title: Na(+)-H+ antiporter and Na(+)-(HCO3-)n symporter regulate intracellular pH in mouse medullary thick limbs of Henle.
abstract: To determine mechanisms of intracellular pH (pHi) regulation in mouse medullary thick limbs (MTAL), pHi was measured in MTAL suspensions and in the isolated perfused MTAL by use of 2',7'-bis(carboxyethyl)-5(6)carboxyfluorescein (BCECF). A method to obtain MTAL suspensions from the mouse outer medulla is reported. Characterization of suspensions with microscopy, anti-Tamm-Horsfall antibody labeling, measurement of O2 consumption, and adenosine 3',5'-cyclic monophosphate responses to antidiuretic hormone indicated that these suspensions were highly purified for viable MTAL tubules. The resting pHi was 7.41 +/- 0.02 (means +/- SE) in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered media and 7.23 +/- 0.02 in CO2- HCO3(-)-buffered media, both at extracellular pH 7.4. MTAL tubules exhibited rapid pHi recovery from intracellular acidification. Recovery of pHi was dependent on luminal Na+ (apparent Km = 13.2 +/- 3.2 mM) and was inhibited by amiloride (apparent Ki = 10.6 microM), consistent with the activity of an apical Na(+)-H+ antiporter. Antiporter activity was enhanced by acidification and was diminished at the resting pHi. Recovery from intracellular alkalinization (rapid withdrawal of CO2- HCO3-) was sensitive to the stilbene anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, Cl(-)-insensitive, and Na(+)-sensitive, consistent with the activity of a Na(+)-(HCO3-)n symporter. Both transporters were significantly involved in steady-state pHi regulation in the presence of CO2- HCO3-. In contrast, the Na(+)-H+ antiporter played the dominant role in steady-state pHi regulation in the absence of CO2- HCO3-.
authors.affiliation: Harvard Center for the Study of Kidney Diseases, Harvard Medical School, Boston, Massachusetts.
authors.forename: D
authors.initials: D
authors.lastname: Kikeri
authors.forename: S
authors.initials: S
authors.lastname: Azar
authors.forename: A
authors.initials: A
authors.lastname: Sun
authors.forename: M L
authors.initials: ML
authors.lastname: Zeidel
authors.forename: S C
authors.initials: SC
authors.lastname: Hebert
mesh_terms: D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology; D001639:Bicarbonates; D002352:Carrier Proteins / Q000502:physiology*; D000242:Cyclic AMP / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism*; D007679:Kidney Medulla; D007684:Kidney Tubules / Q000378:metabolism*; D008138:Loop of Henle / Q000378:metabolism*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D010101:Oxygen Consumption; D011522:Protons; D027982:Sodium-Bicarbonate Symporters; D017923:Sodium-Hydrogen Exchangers; D013535:Suspensions
chemical_list: D001639:Bicarbonates; D002352:Carrier Proteins; D011522:Protons; D027982:Sodium-Bicarbonate Symporters; D017923:Sodium-Hydrogen Exchangers; D013535:Suspensions; D001127:Arginine Vasopressin; D000242:Cyclic AMP