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.