9415710

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6

Activation of alpha 1-adrenergic receptors (alpha 1-AR) increases Na+/H+ exchange (NHE) in proximal tubule. NHE mediates the majority of active Na+ absorption in the proximal tubule. Three alpha 1-AR subtypes have been detected in kidney by molecular and binding techniques. We detected message for all three alpha 1-AR subtypes in mouse proximal tubule cells through reverse transcription-polymerase chain reaction and Northern analysis. To determine the alpha 1-AR subtypes that regulate NHE in mouse proximal tubule cells, two strategies were used: (i) antisense oligodeoxynucleotides (ODNs) to selectively inhibit expression of alpha 1A-, alpha 1B-, and alpha 1D-AR subtypes and (ii) subtype-selective alpha 1-AR antagonists. Streptolysin-O permeabilization was used to introduce antisense and sense ODNs into cells three times over 72 hr. Western blot analysis of membranes prepared from cells treated with alpha 1B-AR antisense ODN demonstrated that alpha 1B-AR protein expression was reduced by 90% at 72 hr compared with control or sense ODN treatments. Functional regulation of NHE by alpha 1-ARs was determined by alpha 1-AR agonist changes in intracellular pH (pHi) in cells grown on coverslips and loaded with 2',7'-bis(2-carboxyethyl)-5(6)carboxyfluorescein-acetoxymethyl ester. Antisense ODNs for alpha 1B-AR significantly reduced phenylephrine (PHE)-induced maximal changes in pHi by 49%. The PHE-induced changes in pHi observed in cells treated with alpha 1A-AR antisense ODNs was reduced by 42%. The selective alpha 1A-AR antagonist WB-4101 and the alpha 1B-AR antagonist spiperone reduce PHE-induced pHi increases to a comparable extent. No significant changes in pHi were observed with cells treated with alpha 1D-AR antisense ODNs or the alpha 1D-AR antagonist BMY 7378 compared with untreated cells. Combined treatment with alpha 1A- and alpha 1B-AR antisense ODNs and antagonists additively inhibits PHE-induced delta pHi by 90%. We conclude that alpha 1A and alpha 1B-AR but not alpha 1D-ARs regulate NHE in proximal tubule cells.

eng

IM

MEDLINE

0026-895X

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1010-8

pubmed-meshheading:9415710-Adrenergic alpha-Agonists, pubmed-meshheading:9415710-Adrenergic alpha-Antagonists, pubmed-meshheading:9415710-Animals, pubmed-meshheading:9415710-Cells, Cultured, pubmed-meshheading:9415710-DNA, Complementary, pubmed-meshheading:9415710-Dioxanes, pubmed-meshheading:9415710-Hydrogen-Ion Concentration, pubmed-meshheading:9415710-Kidney Tubules, Proximal, pubmed-meshheading:9415710-Mice, pubmed-meshheading:9415710-Nephrons, pubmed-meshheading:9415710-Oligonucleotides, Antisense, pubmed-meshheading:9415710-Piperazines, pubmed-meshheading:9415710-Polymerase Chain Reaction, pubmed-meshheading:9415710-RNA, pubmed-meshheading:9415710-Rats, pubmed-meshheading:9415710-Receptors, Adrenergic, alpha-1, pubmed-meshheading:9415710-Sodium-Hydrogen Antiporter, pubmed-meshheading:9415710-Spiperone, pubmed-meshheading:9415710-Transcription, Genetic

Proximal nephron Na+/H+ exchange is regulated by alpha 1A- and alpha 1B-adrenergic receptor subtypes.

Journal Article, Research Support, U.S. Gov't, P.H.S.