Fluorescence-based functional assay for sarcolemmal ATP-sensitive potassium channel activation in cultured neonatal rat ventricular myocytes.
BACKGROUND
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 (DiBAC4(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 DiBAC4(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.


CONCLUSIONS
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.
Whiteaker
K L
KL
Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064, USA. kristi.l.whiteaker@abbott.com
Davis-Taber
R
R
Scott
V E
VE
Gopalakrishnan
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D001577:Benzophenones / Q000494:pharmacology; D002478:Cells, Cultured; D019806:Cromakalim / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005453:Fluorescence; D005905:Glyburide / Q000494:pharmacology; D006146:Guanidines / Q000494:pharmacology; D006352:Heart Ventricles / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D020110:Pinacidil / Q000494:pharmacology; D026902:Potassium Channel Blockers / Q000494:pharmacology; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012508:Sarcolemma / Q000187:drug effects / Q000378:metabolism*; D014665:Vasodilator Agents
D000577:Amides; D001577:Benzophenones; D006146:Guanidines; D026902:Potassium Channel Blockers; D024661:Potassium Channels, Inwardly Rectifying; D011725:Pyridines; D014665:Vasodilator Agents; C095007:Zeneca ZD 6169; D019806:Cromakalim; C073896:N-cyano-N'-(1,1-dimethylpropyl)-N''-(3-pyridinyl)guanidine; D020110:Pinacidil; D000255:Adenosine Triphosphate; D005905:Glyburide