Intracellular cGMP and Ca2+ regulate vascular smooth muscle (VSM) function in health and disease. The emerging view is that intracellular Ca2+ signals are highly dynamic, and that patterning of Ca2+ signals determines VSM function. Understanding of intracellular cGMP signaling dynamics, subsequent activation of cGMP-dependent protein kinase (PKG), and its relationship to Ca2+ signals has lagged. Here, using novel cGMP biosensors and PKG inhibitors, we will provide evidence that cGMP, like Ca2+, is spatially and temporally dynamic, and dependent on multiple interrelated control mechanisms. Our approach will involve high resolution measurements of Ca2+ and cGMP, and provide an unparalleled view of the interactive control of VSM function by these messenger molecules.
In Specific Aim 1 we will test the theory that membrane (pGC) and cytosolic (sGC) guanylyl cyclases engage fundamentally different patterns of cGMP formation. We propose that NO and ANP, which primarily activate sGC and pGC, respectively, create discrete pools of intracellular cGMP, and thus provide the structural basis for the functional compartmentalization of cGMP signals. We will also test the mechanisms by which PKG controls vascular tone by serving as both a negative and a positive feedback regulator for cGMP pools. These assessments have been made possible by our recent development of novel cGMP-biosensors, which allow direct measurement of cellular cGMP with high temporal and spatial resolution.
In Specific Aim 2 we will elucidate the interplay between cGMP/PKG and calcium signaling. We propose that cGMP and PKG act in part through modulation of Ca2+ sparks, BKCa channels, and global calcium to regulate vascular function.
In Specific Aim 3 we will determine the contributions of PKG to vascular control in vivo by studying the efficacy of PKG inhibitors to increase blood pressure and vascular resistance in the intact animal. Our approach to test the three aims of this proposal will be multidisciplinary, employing state-of-the-art techniques from physiology (high speed calcium imaging, patch clamp techniques, resistance artery myography, blood pressure and blood flow measurements), cell biology (confocal fluorescence microscopy, ratiometric fluorescence microscopy, smooth muscle cell culture), molecular biology (insect cell culture, mutagenesis, adenovirus) and biochemistry (enzyme kinetic techniques, fluorescence energy transfer, cellular protein delivery systems). This work will provide an integrated view of the factors that modulate PKG activity in vascular smooth muscle and thereby significantly enhance our understanding of arterial functions in health and disease.

Public Health Relevance

The cGMP-dependent protein kinase (PKG) is an essential regulator of cellular function in blood vessels throughout the body. This proposal seeks to ascertain the molecular mechanisms of vascular control involving PKG and its signaling partners. Understanding how blood vessels constrict and dilate is critical for the development of new strategies and therapeutic agents aimed at prevention and treatment of vascular disorders such as hypertension, stroke and coronary artery disease.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL068991-06
Application #
7591107
Study Section
Vascular Cell and Molecular Biology Study Section (VCMB)
Program Officer
Goldman, Stephen
Project Start
2002-01-01
Project End
2013-03-31
Budget Start
2009-04-01
Budget End
2010-03-31
Support Year
6
Fiscal Year
2009
Total Cost
$376,250
Indirect Cost
Name
University of Vermont & St Agric College
Department
Pharmacology
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
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Arora, Kavisha; Sinha, Chandrima; Zhang, Weiqiang et al. (2015) Altered cGMP dynamics at the plasma membrane contribute to diarrhea in ulcerative colitis. Am J Pathol 185:2790-804
Held, Kara F; Dostmann, Wolfgang R (2013) Real-time monitoring the spatiotemporal dynamics of intracellular cGMP in vascular smooth muscle cells. Methods Mol Biol 1020:131-45
Moon, Thomas M; Osborne, Brent W; Dostmann, Wolfgang R (2013) The switch helix: a putative combinatorial relay for interprotomer communication in cGMP-dependent protein kinase. Biochim Biophys Acta 1834:1346-51
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Osborne, Brent W; Wu, Jian; McFarland, Caitlin J et al. (2011) Crystal structure of cGMP-dependent protein kinase reveals novel site of interchain communication. Structure 19:1317-27
Nickl, Christian K; Raidas, Shiv Kumar; Zhao, Hong et al. (2010) (D)-Amino acid analogues of DT-2 as highly selective and superior inhibitors of cGMP-dependent protein kinase Ialpha. Biochim Biophys Acta 1804:524-32
Lavogina, Darja; Nickl, Christian K; Enkvist, Erki et al. (2010) Adenosine analogue-oligo-arginine conjugates (ARCs) serve as high-affinity inhibitors and fluorescence probes of type I cGMP-dependent protein kinase (PKGIalpha). Biochim Biophys Acta 1804:1857-68
Bove, Peter F; Wesley, Umadevi V; Greul, Anne-Katrin et al. (2007) Nitric oxide promotes airway epithelial wound repair through enhanced activation of MMP-9. Am J Respir Cell Mol Biol 36:138-46

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