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Acute hypoxia dilates most systemic arteries leading to increased tissue perfusion. We have previously shown that at high-stimulus conditions, porcine coronary artery was relaxed by hypoxia without a change in intracellular [[Ca.sup.2+]] (27). This [Ca.sup.2+]-desensitizing hypoxic relaxation (CDHR) was validated in permeabilized porcine coronary artery smooth muscle (PCASM) in which hypoxia decreased force and myosin regulatory light chain phosphorylation (p-MRLC) despite fixed [[Ca.sup.2+]] (10). Rho kinase-dependent phosphorylation of myosin phosphatase-targeting subunit 1 (p-MYPT1) is associated with decreased MRLC phosphatase activity and increased [Ca.sup.2+] sensitivity of both p-MRLC and force. We recently reported that p-MYPT1 dephosphorylation was a key effector in CDHR (33). in the current study, we tested the hypothesis that Rho kinase and not p-MYPT1 phosphatase is the regulated enzyme involved in CDHR. We used [alpha]-toxin to permeabilize deendothelialized PCASM. CDHR was attenuated in contractions attributable to myosin light chain kinase (MLCK, in the presence of the Rho kinase inhibitor Y-27632). In contrast, hypoxia relaxed contractions attributable to Rho kinase phosphorylation of MYPT1 and MRLC or MRLC alone (in the presence of the MLCK inhibitor ML7). Using an in situ assay, we showed that Rho kinase activity, measured as thiophosphorylation of MYPTI and MRLC, was nearly abolished by hypoxia. The in vitro activity of the catalytically active fragment of Rho kinase was not affected by hypoxia. Our evidence strongly implicates that hypoxia directly inhibits Rho kinase-dependent phosphorylation of MYPT1. This underlies the decreases in both p-MYPT1 and p-MRLC and thereby leads to the [Ca.sup.2+]-desensitizing hypoxic relaxation. smooth muscle; oxygen sensing; RhoA; contractility doi:10.1152/ajplung.01158.2006

We previously showed that plasma membrane [Ca.sup.2+]-ATPase (PMCA) activity accounted for 25-30% of relaxation in bladder smooth muscle (8). Among the four PMCA isoforms only PMCA1 and PMCA4 are expressed in smooth muscle. To address the role of these isoforms, we measured cytosolic [Ca.sup.2+] ([[[Ca.sup.2+]].sub.i]) using fura-PE3 and simultaneously measured contractility in bladder smooth muscle from wild-type (WT), Pmca[1.sup.+/-], Pmca[4.sup.+/-], Pmca[4.sup.-/-], and Pmca[1.sup.-/-] Pmca[4.sup.-/-] mice. There were no differences in basal [[[Ca.sup.2+]].sub.1] values between bladder preparations. KC1 (80 mM) elicited both larger forces (150-190%) and increases in [[[Ca.sup.2+]].sub.i] (130-180%) in smooth muscle from Pmca[1.sup.+/-] and Pmca[1.sup.+/-] Pmca[4.sup.-/-] bladders than those in WT or Pmca[4.sup.-/-]. The responses to carbachol (CCh: 10 [micro]M) were also greater in Pmca[1.sup.+/-] (120-150%) than in WT bladders. In contrast, the responses in Pmca[4.sup.-/-] and Pmca[1.sup.+/-] Pmca[4.sup.-/-] bladders to CCh were significantly smaller (40-50%) than WT. The rise in half-times of force and [[[Ca.sup.2+]].sub.i] increases in response to KCl and CCh, and the concomitant half-times of their decrease upon washout of agonist were prolonged in Pmca[4.sup.-/-] (130-190%) and Pmca[1.sup.+/-] Pmca[4.sup.-/-] (120-250%) bladders, but not in Pmca[1.sup.+/-] bladders with respect to WT. Our evidence indicates distinct isoform functions with the PMCA1 isoform involved in overall [Ca.sup.2+] clearance, while PMCA4 is essential for the [[[Ca.sup.2+]].sub.i] increase and contractile response to the CCh receptor-mediated signal transduction pathway. PMCA; bladder smooth muscle; gene-altered mice

We investigated the roles and relationships of plasma membrane [Ca.sup.2+]-ATPase (PMCA), sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase (SERCA)2, and [Na.sup.+]/[Ca.sup.2+] exchanger (NCX) in bladder smooth muscle contractility in Pmca-ablated mice: Pmca4-null mutant ([Pima4.sup.-/-]) and heterozygous Pmcal and homozygous Pmca4 double gene-targeted ([Pmcal.sup.+/-] [Pima4.sup.-/-]) mice. Gene manipulation did not alter the amounts of PMCA1, SERCA2, and NCX. To study the role of each[Ca.sup.2+] transport system, contraction of circular ring preparations was elicited with KC1 (80 mM) plus atropine, and then the muscle was relaxed with [Ca.sup.2+]-free physiological salt solution containing EGTA. We measured the contributions of [Ca.sup.2+] clearance components by inhibiting SERCA2 (with 10 [micro]M cyclopiazonic acid) and/or NCX (by replacing NaC1 with N-methyl-D-glucamine/HC1 plus 10 [micro]M KB-R7943). Contraction halftime (time to 50% of maximum tension) was prolonged in the gene-targeted muscles but marginally shortened when SERCA2 or NCX was inhibited. The inhibition of NCX significantly inhibited this prolongation, suggesting that NCX activity might be augmented to compensate for PMCA4 function in the gene-targeted muscles under nonstimulated conditions. Inhibition of SERCA2 and NCX as well as gene targeting all prolonged the relaxation half-time. The contribution of PMCA to relaxation was calculated to be ~25-30%, with that of SERCA2 being 20% and that of NCX being 70%. PMCA and SERCA2 appeared to function additively, but the function of NCX might overlap with those of other components. In summary, gene manipulation of PMCA indicates that PMCA, in addition to SERCA2 and NCX, plays a significant role in both excitation-contraction coupling and the [Ca.sup.2+] extrusion-relaxation relationship, i.e., [Ca.sup.2+] homeostasis, of bladder smooth muscle. ATP2B; sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase 2; [Na.sup.+]/[Ca.sup.2+] exchanger; homeostasis

The structure-activity correlates of the sodium channel blocker peptidemu-conotoxin GIIIA was investigated by nuclear magnetic resonance studies and simulated annealing computations of an inactive analog. Results indicate that activity of the peptide was influenced by direct interaction of an important arginine residue with the sodium channel molecules. The active site was characterized by a cluster of arginine, lysine and hydroxyproline which interacts with the receptor site of the sodium channel. The hydroxyl group of hydroxyproline shares a common channel site with tetrodoxin and saxitoxin.