Kuroki T, Inoguchi T, Umeda F, Ueda F, Nawata H
Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Diabetes. 1998 Jun;47(6):931-6. doi: 10.2337/diabetes.47.6.931.
Gap junction is thought to have a crucial role in maintaining tissue homeostasis. We examined the effect of a high glucose level on gap junctional intercellular communication (GJIC) activity in cultured vascular smooth muscle cells (VSMCs) using the fluorescent dye transfer method. After a 48-h incubation with 22 mmol/l glucose (high glucose level), GJIC activity of VSMCs was significantly reduced compared with incubation with 5.5 mmol/l glucose (normal glucose level) (P < 0.05). Treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate (TPA; 5 x 10(-8) mol/l), a protein kinase C (PKC) activator, for 1 h also reduced GJIC activity (P < 0.01). In addition, treatment of the cells with calphostin C, a specific PKC inhibitor, for 3 h completely restored the GJIC activity inhibited by the high glucose level. Western blot analysis showed that connexin 43 (Cx43), which is the major functional protein of gap junction, is present in multiphosphorylated forms: a nonphosphorylated form (P0) and phosphorylated forms (P1, P2, and P3). Incubation of VSMCs with a high glucose level significantly increased the density ratio of P3/P0 compared with a normal glucose level (P < 0.05). Similarly, treatment of the cells with TPA significantly increased the P3/P0 ratio compared with controls (P < 0.01). In addition, the increase in the P3/P0 density ratio induced by a high glucose level was restored to the control level by both staurosporine and calphostin C. These results suggest that the high glucose level induced the inhibition of GJIC activity in cultured VSMCs through excessive phosphorylation of Cx43, mediated by PKC activation. This may contribute to the development of the macroangiopathy associated with diabetes.
缝隙连接被认为在维持组织内环境稳定中起关键作用。我们使用荧光染料转移法研究了高糖水平对培养的血管平滑肌细胞（VSMC）中缝隙连接细胞间通讯（GJIC）活性的影响。在22 mmol/L葡萄糖（高糖水平）孵育48小时后，与5.5 mmol/L葡萄糖（正常糖水平）孵育相比，VSMC的GJIC活性显著降低（P < 0.05）。用蛋白激酶C（PKC）激活剂12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA；5×10⁻⁸ mol/L）处理细胞1小时也降低了GJIC活性（P < 0.01）。此外，用特异性PKC抑制剂钙泊三醇C处理细胞3小时可完全恢复高糖水平抑制的GJIC活性。蛋白质印迹分析表明，缝隙连接的主要功能蛋白连接蛋白43（Cx43）以多种磷酸化形式存在：非磷酸化形式（P0）和磷酸化形式（P1、P2和P3）。与正常糖水平相比，高糖水平孵育VSMC显著增加了P3/P0的密度比（P < 0.05）。同样，与对照组相比，用TPA处理细胞显著增加了P3/P0比值（P < 0.01）。此外，星形孢菌素和钙泊三醇C均将高糖水平诱导的P3/P0密度比增加恢复到对照水平。这些结果表明，高糖水平通过PKC激活介导的Cx43过度磷酸化诱导培养的VSMC中GJIC活性的抑制。这可能有助于糖尿病相关大血管病变 的发展。
