The present investigation tested the hypothesis that nitric oxide (NO) potentiates ATP-sensitive K(+) (K(ATP)) channels by protein kinase G (PKG)-dependent phosphorylation in rabbit ventricular myocytes with the use of patch-clamp techniques. Sodium nitroprusside (SNP; 1 mM) potentiated K(ATP) channel activity in cell-attached patches but failed to enhance the channel activity in either inside-out or outside-out patches. The 8-(4-chlorophenylthio)-cGMP Rp isomer (Rp-CPT-cGMP, 100 microM) suppressed the potentiating effect of SNP. 8-(4-Chlorophenylthio)-cGMP (8-pCPT-cGMP, 100 microM) increased K(ATP) channel activity in cell-attached patches. PKG (5 U/microl) added together with ATP and cGMP (100 microM each) directly to the intracellular surface increased the channel activity. Activation of K(ATP) channels was abolished by the replacement of ATP with ATPgammaS. Rp-pCPT-cGMP (100 microM) inhibited the effect of PKG. The heat-inactivated PKG had little effect on the K(ATP) channels. Protein phosphatase 2A (PP2A, 1 U/ml) reversed the PKG-mediated K(ATP) channel activation. With the use of 5 nM okadaic acid (a PP2A inhibitor), PP2A had no effect on the channel activity. These results suggest that the NO-cGMP-PKG pathway contributes to phosphorylation of K(ATP) channels in rabbit ventricular myocytes.
This investigation used a patch clamp technique to test the hypothesis that protein kinase G (PKG) contributes to the phosphorylation and activation of ATP-sensitive K ؉ (K ATP ) channels in rabbit ventricular myocytes. Nitric oxide donors and PKG activators facilitated pinacidilinduced K ATP channel activities in a concentration-dependent manner, and a selective PKG inhibitor abrogated these effects. In contrast, neither a selective protein kinase A (PKA) activator nor inhibitor had any effect on K ATP channels at concentrations up to 100 and 10 M, respectively. Exogenous PKG, in the presence of both cGMP and ATP, increased channel activity, while the catalytic subunit of PKA had no effect. PKG activity was prevented by heat inactivation, replacing ATP with adenosine 5-O-(thiotriphosphate) (a nonhydrolyzable analog of ATP), removing Mg 2؉ from the internal solution, applying a PKG inhibitor, or by adding exogenous protein phosphatase 2A. The effects of cGMP analogs and PKG were observed under conditions in which PKA was repressed by a selective PKA inhibitor. The results suggest that K ATP channels are regulated by a PKG-signaling pathway that acts via PKG-dependent phosphorylation. This mechanism may, at least in part, contribute to a signaling pathway that induces ischemic preconditioning in rabbit ventricular myocytes.Protein phosphorylation is a putative effector mechanism in the infarct size-limiting effect of ischemic preconditioning (1), a phenomenon whereby a brief period of ischemia and reperfusion can protect the heart against subsequent prolonged ischemia and reperfusion injury (2). Indeed, it was shown that phosphorylation levels decrease during ischemia in nonpreconditioned hearts, whereas they increase during ischemia in preconditioned hearts (3). A number of cellular components, including cytoskeletal and stress proteins, have been proposed as potential phosphorylation targets in the ischemic preconditioned heart (4 -6).ATP-sensitive K ϩ channels (K ATP channels) 1 in sarcolemma and mitochondria are also modulated by phosphorylation (7, 8).The majority of the studies on the contribution of phosphorylation to K ATP channel activity to the cardioprotective effects of ischemic preconditioning have centered on the role of protein kinase C. Protein kinase C may act as a link between one or more receptor-mediated pathways and increased K ATP channel activity and thus lead to ischemic preconditioning (9). The release of endogenous substances, such as adenosine, bradykinin, nitric oxide (NO), and prostacyclin (10 -12), has been proposed as a potential mechanism of ischemic preconditioning. These substances increase cGMP via direct stimulation of myocardial cells or via the endothelium. It was reported that the cGMP levels in preconditioned hearts are higher than in nonpreconditioned hearts (13-15). Since cGMP can induce protein phosphorylation via protein kinase G (PKG) activation, the involvement of PKG-dependent phosphorylation in ischemic preconditioning is expected. To date, however, the role of PKG ...
The pacemaker activity of interstitial cells of Cajal (ICCs) has been known to initiate the propagation of slow waves along the whole gastrointestinal tract through spontaneous and repetitive generation of action potentials. We studied the mechanism of the pacemaker activity of ICCs in the mouse small intestine and tested it using a mathematical model. The model includes ion channels, exchanger, pumps and intracellular machinery for Ca2+ regulation. The model also incorporates inositol 1,4,5-triphosphate (IP3) production and IP3-mediated Ca2+ release activities. Most of the parameters were obtained from the literature and were modified to fit the experimental results of ICCs from mouse small intestine. We were then able to compose a mathematical model that simulates the pacemaker activity of ICCs. The model generates pacemaker potentials regularly and repetitively as long as the simulation continues. The frequency was set at 20 min(-1) and the duration at 50% repolarization was 639 ms. The resting and overshoot potentials were -78 and +1.2 mV, respectively. The reconstructed pacemaker potentials closely matched those obtained from animal experiments. The model supports the idea that cyclic changes in [Ca2+]i and [IP3] play key roles in the generation of ICC pacemaker activity in the mouse small intestine.
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