University of SaskatchewanHARVEST
  • Login
  • Submit Your Work
  • About
    • About HARVEST
    • Guidelines
    • Browse
      • All of HARVEST
      • Communities & Collections
      • By Issue Date
      • Authors
      • Titles
      • Subjects
      • This Collection
      • By Issue Date
      • Authors
      • Titles
      • Subjects
    • My Account
      • Login
      JavaScript is disabled for your browser. Some features of this site may not work without it.
      View Item 
      • HARVEST
      • Electronic Theses and Dissertations
      • Graduate Theses and Dissertations
      • View Item
      • HARVEST
      • Electronic Theses and Dissertations
      • Graduate Theses and Dissertations
      • View Item

      Modulation of ATP-sensitive potassium channels by hydrogen sulfide and hydroxylamine

      Thumbnail
      View/Open
      PhD-thesis-Tang.pdf (2.142Mb)
      Date
      2004-12-10
      Author
      Tang, Guanghua
      Type
      Thesis
      Degree Level
      Doctoral
      Metadata
      Show full item record
      Abstract
      ATP-sensitive potassium (K+) channels (KATP) in vascular smooth muscle cells (VSMC) play a major role in the regulation of vascular tone by coupling cell contractility and K+ fluxes to cellular metabolism. They are composed of the regulatory sulphonylurea receptors (SUR) and the pore-forming inwardly rectifying K+ (Kir) channels. SUR subunits interact closely with Kir subunits by conferring their sensitivity to nucleotide or sulphonylurea. However, the modulatory mechanisms of KATP channels in VSMC are largely unknown. In particular, the effects of hydrogen sulfide (H2S) and hydroxylamine (HA) on KATP channels and underlying mechanisms have not been addressed in VSMC of resistance arteries. The combined approaches including molecular biology, biochemical assays, and patch-clamp techniques were applied. The electrophysiological and pharmacological features of native KATP channels in VSMC and cloned KATP channels in HEK-293 cells, and the modulation of KATP channels by H2S and HA in single freshly isolated VSMC from rat mesenteric arteries were characterized. In the present study, only small conductance KATP channels of 13 pS were found in rat mesenteric artery VSMC. The recorded macroscopic and unitary KATP currents were activated by nucleoside diphosphate in the presence of magnesium and K+ channel openers, inhibited by a specific KATP channel blocker glibenclamide, but were insensitive to ATP inhibition. The reversal potential shifted rightward in response to the elevation of extracellular K+ and matched the calculated K+ equilibrium potential, indicating the basal currents in both VSMC and HEK-293 cells are carried by K+ ions. Heterologous expression of Kir6.1 with SUR2B in HEK-293 cells formed functional channels and elicited whole-cell K+ currents, which shared some similar biophysical characteristics of native KATP channels in VSMC. Basal KATP currents and resting membrane potential in VSMC were reduced by glibenclamide, demonstrating that KATP channels contribute to background K+ conductance and in the setting of resting membrane potential in this resistance artery. Exogenous H2S enhanced macroscopic and unitary KATP currents with an EC50 of 116 ± 8.3 µM and hyperpolarized membrane potential. H2S activated KATP channels by increasing the open probability of single channels, but not single channel conductance. The reduced endogenous H2S production by D, L-propargylglycine resulted in the attenuation of KATP currents. H2S-induced activation of KATP channels and resultant hyperpolarization were not mediated by cGMP signaling pathway. HA enhanced reversibly KATP currents in a dose-dependent fashion with an EC50 of 54±3.4 µM and also hyperpolarized the cell membrane. HA-stimulated KATP currents were blocked by free radical scavengers (superoxide dismutase and N-acetyl-L-cysteine), and KATP channels were stimulated by a free radical generating system (hypoxanthine/xanthine oxidase), indicating the involvement of superoxide (O2-) in HA effects. Sodium nitroprusside and 8-Br-cGMP did not affect basal KATP currents and HA-stimulated KATP currents, disproving the involvement of NO-sGC-cGMP-mediated signaling pathway in the HA effects. Therefore, HA-induced KATP channel activation and hyperpolarization are likely due to the generation of O2-. In conclusion, KATP channels in resistance artery VSMC serve as the regulatory targets of H2S and HA. These two endogenous molecules modulate KATP channels via different mechanisms. H2S may directly act on KATP channel proteins while HA oxidized them via the formation of O2-, leading to the activation of KATP channels.
      Degree
      Doctor of Philosophy (Ph.D.)
      Department
      Physiology
      Program
      Physiology
      Supervisor
      Wang, Rui
      Committee
      Thornhill, James; Sulakhe, Prakash; Sauve, Remy; Gopalakrishnan, Venkat; Fisher, Thomas E.; Wollin, Armin
      Copyright Date
      December 2004
      URI
      http://hdl.handle.net/10388/etd-12232004-155703
      Subject
      Smooth muscle
      hydrogen sulfide
      elcetrophysiology
      nitric oxide
      KATP channels
      Collections
      • Graduate Theses and Dissertations
      University of Saskatchewan

      University Library

      © University of Saskatchewan
      Contact Us | Disclaimer | Privacy