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Pumping ions: Regulation of intracellular pH in hepatocytes
Author(s) -
Fitz Greg
Publication year - 1992
Publication title -
hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.488
H-Index - 361
eISSN - 1527-3350
pISSN - 0270-9139
DOI - 10.1002/hep.1840160140
Subject(s) - dids , chemistry , intracellular , cotransporter , intracellular ph , kinetics , bumetanide , membrane potential , nuclear chemistry , biophysics , ion transporter , chromatography , analytical chemistry (journal) , membrane , sodium , biochemistry , biology , physics , organic chemistry , quantum mechanics
In rat hepatocytes, basolateral Na + ‐H + exchange and Na + ‐HCO 3 − cotransport function as acid extruders. To assess mechanisms of acid loading, intracellular pH (pH i ) recovery from an alkaline load was analyzed in short‐term cultured rat hepatocyte monolayers using the pH‐sensitive dye BCECF. Electrophysiological techniques were also used to assess the role of the membrane potential (V m ). Cells were alkaline loaded by suddenly reducing external CO 2 and HCO 3 − (from 10% and 50 mM, respectively, to 5% and 25 mM) at constant pH. After this maneuver, pH i rapidly rose by 0.13 ± 0.03 pH units (pH u ) and recovered to baseline at an initial rate of 0.026 ± 0.009 pH u /min. Intracellular buffering power was estimated from the dependence of pH i on [NH 4 − ] o and varied between 70 and 10.5 mM/pH u in a pH i range of 6.5‐7.6. Initial pH i recovery corresponded to a rate of OH − efflux ( J OH ) of 1.76 ± 0.71 mM/min and was blocked by 0.5 mM DIDS (0.003 ± 0.002; J OH = 0.18 ± 0.06) or by 1 mM H 2 DIDS (0.001 ± 0.002; J OH = 0.26 ± 0.08) and by removal of [Cl − ] o (0.003 ± 0.007; J OH = 0.28 ± 0.07). The dependence of J OH on [Cl − ] o exhibited saturation kinetics with an apparent K m for [Cl − ] o of 5.1 mM. pH i recovery was Na + independent and was not inhibited by substitution of Na + with NMDG (0.045 ± 0.09; J OH = 2.94 ± 0.59). During an alkaline load, cell V m hyperpolarized from –33.4 ± 1.8 to –43.4 ± 2.8 mV, mainly due to an increase in K + conductance by a factor of 2.8 ± 0.3. Ba 2+ blocked these changes and depolarized V m by 12.1 ± 1.2 mV but had no effect on pH i recovery (0.025 ± 0.007; J OH = 1.46 ± 0.60), excluding that V m functions as a major force for HCO 3 − extrusion after an alkaline load. The rate of J OH was directly proportional to the pH i reached after the alkaline load and varied fourfold over the pH i range of 7.25‐7.46. These data indicate that pH i recovery from an alkaline load in rat hepatocytes is mediated by an electroneutral Na + ‐independent Cl − ‐HCO 3 − exchanger.