Ca Waves Research Articles

Regulation of Sarcoplasmic Reticulum Calcium Leak by Cytosolic Calcium in Rabbit Ventricular Myocytes

Diastolic sarcoplasmic reticulum (SR) Ca leak determines Ca content and, therefore, the amplitude of action potential-induced global Ca transients in ventricular myocytes. However, the pathways and properties of SR Ca leak have been poorly described. Here, we studied the effects of cytosolic [Ca] ([Ca]i) on SR Ca leak in permeabilized rabbit ventricular myocytes. Using confocal microscopy we simultaneously measured intra-SR free Ca ([Ca]SR) with fluo-5N and cytosolic Ca sparks with rhod-2, and monitored SR Ca leak as the change in [Ca]SR over time after complete SERCA inhibition with thapsigargin (10 μM). Increasing [Ca]i from 150 to 250 nM significantly increased SR Ca leak (by ∼30%) over the entire range of [Ca]SR. This increase in SR Ca leak associated with an increase in Ca spark frequency. Further increasing [Ca]i to 350 nM led to rapid [Ca]SR depletion due to the occurrence of spontaneous Ca waves. In contrast, lowering [Ca]i to 50 nM markedly decreased SR Ca leak rate (by ∼60%) and nearly abolished Ca spark activity. When the ryanodine receptor (RyR) was completely inhibited with ruthenium red (50 μM), changes in [Ca]i between 50 and 350 nM did not produce any significant effect on SR Ca leak, showing that changes of [Ca]i over a physiological range alter SR Ca leak solely by regulating RyR activity. However, decreasing [Ca]i to a lower, non-physiologically level (5 nM) activated additional SR Ca leak pathway(s) that were insensitive to RyR or SR Ca ATPase inhibition. In summary, [Ca]i plays an important role in regulating SR Ca leak by activating RyR and preventing Ca leak through unspecified pathways.

Nitric Oxide Can Mediate Beta-Adrenergic- and CaMKII-Dependent Spontaneous Ca2+ Waves in Cardiac Myocytes, Independent of PKA Activation

Increased diastolic SR Ca leak can initiate spontaneous Ca waves (SCaWs). SCaWs activate inward Na/Ca exchanger current causing an arrhythmogenic delayed afterdepolarization. Here we examine SCaWs in ventricular myocytes isolated from rabbit hearts. Myocytes did not exhibit SCaWs at baseline conditions, but 43% did when exposed to isoproterenol (ISO). This ISO-induced increase in activity was reversed by inhibition of CaMKII by KN93, but not with PKA inhibition by H89. At similar [Ca]SRT (121 μM) 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 & 1.29±0.39 SCaWs cell−1, respectively). We attribute this increase in activity to the previously characterized CaMKII-dependent increase in RyR-dependent leak. We also find that SR Ca leak is increased by the nitric oxide (NO) donor, SNAP; and this NO-dependent effect is also completely reversed by KN-93. We also show the increase in leak to be dependent on nitric oxide synthase 1 (NOS1) activity. At comparable SR Ca load (132 μM) ISO treated myocytes have significantly higher leak vs. control (8.4 vs. 3.8 μM). The ISO-induced leak (at constant SR Ca load) was attenuated by the NOS1 inhibitor, SMLT, but not the NOS3 inhibitor, L-NIO (3.5 vs. 6.8 μM). Moreover, ISO causes an upward trend in myocyte [NO] (sensed by the NO-dependent dye, DAF-2 A), and the NOS inhibitor, L-NAME significantly attenuated the development of SCaWs. Together this data suggests a novel pathway in which β1-adrenergic receptor activation stimulates NO production via NOS1, which in turn activates CaMKII to increase RyR gating, SR Ca leak, SCaWs and delayed afterdepolarizations.

