Houslay M D
Department of Biochemistry, University of Glasgow, Scotland, U.K.
Cell Signal. 1990;2(1):85-98. doi: 10.1016/0898-6568(90)90036-a.
Using experimentally derived data for the activities and kinetic constants of hepatocyte cyclic AMP phosphodiesterase isoenzymes together with the derived changes in adenylate cyclase activity, due to stimulation and subsequent desensitization by glucagon, a computer model was established to simulate hepatocyte cyclic AMP metabolism. The established ability of glucagon to activate the 'dense-vesicle' cyclic AMP phosphodiesterase by eliciting its cyclic AMP-dependent phosphorylation was shown on the model to be capable of eliciting a profound reduction in the glucagon-stimulated increase in intracellular cyclic AMP. This was consistent with experimentally derived observations using the compound ICI 118233 which was used to inactivate the 'dense-vesicle' enzyme selectively. The non-hydrolysable adenosine agonist N6 (phenylisopropyl)-adenosine (PIA), which prevents glucagon pre-treatment of hepatocytes blocking the ability of insulin to stimulate the peripheral plasma membrane cyclic AMP phosphodiesterase, is shown here to accentuate the ability of insulin to decrease glucagon-elevated intracellular cyclic AMP concentrations. This effect was obliterated using the compound ICI 63197, a selective inhibitor of the peripheral plasma membrane phosphodiesterase. Computer modelling studies, taking into account experimentally derived actions in insulin in activating the peripheral plasma membrane phosphodiesterase, confirmed the potential of this enzyme to decrease intracellular cyclic AMP concentrations. Modelling of the putative effect of an insulin 'mediator' in activating the two cyclic GMP-stimulated cyclic AMP phosphodiesterase isoenzymes was shown to elicit a decrease in intracellular cyclic AMP concentrations which was comparable to that caused by insulin's action on intact hepatocytes. The relative contribution of each phosphodiesterase form to the metabolism of hepatocyte intracellular cyclic AMP, together with an assessment of the potential effect of inhibition and activation of specific species, was evaluated using the computer model. These experimental and stimulation studies indicate that alterations in the phosphodiesterase activity of the 'dense-vesicle' enzyme, the peripheral plasma membrane enzyme, the cyclic GMP-stimulated cyclic AMP isoforms and the IBMX-insensitive PDE-MQ-II can elicit profound effects upon hepatocyte intracellular cyclic AMP concentrations.
利用通过实验得出的肝细胞环磷酸腺苷磷酸二酯酶同工酶的活性和动力学常数数据，以及由于胰高血糖素刺激和随后脱敏导致的腺苷酸环化酶活性的变化，建立了一个计算机模型来模拟肝细胞环磷酸腺苷代谢。模型显示，胰高血糖素通过引发其环磷酸腺苷依赖性磷酸化来激活“致密囊泡”环磷酸腺苷磷酸二酯酶的既定能力，能够显著降低胰高血糖素刺激引起的细胞内环磷酸腺苷增加。这与使用化合物ICI 118233进行实验得出的观察结果一致，该化合物用于选择性地使“致密囊泡”酶失活。非水解性腺苷激动剂N6（苯异丙基）腺苷（PIA）可防止胰高血糖素预处理肝细胞阻断胰岛素刺激外周质膜环磷酸腺苷磷酸二酯酶的能力，此处显示其可增强胰岛素降低胰高血糖素升高的细胞内环磷酸腺苷浓度的能力。使用化合物ICI 63197（外周质膜磷酸二酯酶的选择性抑制剂）可消除这种效应。计算机建模研究考虑了胰岛素激活外周质膜磷酸二酯酶的实验得出的作用，证实了该酶降低细胞内环磷酸腺苷浓度的潜力。胰岛素“介质”激活两种环鸟苷酸刺激的环磷酸腺苷磷酸二酯酶同工酶的假定作用的建模显示，细胞内环磷酸腺苷浓度降低，这与胰岛素对完整肝细胞的作用所引起的降低相当。使用计算机模型评估了每种磷酸二酯酶形式对肝细胞细胞内环磷酸腺苷代谢的相对贡献，以及对特定类型抑制和激活的潜在影响的评估。这些实验和刺激研究表明，“致密囊泡”酶、外周质膜酶、环鸟苷酸刺激的环磷酸腺苷同工型以及IBMX不敏感的PDE-MQ-II的磷酸二酯酶活性改变可对肝细胞细胞内环磷酸腺苷浓度产生深远影响。
