Capacitative calcium entry and TRPC channel proteins are expressed in rat distal pulmonary arterial smooth muscle
Capacitative calcium entry and TRPC channel proteins are expressed in rat distal pulmonary arterial smooth muscle
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DOI：
10.1152/ajplung.00319.2003
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发表时间：
2004-04-01
影响因子：
4.9
通讯作者：
Sylvester, JT
中科院分区：
文献类型：
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作者：
Wang, J;Shimoda, LA;Sylvester, JT
果蝇瞬时受体电位（TRP）基因的哺乳动物同源物编码TRPC蛋白，其构成阳离子通道，发挥几种假定的作用，包括由内质网（ER）中Ca 2+储存耗尽触发的Ca 2+进入。这种容量性钙内流（CCE）被认为补充了Ca 2+储存并有助于许多组织中的信号传导，包括来自主肺动脉的平滑肌细胞（PASMCs）;然而，CCE和TRPC蛋白在来自远端肺动脉的PASMCs中的作用仍然不确定，这被认为是肺血管反应性的主要部位。作为TRPC通道对远端PASMCs中CCE和Ca 2+信号传导的可能性的初步测试，我们通过从大鼠肺内动脉（>第4代）分离的肌细胞的原代培养物中的fura-2荧光测量[Ca 2 +]（i）。在用含有cyclopiazonic acid（10 μ M）和nifedipine（5 μ M）的无钙培养基灌注细胞以耗尽ER Ca 2+储存并阻断电压依赖性Ca 2+通道的细胞中，细胞外Ca 2+（2.5 mM）的恢复引起[Ca 2 +]（i）的显著增加，而MnCl 2（200 μ M）淬灭了fura-2荧光，表明CCE。SKF-96365、LaCl 3和NiCl 2在不改变Ca 2+对60 mM KCl反应的浓度下阻断CCE（IC 50分别为6.3、40.4和191 μ M）。从远端肺内动脉和PASMCs分离的RNA和蛋白质进行RT-PCR和Western印迹显示TRPC 1，- 4和- 6的mRNA和蛋白质表达，但不TRPC 2，- 3，- 5或- 7。我们的研究结果表明，CCE通过TRPC编码的Ca 2+通道可能有助于远端肺内动脉肌细胞的Ca 2+信号。
Mammalian homologs of transient receptor potential (TRP) genes in Drosophila encode TRPC proteins, which make up cation channels that play several putative roles, including Ca2+ entry triggered by depletion of Ca2+ stores in endoplasmic reticulum (ER). This capacitative calcium entry (CCE) is thought to replenish Ca2+ stores and contribute to signaling in many tissues, including smooth muscle cells from main pulmonary artery (PASMCs); however, the roles of CCE and TRPC proteins in PASMCs from distal pulmonary arteries, which are thought to be the major site of pulmonary vasoreactivity, remain uncertain. As an initial test of the possibility that TRPC channels contribute to CCE and Ca2+ signaling in distal PASMCs, we measured [Ca2+](i) by fura-2 fluorescence in primary cultures of myocytes isolated from rat intrapulmonary arteries (>4th generation). In cells perfused with Ca2+-free media containing cyclopiazonic acid ( 10 muM) and nifedipine ( 5 muM) to deplete ER Ca2+ stores and block voltage-dependent Ca2+ channels, restoration of extracellular Ca2+ (2.5 mM) caused marked increases in [Ca2+](i) whereas MnCl2 (200 muM) quenched fura-2 fluorescence, indicating CCE. SKF-96365, LaCl3, and NiCl2, blocked CCE at concentrations that did not alter Ca2+ responses to 60 mM KCl (IC50 6.3, 40.4, and 191 muM, respectively). RT-PCR and Western blotting performed on RNA and protein isolated from distal intrapulmonary arteries and PASMCs revealed mRNA and protein expression for TRPC1, - 4, and - 6, but not TRPC2, - 3, - 5, or - 7. Our results suggest that CCE through TRPC-encoded Ca2+ channels could contribute to Ca2+ signaling in myocytes from distal intrapulmonary arteries.