Simultaneous Phosphorylation of RyR2 by PKA and Camkii is Required for Induction of CA-Dependent Arrhythmia Caused by MIR-1 Overexpression

We recently showed that disruption of localization of PP2A phosphatase activity to the ryanodine receptor (RyR2) complex by overexpression of the muscle-specific microRNA, miR-1, stimulates excitation-contraction coupling and results in increased arrhythmogenic potential in cardiac myocytes. In the present study, we examined the role of PKA and CAMKII as mediators of the effects miR-1 on Ca signaling and arrhythmogenesis using cellular electrophysiology and Ca imaging complemented with quantitative measurements of RyR2 phosphorylation at PKA site S-2808, and at CAMKII site S-2814. Adenovirally-mediated 2-fold overexpression of miR-1 resulted in 10-fold increase in arrhythmogenic potential measured as a frequency of spontaneous Ca waves and DADs in myocytes exposed to 100 nM isoproterenol (ISO). Quantitative imminoblotting using site-directed phosphospecific-antibodies showed that RyR2 phosphorylation in miR-1 overexpressing cells was low at S-2808 under basal conditions. Exposure of myocytes to ISO maximized phosphorylation at S-2808. Phosphorylation at S-2814 under basal conditions was maximal and did not further increase in the presence of ISO. Additionally, ISO increased SERCa-mediated SR Ca uptake and SR Ca load through phosphorylation of phospholamban (PLB). To define which of these factors (increased CaSR content or increased RyR2 PKA phosphorylation) mediated the increased arrhythmogenic potential we infected myocytes with viral constructs of a dominant-negative PLB mutant that accelerates SERCa-mediated SR Ca uptake by displacing endogenous PLB from SERCa. Myocytes coexpressing miR-1 and dnPLB did not exhibit enhanced predisposition to Ca-dependent arrhythmia in the presence of ISO despite maximal SR Ca load. Importantly treatment of cells with either PKA or CAMK inhibitors completely abolished increased arrhythmogenic activity. We conclude that neither CAMKII nor PKA phosphorylation alone is sufficient to produce the changes in RyR2 activity that underlies the arrhythmogenic disturbances caused by miR-1 overexpression.

Reduced SERCA2 abundance decreases the propensity for Ca2+ wave development in ventricular myocytes

To describe the overall role of reduced sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) for Ca(2+) wave development. SERCA2 knockout [Serca2(flox/flox) Tg(alphaMHC-MerCreMer); KO] mice allowing inducible cardiomyocyte-specific disruption of the Serca2 gene in adult mice were compared with Serca(flox/flox) (FF) control mice. Six days after Serca2 gene disruption, SERCA2 protein abundance was reduced by 53% in KO compared with FF, whereas SERCA2 activity in field-stimulated, Fluo-5F AM-loaded cells was reduced by 42%. Baseline Ca(2+) content of the sarcoplasmic reticulum (SR) and Ca(2+) transient amplitude and rate constant of decay measured in whole-cell voltage-clamped cells were decreased in KO to 75, 81, and 69% of FF values. Ca(2+) waves developed in only 31% of KO cardiomyocytes compared with 57% of FF when external Ca(2+) was raised (10 mM), although SR Ca(2+) content needed for waves to develop was 79% of FF values. In addition, waves propagated at a 15% lower velocity in KO cells. Ventricular extrasystoles (VES) occurred with lower frequency in SERCA2 KO mice (KO: 3 +/- 1 VES/h vs. FF: 8 +/- 1 VES/h) (P < 0.05 for all results). Reduced SERCA2 abundance resulted in decreased amplitude and decay rate of Ca(2+) transients, reduced SR Ca(2+) content, and decreased propensity for Ca(2+) wave development.

Novel Excitatory Effects of Adenosine Triphosphate on Contractile and Pacemaker Activity in Rabbit Urethral Smooth Muscle

Adenosine triphosphate is thought to be an important neurotransmitter in urethral smooth muscle but its physiological role is still unclear. We characterized the effects of adenosine triphosphate on contractile and pacemaker activity in rabbit urethral smooth muscle. Tension recordings were made from strips of rabbit proximal urethral smooth muscle. Membrane currents from freshly isolated smooth muscle cells and interstitial cells of Cajal were recorded using the patch clamp technique. Intracellular Ca(2+) was measured using confocal microscopy. Exogenous application of adenosine triphosphate (10 microM) evoked robust contractions that were inhibited by the type 2 purinergic receptor blocker suramin (100 microM) and the selective type 2 purinergic Y1 receptor antagonist MRS2500 (Tocris Bioscience, Ellisville, Missouri) (100 nM). Application of the type 2 purinergic Y receptor agonist 2-MeSADP (1 microM) mimicked the effects of adenosine triphosphate. When smooth muscle cells were studied under voltage clamp at -60 mV, adenosine triphosphate evoked a large single inward current (greater than 1.2 nA) but 2-MeSADP produced a small current (about 16 pA). In contrast, when interstitial cells of Cajal were held at -60 mV, they showed spontaneous transient inward currents that were increased in frequency by adenosine triphosphate and 2-MeSADP. These excitatory effects were inhibited by suramin and MRS2500. Interstitial cells of Cajal showed spontaneous Ca(2+) waves that were increased in frequency by adenosine triphosphate and 2-MeSADP. These effects were also inhibited by suramin and MRS2500. Contractile effects of adenosine triphosphate in urethral smooth muscle are mediated by the activation of type 2 purinergic Y receptors on interstitial cells of Cajal.

