The general aim of this project is to elucidate the mechanisms by which agonists and growth states affect signalling pathways in vascular smooth muscle. [Ca2+]i will be measured both by aequorin luminescence and fura- 2 fluorescence. Myosin light chain (MLC) phosphorylation will be determined by two-dimensional polyacrylamide gel electrophoresis. Stiffness will be measured by small sinusoidal force oscillations. Digital imaging will be enhanced by mathematical deconvolution. Specifically: 1) the mechanism of alpha2-mediated signal transduction in rabbit saphenous vein will be determined by quantitating the relationship between [Ca2+]i, MLC phosphorylation and force levels during stimulation with specific alpha2 agonists in comparison to other stimulants. 2) The mechanism of the decreased [Ca2+]i sensitivity of force during K depolarization in ferret aorta will be determined by quantitating [Ca2+]i, MLC phosphorylation, and force relationships during a hysteresis protocol and by using a CaM KinaseII inhibitor. 3) The hypothesis that alpha1 stimulation in ferret aorta causes a shift in the relationship between MLC phosphorylation and force generation will be tested by hyperpermeabilization with beta-escin to clamp [Ca2+]i at known levels, allowing the true steady state relationships between [Ca2+]i, MLC phosphorylation, and force to be determined. 4) The determination of the mechanism of active intrinsic tone in resting ferret aorta will be completed by measuring changes in [Ca2+]i, MLC phosphorylation, and active stiffness during the development of intrinsic tone on warming in the presence and absence of MLC kinase inhibition and vasodilators. 5) The hypothesis that changes in muscle length result in changes in [Ca2+]i or [Ca2+]i sensitivity will be tested by measuring [Ca2+]i, MLC phosphorylation and force at different lengths. 6) The hypothesis that hypertrophy of rat aorta results in an increased [Ca2+]nucleus/[Ca2+]cytosol ratio will be tested by more accurately measuring intranuclear [Ca2+]. It is expected that by determining the details of these signalling pathways, not only will we better understand how the vascular smooth muscle cell contracts, but also some insight will be gained as to the mechanisms of growth regulation. In the long run, these findings should have considerable significance for understanding pathophysiological states of increased vascular tone (such as hypertension and spasm) and of increased vascular growth (such as atherosclerosis and restenosis).

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL031704-10A1
Application #
3342923
Study Section
Experimental Cardiovascular Sciences Study Section (ECS)
Project Start
1983-07-01
Project End
1995-12-31
Budget Start
1993-01-01
Budget End
1993-12-31
Support Year
10
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Beth Israel Deaconess Medical Center
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02215
Poythress, Ransom H; Gallant, Cynthia; Vetterkind, Susanne et al. (2013) Vasoconstrictor-induced endocytic recycling regulates focal adhesion protein localization and function in vascular smooth muscle. Am J Physiol Cell Physiol 305:C215-27
Yilmaz, Mehtap; Gangopadhyay, Samudra S; Leavis, Paul et al. (2013) Phosphorylation at Ser²? in the ATP-binding site of Ca²?/calmodulin-dependent kinase II as a mechanism for switching off the kinase activity. Biosci Rep 33:
Min, Jianghong; Reznichenko, Maya; Poythress, Ransom H et al. (2012) Src modulates contractile vascular smooth muscle function via regulation of focal adhesions. J Cell Physiol 227:3585-92
Gallant, Cynthia; Appel, Sarah; Graceffa, Philip et al. (2011) Tropomyosin variants describe distinct functional subcellular domains in differentiated vascular smooth muscle cells. Am J Physiol Cell Physiol 300:C1356-65
Appel, Sarah; Allen, Philip G; Vetterkind, Susanne et al. (2010) h3/Acidic calponin: an actin-binding protein that controls extracellular signal-regulated kinase 1/2 activity in nonmuscle cells. Mol Biol Cell 21:1409-22
Vetterkind, Susanne; Lee, Eunhee; Sundberg, Eric et al. (2010) Par-4: a new activator of myosin phosphatase. Mol Biol Cell 21:1214-24
Vetterkind, Susanne; Morgan, Kathleen G (2009) The pro-apoptotic protein Par-4 facilitates vascular contractility by cytoskeletal targeting of ZIPK. J Cell Mol Med 13:887-95
Gangopadhyay, Samudra S; Kengni, Edouard; Appel, Sarah et al. (2009) Smooth muscle archvillin is an ERK scaffolding protein. J Biol Chem 284:17607-15
Kim, H R; Appel, S; Vetterkind, S et al. (2008) Smooth muscle signalling pathways in health and disease. J Cell Mol Med 12:2165-80
Gangopadhyay, Samudra S; Gallant, Cynthia; Sundberg, Eric J et al. (2008) Regulation of Ca2+/calmodulin kinase II by a small C-terminal domain phosphatase. Biochem J 412:507-16

Showing the most recent 10 out of 11 publications