Ca2+ channels, 'quantized' Ca2+ release, and differentiation of myocytes in the cardiovascular system

The application of confocal microscopy to cardiac and skeletal muscle has resulted in the observation of transient, spatially localized elevations in [Ca2+]i, termed 'Ca2+ sparks'. Ca2+ sparks are thought to represent 'elementary' Ca2+ release events, which arise from one or more ryanodine receptor (RyR) channels in the sarcoplasmic reticulum. In cardiac muscle, Ca2+ sparks appear to be key elements of excitation-contraction coupling, in which the global [Ca2+]i transient is thought to involve the recruitment of Ca2+ sparks, each of which is controlled locally by single coassociated L-type Ca2+ channels. Recently, Ca2+ sparks have been detected in smooth muscle cells of arteries. In this review, we analyse the complex relationship of Ca2+ influx and Ca2+ release with local, subcellular Ca2+ microdomains in light of recent studies on Ca2+ sparks in cardiovascular cells. We performed a comparative analysis of 'elementary' Ca2+ release units in mouse, rat and human arterial smooth muscle cells, using measurements of Ca2+ sparks and plasmalemmal K(Ca) currents activated by Ca2+ sparks (STOCs). Furthermore, the appearance of Ca2+ sparks during ontogeny of arterial smooth muscle is explored. Using intact pressurized arteries, we have investigated whether RyRs causing Ca2+ sparks (but not smaller 'quantized' Ca2+ release events, e.g. hypothetical 'Ca2+ quarks') function as key signals that, through membrane potential and global cytoplasmic [Ca2+], oppose arterial myogenic tone and influence vasorelaxation. We believe that voltage-dependent Ca2+ channels and local RyR-related Ca2+ signals are important in differentiation, proliferation, and gene expression. Our findings suggest that 'elementary' Ca2+ release units may represent novel potent therapeutic targets for regulating function of intact arterial smooth muscle tissue.     

TRPV4 forms a novel Ca2+ signaling complex with ryanodine receptors and BKCa channels

Vasodilatory factors produced by the endothelium are critical for the maintenance of normal blood pressure and flow. We hypothesized that endothelial signals are transduced to underlying vascular smooth muscle by vanilloid transient receptor potential (TRPV) channels. TRPV4 message was detected in RNA from cerebral artery smooth muscle cells. In patch-clamp experiments using freshly isolated cerebral myocytes, outwardly rectifying whole-cell currents with properties consistent with those of expressed TRPV4 channels were evoked by the TRPV4 agonist 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) (5 micromol/L) and the endothelium-derived arachidonic acid metabolite 11,12 epoxyeicosatrienoic acid (11,12 EET) (300 nmol/L). Using high-speed laser-scanning confocal microscopy, we found that 11,12 EET increased the frequency of unitary Ca2+ release events (Ca2+ sparks) via ryanodine receptors located on the sarcoplasmic reticulum of cerebral artery smooth muscle cells. EET-induced Ca2+ sparks activated nearby sarcolemmal large-conductance Ca2+-activated K+ (BKCa) channels, measured as an increase in the frequency of transient K+ currents (referred to as "spontaneous transient outward currents" [STOCs]). 11,12 EET-induced increases in Ca2+ spark and STOC frequency were inhibited by lowering external Ca2+ from 2 mmol/L to 10 micromol/L but not by voltage-dependent Ca2+ channel inhibitors, suggesting that these responses require extracellular Ca2+ influx via channels other than voltage-dependent Ca2+ channels. Antisense-mediated suppression of TRPV4 expression in intact cerebral arteries prevented 11,12 EET-induced smooth muscle hyperpolarization and vasodilation. Thus, we conclude that TRPV4 forms a novel Ca2+ signaling complex with ryanodine receptors and BKCa channels that elicits smooth muscle hyperpolarization and arterial dilation via Ca2+-induced Ca2+ release in response to an endothelial-derived factor.     

