Canaff L, Petit J L, Kisiel M, Watson P H, Gascon-Barré M, Hendy G N
Departments of Medicine, Physiology and Human Genetics, McGill University and Royal Victoria Hospital, Montreal, Quebec H3A 1A1, Canada.
J Biol Chem. 2001 Feb 9;276(6):4070-9. doi: 10.1074/jbc.M009317200. Epub 2000 Nov 8.
Liver cells respond to changes in Ca(2+)(o). The hepatic functions affected include bile secretion, metabolic activity, liver regeneration, and the response to xenobiotics. In the present study, we demonstrate the presence, in the liver, of the extracellular calcium-sensing receptor (CASR), described previously in the parathyroid and thyroid glands and kidney. CASR mRNA was specifically expressed in hepatocytes and was absent in nonparenchymal liver cells (stellate, endothelial, and Kupffer cells). Western blot analysis using a specific CASR antibody showed staining in both whole liver and hepatocyte extracts. Immunohistochemistry and in situ hybridization of rat liver sections showed expression of CASR protein and mRNA by a subset of hepatocytes. The known agonists of the CASR, gadolinium (Gd(3+); 0.5-3.0 mm) and spermine (1.25-20 mm), in the absence of Ca(2+)(o), elicited dose-related increases in Ca(2+)(i) in isolated rat hepatocytes loaded with Fura-2/acetoxymethyl ester. There was a greatly attenuated response to a second challenge with either agonist. The response was also abrogated when inositol 1,4,5-trisphosphate (IP(3))-sensitive calcium pools had been depleted by pretreatment with either thapsigargin or phenylephrine, an alpha(1)-adrenergic receptor agonist known to mobilize Ca(2+)(i) from IP(3)-sensitive pools. Addition of the deschloro-phenylalkylamine compound, NPS R-467, but not the S enantiomer, NPS S-467, increased the sensitivity of the Ca(2+)(i) mobilization response to 1.25 mm spermine. Bile flow ceased after Ca(2+)(o) withdrawal, and its recovery was enhanced by spermine in isolated perfused liver preparations. The CASR agonists Ca(2+) and Gd(3+) increased bile flow, and the response to a submaximal Ca(2+) concentration was enhanced by NPS R-467 but not the S compound. Thus, the data indicate that rat hepatocytes harbor a CASR capable of mobilizing Ca(2+)(i) from IP(3)-sensitive stores and that activation of the CASR stimulates bile flow.
肝细胞会对细胞外钙离子（Ca(2+)(o)）的变化做出反应。受影响的肝脏功能包括胆汁分泌、代谢活性、肝脏再生以及对外源生物的反应。在本研究中，我们证明了肝脏中存在细胞外钙敏感受体（CASR），该受体先前已在甲状旁腺、甲状腺和肾脏中被描述。CASR信使核糖核酸（mRNA）在肝细胞中特异性表达，而在非实质肝细胞（星状细胞、内皮细胞和库普弗细胞）中不存在。使用特异性CASR抗体的蛋白质印迹分析显示，在全肝提取物和肝细胞提取物中均有染色。大鼠肝脏切片的免疫组织化学和原位杂交显示，一部分肝细胞表达CASR蛋白和mRNA。在无细胞外钙离子（Ca(2+)(o)）的情况下，已知的CASR激动剂钆（Gd(3+)；0.5 - 3.0毫米）和精胺（1.25 - 20毫米），可使负载有Fura - 2/乙酰氧甲酯的分离大鼠肝细胞中的细胞内钙离子（Ca(2+)(i)）呈剂量相关增加。对用任一激动剂进行的第二次刺激的反应大大减弱。当用毒胡萝卜素或苯肾上腺素预处理耗尽了1,4,5 - 三磷酸肌醇（IP(3)）敏感的钙库时，反应也被消除，苯肾上腺素是一种已知可从IP(3)敏感库中动员细胞内钙离子（Ca(2+)(i)）的α(1) - 肾上腺素能受体激动剂。添加去氯苯烷基胺化合物NPS R - 467（而非其S对映体NPS S - 467）可增加细胞内钙离子（Ca(2+)(i)）动员反应对1.25毫米精胺的敏感性。在分离的灌注肝脏制剂中，去除细胞外钙离子（Ca(2+)(o)）后胆汁流动停止，而精胺可增强其恢复。CASR激动剂钙离子（Ca(2+)）和钆（Gd(3+)）可增加胆汁流动，并且NPS R - 467可增强对次最大钙离子（Ca(2+)）浓度的反应，而S化合物则不能。因此，数据表明大鼠肝细胞含有一种能够从IP(3)敏感储存库中动员细胞内钙离子（Ca(2+)(i)）的CASR，并且CASR的激活会刺激胆汁流动。
