Pressor effects of the vasoconstrictor hormone arginine vasopressin (AVP), observed when systemic AVP concentrations are less than 100 pM, are important for the physiological maintenance of blood pressure, and they are also the basis for therapeutic use of vasopressin to restore blood pressure in hypotensive patients. However, the mechanisms by which circulating AVP induces arterial constriction are unclear. We examined the novel hypothesis that KCNQ potassium channels mediate the physiological vasoconstrictor actions of AVP. Reverse transcriptase polymerase chain reaction revealed expression of KCNQ1, KCNQ4, and KCNQ5 in rat mesenteric artery smooth muscle cells (MASMCs). Whole-cell perforated patch recordings of voltage-sensitive K ϩ (K v ) currents in freshly isolated MASMCs revealed 1,3-dihydro-1-phenyl-3,3-bis(4-pyridinylmethyl)-2H-indol-2-one (linopirdine)-and 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone (XE-991)-sensitive KCNQ currents that were electrophysiologically and pharmacologically distinct from other K v currents. Suppression of KCNQ currents by AVP (100 pM) was associated with significant membrane depolarization, and it was abolished by the protein kinase C (PKC) inhibitor calphostin C (250 nM). The KCNQ channel blocker linopirdine (10 M) inhibited KCNQ currents in MASMCs, and it induced constriction of isolated rat mesenteric arteries. The vasoconstrictor responses were not additive when combined with 30 pM AVP, and they were prevented by the L-type Ca 2ϩ channel blocker verapamil. Ethyl-N-[2-amino-6-(4-fluorophenylmethylamino)pyridin-3-yl] carbamic acid (flupirtine) significantly enhanced KCNQ currents, and it reversed constrictor responses to 30 pM AVP. In vivo, i.v. administration of linopirdine induced a dose-dependent increase in mesenteric artery resistance and blood pressure, whereas flupirtine had the opposite effects. We conclude that physiological concentrations of AVP induce mesenteric artery constriction via PKCdependent suppression of KCNQ currents and L-type Ca 2ϩ channel activation in MASMCs.Membrane voltage (V m ) determines the open probability of L-type Ca 2ϩ channels in vascular smooth muscle cells (VSMCs), and K ϩ channels represent a primary effector for adjusting V m . To the extent that Kϩ channels are open in resting VSMCs, the outward flux of K ϩ through these channels (measured as K ϩ current) will tend to stabilize the resting V m at negative (hyperpolarized) voltages and prevent opening of voltage-sensitive Ca 2ϩ channels. In contrast, reduction of outward K ϩ currents in VSMCs results in a shift to more positive V m (membrane depolarization) leading to activation of L-type Ca 2ϩ channels and entry of Ca 2ϩ into the cell. Elevation of the cytosolic Ca 2ϩ concentration in this manner can trigger VSMC contraction and vasoconstriction.KCNQ channels (Kv7 family) are voltage-sensitiveThis work was supported by the National Heart Lung and Blood Institute Grant R01 HL070670 (to K.L.B.) and the American Heart Association Grant 0715618Z (to A.R.M.).The chemical st...

Abstract-Reperfusion of cardiac tissue after an ischemic episode is associated with metabolic and contractile dysfunction, including reduced tension development and activation of the Na ϩ -H ϩ exchanger (NHE). Oxygen-derived free radicals are key mediators of reperfusion abnormalities, although the cellular mechanisms involved have not been fully defined. In the present study, the effects of free radicals on mitogen-activated protein (MAP) kinase function were investigated using cultured neonatal rat ventricular myocytes. Acute exposure of spontaneously beating myocytes to 50 mol/L hydrogen peroxide (H 2 O 2 ) caused a sustained decrease in contraction amplitude (80% of control). MAP kinase activity was measured by in-gel kinase assays and Western blot analysis. Acute exposure to H 2 O 2 (100 mol/L, 5 minutes) resulted in sustained MAP kinase activation that persisted for 60 minutes. Catalase, but not superoxide dismutase, completely inhibited MAP kinase activation by H 2 O 2 . Pretreatment with chelerythrine (10 mol/L, 45 minutes), a protein kinase C inhibitor, or genistein (75 mol/L, 45 minutes) or herbimycin A (3 mol/L, 45 minutes), tyrosine kinase inhibitors, caused significant inhibition of H 2 O 2 -stimulated MAP kinase activity (51%, 78%, and 45%, respectively, at 20 minutes). Brief exposure to H 2 O 2 also stimulated NHE activity. This effect was completely abolished by pretreatment with the MAP kinase kinase inhibitor PD 98059 (30 mol/L, 60 minutes). These results suggest that low doses of H 2 O 2 induce MAP kinase-dependent pathways that regulate NHE activity during reperfusion injury.

