Molecular Mechanism and its Clinical Significance of Abnormalities in Voltage-Sensitive Calcium Channel in the Diabetic Heart.

本实验观察了糖尿病大鼠心肌细胞电压操纵性钙通道（VOCC）和细胞内钙沉积，以确定糖尿病心肌细胞内钙超载。(1)糖尿病心室肌细胞线粒体内钙沉积：与Shionogi研究实验室的Mizuhira博士合作，使用磷酸盐-焦锑酸盐沉淀法、微波固定法和电子显微镜上的能量分散X射线显微分析来测量细胞内钙沉积。糖尿病组心肌线粒体内钙含量较对照组增加48%，以线粒体异常肿胀、嵴崩解为特征。(2)糖尿病患者心脏膜VOCC的减少：使用[3 H] PN 220 -110（一种二氢吡啶类钙通道阻滞剂）测量心脏质膜VOCC。[3 H]P最大结合位点增加 ...更多信息 N200-110在6周糖尿病大鼠中被发现，在10周糖尿病大鼠中比对照组增加64%，而结合的Kd没有任何变化。[3 H] PN 200 -110与糖尿病大鼠心肌细胞膜组分的结合对维拉帕米的作用不敏感。(3)骨骼肌细胞膜VOCC和细胞内钙沉积的减少：能量色散X射线微区分析显示，糖尿病大鼠比目鱼肌线粒体内和肌浆网钙含量增加。此外，通过原子吸收分析测定的糖尿病大鼠比目鱼肌组织钙含量与对照组相比也显著增加。在糖尿病组中，[3 H] PN 200 -110与骨骼肌膜部分的最大结合显著增加，比对照组高91%，而结合亲和力没有任何变化。(4)钙通道阻滞剂对糖尿病心肌蛋白激酶C（PKC）活性的影响：糖尿病心肌膜结合型和可溶性PKC活性分别比对照组增加41%和94%。糖尿病心肌中PKC的增加通过体内维拉帕米治疗完全正常化。提示糖尿病心肌病可能与心肌细胞内钙超载有关。少

Voltage operated calcium channel (VOCC) and intracellular calcium deposition were evaluated in cardiac muscle isolated from diabetic rats in order to identify calcium over-loading in diabetic cardiac myocytes. (1) Intramitochondrial calcium deposition in diabetic ventricular myocytes : collaboration with Dr Mizuhira of the Shionogi research laboratory, intracelluar calcium deposition was measured using the oxalate-pyroantimonate precipitation method , the microwave fixation method and energy-disperse X-ray microanalysis on electron microscope. Intramitochondrial calcium content in the diabetic group increased by 48% as compared with the control and the increase was marked in abnormally swollen mitochondria with disintegrated cristae which were marked in diabetic cardiac muscle. (2) Abnormalities in cardiac membrane VOCC in diabetes : Cardiac plasma membrane VOCC was measured using [3H]PN220-110, a dihydropyridine calcium channel blocker. An increase in the maximum binding site of [3H]P … More N200-110 was found in 6-wk-diabetic rats and the increase reached to 64% above the control in 10-wk-diabetic rats without any change in Kd for the binding. The [3H]PN200-110 binding to cardiac membrane fraction isolated from diabetic rats was less sensitive to verapamil effect. (3) Abnormalities in skeletal muscle membrane VOCC and intracellular calcium deposition : Using energy-disperse X-ray microanalysis, the increases in both intra-mitochondria and sarcoplasmic reticular calcium content were shown in soleus muscle obtained from diabetic rats. Furthermore, tissue calcium content which was measured by atomic absorption analysis also significantly increased in soleus muscle in diabetic rats as compared to the control. The maximum [3H]PN200-110 binding to skeletal muscle membrane fraction in the diabetic group significantly increased by 91% above the control without any change in the affinity of the binding. (4) The effect of calcium channel blocker on protein kinase C (PKC) activity in diabetic cardiac muscle : Both membrane-bound and soluble PKC activities in diabetic cardic muscle increased by 41 and 94% above the control, respectively. The increase in PKC in diabetic cardiac muscle was completely normalized by in vivo verapamil treatment. These results indicate that intracelluar calcium overloading in cardiac muscle may be associated with diabetic myocardiopathy. Less