Major features of the transcellular signaling mechanism responsible for endothelium-dependent regulation of vascular smooth muscle tone are unresolved. We identified local calcium (Ca2+) signals (“sparklets”) in the vascular endothelium of resistance arteries that represent Ca2+ influx through single TRPV4 cation channels. Gating of individual TRPV4 channels within a four-channel cluster was cooperative, with activation of as few as three channels per cell causing maximal dilation through activation of endothelial cell intermediate (IK)- and small (SK)-conductance, Ca2+-sensitive potassium (K+) channels. Endothelial-dependent muscarinic receptor signaling also acted largely through TRPV4 sparklet-mediated stimulation of IK and SK channels to promote vasodilation. These results support the concept that Ca2+ influx through single TRPV4 channels is leveraged by the amplifier effect of cooperative channel gating and the high Ca2+ sensitivity of IK and SK channels to cause vasodilation.

Calcium (Ca 2؉ ) release through inositol 1,4,5-trisphosphate receptors (IP 3Rs) regulates the function of virtually every mammalian cell. Unlike ryanodine receptors, which generate local Ca 2؉ events (''sparks'') that transmit signals to the juxtaposed cell membrane, a similar functional architecture has not been reported for IP 3Rs. Here, we have identified spatially fixed, local Ca 2؉ release events (''pulsars'') in vascular endothelial membrane domains that project through the internal elastic lamina to adjacent smooth muscle membranes. Ca 2؉ pulsars are mediated by IP3Rs in the endothelial endoplasmic reticulum of these membrane projections. Elevation of IP 3 by the endothelium-dependent vasodilator, acetylcholine, increased the frequency of Ca 2؉ pulsars, whereas blunting IP3 production, blocking IP3Rs, or depleting endoplasmic reticulum Ca 2؉ inhibited these events. The elementary properties of Ca 2؉ pulsars were distinct from ryanodine-receptor-mediated Ca 2؉ sparks in smooth muscle and from IP3-mediated Ca 2؉ puffs in Xenopus oocytes. The intermediate conductance, Ca 2؉ -sensitive potassium (K Ca3.1) channel also colocalized to the endothelial projections, and blockage of this channel caused an 8-mV depolarization. Inhibition of Ca 2؉ pulsars also depolarized to a similar extent, and blocking K Ca3.1 channels was without effect in the absence of pulsars. Our results support a mechanism of IP 3 signaling in which Ca 2؉ release is spatially restricted to transmit intercellular signals.calcium ͉ endothelium ͉ calcium biosensor ͉ intermediate conductance Ca 2ϩ -sensitive potassium channel ͉ calcium pulsar

Different calcium signals in the endothelium and smooth muscle target different types of Ca2+-sensitive K+ channels to modulate vascular function. These differential calcium signals and targets represent multilayered opportunities for prevention and/or treatment of vascular dysfunctions.

Ca2+‐activated chloride currents (ICl(Ca)) were recorded from smooth muscle cells isolated from rabbit pulmonary (PA) and coronary artery (CA) as well as rabbit portal vein (PV). The characteristics and regulation by Ca2+‐calmodulin‐dependent kinase II (CaMKII) were compared between the three cell types. In PA and CA myocytes dialysed and superfused with K+‐free media, pipette solutions containing fixed levels of free Ca2+ in the range of 250 nm to 1 μm evoked well sustained, outwardly rectifying ICl(Ca) currents in about 90 % of cells. The CaMKII inhibitor KN‐93 (5 μm) increased the amplitude of ICl(Ca) in PA and CA myocytes. However, the threshold intracellular Ca2+ concentration for detecting this effect was different in the two arterial cell types. KN‐93 also enhanced the rate of activation of the time‐dependent current during depolarising steps, slowed the kinetics of the tail current following repolarisation, and induced a negative shift of the steady‐state activation curve. In PA myocytes, the effects of KN‐93 were not mirrored by its inactive analogue KN‐92 but were reproduced by the inclusion of autocamtide‐2‐related CaMKII inhibitory peptide (ARIP) in the pipette solution. Cell dialysis with constitutively active CaMKII (30 nm) significantly reduced ICl(Ca) evoked by 500 nm Ca2+. In PV myocytes, ICl(Ca) was evoked by pipette solutions containing up to 1 μm free Ca2+ in less than 40 % of cells. Application of KN‐93 to cells where ICl(Ca) was sustained produced a small inhibition (≈25 %) of the current in 70 % of the cells. The present study shows that regulation of Ca2+‐dependent Cl− channels by CaMKII differs between arterial and portal vein myocytes.

