ACETYL-COENZYME-A CARBOXYLASE ACTIVITY IN NEONATAL RAT CARDIAC MYOCYTES IN CULTURE - CITRATE DEPENDENCE AND EFFECTS OF HYPOXIA

The presence of a 280,000 M(r) isoform of acetyl-CoA carboxylase (ACC- 280) in the cardiac myocyte suggests that heart muscle is capable of m alonyl-CoA synthesis, Cellular factors which regulate activity of ACC- 280 are unknown. We have employed a neonatal rat cardiac myocyte cultu re (where the majority of ACC is present as ACC-280) to examine the ef fects of hypoxia and decreased cellular ATP on the activity of ACC in the cells. The myocyte culture has the following advantages over simil ar studies in the intact rat heart: the presence of a pure population of myocytes and the ability to measure cytosolic ACC fi ee from contam ination by mitochondrial carboxylases, ACC activity in cultured cardia c myocytes is completely dependent on the presence of citrate (A(0.5) = 3.8 mM). Under control conditions, the cytosolic citrate concentrati on in situ is determined to be less than 1 mM. With 5 h of hypoxia, cy tosolic ATP decreases from 9.85 +/- 0.23 to 2.83 +/- 0.25 mM and cytos olic AMP increases from undetectable levels to 40 +/- 0.4 mu M. With h ypoxia, a significant portion of the total ACC activity is now express ed in the absence of citrate and the amount of activity which is stimu lated by 10 mM citrate is significantly less (1.268 +/- 0.106 nmol/4 x 10(5) cells) than is seen under control conditions (3.042 +/- 0.048), There are no significant changes in the total amount of cellular prot ein on the plates after 5 h of hypoxia. Consistent with net ACC activa tion in hypoxia, malonyl-CoA levels increase in the cells by 7 h of hy poxia, Decreased radioactive phosphate content of immunopurified ACC-2 80 after 5 h of hypoxia is consistent with net dephosphorylation of AC C-280 and increased citrate-independent activity. (C) 1996 Academic Pr ess, Inc.