Transfection-mediated expression of a dominant cAMP-resistant phenotype in the opossum kidney (OK) cell line prevents parathyroid hormone-induced inhibition of Na-phosphate cotransport. A protein kinase-A-mediated event.
Sodium-phosphate cotransport in the PTH-responsive opossum kidney (OK) cell line is inhibited by PTH, cAMP, and activators of protein kinase C. In order to probe the role of cAMP, we stably transfected OK cells with an expression vector for a cAMP-binding mutation of the murine protein kinase A regulatory subunit. Two-dimensional electrophoresis of cAMP-binding proteins from transfected cells indicated a 20-fold overexpression of the mutant regulatory unit. Protein kinase A from these cells had a 20-fold increase in the concentration of cAMP required for half-maximal activation, 2.8 microM vs. 0.15 microM for wild type cells. In the transfected cells, Na-phosphate cotransport was insensitive to up to 1 mM 8-Br-cAMP and 1 microM PTH, while these same agonists caused a significant inhibition of transport in the wild type cells. The effects on Na-phosphate cotransport of the protein kinase C activators oleoyl-acetyl glycerol and tetradecanoyl-phorbol acetate, which were marked in the wild type cells, were still present, although attenuated, in the transfected mutants. With prolonged passage, the cAMP-insensitive phenotype reverted to wild type cAMP sensitivity despite continued selection for the cotransfected neo marker. The revertant cells had a normal cAMP requirement for half-maximal activation of protein kinase A, 0.13 microM, and the PTH and cAMP-sensitive inhibition of Na-phosphate cotransport was restored. We suggest that an intact and normally cAMP-sensitive protein kinase A pathway is an absolute requirement for PTH inhibition of Na-phosphate cotransport in the OK cell.
Segal
J H
JH
Department of Medicine, University of California, San Francisco.
Pollock
A S
AS
D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D009893:Opossums; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010641:Phenotype; D010710:Phosphates / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; D014162:Transfection
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D010281:Parathyroid Hormone; D010710:Phosphates; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; C015482:protein kinase modulator; D012964:Sodium; D000242:Cyclic AMP; D011494:Protein Kinases