Search

Background & Aims: The Na + /H + exchanger is the main intracellular pH regulator in hepatic stellate cells (HSCs), and its activity is increased by platelet-derived growth factor (PDGF). Amiloride, an Na + /H + exchange inhibitor, reduces PDGF-induced HSC proliferation, suggesting that the Na + /H + exchanger plays a role in regulating HSC proliferative response. The aim of this study was to characterize the intracellular pathways mediating activation of the Na + /H + exchanger by PDGF in HSCs. Methods: The activity of the Na + /H + exchanger and HSC proliferation rate were evaluated under control condition and after incubation with PDGF in the absence or presence of specific inhibitors of the main intracellular pathways of signal transduction. Na + /H + exchange protein expression was evaluated by means of Western blot. Results: PDGF induced a significant increase in the activity of the Na + /H + exchanger without modifying protein expression. Inhibition of the calcium/calmodulin– and protein kinase C–dependent pathways resulted in a significant inhibition of both Na + /H + exchange activity and of PDGF-induced HSC proliferation. The involvement of the two pathways was confirmed by showing that incubation of HSCs with both phorbol-12-myristate-13-acetate, a potent protein kinase C activator, and thapsigargin, which increases intracellular calcium levels, significantly increased both the Na + /H + exchanger activity and HSC proliferation rate. Inhibition of the protein kinase A pathway did not modify either PDGF-induced Na + /H + exchange activation or PDGF-induced HSC proliferation. On the contrary, inhibition of the mitogen-activated protein kinase– and of phosphatidylinositol 3-kinase–dependent pathways significantly reduced PDGF-induced HSC proliferation without affecting the activity of the Na + /H + exchanger. Conclusions: Activation of the Na + /H + exchanger by PDGF in HSCs is mediated by calcium/calmodulin- and protein kinase C–dependent pathways. PDGF-induced HSC proliferation is mediated by Na + /H + exchange–dependent and –independent pathways. GASTROENTEROLOGY 1999;116:1155-1166

Background & Aims: We evaluated the expression of neurotrophins in rat cholangiocytes and the role and mechanisms by which nerve growth factor (NGF) modulates cholangiocyte proliferation. Methods: The expression of neurotrophins and their receptors was investigated by immunohistochemistry in liver sections and reverse-transcription polymerase chain reaction and immunoblots in isolated cholangiocytes. In vitro, the effect of NGF on cholangiocyte proliferation and signal transduction was investigated by immunoblotting for proliferating cell nuclear antigen, phosphorylated AKT (p-AKT), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), phosphorylated c-jun-N-terminal kinase, and phosphorylated p38. In vivo, rats that had undergone bile duct ligation (BDL) were treated with an anti-NGF antibody to immunoneutralize NGF and bile duct mass, proliferation, apoptosis, and inflammation were investigated by immunohistochemistry. Results: NGF and its TrkA receptor were expressed by normal rat cholangiocytes and up-regulated following BDL. Cholangiocytes secrete NGF, and secretion is increased in proliferating BDL cholangiocytes. In vitro, NGF stimulated cholangiocyte proliferation, which was associated with enhanced p-AKT and p-ERK1/2 expression. NGF proliferation in vitro was partially blocked by the MEK inhibitor (UO126) and completely ablated by the phosphatidylinositol 3-kinase inhibitor (wortmannin). In vitro, NGF and estrogens have an additive effect on cholangiocyte proliferation by acting on phosphorylated TrkA and p-ERK1/2. In vivo, immunoneutralization of NGF decreased bile duct mass in BDL rats, which was associated with depressed proliferation and enhanced apoptosis and with increased portal inflammation. Conclusions: Cholangiocytes secrete NGF and express NGF receptors. NGF induces cholangiocyte proliferation by activating the ERK and, predominantly, the phosphatidylinositol 3-kinase pathway and exerts an additive effect in combination with estrogens on proliferation.