GABAAand GABABreceptors of distinct properties affect oppositely the proliferation of mouse embryonic stem cells through synergistic elevation of intracellular Ca2+

Gamma-amminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system of vertebrates, serves as an autocrine/paracrine signaling molecule during development, modulating a number of calcium (Ca(2+))-dependent processes, including proliferation, migration, and differentiation, acting via 2 types of GABA receptors (GABARs): ionotropic GABA(A)Rs and metabotropic GABA(B)Rs. Here, we demonstrate that mouse embryonic stem cells (mESCs), which possess the capacity for virtually unlimited self-renewal and pluripotency, synthesize GABA and express functional GABA(A)Rs and GABA(B)Rs, as well as voltage-gated calcium channels (VGCCs), ryanodine receptors (RyRs), and inwardly rectifying potassium (GIRK) channels. On activation, both GABAR types triggered synergistically intracellular calcium rise. Muscimol (a GABA(A)R agonist) induced single Ca(2+) transients involving both VGCC-mediated Ca(2+) influx and intracellular stores, while baclofen (a GABA(B)R agonist) evoked Ca(2+) transients followed by intercellular Ca(2+) waves and oscillations that were resistant to antagonists and entirely dependent on Ca(2+) release from intracellular stores. Prolonged treatment with muscimol slightly inhibited, while baclofen or SR95531 (a GABA(A)R antagonist) significantly facilitated, mESC proliferation. GABA(A)R-specific ligands also induced morphological and gene expression changes indicating a differentiation shift. Our data suggest that the interplay between GABARs and downstream (coupled) effectors differentially modulates mESC proliferation/differentiation through selective activation of second messenger signaling cascades.-Schwirtlich, M., Emri, Z., Antal, K., Máté, Z., Katarova, Z., Szabó, G. GABA(A) and GABA(B) receptors of distinct properties affect oppositely the proliferation of mouse embryonic stem cells through synergistic elevation of intracellular Ca(2+).

Human Airway Contraction and Formoterol-Induced Relaxation Is Determined by Ca2+ Oscillations and Ca2+ Sensitivity

The etiology of airway hyperresponsiveness associated with asthma requires an understanding of the regulatory mechanisms mediating human airway smooth muscle cell (SMC) contraction. The objective of this study was to determine how human airway SMC contraction (induced by histamine) and relaxation (induced by formoterol) are regulated by Ca(2+) oscillations and Ca(2+) sensitivity. The responses of human small airways and their associated SMCs were studied in human lung slices cut from agarose-inflated lungs. Airway contraction was measured with phase-contrast video microscopy. Ca(2+) signaling and Ca(2+) sensitivity of airway SMCs were measured with two-photon fluorescence microscopy and Ca(2+)-permeabilized lung slices. The agonist histamine induced contraction of human small airways by stimulating both an increase in intracellular Ca(2+) concentration in the SMCs in the form of oscillatory Ca(2+) waves and an increase in Ca(2+) sensitivity. The frequency of the Ca(2+) oscillations increased with histamine concentration, and correlated with increased contraction. Formoterol induced airway relaxation at low concentrations by initially decreasing SMC Ca(2+) sensitivity. At higher concentrations, formoterol additionally slowed or inhibited the Ca(2+) oscillations of the SMCs to relax the airways. The action of formoterol was only slowly reversed. Human lung slices provide a powerful experimental assay for the investigation of small airway physiology and pharmacology. Histamine induces contraction by simultaneously increasing SMC Ca(2+) signaling and Ca(2+) sensitivity. Formoterol induces long-lasting relaxation by initially reducing the Ca(2+) sensitivity and, subsequently, the frequency of the Ca(2+) oscillations of the airway SMCs.

