Expression of Na-K-ATPase alpha- and beta-subunit mRNA and protein isoforms in the rat nephron.
Na-K-ATPase is a heterodimeric complex composed of an alpha-catalytic and a glycosylated beta-subunit. Previous studies using in situ hybridization and Northern blot analysis to determine alpha- and beta-subunit mRNA isoform expression in the rat kidney have given conflicting results. This heterogeneity may be due to detection of alpha 2- or alpha 3-isoforms arising from nonrenal epithelial sources such as peripheral nerves or vascular smooth muscle. To address this possibility, we investigated alpha-subunit mRNA isoform expression in different nephron segments using tubule microdissection and reverse transcription-polymerase chain reaction amplification. Southern blot analysis of polymerase chain reaction products using isoform-specific primers and probes detected the expression of alpha 1- and alpha 3-mRNA isoforms in whole kidney, cortical collecting ducts (CCD), and proximal tubule S2 subsegments (S2). No evidence for alpha 2 was found in kidney or microdissected CCD or S2. To determine whether alpha 1- or alpha 3-mRNA in the CCD or S2 is translated into protein, Western blot analysis of total protein from microdissected S2, CCD, medullary thick ascending limb (MTAL), or outer medullary collecting duct outer stripe (OMCDos) was performed. Total protein was separated according to size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then probed using polyclonal antibodies specific for the alpha 1-, alpha 2-, alpha 3-, beta 1-, and beta 2-protein isoforms. Rat brain was used as a positive control and demonstrated that the antibodies could detect a single 97- and 35-kDa band for the alpha- and beta-isoforms, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Tumlin
J A
JA
Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322.
Hoban
C A
CA
Medford
R M
RM
Sands
J M
JM
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism; D017931:DNA Primers; D015870:Gene Expression; D017403:In Situ Hybridization; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000096:biosynthesis* / Q000302:isolation & purification; D007668:Kidney / Q000201:enzymology; D007684:Kidney Tubules / Q000201:enzymology; D046911:Macromolecular Substances; D008297:Male; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000201:enzymology; D009399:Nephrons / Q000201:enzymology*; D009928:Organ Specificity; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis* / Q000302:isolation & purification
D017931:DNA Primers; D007527:Isoenzymes; D046911:Macromolecular Substances; D012333:RNA, Messenger; D004247:DNA; D000254:Sodium-Potassium-Exchanging ATPase