title: Characterization of ion transport mechanisms regulating intracellular pH in hepatic stellate cells.
abstract: The aim of this study was to evaluate intracellular pH (pHi) regulation in nonactivated and activated rat hepatic stellate cells (HSC). The fluorescent pHi indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein was used to measure pHi in the presence and absence of HCO3-. In the absence of HCO3-, baseline pHi was significantly higher (P < 0.001) in activated than in nonactivated HSC (7.1 +/- 0.1 vs. 6.9 +/- 0.2) and decreased, in both groups, after amiloride administration and after Na+ removal. After an acid-loading maneuver, pHi recovery was significantly higher (P < 0.03) in activated than in nonactivated HSC (H+ flux = 11.0 +/- 3.8 vs. 7.7 +/- 2.9 mM/min at pHi 6.6) and was inhibited by amiloride and Na+ removal. In the presence of HCO3-, baseline pHi was higher in both groups and decreased after amiloride administration. Amiloride and Na+ removal inhibited pHi recovery after an intracellular acid load by 77 and 93%, respectively, in nonactivated and by 82 and 92%, respectively, in activated HSC, whereas 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibited pHi recovery by only 27%. Acute Cl- removal increased pHi by 0.07 +/- 0.01 pH unit/min in the absence but not in the presence of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid in nonactivated and activated HSC in an Na(+)-independent manner. In activated HSC, 24 h of incubation with 25 ng/ml platelet-derived growth factor (PDGF)-BB (in 0.5% serum) did not modify baseline pHi (7.07 +/- 0.1 vs. 7.08 +/- 0.1 in HSC cultured in 0.5% serum only) but significantly (P < 0.02) increased, with respect to controls, pHi recovery after an acute acid load. Incubation with PDGF for 24 h induced a fivefold increase in HSC proliferation expressed as percentage of bromodeoxyuridine-positive cells (30.8 +/- 6.7 vs. 6.1 +/- 1.9% in controls). When amiloride (0.1 mM) was present, PDGF-induced HSC proliferation was significantly inhibited (8.1 +/- 0.4%, P < 0.001). Our results show that 1) the Na+/H+ exchanger is the main pHi regulator in rat HSC, 2) activation of HSC is associated with an increase in pHi and in the activity of the Na+/H+ exchanger, 3) PDGF increases the activity of this exchanger, and 4) amiloride is able to inhibit HSC proliferation induced by PDGF.
authors.affiliation: Department of Gastroenterology, University of Ancona, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Di Sario
authors.forename: G S
authors.initials: GS
authors.lastname: Baroni
authors.forename: E
authors.initials: E
authors.lastname: Bendia
authors.forename: L
authors.initials: L
authors.lastname: D'Ambrosio
authors.forename: F
authors.initials: F
authors.lastname: Ridolfi
authors.forename: J R
authors.initials: JR
authors.lastname: Marileo
authors.forename: A M
authors.initials: AM
authors.lastname: Jezequel
authors.forename: A
authors.initials: A
authors.lastname: Benedetti
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002021:Buffers; D002455:Cell Division; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D008297:Male; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism
chemical_list: D001639:Bicarbonates; D002021:Buffers; D002712:Chlorides; D005452:Fluoresceins; D005456:Fluorescent Dyes; D010982:Platelet-Derived Growth Factor; D017923:Sodium-Hydrogen Exchangers; D000643:Ammonium Chloride; D000584:Amiloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid