Proximal Nephron Na⁺/H⁺ Exchange Is Regulated by α_1A- and α_1B-Adrenergic Receptor Subtypes

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 α_1A-, α_1B-, and α_1D-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 α_1B-AR antisense ODN demonstrated that α_1B-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 (pH_i) in cells grown on coverslips and loaded with 2′,7′-bis(2-carboxyethyl)-5(6)carboxyfluorescein-acetoxymethyl ester. Antisense ODNs for α_1B-AR significantly reduced phenylephrine (PHE)-induced maximal changes in pH_i by 49%. The PHE-induced changes in pH_i observed in cells treated with α_1A-AR antisense ODNs was reduced by 42%. The selective α_1A-AR antagonist WB-4101 and the α_1B-AR antagonist spiperone reduce PHE-induced pH_i increases to a comparable extent. No significant changes in pH_i were observed with cells treated with α_1D-AR antisense ODNs or the α_1D-AR antagonist BMY 7378 compared with untreated cells. Combined treatment with α_1A- and α_1B-AR antisense ODNs and antagonists additively inhibits PHE-induced ΔpH_i by 90%. We conclude that α_1A and α_1B-AR but not α_1D-ARs regulate NHE in proximal tubule cells.