Bischof G, Brenman J, Bredt D S, Machen T E
Department of Molecular & Cell Biology, University of California, Berkeley, USA.
Cell Calcium. 1995 Apr;17(4):250-62. doi: 10.1016/0143-4160(95)90071-3.
A possible role of the nitric oxide (NO)/cGMP pathway in the regulation of Ca2+ entry into HT29/B6 human colonic epithelial cells was investigated using digital image processing of Fura-2 fluorescence and immunoblotting for nitric oxide synthase (NOS). We tested the hypothesis that Ca2+ store depletion causes increased NOS activity and [NO], which is stimulatory to Ca2+ entry by increasing guanylate cyclase (GC) and [cGMP]. Cells were incubated in 95 mM K(+)-containing solutions to depolarize the cell membrane potential and thereby exclude effects of NO and CGMP on K+ or Cl- channels, which might affect Ca2+ entry. Sodium nitroprusside (SNP, 0.5 microM and 30 microM), a NO donor, only slightly raised intracellular [Ca2+] ([Ca2+]i) in resting cells, but in 100 microM carbachol-stimulated cells the sustained, elevated Ca2+ plateau (reflecting Ca2+ entry) as well as Ba2+ entry were increased by 0.5 microM SNP, while 5, 10 or 30 microM SNP either had no effect or were inhibitory. Pretreatment of cells with the NOS inhibitor N-nitro-L-arginine (1 mM) reduced carbachol-stimulated Ca2+ entry, and simultaneous treatment with 0.5 microM (but not 30 microM) SNP restored Ca2+ influx. 8-Br-cGMP (1 mM) had little effect on [Ca2+]i or on rates of Ca2+ or Ba2+ influx into resting cells, but there were large effects on cells in which capacitative Ca2+ entry was activated by carbachol or cyclopiazonic acid (10 microM). The GC inhibitor LY83583 (10 microM) reduced carbachol-stimulated Ca2+ entry, and this entry was restored with 8-Br-cGMP. Western blotting revealed that endothelial-type NOS was present in the particulate fraction of cells. The data are consistent with the notion that Ca2+ entry into HT29/B6 cells is regulated by endothelial NOS/NO and GC/cGMP, but effects are most pronounced in store-depleted cells. Thus, NO and cGMP appear to potentiate the action of messengers released from the store during the emptying process, but NO and cGMP have only small effects of their own to open the Ca2+ channel in the plasma membrane. High [SNP] appeared to be inhibitory while low [SNP] was stimulatory, indicating that a precise range of [NO] may be required for effective stimulation of Ca2+ entry.
利用Fura-2荧光的数字图像处理和一氧化氮合酶（NOS）的免疫印迹法，研究了一氧化氮（NO）/环鸟苷酸（cGMP）途径在调节Ca2+进入HT29/B6人结肠上皮细胞中的可能作用。我们检验了以下假设：Ca2+储存耗尽会导致NOS活性和[NO]增加，而[NO]通过增加鸟苷酸环化酶（GC）和[cGMP]来刺激Ca2+进入。将细胞置于含95 mM K+的溶液中孵育，以使细胞膜电位去极化，从而排除NO和CGMP对K+或Cl-通道的影响，因为这些影响可能会影响Ca2+进入。NO供体硝普钠（SNP，0.5 microM和30 microM）仅使静息细胞中的细胞内[Ca2+]（[Ca2+]i）略有升高，但在100 microM卡巴胆碱刺激的细胞中，0.5 microM SNP增加了持续升高的Ca2+平台期（反映Ca2+进入）以及Ba2+进入，而5、10或30 microM SNP要么没有影响，要么具有抑制作用。用NOS抑制剂N-硝基-L-精氨酸（1 mM）预处理细胞可降低卡巴胆碱刺激的Ca2+进入，同时用0.5 microM（但不是30 microM）SNP处理可恢复Ca2+内流。8-溴-cGMP（1 mM）对[Ca2+]i或Ca2+或Ba2+进入静息细胞的速率几乎没有影响，但对由卡巴胆碱或环匹阿尼酸（10 microM）激活的钙池操纵性Ca2+进入的细胞有很大影响。GC抑制剂LY83583（10 microM）降低了卡巴胆碱刺激的Ca2+进入，而8-溴-cGMP可恢复这种进入。蛋白质免疫印迹法显示内皮型NOS存在于细胞的微粒部分。这些数据与以下观点一致：Ca2+进入HT29/B6细胞受内皮NOS/NO和GC/cGMP调节，但在储存耗尽的细胞中作用最为明显。因此，NO和cGMP似乎增强了排空过程中从储存库释放的信使的作用，但NO和cGMP自身对打开质膜中的Ca2+通道的作用很小。高[SNP]似乎具有抑制作用，而低[SNP]具有刺激作用，这表明有效刺激Ca2+进入可能需要精确范围的[NO]。
