Jope R S, Song L, Powers R
Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham 35294-0017.
Biochem J. 1994 Dec 1;304 ( Pt 2)(Pt 2):655-9. doi: 10.1042/bj3040655.
A method utilizing exogenously added [3H]PtdIns incubated with membranes prepared from postmoretem human brain has been shown to provide a means of measuring agonist-induced, guanosine 5'-O-(thiotriphosphate) (GTP[S])-dependent hydrolysis of [3H]PtdIns, thus allowing investigations of the activity of the phosphoinositide second-messenger system in accessible human brain tissue. Agonists inducing [3H]PtdIns hydrolysis include carbachol, trans-1-aminocyclopentyl-1,3-dicarboxylate (ACPD; a glutamatergic metabotropic receptor agonist), serotonin and ATP, with the latter two agonists producing the largest responses. In addition to ATP, [3H]PtdIns hydrolysis was induced by ADP and by 2-methylthio-ATP, indicating that P2-purinergic receptors mediate this process. Subtype-selective antibodies we used to identify Gq/11 and phospholipase C-beta as the G-protein and phospholipase C subtypes that mediated GTP[S]-induced and agonist-induced [3H]PtdIns hydrolysis. These results demonstrate that this method reveals that agonist-induced, GTP[S]-dependent [3H]PtdIns hydrolysis is retained in postmortem human brain membranes with properties similar to rat brain. This method should allow studies of the modulation of phosphoinositide hydrolysis in human brain and investigations of potential alterations in postmortem brain from subjects with neurological and psychiatric diseases.
一种利用外源性添加的[3H]磷脂酰肌醇与死后人类大脑制备的膜一起孵育的方法，已被证明能提供一种测量激动剂诱导的、鸟苷5'-O-(硫代三磷酸)(GTP[S])依赖性[3H]磷脂酰肌醇水解的手段，从而能够研究可获取的人类脑组织中磷酸肌醇第二信使系统的活性。诱导[3H]磷脂酰肌醇水解的激动剂包括卡巴胆碱、反式-1-氨基环戊基-1,3-二羧酸(ACPD；一种谷氨酸能代谢型受体激动剂)、5-羟色胺和ATP，后两种激动剂产生的反应最大。除了ATP，ADP和2-甲硫基-ATP也能诱导[3H]磷脂酰肌醇水解，表明P2-嘌呤能受体介导了这一过程。我们使用亚型选择性抗体来鉴定Gq/11和磷脂酶C-β作为介导GTP[S]诱导的和激动剂诱导的[3H]磷脂酰肌醇水解的G蛋白和磷脂酶C亚型。这些结果表明，该方法揭示了激动剂诱导的、GTP[S]依赖性的[3H]磷脂酰肌醇水解在死后人类脑膜中得以保留，其性质与大鼠脑相似。该方法应有助于研究人类大脑中磷酸肌醇水解的调节，并有助于研究患有神经和精神疾病的受试者死后大脑中的潜在变化。