Spatiotemporal characteristics of junctional and nonjunctional focal Ca2+ release in rat atrial myocytes

Atrial myocytes have two functionally separate Ca2+ release sites: those in peripheral sarcoplasmic reticulum (SR) adjacent to the Ca2+ channels of surface membrane and those in central SR not associated with Ca2+ channels. Recently, we have reported on the gating of these two different Ca2+ release sites by Ca2+ current. In the present study, we report on the spatiotemporal properties of focal Ca2+ releases (sparks) occurring spontaneously in central and peripheral sites of voltage-clamped rat atrial myocytes, using rapid 2-dimensional (2-D) confocal Ca2+ imaging. Peripheral and central sparks were similar in size and release time (approximately 300 000 Ca2+ ions for congruent with 12 ms), but significantly larger and longer than ventricular sparks. Both sites were resistant to Cd2+ and inhibited by ryanodine. Peripheral sparks were brighter and flattened against surface membrane, had approximately 5-fold higher frequency, approximately 2 times faster diffusion coefficient, and dissipated abruptly. Central sparks, in contrast, occurred less frequently, were elongated along the cellular longitudinal axis, and dissipated slowly. Compound sparks (composed of 2 to 5 unitary focal releases) aligned longitudinally and occurred more frequently at the center. The diversity of peripheral and central sparks with respect to shape, frequency, and speed of spatial development and decay is consistent with regional ultrastructural heterogeneity of SR. The retarded dissipation of central atrial sparks, and high prevalence of compound sparks in cell center may be critical in facilitating the propagation of Ca2+ waves in atrial myocytes lacking t-tubular system and provide the atrial myocytes with functional Ca2+ signaling diversity. The full text of this article is available at http://www.circresaha.org.     

Contribution of sarcoplasmic reticulum Ca2+ to the activation of Ca2+ -activated K+ channels in the resting state of arteries from spontaneously hypertensive rats

OBJECTIVE : Localized release of Ca2+ from the sarcoplasmic reticulum (SR) toward the plasmalemma, sometimes visualized as Ca2+ sparks, can activate Ca2+-activated K+ (KCa) channels. We have already reported that the addition of charybdotoxin (ChTX), a blocker of KCa channels, to the resting state of arteries from spontaneously hypertensive rats (SHR) caused a powerful contraction, suggesting that KCa channels were active in the resting state. This study aimed to determine whether the Ca2+ responsible for activity of KCa channels was derived from SR. METHODS : Possible mechanisms underlying the ChTX-induced contractions were examined in endothelium-denuded strips of femoral, mesenteric, small mesenteric and carotid arteries from 13-week-old SHR and normotensive Wistar-Kyoto (WKY) rats by using selective inhibitors of the Ca2+ spark process. RESULTS : ChTX (100 nmol/l) induced a contraction in the SHR arteries. The ChTX-induced contractions were increased by a moderate membrane depolarization by 15.9 mmol/l K+ and were abolished by nifedipine (100 nmol/l). When SR Ca2+ was depleted by treatment of the strips with ryanodine (10 mumol/l) plus caffeine (20 mmol/l) or with thapsigargin (100 nmol/l), the ChTX-induced contraction was decreased in femoral, mesenteric and small mesenteric arteries and was almost abolished in the carotid artery. A similar phenomenon can be observed in arteries from WKY rats after a moderate membrane depolarization. In both SHR and WKY rats, SR Ca2+-dependent ChTX-induced contraction always represents 20-30% of the maximal K+-induced contraction. CONCLUSIONS : We conclude that activation of KCa channels depended upon influx of Ca2+ through L-type Ca2+ channels and release of Ca2+ from the SR, suggesting that recycling of entering Ca2+ from the superficial SR toward the plasmalemma sufficiently elevated Ca2+ near these channels to activate them.     

Ca2+ channels, Ca2+ sparks, and regulation of arterial smooth muscle function

Summary In cardiac, skeletal, and arterial muscle, transient, spatially localized elevations in [Ca²⁺]_i, termed "Ca²⁺ sparks", have been observed using confocal laser scanning microscopy. Ca²⁺ sparks are thought to represent "elementary" Ca²⁺ release events, which arise from one or more ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). In striated muscle, Ca²⁺ sparks are thought to be key elements of excitation-contraction coupling. In arterial smooth muscle, Ca²⁺ sparks have been suggested to oppose myogenic vasoconstriction and to influence vasorelaxation. Using a developmental model, we have investigated whether RyRs causing Ca²⁺ sparks and activation of Ca²⁺-activated K⁺ (K_Ca) channels (STOCs) function as "elementary" Ca²⁺ release units that regulate arterial mygenic tone. Whereas increases in the global [Ca²⁺]_i induce sustained constriction of arterial smooth muscle, Ca²⁺ sparks induce vasodilation through the local activation of K_Ca channels. In cerebral arteries, the global bulk [Ca²⁺]_i and a Ca²⁺ spark frequency < 10^-2 Hz/cell do not cause sufficient K_Ca channel activity to regulate membrane potential of smooth muscle cells and myogenic tone. The frequency of Ca²⁺ sparks and STOCs is regulated by agents that modulate protein kinase G and protein kinase A activity. Our findings suggest that "elementary" Ca²⁺ release units may represent novel, important therapeutic targets for regulating function of the intact arterial smooth muscle tissue.     

