Iwamuro Y, Zhang X F, Okamoto Y, Miwa S, Masaki T
Department of Pharmacology, Kyoto University Faculty of Medicine, Japan.
Nihon Yakurigaku Zasshi. 1999 Oct;114 Suppl 1:96P-102P. doi: 10.1254/fpj.114.supplement_96.
To clarify Ca2+ entry channels involved in the endothelin-1 (ET-1)-induced increase in the intracellular concentration ([Ca2+]i), we performed whole-cell recordings of patch-clamp techniques and monitoring of [Ca2+]i with Ca2+ indicators fura-2 and fluo-3 in A7r5 cells (a cell line derived from rat thoracic aortic smooth muscle cells). With whole-cell recordings, lower concentrations (< or = 1 nM) of ET-1 activated a Ca(2+)-permeable nonselective cation channel (designated NSCC-1). In contrast, higher concentrations (> or = 1 nM) of ET-1 activated two types of Ca(2+)-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) and store-operated Ca2+ channel (SOCC). Importantly, we found that these Ca2+ channels can be pharmacologically discriminated using blockers of the so-called receptor operated Ca2+ influx such as SK&F 96365 and LOE 908. That is, NSCC-1 is resistant to SK&F 96365 but sensitive to LOE 908; NSCC-2 is sensitive to both SK&F 96365 and LOE 908; SOCC is sensitive to SK&F 96365 but resistant to LOE 908. Using these blockers, we analyzed the ET-1-induced increase in [Ca2+]i. The increase in [Ca2+]i induced by lower concentrations of ET-1 was resistant to SK&F 96365 but sensitive to LOE 908. In contrast, the increase in [Ca2+]i induced by higher concentrations of ET-1 was partially suppressed to approximately 30% of controls by either SK&F 96365 or LOE 908 alone, and it was abolished by their combination. These results show that the increase in [Ca2+]i induced by lower concentrations (< or = 1 nM) of ET-1 results from Ca2+ influx through NSCC-1, whereas the increase in [Ca2+]i induced by higher concentrations (> or = 10 nM) of ET-1 results from Ca2+ influx through NSCC-1, NSCC-2 and SOCC.
为了阐明参与内皮素 -1（ET -1）诱导的细胞内钙离子浓度（[Ca2+]i）升高的钙离子进入通道，我们采用膜片钳技术进行全细胞记录，并使用钙离子指示剂fura -2和fluo -3监测A7r5细胞（一种源自大鼠胸主动脉平滑肌细胞的细胞系）中的[Ca2+]i。通过全细胞记录发现，较低浓度（≤1 nM）的ET -1激活了一种钙离子通透的非选择性阳离子通道（命名为NSCC -1）。相反，较高浓度（≥1 nM）的ET -1激活了两种类型的钙离子通透的非选择性阳离子通道（命名为NSCC -1和NSCC -2）以及储存 - 操作性钙离子通道（SOCC）。重要的是，我们发现可以使用所谓的受体操纵性钙离子内流阻滞剂（如SK&F 96365和LOE 908）从药理学上区分这些钙离子通道。也就是说，NSCC -1对SK&F 96365有抗性，但对LOE 908敏感；NSCC -2对SK&F 96365和LOE 908均敏感；SOCC对SK&F 96365敏感，但对LOE 908有抗性。使用这些阻滞剂，我们分析了ET -1诱导的[Ca2+]i升高情况。较低浓度的ET -1诱导的[Ca2+]i升高对SK&F 96365有抗性，但对LOE 908敏感。相反，较高浓度的ET -1诱导的[Ca2+]i升高单独使用SK&F 96365或LOE 908时被部分抑制至对照的约30%，而两者联合使用则可将其完全消除。这些结果表明，较低浓度（≤1 nM）的ET -1诱导的[Ca2+]i升高是由钙离子通过NSCC -1内流所致，而较高浓度（≥10 nM）的ET -1诱导的[Ca2+]i升高是由钙离子通过NSCC -1、NSCC -2和SOCC内流所致。
