Miyashita T, Kubo Y
Department of Neurophysiology, Tokyo Metropolitan Institute for Neuroscience, Fuchu-city, Japan.
Recept Channels. 2000;7(2):77-91.
We previously reported that the metabotropic glutamate receptor R1alpha (mGluR1alpha) can be activated not only by applying glutamate but also by raising extracellular Ca2+ (Ca2+o) concentration, and that the constant stimulation by Ca2+o causes morphological change of transfected Chinese Hamster Ovary (CHO) cells (Kubo Y Miyashita T and Murata Y (1998) Science 279, 1722-1725). The physiological role of the Ca2+o-sensing function of mGluR1alpha, however, is not fully clear yet, especially because Ca2+ is constitutively present in the extracellular space unlike other neurotransmitters. In this work, we aimed to elucidate the physiological significance of the Ca2+o-sensing function of mGluR1alpha. The effect of mGluR1alpha activation by Ca2+o on the morphological change of CHO cells was mimicked by forskolin. The effect of mGluR1alpha activation on the morphological change was suppressed by the inhibitors of adenylate cyclase, protein kinase A (PKA) and MAP kinase kinase (MAPKK), and the effect of forskolin was also decreased by the inhibitors of PKA and MAPKK. These results demonstrate the involvement of cAMP, PKA, MAPKK, MAPK pathway in the morphological change. We actually confirmed that the Ca2+o stimulation of mGluR1alpha increased the basal cAMP level of transfected CHO cells. This increase in cAMP was observed even when only the membrane fraction of mGluR1alpha transfected CHO cells were used, and the increase was inhibited by anti-Gs alpha antibody. Taken together, we concluded that the Ca2+o-sensing function of mGluR1alpha and the continuous stimulation by Ca2+o caused the increase in the basal cAMP level by direct coupling with Gs, and triggered the subsequent activation of PKA, MAPKK, and MAPK cascade which resulted in the morphological change of transfected CHO cells.
我们之前报道过，代谢型谷氨酸受体R1α（mGluR1α）不仅可以通过应用谷氨酸来激活，还可以通过提高细胞外Ca2+（Ca2+o）浓度来激活，并且Ca2+o的持续刺激会导致转染的中国仓鼠卵巢（CHO）细胞发生形态变化（久保洋、宫下哲和村田洋（1998年）《科学》279卷，1722 - 1725页）。然而，mGluR1α的Ca2+o感知功能的生理作用尚未完全明确，特别是因为与其他神经递质不同，Ca2+在细胞外空间中是持续存在的。在这项研究中，我们旨在阐明mGluR1α的Ca2+o感知功能的生理意义。毛喉素模拟了Ca2+o对mGluR1α的激活对CHO细胞形态变化的影响。腺苷酸环化酶、蛋白激酶A（PKA）和丝裂原活化蛋白激酶激酶（MAPKK）的抑制剂抑制了mGluR1α激活对形态变化的影响，并且毛喉素的作用也被PKA和MAPKK的抑制剂降低。这些结果证明了cAMP、PKA、MAPKK、MAPK信号通路参与了形态变化。我们实际上证实，Ca2+o对mGluR1α的刺激增加了转染的CHO细胞的基础cAMP水平。即使仅使用mGluR1α转染的CHO细胞的膜部分，也观察到了这种cAMP的增加，并且这种增加被抗Gsα抗体抑制。综上所述，我们得出结论，mGluR1α的Ca2+o感知功能以及Ca2+o的持续刺激通过与Gs直接偶联导致基础cAMP水平升高，并引发随后的PKA、MAPKK和MAPK级联反应的激活，从而导致转染的CHO细胞发生形态变化。
