PHOSPHATIDYL INOSITOL, PROSTAGLANDINS, AND EXCITATION

广泛的长期目标是进一步阐明 磷酸肌醇的生物化学和生物学意义 信号系统 更具体地说，（1） 磷酸肌醇中的肌醇1，2-环4，5-三磷酸（cIP 3） 信号过程？ 名单上的首要任务将是寻找一个 cIP 3受体是三磷酸肌醇的独特受体 (I(1 4，5）P3）受体，因为这种受体的发现 为第二信使的功能提供了强有力的证据 对于cIP 3。 可能的第二信使或调节作用将是 在透化的腺泡和无细胞级分中测试，例如（a） 抑制I（1，4，5）P3与其受体的结合。 这可能 在其初始尖峰之后抑制Ca 2+动员;（B）协同作用 cIP 3与I（1，4，5）P3或I（1，3，4，5）P3对Ca 2+内流的影响;（c） 通过cIP 3激活内质网中的PI水解 （ER）。 (2)什么是游离花生四烯酸（AA）的功能， 激动剂刺激的细胞中的代谢物？ 我们会研究 AA从透化的和 未透化的胰腺腺泡细胞。 我们亦会研究 AA单独或联合激活蛋白激酶C（PKC） 与PKC的激活剂，如Ca 2+，二酰基甘油（DAG），和 磷脂酰丝氨酸。 (3)是否存在直接磷酸二酯 ER中PI的裂解，或者是激动剂刺激的PI损失 严格地通过PI的磷酸化形成PIP，然后在细胞中形成PIP 2。 质膜（PM）？来自ER的微粒体将被 与cIP 3、I（1，4，5）P3、DAG、Ca 2+等孵育，其可以是 可以直接触发PI崩溃的信使。 此外，直接 如果我们能证明一个形成的 I（1）P的量超过I（1，4，5）P3所能形成的量 降解途径 (4)我们将研究cIP 3的形成， 中枢神经系统中的AA。 我们将主要集中在 小脑，其具有最高密度的IP 3受体， 它会释放大量的甘草素 低浓度的乙酰胆碱。 (5)我们正在启动 磷脂酰肌醇信号转导的分子生物学研究 系统 我们基本上纯化了53 Kd PIP-激酶带， SDS-PAGE从人红细胞，并正在制备抗体， 筛选表达载体λ中的人脑cDNA文库， GT 11

The broad, long-term objectives are to throw further light on the biochemistry and biological significance of the phosphoinositide signalling system. More specifically, (1) What is the role of inositol 1,2-cyclic 4,5-trisphosphate (cIP3) in the phosphoinositde signalling process? Paramount on the list will be a search for a cIP3 receptor unique from the inositol (1,4,5)-trisphosphate (I(1,4,5)P3) receptor because the discovery of such a receptor would provide very strong evidence for a second messenger function for cIP3. Possible second messenger or regulatory roles will be tested in permeabilized acini and cell-free fractions, such as (a) inhibition of binding of I(1,4,5)P3 at its receptor. This could dampen Ca2+ mobilization after its initial spike; (b) synergism between cIP3 and I(1,4,5)P3 or I(1,3,4,5)P3 on Ca2+ influx; (c) activation by cIP3 of PI hydrolysis in the endoplasmic reticulum (ER). (2) What is the function of free arachidonic acid (AA) and metabolites in agonist-stimulated cells? We will examine the capability of AA to release Ca2+ from permeabilized and unpermeabilized pancreatic acinar cells. We will also study the activation of protein kinase C (PKC) by AA alone or in combination with activators of PKC, such as Ca2+, diacylglycerol (DAG), and phosphatidylserine. (3) Is there direct phosphodiesteratic cleavage of PI in the ER, or is the agonist-stimulated loss of PI strictly by phosphorylation of PI to form PIP and then PIP2 in the plasma membrane (PM)? Microsomes derived from the ER will be incubated with cIP3, I(1,4,5)P3, DAG, Ca2+ etc., which may be messengers which could trigger direct PI breakdown. Also, direct breakdown could be established if we are able to show a formation of I(l)P in excess of that which could be formed by the I(1,4,5)P3 degradation pathway. (4) We will study the formation of cIP3 and AA in the central nervous system. We will concentrate mainly on the cerebellum, which has the highest density of IP3 receptors and which releases substantial amounts of prostaglandins in response to low concentrations of acetylcholine. (5) We are initiating molecular-biological studies on the phosphoinositide signalling system. We have essentially purified the 53 Kd PIP-kinase band on SDS-PAGE from human red cells and are preparing antibodies to screen a human brain cDNA library in the expression vector, lambda GT11.