Fluorescence-based functional assay for sarcolemmal ATP-sensitive potassium channel activation in cultured neonatal rat ventricular myocytes

Abstract

Introduction: Activation of ATP-sensitive K + channels (K ATP) has been shown to induce ischemic preconditioning that serves as a protective mechanism in the heart. A high throughput assay for identifying K ATP channel openers would therefore be desirable. Methods: We describe a cell-based 96-well format fluorescence assay using bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC 4(3)) to evaluate membrane potential changes evoked by K ATP channel openers and blockers in cultured neonatal rat ventricular myocytes. Results: Pinacidil and its analog P1075 ( N-cyano- N′-(1,1-dimethylpropyl)- N″-3-pyridylguanidine), ZD6169 ( N-(4-benzoylphenyl)-3,3,3,-trifluoro-2-hydroxy-2-methyl propionamide), and the enantiomers of cromakalim evoked concentration-dependent decreases in DiBAC 4(3) fluorescence responses. Pretreatment with the K ATP channel blocker, glyburide attenuated opener-evoked decreases in fluorescence responses in a concentration-dependent manner. The rank order potency of openers in cardiac myocytes correlated well, but showed 6–10-fold higher potency in activating vascular smooth muscle K ATP channels in A10 cells. Discussion: Our studies demonstrate that the pharmacological modulation of sarcolemmal K ATP channels can be readily assessed in a high throughput manner by measuring glyburide-sensitive fluorescence changes in cardiac ventricular myocytes.