Cl(-)-HCO3- exchanger in isolated rat hepatocytes: role in regulation of intracellular pH.
In rat hepatocytes, basolateral Na(+)-H+ exchange and Na(+)-HCO3- cotransport function as acid extruders. To assess mechanisms of acid loading, intracellular pH (pHi) 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 (Vm). Cells were alkaline loaded by suddenly reducing external CO2 and HCO3- (from 10% and 50 mM, respectively, to 5% and 25 mM) at constant pHo. After this maneuver, pHi rapidly rose by 0.13 +/- 0.03 pH units (pHu) and recovered to baseline at an initial rate of 0.026 +/- 0.009 pHu/min. Intracellular buffering power was estimated from the dependence of pHi on [NH4+]o and varied between 70 and 10.5 mM/pHu in a pHi range of 6.5-7.6. Initial pHi recovery corresponded to a rate of OH- efflux (JOH) of 1.76 +/- 0.71 mM/min and was blocked by 0.5 mM DIDS (0.003 +/- 0.002; JOH = 0.18 +/- 0.06) or by 1 mM H2DIDS (0.001 +/- 0.002; JOH = 0.26 +/- 0.08) and by removal of [Cl-]o (0.003 +/- 0.007; JOH = 0.28 +/- 0.07). The dependence of JOH on [Cl-]o exhibited saturation kinetics with an apparent Km for [Cl-]o of 5.1 mM. pHi recovery was Na+ independent and was not inhibited by substitution of Na+ with NMDG (0.045 +/- 0.09; JOH = 2.94 +/- 0.59). During an alkaline load, cell Vm 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Benedetti
A
A
Department of Medicine and Liver Center, Yale University School of Medicine, New Haven, Connecticut 06510.
Strazzabosco
M
M
Corasanti
J G
JG
Haddad
P
P
Graf
J
J
Boyer
J L
JL
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000641:Ammonia / Q000494:pharmacology; D000818:Animals; D001464:Barium / Q000494:pharmacology; D001639:Bicarbonates / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides / Q000378:metabolism* / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains
D001639:Bicarbonates; D002352:Carrier Proteins; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides; D001464:Barium; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D000641:Ammonia; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid