Kinetics of receptor-mediated endocytosis of albumin in cells derived from 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 fluid-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 substrates for RME and FPE, respectively. Removal of extracellular Ca2+ led to a dramatic decrease of the apparent affinity of RME, but did not influence the maximum endocytotic uptake rate (Jmax). Reduction of extracellular Ca2+ to 1 mumol/1 had no effect. Apparent affinity of specific binding of albumin to the plasma membrane was increased to 200% of control in the absence of extracellular Ca2+, whereas maximum binding capacity was slightly decreased. FPE was not affected by removal of extracellular Ca2+. Additional removal of cytoplasmic Ca2+, using ionomycin, had no further effect on RME and did not affect FPE. Increases of cytoplasmic (using ionomycin at extracellular Ca2+ concentrations of 1 mumol/l or 1.2 mmol/l) or extracellular Ca2+ did not alter the kinetics of RME or FPE. Dibutyryl-cAMP reduced Jmax but left the apparent affinity of RME unchanged. FPE and albumin binding to the plasma membrane were not changed in the presence of cAMP. Removal of extracellular Ca2+ and addition of cAMP led to an alkalinization of endocytotic vesicles. Yet the alkalinization induced by removal of Ca2+ was significantly greater as compared to the alkalinization in the presence of cAMP. Endosomal alkalinization with bafilomycin A1 had no further effect in the absence of Ca2+, but reduced RME in the presence of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)
D000418:Albumins / Q000378:metabolism*; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000502:physiology*; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D016650:Fluorescein-5-isothiocyanate; D006863:Hydrogen-Ion Concentration; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D009893:Opossums / Q000378:metabolism*
D000418:Albumins; D003994:Bucladesine; D000242:Cyclic AMP; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium