PHARMACOLOGICAL EVIDENCE FOR REGULATION OF NA+-CA++ EXCHANGE BY CA++/CALMODULIN-DEPENDENT PROTEIN-KINASE IN ISOLATED BOVINE ADRENAL-MEDULLARY CELLS

The mechanism of the regulation of Ca++ influx via Na+-Ca++ exchange in response to Na+ deprivation was studied in bovine adrenal medullary cells. Protein kinase inhibitors staurosporine and (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cycloocta[c,d,e]trinden-1-one depressed Na+ deprivation-induced Ca-45(++) uptake and catecholamine secretion in a concentration-dependent manner. However, 1 mM dibutyryl cyclic AMP and 1 mu M forskolin, an activator of adenylate cyclase, had little effect on Na+ deprivation-induced Ca-45(++) uptake and catecholamine secretion. Dibutyryl cyclic GMP(I mM) and muscarine (30 mu M), which increased intracellular cyclic GMP level via stimulation of muscarinic receptors, had also little effect on the responses. Although the phorbol esters 12-O-tetradecanoylphorbol-13-acetate and phorbol 12,13-dibutyrate, activators of protein kinase C, enhanced Na+ deprivation-induced catecholamine secretion, these compounds failed to affect Na+ deprivation-induced Ca-45(++) uptake. On the other hand, a variety of calmodulin antagonists such as calmidazolium, trifluoperazine, pimozide and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide inhibited Na+ deprivation-induced Ca-45(++) uptake and catecholamine secretion in a concentration-dependent manner. Furthermore, 1-[N,O-bis(S-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine, which is known as an inhibitor of Ca++/calmodulin-dependent protein kinase II, also reduced Na+ deprivation-induced Ca-45(++) uptake and catecholamine secretion. Chelation of intracellular Ca++ with Quin-2 acetoxymethyl ester resulted in a decrease in Na+ deprivation-induced Ca-45(++) uptake. However, these compounds that inhibited the Na+ deprivation-induced responses in the cells did not cause solely nonspecific and direct inhibition on Na+-Ca++ exchanger. These pharmacological observations suggest that Ca++/calmodulin-dependent protein kinase is involved in the regulation of Na+-Ca++ exchange in bovine adrenal medullary cells.