Calmodulin kinase II initiates arrhythmogenicity during metabolic acidification in murine hearts

Aim:The multifunctional signal molecule calmodulin kinase II (CaMKII) has been associated with cardiac arrhythmogenesis under conditions where its activity is chronically elevated. Recent studies report that its activity is also acutely elevated during acidosis. We test a hypothesis implicating CaMKII in the arrhythmogenesis accompanying metabolic acidification.Methods:We obtained monophasic action potential recordings from Langendorff-perfused whole heart preparations and single cell action potentials (AP) using whole-cell patch-clamped ventricular myocytes. Spontaneous sarcoplasmic reticular (SR) Ca2+release events during metabolic acidification were investigated using confocal microscope imaging of Fluo-4-loaded ventricular myocytes.Results:In Langendorff-perfused murine hearts, introduction of lactic acid into the Krebs-Henseleit perfusate resulted in abnormal electrical activity and ventricular tachycardia. The CaMKII inhibitor, KN-93 (2 μm), reversibly suppressed this spontaneous arrhythmogenesis during intrinsic rhythm and regular 8 Hz pacing. However, it failed to suppress arrhythmia evoked by programmed electrical stimulation. These findings paralleled a CaMKII-independent reduction in the transmural repolarization gradients during acidosis, which previously has been associated with the re-entrant substrate under other conditions. Similar acidification produced spontaneous AP firing and membrane potential oscillations in patch-clamped isolated ventricular myocytes when pipette solutions permitted cytosolic Ca2+ to increase following acidification. However, these were abolished by both KN-93 and use of pipette solutions that held cytosolic Ca2+ constant during acidosis. Acidosis also induced spontaneous Ca2+ waves in isolated intact Fluo-4-loaded myocytes studied using confocal microscopy that were abolished by KN-93.Conclusion:These findings together implicate CaMKII-dependent SR Ca2+ waves in spontaneous arrhythmic events during metabolic acidification.

IP3 mobilization and diffusion determine the timing window of Ca2+ release by synaptic stimulation and a spike in rat CA1 pyramidal cells

Synaptically activated calcium release from internal stores in CA1 pyramidal neurons is generated via metabotropic glutamate receptors by mobilizing IP(3). Ca(2+) release spreads as a large amplitude wave in a restricted region of the apical dendrites of these cells. These Ca(2+) waves have been shown to induce certain forms of synaptic potentiation and have been hypothesized to affect other forms of plasticity. Pairing a single backpropagating action potential (bAP) with repetitive synaptic stimulation evokes Ca(2+) release when synaptic stimulation alone is subthreshold for generating release. We examined the timing window for this synergistic effect under conditions favoring Ca(2+) release. The window, measured from the end of the train, lasted 250-500 ms depending on the duration of stimulation tetanus. The window appears to correspond to the time when both IP(3) concentration and [Ca(2+)](i) are elevated at the site of the IP(3) receptor. Detailed analysis of the mechanisms determining the duration of the window, including experiments using different forms of caged IP(3) instead of synaptic stimulation, suggest that the most significant processes are the time for IP(3) to diffuse away from the site of generation and the time course of IP(3) production initiated by activation of mGluRs. IP(3) breakdown, desensitization of the IP(3) receptor, and the kinetics of IP(3) unbinding from the receptor may affect the duration of the window but are less significant. The timing window is short but does not appear to be short enough to suggest that this form of coincidence detection contributes to conventional spike timing-dependent synaptic plasticity in these cells.

Role of coupled gating between cardiac ryanodine receptors in the genesis of triggered arrhythmias

Mutations in the ryanodine receptor (RyR) have been linked to exercise-induced sudden cardiac death. However, the precise sequence of events linking RyR channel mutations to a whole heart arrhythmia is not completely understood. In this paper, we apply a detailed, mathematical model of subcellular calcium (Ca) release, coupled to membrane voltage, to study how defective RyR channels can induce arrhythmogenic-triggered activity. In particular, we show that subcellular Ca activity, such as spontaneous Ca sparks and Ca waves, is highly sensitive to coupled gating between RyR channels in clusters. We show that small changes in coupled gating can induce aberrant Ca release activity, which, under Ca overload conditions, can induce delayed afterdepolarization (DAD). We systematically investigate the properties of subcellular Ca during DAD induction and show that the voltage time course during a DAD is dependent on the timing and number of spontaneous Ca sparks that transition to Ca waves. These results provide a detailed mechanism for the role of coupled gating in the genesis of triggered arrhythmias.

