activities exhibited several distinct potentially arrhythmogenic patterns, including: 1) delayed afterdepolarizations and extrasystolic action potentials (APs) linked to diastolic spontaneous Ca waves; 2) intermittent prolongations of AP duration associated with pre-systolic spontaneous Ca waves and post-systolic triggered Ca waves; and 3) disorganized release uncoupled from myocyte electrical activity. The level of [Ca]SR threshold for spontaneous Ca waves and the time to attain the threshold during the pacing cycle were critical in determining the type of arrhythmogenic abnormality. These experiments suggest a common mechanistic framework for apparently different arrhythmic phenotypes and provide new insights into the relationship between abnormal Ca release and arrhythmogenesis in HF.

Synchronization of Spontaneous Calcium Release Waves Among Myocytes in Intact Heart Determines the Magnitude of Delayed Afterdepolarizations and Triggered Activity

Intracellular Ca waves occur as the result of spontaneous Ca release (SCR) during Ca overload. Ca waves activate Na-Ca exchange, causing delayed afterdepolarizations (DAD) which can achieve threshold and produce triggered extrasystoles. It is not known how these single cell events depolarize enough myocytes in intact heart to produce a triggered beat. We combined experimental observations with computer simulations to explain how SCR synchronization among myocytes brings a critical tissue mass to threshold. Confocal microscopy was used to measure SCR waves in groups of myocytes in the LV epicardium of rat hearts loaded with fluo-4AM. Contraction was abolished with cytochalasin-D and blebbistatin. Raising extracellular [Ca] and rapid pacing protocols were used to increase sarcoplasmic reticulum (SR) Ca load and induce Ca waves. As Ca load increased, the number of myocytes giving waves increased. Both the wave latency and the variability (SD) of wave latency decreased with increasing Ca load. Similar results were obtained in isolated rat ventricular myocytes, indicating that the reduction in latency interval and variability represent intrinsic properties of SR release in Ca overload. Computer simulations demonstrate that decreasing wave latency and variability determine the rate and magnitude of increased cytoplasmic [Ca] and therefore determine the timing and magnitude of the DAD. The synchrony of SCR waves among myocytes therefore determines the likelihood of achieving threshold and producing a triggered beat. These results demonstrate that intrinsic properties of SR Ca release are responsible for Ca wave synchronization during Ca overload, causing DADs to reach threshold and produce triggered arrhythmias.

Mechanisms of Spontaneous Calcium Wave Generation During Beta-Adrenergic Stimulation in Rabbit Ventricular Myocytes

The beta-adrenergic signaling pathway represents the principal positive inotropic mechanism of the heart. While the effects of beta-adrenergic stimulation on L-type Ca channel Ca influx and SERCA-mediated sarcoplasmic reticulum (SR) Ca uptake are well established, the effects on SR Ca release through ryanodine receptor (RyR) release clusters remains highly controversial. Here, we examine SR Ca release in rabbit ventricular myocytes in the form of spontaneous Ca waves during beta-adrenergic stimulation with isoproterenol under controlled cytosolic and SR [Ca]. Cytosolic Ca was monitored using high-affinity Ca indicators indo-1 or rhod-2, while SR Ca was measured directly using the low-affinity Ca indicator fluo-5N or indirectly using the amplitude of the cytosolic Ca transient in response to 10 mM caffeine. Under control conditions, Ca waves were not observed following rest from 0.75 Hz pacing. In the presence of isoproterenol (500 nM), SR Ca content increased by 34% and spontaneous Ca waves were observed in 67% of cells during rest after pacing. However, when post-rest cytosolic Ca and SR Ca content were experimentally matched to control conditions using low extracellular Ca (100 uM versus 2 mM) and SERCA inhibition (7.5 uM cyclopiazonic acid), spontaneous Ca waves were never observed in the presence of isoproterenol. In contrast, pharmacological sensitization of the RyR with 250 uM caffeine induced Ca waves under control conditions (8/12 cells) and in the presence of isoproterenol at matched cytosolic Ca and SR Ca content (7/12). Together, these data suggest that spontaneous Ca release during beta-adrenergic stimulation is a result of increased RyR sensitivity in response to increased SR Ca content, and is not due to direct alterations in RyR function by the beta-adrenergic signaling cascade.