Ignition of calcium sparks in arterial and cardiac muscle through caveolae

Ca(2+) sparks are localized intracellular Ca(2+) events released through ryanodine receptors (RyRs) that control excitation-contraction coupling in heart and smooth muscle. Ca(2+) spark triggering depends on precise delivery of Ca(2+) ions through dihydropyridine (DHP)-sensitive Ca(2+) channels to RyRs of the sarcoplasmic reticulum (SR), a process requiring a very precise alignment of surface and SR membranes containing Ca(2+) influx channels and RyRs. Because caveolae contain DHP-sensitive Ca(2+) channels and may colocalize with SR, we tested the hypothesis that caveolae are the structural element necessary for the generation of Ca(2+) sparks. Using methyl-ss-cyclodextrin (dextrin) to deplete caveolae, we found that dextrin dose-dependently decreased the frequency, amplitude, and spatial size of Ca(2+) sparks in arterial smooth muscle cells and neonatal cardiomyocytes. However, temporal characteristics of Ca(2+) sparks were not significantly affected. We ruled out the possibility that the decreases in Ca(2+) spark frequency and size are caused by changes in DHP-sensitive L-type channels, SR Ca(2+) load, or changes in membrane potential. Our results suggest a novel signaling model that explains the formation of Ca(2+) sparks in a caveolae microdomain. The transient elevation in [Ca(2+)](i) at the inner mouth of a single caveolemmal Ca(2+) channel induces simultaneous activation and thus opens several RyRs to generate a local Ca(2+) release event, a Ca(2+) spark. Alterations in the molecular assembly and ultrastructure of caveolae may lead to pathophysiological changes in Ca(2+) signaling. Thus, caveolae may be intimately involved in cardiovascular cell dysfunction and disease.     

Imaging microdomain Ca2+ in muscle cells

Ca2+ ions passing through a single or a cluster of Ca2+-permeable channels create microscopic, short-lived Ca2+ gradients that constitute the building blocks of cellular Ca2+ signaling. Over the last decade, imaging microdomain Ca2+ in muscle cells has unveiled the exquisite spatial and temporal architecture of intracellular Ca2+ dynamics and has reshaped our understanding of Ca2+ signaling mechanisms. Major advances include the visualization of "Ca2+ sparks" as the elementary events of Ca2+ release from the sarcoplasmic reticulum (SR), "Ca2+ sparklets" produced by openings of single Ca2+-permeable channels, miniature Ca2+ transients in single mitochondria ("marks"), and SR luminal Ca2+ depletion transients ("scraps"). As a model system, a cardiac myocyte contains a 3-dimensional grid of 104 spark ignition sites, stochastic activation of which summates into global Ca2+ transients. Tracking intermolecular coupling between single L-type Ca2+ channels and Ca2+ sparks has provided direct evidence validating the local control theory of Ca2+-induced Ca2+ release in the heart. In vascular smooth muscle myocytes, Ca2+ can paradoxically signal both vessel constriction (by global Ca2+ transients) and relaxation (by subsurface Ca2+ sparks). These findings shed new light on the origin of Ca2+ signaling efficiency, specificity, and versatility. In addition, microdomain Ca2+ imaging offers a novel modality that complements electrophysiological approaches in characterizing Ca2+ channels in intact cells.     