Background Fibroblast proliferation and differentiation are central in atrial fibrillation (AF)–promoting remodeling. Here, we investigated fibroblast regulation by Ca2+-permeable transient receptor potential canonical-3 (TRPC3) channels. Methods and Results Freshly isolated rat cardiac fibroblasts abundantly expressed TRPC3 and had appreciable nonselective cation currents (INSC) sensitive to a selective TPRC3 channel blocker, pyrazole-3 (3 μmol/L). Pyrazole-3 suppressed angiotensin II-induced Ca2+ influx, proliferation, and α-smooth muscle actin protein expression in fibroblasts. Ca2+ removal and TRPC3 blockade suppressed extracellular signal-regulated kinase phosphorylation, and extracellular signal-regulated kinase phosphorylation inhibition reduced fibroblast proliferation. TRPC3 expression was upregulated in atria from AF patients, goats with electrically maintained AF, and dogs with tachypacing-induced heart failure. TRPC3 knockdown (based on short hairpin RNA [shRNA]) decreased canine atrial fibroblast proliferation. In left atrial fibroblasts freshly isolated from dogs kept in AF for 1 week by atrial tachypacing, TRPC3 protein expression, currents, extracellular signal-regulated kinase phosphorylation, and extracellular matrix gene expression were all significantly increased. In cultured left atrial fibroblasts from AF dogs, proliferation rates, α-smooth muscle actin expression, and extracellular signal-regulated kinase phosphorylation were increased and were suppressed by pyrazole-3. MicroRNA-26 was downregulated in canine AF atria; experimental microRNA-26 knockdown reproduced AF-induced TRPC3 upregulation and fibroblast activation. MicroRNA-26 has NFAT (nuclear factor of activated T cells) binding sites in the 5′ promoter region. NFAT activation increased in AF fibroblasts, and NFAT negatively regulated microRNA-26 transcription. In vivo pyrazole-3 administration suppressed AF while decreasing fibroblast proliferation and extracellular matrix gene expression. Conclusions TRPC3 channels regulate cardiac fibroblast proliferation and differentiation, likely by controlling the Ca2+ influx that activates extracellular signal-regulated kinase signaling. AF increases TRPC3 channel expression by causing NFAT-mediated downregulation of microRNA-26 and causes TRPC3-dependent enhancement of fibroblast proliferation and differentiation. In vivo, TRPC3 blockade prevents AF substrate development in a dog model of electrically maintained AF. TRPC3 likely plays an important role in AF by promoting fibroblast pathophysiology and is a novel potential therapeutic target.

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Calcium-activated chloride channels (ClCa) are ligand-gated anion channels as they have been shown to be activated by a rise in intracellular Ca2+ concentration in various cell types including cardiac, skeletal and vascular smooth muscle cells, endothelial and epithelial cells, as well as neurons. Because ClCa channels are normally closed at resting, free intracellular Ca2+ concentration (approximately 100 nmol/L) in most cell types, they have generally been considered excitatory in nature, providing a triggering mechanism during signal transduction for membrane excitability, osmotic balance, transepithelial chloride movements, or fluid secretion. Unfortunately, the genes responsible for encoding this class of ion channels is still unknown. This review centers primarily on recent findings on the properties of these channels in smooth muscle cells. The first section discusses the functional significance and biophysical and pharmacological properties of ClCa channels in smooth muscle cells, and ends with a description of 2 candidate gene families (i.e., CLCA and Bestrophin) that are postulated to encode for these channels in various cell types. The second section provides a summary of recent findings demonstrating the regulation of native ClCa channels in vascular smooth muscle cells by calmodulin-dependent protein kinase II and calcineurin and how their fine tuning by these enzymes may influence vascular tone.

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