The KATP channel opener diazoxide protects cardiac myocytes during metabolic inhibition without causing mitochondrial depolarization or flavoprotein oxidation.
1. The K(ATP) channel opener diazoxide has been proposed to protect cardiac muscle against ischaemia by opening mitochondrial K(ATP) channels to depolarize the mitochondrial membrane potential, DeltaPsi(m). We have used the fluorescent dye TMRE to measure DeltaPsi(m) in adult rat freshly isolated cardiac myocytes exposed to diazoxide and metabolic inhibition. 2. Diazoxide, at concentrations that are highly cardioprotective (100 or 200 microM), caused no detectable increase in TMRE fluorescence (n=27 cells). However, subsequent application of the protonophore FCCP, which should collapse DeltaPsi(m), led to large increases in TMRE fluorescence (>300%). 3. Metabolic inhibition (MI: 2 mM NaCN+1 mM iodoacetic acid (IAA) led to an immediate partial depolarization of DeltaPsi(m), followed after a few minutes delay by complete depolarization which was correlated with rigor contracture. Removal of metabolic inhibition led to abrupt mitochondrial repolarization followed in many cells by hypercontracture, indicated by cell rounding and loss of striated appearance. 4. Prior application of diazoxide (100 microM) reduced the number of cells that hypercontracted after metabolic inhibition from 63.7+/-4.7% to 24.2+/-1.8% (P< 0.0001). 5-hydroxydeanoate (100 microM) reduced the protection of diazoxide (46.8+/-2.7% cells hypercontracted, P< 0.0001 vs diazoxide alone). 5. Diazoxide caused no detectable change in flavoprotein autofluorescence (n=26 cells). 6. Our results suggest that mitochondrial depolarization and flavoprotein oxidation are not inevitable consequences of diazoxide application in intact cardiac myocytes, and that they are also not essential components of the mechanism by which it causes protection.
Lawrence
C L
CL
Ion Channel Group, Department of Cell Physiology and Pharmacology, University of Leicester, P.O. Box 138, Leicester, LE1 9HN.
Billups
B
B
Rodrigo
G C
GC
Standen
N B
NB
D000818:Animals; D002469:Cell Separation; D003981:Diazoxide / Q000494:pharmacology*; D005420:Flavoproteins / Q000378:metabolism*; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008929:Mitochondria, Heart / Q000187:drug effects* / Q000378:metabolism; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism*; D010084:Oxidation-Reduction; D015221:Potassium Channels / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D014665:Vasodilator Agents / Q000494:pharmacology*
D005420:Flavoproteins; D015221:Potassium Channels; D014665:Vasodilator Agents; D003981:Diazoxide