Downregulation of the BK channel beta1 subunit in genetic hypertension

The molecular mechanisms underlying increased arterial tone during hypertension are unclear. In vascular smooth muscle, localized Ca2+ release events through ryanodine-sensitive channels located in the sarcoplasmic reticulum (Ca2+ sparks) activate large-conductance, Ca2+-sensitive K+ (BK) channels. Ca2+ sparks and BK channels provide a negative feedback mechanism that hyperpolarizes smooth muscle and thereby opposes vasoconstriction. In this study, we examined Ca2+ sparks and BK channel function in Wistar-Kyoto (WKY) rats with borderline hypertension and in spontaneously hypertensive rats (SHR), a widely used genetic model of severe hypertension. We found that the amplitude of spontaneous BK currents in WKY and SHR cells were smaller than in normotensive cells even though Ca2+ sparks were of similar magnitude. BK channels in WKY and SHR cells were less sensitive to physiological changes in intracellular Ca2+ than normotensive cells. Our data indicate that decreased expression of the BK channel beta1 subunit underlies the lower Ca2+ sensitivity of BK channels in SHR and WKY myocytes. We conclude that the lower expression of the beta1 subunit during genetic borderline and severe hypertension reduced BK channel activity by decreasing the sensitivity of these channels to physiological changes in Ca2+. These results support the view that changes in the molecular composition of BK channels may be a fundamental event contributing to the development of vascular dysfunction during hypertension.     

Luminal Ca2+ controls termination and refractory behavior of Ca2+-induced Ca2+ release in cardiac myocytes

Despite extensive research, the mechanisms responsible for the graded nature and early termination of Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) in cardiac muscle remain poorly understood. Suggested mechanisms include cytosolic Ca2+-dependent inactivation/adaptation and luminal Ca2+-dependent deactivation of the SR Ca2+ release channels/ryanodine receptors (RyRs). To explore the importance of cytosolic versus luminal Ca2+ regulatory mechanisms in controlling CICR, we assessed the impact of intra-SR Ca2+ buffering on global and local Ca2+ release properties of patch-clamped or permeabilized rat ventricular myocytes. Exogenous, low-affinity Ca2+ buffers (5 to 20 mmol/L ADA, citrate or maleate) were introduced into the SR by exposing the cells to "internal" solutions containing the buffers. Enhanced Ca2+ buffering in the SR was confirmed by an increase in the total SR Ca2+ content, as revealed by application of caffeine. At the whole-cell level, intra-SR [Ca2+] buffering dramatically increased the magnitude of Ca2+ transients induced by I(Ca) and deranged the smoothly graded I(Ca)-SR Ca2+ release relationship. The amplitude and time-to-peak of local Ca2+ release events, Ca2+ sparks, as well as the duration of local Ca2+ release fluxes underlying sparks were increased up to 2- to 3-fold. The exogenous Ca2+ buffers in the SR also reduced the frequency of repetitive activity observed at individual release sites in the presence of the RyR activator Imperatoxin A. We conclude that regulation of RyR openings by local intra-SR [Ca2+] is responsible for termination of CICR and for the subsequent restitution behavior of Ca2+ release sites in cardiac muscle.     

Ca2+/Calmodulin-dependent protein kinase II phosphorylation of ryanodine receptor does affect calcium sparks in mouse ventricular myocytes

Previous studies in transgenic mice and with isolated ryanodine receptors (RyR) have indicated that Ca2+-calmodulin-dependent protein kinase II (CaMKII) can phosphorylate RyR and activate local diastolic sarcoplasmic reticulum (SR) Ca2+ release events (Ca2+ sparks) and RyR channel opening. Here we use relatively controlled physiological conditions in saponin-permeabilized wild type (WT) and phospholamban knockout (PLB-KO) mouse ventricular myocytes to test whether exogenous preactivated CaMKII or endogenous CaMKII can enhance resting Ca2+ sparks. PLB-KO mice were used to preclude ancillary effects of CaMKII mediated by phospholamban phosphorylation. In both WT and PLB-KO myocytes, Ca2+ spark frequency was increased by both preactivated exogenous CaMKII and endogenous CaMKII. This effect was abolished by CaMKII inhibitor peptides. In contrast, protein kinase A catalytic subunit also enhanced Ca2+ spark frequency in WT, but had no effect in PLB-KO. Both endogenous and exogenous CaMKII increased SR Ca2+ content in WT (presumably via PLB phosphorylation), but not in PLB-KO. Exogenous calmodulin decreased Ca2+ spark frequency in both WT and PLB-KO (K0.5 approximately 100 nmol/L). Endogenous CaMKII (at 500 nmol/L [Ca2+]) phosphorylated RyR as completely in <4 minutes as the maximum achieved by preactivated exogenous CaMKII. After CaMKII activation Ca2+ sparks were longer in duration, and more frequent propagating SR Ca2+ release events were observed. We conclude that CaMKII-dependent phosphorylation of RyR by endogenous associated CaMKII (but not PKA-dependent phosphorylation) increases resting SR Ca2+ release or leak. Moreover, this may explain the enhanced SR diastolic Ca2+ leak and certain triggered arrhythmias seen in heart failure.     

