KINETICS OF RECEPTOR-MEDIATED ENDOCYTOSIS OF ALBUMIN IN CELLS DERIVEDFROM THE PROXIMAL TUBULE OF THE KIDNEY (OPOSSUM KIDNEY-CELLS) INFLUENCE OF CA2+ AND CAMP

In this study we investigated the effects of Ca2+ and cyclic adenosine monophosphate (cAMP) on the kinetic of receptor mediated (RME) and fl uid-phase (FPE) endocytosis in opossum kidney (OK) cells, derived from the proximal tubule of the kidney. We used fluorescein isothiocyanate (FITC)-labelled albumin and FITC-labelled dextran as endocytotic subs trates for RME and FPE, respectively. Removal of extracellular Ca2+ le d to a dramatic decrease of the apparent affinity of RME, but did not influence the maximum endocytotic uptake rate (J(max)). Reduction of e xtracellular Ca2+ to 1 mu mol/l had no effect. Apparent affinity of sp ecific binding of albumin to the plasma membrane was increased to 200% of control in the absence of extracellular Ca2+, whereas maximum bind ing capacity was slightly decreased, FPE was not affected by removal o f extracellular Ca2+. Additional removal of cytoplasmic Ca2+, using io nomycin, had no further effect on RME and did not affect FPE. Increase d of cytoplasmic (using ionomycin at extracellular Ca2+ concentrations of 1 mu mol/l or 1.2 mmol/l) or extracellular Ca2+ did not alter the kinetics of RME or FPE. Dibutyryl-cAMP reduced J(max) but left the app arent affinity of RME unchanged. FPE and albumin binding to the plasma membrane were not changed in the presence of cAMP. Removal of extrace llular Ca2+ and addition of cAMP led to an alkalinization of endocytot ic vesicles. Yet the alkalinization induced by removal of Ca2+ was sig nificantly greater as compared to the alkalinization in the presence o f cAMP. Endosomal alkalinization with bafilomycin A(1) had no further effect in the absence of Ca2+, but reduced RME in the presence of cAMP . From these results we conclude that cAMP and extracellular Ca2+ infl uence the kinetics of RME in proximal tubular cells. The action of Ca2 + removal seems to be mediated partially by changes of endosomal pH. C a2+ seems to be a prerequisite for RME but not a regulator, whereas cA MP may act as physiological inhibitor of RME.