Several recent studies have implicated the RhoA-Rho kinase pathway in arterial myogenic behavior. The goal of this study was to determine the effects of Rho kinase inhibition (Y-27632) on cerebral artery calcium and diameter responses as a function of transmural pressure. Excised segments of rat posterior cerebral arteries (100-200 microm) were cannulated and pressurized in an arteriograph at 37 degrees C. Increasing pressure from 10 to 60 mmHg triggered an elevation of cytosolic calcium concentration ([Ca(2+)](i)) from 113 +/- 9 to 199 +/- 12 nM and development of myogenic tone. Further elevation of pressure to 120 mmHg induced only a minor additional increase in [Ca(2+)](i) and constriction. Y-27632 (0.3-10 microM) inhibited myogenic tone in a concentration-dependent manner at 60 and 120 mmHg with comparable efficacy; conversely, sensitivity was decreased at 120 vs. 60 mmHg (50% inhibitory concentration: 2.5 +/- 0.3 vs. 1.4 +/- 0.1 microM; P < 0.05). Dilation was accompanied by further increases in [Ca(2+)](i) and an enhancement of Ca(2+) oscillatory activity. Y-27632 also effectively dilated the vessels permeabilized with alpha-toxin in a concentration-dependent manner. However, dilator effects of Y-27632 at low concentrations were larger at 60 vs. 100 mmHg. In summary, the results support a significant role for RhoA-Rho kinase pathway in cerebral artery mechanotransduction of pressure into sustained vasoconstriction (myogenic tone and reactivity) via mechanisms that augment smooth muscle calcium sensitivity. Potential downstream events may involve inhibition of myosin phosphatase and/or stimulation of actin polymerization, both of which are associated with increased smooth muscle force production.

STIM1 has been recently identified as a Ca 2؉ sensor in endoplasmic reticulum (ER) and an initiator of the store-operated Ca 2؉ entry (SOCE) pathway, but the mechanism of SOCE activation remains controversial. Here we focus on the early ERdelimited steps of the SOCE pathway and demonstrate that STIM1 is critically involved in initiating of production of calcium influx factor (CIF), a diffusible messenger that can deliver the signal from the stores to plasma membrane and activate SOCE. We discovered that CIF production is tightly coupled with STIM1 expression and requires functional integrity of its intraluminal sterile ␣-motif (SAM) domain. We demonstrate that 1) molecular knockdown or overexpression

Park KM, Trucillo M, Serban N, Cohen RA, Bolotina VM. Role of iPLA2 and store-operated channels in agonist-induced Ca 2ϩ influx and constriction in cerebral, mesenteric, and carotid arteries. Am J Physiol Heart Circ Physiol 294: H1183-H1187, 2008. First published December 21, 2007 doi:10.1152/ajpheart.01148.2007.-Store-operated channels (SOC) and store-operated Ca 2ϩ entry are known to play a major role in agonist-induced constriction of smooth muscle cells (SMC) in conduit vessels. In microvessels the role of SOC remains uncertain, in as much as voltage-gated L-type Ca 2ϩ (Ca L 2ϩ ) channels are thought to be fully responsible for agonist-induced Ca 2ϩ influx and vasoconstriction. We present evidence that SOC and their activation via a Ca 2ϩ -independent phospholipase A2 (iPLA2)-mediated pathway play a crucial role in agonist-induced constriction of cerebral, mesenteric, and carotid arteries. Intracellular Ca 2ϩ in SMC and intraluminal diameter were measured simultaneously in intact pressurized vessels in vitro. We demonstrated that 1) Ca 2ϩ and contractile responses to phenylephrine (PE) in cerebral and carotid arteries were equally abolished by nimodipine (a Ca L 2ϩ inhibitor) and 2-aminoethyl diphenylborinate (an inhibitor of SOC), suggesting that SOC and Ca L 2ϩ channels may be involved in agonist-induced constriction of cerebral arteries, and 2) functional inhibition of iPLA2␤ totally inhibited PE-induced Ca 2ϩ influx and constriction in cerebral, mesenteric, and carotid arteries, whereas K ϩ -induced Ca 2ϩ influx and vasoconstriction mediated by Ca L 2ϩ channels were not affected. Thus iPLA2-dependent activation of SOC is crucial for agonistinduced Ca 2ϩ influx and vasoconstriction in cerebral, mesenteric, and carotid arteries. We propose that, on PE-induced depletion of Ca 2ϩ stores, nonselective SOC are activated via an iPLA2-dependent pathway and may produce a depolarization of SMC, which could trigger a secondary activation of Ca L 2ϩ channels and lead to Ca 2ϩ entry and vasoconstriction.