The overall thrust of this project is to isolate those subcellular structures, sarcoplasmic reticulum and sarcolemma, which are apparently most responsible for the immediate regulation of sarcoplasmic calcium levels in vascular smooth muscle and to examine the biochemical and physiological mechanisms which underlie the calcium transport properties of these structures with the intention of furthering our understanding of the role of these structures in excitation-contraction-relaxation coupling in vascular smooth muscle. Specifically we will seek to answer the following questions: 1. Can a "cleaner" preparation of fragmented sarcoplasmic reticulum (FSR) be obtained from bovine aorta by isopycnic centrifugation? 2. What is the influence of (K ion), (H ion) and (ATP) on both the transport and ATPase activities of the FSR? 3. Is there a phosphorylated protein associated with calcium transport or ATPase activity by FSR from vascular smooth muscle such as has been demonstrated in striated muscle FSR? 4. If such a phosphorylated intermediate exists, what is the correlation between its rate of formation and hydrolysis and the kinetics of transport and ATPase by the intact FSR? 5. Can a subcellular fraction of bovine aorta be prepared which is highly enriched in sarcolemma as evidenced by enrichment of appropriate marker enzymes? 6. What is the geometry of the sarcolemmal preparation, i.e., is it sheets, vesicles or inverted vesicles? 7. What is the capacity and affinity of the calcium transport property of the sarcolemma? 8. What is the influence of (Na ion), K ion), (H ion) and (ATP) on calcium transport by the sarcolemma? 9. Are there any means, either biochemical or physical, by which the calcium transport properties of either the FSR or sarcolemma can be altered to produce an apparent increase in sarcoplasmic calcium levels, i.e., can calcium release be demonstrated?
NIH ExPorter
NIH ExPorter