| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1991: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1990: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
1 I made use of front-surface fluorometry and strips of the pig coronary artery loaded with fura-2 to determine the effects of endothelin-1(ET-1) on cytosolic free Ca concentrations([Ca]i) and on contractions. The ratio of phosphorylated to non-phosphorylated myosin light chain(MLC) of the strips was determined using two dimensional electrophoresis. In the presence of extracellular Ca, ET-1 induced early rapid and then sustained increase in [Ca]i and contraction. Phosphorylated MLC rapidly increased to reach the maximum at 30 sec, then gradually declined, but remained at a level higher than the resting level. In the absence of extracellular Ca, ET-1 induced only transient increases in [Ca]i and contraction, with no sustained phase. However, ET-1 induced a similar extent of phosphorylation, with a time course similar to that seen in the presence of extracellular Ca. These findings suggested that phosphorylation of MLC relates to the early rapid increases in [Ca]i and tension development in ET-induced contractions, both in the presence and absence of extracellular Ca and that the sustained contraction is maintained by Ca-dependent mechanisms but not mediated by the phosphorylation of MLC.
2 Using front-surface fluorometry of fura-2 and valvular strips of the pig aorta, I recorded changes in [Ca]i of endothelial cells. Both ET71 and ET-3 elevated [Ca]i of a peak (the first phase) and sustained type. The first phase was considered to be due to a release of Ca from intracellular storage sites. The sustained phase depended on extracellular Ca and was considered to be due to an influx of Ca through the plasma membrane. At equimolar concentrations, the peak elevations of [Ca]i induced by ET-1 were much higher than those induce d by endothelin-3. The results suggested that, in endothelial cells in situ, ET-1 mobilizes stored Ca and may activate Ca-sensitive pathways, including the release of prostacyclin and endothelium-derived relaxing factors, more potently than does ET-3.
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