KINETICS OF ATP-SENSITIVE K-87 NMR-SPECTROSCOPY( CHANNELS IN ISOLATEDRAT HEARTS ASSESSED BY RB)

An experimental model was developed to evaluate the effects of activat ors and inhibitors of K-ATP channels on unidirectional K+ fluxes in th e whole heart. Isolated rat hearts perfused in the Langendorff mode we re equilibrated with P-i-free Krebs-Henseleit buffer (KH buffer) conta ining 0.94-2.14 mM RbCl and 3.76 mM KCl (20-36% of K+ substituted by R b+). Rb+ efflux was initiated by removing Rb+ from the perfusate and R b-87 spectra were acquired continuously with a 1-2 min time resolution . In hearts with normal energetics, the efflux of Rb+ fit a monoexpone ntial 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 mu M carbachol (carb) and 20 mM MgSO4, inhibit ed Rb+ efflux such that the rate constant, k (10(3)/min), decreased fr om 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 bumet anide (Bum, 30 mu M) and glibenclamide (Glib, 5 mu M). Dinitrophenol ( DNP, 0.2 mM) added in the presence of high [K+] + Bum increased k thre e-fold, to 160 +/- 5. This effect was associated with a significant de crease in phosphocreatine (PCr, <10% of initial) and ATP ( 15%) levels , and a 7-fold increase in the P-i level, assessed by P-31-NMR spectro scopy. Glib completely reversed the effect of DNP. Pinacidil (Pin, 20- 80 mu M) did not affect the k value either in beating control hearts o r 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 Rbf efflux. We conclude that:(1) heart rate-independ ent 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 K-ATP channels in whole hearts; and (3) Pin is not an effective K-ATP channel opener in the rat heart model. (C) 1998 John Wiley & Sons, Ltd.