Carbon monoxide dilates cerebral arterioles by enhancing the coupling of Ca2+ sparks to Ca2+-activated K+ channels

Carbon monoxide (CO) is generated endogenously by the enzyme heme oxygenase. Although CO is a known vasodilator, cellular signaling mechanisms are poorly understood and are a source of controversy. The goal of the present study was to investigate mechanisms of CO dilation in porcine cerebral arterioles. Data indicate that exogenous or endogenously produced CO is a potent activator of large-conductance Ca2+-activated K+ (K(Ca)) channels and Ca2+ spark-induced transient K(Ca) currents in arteriole smooth muscle cells. In contrast, CO is a relatively poor activator of Ca2+ sparks. To understand the apparent discrepancy between potent effects on transient K(Ca) currents and weak effects on Ca2+ sparks, regulation of the coupling relationship between these events by CO was investigated. CO increased the percentage of Ca2+ sparks that activated a transient K(Ca) current (ie, the coupling ratio) from approximately 62% in the control condition to 100% and elevated the slope of the amplitude correlation between these events approximately 2.6-fold, indicating that Ca2+ sparks induced larger amplitude transient K(Ca) currents in the presence of CO. This signaling pathway for CO is physiologically relevant because ryanodine, a ryanodine-sensitive Ca2+ release channel blocker that inhibits Ca2+ sparks, abolished CO dilation of pial arterioles in vivo. Thus, CO dilates cerebral arterioles by priming K(Ca) channels for activation by Ca2+ sparks. This study presents a novel dilatory signaling pathway for CO in the cerebral circulation and appears to be the first demonstration [corrected] of a vasodilator that acts by increasing the effective coupling of Ca2+ sparks to K(Ca) channels.     

Role of cyclic ADP-ribose in Ca2+-induced Ca2+ release and vasoconstriction in small renal arteries

Cyclic-ADP-ribose (cADPR) has been reported to serve as a second messenger to mobilize intracellular Ca2+ independent of IP3 in a variety of mammalian cells. This cADPR-mediated Ca2+ signaling pathway importantly participates in the regulation of various cell functions. The present study determined the role of endogenous cADPR in mediating ryanodine-sensitive Ca2+-induced Ca2+ release (CICR) in vascular myocytes from small renal arteries and vasomotor response of these arteries. In freshly-isolated renal arterial myocytes, addition of CaCl2 (0.01, 0.1, and 1 mM) into the Ca2+-free bath solution produced a rapid Ca2+ release response from the sarcoplasmic reticulum (SR), with a maximal increase of 237+/-25 nM at 1 mM CaCl2. This CaCl2 response was significantly blocked by a cell-membrane permeant cADPR antagonist, 8-bromo-cADP-ribose (8-br-cADPR) (30 microM) or ryanodine (50 microM). Caffeine, a classical CICR or ryanodine receptor activator was found to stimulate the SR Ca2+ release (Delta[Ca2+]i: 253+/-35 nM), which was also attenuated by 8-br-cADPR or ryanodine. Using isolated and pressurized small renal arteries bathed with Ca2+-free solution, both CaCl2 and caffeine-induced vasoconstrictions were significantly attenuated by either 8-br-cADPR or ryanodine. Biochemical analyses demonstrated that CaCl2 and caffeine did not increase cADPR production in these renal arterial myocytes, but confocal microscopy showed that a dissociation of the accessory protein, FK506 binding protein 12.6 (FKBP12.6) from ryanodine receptors was induced by CaCl2. We conclude that cADPR importantly contributes to CICR and vasomotor responses of small renal arteries through enhanced dissociation of ryanodine receptors from their accessory protein.     