Recent evidence suggests that chemokine (C-X-C motif) receptor 4 (CXCR4) contributes to the regulation of blood pressure through interactions with α 1 -adrenergic receptors (ARs) in vascular smooth muscle. The underlying molecular mechanisms, however, are unknown. Using proximity ligation assays to visualize single-molecule interactions, we detected that α 1A/B -ARs associate with CXCR4 on the cell surface of rat and human vascular smooth muscle cells (VSMC). Furthermore, α 1A/B -AR could be coimmunoprecipitated with CXCR4 in a HeLa expression system and in human VSMC. A peptide derived from the second transmembrane helix of CXCR4 induced chemical shift changes in the NMR spectrum of CXCR4 in membranes, disturbed the association between α 1A/B -AR and CXCR4, and inhibited Ca 2+ mobilization, myosin light chain (MLC) 2 phosphorylation, and contraction of VSMC upon α 1 -AR activation. CXCR4 silencing reduced α 1A/B -AR:CXCR4 heteromeric complexes in VSMC and abolished phenylephrine-induced Ca 2+ fluxes and MLC2 phosphorylation. Treatment of rats with CXCR4 agonists (CXCL12, ubiquitin) reduced the EC 50 of the phenylephrineinduced blood pressure response three-to fourfold. These observations suggest that disruption of the quaternary structure of α 1A/B -AR:CXCR4 heteromeric complexes by targeting transmembrane helix 2 of CXCR4 and depletion of the heteromeric receptor complexes by CXCR4 knockdown inhibit α 1 -AR-mediated function in VSMC and that activation of CXCR4 enhances the potency of α 1 -AR agonists. Our findings extend the current understanding of the molecular mechanisms regulating α 1 -AR and provide an example of the importance of G protein-coupled receptor (GPCR) heteromerization for GPCR function. Compounds targeting the α 1A/B -AR:CXCR4 interaction could provide an alternative pharmacological approach to modulate blood pressure.CXCL12 | ubiquitin | AMD3100 | phenylephrine | blood pressure

[Arg 8 ]-vasopressin (AVP), at low concentrations (10-500 pM), stimulates oscillations in intracellular Ca 2+ concentration (Ca 2+ spikes) in A7r5 rat aortic smooth muscle cells. Our previous studies provided biochemical evidence that protein kinase C (PKC) activation and phosphorylation of voltage-sensitive K + (K v ) channels are crucial steps in this process. In the present study, K v currents (I Kv ) and membrane potential were measured using patch clamp techniques. Treatment of A7r5 cells with 100 pM AVP resulted in significant inhibition of I Kv . This effect was associated with gradual membrane depolarization, increased membrane resistance, and action potential (AP) generation in the same cells. The AVP-sensitive I Kv was resistant to 4-aminopyridine, iberiotoxin, and glibenclamide but was fully inhibited by the selective KCNQ channel blockers linopirdine (10 μM) and XE-991 (10 μM) and enhanced by the KCNQ channel activator flupirtine (10 μM). BaCl 2 (100 μM) or linopirdine (5 μM) mimicked the effects of AVP on K + currents, AP generation, and Ca 2+ spiking. Expression of KCNQ5 was detected by RT-PCR in A7r5 cells and freshly isolated rat aortic smooth muscle. RNA interference directed toward KCNQ5 reduced KCNQ5 protein expression and resulted in a significant decrease in I Kv in A7r5 cells. I Kv was also inhibited in response to the PKC activator 4β-phorbol 12-myristate 13-acetate (10 nM), and the inhibition of I Kv by AVP was prevented by the PKC inhibitor calphostin C (250 nM). These results suggest that the stimulation of Ca 2+ spiking by physiological concentrations of AVP involves PKC-dependent inhibition of KCNQ5 channels and increased AP firing in A7r5 cells.Keywords potassium channel; signal transduction; membrane potential; calcium; vascular smooth muscle; M current Vasoconstrictor hormones cause contraction of vascular smooth muscle (VSM) cells by increasing cytosolic free Ca 2+ concentration ([Ca 2+ ] i ), which in turn activates the cells' contractile apparatus. Voltage-sensitive L-type Ca 2+ channels are known to be important in vasoconstrictor action (27), although the signaling pathways leading to activation of L-type channels are not well characterized. Influx of Ca 2+ via L-type channels is enhanced by NIH Public Access Author ManuscriptAm J Physiol Heart Circ Physiol. Author manuscript; available in PMC 2008 November 3. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript membrane depolarization, which may result from activation of nonselective cation currents (34,47,54) or Cl − currents (30). Alternatively, inhibition of outward K + currents could provide a depolarizing stimulus for activation of L-type Ca 2+ channels (38).We have previously demonstrated that concentrations of [Arg 8 ]-vasopressin (AVP) that may be found in the systemic circulation (10-500 pM) modulate the frequency of L-type Ca 2+ channel-dependent Ca 2+ spikes in A7r5 rat aortic smooth muscle cells. The stimulation of Ca 2+ -dependent action potentials, which underlie AVP-stimulated...

