Howe P H, Akhtar R A, Naderi S, Abdel-Latif A A
J Pharmacol Exp Ther. 1986 Nov;239(2):574-83.
Previously, we have reported that activation of muscarinic cholinergic receptors in the iris smooth muscle results in a rapid breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) into 1,2-diacylglycerol and myo-inositol trisphosphate (IP3) and that the stimulated hydrolysis of this phospholipid correlates well with contraction. To determine whether or not there is a causal relationship between PIP2 breakdown and contraction, we have conducted correlative studies on the effects of carbachol (CCh) on PIP2 breakdown, measured as IP3 accumulation, myosin light chain (MLC) phosphorylation and contraction in the rabbit iris sphincter. We have also investigated the effects of time, temperature, atropine antagonism, Ca++ and C-kinase activators on the three measured responses. The data obtained can be summarized as follows: dose-response studies for IP3 accumulation, MLC phosphorylation and contraction revealed a close correlation between these responses; kinetic data on atropine antagonism showed that the three measured responses are competitively inhibited by the muscarinic antagonist; time course studies conducted at low temperature showed that the CCh-induced IP3 accumulation and MLC phosphorylation may precede contraction; time course studies on the effect of Ca++ on the three measured responses showed that IP3 release may account for the rapid phase of CCh-induced contraction and that extracellular Ca++ is essential for sustained MLC phosphorylation and the slow phase of contraction; the activity of phospholipase C, the enzyme involved in PIP2 hydrolysis, in membrane fragments from 32P-labeled sphincter muscle was found to be highly sensitive to Ca++, with half-maximal stimulation at about 1.1 microM Ca++; and phorbol 12,13-dibutyrate, but not phorbol 12-myristate 13-acetate, induced MLC phosphorylation and muscle contraction in a dose- and time-dependent manner. Phorbol 12,13-dibutyrate and ionomycin acted in a synergistic manner to elicit contraction. In conclusion, contractions by CCh in the iris sphincter may be explained on the basis of enhanced PIP2 turnover and its derived second messenger molecule(s); that there are consistent correlations, using different concentrations of CCh, atropine antagonism, time, temperature and Ca++, between the stimulated hydrolysis of PIP2, MLC phosphorylation and contraction. Finally, whereas the data presented favor the involvement of IP3 in the phasic component of the contractile response, the studies with phorbol 12,13-dibutyrate suggest that contractile regulation by 1,2-diacylglycerol, through activation of C-kinase, may be important during the tonic component of smooth muscle contraction.
此前，我们曾报道，虹膜平滑肌中毒蕈碱胆碱能受体的激活会导致磷脂酰肌醇4,5 -二磷酸（PIP2）迅速分解为1,2 -二酰甘油和肌醇三磷酸（IP3），且这种磷脂的刺激水解与收缩密切相关。为了确定PIP2分解与收缩之间是否存在因果关系，我们进行了相关研究，考察了卡巴胆碱（CCh）对PIP2分解（以IP3积累来衡量）、肌球蛋白轻链（MLC）磷酸化及兔虹膜括约肌收缩的影响。我们还研究了时间、温度、阿托品拮抗、Ca++和C激酶激活剂对这三种测量反应的影响。所获数据可总结如下：IP3积累、MLC磷酸化和收缩的剂量反应研究表明这些反应之间存在密切相关性；阿托品拮抗的动力学数据显示，这三种测量反应均受到毒蕈碱拮抗剂的竞争性抑制；在低温下进行的时间进程研究表明，CCh诱导的IP3积累和MLC磷酸化可能先于收缩；关于Ca++对这三种测量反应影响的时间进程研究表明，IP3释放可能是CCh诱导收缩快速相的原因，且细胞外Ca++对于MLC持续磷酸化和收缩的缓慢相至关重要；发现32P标记的括约肌肌肉膜片段中参与PIP2水解的磷脂酶C活性对Ca++高度敏感，在约1.1 microM Ca++时达到最大刺激的一半；佛波醇12,13 -二丁酸酯而非佛波醇12 -肉豆蔻酸酯13 -乙酸酯以剂量和时间依赖性方式诱导MLC磷酸化和肌肉收缩。佛波醇12,13 -二丁酸酯和离子霉素以协同方式引发收缩。总之，CCh在虹膜括约肌中的收缩作用可基于增强的PIP2周转及其衍生第二信使分子来解释；在不同浓度的CCh、阿托品拮抗、时间、温度和Ca++条件下，PIP2的刺激水解、MLC磷酸化和收缩之间存在一致的相关性。最后，尽管所呈现的数据支持IP3参与收缩反应的时相成分，但佛波醇12,13 -二丁酸酯的研究表明，1,2 -二酰甘油通过激活C激酶进行的收缩调节在平滑肌收缩的紧张成分中可能很重要。
