Masgrau R, Servitja J M, Young K W, Pardo R, Sarri E, Nahorski S R, Picatoste F
Departament de Bioquímica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Catalonia, Spain.
Eur J Neurosci. 2001 Jan;13(2):248-56. doi: 10.1046/j.0953-816x.2000.01384.x.
In this study we have determined the metabotropic glutamate receptors (mGluRs) involved in the glutamate activation of phospholipase C (PLC) and Ca(2+) mobilization in cerebellar granule cells at 9 days in vitro; and studied the Ca(2+) modulation of the PLC response. Both PLC activation and Ca(2+) signalling were found to be mediated exclusively by the mGluR1 subtype, although both group I mGluRs, mGluR1 alpha and mGluR5, could be detected in cell extracts. Exposure of cells to medium devoid of Ca(2+) for various times before agonist stimulation reduced the PLC response, which was quickly recovered following the re-exposure of cells to Ca(2+)-containing medium. The extent of the glutamate response correlated well with changes in the cytosolic Ca(2+) concentration. On the other hand, loading of the intracellular Ca(2+) stores by a transient depolarization followed by washing in nondepolarizing buffer, allowed glutamate to release stored Ca(2+) in the majority of cells and enhanced glutamate activation of PLC. Under such conditions, the absence of extracellular Ca(2+) during stimulation and the chelation of cytosolic Ca(2+) with BAPTA/AM inhibited both glutamate-elicited Ca(2+) response and PLC activation. Overall, these results indicate that the mGluR-mediated activation of PLC depends on the presence of extracellular Ca(2+) and can be modulated by moderate changes of cytosolic Ca(2+). Furthermore, ryanodine reduced PLC stimulation by glutamate in predepolarized cells but not in control cells, suggesting that ryanodine receptors could play a role in the potentiation of the mGluR-mediated activation of PLC by Ca(2+) release in predepolarized cells.
在本研究中，我们确定了体外培养9天的小脑颗粒细胞中，参与谷氨酸激活磷脂酶C（PLC）和Ca²⁺动员的代谢型谷氨酸受体（mGluRs）；并研究了Ca²⁺对PLC反应的调节作用。尽管在细胞提取物中可检测到I组mGluRs，即mGluR1α和mGluR5，但发现PLC激活和Ca²⁺信号传导均仅由mGluR1亚型介导。在激动剂刺激前，将细胞暴露于无Ca²⁺的培养基中不同时间，会降低PLC反应，而在细胞重新暴露于含Ca²⁺的培养基后，该反应会迅速恢复。谷氨酸反应的程度与胞质Ca²⁺浓度的变化密切相关。另一方面，通过短暂去极化加载细胞内Ca²⁺储存，然后在非去极化缓冲液中冲洗，可使谷氨酸在大多数细胞中释放储存的Ca²⁺，并增强谷氨酸对PLC的激活作用。在这种情况下，刺激期间细胞外Ca²⁺的缺失以及用BAPTA/AM螯合胞质Ca²⁺，会抑制谷氨酸引发的Ca²⁺反应和PLC激活。总体而言，这些结果表明，mGluR介导的PLC激活依赖于细胞外Ca²⁺的存在，并且可由胞质Ca²⁺的适度变化调节。此外，ryanodine降低了预去极化细胞中谷氨酸对PLC的刺激，但对对照细胞无此作用，这表明ryanodine受体可能在预去极化细胞中通过Ca²⁺释放增强mGluR介导的PLC激活过程中发挥作用。