Pharmacological Characteristics and Clinical Applications of K201

K201 is a 1,4-benzothiazepine derivative that is a promising new drug with a strong cardioprotective effect. We initially discovered K201 as an effective suppressant of sudden cardiac cell death due to calcium overload. K201 is a nonspecific blocker of sodium, potassium and calcium channels, and its cardioprotective effect is more marked than those of nicorandil, prazosine, propranolol, verapamil and diltiazem. Recently, K201 has also been shown to have activities indicated for treatment of atrial fibrillation, ventricular fibrillation, heart failure and ischemic heart disease, including action as a multiple-channel blocker, inhibition of diastolic Ca2+ release from the sarcoplasmic reticulum, suppression of spontaneous Ca2+ sparks and Ca2+ waves, blockage of annexin V and provision of myocardial protection, and improvement of norepinephrine-induced diastolic dysfunction. Here, we describe the pharmacological characteristics and clinical applications of K201.

Three‐dimensional high‐resolution imaging of cardiac proteins to construct models of intracellular Ca2+ signalling in rat ventricular myocytes

Quantitative understanding of the Ca(2+) handling in cardiac ventricular myocytes requires accurate knowledge of cardiac ultrastructure and protein distribution. We have therefore developed high-resolution imaging and analysis approaches to measure the three-dimensional distribution of immunolabelled proteins with confocal microscopy. Labelling of single rat cardiac myocytes with an antibody to the Z-line marker alpha-actinin revealed a complex architecture of sarcomere misalignment across single cells. Double immunolabelling was used to relate the Z-line structure to the distribution of ryanodine receptors (RyRs, the intracellular Ca(2+) release channels) and the transverse tubular system. Both RyR and transverse tubular system distributions exhibited frequent dislocations from the simple planar geometry generally assumed in existing mathematical models. To investigate potential effects of these irregularities on Ca(2+) dynamics, we determined the three-dimensional distribution of RyR clusters within an extended section of a single rat ventricular myocyte to construct a model of stochastic Ca(2+) dynamics with a measured Ca(2+) release unit (CRU) distribution. Calculations with this model were compared with a second model in which all CRUs were placed on flat planes. The model with a realistic CRU distribution supported Ca(2+) waves that spread axially along the cell at velocities of approximately 50 mum s(-1). By contrast, in the model with planar CRU distribution the axial wave spread was slowed roughly twofold and wave propagation often nearly faltered. These results demonstrate that spatial features of the CRU distribution on multiple length scales may significantly affect intracellular Ca(2+) dynamics and must be captured in detailed mechanistic models to achieve quantitative as well as qualitative insight.

Spontaneous Ca2+ Waves in Rabbit Corpus Cavernosum: Modulation by Nitric Oxide and cGMP

Detumescent tone and subsequent relaxation by nitric oxide (NO) are essential processes that determine the erectile state of the penis. Despite this, the mechanisms involved are incompletely understood. It is often assumed that the tone is associated with a sustained high cytosolic Ca(2+) level in the corpus cavernosum smooth muscle cells, however, an alternative possibility is that oscillatory Ca(2+) signals regulate tone, and erection occurs as a result of inhibition of Ca(2+) oscillations by NO. The aim of this study is to determine if smooth muscle cells displayed spontaneous Ca(2+) oscillations and, if so, whether these were regulated by NO. Male New Zealand white rabbits were euthanized and smooth muscle cells were isolated by enzymatic dispersal for confocal imaging of intracellular Ca(2+) (using fluo-4AM) and patch clamp recording of spontaneous membrane currents. Thin tissue slices were also loaded with fluo-4AM for live imaging of Ca(2+). Cytosolic Ca(2+) was measured in isolated smooth muscle cells and tissue slices. Results. Isolated rabbit corpus cavernosum smooth muscle cells developed spontaneous Ca(2+) waves that spread at a mean velocity of 65 microm/s. Dual voltage clamp/confocal recordings revealed that each of the Ca(2+) waves was associated with an inward current typical of the Ca(2+)-activated Cl(-) currents developed by these cells. The waves depended on an intact sarcoplasmic reticulum Ca(2+) store, as they were blocked by cyclopiazonic acid (Calbiochem, San Diego, CA, USA) and agents that interfere with ryanodine receptors and IP(3)-mediated Ca(2+) release. The waves were also inhibited by an NO donor (diethylamine NO; Tocris Bioscience, Bristol, Avon, UK), 3-(5-hydroxymethyl-2-furyl)-1-benzyl indazole (YC-1) (Alexis Biochemicals, Bingham, Notts, UK), 8-bromo-cyclic guanosine mono-phosphate (Tocris), and sildenafil (Viagra, Pfizer, Sandwich, Kent, UK). Regular Ca(2+) oscillations were also observed in whole tissue slices where they were clearly seen to precede contraction. This activity was also markedly inhibited by sildenafil, suggesting that it was under NO regulation. These results provide a new basis for understanding detumescent tone in the corpus cavernosum and its inhibition by NO.

