Menè P, Pugliese F, Cinotti G A
Division of Nephrology, University of Rome La Sapienza, Rome, Italy.
J Am Soc Nephrol. 1996 Jul;7(7):983-90. doi: 10.1681/ASN.V77983.
Sustained Ca2+ influx follows discharge of intracellularly stored Ca2+ in a variety of cell types previously equilibrated in Ca(2+)-free media, including cultured human mesangial cells. This Ca2+ influx pathway has been referred to as capacitative Ca2+ entry or Ca2+ release-activated Ca2+ influx (iCRAC). This study investigated two cellular mechanisms potentially controlling iCRAC in human mesangial cells, protein kinase C (PKC), a key signalling kinase activated by vasoconstrictors that release Ca2+ from internal stores, and calmodulin, a Ca(2+)-binding protein that may couple Ca2+ release to the putative channel(s). The PKC activator phorbol myristate acetate (PMA) dose-dependently inhibited both Ca2+ influx in resting cells and iCRAC, assessed by microfluorometry in fura-2-loaded monolayers, when added before or after 1 uM angiotensin II (AngII) (Ca2+ influx at 1 mM (Ca2+)e +278 +/- 56%/+80 +/- 8%, at 10 mM + 473 +/- 59%/+250 +/- 24% (Ca2+)e, -/+ PMA, respectively, P < 0.05). PMA did not affect 5 uM ionomycin-induced iCRAC, possibly because it downregulated Ca2+ release by AngII but not by ionomycin, suggesting a key role of released Ca2+ in triggering subsequent Ca2+ influx. This was confirmed by buffering the (Ca2+)i elevation induced by AngII with intracellularly trapped 1,2-bis-(0-Aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), which abolished any subsequent iCRAC. Moreover, the calmodulin inhibitors calmidazolium (10 uM), trifluoperazine (0.1 mM), or W-7 (0.1 mM) significantly inhibited AngII- or ionomycin-activated iCRAC (+106 +/- 38/229 +/- 53, +58 +/- 9/195 +/- 29, +161 +/- 38/180 +/- 40% at 1/10 mM (Ca2+)e, all P < 0.05), but did not affect basal Ca2+ entry, consistent with a direct role of cytoplasmic Ca2+ in the regulation of ion gating. These results indicate that iCRAC is under the control of both PKC and calmodulin, and that the site of regulation is distal to the emptying of Ca2+ stores. iCRAC may represent a key mechanism for the control of Ca(2+)-regulated mesangial functions.
在先前在无钙培养基中平衡的多种细胞类型中，包括培养的人系膜细胞，细胞内储存的Ca2+释放后会持续有Ca2+内流。这种Ca2+内流途径被称为容量性Ca2+内流或Ca2+释放激活的Ca2+内流（iCRAC）。本研究调查了两种可能控制人系膜细胞中iCRAC的细胞机制，蛋白激酶C（PKC），一种由从内部储存中释放Ca2+的血管收缩剂激活的关键信号激酶，以及钙调蛋白，一种可能将Ca2+释放与假定通道偶联的Ca2+结合蛋白。PKC激活剂佛波醇肉豆蔻酸酯乙酸酯（PMA）剂量依赖性地抑制静息细胞中的Ca2+内流和iCRAC，通过在负载fura-2的单层细胞中进行微荧光测定来评估，当在1 μM血管紧张素II（AngII）之前或之后添加时（在1 mM（Ca2+）e时Ca2+内流为+278±56%/+80±8%，在10 mM时为+473±59%/+250±24%（Ca2+）e，分别为-/+PMA，P<0.05）。PMA不影响5 μM离子霉素诱导的iCRAC，可能是因为它下调了AngII诱导的Ca2+释放，但没有下调离子霉素诱导的Ca2+释放，这表明释放的Ca2+在触发随后的Ca2+内流中起关键作用。用细胞内捕获的1,2-双-（邻氨基苯氧基）乙烷-N,N,N',N'-四乙酸（BAPTA）缓冲AngII诱导的（Ca2+）i升高证实了这一点，这消除了任何随后的iCRAC。此外，钙调蛋白抑制剂氯咪达唑（10 μM）、三氟拉嗪（0.1 mM）或W-7（0.1 mM）显著抑制AngII或离子霉素激活的iCRAC（在1/10 mM（Ca2+）e时分别为+106±38/229±53、+58±9/195±29、+161±38/180±40%，所有P<0.05），但不影响基础Ca2+内流，这与细胞质Ca2+在离子门控调节中的直接作用一致。这些结果表明，iCRAC受PKC和钙调蛋白的控制，并且调节位点在Ca2+储存排空的远端。iCRAC可能代表了控制Ca2+调节的系膜功能的关键机制。
