| In 1980, Furchgott and his colleagues presented the first evidence that Acetylcholine (ACh), by acting on endothelial cells, stimulated the release of a relaxing factor (EDRF) that acted on the preconstricted rabbit thoracic aorta to produce relaxation. A research interest on the endothelium-derived regulation of vascular tone, since then, is on the increase. Now, the biological and chemical evidences have been obtained to show that EDRF is nitric oxide (NO). In addition, in the later of 80's, the results obtained in Weston and Suzuki's laboratories and other laboratories showed that a new substance, christened EDHF, could be released by ACh or other vasoactive substances such as substance P. And the EDHF could cause hyperpolarization of the vascular smooth muscle cells in an endothelium-dependent manner. EDHF-mediated relaxation is unaffected by haemoglobin or methylene blue, which indicates that endothelium-derived hyperpolarization is independent of the nitric oxide pathway. Since isolated vascular endothelial cells were found to hyperpolarize in response to ACh. the existence of EDHF has been challenged by the suggestion that the hyperpolarization in smooth muscle cells is merely conducted from the endothelial cells through myoendothelial gap junctions because smooth muscle and endothelial cells are found to be coupled. The objectives of this study were: (1) to establish a model of co-culture of endothelial and smooth muscle cells; (2)-5to ascertain the effects of the ACh on the membrane potential of the rabbit aortic smooth muscle cells and bovine aortic endothelial cells; (3) to clarify the role of gap junctions in ACh-induced endothelium-derived hyperpolarization in rabbit aortic smooth muscle cells; (4) to examine the contribution of both NO and gap junction to the ACh-induced endothelium-derived hyperpolarization in rabbit aortic smooth muscle cells.1. Development of a co-culture method of endothelial and smooth muscle cellsBy using the microporous polycarbonate filter membrane as the carrier, endothelial and smooth muscle cells were inoculated on the opposite sides of the carrier. The interaction between the endothelial and smooth muscle cells were examined by light and transmission electron microscopy. Relative to the endothelial cells monolayer, the smooth muscle cells were multilayers. There were a lot of gap-junctions between endothelial cells, smooth muscle cells and endothelial cells and smooth muscle cells. The number of the myoendothelial junctions had respect to the co-culturing time. After smooth muscle cells were seeded on the filter, the endothelial cells and smooth muscle cells can form the gap junctions through the micropores within 24 h. This new co-culture method might be a promising in vitro model for studies of interactions between endothelial and smooth muscle cells.2. The effect of ACh on the bovine aortic endothelial cellsThe effects of ACh on the membrane potential of endothelial cells of bovine aorta were studied using the conventional whole-cell patch-clamp method. The resting membrane potential observed in 90% of endothelial cells obtained from the bovine aorta was -72?mV(n=42); in the remaining cells (n=6), the cell membrane potential observed was more depolarized (-32 + 5), existing a bimodal distribution. And the current type existing in resting bovine aortic endothelial cells was an inwardly rectifying K+ current. Membrane potentials of the bovine aortic endothelial cells were recorded in current-clamp mode and lOuM ACh was applied. ACh induced hyperpolarizing responses with two phases, an initial fast phase followed by a slower second phase. Using the whole-cell voltage-clamp procedure, ACh induced an outward current. Application of TEA could block the outward current, and decrease the mean amplitudes of the initial and second phases of ACh-induced hyperpolarization. The results indicated that under resting conditions, the main conductance observed in bovine aortic endothelial cells was inwardly rectifying K" current. The endothelial cells' hype... |
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