The effects of phosphatidylinositol 3,5‐bisphosphate on cardiac muscle

The downstream product of phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P2), inositol 1,4,5‐trisphosphate (IP3), is known to induce calcium (Ca) release from the sarcoplasmic reticulum and elicit alterations in contraction of the heart and arrhythmias. What has not been studied is the effects of other phosphoinositides (PIs) such as phosphatidylinositol 3,5‐bisphosphate (PI(3,5)P2) and phosphatidylinositol 3‐phosphate (PI(3)P) on the heart. A striated muscle specific phosphatase (MIP/MTMR14) dephosphorylates PI(3,5)P2, and is present in skeletal and cardiac muscle. MIP knockout animals display reduced exercise performance, providing a strong rationale that PI(3,5)P2 may be involved in heart function. We tested 0.5 µM PI(3,5)P2, PI(4,5)P2, and PI(3)P on left ventricular muscle strips from adult mice paced at 1 Hz. All PIs tested induced an increase in the magnitude of the twitch‐force of the left ventricle. PI(3,5)P2 was also applied to single isolated cardiac myocytes and induced an increase in intracellular Ca levels and altered spontaneous Ca waves. While the mechanism of these actions remains to be determined, these data suggest that PI(3,5)P2, PI(4,5)P2, and PI(3)P may play a role in altering cardiac function. It will be essential to determine the potential role of this novel signaling mechanism in heart‐related diseases where Ca homeostasis is known to be modified.

Inositol‐1,4,5‐trisphosphate receptor‐mediated Ca2+ waves in pyramidal neuron dendrites propagate through hot spots and cold spots

We studied inositol-1,4,5-trisphosphate (IP(3)) receptor-dependent intracellular Ca(2+) waves in CA1 hippocampal and layer V medial prefrontal cortical pyramidal neurons using whole-cell patch-clamp recordings and Ca(2+) fluorescence imaging. We observed that Ca(2+) waves propagate in a saltatory manner through dendritic regions where increases in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) were large and fast ('hot spots') separated by regions where increases in [Ca(2+)](i) were comparatively small and slow ('cold spots'). We also observed that Ca(2+) waves typically initiate in hot spots and terminate in cold spots, and that most hot spots, but few cold spots, are located at dendritic branch points. Using immunohistochemistry, we found that IP(3) receptors (IP(3)Rs) are distributed in clusters along pyramidal neuron dendrites and that the distribution of inter-cluster distances is nearly identical to the distribution of inter-hot spot distances. These findings support the hypothesis that the dendritic locations of Ca(2+) wave hot spots in general, and branch points in particular, are specially equipped for regenerative IP(3)R-dependent internal Ca(2+) release. Functionally, the observation that IP(3)R-dependent [Ca(2+)](i) rises are greater at branch points raises the possibility that this novel Ca(2+) signal may be important for the regulation of Ca(2+)-dependent processes in these locations. Futhermore, the observation that Ca(2+) waves tend to fail between hot spots raises the possibility that influences on Ca(2+) wave propagation may determine the degree of functional association between distinct Ca(2+)-sensitive dendritic domains.

