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

Abstract-The endothelium is a critical regulator of vascular tone, and dysfunction of the endothelium contributes to numerous cardiovascular pathologies. Recent studies suggest that apamin-sensitive, small-conductance, Ca 2ϩ -activated K ϩ channels may play an important role in active endothelium-dependent vasodilations, and expression of these channels may be altered in disease states characterized by vascular dysfunction. Here, we used a transgenic mouse (SK3 T/T ) in which SK3 expression levels can be manipulated with dietary doxycycline (DOX) to test the hypothesis that the level of expression of the SK subunit, SK3, in endothelial cells alters arterial function and blood pressure. SK3 protein was elevated in small mesenteric arteries from SK3 T/T mice compared with wild-type mice and was greatly suppressed by dietary DOX. SK3 was detected in the endothelium and not in the smooth muscle by immunohistochemistry. In whole-cell patch-clamp experiments, SK currents in endothelial cells from SK3 T/T mice were almost completely suppressed by dietary DOX. In intact arteries, SK3 channels contributed to sustained hyperpolarization of the endothelial membrane potential, which was communicated to the arterial smooth muscle. Pressure-and phenylephrine-induced constrictions of SK3 T/T arteries were substantially enhanced by treatment with apamin, suppression of SK3 expression with DOX, or removal of the endothelium. In addition, suppression of SK3 expression caused a pronounced and reversible elevation of blood pressure. These results indicate that endothelial SK3 channels exert a profound, tonic, hyperpolarizing influence in resistance arteries and suggest that the level of SK3 channel expression in endothelial cells is a fundamental determinant of vascular tone and blood pressure. Key Words: endothelium Ⅲ potassium channels Ⅲ vascular tone Ⅲ blood pressure B lood pressure and flow are regulated by the constriction and dilation of resistance arteries, generally with internal diameters Ͻ300 m. 1 Physiological stimulation through elevations in intravascular pressure or increased sympathetic activity promotes smooth muscle depolarization, intracellular Ca 2ϩ influx, and vasoconstriction. The resulting increase in total peripheral resistance within the vasculature increases blood pressure. 2 The endothelium exerts a dilating influence that opposes arterial constriction. Activation of K ϩ channels is thought to contribute to this influence through increased release of relaxing factors such as NO and prostacyclin (PGI 2 ) and through smooth muscle hyperpolarization. [3][4][5] The smallconductance Ca 2ϩ -activated K ϩ (SK) channel has received considerable attention as a potential mediator of these responses. SK channels are opened by intracellular Ca 2ϩ via an association with calmodulin 6 and are believed to play a role in the modulation of tissue excitability. 7 Of the three characterized SK channel isoforms (SK1, SK2, and SK3), 8 mRNA for SK2 and SK3 has been identified in endothelial cells. 9 Apamin, a toxin blocker of S...

When arteries constrict to agonists, the endothelium inversely responds, attenuating the initial vasomotor response. The basis of this feedback mechanism remains uncertain, although past studies suggest a key role for myoendothelial communication in the signaling process. The present study examined whether second messenger flux through myoendothelial gap junctions initiates a negative-feedback response in hamster retractor muscle feed arteries. We specifically hypothesized that when agonists elicit depolarization and a rise in second messenger concentration, inositol trisphosphate (IP(3)) flux activates a discrete pool of IP(3) receptors (IP(3)Rs), elicits localized endothelial Ca(2+) transients, and activates downstream effectors to moderate constriction. With use of integrated experimental techniques, this study provided three sets of supporting observations. Beginning at the functional level, we showed that blocking intermediate-conductance Ca(2+)-activated K(+) channels (IK) and Ca(2+) mobilization from the endoplasmic reticulum (ER) enhanced the contractile/electrical responsiveness of feed arteries to phenylephrine. Next, structural analysis confirmed that endothelial projections make contact with the overlying smooth muscle. These projections retained membranous ER networks, and IP(3)Rs and IK channels localized in or near this structure. Finally, Ca(2+) imaging revealed that phenylephrine induced discrete endothelial Ca(2+) events through IP(3)R activation. These events were termed recruitable Ca(2+) wavelets on the basis of their spatiotemporal characteristics. From these findings, we conclude that IP(3) flux across myoendothelial gap junctions is sufficient to induce focal Ca(2+) release from IP(3)Rs and activate a discrete pool of IK channels within or near endothelial projections. The resulting hyperpolarization feeds back on smooth muscle to moderate agonist-induced depolarization and constriction.

Arrays of octameric peptide libraries on cellulose paper were screened by using 32 protein kinase inhibitor ͉ combinatorial libraries ͉ SPOT method ͉ membrane translocation signal ͉ smooth muscle T he cGMP-dependent protein kinases type I␣ and I␤ (cGPK) act directly downstream in the NO-mediated signaling pathway, controlling a variety of cellular responses, ranging from smooth muscle cell relaxation to neuronal synaptic plasticity (1, 2). The structural similarity of cGPK and its closest relative, the cAMP-dependent protein kinase (cAPK), has made it difficult to study cGPK pathways independent of those mediated by cAPK, primarily because of the lack of potent and selective cGPK inhibitors. Because recent studies have suggested that cAMP and cGMP are each able to cross-activate either cGPK or cAPK under physiological conditions, the specific role for cGPK within the NO͞cGMP-mediated signaling pathway remains obscure (for a review see ref. 1). However, recent advances have clearly identified specific intracellular targets for the cGPK isozymes I␣ and I␤ (3, 4). Also, inactivation of the genes for cGPK I␣͞I␤ and cGPK II showed that the cGPK isozymes regulate distinct cellular functions by pathways separate from those mediated by cAPK (5, 6).Attempts to identify cGPK-selective inhibitor peptides based on the autoinhibitory domain of the enzyme or in vivo substrates have been tedious at best, because of the lack of a well defined consensus sequence. Only a relative preference for basic residues surrounding the phosphate acceptor site has been established (7). Various synthetic peptides have been used to analyze the sequence requirements for cGPK substrates (8-11). Recently, we developed an iterative approach using phosphorylation of peptide libraries on cellulose paper to determine a priori the substrate specificity of cGPK versus cAPK. Consequently, we identified the cGPK substrate sequence TQAKRKKSLAM-FLR, in which the serine represents the phosphate-acceptor site (12, 13). Substitution of this serine by alanine yielded cGPK inhibitors with K i values of 7.5-22 M (13) and improved cGPK͞cAPK selectivity, as has been reported with other synthetic peptide derivatives (14,15). However, all cGPK peptide inhibitors known so far lack satisfactory potency and selectivity.Here we report a peptide library screen specifically designed to select for tight binding peptides rather than substrate peptides. First, we took advantage of the autophosphorylation properties of cGPK, which provides the means to study the transient enzyme-peptide interactions. Second, we used peptide libraries that lack the phosphate acceptor residues serine and threonine to select for peptide binding over phosphorylation. Linking the best sequence from this screen to membrane translocation signals (MTS) for intracellular delivery resulted in the highly effective cGPK I␣ inhibitors DT-2 and DT-3. Finally, we have demonstrated that both peptides are powerful tools for studying the specific functional roles of cGPK in smooth muscle. MethodsEnzyme Prepa...