REGULATION OF CL- TRANSPORT BY IBMX IN RENAL A6 EPITHELIUM

We studied regulation of Cl- transport by cAMP and Ca2+ in renal epith elial A6 cells. Stimulation of A6 cells by 1 mM 3-isobutyl-1-methylxan thine (IBMX, an inhibitor of phosphodiesterase), which increased cytos olic cAMP, elicited biphasic increases in short-circuit current (I-sc) , i.e., a transient phase followed by a sustained one. Apical applicat ion of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, a Cl- channel b locker) markedly and dose-dependently inhibited the IBMX-induced Pretr eatment with nifedipine (100 mu M, a Ca2+ channel blocker) or 1,2-bis (o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethy l)-ester (BAPTA/AM, 10 mu M, a Ca2+ chelator) partially but markedly i nhibited the I-sc. On the other hand, a cAMP-dependent protein kinase inhibitor, H89 (0.5 mu M for 1 h), also reduced the IBMX-induced I-sc to a level similar to that following nifedipine or BAPTA pretreatment. Nifedipine had no synergistic effects on the IBMX-induced I-sc in cel ls treated with H89. Ionomycin (a Ca2+ ionophore) could mimic the tran sient increase dose dependently, and H89 did not block the ionomycin-i nduced I-sc. Taken together our observations suggest that: (1) part of the IBMX-stimulated Cl- release is regulated by an increased cytosoli c Ca2+ through nifedipine-sensitive Ca2+ influx; (2) cAMP-dependent ph osphorylation may be required for elevation of the cytosolic Ca2+ conc entration but not for activation of Cl- channels, which are directly a ctivated by cytosolic Ca2+; and (3) the IBMX-induced sustained Cl- rel ease requires cAMP elevation in addition to an increase in the cytosol ic Ca2+ concentration.