Expression of P2Y nucleotide receptors and ectonucleotidases in quiescent and activated rat hepatic stellate cells
Expression of P2Y nucleotide receptors and ectonucleotidases in quiescent and activated rat hepatic stellate cells
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DOI：
10.1152/ajpgi.00294.2003
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发表时间：
2004-08-01
影响因子：
4.5
通讯作者：
Wells, RG
中科院分区：
文献类型：
--
作者：
Dranoff, JA;Ogawa, M;Wells, RG
细胞外核苷酸调节多种细胞活动，包括肝外纤维化细胞的增殖。然而，细胞外核苷酸受体在肝星状细胞（HSC）中的表达尚不清楚。因此，我们的目的是研究核苷酸信号介质在HSC中的表达，并确定细胞外核苷酸是否调节HSC的功能。用共聚焦视频显微镜观察活HSC中胞浆Ca2+ (Ca-i(2+))的变化。采用RT-PCR、Northern blot、免疫blot和共聚焦免疫荧光检测静止和活化HSC中P2Y受体亚型表达和外核苷酶表达。用比色法测定功能性外核苷酸酶活性。采用实时RT-PCR技术评估活化HSC中核苷酸敏感的前胶原-1 mRNA表达。细胞外ATP增加HSC中Ca-i(2+)；这被P2受体抑制剂苏拉明所抑制。静止状态的HSC表达P2Y亚型P2Y(2)和P2Y(4)，可被ATP和UTP激活，而激活的HSC表达P2Y亚型P2Y(6)，可被UDP和ATP激活。激活的而非静止的HSC表达外核苷酸酶三磷酸核苷二磷酸水解酶2，活化的HSC细胞外UDP增加了原胶原-1 mRNA的表达，这被P2Y受体抑制剂苏拉明抑制。HSC表达功能性P2Y受体，并在激活时切换P2Y受体亚型的表达。此外，HSC在激活后对核苷三磷酸二磷酸水解酶的表达有差异调节。由于活化的HSC中P2Y受体的激活调节了原胶原-1的转录，P2Y受体可能是预防或治疗肝纤维化的一个有吸引力的靶点。
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 Ca2+ (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.