Xu X, Star R A, Tortorici G, Muallem S
Department of Physiology, University of Texas Southwestern Medical Center, Dallas 75235-9040.
J Biol Chem. 1994 Apr 29;269(17):12645-53.
The mechanism of activation of the agonist-stimulated Ca2+ entry pathway in the plasma membrane is not known. To determine the role of nitric-oxide synthase (NOS) and cGMP in the regulation of this pathway, we used intact and streptolysin O (SLO)-permeable pancreatic acini and measured the relationship between Ca2+ release from internal stores, the NO metabolic pathway, generation of cGMP, and activation of Ca2+ entry. We found that agonist- or thapsigargin (Tg)-activated Ca2+ entry is inhibited by L-NA, a specific inhibitor of NOS, and by LY83583, an inhibitor of guanylyl cyclase. Inhibition of Ca2+ entry by inhibition of NOS was reversed by the NO releasing molecules NO2- and sodium nitroprusside (SNP) and by Bt2cGMP. Inhibition of Ca2+ entry by inhibition of guanylyl cyclase was reversed by Bt2cGMP, but not by the NO releasing agents. The use of L-NA-treated cells and different concentrations of SNP revealed that cGMP has a dual effect on Ca2+ entry. Increasing cGMP up to 10-fold above control activated Ca2+ entry. Further increase in cGMP up to 80-fold above control inhibited Ca2+ entry in a concentration-dependent manner. Measurement of cellular cGMP in intact cells showed that carbachol, Tg, and NO2- increased cGMP to similar levels. The effects of carbachol and Tg were inhibited by L-NA and LY83586, whereas the effect of NO2- was inhibited only by LY83583. SLO-permeabilized cells were shown to be agonist-competent in that the agonist induced Ca2+ release from the inositol 1,4,5-trisphosphate (IP3) pool and activated a NO-dependent generation of cGMP. These cells were used to study the regulation of NOS by Ca2+ and by Ca2+ content of the internal stores. When internal stores were maintained loaded with Ca2+, increasing medium [Ca2+] up to 2.5 microM only modestly increased NOS activity. In contrast, the depletion of Ca2+ from internal stores markedly increased NOS activity independent of medium [Ca2+]. Thus, NOS senses both cytosolic [Ca2+]i and internal store Ca2+ load. We propose that activation of Ca2+ entry involves an agonist-mediated Ca2+ release from internal stores which activates a cellular pool of NOS to generate cGMP, which then modulates Ca2+ entry pathway in the plasma membrane. This mechanism can explain the capacitative nature of Ca2+ entry. The biphasic effect of cGMP provides the cells with a negative feedback mechanism which inhibits Ca2+ entry during periods of high cell [Ca2+]i. This could allow oscillatory behavior of Ca2+ entry.
质膜中激动剂刺激的Ca2+内流途径的激活机制尚不清楚。为了确定一氧化氮合酶（NOS）和cGMP在该途径调节中的作用，我们使用了完整的和经链球菌溶血素O（SLO）通透的胰腺腺泡，并测量了从内部储存库释放Ca2+、NO代谢途径、cGMP的生成以及Ca2+内流激活之间的关系。我们发现，NOS的特异性抑制剂L-NA和鸟苷酸环化酶抑制剂LY83583可抑制激动剂或毒胡萝卜素（Tg）激活的Ca2+内流。通过释放NO的分子NO2-和硝普钠（SNP）以及Bt2cGMP可逆转抑制NOS对Ca2+内流的抑制作用。抑制鸟苷酸环化酶对Ca2+内流的抑制作用可被Bt2cGMP逆转，但不能被释放NO的试剂逆转。使用经L-NA处理的细胞和不同浓度的SNP表明，cGMP对Ca2+内流具有双重作用。将cGMP增加至比对照高10倍可激活Ca2+内流。cGMP进一步增加至比对照高80倍则以浓度依赖性方式抑制Ca2+内流。对完整细胞中细胞内cGMP的测量表明，卡巴胆碱、Tg和NO2-可使cGMP增加至相似水平。卡巴胆碱和Tg的作用可被L-NA和LY83586抑制，而NO2-的作用仅被LY83583抑制。SLO通透的细胞显示对激动剂有反应，因为激动剂可诱导从肌醇1,4,5-三磷酸（IP3）池中释放Ca2+并激活NO依赖性的cGMP生成。这些细胞用于研究Ca2+和内部储存库的Ca2+含量对NOS的调节作用。当内部储存库保持Ca2+负载时，将培养基[Ca2+]增加至2.5μM仅适度增加NOS活性。相反，从内部储存库中耗尽Ca2+可显著增加NOS活性，而与培养基[Ca2+]无关。因此，NOS可感知胞质[Ca2+]i和内部储存库的Ca2+负载。我们提出，Ca2+内流的激活涉及激动剂介导的从内部储存库释放Ca2+，这激活了细胞内的NOS池以生成cGMP，然后cGMP调节质膜中的Ca2+内流途径。该机制可以解释Ca2+内流的容量性质。cGMP的双相作用为细胞提供了一种负反馈机制，可在细胞[Ca2+]i较高时抑制Ca2+内流。这可能允许Ca2+内流的振荡行为。
