SUBCELLULAR-DISTRIBUTION OF ENDOGENOUS LONG-CHAIN ACYLCARNITINES DURING HYPOXIA IN ADULT CANINE MYOCYTES

Objective: Previous studies from our laboratory showed a pronounced in crease in the sarcolemmal accumulation of long chain acylcarnitines in isolated neonatal rat myocytes after a prolonged (60 minute) hypoxic interval. Because much shorter intervals of hypoxia are associated wit h electrophysiological alterations in adult cells, the present study w as performed to assess the extent of sarcolemmal accumulation of long chain acylcamitines during hypoxia in adult canine myocytes. Methods: Cells were incubated (for 24 hours) with H-3-carnitine and the uniform ity of incorporation into carnitine fractions was verified biochemical ly. Cells were exposed to hypoxia (Po2<15 mm Hg) for 10 or 20 minutes in the presence or absence of sodium -[5-(4-chlorophenyl)-pentyl]-oxir ane-2-carboxylate (POCA; 10 muM), an inhibitor of carnitine acyltransf erase I. Cells were processed for electron microscopical autoradiograp hy with a technique to spatially fix endogenous long chain acylcamitin es with selective and complete removal of short chain and free carniti ne. Grain distributions were analysed by the maximum likelihood method from digitised micrographs. Results: Total mass of long chain acylcam itines increased ninefold [42.3(3.3) to 374(42) pmol.mg-1 protein] by 10 minutes of hypoxia and 15-fold [to 632(36) nmol.mg-1 protein] by 20 minutes of hypoxia. Normoxic cells exhibited little long chain acylca rnitines in the sarcolemma, and modest amounts in mitochondria and cyt oplasm. In hypoxic cells, content of long chain acylcarnitines in mito chondria and cytoplasm increased by a maximum of twofold. By contrast, long chain acylcarnitines increased 100-fold in the sarcolemma to 4.1 8 X 10(6) molecules.mum-3 after 10 minutes of hypoxia. The increase in long chain acylcamitines with hypoxia was completely prevented by pre treatment with POCA. Conclusion: Hypoxia in adult ventricular myocytes induces a rapid and preferential increase in endogenous long chain ac ylcamitines within the sarcolemma.