Abstract-Vasoconstrictors that bind to phospholipase C-coupled receptors elevate inositol-1,4,5-trisphosphate (IP 3 The mechanism by which IP 3 regulates arterial contractility is generally well accepted. 1 Indeed, IP 3 -induced SR Ca 2ϩ release is considered to be the only mechanism by which this second messenger regulates arterial diameter. However, the physiological mechanisms by which IP 3 regulates intracellular Ca 2ϩ signaling and arterial diameter are poorly understood, and few studies have directly tested the accepted view. Arterial contractility regulation by IP 3 has primarily been studied by using vasoconstrictors that activate PLC. Because PLC activation elevates both DAG and IP 3 and reduces PIP 2 , mechanisms by which IP 3 specifically modulates arterial [Ca 2ϩ ] i signaling and diameter require additional study. Here, we investigated IP 3 regulation of ion channel activity, intracellular Ca 2ϩ signaling, and contractility in cerebral artery myocytes and pressurized arteries. We show that IP 3 activates a nonselective cation current (I Cat ) in myocytes and induces vasoconstriction via a mechanism that does not require the release of SR Ca 2ϩ but involves IP 3 R and TRPC3 (canonical transient receptor potential 3) channel activation. IP 3 -induced Na ϩ influx produces membrane depolarization, voltage-dependent Ca 2ϩ channel activation, an [Ca 2ϩ ] i elevation, and vasoconstriction. We also show that TRPC3 channel Original

Thomas-Gatewood C, Neeb ZP, Bulley S, Adebiyi A, Bannister JP, Leo MD, Jaggar JH. TMEM16A channels generate Ca 2ϩ -activated Cl Ϫ currents in cerebral artery smooth muscle cells.

Rationale Voltage-dependent L-type (CaV1.2) Ca2+ channels are a heteromeric complex formed from pore forming α1 and auxiliary α2δ and β subunits. CaV1.2 channels are the principal Ca2+ influx pathway in arterial myocytes and regulate multiple physiological functions, including contraction. The macromolecular composition of arterial myocyte CaV1.2 channels remains poorly understood, with no studies having examined the molecular identity or physiological functions of α2δ subunits. Objective Investigate the functional significance of α2δ subunits in myocytes of resistance-size (100–200 μm diameter) cerebral arteries. Methods and Results α2δ-1 was the only α2δ isoform expressed in cerebral artery myocytes. Pregabalin, an α2δ-1/-2 ligand, and an α2δ-1 antibody, inhibited CaV1.2 currents in isolated myocytes. Acute pregabalin application reversibly dilated pressurized arteries. Using a novel application of surface biotinylation, data indicated that >95 % of CaV1.2 α1 and α2δ-1 subunits are present in the arterial myocyte plasma membrane. α2δ-1 knockdown using shRNA reduced plasma membrane-localized CaV1.2 α1 subunits, caused a corresponding elevation in cytosolic CaV1.2 α1 subunits, decreased intracellular Ca2+ concentration, inhibited pressure-induced vasoconstriction (“myogenic tone”), and attenuated pregabalin-induced vasodilation. Prolonged (24 hour) pregabalin exposure did not alter total α2δ-1 or CaV1.2 α1 proteins, but decreased plasma membrane expression of each subunit, which reduced myogenic tone. Conclusions α2δ-1 is essential for plasma membrane expression of arterial myocyte CaV1.2 α1 subunits. α2δ-1 targeting can block CaV1.2 channels directly and inhibit surface expression of CaV1.2 α1 subunits, leading to vasodilation. These data identify α2δ-1 as a novel molecular target in arterial myocytes, manipulation of which regulates contractility.

