In situ phosphorylation of proteins in MCTs microdissected from rat kidney: effect of AVP.
Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein phosphorylation is considered a key step in the cellular action of vasopressin (AVP) to regulate water permeability in collecting tubules. However, the proteins serving as a substrate(s) for phosphorylation in undisrupted cells have not yet been identified. In the present study, we developed a method for investigation of in situ phosphorylation of microdissected segments of medullary collecting tubules (MCT) from rat kidney. Incubation of microdissected MCT segments with low concentrations of saponin, "semipermeabilization," increased permeability of the membrane for ATP but did not allow leakage of macromolecules such as lactate dehydrogenase. This treatment also did not cause major disruption of cell structure, or impairment of AVP-sensitive adenylate cyclase. Incubation of semipermeabilized MCT with gamma-[32P]ATP resulted in incorporation of 32Pi into two major protein bands [band "A" of apparent molecular mass (Mr) approximately equal to 66 kDa, and band "B" of Mr approximately equal to 45 kDa] detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent autoradiography. Similar incubation of tubules disrupted by hyposmotic solutions and a stronger detergent Triton X-100 resulted in 32Pi incorporation into multiple protein bands. Incubation of MCT with 1 microM AVP resulted in increased 32Pi radioactivity in band A and decreased 32Pi radioactivity in band B. These findings demonstrate a novel method for identification of endogenous protein substrate(s) for cAMP-dependent protein kinase and other protein kinases and phosphatases that are probably involved in post-cAMP steps in the cellular action of AVP in the intact cells of collecting tubules.
Homma
S
S
Department of Medicine, Mayo Clinic and Foundation, Rochester, Minnesota 55905.
Gapstur
S M
SM
Yusufi
A N
AN
Dousa
T P
TP
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology*; D002463:Cell Membrane Permeability; D000242:Cyclic AMP / Q000378:metabolism; D066298:In Vitro Techniques; D007679:Kidney Medulla / Q000378:metabolism*; D007684:Kidney Tubules / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012503:Saponins / Q000494:pharmacology
D010750:Phosphoproteins; D011506:Proteins; D012503:Saponins; D001127:Arginine Vasopressin; D000255:Adenosine Triphosphate; D000242:Cyclic AMP