CHARACTERIZATION OF ION-TRANSPORT MECHANISMS REGULATING INTRACELLULARPH IN HEPATIC STELLATE CELLS

The aim of this study was to evaluate intracellular pH (pH(i)) regulat ion in nonactivated and activated rat hepatic stellate cells (HSC). Th e fluorescent pH(i) indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluo rescein was used to measure pH(i) in the presence and absence of HCO3- . In the absence of HCO3-, baseline pH(i) 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 a nd after Na+ removal. After an acid-loading maneuver, pH(i) recovery w as significantly higher (P < 0.03) in activated than in nonactivated H SC (H+ flux = 11.0 +/- 3.8 vs. 7.7 +/- 2.9 mM/min at pH(i) 6.6) and wa s inhibited by amiloride and Na+ removal. In the presence of HCO3-, ba seline pH(i) was higher in both groups and decreased after amiloride a dministration. Amiloride and Na+ removal inhibited pH(i) recovery afte r an intracellular acid load by 77 and 93%, respectively, in nonactiva ted and by 82 and 92%, respectively, in activated HSC, whereas 4,4'-di isothiocyanostilbene-2,2'-disulfonic acid inhibited pH(i) recovery by only 27%. Acute Cl- removal increased pH(i) by 0.07 +/- 0.01 pH unit/m in in the absence but not in the presence of 4,4'-diisothiocyanostilbe ne-2,2'-disulfonic acid in nonactivated and activated HSC in an Na+-in dependent manner. In activated HSC, 24 h of incubation with 25 ng/ml p latelet-derived growth factor (PDGF)-BB (in 0.5% serum) did not modify baseline pH(i) (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 c ontrols, pH(i) recovery after an acute acid load. Incubation with PDGF for 24 h induced a fivefold increase in HSC proliferation expressed a s percentage of bromodeoxyuridine-positive cells (30.8 +/- 6.7 vs. 6.1 +/- 1.9% in controls). When amiloride (0.1 mM) was present, PDGF-indu ced HSC proliferation was significantly inhibited (8.1 +/- 0.4%, P < 0 .001). Our results show that 1) the Na+/H+ exchanger is the main pH(i) regulator in rat HSC, 2) activation of HSC is associated with an incr ease in pH(i) and in the activity of the Na+/H+ exchanger, 3) PDGF inc reases the activity of this exchanger, and 4) amiloride is able to inh ibit HSC proliferation induced by PDGF.