ANALYSIS OF ENDOTHELIUM-DERIVED CONTRACTILE OR RELAXING FACTORS IN LYMPHATIC ENOTHELIAL CELLS

乙酰胆碱诱导淋巴管内皮细胞释放一氧化氮：在预先收缩的有内皮的狗胸导管中，乙酰胆碱(ACh)产生剂量依赖性的松弛。机械摩擦血管内皮细胞可消除ACh的松弛作用。氧合血红蛋白、N、G-单甲基-L-精氨酸(L-NMMA)和亚甲蓝预处理可明显抑制ACh所致的胸主动脉内皮细胞的松弛。ACh还引起了所谓的夹心式制剂的显著松弛，表明ACh反应的纵条内皮细胞一定释放了SOMO可转移底物(S)，当扩散到环段时，激活了预收缩的淋巴肌细胞的松弛。以上结果提示ACh的舒缩作用主要是通过内皮细胞释放一氧化氮或其相关化合物来实现的。ACh对牛肠系膜…自发性收缩的影响更多的肢体运动：在内皮完整的牛肠系膜淋巴管中，ACh对自发收缩产生剂量依赖的负变时性和变力性效应。用L-NMMA机械摩擦血管内皮细胞，可完全消除ACh对淋巴管自发性收缩的抑制作用。再加L-精氨酸处理，可明显逆转L-NMMA诱导的抑制反应。结果提示，淋巴管内皮细胞释放的一氧化氮或相关化合物可能抑制自发性收缩的起搏活动，降低淋巴管的肌源性传导和/或机械活动。犬胸段淋巴管内淋巴内皮细胞的培养：淋巴管远端经无菌聚乙烯导管插管，然后用含有250U/ml冷却酶和0.5 mM EGTA的Rinaldini溶液灌流，与胶原酶孵育10min后，用含有20%胎牛血清的最低限度的基本培养液灌流胸腔内皮细胞，然后将灌流液离心得到去除的淋巴管内皮细胞。细胞颗粒在37ﾟC、空气和5%CO_2的加湿培养箱中培养，融合时细胞呈典型的鹅卵石状。较少

Acetylcholine-induced release of nitric oxide from lymphatic endothelial cells : In precontracted dog thoracic ducts with endothelium, acetylcholine (ACh) produced dose-dependent relaxations. The ACh-induced relaxation was abolished by mechanical rubbing of the endothelium. Pretreatment with oxyhemoglobin, N^G-monomethy-L-arginine (L-NMMA) and methlene blue markedly supperssed the ACh-induced relaxation in the thoracic ducts with endothelium. ACh also caused a marked relaxation in the so-called sandwich-mounted preparation, demonstrating that the endothelial cells of the longitudinal strip in response to ACh must have released somo transferable substrance(s) that, on diffusion into the ring segment, activated relaxation of the precontracted lymphatic muscle cells. All these findings suggest that the ACh-induced relaxation is mainly through release of nitric oxide or its related compounds from the endothelial cells.Effects of ACh on spontaneous contractions in isolated bovine mesenteric … More limphatics : In isolated bovine mesenteric lymphatics with intact endothelium, ACh produced dose-dependent negative chronotropic and inotropic effects on the spontaneous contractions. The both effect induced by ACh were abolished by mechanical rubbing of the endothelium pretreatment with L-NMMA markedly supperessed the ACh-induced effects on spontaneous contractions in the lymph vessels. An additional treatment with L-arginine caused a marked reversal of the L-NMMA-induced inhibitory responses. The findings suggest that nitric oxide or its related compounds liberated from lymphatic endothelial cells seems to inhibit pacemaker activity of the spontaneous contractions and reduce the myogenic conduction and/or the mechanical activity of lymphatic smooth muscles.Tissue culture of lympatic endothelial cells in dog toracic ducts :The distal end of the lymph vessel was cannulated by a sterilized polyethylene catheter and then perfused with Rinaldini soultion containing 250 U/ml coolagenase and 0.5 mM EGTA.After 10 min incubation with collagenase, intraluminal space of the thoracic duct was perfused with minimum essential mediam containing 20 % fetal bovine serum, and then the perfusate was centrifuged to obtain the denuded lymphatic endothelial cells. The pellets of the cells were cultured at 37ﾟC in a humidified incubator with air and 5 % CO_2. The polygonal cells had a classic "cobblestone" morphology at confluence. Less