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

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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