This proposal represents the next five years of a long-standing research program, the general focus of which continues to be a determination of the mechanism of protein kinase C (PKC)-dependent contraction of vascular smooth muscle cells. The general aim of the next five years will be to test the specific hypothesis that PKC-mediated contraction of vascular smooth muscle cells involves thin filament regulation.
The specific aims are: (1) to test the hypothesis that calponin (CaP) is a physiologically important regulator of vascular tone by determining the effects of fragments and peptides of CaP in permeabilized single freshly dissociated cells; (2) to test the hypothesis that PKC and CaP are linked in a signaling cascade involving cytoskeIetal elements that results in the observed PKC-dependent redistribution of CaP by: (a) determining the structures from which and to which CaP associates upon PKC activations and, (b) determining what other signaling molecules are involved in the cascade; (3) to test the hypothesis that caldesmon (CaD) is a physiologically important regulator of vascular tone b: (a) monitoring its phosphorylation levels, (b) extending past studies on a C terminal CaD peptide, and (c) determining the effect of the fragments of the N- terminus of CaD on tone development, maintenance, relaxation and cytoskeletal architecture; (4) to test the hypothesis that signaling steps between PKC activation and mitogen-activated protein kinase (MAPK) redistribution/activation involve recruitment of Raf kinase and MAPK kinase (MEK) and to test whether phosphorylation/activation of MAPK plays a role in its biphasic subcellular targeting; (5) to determine the degree to which the observed MAP kinase-related contraction is selectively caused by upstream activation of PKC-epsilon as opposed to other isoforms. Experiments will be performed using two prototypical vascular smooth muscles from the ferret, the phasic portal vein, and the tonic aorta. The proposal is based on progress made in this laboratory, as well as other laboratories over the last grant period demonstrating a role for PKC-mediated contraction of differentiated vascular smooth muscle cells. The studies outlined are aimed at elucidating the details of the mechanisms involved in this contraction. The results obtained will either help to prove, or disprove, the hypothesis of PKC-mediated thin filament regulation. In either case, the information will be of broad general interest.
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