Influence of Cl- on organic anion transport in short-term cultured rat hepatocytes and isolated perfused rat liver.

Transport of 35S-labeled sulfobromophthalein [35S]BSP was studied in short-term cultured rat hepatocytes incubated in bovine serum albumin. At 37 degrees C, initial uptake of [35S]BSP was 5-10-fold that at 4 degrees C, linear for at least 15 min, saturable, inhibited by bilirubin, and reduced by greater than 70% after ATP depletion or isosmotic substitution of sucrose for NaCl in medium. Replacement of Na+ by K+ or Li+ did not alter uptake, whereas replacement of Cl- by HCO-3 or gluconate- reduced uptake by approximately 40%. Substitution of Cl- by the more permeant NO-3 enhanced initial BSP uptake by 30%. Efflux of [35S]BSP from cells to media was inhibited by 40% after ATP depletion or sucrose substitution. To confirm these results in a more physiologic system, transport of [3H]bilirubin was studied in isolated livers perfused with control medium or medium in which Cl- was replaced by gluconate-. Perfusion data analyzed by the model of Goresky, revealed 40-50% reductions in influx and efflux with gluconate- substitution. These results are consistent with existence of a Cl-/organic anion-exchange mechanism similar to that described by others in renal tubules.

Influence of cytosolic pH on receptor-mediated endocytosis of asialoorosomucoid.

Inhibitors of specific steps in the endocytosis of galactose-terminating glycoproteins (asialoglycoproteins) by cultured rat hepatocytes have been described (J. Cell Biol. 98: 375-381, 1984). In particular, substitution of K+ for Na+ in the culture medium results in reduced delivery of ligand to lysosomes; ligand-receptor internalization, dissociation, and segregation remain normal. We have now demonstrated by direct microelectrode measurement that incubation of hepatocytes in K+-substituted medium results in a reduction of intracellular pH by greater than or equal to 0.5 U. In addition, we have shown that reduced intracellular pH in these cells produced by either direct (CO2 diffusion) or indirect (K+ substitution) acidification inhibits ligand delivery to lysosomes. Return of internalized ligand-receptor complex to the cell surface (diacytosis) is also inhibited by these manipulations. These studies suggest that intracellular pH may modulate specific steps involving vesicle translocation and fusion in the receptor-mediated endocytosis of asialoglycoproteins. Similar effects of direct acidification of hepatocytes by CO2 diffusion and indirect acidification by K+ substitution for Na+, on diacytosis and ligand delivery to lysosomes, suggest that K+ substitution may influence these events by altering intracellular pH.