Proximal nephron Na+/H+ exchange is regulated by alpha 1A- and alpha 1B-adrenergic receptor subtypes.
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.
Liu
F
F
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Nesbitt
T
T
Drezner
M K
MK
Friedman
P A
PA
Gesek
F A
FA
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000378:metabolism; D004146:Dioxanes / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007687:Kidney Tubules, Proximal / Q000378:metabolism* / Q000648:ultrastructure*; D051379:Mice; D009399:Nephrons / Q000378:metabolism* / Q000648:ultrastructure*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D012313:RNA / Q000378:metabolism; D051381:Rats; D018340:Receptors, Adrenergic, alpha-1 / Q000145:classification / Q000187:drug effects / Q000502:physiology*; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D013134:Spiperone / Q000494:pharmacology; D014158:Transcription, Genetic
C547714:Adra1a protein, mouse; C547717:Adra1b protein, mouse; D000316:Adrenergic alpha-Agonists; D000317:Adrenergic alpha-Antagonists; D018076:DNA, Complementary; D004146:Dioxanes; D016376:Oligonucleotides, Antisense; D010879:Piperazines; D018340:Receptors, Adrenergic, alpha-1; D017923:Sodium-Hydrogen Exchangers; D013134:Spiperone; D012313:RNA; C010654:(2-(2',6'-dimethoxy)phenoxyethylamino)methylbenzo-1,4-dioxane; C053428:BMY 7378