The action of ouabain upon chloride secretion in cultured MDCK epithelium

Abstract

Net Na+ loss from confluent monolayers of cultured epithelial cells grown on plastic petri dishes into choline chloride is consistent with loss from two separate pools (t 1/2 2.4 and 43.7 min). Tissue K+ is lost with a single time constant (t 1/2 76.9 min). Since tissue equilibration of [14C]inulin is also rapid (t 1/2 approx. 1 min), it is inferred that the fast component of Na+ loss comprises loss from extracellular pools, whereas the slow component comprises intracellular loss. By washing extracellular cations from cell monolayers and directly measuring cell numbers and volumes by Coulter Counter, intracellular Na+ and K+ concentrations were estimated to be 16 +/- 2 (S.E.) and 151 +/- 2 (S.E.) mM. Ouabain at high concentrations (1 x 10(-5) to 1 x 10(-3) M) raised intracellular Na+, and lowered intracellular K+. The t 1/2 for cation equilibration with the external medium was approx. 70 min (+ ouabain). Ouabain inhibited ATP-stimulated Cl- secretion by epithelial MDCK monolayers mounted in Ussing chambers. The inhibition was time-dependent and consistent with dissipation of intracellular cation gradients. The ATP-dependent increase in monolayer conductance, observed in control tissues, was largely unaffected by ouabain.