Abstract-Pulmonary vascular tone is strongly influenced by the resting membrane potential of smooth muscle cells, depolarization promoting Ca 2ϩ influx, and contraction. The resting potential is determined largely by the activity of K ϩ -selective ion channels, the molecular nature of which has been debated for some time. In this study, we provide strong evidence that the two-pore domain K ϩ channel, TASK-1, mediates a noninactivating, background K ϩ current (I KN ), which sets the resting membrane potential in rabbit pulmonary artery smooth muscle cells (PASMCs). TASK-1 mRNA was found to be present in PASMCs, and the membranes of PASMCs contained TASK-1 protein. Both I KN and the resting potential were found to be exquisitely sensitive to extracellular pH, acidosis inhibiting the current and causing depolarization. Moreover, I KN and the resting potential were enhanced by halothane (1 mmol/L), inhibited by Zn 2ϩ (100 to 200 mol/L) and anandamide (10 mol/L), but insensitive to cytoplasmic Ca 2ϩ . These properties are all diagnostic of TASK-1 channels and add to previously identified features of I KN that are shared with TASK-1, such as inhibition by hypoxia, low sensitivity to 4-aminopyridine and quinine and insensitivity to tetraethylammonium ions. It is therefore concluded that TASK-1 channels are major contributors to the resting potential in pulmonary artery smooth muscle. They are likely to play an important role in mediating pulmonary vascular responses to changes in extracellular pH, and they could be responsible for the modulatory effects of pH on hypoxic pulmonary vasoconstriction.

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Background and purpose:The sarcoplasmic reticulum (SR) releases Ca 2+ via inositol 1,4,5-trisphosphate receptors (IP3R) in response to IP3-generating agonists. Ca 2+ release subsequently propagates as Ca 2+ waves. To clarify the role of IP3 production in wave generation, the contribution of a key enzyme in the production of IP3 was examined using a phosphoinositide-specific phospholipase C (PI-PLC) inhibitor, U-73122. Experimental approach: Single colonic myocytes were voltage-clamped in whole-cell configuration and cytosolic Ca 2+ concentration ([Ca 2+ ]cyto) measured using fluo-3. SR Ca 2+ release was evoked either by activation of IP3Rs (by carbachol or photolysis of caged IP3) or ryanodine receptors (RyRs; by caffeine). Key results: U-73122 inhibited carbachol-evoked [Ca 2+ ]cyto transients. The drug also inhibited [Ca 2+ ]cyto increases, evoked by direct IP3R activation (by photolysis of caged IP3) and RyR activation (by caffeine), which do not require PI-PLC activation. U-73122 also increased steady-state [Ca 2+ ]cyto and slowed the rate of Ca 2+ removal from the cytoplasm. Smith et al., 1990) and is used widely. U-73122 inhibited PI hydrolysis and IP3 synthesis in broken cell systems and reduced agonist-evoked [Ca 2+ ]cyto rises in intact cells, such as neutrophils Smith et al., 1990), neuroblastoma cells (Thompson et al., 1991), acinar cells (Yule and Williams, 1992) and platelets . U-73122 has thus gained general acceptance as a specific PI-PLC inhibitor and inhibition of [Ca 2+ ]cyto rises in intact cells by U-73122 has been interpreted as evidence for a contribution of PI-PLC in the response in a number of studies (Yule and Williams, 1992;Hansen et al., 1995;Heemskerk et al., 1997), including smooth muscle (Zizzo et al., 2008;Frei et al., 2009).In the present study, which examined the role of PI-PLC in IP3-mediated Ca 2+ regulation in smooth muscle, U-73122 potently inhibited IP3-mediated Ca 2+ release independently of PI-PLC activity. Methods Colonic myocyte isolationAll animal care and experimental procedures complied with the Animal (Scientific Procedures) Act UK 1986. Male guinea pigs (500-700 g) were humanely killed by cervical dislocation and immediate exsanguination. The colon was immediately removed and transferred to an oxygenated (95% O2/5% CO2) physiological saline solution of the following composition (mM): NaCl 118.4, NaHCO3 25, KCl 4.7, NaH2PO4 1.13, MgCl2 1.3, CaCl2 2.7 and glucose 11 (pH 7.4). From this tissue single smooth muscle cells were enzymically isolated (McCarron and Muir, 1999), stored at 4°C and used the same day. All experiments were conducted at room temperature (20-22°C). Electrophysiological experimentsCells were voltage-clamped using conventional tight seal whole-cell recording (MacMillan et al., 2005). The composition of the extracellular solution was (mM): sodium glutamate 80, NaCl 40, tetraethylammonium chloride (TEA) 20, MgCl2 1.1, CaCl2 3, HEPES 10 and glucose 30 (pH 7.4 adjusted with NaOH 1 M). The Ca 2+ -free bathing solution additionally...

Ca2+ release from the sarcoplasmic reticulum (SR) by the IP3 receptors (IP3Rs) crucially regulates diverse cell signalling processes from reproduction to apoptosis. Release from the IP3R may be modulated by endogenous proteins associated with the receptor, such as the 12 kDa FK506-binding protein (FKBP12), either directly or indirectly by inhibition of the phosphatase calcineurin. Here, we report that, in addition to calcineurin, FKPBs modulate release through the mammalian target of rapamycin (mTOR), a kinase that potentiates Ca2+ release from the IP3R in smooth muscle. The presence of FKBP12 was confirmed in colonic myocytes and co-immunoprecipitated with the IP3R. In aortic smooth muscle, however, although present, FKBP12 did not co-immunoprecipitate with IP3R. In voltage-clamped single colonic myocytes rapamycin, which together with FKBP12 inhibits mTOR (but not calcineurin), decreased the rise in cytosolic Ca2+ concentration ([Ca2+]c) evoked by IP3R activation (by photolysis of caged IP3), without decreasing the SR luminal Ca2+ concentration ([Ca2+]l) as did the mTOR inhibitors RAD001 and LY294002. However, FK506, which with FKBP12 inhibits calcineurin (but not mTOR), potentiated the IP3-evoked [Ca2+]c increase. This potentiation was due to the inhibition of calcineurin; it was mimicked by the phosphatase inhibitors cypermethrin and okadaic acid. The latter two inhibitors also prevented the FK506-evoked increase as did a calcineurin inhibitory peptide (CiP). In aortic smooth muscle, where FKBP12 was not associated with IP3R, the IP3-mediated Ca2+ release was unaffected by FK506 or rapamycin. Together, these results suggest that FKBP12 has little direct effect on IP3-mediated Ca2+ release, even though it is associated with IP3R in colonic myocytes. However, FKBP12 might indirectly modulate Ca2+ release through two effector proteins: (1) mTOR, which potentiates and (2) calcineurin, which inhibits Ca2+ release from IP3R in smooth muscle

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Smooth muscle responds to IP] c is critically affected by the activity of the intracellular store (the sarcoplasmic reticulum, SR) from which Ca 2+ release is controlled by two major channel/receptor complexes, the inositol trisphosphate receptor (IP 3 R) and the ryanodine receptor (RyR). IP 3 Rs are activated by IP 3 generated in response to many G-protein-or tyrosine kinase-linked receptor activators, including drugs, and by certain