Jope R S, Casebolt T L, Johnson G V
Neurochem Res. 1987 Aug;12(8):693-700. doi: 10.1007/BF00970524.
Cortical slices from rat brain were used to study carbachol-stimulated inositol phospholipid hydrolysis. Omission of calcium during incubation of slices with [3H]inositol increased its incorporation into receptor-coupled phospholipids. Carbachol-stimulated hydrolysis of [3H]inositol phospholipids in slices was dose-dependent, was affected by the concentrations of calcium and lithium present and resulted in the accumulation of mostly [3H]inositol-1-phosphate. Incubation of slices with N-ethylmaleimide or a phorbol ester reduced the response to carbachol. Membranes prepared from cortical slices labeled with [3H]inositol retained the receptor-stimulated inositol phospholipid hydrolysis reaction. The basal rate of inositol phospholipid hydrolysis was higher than in slices and addition of carbachol further stimulated the process. Addition of GTP stimulated inositol phospholipid hydrolysis, suggesting the presence of a guanine nucleotide-binding protein coupled to phospholipase C. Carbachol and GTP-stimulated inositol phospholipid hydrolysis in membranes was detectable following a 3 min assay period. In contrast to slices, increased levels of inositol bisphosphate and inositol trisphosphate were detected following incubation of membranes with carbachol. These results demonstrate that agonist-responsive receptors are present in cortical membranes, that the receptors may be coupled to phosphatidylinositol 4, 5-bisphosphate, rather than phosphatidylinositol, hydrolysis and that a guanine nucleotide-binding protein may mediate the coupling of receptor activation to inositol phospholipid hydrolysis in brain.
用大鼠脑皮质切片研究卡巴胆碱刺激的肌醇磷脂水解。在用[3H]肌醇孵育切片期间省略钙会增加其掺入受体偶联磷脂的量。卡巴胆碱刺激的切片中[3H]肌醇磷脂水解呈剂量依赖性，受存在的钙和锂浓度影响，并导致主要是[3H]肌醇-1-磷酸的积累。用N-乙基马来酰亚胺或佛波酯孵育切片会降低对卡巴胆碱的反应。用[3H]肌醇标记的皮质切片制备的膜保留了受体刺激的肌醇磷脂水解反应。肌醇磷脂水解的基础速率高于切片，添加卡巴胆碱进一步刺激该过程。添加GTP刺激肌醇磷脂水解，表明存在与磷脂酶C偶联的鸟嘌呤核苷酸结合蛋白。在3分钟的测定期后可检测到卡巴胆碱和GTP刺激的膜中肌醇磷脂水解。与切片相反，在用卡巴胆碱孵育膜后检测到肌醇二磷酸和肌醇三磷酸水平升高。这些结果表明皮质膜中存在激动剂反应性受体，这些受体可能与磷脂酰肌醇4,5-二磷酸偶联，而不是与磷脂酰肌醇水解偶联，并且鸟嘌呤核苷酸结合蛋白可能介导受体激活与脑中肌醇磷脂水解的偶联。
