Dependence on Ca2+ of the activities of phosphatidylinositol 4,5-bisphosphate phosphodiesterase and inositol 1,4,5-trisphosphate phosphatase in smooth muscles of the porcine coronary artery.
Dependence on Ca2+ of the activities of phosphatidylinositol 4,5-bisphosphate phosphodiesterase and inositol 1,4,5-trisphosphate phosphatase in smooth muscles of the porcine coronary artery.
复制标题
猪冠状动脉平滑肌中磷脂酰肌醇4,5-二磷酸磷酸二酯酶和肌醇1,4,5-三磷酸磷酸酶活性对Ca2+的依赖性。
作者：
T. Sasaguri;M. Hirata;H. Kuriyama
用~（32）P标记的磷脂酰肌醇4，5-二磷酸（PIP_2）和肌醇1，4，5-三磷酸（IP_3）分别作底物，测定了猪冠状动脉平滑肌颗粒和胞浆中PIP_2磷酸二酯酶（PDE）和IP_3磷酸酶的活性。如从IP 3的产生评估的，在胞质溶胶级分中的PIP 2 PDE的活性比在颗粒级分中的活性高约10倍。在无MgCl_2存在下，两个组分中的PIP 2 PDE活性在10（-7）-10（-5）M的生理范围内与游离Ca ~（2+）浓度无因果关系，但10（-4）M的游离Ca ~（2+）显著增强PIP 2 PDE活性。在有游离Ca ~（2+）存在的情况下，向测定培养基中加入1 mM-MgCl_2可显著抑制两种组分中PIP 2 PDE的活性（10（-8）-10（-5）M）。在无MgCl_2存在下，10（-5）M-乙酰胆碱（ACh）产生IP_3，此作用可被3 × 10（-6）M-阿托品阻断。乙酰胆碱诱导的PIP 2 PDE激活在1 mM-MgCl 2存在下停止;然而，在加入10 μ M-鸟苷5 '-[γ-硫代]三磷酸时发生的再激活不依赖于游离Ca 2+浓度（10（-7）-10（-5）M）。IP 3磷酸酶的活性（根据颗粒和细胞质组分中IP 3含量的减少来确定）在两种组分中具有大致相同的效力。MgCl 2以浓度依赖性方式增强胞质组分中的IP 3磷酸酶活性，最大值出现在1 mM-MgCl 2时，并且在生理浓度的游离Ca 2+（10（-7）-10（-6）M）存在下也增强。这些发现表明，在鸟嘌呤核苷酸和1 mM-MgCl 2存在下，ACh的应用所发生的PIP 2 PDE的激活与游离Ca 2+浓度无关，并且磷酸酶对IP 3的水解增加，这取决于游离Ca 2+的浓度。
The activities of phosphatidylinositol 4,5-bisphosphate (PIP2) phosphodiesterase (PDE) and inositol 1,4,5,-trisphosphate (IP3) phosphatase in the particulate and cytosol fractions prepared from porcine coronary artery smooth muscles were examined using 32P-labelled PIP2 and IP3 as substrates, respectively. The activity of PIP2 PDE, as assessed from the production of IP3, in the cytosol fraction was about 10-fold higher than that in the particulate fraction. In the absence of MgCl2, the activity of PIP2 PDE in both fractions showed no causal relation to the free Ca2+ concentration in the physiological range of 10(-7)-10(-5) M, but was enhanced remarkably by 10(-4) M free Ca2+. The addition of 1 mM-MgCl2 to the assay medium markedly inhibited the activity of PIP2 PDE in both fractions in the presence of free Ca2+ (10(-8)-10(-5) M). In the absence of MgCl2, 10(-5)M-acetylcholine (ACh) produced IP3, and this action was blocked by 3 X 10(-6) M-atropine. The ACh-induced activation of PIP2 PDE ceased in the presence of 1 mM-MgCl2; however, the reactivation occurring on the addition of 10 microM-guanosine 5'-[gamma-thio]triphosphate did not depend on the free Ca2+ concentrations (10(-7)-10(-5)M). The activities of IP3 phosphatase, determined from decrease in the amount of IP3 in the particulate and cytosol fractions, had much the same potency in both fractions. The activity of IP3 phosphatase in the cytosol fraction was enhanced by MgCl2 in a concentration-dependent manner, the maximal value occurring at 1 mM-MgCl2, and was also enhanced in the presence of physiological concentrations of free Ca2+ (10(-7)-10(-6) M). These findings suggest that the activation of PIP2 PDE which occurs with application of ACh in the presence of guanine nucleotides and 1 mM-MgCl2 is independent of the free Ca2+ concentration, and that the hydrolysis of IP3 by phosphatase increases, depending on the concentration of free Ca2+.