Abstract
Activation of α1-adrenergic receptors (α1-AR) increases Na+/H+ exchange (NHE) in proximal tubule. NHE mediates the majority of active Na+ absorption in the proximal tubule. Three α1-AR subtypes have been detected in kidney by molecular and binding techniques. We detected message for all three α1-AR subtypes in mouse proximal tubule cells through reverse transcription-polymerase chain reaction and Northern analysis. To determine the α1-AR subtypes that regulate NHE in mouse proximal tubule cells, two strategies were used: (i) antisense oligodeoxynucleotides (ODNs) to selectively inhibit expression of α1A-, α1B-, and α1D-AR subtypes and (ii) subtype-selective α1-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 α1B-AR antisense ODN demonstrated that α1B-AR protein expression was reduced by 90% at 72 hr compared with control or sense ODN treatments. Functional regulation of NHE by α1-ARs was determined by α1-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 α1B-AR significantly reduced phenylephrine (PHE)-induced maximal changes in pHi by 49%. The PHE-induced changes in pHi observed in cells treated with α1A-AR antisense ODNs was reduced by 42%. The selective α1A-AR antagonist WB-4101 and the α1B-AR antagonist spiperone reduce PHE-induced pHi increases to a comparable extent. No significant changes in pHi were observed with cells treated with α1D-AR antisense ODNs or the α1D-AR antagonist BMY 7378 compared with untreated cells. Combined treatment with α1A- and α1B-AR antisense ODNs and antagonists additively inhibits PHE-induced ΔpHi by 90%. We conclude that α1A and α1B-AR but not α1D-ARs regulate NHE in proximal tubule cells.
Footnotes
- Received April 11, 1997.
- Accepted September 5, 1997.
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Send reprint requests to: Frank A. Gesek, Dept. of Pharmacology & Toxicology, Dartmouth Medical School, 7650 Remsen, Room 611, Hanover, NH 03755-3835. E-mail:fg{at}dartmouth.edu
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This work was supported by National Institutes of Health Grants DK46064, DK07301, and AR27032.
- The American Society for Pharmacology and Experimental Therapeutics
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