Qualitative level of GTP-binding proteins in diabetic rat myocardium and its biological significance in alteration of beta-adrenoceptor mediated cellular response.

本研究旨在明确ADP核糖化法和免疫印迹法中哪种方法更适合于糖尿病大鼠心肌GTP结合蛋白(G)的检测。ADP核糖化法测得Gs和GI水平均升高，但抗Gsα和Giα亚基抗血清免疫印迹显示GI水平降低，Gs水平不变。在糖尿病大鼠心肌中，我们观察到：1.在β-肾上腺素能受体(R)、GppNHp、NaF和Forsklin刺激下，腺苷酸环化酶(AC)活性增强；2.GimRNA水平下降，Gs mRNA水平不变。在假设糖尿病状态下Gi蛋白水平降低对Gs-AC系统产生紧张性抑制作用，导致AC活性改变的假设下，我们观察了Gi蛋白功能缺失对PTX处理的兔心肌R-Gs-AC系统的影响。我们发现，PTX处理的心肌对β-肾上腺素能受体刺激和GppNHp反应产生AC活动增强，但对NaF和Forsklin没有反应。因此，即使有一些差异，糖尿病心肌中AC活动的改变反应可以很好地解释为GI水平的下降。这些结果表明，免疫印迹法估计的G蛋白水平很好地反映了糖尿病心肌R-Gs-AC偶联的变化。

This research project has aimed to clarify which of ADP-ribosylation method or immunoblotting method is adequate for estimating GTP binding proteins (G) in diabetic rat myocardium. When estimated by ADP-ribosylation method, we found increased level of both Gs and Gi, but immunoblotting with anti-Gs alpha-and Gi alpha-subunit antiserum indicated decreased level of Gi and unchanged level of Gs. In diabetic rat myocardium, we observed : 1. augmented activation of adenylate cyclase (AC) , when stimulated by beta-adrenoceptors (R) , GppNHp, NaF and forskolin in comparison with the AC activities in control normal rat myocardium, 2. decreased level of GimRNA and unchanged level of Gs mRNA level. Under the hypothesis that the decreased level of Gi protein which gives tonic inhibitory influence on Gs-AC system, causes the alterations of AC activities in diabetic state, we examined influence of functional abolishment of Gi protein on R-Gs-AC system in PTX-treated rabbit myocardium. we found that PTX-treated myocardium produced augmentation of AC activities in response to beta-adrenoceptor stimulation and GppNHp, but, not to NaF and forskolin. Thus, even though there are a few discrepancy, the altered response of AC activities in diabetic myocardium is well explained by the decreased level of Gi. From these results, we conclude that G protein level estimated by immnoblotting method reflects well alterations in R-Gs-AC coupling in diabetic myocardium.