,4,5-trisphosphate receptors (IP3Rs) regulate diverse physiological functions, including contraction and proliferation. There are three IP3R isoforms, but their functional significance in arterial smooth muscle cells is unclear. Here, we investigated relative expression and physiological functions of IP3R isoforms in cerebral artery smooth muscle cells. We show that 2-aminoethoxydiphenyl borate and xestospongin C, membrane-permeant IP3R blockers, reduced Ca 2ϩ wave activation and global intracellular Ca 2ϩ ([Ca 2ϩ ]i) elevation stimulated by UTP, a phospholipase C-coupled purinergic receptor agonist. Quantitative PCR, Western blotting, and immunofluorescence indicated that all three IP3R isoforms were expressed in acutely isolated cerebral artery smooth muscle cells, with IP3R1 being the most abundant isoform at 82% of total IP3R message. IP3R1 knockdown with short hairpin RNA (shRNA) did not alter baseline Ca 2ϩ wave frequency and global [Ca 2ϩ ]i but abolished UTP-induced Ca 2ϩ wave activation and reduced the UTP-induced global [Ca 2ϩ ]i elevation by ϳ61%. Antibodies targeting IP3R1 and IP3R1 knockdown reduced UTP-induced nonselective cation current (Icat) activation. IP3R1 knockdown also reduced UTP-induced vasoconstriction in pressurized arteries with both intact and depleted sarcoplasmic reticulum (SR) Ca 2ϩ by ϳ45%. These data indicate that IP 3R1 is the predominant IP3R isoform expressed in rat cerebral artery smooth muscle cells. IP 3R1 stimulation contributes to UTP-induced I cat activation, Ca 2ϩ wave generation, global [Ca 2ϩ ]i elevation, and vasoconstriction. In addition, IP 3R1 activation constricts cerebral arteries in the absence of SR Ca 2ϩ release by stimulating plasma membrane Icat.cerebral artery smooth muscle cells; calcium wave; short hairpin RNA INOSITOL 1,4,5-trisphosphate (IP 3 ) receptors (IP 3 Rs) are expressed in many cell types and regulate several physiological functions, including development, muscle contraction, cell proliferation, and differentiation (1,45,47,56 Rs (7,43,54). IP 3 also activates cation channels in vascular smooth muscle cells through mechanisms that do not require the stimulation of sarcoplasmic reticulum (SR) Ca 2ϩ release (27, 54). In cerebral artery smooth muscle cells, IP 3 R activation stimulates TRPC3 channels, leading to Na ϩ influx, membrane depolarization, voltage-dependent Ca 2ϩ channel activation, and vasoconstriction (54). Thus IP 3 Rs regulate ion channel activity, Ca 2ϩ signals, and physiological functions in the vasculature. However, despite the functional importance of IP 3 Rs in arterial smooth muscle cells, specific IP 3 R isoforms that are expressed in this cell type and their functional significance are poorly understood.Three IP 3 R isoforms, designated IP 3 R1, IP 3 R2, and IP 3 R3, are encoded by distinct genes (6, 37). IP 3 R isoform expression varies widely between different cell types. Cerebellar Purkinje neurons express predominantly IP 3 R1, pancreatic ␤-cells primarily express IP 3 R3, and cardiac myocytes express IP 3 R2 (14, 48...

Hydrogen sulfide (H(2)S) is a gaseous signaling molecule that appears to contribute to the regulation of vascular tone and blood pressure. Multiple potential mechanisms of vascular regulation by H(2)S exist. Here, we tested the hypothesis that piglet cerebral arteriole smooth muscle cells generate ATP-sensitive K(+) (K(ATP)) currents and that H(2)S induces vasodilation by activating K(ATP) currents. Gas chromatography/mass spectrometry data demonstrated that after placing Na(2)S, an H(2)S donor, in solution, it rapidly (1 min) converts to H(2)S. Patch-clamp electrophysiology indicated that pinacidil (a K(ATP) channel activator), Na(2)S, and NaHS (another H(2)S donor) activated K(+) currents at physiological steady-state voltage (-50 mV) in isolated cerebral arteriole smooth muscle cells. Glibenclamide, a selective K(ATP) channel inhibitor, fully reversed pinacidil-induced K(+) currents and partially reversed (∼58%) H(2)S-induced K(+) currents. Western blot analysis indicated that piglet arterioles expressed inwardly rectifying K(+) 6.1 (K(ir)6.1) channel and sulfonylurea receptor 2B (SUR2B) K(ATP) channel subunits. Pinacidil dilated pressurized (40 mmHg) piglet arterioles, and glibenclamide fully reversed this effect. Na(2)S also induced reversible and repeatable vasodilation with an EC(50) of ∼30 μM, and this effect was partially reversed (∼55%) by glibenclamide. Vasoregulation by H(2)S was also studied in pressurized resistance-size cerebral arteries of mice with a genetic deletion in the gene encoding SUR2 (SUR2 null). Pinacidil- and H(2)S-induced vasodilations were smaller in arterioles of SUR2 null mice than in wild-type controls. These data indicate that smooth muscle cell K(ATP) currents control newborn cerebral arteriole contractility and that H(2)S dilates cerebral arterioles by activating smooth muscle cell K(ATP) channels containing SUR2 subunits.

