Expression of P2Y nucleotide receptors and ectonucleotidases in quiescent and activated rat hepatic stellate cells.
Extracellular nucleotides regulate a variety of cellular activities, including proliferation of fibrogenic cells outside of the liver. However, the expression of receptors for extracellular nucleotides in hepatic stellate cells (HSC) is unknown. Thus our aims were to investigate the expression of mediators of nucleotide signaling in HSC and to determine whether extracellular nucleotides regulate HSC function. Confocal video microscopy was used to observe nucleotide-induced changes in cytosolic Ca(2+) (Ca(i)(2+)) in live HSC. P2Y receptor subtype expression and ectonucleotidase expression in quiescent and activated HSC were determined using RT-PCR, Northern blot, immunoblot, and confocal immunofluorescence. Functional ectonucleotidase activity was assessed using a colorimetric method. Nucleotide-sensitive procollagen-1 mRNA expression in activated HSC was assessed using real-time RT-PCR. Extracellular ATP increased Ca(i)(2+) in HSC; this was inhibited by the P2 receptor inhibitor suramin. Quiescent HSC expressed the P2Y subtypes P2Y(2) and P2Y(4) and were activated by ATP and UTP, whereas activated HSC expressed the P2Y subtype P2Y(6) and were activated by UDP and ATP. Activated but not quiescent HSC expressed the ectonucleotidase nucleoside triphosphate diphosphohydrolase 2, extracellular UDP tripled procollagen-1 mRNA expression in activated HSC, and this was inhibited by the P2Y receptor inhibitor suramin. HSC express functional P2Y receptors and switch the expression of P2Y receptor subtypes on activation. Moreover, HSC differentially regulate nucleoside triphosphate diphosphohydrolase expression after activation. Because activation of P2Y receptors in activated HSC regulates procollagen-1 transcription, P2Y receptors may be an attractive target to prevent or treat liver fibrosis.
Dranoff
Jonathan A
JA
Yale Univ. School of Medicine, Section of Digestive Diseases, 333 Cedar St. LMP 1080, New Haven, CT 06520, USA. jonathan.dranoff@yale.edu
Ogawa
Mika
M
Kruglov
Emma A
EA
Gaça
Marianna D A
MD
Sévigny
Jean
J
Robson
Simon C
SC
Wells
Rebecca G
RG
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D024042:Collagen Type I / Q000235:genetics; D003600:Cytosol / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D043583:Nucleoside-Triphosphatase / Q000378:metabolism; D009708:Nucleotidases / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058473:Receptors, Purinergic P2Y2; D014530:Uridine Diphosphate / Q000494:pharmacology; D014544:Uridine Triphosphate / Q000494:pharmacology
D024042:Collagen Type I; C546387:P2ry2 protein, rat; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; C104749:purinoceptor P2Y4; C099845:purinoceptor P2Y6; D014530:Uridine Diphosphate; D000255:Adenosine Triphosphate; D009708:Nucleotidases; D043583:Nucleoside-Triphosphatase; D002118:Calcium; D014544:Uridine Triphosphate