Modulation of intracellular Ca ion concentrations by endothelium-derived hyperpolarizing factor in vascular smooth muscle

(1)在豚鼠主动脉中成功制备了用于测量细胞内钙离子浓度和机械反应的血管平滑肌。EDHF使该动脉平滑肌松弛。(2)采用酶解法分离牛胸主动脉内皮细胞，在微载体床上培养。初步实验表明，这些细胞（3 × 10^7个细胞/每次实验）可以产生几种类型的血管活性物质，以响应bradikinine （Bk）。(3)利用离体豚鼠胸主动脉环估计内皮细胞产生EDHF。体外培养的内皮细胞中含有Bk的过盛液使Bk收缩的主动脉环制剂松弛，且松弛对硝基精氨酸和吲哚美辛有抗性，但对含硝基精的高钾溶液敏感。结果表明，Bk刺激培养的内皮细胞释放血管松弛因子，这可能与EDRF或PG12不同。这些未知因素的影响在高钾溶液中降低，表明与膜超极化有关。通过原代培养的豚鼠主动脉平滑肌细胞中负载的钙敏感染料Fura-2的荧光强度，估计EDHF对细胞内钙离子的调节浓度（Fura-2以Fura-2 AM的形式加载到细胞中）。Bk将荧光从静止的约50nM瞬间升高到相当于300-500nM Ca的水平，并达到约200nM的稳定水平。从培养的内皮细胞中分离出含bk的过清液，使Fura-2荧光降低到静息水平，与硝基精氨酸和吲哚那辛的存在与否无关。在高钾溶液中，内皮细胞流出物对荧光强度的降低不明显。结果表明，内皮因子的超极化可能是导致荧光减弱的原因。因此，EDHF通过降低钙浓度放松动脉平滑肌。少

(1) Vascular smooth muscles for the measurement of intracellular Ca ion concentrations and also for mechanical responses were successfully prepared in the guinea-pig aorta. Smooth muscle of this artery was confirmed to be relaxd by EDHF.(2) Endothelial cells isolated from bovine thoracic aorta using enzymatic dispersion were cultured on microcarrier beeds. Preliminary experiments showed that these cells (3x10^7 cells/each experiment) can produce several types of vasoactive substance in response to bradikinine (Bk).(3) Production of EDHF from the cultured endothelial cells was estimated by using isolated thoracic aortic ring of the guinea-pig. Superfusates of the cultured endothelial cells with Bk relaxd the Bk-contracted aortic ring preparations, and the relaxation was resistant to nitroarginine and indomethacin, but sensitive to high-K solution containing nitroaginine. The results suggested that Bk stimulates the cultured endothelial cells to release vesorelaxtant factors which may be … More different from EDRF or PG12. The effects of these unidentified factors reduced in high-K solution, indicated an involvement of membrane hyperpolarization.4. Intracellular Ca ion concentrations modulated by EDHF were estimated from the fluorescent intensities of Ca-sensitive dye, Fura-2 loaded in primary cultured smooth muscle cells of the guinea-pig aorta (Fura-2 was loaded into the cell in a form of Fura-2 AM). Bk elevated the fluorescent transiently to level equivalent to 300-500nM Ca from the resting level of about 50nM, and them reached the steady level of about 200nM.Bk-containing superfusate elluted from the cultured endothelial cells reduced the Fura-2 fluorescent to the resting level, irrespective to the absence and presence of nitroarginine and indonethacin. The reduction of fluorescent intensity by the endothelial effluents was not clearly detected in high-K solution. The results suggested that hyperpolarization by endothelial factor may be responsible to the reduction of fluorescent. Thus, EDHF relax arterial smooth muscles with reduced Ca concentrations. Less