Kinetics of ATP-sensitive K+ channels in isolated rat hearts assessed by 87Rb NMR spectroscopy.
An experimental model was developed to evaluate the effects of activators and inhibitors of K(ATP) channels on unidirectional K+ fluxes in the whole heart. Isolated rat hearts perfused in the Langendorff mode were equilibrated with Pi-free Krebs-Henseleit buffer (KH buffer) containing 0.94-2.14 mM RbCl and 3.76 mM KCl (20-36% of K+ substituted by Rb+). Rb+ efflux was initiated by removing Rb+ from the perfusate and 87Rb spectra were acquired continuously with a 1-2 min time resolution. In hearts with normal energetics, the efflux of Rb+ fit a monoexponential function, and the rate constant did not depend on intracellular [Rb+]. Agents depressing excitability and heart rate (HR), such as 0.6 mM lidocaine (Lido), 10 microM carbachol (carb) and 20 mM MgSO4, inhibited Rb+ efflux such that the rate constant, k (10(3)/min), decreased from 50+/-1.2 in the beating heart to 26+/-1, 40+/-1.1 and 19+/-1.2, respectively. In contrast, high [K+] (21 mM) did not affect the k value (50+/-4.5), independently of the presence or absence of bumetanide (Bum, 30 microM) and glibenclamide (Glib, 5 microM). Dinitrophenol (DNP, 0.2 mM) added in the presence of high [K+] + Bum increased k three-fold, to 160+/-5. This effect was associated with a significant decrease in phosphocreatine (PCr, <10% of initial) and ATP ( 15%) levels, and a 7-fold increase in the Pi level, assessed by 31P-NMR spectroscopy. Glib completely reversed the effect of DNP. Pinacidil (Pin, 20-80 microM) did not affect the k value either in beating control hearts or in the presence of Carb or KCl + Bum. Moreover, under conditions of moderate metabolic stress induced by 0.05 mM DNP (PCr, 35%; ATP, 65%), where half-maximal activation of K(ATP) channels occurred, Pin did not further activate Rb+ efflux. We conclude that:(1) heart rate-independent Rb+ efflux accounts for 40-80% of the total Rb+ efflux in beating (300 bpm) rat hearts;(2) DNP-activated Rb+ efflux is a good model for testing inhibitors of KATP channels in whole hearts; and (3) Pin is not an effective K(ATP) channel opener in the rat heart model.
Institute for Biodiagnostics, National Research Council, Winnipeg, Canada.
V V
VV
Kupriyanov
E
E
Yushmanov
B
B
Xiang
R
R
Deslauriers
D019297:2,4-Dinitrophenol / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000502:physiology*; D000818:Animals; D006339:Heart Rate / Q000187:drug effects / Q000502:physiology; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008297:Male; D009206:Myocardium / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012413:Rubidium / Q000493:pharmacokinetics; D012414:Rubidium Radioisotopes; D014475:Uncoupling Agents / Q000494:pharmacology
D026902:Potassium Channel Blockers; D015221:Potassium Channels; D012414:Rubidium Radioisotopes; D014475:Uncoupling Agents; D000255:Adenosine Triphosphate; D012413:Rubidium; D019297:2,4-Dinitrophenol; D011188:Potassium