Role of K+ Channels in Regulating Spontaneous Activity in Detrusor Smooth Muscle In Situ in the Mouse Bladder

We investigated the functional role of K(+) channels for regulating spontaneous activity in mouse bladder detrusor smooth muscle. The effects of different K(+) channels blockers on spontaneous changes in membrane potential and intracellular Ca(2+) dynamics were examined using intracellular recording techniques and Ca(2+) imaging with fluo-4 fluorescence, respectively. Detrusor smooth muscle generated spontaneous action potentials and Ca(2+) transients. Iberiotoxin (0.1 microM), charybdotoxin (0.1 microM) or tetraethylammonium (1 mM) increased the amplitude of action potentials and prolonged their repolarizing phase without inhibiting their after-hyperpolarization. Tetraethylammonium (10 mM) but not stromatoxin (0.1 microM) suppressed after-hyperpolarization and further increased the amplitude and half duration of action potentials. Apamin (0.1 microM) increased the frequency of action potentials but had no effect on their configuration. Spontaneous Ca(2+) transients were generated in individual detrusor smooth muscle cells and occasionally propagated to neighboring cells to form intercellular Ca(2+) waves. Transmural nerve stimulations invariably initiated synchronous Ca(2+) transients within and across muscle bundles. Charybdotoxin (0.1 microM) increased the amplitude of spontaneous Ca(2+) transients, while the subsequent application of tetraethylammonium (10 mM) increased their half duration. In addition, tetraethylammonium increased the synchronicity of Ca(2+) transients in muscle bundles. These results suggest that large and intermediate conductance Ca(2+) activated K(+) channels contribute to action potential repolarization and restrict the excitability of detrusor smooth muscle in the mouse bladder. In addition, the activation of voltage dependent K(+) channels is involved in repolarization and after-hyperpolarization, and it has a fundamental role in stabilizing detrusor smooth muscle excitability.

The effects of membrane potential, SR Ca2+ content and RyR responsiveness on systolic Ca2+ alternans in rat ventricular myocytes

Previous work has shown that small depolarizing pulses produce a beat to beat alternation in the amplitude of the systolic Ca(2+) transient in ventricular myocytes. The aim of the present work was to investigate the role of changes of SR Ca(2+) content and L-type Ca(2+) current in this alternans. As the amplitude of the depolarizing pulse was increased from 10 to 30 mV the magnitude of alternans decreased. Confocal linescan studies showed that this was accompanied by an increase in the number of sites from which Ca(2+) waves propagated. A sudden decrease in the depolarisation amplitude resulted in three classes of behaviour: (1) a gradual decrease in Ca(2+) transient amplitude before alternans developed accompanied by a loss of SR Ca(2+), (2) a gradual increase in Ca(2+) transient amplitude before alternans accompanied by a gain of SR Ca(2+), and (3) immediate development of alternans with no change of SR content. We conclude that alternans develops if the combination of decreased opening of L-type channels and change of SR Ca(2+) content results in spatially fragmented release from the SR as long as there is sufficient Ca(2+) in the SR to sustain wave propagation. Potentiation of the opening of the ryanodine receptor (RyR) by low concentrations of caffeine (100 microm) abolished alternans for a few pulses but the alternans then redeveloped once SR Ca(2+) content fell to the new threshold for wave propagation. Finally we show evidence that inhibiting L-type Ca(2+) current with 200 mum Cd(2+) produces alternans by means of a similar fragmentation of the Ca(2+) release profile and propagation of mini-waves of Ca(2+) release.

Heterogeneities in ICC Ca2+ Activity Within Canine Large Intestine

In human and canine colon, both slow (slow waves, 2-8/min) and fast (myenteric potential oscillations [MPOs]; 16-20/min) electrical rhythms in the smooth muscle originate at the submucosal and myenteric borders, respectively. We used Ca(2+) imaging to investigate whether interstitial cells of Cajal (ICCs) at these borders generated distinct rhythms. Segments of canine colon were pinned with submucosal or myenteric surface uppermost or cut in cross section. Tissues were loaded with a Ca(2+) indicator (fluo-4), and activity was monitored at 36.5 +/- 0.5 degrees C using an electron multiplying charge coupled device (EMCCD). Rhythmic, biphasic Ca(2+) transients (5-8/min), similar in waveform to electrical slow waves, propagated without decrement as a wave front (2-5 mm/s) through the ICC-SM network lying along the submucosal surface of the circular muscle (CM). In contrast, rhythmic intracellular Ca(2+) waves (approximately 16/min) and spontaneous reductions in Ca(2+) were observed in ICCs at the myenteric border (ICC-MY). Normally, intracellular Ca(2+) waves were unsynchronized between adjacent ICC-MY, although excitatory nerve activity synchronized activity. In addition, spontaneous reductions in Ca(2+) were observed that inhibited Ca(2+) waves. N omega-nitro-L-arginine (100 micromol/L; nitric oxide antagonist) blocked the reductions in Ca(2+) and increased the frequency (approximately 19/min) of intracellular Ca(2+) waves within ICC-MY. ICC-SMs form a tightly coupled network that is able to generate and propagate slow waves. In contrast, Ca(2+) transients in ICC-MYs, which are normally not synchronized, have a similar duration and frequency as MPOs. Like MPOs, their activity is inhibited by nitrergic nerves and synchronized by excitatory nerves.

