Fukuda N, Honda M, Hatano M
2nd Department of Internal Medicine, Nihon University School of Medicine, Tokyo, Japan.
Jpn Circ J. 1989 Mar;53(3):270-7. doi: 10.1253/jcj.53.270.
In order to evaluate the relationship between the calcium-calmodulin system and the adenylate cyclase activity of vascular smooth muscle, we examined the effects of several calcium effectors on the basal and stimulated adenylate cyclase activity. Thoracic aortae were removed from Wistar rats and the tissues were homogenated with cold homogenizing buffer containing 1 nM EDTA. Membrane protein fraction of the smooth muscle was prepared by centrifugation at 37,000 g. In this procedure, endogenous guanine nucleotides and contractile proteins remained. The protein fraction was incubated with 2 mM EGTA, 50 microM trifluoperazine, 0.1 microM A23187 or 25 microM calmodulin under basal and stimulated (50 microM isoproterenol, 100 microM GTP and 50 microM forskolin) conditions. The adenylate cyclase activity was determined by a method modified in our laboratory using double isotope counting. Trifluoperazine reduced the basal adenylate cyclase activity significantly (p less than 0.01) as well as the stimulated enzyme activities. A23187 did not affect the basal enzyme activity, but elevated the isoproterenol stimulated enzyme activity significantly (p less than 0.05). EGTA did not affect the basal and stimulated adenylate cyclase activities. Calmodulin elevated the basal enzyme activity significantly (p less than 0.02), but did not affect the stimulated enzyme activities. These results suggest that the calcium-calmodulin system is necessary for maintenance of the adenylate cyclase activity of vascular smooth muscle cells. The calmodulin acting site is considered to be the catalytic subunit, and stimulation of the enzyme is accelerated by calcium ion.
为了评估钙 - 钙调蛋白系统与血管平滑肌腺苷酸环化酶活性之间的关系，我们研究了几种钙效应剂对基础和刺激状态下腺苷酸环化酶活性的影响。从Wistar大鼠身上取下胸主动脉，将组织用含有1 nM乙二胺四乙酸（EDTA）的冷匀浆缓冲液匀浆。通过在37,000 g下离心制备平滑肌的膜蛋白组分。在此过程中，内源性鸟嘌呤核苷酸和收缩蛋白得以保留。将蛋白组分在基础和刺激（50 microM异丙肾上腺素、100 microM鸟苷三磷酸（GTP）和50 microM福斯高林）条件下与2 mM乙二醇双乙醚二胺四乙酸（EGTA）、50 microM三氟拉嗪、0.1 microM A23187或25 microM钙调蛋白一起孵育。腺苷酸环化酶活性通过我们实验室改进的使用双同位素计数的方法来测定。三氟拉嗪显著降低基础腺苷酸环化酶活性（p小于0.01）以及刺激后的酶活性。A23187不影响基础酶活性，但显著提高异丙肾上腺素刺激后的酶活性（p小于0.05）。EGTA不影响基础和刺激状态下的腺苷酸环化酶活性。钙调蛋白显著提高基础酶活性（p小于0.02），但不影响刺激后的酶活性。这些结果表明，钙 - 钙调蛋白系统对于维持血管平滑肌细胞的腺苷酸环化酶活性是必要的。钙调蛋白的作用位点被认为是催化亚基，并且酶的刺激作用由钙离子加速。
