Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, Complutense University of Madrid, E-28040 Madrid, Spain.
Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, Complutense University of Madrid, E-28040 Madrid, Spain.
Free Radic Biol Med. 2014 Apr;69:390-402. doi: 10.1016/j.freeradbiomed.2014.01.029. Epub 2014 Jan 28.
Previous work from our group stated that nitric oxide (NO), via cytokines, induces apoptosis in chromaffin cells by a mechanism involving iNOS, nNOS, and NF-κB. In this paper the involvement of glutamate as a possible intracellular trigger of neurosecretion and NO-mediated apoptosis has been evaluated. We show that chromaffin cells express different ionotropic and metabotropic glutamate receptors, this exerting different effects on the regulation of basal and glutamate-induced catecholamine secretion, via NO/cGMP. In addition, we studied the effects of endogenously generated NO, both basal and glutamate-stimulated, on apoptosis of chromaffin cells. Our results show that glutamate agonists are able to induce cell death and apoptosis in bovine chromaffin cells, parallel to an increase in NO production. Such effects were reversed by NOS inhibitors and glutamate receptor antagonists. Under basal conditions, iNOS inhibitors did not have any effect on apoptosis, whereas nNOS inhibitors induced apoptosis, indicating a neuroprotective effect of constitutive nNOS-generated NO. In contrast, glutamate-induced apoptosis was strongly reversed by nNOS inhibitors and weakly by iNOS inhibitors, thus indicating nNOS involvement in glutamate-mediated apoptosis. These results were confirmed by the fact that nNOS expression, but not iNOS, is specifically activated by glutamate. Finally, our results suggest the participation of PKG, PKA, PKC, and MAPK pathways in glutamate-mediated nNOS activation in chromaffin cells and point out the involvement of both PKA and PKC signaling pathways in the apoptotic effect of glutamate.
先前我们的研究小组指出，一氧化氮（NO）通过细胞因子诱导嗜铬细胞凋亡的机制涉及诱导型一氧化氮合酶（iNOS）、神经元型一氧化氮合酶（nNOS）和核因子-κB（NF-κB）。在本文中，评估了谷氨酸作为神经分泌和 NO 介导的细胞凋亡的可能细胞内触发因素的作用。我们表明，嗜铬细胞表达不同的离子型和代谢型谷氨酸受体，这通过 NO/cGMP 对基础和谷氨酸诱导儿茶酚胺分泌的调节产生不同的影响。此外，我们研究了内源性产生的 NO（无论是基础水平还是谷氨酸刺激水平）对嗜铬细胞凋亡的影响。我们的结果表明，谷氨酸激动剂能够诱导牛嗜铬细胞的细胞死亡和凋亡，同时增加 NO 的产生。这种作用可以通过 NOS 抑制剂和谷氨酸受体拮抗剂逆转。在基础条件下，iNOS 抑制剂对凋亡没有任何影响，而 nNOS 抑制剂诱导凋亡，表明组成型 nNOS 产生的 NO 具有神经保护作用。相反，谷氨酸诱导的凋亡被 nNOS 抑制剂强烈逆转，而被 iNOS 抑制剂弱逆转，因此表明 nNOS 参与了谷氨酸介导的凋亡。nNOS 表达而不是 iNOS 被谷氨酸特异性激活这一事实证实了这些结果。最后，我们的结果表明 PKG、PKA、PKC 和 MAPK 途径参与了谷氨酸诱导的 nNOS 在嗜铬细胞中的激活，并且指出 PKA 和 PKC 信号通路都参与了谷氨酸的凋亡作用。