Refractoriness of Ryanodine Receptors During Calcium Alternans in Rabbit Atrial Myocytes

Electro-mechanical and Ca alternans is a known pro-arrhythmic factor. At the cellular level Ca alternans appears as cytosolic Ca transients of alternating amplitude at regular beating frequency. Direct intra-sarcoplasmic reticulum (SR) [Ca] measurements with the low affinity Ca indicator fluo-5N entrapped in the SR revealed that alternans in diastolic SR content are not a prerequisite for cytosolic Ca alternans, and thus SR Ca content is not the sole determinant of alternans. The goal of this study was to determine whether alternans of the kinetics of recovery from inactivation of ryanodine receptors and refractoriness of release represent a key factor underlying cytosolic Ca alternans. Alternans was induced by electrical pacing (1.6 to 2.5 Hz). After Ca alternans was established, pacing was stopped and the occurrence of spontaneous Ca waves and Ca sparks was quantified. The time interval from cessation of stimulation to the appearance of the first Ca waves was significantly shorter and the frequency of Ca sparks was higher after the small Ca transient compared to the large transient. Application of 0.1 mM caffeine or 10 μM isoproterenol rescued Ca alternans and shortened the rest interval until appearance of Ca waves. Photolysis of caged Ca (DM-nitrophen) to produce photolytically triggered Ca release (PTCR) from the SR was used to probe the refractoriness of SR Ca release during alternans. During the decay phase of the Ca transient PTCR was significantly less during the large Ca transient. During the rising phase of the Ca transient PTCR was greater during the large Ca transient, and was capable of inducing a phase reversal of Ca alternans. We conclude that alternating ryanodine receptor inactivation recovery intervals, together with alternations in SR Ca load, represent key determinants of Ca alternans. (VMS and CL contributed equally).

Increased Ca Sparks and Waves Frequency and Unchanged [Na]i in Hearts from Ankyrin-B Heterozygous Mice

The interplay between Na/Ca exchanger (NCX) and Na/K-ATPase (NKA) is essential in regulating cardiac [Na]i, [Ca]i and contractility. The membrane targeting and stability of both NCX and NKA require direct interaction with cytoskeletal protein ankyrin B (AnkB). Humans with AnkB loss-of-function mutations and AnkB heterozygous mice (AnkB+/-) display a complex cardiac phenotype that includes ventricular arrhythmias and sudden death. Cardiac myocytes from AnkB+/- mice show reduced NCX and NKA expression at the T-tubules, larger cellular and SR Ca load and increased frequency of delayed afterdepolarizations (DADs). How the coordinated loss of NCX and NKA affects SR Ca release and [Na]i is poorly understood. We measured Ca sparks and waves in myocytes from AnkB+/- and wild-type (WT) mice with and without 1 μM isoproterenol. Under control conditions, Ca transients, SR Ca load and Ca sparks frequency (CaSpF), even normalized to SR Ca content, were higher in AnkB+/- mice at all frequencies. With isoproterenol, Ca transient amplitude and SR Ca content were similar in myocytes from WT and AnkB+/- mice, but CaSpF was still elevated in AnkB+/- mice. Ca waves occurred preponderantly in AnkB+/- mice. In some cases, Ca waves and sparks evolved into synchronous after-Ca transients that are indicative of DADs. This data suggest that SR Ca release is enhanced in AnkB+/- mice. We measured [Na]i to determine whether the increased CaSpF in AnkB+/- mice is due to higher [Na]i, which may elevate diastolic Ca. [Na]i was slightly, but not significantly, lower in AnkB+/- vs. WT mice (11.4±1.1 vs. 13.2±1.7 mM in WT). Isoproterenol lowered [Na]i by a comparable amount in both AnkB+/- and WT mice (to 8.1±0.9 mM and 11.1±1.5 mM). Thus, altered [Na]i does not explain the increased Ca sparks frequency in AnkB+/- mice.

Sarcoplasmic Reticulum Function in Smooth Muscle

The sarcoplasmic reticulum (SR) of smooth muscles presents many intriguing facets and questions concerning its roles, especially as these change with development, disease, and modulation of physiological activity. The SR's function was originally perceived to be synthetic and then that of a Ca store for the contractile proteins, acting as a Ca amplification mechanism as it does in striated muscles. Gradually, as investigators have struggled to find a convincing role for Ca-induced Ca release in many smooth muscles, a role in controlling excitability has emerged. This is the Ca spark/spontaneous transient outward current coupling mechanism which reduces excitability and limits contraction. Release of SR Ca occurs in response to inositol 1,4,5-trisphosphate, Ca, and nicotinic acid adenine dinucleotide phosphate, and depletion of SR Ca can initiate Ca entry, the mechanism of which is being investigated but seems to involve Stim and Orai as found in nonexcitable cells. The contribution of the elemental Ca signals from the SR, sparks and puffs, to global Ca signals, i.e., Ca waves and oscillations, is becoming clearer but is far from established. The dynamics of SR Ca release and uptake mechanisms are reviewed along with the control of luminal Ca. We review the growing list of the SR's functions that still includes Ca storage, contraction, and relaxation but has been expanded to encompass Ca homeostasis, generating local and global Ca signals, and contributing to cellular microdomains and signaling in other organelles, including mitochondria, lysosomes, and the nucleus. For an integrated approach, a review of aspects of the SR in health and disease and during development and aging are also included. While the sheer versatility of smooth muscle makes it foolish to have a "one model fits all" approach to this subject, we have tried to synthesize conclusions wherever possible.

