Increased cytosolic Ca2+ inhibits AVP-stimulated adenylyl cyclase activity in rat IMCT cells by activation of PKC.
In rat inner medullary collecting tubule (RIMCT) cells increasing cytosolic Ca2+ with a calcium ionophore inhibits arginine vasopressin (AVP)-stimulated adenylyl cyclase (AC). Inhibition by Ca2+ is not observed in pertussis toxin (PT)-treated cells, indicating a role for the inhibitory G protein, Gi. The mechanism of activation of Gi remains to be determined. We examined the hypothesis that inhibition of AVP-stimulated AC by increased cytosolic Ca2+ is due to activation of protein kinase C (PKC). Preincubation of RIMCT cells with ionophore results in inhibition of AVP-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) formation. To assess whether stimulation of phospholipase C (PLC) and therefore activation of PKC occurs with ionophore and AVP, inositol trisphosphate (IP3) production was measured. Incubation of RIMCT cells with either 10(-7) M AVP or ionophore results in IP3 production that is no different from basal. However, simultaneous exposure to 100 nM AVP with ionophore results in marked enhancement of IP3 production clearly reflecting stimulation of PLC in this setting. Stimulation of PLC is not observed in PT-treated cells. Likewise, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of PKC, mimics the effect of PT to prevent inhibition of AVP-stimulated AC by ionophore, but N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide (H-8), an inhibitor of protein kinase A (PKA), does not. As is the case when PKC is stimulated directly with a phorbol ester, exposure to ionomycin inhibits the response to AVP but does not alter the response to isoproterenol. These studies demonstrate that increased cytosolic Ca2+ does not, as previously postulated, inhibit AC by a direct effect on Gi. Rather, when cytosolic Ca2+ is increased, AVP stimulates PLC; the ensuring activation of PKC inhibits cAMP formation.
Teitelbaum
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I
Department of Medicine, University of Colorado School of Medicine, Denver 80262.
Berl
T
T
D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D000242:Cyclic AMP / Q000096:biosynthesis; D003600:Cytosol / Q000378:metabolism*; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000502:physiology; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis; D015759:Ionomycin / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D007679:Kidney Medulla; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats
D000067956:Adenylyl Cyclase Inhibitors; D001127:Arginine Vasopressin; D015759:Ionomycin; D015544:Inositol 1,4,5-Trisphosphate; D000242:Cyclic AMP; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D007545:Isoproterenol; D002118:Calcium