DISTINCT REGULATION OF NA+ REABSORPTION AND CL- SECRETION BY ARGININE-VASOPRESSIN IN THE AMPHIBIAN CELL-LINE A6

The neurohypophysial peptide arginine vasopressin (AVP) increases Na+ absorption across A6 epithelia. In addition to the positive natriferic response, AVP increases net basolateral to apical Cl- flux. The time course of activation of electrogenic ion transport in A6 epithelia was examined by measuring transepithelial short-circuit current (I(SC)). Basolateral application of AVP (0.1 U/ml) or forskolin (10 muM) affects I(SC) in a biphasic manner. Shortly after addition of AVP, an early (transient) phase is observed in which I(SC) is rapidly stimulated, reaching a peak value at 1.4 +/- 0.1 min. A subsequent decrease in current is interrupted by a slower, late phase in which I(SC) reaches a peak 23 +/- 3 min after addition of AVP. The late increase in I(SC) is sustained over the remainder of the 40-min period of observation. The time course of I(SC) stimulation by forskolin is qualitatively similar. Replacement of external Cl- by aspartate lowers baseline transport nearly 40%, strongly blunts the early phase of I(SC) stimulation, and retains the late increase. Addition of amiloride (10 muM) to the apical bath before AVP or forskolin stimulation of I(SC) eliminates the late increase of I(SC). Steady-state amiloride-insensitive I(SC) activated under these conditions was sensitive to apical application of the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoate (20 muM) and niflumic acid (100 muM). 4, 4'-Diisothiocyanostilbene-2,2'-disulfonic acid (1 mM) was not an effective inhibitor of this current. Basolateral bumetanide (100 muM) inhibited baseline I(SC) and reduced both the peak transient and steady-state amiloride-insensitive I(SC). These data suggest that the early phase of I(SC) activation across A6 monolayers by AVP or forskolin represents rapid regulation of Cl- secretion. The late phase represents both steady-state Cl- secretion and gradual upregulation of Na+ reabsorption.