ENDOTHELIUM INACTIVATION IN INVITRO PERFUSED VASCULAR BEDS COMPARISONOF METHODS

In order to choose the best procedure to inactive the endothelium from vascular beds perfused in vitro, we compared four methods: perfusion with sodium deoxycholate 0.3% for 30 sec; 3-[(3-cholamidopropyl)dimeth ylammonio]-1-propane sulfonate 0.3% (CHAPS) for 2.5 min; collagenase 0 .2% for 15 min, and distilled water for 10 min, using the mesenteric a rterial bed (MAB) of the rat. The effectiveness of the treatments used to inactivate the endothelium was assessed functionally by using acet ylcholine and sodium nitroprusside and histologically using light micr oscopy. Phenylephrine was used to test the contractile properties of t he preparations after each treatment. After collagenase, distilled wat er, and CHAPS treatment, a potentiated response to phenylephrine was o bserved, whereas sodium deoxycholate treatment did not modify phenylep hrine-induced responses. Acetylcholine-induced responses were reduced by collagenase (60% reduction), CHAPS (30% reduction), and distilled w ater (52% reduction) treatment, and sodium deoxycholate completely abo lished acetylcholine-induced responses. Except after collagenase treat ment, smooth muscle relaxant responses were not altered. Medial smooth muscle cells displayed an unchanged morphology, appearing similar to those in control mesenteric arterial beds, except for collagenase and distilled water. Despite the fact that sodium deoxycholate treatment c ompletely abolished acetylcholine-induced response, endothelial cells were still found. No treatment totally removed endothelial cells. In c onclusion, we suggest that sodium deoxycholate treatment is the best p rocedure to inactivate endothelial cells from vascular beds perfused i n vitro since it completely abolished endothelium-dependent relaxation and did not interfere with smooth muscle vasodilating and contracting properties.