Mechanism of asynchronous Ca2+ waves underlying agonist‐induced contraction in the rat basilar artery

Uridine 5'-triphosphate (UTP) is a potent vasoconstrictor of cerebral arteries and induces Ca(2+) waves in vascular smooth muscle cells (VSMCs). This study aimed to determine the mechanisms underlying UTP-induced Ca(2+) waves in VSMCs of the rat basilar artery. Isometric force and intracellular Ca(2+) ([Ca(2+)](i)) were measured in endothelium-denuded rat basilar artery using wire myography and confocal microscopy respectively. Uridine 5'-triphosphate (0.1-1000 micromol.L(-1)) concentration-dependently induced tonic contraction (pEC(50) = 4.34 +/- 0.13), associated with sustained repetitive oscillations in [Ca(2+)](i) propagating along the length of the VSMCs as asynchronized Ca(2+) waves. Inhibition of Ca(2+) reuptake in sarcoplasmic reticulum (SR) by cyclopiazonic acid abolished the Ca(2+) waves and resulted in a dramatic drop in tonic contraction. Nifedipine reduced the frequency of Ca(2+) waves by 40% and tonic contraction by 52%, and the nifedipine-insensitive component was abolished by SKF-96365, an inhibitor of receptor- and store-operated channels, and KB-R7943, an inhibitor of reverse-mode Na(+)/Ca(2+) exchange. Ongoing Ca(2+) waves and tonic contraction were also abolished after blockade of inositol-1,4,5-triphosphate-sensitive receptors by 2-aminoethoxydiphenylborate, but not by high concentrations of ryanodine or tetracaine. However, depletion of ryanodine-sensitive SR Ca(2+) stores prior to UTP stimulation prevented Ca(2+) waves. Uridine 5'-triphosphate-induced Ca(2+) waves may underlie tonic contraction and appear to be produced by repetitive cycles of regenerative Ca(2+) release from the SR through inositol-1,4,5-triphosphate-sensitive receptors. Maintenance of Ca(2+) waves requires SR Ca(2+) reuptake from Ca(2+) entry across the plasma membrane via L-type Ca(2+) channels, receptor- and store-operated channels, and reverse-mode Na(+)/Ca(2+) exchange.

Ca Alternans in Cardiac Myocytes: Relating Macroscopic Behavior to Microscopic Ca Release Properties

Beat-to-beat alternation in the intracellular Ca (Cai) transient (Cai alternans) causes pulsus alternans and electrocardiographic T-wave alternans, conditions associated with cardiac arrhythmias and sudden death. The whole cell Cai transient represents the summed activity of thousands of individual Ca sparks, i.e. Ca released locally by Ca release units (CRU). However, the extent to which the global behavior of the whole cell Cai transient mirrors the microscopic behavior of individual CRU units is unclear. We derived a one-dimensional iterated map of CRU behavior in which we could independently adjust the probabilities of random triggering of Ca sparks, recruitment of Ca sparks from adjacent CRUs, and CRU refractoriness following a Ca spark. After verifying that these three local properties (randomness, recruitment and refractoriness) could sustain an ensemble (global) alternans in a two-dimensional cellular automata network, we developed a physiologically-detailed subcellular Ca cycling model containing a network of coupled stochastic CRU which replicated the iterated map predictions. We find that a number of experimentally-reported phenomena, including whole cell Cai alternans, Ca waves in the presence of high spatial cooperativity, graded whole-cell Ca release, and a steep dependence of fractional SR Ca release on SR Ca load, emerge naturally from the collective behavior of individual CRUs depending on the balance of these three properties. A striking prediction is that microscopic CRU behavior does not always mirror collective CRU behavior, e.g. during whole cell Cai alternans, Ca sparks from individual CRU do not consistently alternate. In addition, whole cell Cai alternans is not generally dependent on alternans of diastolic SR Ca content. The findings provide novel multiscale insights into how global Ca signaling properties emerge from simple microscopic CRU properties.