Phospholipase D in heart: basal activity and stimulation by phorbol esters and aluminum fluoride

Summary Evidence for a general role of phospholipase D in signal transduction is accumulating. In the present study, the activity of the enzyme was investigated in heart tissue under basal conditions and after addition of phorbol esters or aluminum fluoride (AlF_inf4^sup–; 10 mM NaF plus 10 M AlCl₃). Atria of rats and chickens were incubated with [³H]-myristic acid in order to label preferentially phosphatidylcholine. Under basal conditions, the tissues generated choline and phosphatidic acid (PtdOH), the primary catalytic products of phospholipase D. When 0.5 or 2.0% ethanol was present, [³H]-phosphatidyl-ethanol (PETH) was rapidly formed at the expense of [³H]-PtdOH. This transphosphatidylation reaction is specific for phospholipase D activity. The basal formation of PETH was not inhibited by a Ca²⁺-free, EGTA-containing medium. - The phorbol ester 4-phorbol-12,13-dibutyrate (PDB), which is known to activate protein kinase C, enhanced the net formation of choline, whereas the inactive 4-phorbol-13-acetate (PAc) was ineffective. PDB (0.2 M), in contrast to PAc, also increased the formation of [³H]-PtdOH and, in the presence of ethanol, of [³H]-PETH. The PDB-evoked formation of PETH occurred again at the expense of PtdOH. Treshold and maximum effective concentrations of PDB were 10 nM and 0.2–0.6 M, respectively. The effects of PDB on either choline efflux and generation of PETH showed the same Ca^t+-dependency, i.e., both effects were blocked by a Ca²⁺-free, EGTA-containing medium, but not by a Ca²⁺-free medium without EGTA. In protein kinase C-deficient tissue which was prepared by pretreatment with 0.61 M PDB for 27 h, PDB failed to enhance the formation of PtdOH and PETH. - A1F4–, a known activator of G-proteins, increased not only the tissue content of inositol phosphates, but also markedly enhanced choline efflux and formation of [³H]-PtdOH and PETH. In conclusion, in mammalian and avian atria a high phospholipase D activity was found even under basal conditions. The enzyme was stimulated by protein kinase C and presumably by a G protein.Abbreviations IP inositol phosphate - DAG diacylglycerol - PL phospholipase - PtdOH phosphatidic acid - PETH phosphatidylethanol - PDB 4-phorbol-12,13-dibutyrate - PAc 4-phorbol-13-acetate - AlF_inf4^sup– aluminum fluoride - DMSO dimethylsulfoxideCorrespondence to K. Löffelholz at the above address