Preserved BK channel function in vasospastic myocytes from a dog model of subarachnoid hemorrhage

Cerebral vasospasm after subarachnoid hemorrhage (SAH) is due to contraction of smooth muscle cells in the cerebral arteries. The mechanism of this contraction, however, is not well understood. Smooth muscle contraction is regulated in part by membrane potential, which is determined by K+ conductance in smooth muscle. Voltage-gated (Kv) and large-conductance, Ca2+-activated K+ (BK) channels dominate arterial smooth muscle K+ conductance. Vasospastic smooth muscle cells are depolarized relative to normal cells, but whether this is due to altered Kv or BK channel function has not been determined. This study determined if BK channels are altered during vasospasm after SAH in dogs. We first characterized BK channels in basilar-artery smooth muscle using whole-cell patch clamping and single-channel recordings. Next, we compared BK channel function between normal and vasospastic cells. There were no significant differences between normal and vasospastic cells in BK current density, kinetics, Ca2+ and voltage sensitivity, single-channel conductance or apparent Ca2+ affinity. Basilar-artery myocytes had no, small- or intermediate-conductance, Ca2+-activated K+ channels. The lack of difference in BK channels between vasospastic and control cells suggests alteration(s) in other K+ channels or other ionic conductances may underlie the membrane depolarization and vasoconstriction observed during vasospasm after SAH.     

Effects of adenovirus-mediated sorcin overexpression on excitation-contraction coupling in isolated rabbit cardiomyocytes

To evaluate the effect of sorcin on cardiac excitation-contraction coupling, adult rabbit ventricular myocytes were transfected with a recombinant adenovirus coding for human sorcin (Ad-sorcin). A beta-galactosidase adenovirus (Ad-LacZ) was used as a control. Fractional shortening in response to 1-Hz field stimulation (at 37 degrees C) was significantly reduced in Ad-sorcin-transfected myocytes compared with control myocytes (2.10+/-0.05% [n=311] versus 2.42+/-0.06% [n=312], respectively; P<0.001). Action potential duration (at 20 degrees C) was significantly less in the Ad-sorcin group (458+/-22 ms, n=11) compared with the control group (520+/-19 ms, n=10; P<0.05). In voltage-clamped, fura 2-loaded myocytes (20 degrees C), a reduced peak-systolic and end-diastolic [Ca2+]i was observed after Ad-sorcin transfection. L-type Ca2+ current amplitude and time course were unaffected. Caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR) and the accompanying inward Na+-Ca2+ exchanger (NCX) current revealed a significantly lower SR Ca2+ content and faster Ca2+-extrusion kinetics in Ad-sorcin-transfected cells. Higher NCX activity after Ad-sorcin transfection was confirmed by measuring the NCX current-voltage relationship. beta-Escin-permeabilized rabbit cardiomyocytes were used to study the effects of sorcin overexpression on Ca2+ sparks imaged with fluo 3 at 145 to 160 nmol/L [Ca2+] using a confocal microscope. Under these conditions, caffeine-mediated SR Ca2+ release was not different between the two groups. Spontaneous spark frequency, duration, width, and amplitude were lower in sorcin-overexpressing myocytes. In summary, sorcin overexpression in rabbit cardiomyocytes decreased Ca2+-transient amplitude predominantly by lowering SR Ca2+ content via increased NCX activity. The effect of sorcin overexpression on Ca2+ sparks indicates an effect on the ryanodine receptor that may also influence excitation-contraction coupling.     

Fetal and postnatal development of Ca2+ transients and Ca2+ sparks in rat cardiomyocytes

OBJECTIVE: The aim of this study was to characterize the spatio-temporal dynamics of [Ca(2+)](i) in rat heart in the fetal and neonatal periods. METHODS: Using confocal scanning laser microscopy and the Ca(2+) indicator fluo-3, we investigated Ca(2+) transients and Ca(2+) sparks in single ventricular myocytes freshly isolated from rat fetuses and neonates. T-tubules were labeled with a membrane-selective dye (di-8-ANEPPS). Spatial association of dihydropyridine receptors (DHPR) and ryanodine receptors (RyR) was also examined by double-labeling immunofluorescence. RESULTS: Ca(2+) transients in the fetal myocytes were characterized by slower upstroke and decay of [Ca(2+)](i) compared to those in adult myocytes. The magnitude of fetal Ca(2+) transients was decreased after application of ryanodine (1 microM) or thapsigargin (1 microM). However, Ca(2+) sparks were rarely detected in the fetal myocytes. Frequent ignition of Ca(2+) sparks was established in the 6-9-day neonatal period, and was predominantly observed in the subsarcolemmal region. The developmental change in Ca(2+) sparks coincided with development of the t-tubule network. The immunofluorescence study revealed colocalization of DHPR and RyR in the postnatal period, which was, however, not observed in the fetal period. In the adult myocytes, Ca(2+) sparks disappeared after disruption of t-tubules by glycerol incubation (840 mM). CONCLUSIONS: The sarcoplasmic reticulum (SR) of rat ventricular myocytes already functions early in the fetal period. However, ignition of Ca(2+) sparks depends on postnatal t-tubule formation and resultant colocalization of DHPR and RyR.     

