REGULATORY MECHANISMS OF VASCULAR CONTRACTION AND CELL PROLIFERATION IN DIABETES MELLITUS

本研究旨在探讨糖尿病血管收缩和细胞增殖调节异常的机制。结果如下：1.血管平滑肌细胞的增殖。我建立了在单细胞水平上测定原代培养的血管平滑肌细胞的细胞周期时相的方法。结果发现：(1)血小板衍生生长因子刺激细胞周期从G_0期进入G_1期并不需要细胞内钙瞬变，(2)高糖不刺激G_0细胞周期，而是刺激G_1细胞进入S和M期，细胞内钙浓度([Ca^&lt；2&gt；应用前表面荧光法和原位Fura-2负载瓣膜内皮细胞的方法发现：(1)内皮素-1(ET-1)使[Ca~(2+)]i~(…)升高更多的是刺激细胞内钙内流和释放；2)钙内流受百日咳毒素敏感的G蛋白调节，而细胞内钙释放受百日咳毒素敏感的G蛋白调节。(1)RT-PCR和Fura-2显微荧光检测发现，血管紧张素II(AT-II)、蛋白激酶C和蛋白激酶A调节AT-II受体mRNA的表达，并伴随着生理反应性的增强。(2)应用Fura-2前表面荧光法和猪冠状动脉条，发现ET-3主要通过细胞外钙内流引起血管收缩，并且ET-1和ET-3对血管收缩反应中钙敏感性功能的时间依赖性调节机制不同。(3)去甲肾上腺素使收缩装置的钙敏感性增加，而5-羟色胺对收缩装置的钙敏感性的增强作用不明显。较少

The purpose of this research is to investigate the mechanisms of abnormal regulation of vascular contraction and cell proliferation in diabetes mellitus. The following results were obtained :1.CELL PROLIFERATION OF VASCULAR SMOOTH MUSCLE CELLS.I developed the method to determine the phase of the cell cycle of vascular smooth muscle cells in primary culture at the single cell level. It was found that : (1)cytosolic Ca^<2+> transients are not required for platelet-derived growth factor (PDGF) to stimulate the cell cycle progression from the G_0 phase to the G_1 phase ; and (2)high glucose does not stimulate the cell cycle of the G_0 cells, whereas it stimulates the cell cycle of the PDGF-pretreated G_1 cells to the S and M phases, without any increase of cytosolic Ca^<2+> concentration ([Ca^<2+>]i).2.CALCIUM TRANSIENTS OF ENDOTHELIAL CELLS.Using front-surface fluorometry and fura-2-loaded valvular endothelial cells in situ, it was found that : (1)endothelin-1 (ET-1) elevates [Ca^<2+>]i b … More y the stimulation of both Ca^<2+> influx and intracellular Ca^<2+> release ; and 2)the Ca^<2+>influx is regulated by an pertussis toxin-sensitive G-protein, while the release of intracellular Ca^<2+> is not.3.VASCULAR CONTRACTION.(1)Using RT-PCR and fura-2 microfluorometry, it was found that angiotensin II (AT-II), protein kinase C,and protein Kinase A regulate the expression of AT-II receptor mRNA,and the increase in mRNA level is accompanied by an increase in physiological responsiveness. (2) Using fura-2-front-surface fluorometry and porcine coronary arterial strips, it was found that ET-3 induced vasoconstriction by increasing [Ca^<2+>]i mainly through the influx of extracellular Ca^<2+>, and that distinct mechanisms of time-dependent modulation of the Ca^<2+>-sensitivity function in the vasoconstrictor responses to ET-1 and ET-3. (3)It was found that norepinephrine induced increase in the Ca^<2+>-sensitivity of contractile apparatus, while serotonin demonstrates little enhancement of the Ca^<2+>-sensitivity of contractile apparatus. Less