2ND MESSENGERS MEDIATING ACTIVATION OF CHLORIDE CURRENT BY INTRACELLULAR GTP-GAMMA-S IN BOVINE CHROMAFFIN CELLS

1. Intracellular mechanisms and second messengers involved in chloride current activation by intracellular GTP-gamma-S (guanosine 5'-O-(3-thiotriphosphate)) in bovine chromaffin cells were studied using the whole-cell patch-clamp technique combined with measurements of intracellular calcium [Ca2+]i. 2. No correlation between the time of current activation and the appearance of [Ca2+]i transients was observed; intracellular introduction of sufficient EGTA (10 mM) to suppress the [Ca2+]i transients did not affect the current activation by GTP-gamma-S. 3. The cyclic nucleotides, cyclic AMP or cyclic GMP, did not activate the current when introduced intracellularly (50-250-mu-M). The ability of GTP-gamma-S to activate the current decreased when cyclic GMP (250-mu-M), together with MgATP (2 mM), was added to the perfusate. 4. Neomycin (0.5-1 mM), a presumed inhibitor of phospholipase C effectively prevented the current activation by GTP-gamma-S but it did not prevent [Ca2+]i transients. 5. Modulation of protein kinase C activity using specific inhibitors (H-7, 300-mu-M; polymyxin B, 400 U/ml) or activators (phorbol ester PMA, 100 nM, 20-90 min at 37-degrees-C) did not affect the current activation by GTP-gamma-S nor did it cause current activation in the absence of GTP-gamma-S. 6. Activation of the current by GTP-gamma-S could be prevented by incubating the cells for 10-15 min with 2.5-mu-M p-bromophenacyl bromide (p-BPB), an inhibitor of phospholipase A2 activity. Exogenous arachidonic acid (5-10-mu-M), applied extracellularly or intracellularly, neither activated the current itself nor did it interfere with its activation by GTP-gamma-S. 7. Activation of the current by GTP-gamma-S could also be prevented by incubating the cells with 1-mu-M-nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, but not with indomethacin (2-mu-M), an inhibitor of cyclo-oxygenase pathway of arachidonic acid metabolism. 8. It is suggested that chloride current activation by GTP-gamma-S in bovine chromaffin cells involves G protein-mediated stimulation of phospholipase A2 activity and subsequent formation of lipoxygenase product(s) of arachidonic acid metabolism.