A ctivation of plasma membrane phospholipase (PL)Ccoupled receptors by vasoconstrictor agonists leads to phosphatidylinositol 4,5-bisphosphate (PIP 2 ) hydrolysis and the generation of inositol-1,4,5-trisphosphate (IP 3 ) and diacylglycerol. 1 In vascular myocytes, diacylglycerol (DAG) activates protein kinase (PK)C, leading to the phosphorylation of a wide variety of proteins, including ion channels. 2 IP 3 binds to sarcoplasmic reticulum (SR) IP 3 receptors (IP 3 Rs), resulting in SR Ca 2ϩ release, an elevation in intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ), and vasoconstriction. 3 Recent evidence also indicates that IP 3 -induced vasoconstriction can occur independently of SR Ca 2ϩ release and via the activation of type 1 IP 3 receptors (IP 3 R1) and type 3 canonical transient receptor potential (TRPC) channels. 4,5 However, the functional signaling mechanisms by which IP 3 Rs and TRPC channels communicate in arterial myocytes are unclear.The mammalian TRP channel superfamily is encoded by at least 28 different genes that are subdivided into 7 families. 6 These families encode ion channels with diverse ion selectivity, modes of regulation, and physiological functions. 6 Vascular myocytes express at least 4 TRP families, including TRPC, TRPM, TRPV, and TRPP. [7][8][9][10] These channels regulate arterial myocyte membrane potential, [Ca 2ϩ ] i , contractility, and proliferation, and are implicated in the etiology of vascular diseases. 4,8 -12 Given the diversity of vascular myocyte TRP channels, it has become important to identify signaling pathways that Original received July 15, 2009; resubmission received January 8, 2010; revision received March 22, 2010; accepted March 29, 2010. From the Department of Physiology, University of Tennessee Health Science Center, Memphis. Correspondence to Jonathan H. Jaggar, Department of Physiology, University of Tennessee Health Science Center, 894 Union Ave, Nash Building, Memphis, TN 38139. E-mail jjaggar@uthsc.edu © 2010 American Heart Association, Inc. Thus, TRPC3 and TRPC6 channels perform distinct physiological functions, but signaling pathways that mediate this differential regulation are unclear.Here, we studied mechanisms by which IP 3 R1, the principal molecular and functional arterial myocyte IP 3 R isoform, 5 stimulates TRPC currents in resistance-size cerebral arteries. Data suggest that IP 3 R1 is in close spatial proximity to, and associates with, TRPC3, but not TRPC6 or TRPM4 channels. Endothelin (ET)-1, a PLC-coupled receptor agonist, and IP 3 alter the interaction between the IP 3 R N terminus and the TRPC3 channel C terminus, leading to channel activation and vasoconstriction. Data indicate that IP 3 R1 selectively couples to TRPC3 channels because of the close spatial proximity of these proteins and that this mechanism is essential for mediating ET-1 and IP 3 -induced vasoconstriction. Methods Tissue PreparationAnimal protocols used were reviewed and approved by the Animal Care and Use Committee at the University of Tennessee Health Science ...

