Because cytoplasmic cAMP has been reported to be the secondary messenger mediating K+ transport in marginal cells of freshly isolated stria vascularis, the possible role of cAMP in ion transport processes of an immortalized marginal cell line (MCPV-8) showing evidence of K+ and Na+ reabsorption was evaluated in this study. Confluent MCPV-8 monolayers were mounted into Ussing chambers and perfused on both sides with perilymph-like Ringer's solution. Transepithelial short-circuit current (I(SC)), resistance (R(T)) and open-circuit voltage (V(T)) were measured using voltage clamp technique. The following results were obtained. (1) Addition of forskolin (10(-4) M) to the basolateral perfusate increased I(SC) to 311 +/- 42%; no significant change in RT was observed. Addition of BaCl2 (2 mM) to the apical perfusate at the maximal response of forskolin blocked 50-60% of I(SC) and subsequent addition of amiloride (10(-5) M) to the apical perfusate further blocked I(SC) to a value close to 0. (2) To evaluate the effect of cellular cAMP on Ba2+-sensitive K+ current, amiloride-sensitive Na+ current was blocked first by addition of amiloride (10(-5) M) to the apical perfusate; subsequent addition of 3-isobutyl-1-methylxanthine (IBMX, 1 mM) or N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP, 1 mM) to the basolateral perfusate increased I(SC) to 175 +/- 13 and 411 +/- 32%, respectively. The stimulated I(SC) was blocked to close to 0 by addition of BaCl2 (2 mM) to the apical perfusate. N2,2'-O-Dibutyrylguanosine 3',5'-cyclic monophosphate (dbcGMP, 1 mM) had no effect on I(SC). (3) To assess the effect of cellular cAMP on amiloride-sensitive Na+ current, Ba2+-sensitive K+ current was blocked in advance by addition of BaCl2 to the apical perfusate; subsequent addition of IBMX or dbcAMP to the basolateral perfusate increased I(SC) to 219 +/- 21% and 388 +/- 39%, respectively. The stimulated I(SC) was blocked to close to 0 by addition of amiloride to the apical perfusate. dbcGMP had no effect on I(SC). Hence, these results suggest that cellular cAMP is the secondary messenger that mediates the transepithelial transport of both K+ and Na+ in MCPV-8 monolayers.