Basic characterization of an ouabain-resistant, bumetanide-sensitive K + carrier-mediated transport system in J774.2 mouse macrophage-like cell line and in variants deficient in adenylate cyclase and cAMP-dependent protein kinase activities

Abstract

86Rb(K +) transport across the plasma membrane of macrophage-like cells was studied. The cells used were the wild-type J774.2 and its two variants, CT2 cells, deficient in adenylate cyclase, and J7H1 cells, deficient in cAMP-dependent protein kinase. In the three cell lines about 15% of the total 86Rb(K +) influx is transported by the K + carrier-mediated transport system. The 86Rb(K +) efflux carried by the same transporter is negligible when measured in the absence of ouabain in the medium. Therefore this carrier conducts a net inward flux of K + under the experimental conditions used. The transporter is sensitive to extracellular Na + and inhibited by ‘loop’ diuretics; bumetanide inhibits ouabain-resistant 86Rb(K +) influx with IC 50 of 0.1, 5.0, and 0.05 μM for J774.2, CT2 and J7H1 macrophages, respectively. The membrane potential of the three cells was measured, using the distribution of [ 3H]tetraphenylphosphonium ([ 3H]TPP +) across the plasma membrane, and found to be −80.1, −108.5 and −105.1 mV for J774.2, CT2 and J7H1 cells, respectively. The addition of bumetanide to the cell medium does not alter [ 3H]TPP + uptake indicating that the transporter is electrically silent. It is concluded that despite the differences in cAMP metabolism by the three macrophages, the basic characteristics of K + carrier-mediated transport system of the three cells are very similar.