Physical coupling of sarcoplasmic reticulum (SR) type 1 inositol 1,4,5-trisphosphate receptors (IP 3 R1) to plasma membrane canonical transient receptor potential 3 (TRPC3) channels activates a cation current (I Cat ) in arterial smooth muscle cells that induces vasoconstriction. However, structural components that enable IP 3 R1 and TRPC3 channels to communicate locally are unclear. Caveolae are plasma membrane microdomains that can compartmentalize proteins. Here, we tested the hypothesis that caveolae and specifically caveolin-1 (cav-1), a caveolae scaffolding protein, facilitate functional IP 3 R1 to TRPC3 coupling in smooth muscle cells of resistancesize cerebral arteries. Methyl-␤-cyclodextrin (M␤CD), which disassembles caveolae, reduced IP 3 -induced I Cat activation in smooth muscle cells and vasoconstriction in pressurized arteries. Cholesterol replenishment reversed these effects. Cav-1 knockdown using shRNA attenuated IP 3 -induced vasoconstriction, but did not alter TRPC3 and IP 3 R1 expression. A synthetic peptide corresponding to the cav-1 scaffolding domain (CSD) sequence (amino acids 82-101) also attenuated IP 3 -induced I Cat activation and vasoconstriction. A cav-1 antibody co-immunoprecipitated cav-1, TRPC3, and IP 3 R1 from cerebral artery lysate. ImmunoFRET indicated that cav-1, TRPC3 channels and IP 3 R1 are spatially co-localized in arterial smooth muscle cells. IP 3 R1 and TRPC3 channel spatial localization was disrupted by M␤CD and a CSD peptide. Cholesterol replenishment re-established IP 3 R1 and TRPC3 channel close spatial proximity. Taken together, these data indicate that in arterial smooth muscle cells, cav-1 co-localizes SR IP 3 R1 and plasma membrane TRPC3 channels in close spatial proximity thereby enabling IP 3 -induced physical coupling of these proteins, leading to I Cat generation and vasoconstriction.Several G-protein-coupled receptor agonists activate phospholipase C leading to generation of the second messenger, inositol 1,4,5-trisphosphate (IP 3 ) 2 (1). IP 3 binds to endo/sarcoplasmic reticulum-localized IP 3 receptors leading to Ca 2ϩ release from the endo/sarcoplasmic reticulum (2). In arterial smooth muscle cells, the resultant increase in intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) causes vasoconstriction. IP 3 also activates a non-selective cation current (I Cat ), leading to vasoconstriction (3-7). IP 3 -induced I Cat activation occurs independently of sarcoplasmic reticulum (SR) Ca 2ϩ release and due to a physical interaction between type 1 IP 3 receptors (IP 3 R1) and canonical transient receptor potential (TRPC) 3 channels (4 -6). IP 3 R1 selectively couples to TRPC3 channels due to close spatial proximity of these proteins (4). A wide variety of signal transduction pathways require interacting molecules to form functional macromolecular complexes. These complexes often contain one or more scaffolding proteins that traffic, localize, or compartmentalize signaling molecules (8). However, whether scaffolding proteins are required to assemble IP 3 R1 and TRPC...

Plasma membrane large-conductance Ca2+-activated K+ (BKCa) channels and sarcoplasmic reticulum inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are expressed in a wide variety of cell types, including arterial smooth muscle cells. Here, we studied BKCa channel regulation by IP3 and IP3Rs in rat and mouse cerebral artery smooth muscle cells. IP3 activated BKCa channels both in intact cells and in excised inside-out membrane patches. IP3 caused concentration-dependent BKCa channel activation with an apparent dissociation constant (Kd) of ∼4 µM at physiological voltage (−40 mV) and intracellular Ca2+ concentration ([Ca2+]i; 10 µM). IP3 also caused a leftward-shift in BKCa channel apparent Ca2+ sensitivity and reduced the Kd for free [Ca2+]i from ∼20 to 12 µM, but did not alter the slope or maximal Po. BAPTA, a fast Ca2+ buffer, or an elevation in extracellular Ca2+ concentration did not alter IP3-induced BKCa channel activation. Heparin, an IP3R inhibitor, and a monoclonal type 1 IP3R (IP3R1) antibody blocked IP3-induced BKCa channel activation. Adenophostin A, an IP3R agonist, also activated BKCa channels. IP3 activated BKCa channels in inside-out patches from wild-type (IP3R1+/+) mouse arterial smooth muscle cells, but had no effect on BKCa channels of IP3R1-deficient (IP3R1−/−) mice. Immunofluorescence resonance energy transfer microscopy indicated that IP3R1 is located in close spatial proximity to BKCa α subunits. The IP3R1 monoclonal antibody coimmunoprecipitated IP3R1 and BKCa channel α and β1 subunits from cerebral arteries. In summary, data indicate that IP3R1 activation elevates BKCa channel apparent Ca2+ sensitivity through local molecular coupling in arterial smooth muscle cells.