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

Because cytoplasmic cAMP has been reported to be the secondary messenger me diating K+ transport in marginal cells of freshly isolated stria vascularis , the possible role of cAMP in ion transport processes of an immortalized m arginal cell line (MCPV-8) showing evidence of K+ and Na+ reabsorption was evaluated in this study. Confluent MCPV-8 monolayers were mounted into Ussi ng chambers and perfused on both sides with perilymph-like Ringer's solutio n. Transepithelial short-circuit current (I-SC), resistance (R-T) and open- circuit voltage (V-T) were measured using voltage clamp technique. The foll owing results were obtained. (1) Addition of forskolin (10(-4) M) to the ba solateral perfusate increased I-SC to 311 +/- 42%; no significant change in R-T 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 additio n 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+-sens itive K+ current, amiloride-sensitive Na+ current was blocked first by addi tion of amiloride (10(-5) M) to the apical perfusate; subsequent addition o f 3-isobutyl-1-methylxanthine (IBMX, 1 mM) or N-6,2'-O-dibutyryladenosine 3 ',5'-cyclic monophosphate (dbcAMP, 1 mM) to the basolateral perfusate incre ased I-SC to 175 +/- 13% and 411 +/- 32%, respectively. The stimulated Ise was blocked to close to 0 by addition of BaCl2 (2 mM) to the apical perfusa te. N-2,2'-O-Dibutyrylguanosine 3',5'-cyclic monophosphate (dbcGMP, 1 mM) h ad no effect on I-SC. (3) To assess the effect of cellular cAMP on amilorid e-sensitive Na+ current, Ba2+-sensitive K+ current was blocked in advance b y 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 add ition of amiloride to the apical perfusate. dbcGMP had no effect on I-SC. H ence, these results suggest that cellular cAMP is the secondary messenger t hat mediates the transepithelial transport of both K+ and Na+ in MCPV-8 mon olayers. (C) 1999 Published by Elsevier Science B.V. All rights reserved.