Synchronization of Ca2+ oscillations: involvement of ATP release in astrocytes

Glial cells, especially astrocytes, are not merely supportive cells, but are important partners to neighboring cells, including neurons, vascular cells, and other glial cells. Although glial cells are not excitable in terms of electrophysiology, they have been shown to generate synchronized Ca(2+) transients (Ca(2+) oscillations) through mechanisms of chemical coupling. Until recently, Ca(2+) transients in astrocytes were thought to be totally dependent on neuronal activities, because astrocytes express a large variety of receptors for neurotransmitters and surround almost all synapses at which neurotransmitters are spilled over to stimulate astrocytes. In addition, however, astrocytes have been shown to release diffusible substances, so-called 'gliotransmitters', and Ca(2+) transients in astrocytes are therefore also triggered by astrocytic activities, leading to propagation of Ca(2+) transients or Ca(2+) waves. In these processes, the gliotransmitter ATP and activation of P2Y receptors play central roles. Interestingly, astrocytes evoke Ca(2+) transients when neurons are not present, suggesting that astrocytes themselves can initiate and control Ca(2+) transients. Astrocytic Ca(2+) transients are observed even in vivo, through mechanisms of chemical coupling by gliotransmitters, but they are less frequent and synchronous than those in vitro. Although we have not yet clarified their significance in the central nervous system, astrocytic Ca(2+) transients are dramatically affected by pathological conditions, suggesting that, in addition to physiological events, they might be closely involved in disorders in the central nervous system.

Spatiotemporal dynamics of Ca2+ signaling and its physiological roles

Changes in the intracellular Ca2+ concentration regulate numerous cell functions and display diverse spatiotemporal dynamics, which underlie the versatility of Ca2+ in cell signaling. In many cell types, an increase in the intracellular Ca2+ concentration starts locally, propagates within the cell (Ca2+ wave) and makes oscillatory changes (Ca2+ oscillation). Studies of the intracellular Ca2+ release mechanism from the endoplasmic reticulum (ER) showed that the Ca2+ release mechanism has inherent regenerative properties, which is essential for the generation of Ca2+ waves and oscillations. Ca2+ may shuttle between the ER and mitochondria, and this appears to be important for pacemaking of Ca2+ oscillations. Importantly, Ca2+ oscillations are an efficient mechanism in regulating cell functions, having effects supra-proportional to the sum of duration of Ca2+ increase. Furthermore, Ca2+ signaling mechanism studies have led to the development of a method for specific inhibition of Ca2+ signaling, which has been used to identify hitherto unrecognized functions of Ca2+ signals.

Cardiac Action Potential Model with Spatial Subcellular Calcium Cycling and Physiological Transmembrane Currents

Calcium (Ca) is a key player in excitation-contraction (EC) coupling in the cardiac myocyte. When Ca enters through L-type Ca channels, it opens the ryanodine receptor (RyR) channels on the sarcoplasmic reticulum (SR) which stores a large amount of Ca (Ca induced Ca release). There are about 20,000 SR units and each RyR can sense only the local Ca concentration. Most computer cardiac action potential models use a single Ca concentration, which limits accurate modeling of intracellular Ca cycling. This is because the RyRs do not sense the average Ca concentration of the whole cell. Different aspects limit the utility of various models for investigation of intracellular Ca cycling related phenomena such as delayed afterdepolarizations (DADs) and catecholaminergic polymorphic ventricular tachycardia (CPVT) (since Ca concentrations in these phenomena are strongly heterogeneous within the cell). In order to investigate these phenomena, we developed a mathematical action potential model based on the spatial Ca model by Restrepo et al and physiological transmembrane currents by Shannon et al and Mahajan et al. In order to have more natural sparks, we increased the number of grid points for Ca diffusions from the model by Restrepo et al. Then we tuned the model to exhibit physiological action potential duration (APD) and Ca transients for long and short pacing cycle lengths (PCL). At short PCL, this hybrid model shows both voltage driven alternans due to steep APD restitution and Ca driven alternans due to luminal Ca regulation-mediated mechanism. In addition to alternans, this model exhibits Ca waves and DADs. This mathematical model of the cardiac myocyte provides a sort of minimal model to explore the spatio-temporal aspects of wave initiation, propagation and DAD induction related to normal and pathophysiological conditions.