Dendroaspis natriuretic peptide relaxes gastric antral circular smooth muscle of guinea-pig through the cGMP/cGMP-dependent protein kinase pathway

AIM： To systematically investigate if cGMP/cGMPdependent protein kinase G （PKG） signaling pathway may participate in dendroaspis natriuretic peptide （DNP）-induced relaxation of gastric circular smooth muscle.
METHODS： The content of cGMP in guinea pig gastric antral smooth muscle tissue and perfusion solution were measured using radioimmunoassay; spontaneous contraction of gastric antral circular muscles recorded using a 4-channel physiograph; and Ca^2＋-activated K^＋ currents （IK（Ca）） and spontaneous transient outward currents （STOCs） in isolated gastric antral myocytes were recorded using the whole-cell patch clamp technique.
RESULTS： DNP markedly enhanced cGMP levels in gastric antral smooth muscle tissue and in the perfusion medium. DNP induced relaxation in gastric antral circular smooth muscle, which was inhibited by KT5823, a cGMP-dependent PKG inhibitor. DNP increased IK（Ca）. This effect was almost completely blocked by KT5823, and partially blocked by LY83583, an inhibitor of guanylate cyclase to change the production of cGMP. DNP also increased STOCs. The effect of DNP on STOCs was abolished in the presence of KT5823, but not affected by KT-5720, a PKA-specific inhibitor.
CONCLUSION： DNP activates IK（ca） and relaxes guinea-pig gastric antral circular smooth muscle via the cGMP/PKG-dependent singling axis instead of cAMP/ PKA pathway.     

Ca2+-handling in heart failure--a review focusing on Ca2+ sparks

[Ca2+]i-transients have been shown to be altered in isolated ventricular myocytes from terminally failing human myocardium. It has been demonstrated that one reason for this alteration is a reduction in the Ca2+ content of the sarcoplasmic reticulum (SR). Further investigations were done to investigate, whether there may be an additional defect of the Ca2+-release mechanisms from the SR. These release mechanisms were investigated through the recording of Ca2+ sparks in single human myocytes. In cardiac myocytes, Ca2+ sparks are elementary units of Ca2+ release, which occur spontaneously, or which are triggered by Ca2+ influx through L-type Ca2+-channels (Ca2+-induced Ca2+ release). Ca2+ sparks have been investigated in various animal models of cardiac hypertrophy and cardiac failure and results were conflicting. Discrepancies may be explained by different species and also by the mechanisms underlying hypertrophy and heart failure. This review summarizes our current knowledge on Ca2+ sparks in heart failure.     

Cellular mechanisms of altered contractility in the hypertrophied heart: big hearts, big sparks

To investigate the cellular mechanisms for altered Ca2+ homeostasis and contractility in cardiac hypertrophy, we measured whole-cell L-type Ca2+ currents (ICa,L), whole-cell Ca2+ transients ([Ca2+]i), and Ca2+ sparks in ventricular cells from 6-month-old spontaneously hypertensive rats (SHRs) and from age- and sex-matched Wistar-Kyoto and Sprague-Dawley control rats. By echocardiography, SHR hearts had cardiac hypertrophy and enhanced contractility (increased fractional shortening) and no signs of heart failure. SHR cells had a voltage-dependent increase in peak [Ca2+]i amplitude (at 0 mV, 1330+/-62 nmol/L [SHRs] versus 836+/-48 nmol/L [controls], P<0.05) that was not associated with changes in ICa,L density or kinetics, resting [Ca2+]i, or Ca2+ content of the sarcoplasmic reticulum (SR). SHR cells had increased time of relaxation. Ca2+ sparks from SHR cells had larger average amplitudes (173+/-192 nmol/L [SHRs] versus 109+/-64 nmol/L [control]; P<0.05), which was due to redistribution of Ca2+ sparks to a larger amplitude population. This change in Ca2+ spark amplitude distribution was not associated with any change in the density of ryanodine receptors, calsequestrin, junctin, triadin 1, Ca2+-ATPase, or phospholamban. Therefore, SHRs with cardiac hypertrophy have increased contractility, [Ca2+]i amplitude, time to relaxation, and average Ca2+ spark amplitude ("big sparks"). Importantly, big sparks occurred without alteration in the trigger for SR Ca2+ release (ICa,L), SR Ca2+ content, or the expression of several SR Ca2+-cycling proteins. Thus, cardiac hypertrophy in SHRs is linked with an alteration in the coupling of Ca2+ entry through L-type Ca2+ channels and the release of Ca2+ from the SR, leading to big sparks and enhanced contractility. Alterations in the microdomain between L-type Ca2+ channels and SR Ca2+ release channels may underlie the changes in Ca2+ homeostasis observed in cardiac hypertrophy. Modulation of SR Ca2+ release may provide a new therapeutic strategy for cardiac hypertrophy and for its progression to heart failure and sudden death.     

