Albumin endocytosis in OK cells: dependence on actin and microtubules and regulation by protein kinases.
We used proximal tubule-derived opossum kidney (OK) cells to determine the dependence of albumin endocytosis on regulation by protein kinases and on the cytoskeleton. Uptake was observed only across the apical but not the basolateral membrane and exceeded uptake in collecting duct-derived Madin-Darby canine kidney cells 14-fold. Inhibition of endocytosis via clathrin-coated vesicles but not via caveolae abolished uptake. Cytochalasin D reduced uptake to < 5% of control, and inhibition of microtubule polymerization by nocodazole reduced uptake to approximately 55% of control. Activation of protein kinase A (PKA) by adenosine 3',5'-cyclic monophosphate, forskolin, or parathyroid hormone (PTH) reduced uptake to approximately 65% of control. Protein kinase C (PKC) activation did affect uptake to a similar extent as PKA activation but with a certain delay. Stimulation of PKG by guanosine 3',5'-cyclic monophosphate did not affect albumin endocytosis. The inhibitor of tyrosine kinases (TRK), genistein, induced an increase of uptake to approximately 160% of control. Reexocytosis of albumin was enhanced by PKC activation but not by PKA activation. TRK inhibition reduced the rate of reexocytosis. We conclude that albumin endocytosis in OK cells requires the integrity of the actin cytoskeleton. Microtubules facilitate endocytosis. Uptake is regulated by PKA, PKC, and TRK, yet with different time course and by different mechanisms, e.g., reexocytosis. Possibly TRK activity serves in a negative feedback loop to limit albumin endocytosis via a stimulation of reexocytosis.
Gekle
M
M
Department of Physiology, University of Würzburg, Germany.
Mildenberger
S
S
Freudinger
R
R
Schwerdt
G
G
Silbernagl
S
S
D000199:Actins / Q000502:physiology; D000418:Albumins / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002746:Chlorpromazine / Q000494:pharmacology; D003034:Coated Pits, Cell-Membrane / Q000187:drug effects / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D015638:Cytochalasin D / Q000494:pharmacology; D004285:Dogs; D004705:Endocytosis / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein; D007529:Isoflavones / Q000494:pharmacology; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D008870:Microtubules / Q000187:drug effects / Q000502:physiology; D015739:Nocodazole / Q000494:pharmacology; D009893:Opossums; D011493:Protein Kinase C / Q000502:physiology; D011494:Protein Kinases / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000502:physiology; D013045:Species Specificity
D000199:Actins; D000418:Albumins; D004791:Enzyme Inhibitors; D007529:Isoflavones; D015638:Cytochalasin D; D019833:Genistein; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D015739:Nocodazole; D002746:Chlorpromazine