Decreased protein phosphorylation induced by anoxia in proximal renal tubules.
Anoxia-induced depletion of cellular ATP may affect the degree of protein phosphorylation due to kinase inhibition. In this study, protein phosphorylation was measured in rabbit kidney proximal tubules under normoxic or anoxic conditions in a medium containing 32P. During the first 20 min of normoxia, phosphate incorporation was linear, averaging 17 +/- 5 pmol.mg protein-1.min-1 and was 70% inhibited by the protein kinase C inhibitor chelerythrine chloride. Phosphorylation measurements initiated simultaneously with anoxic conditions (95% N2-5% CO2) significantly reduced the initial rate to 58% of control, saturating after 15 min, and reaching 28 +/- 5% of the normoxic value after 60 min of incubation. The phosphatase inhibitor calyculin A did not affect the initial rate of phosphate incorporation by anoxic tubules but increased phosphate incorporation at 60 min to 43 +/- 4% of normoxia. Addition of 32P after 15 min of anoxia abolished phosphate incorporation, demonstrating that kinase activity was completely inhibited. Cellular phosphate uptake was measured and found not to be rate limiting for phosphorylation. Chelerythrine chloride increased lactate dehydrogenase (LDH) release during normoxia, and calyculin A decreased anoxia-induced LDH release, suggesting that protein phosphorylation events may control plasma membrane permeability.
Kobryn
C E
CE
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710.
Mandel
L J
LJ
D000470:Alkaloids; D000818:Animals; D053119:Benzophenanthridines; D004988:Ethers, Cyclic / Q000494:pharmacology; D000860:Hypoxia / Q000378:metabolism*; D066298:In Vitro Techniques; D007687:Kidney Tubules, Proximal / Q000378:metabolism* / Q000473:pathology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008387:Marine Toxins; D019319:Okadaic Acid; D010080:Oxazoles / Q000494:pharmacology; D010617:Phenanthridines / Q000494:pharmacology; D010710:Phosphates / Q000493:pharmacokinetics; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D012016:Reference Values
D000470:Alkaloids; D053119:Benzophenanthridines; D004988:Ethers, Cyclic; D008387:Marine Toxins; D010080:Oxazoles; D010617:Phenanthridines; D010710:Phosphates; D011506:Proteins; D019319:Okadaic Acid; C059041:calyculin A; C016299:chelerythrine; D007770:L-Lactate Dehydrogenase; D011493:Protein Kinase C; D010744:Phosphoric Monoester Hydrolases