Dendroaspis natriuretic peptide relaxes gastric antral circular smooth muscle of guinea-pig through the cGIvlP/cGMP-dependent protein kinase pathway

AIM: To systematically investigate if cGMP/cGMPdependent protein kinase G (PKG) signaling pathway may participate in dendroaspis natriuretic peptide (DNP)-induced relaxation of gastric circular smooth muscle.METHODS: The content of cGMP in guinea pig gastric antral smooth muscle tissue and perfusion solution were measured using radioimmunoassay,spontaneous contraction of gastric antral circular muscles recorded using a 4-channel physiograph; and Ca2 -activated K currents (Ik(Ca)and spontaneous transient outward currents (STOCs) in isolated gastric antral myocytes were recorded using the whole-cell patch clamp technique.]RESULTS: DNP markedly enhanced cGMP levels in gastric antral smooth muscle tissue and in the perfusion medium.DNP induced relaxation in gastric antral circular smooth muscle,which was inhibited by KT5823,a cGMP-dependent PKG inhibitor.DNP increased IK(Ca)* This effect was almost completely blocked by KT5823,and partially blocked by LY83583,an inhibitor of guanylate cyclase to change the production of cGMP.DNP also increased STOCs.The effect of DNP on STOCs was abolished in the presence of KT5823,but not affected by KT-5720,a PKA-specific inhibitor.CONCLUSION: DNP activates IK(Ca) and relaxes guinea-pig gastric antral circular smooth muscle via the cGMP/PKG-dependent singling axis instead of cAMP/PKA pathway.     

Local Ca2+ entry through L-type Ca2+ channels activates Ca2+-dependent K+ channels in rabbit coronary myocytes.

Large-conductance Ca2+-dependent K+ channels (KCa), which are abundant on the sarcolemma of vascular myocytes, provide negative feedback via membrane hyperpolarization that limits Ca2+ entry through L-type Ca2+ channels (ICaL). We hypothesize that local accumulation of subsarcolemmal Ca2+ during ICaL openings amplifies this feedback. Our goal was to demonstrate that Ca2+ entry through voltage-gated ICaL channels can stimulate adjacent KCa channels by a localized interaction in enzymatically isolated rabbit coronary arterial myocytes voltage clamped in whole-cell or in cell-attached patch clamp mode. During slow-voltage-ramp protocols, we identified an outward KCa current that is activated by a subsarcolemmal Ca2+ pool dissociated from bulk cytosolic Ca2+ pool (measured with indo 1) and is dependent on L-type Ca2+ channel activity. Transient activation of unitary KCa channels in cell-attached patches could be detected during long step depolarizations to +40 mV (holding potential, -40 mV; 219 pS in near-symmetrical K+). This local interaction between the channels required the presence of Ca2+ in the pipette solution, was enhanced by the ICaL agonist Bay K 8644, and persisted after impairment of the sarcoplasmic reticulum by incubation with 10 micromol/L ryanodine and 30 micromol/L cyclopiazonic acid for at least 60 minutes. Furthermore, we provide the first direct evidence of simultaneous openings of single KCa (67 pS) and ICaL (3.9 pS) channels in near-physiological conditions, near resting membrane potential. Our data imply a novel sensitive mechanism for regulating resting membrane potential and tone in vascular smooth muscle.