Second 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 microM). The ability of GTP gamma S to activate the current decreased when cyclic GMP (250 microM), 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 microM; 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 microM p-bromophenacyl bromide (p-BPB), an inhibitor of phospholipase A2 activity. Exogenous arachidonic acid (5-10 microM), 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 microM-nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, but not with indomethacin (2 microM), 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.
Doroshenko
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Max-Planck-Institut für biophysikalische Chemie, Göttingen, Germany.
D000313:Adrenal Medulla / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000502:physiology; D002417:Cattle; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D000242:Cyclic AMP / Q000502:physiology; D006152:Cyclic GMP / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000502:physiology*; D007473:Ion Channels / Q000502:physiology*; D008084:Lipoxygenase / Q000502:physiology; D010741:Phospholipases A / Q000502:physiology; D054467:Phospholipases A2; D011451:Prostaglandin-Endoperoxide Synthases / Q000502:physiology; D011493:Protein Kinase C / Q000502:physiology; D015290:Second Messenger Systems / Q000502:physiology*
D002712:Chlorides; D007473:Ion Channels; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP; D008084:Lipoxygenase; D011451:Prostaglandin-Endoperoxide Synthases; D011493:Protein Kinase C; D010741:Phospholipases A; D054467:Phospholipases A2; D006152:Cyclic GMP; D002118:Calcium