Angiotensin II (Ang II) induces vascular smooth muscle cell (VSMC) growth by activating Gq-protein-coupled AT1 receptors, which leads to elevation of cytosolic Ca2+ ([Ca2+]i) and activation of protein kinase C (PKC) and mitogen-activated protein kinases. To assess the link between these Ang II-induced signaling events, we examined the effect of Ang II on the proline-rich tyrosine kinase (PYK2), previously found to be activated by a variety of stimuli that increase [Ca2+]i or activate PKC. PYK2 distribution was demonstrated in rat aortic tissue and in cultured VSMC by immunohistochemistry, revealing a cytosolic distribution distinct from smooth muscle alpha-actin, focal adhesion kinase, or paxillin. The involvement of PYK2 in Ang II signaling was measured by immunoprecipitation and immune complex kinase assays. Treatment of quiescent VSMC with Ang II resulted in a concentration- and time-dependent increase in PYK2 tyrosine phosphorylation and kinase activity in PYK2 immunoprecipitates. PYK2 phosphorylation was inhibited by AT1 receptor blockade and was attenuated by downregulation of PKC or the chelation of [Ca2+]i. Treatment with either phorbol ester or Ca2+ ionophore also increased PYK2 phosphorylation, suggesting that PKC activation and/or increased [Ca2+]i are both necessary and sufficient to activate PYK2. Activation of PYK2 by Ang II was also associated with increased PYK2-src complex formation, suggesting that PYK2 activation represents a potential link between Ang II-stimulated [Ca2+]i and PKC activation with downstream signaling events such as mitogen-activated protein kinase activation involved in the regulation of VSMC growth.

1. Arg8-vasopressin (AVP)-regulated Ca2' transport pathways were investigated in fura-2-loaded A7r5 cells using both single cell and population measurements. 2. AVP evokes an initial concentration-dependent rise in cytosolic free Ca2+ concentration ([Ca2+]

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Celecoxib, rofecoxib, and diclofenac are clinically used cyclooxygenase-2 (COX-2) inhibitors, which have been under intense scrutiny because long-term rofecoxib (Vioxx; Merck, Whitehouse Station, NJ) treatment was found to increase the risk of adverse cardiovascular events. A differential risk profile for these drugs has emerged, but the underlying mechanisms have not been fully elucidated. We investigated the effects of celecoxib, rofecoxib, and diclofenac on ionic currents and calcium signaling in vascular smooth muscle cells (VSMCs) using patch-clamp techniques and fura-2 fluorescence and on arterial constriction using pressure myography. Celecoxib, but not rofecoxib or diclofenac, dramatically enhanced KCNQ (K v 7) potassium currents and suppressed L-type voltage-sensitive calcium currents in A7r5 rat aortic smooth muscle cells (native KCNQ currents or overexpressed human KCNQ5 currents) and freshly isolated rat mesenteric artery myocytes. The effects of celecoxib were concentration-dependent within the therapeutic concentration range, and were reversed on washout. Celecoxib, but not rofecoxib, also inhibited calcium responses to vasopressin in A7r5 cells and dilated intact or endotheliumdenuded rat mesenteric arteries. A celecoxib analog, 2,5-dimethyl-celecoxib, which does not inhibit COX-2, mimicked celecoxib in its enhancement of vascular KCNQ5 currents, suppression of L-type calcium currents, and vasodilation. We conclude that celecoxib inhibits calcium responses in VSMCs by enhancing KCNQ5 currents and suppressing L-type calcium currents, which ultimately reduces vascular tone. These effects are independent of its COX-2 inhibitory actions and may explain the differential risk of cardiovascular events in patients taking different drugs of this class.Celecoxib (Celebrex; Pfizer, New York, NY) and rofecoxib (Vioxx; Merck, Whitehouse Station, NJ) are nonsteroidal anti-inflammatory drugs (NSAIDs) that selectively inhibit cyclooxygenase-2 (COX-2). They were introduced to the market in 1999 and rapidly became the most frequently prescribed new drugs in the United States. These drugs are used clinically to treat pain and inflammation. COX-1 and COX-2 convert arachidonic acid into prostaglandin H 2 , which is further converted to a variety of prostanoids, including prostaglandins, thromboxanes, and prostacyclins. Thromboxane A 2 , a product of COX-1 activity in platelets, promotes vasoconstriction, smooth muscle proliferation, and platelet aggregation. In contrast, prostacyclin generated by COX-2 in the blood vessel walls promotes vasodilatation and inhibition of platelet aggregation. As analgesic/anti-inflammatory agents, COX-2 inhibitors were considered to be an improvement over less selective COX-1/COX-2 inhibitors because they prevent the generation of prostaglandins involved in inflammation and pain while sparing some beneficial effects of COX-1-generated prostanoids. However, these drugs have been under intense scrutiny since 2004, when Vioxx was voluntarily withdrawn from the market because of a r...