(Verbatim from the application): This is a proposal to fund the next 5 years of a longstanding research program to investigate the molecular mechanisms and physiological significance of calcium dependent signaling pathways in differentiated vascular smooth muscle. The next period of support focuses on calmodulin dependent signaling pathways.
The specific aims are: (1) to test the hypothesis that free calmodulin levels are sufficient in resting or stimulated vascular smooth muscle cells to affect two calmodulin binding targets having relatively low affinity for calmodulin, caldesmon and CaM kinase II; (2) to test the hypothesis that the total calmodulin present in single contractile vascular smooth muscle cells redistributes between cellular compartments during physiological stimulation; (3) to test the hypothesis that free calmodulin levels are a stimulus dependent variable by measuring changes in these levels in the presence and absence of stimuli; and (4) to test the hypothesis that CaMKII participates in signaling cascades in differentiated vascular smooth muscle cells. The experiments outlined in this proposal involve the use of both multicellular strips and freshly isolated single cells from ferret aorta and ferret portal vein. The techniques to be used involve high resolution """"""""digital confocal"""""""" microscopy, quantitative image analysis, microforce recording from single saponin permeabilized cells, calcium indicator measurements, peptide loading methods, 2-dimensional polyacrylamide gels, 1-dimensional IEF gels, western blots, phosphorylation measurements, kinase activity measurements and the use of peptide, compound and oligonucleotide antisense inhibitors. All techniques are established in the investigator's laboratory and the results may be expected to significantly advance our understanding by which vascular tone is maintained. Since novel signal transduction mechanisms may be discovered, these studies may well lead to subsequent development of new therapeutic strategies.
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