Recent studies, including our own, have pointed to cGMP-dependent protein kinase as the mediator of cGMP-induced relaxation of vascular smooth muscle in response to agonists such as atrial natriuretic factor, nitrovasodilators (e.g., nitroglycerin), methylxanthines (e.g., caffeine) and metabolites (e.g., EDRF). This proposal is based primarily on two features of cGMP-dependent protein kinase discovered in this laboratory over the past year: (1) Soluble fractions of vascular smooth muscle tissues contain a novel isozymic form named type I beta, in addition to the well-characterized type I alpha found in other tissues; (2) The cGMP-dependent protein kinase exists as chromatographically separable cGMP-deficient and cGMP-bound forms. The tissue and species distribution of types I alpha and I beta will be examined. The newly discovered type I beta will be purified to homogeneity and determined if it is a different gene product from type I alpha. Other physical and kinetic characteristics of the two enzymes will be compared, including protein and peptide substrate specificity. If it is established that type I beta, like type I alpha, has two different cGMP binding sites on each of two subunits, then a thorough study of cGMP binding kinetics and cGMP analog specificity for each site will be undertaken. Where possible, both isozymes will be utilized to study the function of each cGMP binding site by isolating cGMP-deficient and cGMP-bound forms of the enzymes. For some of these experiments, enzyme containing cGMP bound at particular sites will be characterized following isolation by the cGMP-induced DEAE """"""""charge shift"""""""" procedure recently established. The """"""""charge shift"""""""" will also be used as a novel approach to determine if cGMP or cAMP is bound to each site of type I alpha or I beta in the basal state of vascular smooth muscle, which might act as a """"""""priming"""""""" device for activation; and to determine if the various agents mentioned above which modulate cGMP levels,.as well as agents which modulate cAMP and calcium, will change the amount of cyclic nucleotide bound to each site. The role of intra-versus interchain interactions in the cGMP activation mechanism will also be investigated by studies of the monomeric kinase (the native enzyme is dimeric), found recently to be produced by proteolysis of the native enzyme. Also complementary for sequencing and for future studies of the cGMP binding sites will be the attempted cloning of the cDNA and genomic DNA for type I alpha and I beta.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK040029-01A1
Application #
3240107
Study Section
Biochemistry Study Section (BIO)
Project Start
1989-09-01
Project End
1994-08-31
Budget Start
1989-09-01
Budget End
1990-08-31
Support Year
1
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37203
Henesy, Michelle B; Britain, Andrea L; Zhu, Bing et al. (2012) Calcineurin regulates homologous desensitization of natriuretic peptide receptor-A and inhibits ANP-induced testosterone production in MA-10 cells. PLoS One 7:e41711
Francis, Sharron H; Blount, Mitsi A; Corbin, Jackie D (2011) Mammalian cyclic nucleotide phosphodiesterases: molecular mechanisms and physiological functions. Physiol Rev 91:651-90
Francis, Sharron H; Sekhar, Konjeti R; Ke, Hengming et al. (2011) Inhibition of cyclic nucleotide phosphodiesterases by methylxanthines and related compounds. Handb Exp Pharmacol :93-133
Francis, Sharron H (2010) The role of cGMP-dependent protein kinase in controlling cardiomyocyte cGMP. Circ Res 107:1164-6
Francis, Sharron H; Busch, Jennifer L; Corbin, Jackie D et al. (2010) cGMP-dependent protein kinases and cGMP phosphodiesterases in nitric oxide and cGMP action. Pharmacol Rev 62:525-63
Corbin, Jackie D; Zoraghi, Roya; Francis, Sharron H (2009) Allosteric-site and catalytic-site ligand effects on PDE5 functions are associated with distinct changes in physical form of the enzyme. Cell Signal 21:1768-74
Weeks 2nd, James L; Corbin, Jackie D; Francis, Sharron H (2009) Interactions between cyclic nucleotide phosphodiesterase 11 catalytic site and substrates or tadalafil and role of a critical Gln-869 hydrogen bond. J Pharmacol Exp Ther 331:133-41
Vandeput, Fabrice; Krall, Judith; Ockaili, Ramzi et al. (2009) cGMP-hydrolytic activity and its inhibition by sildenafil in normal and failing human and mouse myocardium. J Pharmacol Exp Ther 330:884-91
Liu, Yu-Ting; Matte, Suzanne L; Corbin, Jackie D et al. (2009) Probing the catalytic sites and activation mechanism of photoreceptor phosphodiesterase using radiolabeled phosphodiesterase inhibitors. J Biol Chem 284:31541-7
Steiner, Jennifer A; Carneiro, Ana Marin D; Wright, Jane et al. (2009) cGMP-dependent protein kinase Ialpha associates with the antidepressant-sensitive serotonin transporter and dictates rapid modulation of serotonin uptake. Mol Brain 2:26

Showing the most recent 10 out of 72 publications