CAMP mediates transepithelial K + and Na + transport in a strial marginal cell line

Abstract

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 R T was observed. Addition of BaCl 2 (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 Ba 2+-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 N 6,2′- 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 BaCl 2 (2 mM) to the apical perfusate. N 2,2′- 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, Ba 2+-sensitive K + current was blocked in advance by addition of BaCl 2 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.