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Bidirectional transepithelial K+ flux measurements across 'high-resistance' epithelial monolayers of MDCK cells grown upon millipore filters show no significant net K+ flux. Measurements of influx and efflux across the basal-lateral and apical cell membranes demonstrate that the apical membranes are effectively impermeable to K+. K+ influx across the basal-lateral cell membranes consists of an ouabain-sensitive component, an ouabain-insensitive component, an ouabain-insensitive but furosemide-sensitive component, and an ouabain- and furosemide-insensitive component. The action of furosemide upon K+ influx is independent of (Na+ - K+)-pump inhibition. The furosemide-sensitive component is markedly dependent upon the medium K+, Na+ and Cl- content. Acetate and nitrate are ineffective substitutes for Cl-, whereas Br- is partially effective. Partial Cl- replacement by NO3 gives a roughly linear increase in the furosemide-sensitive component. Na+ replacement by choline abolishes the furosemide-sensitive component, whereas Li+ is a partially effective replacement. Partial Na+ replacement by choline abolishes the furosemide-sensitive component, whereas Li+ is a partially effective replacement. Partial Na+ replacement with choline gives an apparent affinity of approximately 7 mM Na, whereas variation of the external K+ content gives an affinity of the furosemide-sensitive component of 1.0 mM. Furosemide inhibition is of high affinity (K1/2 = 3 micrometer). Piretanide, ethacrynic acid, and phloretin inhibit the same component of passive K+ influx as furosemide; amiloride, 4,-aminopyridine, and 2,4,6-triaminopyrimidine partially so. SITS was ineffective. Externally applied furosemide and Cl- replacement by NO3- inhibit K+ efflux across the basal-lateral membranes indicating that the furosemide-sensitive component consists primarily of K:K exchange.

SUMMARYThe cardiac glycosides, ouabain or digoxin, when applied to HeLa cells at a concentration of 1 /SM, block all Na/K pumping. The recovery of the cells from such a block involves a process of internalization of the glycoside, with its subsequent excretion and the appearance of a working Na pump at the plasma membrane. The rate of recovery of pump function probably does not depend on the direct dissociation of the glycoside from the sodium pumps on the plasma membrane. Low temperature and vinblastine slow the rate of glycoside loss from the cells (the t1 is normally about 20 h). No other agent examined altered the excretion rate. The recovery ofpump function (ti of 6 h) was reduced by all factors examined, i.e. low temperature, vinblastine, low serum concentration, cycloheximide and chloropromazine. We think this means that glycoside excretion and pump recovery are not directly related, though recovery may ultimately depend on glycoside excretion. The recovery in pump function which does occur in the absence of protein synthesis is thought to be due to a store of preformed sodium pumps held in the cell. Fresh HeLa cells 'turnover' their sodium pumps at a slow rate, i.e. they internalize their pumps and replace them with new ones at a rate of about 1 % per hour. When pumps on the plasma membrane are blocked with cardiac glycosides, the rate of replacement can be increased by a factor of at least 4. This, therefore, constitutes a repair process. It is shown that HeLa cells provide a good model for the detoxification mechanism present in the human heart treated with therapeutic concentrations of glycosides.

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HeLa cells and primary cultures of embryonic chick heart cells were grown in medium containing low concentrations of ouabain for 24 h. Compared with normal cells, cells grown in ouabain have fewer free sodium pump sites, an increased intracellular sodium concentration and a decreased intracellular potassium concentration. The cells are able to maintain their intracellular ion contents because the remaining pump sites have an increased turnover rate. When cells that have been chronically exposed to ouabain are returned to normal growth medium, the sodium pump site numbers increase; the recovery process begins within 6 to 8 h and is complete within 24 h. Recovery of pump site numbers is primarily dependent upon de novo protein synthesis since the protein synthesis inhibitor, cycloheximide, prevents recovery.