Identification of PTH-responsive Na/H-exchanger isoforms in a rabbit proximal tubule cell line (RKPC-2).
Renal epithelial cells may express apical and basolateral Na/H exchangers which are different in their physiological regulation and different in their sensitivities to the inhibitor amiloride. In the present study RKPC-2 cells [a Simian virus 40 (SV-40) transformed cell line of rabbit S2 proximal tubular origin] were examined for localization (apical vs basolateral) and regulation of Na/H-exchange activity(ies) by parathyroid hormone (PTH). In addition, using specific cDNA probes we determined the expression of multiple isoforms of Na/H exchangers in RKPC-2 cells. By the use of BCECF [2',7',bis(2-carboxyethyl)-5,6-carboxyfluorescein intracellular pH (pHi) indicator] and single cell fluorescence microscopy, Na/H-exchange activities (defined as initial rate of Na-dependent pHi recovery) were found on the apical and basolateral membrane of RKPC-2 cells; apical and basolateral transport activities differed in sensitivity to dimethylamiloride, the basolateral being more sensitive. Northern blot analysis demonstrated the presence of a 5.2-kb transcript, related to Na/H-exchanger activity NHE-1, and a 3.2-kb transcript, related to Na/H-exchanger activity NHE-2. PTH (10(-8) M) inhibited apically and basolaterally located Na/H-exchanger activities. The inhibitory effect of PTH was mimicked by 8-bromo-adenosine 3'5'-cyclic monophosphate (cAMP); it was blunted in the presence of H-89 (inhibitor of protein kinase A) and was unaffected by calphostin C (inhibitor of protein kinase C). In contrast to 8-bromo-cAMP (and PTH), exposure of RKPC-2 cells to phorbol 12-myristate 13-acetate (TPA) caused a significant stimulation of both Na/H-exchange activities.(ABSTRACT TRUNCATED AT 250 WORDS)
Mrkic
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Department of Physiology, University of Zürich, Switzerland.
Tse
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Forgo
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Helmle-Kolb
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Donowitz
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Murer
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D000262:Adenylyl Cyclases / Q000378:metabolism; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002461:Cell Line, Transformed; D005576:Colforsin / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D010281:Parathyroid Hormone / Q000502:physiology*; D011817:Rabbits; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000378:metabolism*
D010281:Parathyroid Hormone; D017923:Sodium-Hydrogen Exchangers; D005576:Colforsin; D000584:Amiloride; D000262:Adenylyl Cyclases