Intracellular pathways mediating Na+/H+ exchange activation by platelet-derived growth factor in rat hepatic stellate cells.
OBJECTIVE
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.
Di Sario
A
A
Department of Gastroenterology, University of Ancona, Ancona, Italy.
Bendia
E
E
Svegliati Baroni
G
G
Ridolfi
F
F
Bolognini
L
L
Feliciangeli
G
G
Jezequel
A M
AM
Orlandi
F
F
Benedetti
A
A
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007424:Intracellular Fluid / Q000378:metabolism*; D017136:Ion Transport / Q000187:drug effects; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology
D002147:Calmodulin; D010982:Platelet-Derived Growth Factor; D017923:Sodium-Hydrogen Exchangers; D019284:Thapsigargin; D019869:Phosphatidylinositol 3-Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium