EFFECT OF ARACHIDONIC-ACID ON THE L-TYPE CALCIUM CURRENT IN FROG CARDIAC MYOCYTES

1. External application of the unsaturated fatty acid arachidonic acid (AA) to frog ventricular cells caused a large inhibition (similar to 85%) of the L-type calcium current (I-Ca,I-L) previously stimulated by the beta-adrenergic agonist isoprenaline (Iso). The concentration pro ducing half-maximal inhibition (K-1/2) was 1.52 mu M. The inhibitory e ffect did not affect the peak current-voltage relationship but produce d a negative shift in the inactivation curve. 2. The inhibitory effect of AA also occurred in cells internally perfused with cAMP and non-hy drolysable analogues of cAMP. These data suggest that AA is acting by a mechanism located beyond adenylyl cyclase and does not involve chang es in intracellular cAMP levels. 3. AA also inhibited the calcium curr ent stimulated by internal perfusion with the catalytic subunit of pro tein kinase A (PKA), suggesting that AA acts downstream of channel pho sphorylation. 4. The inhibitory effect of AA on the isoprenaline- or c AMP -stimulated I-Ca,I-L is largely reduced in cells internally perfus ed with the thiophosphate donor analogue of ATP, ATP(gamma)S, or prote in phosphatase 1 and 2A inhibitors like microcystin (MC) or okadaic ac id (OA). External application of the phosphatase inhibitor calyculin ( Caly) also reduced the AA effect. These data suggested that the AA eff ect on I-Ca,I-L involves activation of protein phosphatase activity. 5 . The effect of AA on I-Ca,I-L was not affected by staurosporine, an i nhibitor of protein kinases. It was also unaffected in cells internall y perfused with GTP(gamma)S. These results suggest that neither a PKC- nor a G-protein-mediated mechanism are likely to be involved in the e ffect of AA on I-Ca,I-L. 6. A saturated fatty acid, myristic acid (MA) , had no inhibitory effect on the isoprenaline-stimulated Ca2+ current , whereas, in the same cells arachidonic acid produced similar to 85% inhibition of I-Ca,I-L. 7. The inhibitory effect of AA was not affecte d by exposing the cells to indomethacin (Indo), an inhibitor of the me tabolism of AA by cyclo-oxygenase, nor nordihydroguaiaretic acid (NDGA ), an inhibitor of the lipoxygenase pathway. However, the non-metaboli zable analogue of AA, 5,8,11,14-eicosatetraynoic acid (ETYA), was with out effect on the isoprenaline-stimulated I-Ca,I-L. 8. These results s uggest that AA inhibits I-Ca,I-L via a mechanism which involves, in pa rt, stimulation of protein phosphatase activity This process could pro vide a new mechanism in the modulation of calcium channel activity.