Background & Aims: The ligand-dependent transcription factor peroxisome proliferator-activated receptor γ (PPARγ) is expressed in hepatic stellate cells (HSC), and its transcriptional activity is reduced during cell transdifferentiation in culture. PPARγ transcriptional activation decreases platelet-derived growth factor–induced proliferation and inhibits α-smooth muscle actin expression in cultured HSC. The aim of our study was to evaluate whether oral administration of synthetic PPARγ ligands, thiazolidinediones (TZD), might affect collagen deposition in animal models of liver fibrosis. Methods: The effect of 2 TZD (pioglitazone or rosiglitazone) was tested on liver fibrosis induced in rats by either toxin administration (dimethylnitrosamine or carbon tetrachloride) or bile duct ligation. In vivo PPARγ activation was evaluated by gel shift assay using nuclear extracts from HSC isolated from control and treated rats. Results: Oral administration of TZD reduced extracellular matrix deposition and HSC activation in both toxic and cholestatic models of liver fibrosis. PPARγ-specific DNA binding was significantly impaired in nuclear extracts of HSC isolated from fibrotic rats compared with HSC from control rats. TZD administration restored PPARγ DNA binding in HSC nuclei. In vitro, TZD-induced PPARγ activation inhibited collagen and fibronectin synthesis induced by transforming growth factor (TGF)-β1 in human HSC, as measured by enzyme-linked immunosorbent assay and Northen blotting. TZD also reduced the TGF-β1–induced activity of a 3.5-kilobase procollagen type I promoter transfected in human HSC. Conclusions: These findings indicate that PPARγ activation in HSC retards fibrosis in vivo and suggest the use of TZD for the treatment of liver fibrosis. GASTROENTEROLOGY 2002;122:1924-1940

Background & Aims: The Na + /H + exchanger is the main intracellular pH (pH i ) regulator in hepatic stellate cells (HSCs) and plays a key role in regulating proliferation and gene expression. We evaluated the effect of specific inhibition of this exchanger on HSC proliferation and collagen synthesis in vivo and in vitro. Methods: Rat HSCs were incubated in the presence of platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β1, iron ascorbate (FeAsc), and ferric nitrilotriacetate solution (FeNTA) with or without the Na + /H + exchanger inhibitor 5- N -ethyl- N -isopropyl-amiloride (EIPA). pH i and Na + /H + exchanger activity, cell proliferation, and type I collagen accumulation were measured by using the fluorescent dye 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein, by immunohistochemistry for bromodeoxyuridine, and by enzyme-linked immunosorbent assay, respectively. In vivo liver fibrosis was induced by dimethylnitrosamine administration and bile duct ligation (BDL) in rats treated or not treated with amiloride. Results: PDGF, FeAsc, and FeNTA increased Na + /H + exchange activity and induced HSC proliferation. TGF-β1 had no effect on the Na + /H + exchanger and was able, as for FeAsc and FeNTA, to induce type I collagen accumulation. EIPA inhibited all the effects determined by PDGF, FeAsc, and FeNTA and had no effect on TGF-β1–induced collagen accumulation. In vivo, amiloride reduced HSC proliferation, activation, collagen deposition, and collagen synthesis. Conclusions: The Na + /H + exchanger can play a key role in the development of liver fibrosis and in HSC activation in vivo. GASTROENTEROLOGY 2001;120:545-556

Liver acinus shows a well-known metabolic zonation. The aim of this study was to investigate intracellular pH (pHi) regulation in isolated periportal (PP) and perivenular (PV) rat hepatocytes.2,7-bis(carboxyethyl)-5(6)-carboxy-fluorescein was used as pH-sensitive dye. Hepatocyte subconfluent monolayers were acid-loaded by exposure to 20 mmol/L NH4Cl and alkali-loaded by reducing external CO2 and HCO3- at an external pH of 7.4.In the presence of HCO3-/CO2, (1) baseline pHi was higher in PP (7.25 +/- 0.018) than in PV hepatocytes (7.20 +/- 0.013) (P0.05); (2) pHi recovery from an acid load was 40% higher in PP than in PV hepatocytes (P0.02) and was inhibited by amiloride by 36% in PV and 7% in PP hepatocytes; (3) DIDS inhibited amiloride-independent pHi recovery from an acid load by 65% in PP and 52% in PV cells. In the absence of HCO3-/CO2, baseline pHi and pHi recovery from an acid load were not significantly different in PP and PV hepatocytes. pHi recovery from an alkali load was 30% higher in PV than in PP cells (P0.02).Our data suggest that isolated PP rat hepatocytes show higher activity for Na(+)-HCO3- cotransport, whereas PV cells show greater activity for Cl-/HCO3- exchanger.