This is a proposal to pursue studies on the mechanisms of relaxation of gastrointestinal smooth muscle by enteric neurotransmitters, chiefly NO and VIP, which induce relaxation of muscle tone through generation of cAMP and/or cGMP and activation of PKA and/or PKG. Our recent studies have shown: (i) that the levels of cAMP and cGMP are regulated via feedback phosphorylation of cyclases (AC V/VI and sGC) and phosphodiesterases (PDE3A, PDE4D5, and PDE5) by PKA and PKG. and (ii) that both PKA and PKG induce relaxation by inhibiting specific targets (RGS4, IP3R-I, RhoA, and MYPT1) in the signaling pathways mediating contraction. In preliminary studies, we have now identified novel targets of PKA and PKG that play equally important roles in mediating relaxation (Specific Aim I), and demonstrated the significance of scaffolding proteins, namely, AKAP79, caveolin-1, heat-shock protein (Hsp90), and PRIP-2 in modulating signaling by cAMP/PKA (Specific Aim II) and cGMP/PKG (Specific Aim III). The novel targets whose phosphorylation by PKA and/or PKG leads to relaxation include: (i) PLC-?3, which mediates initial muscle contraction by Gi-coupled receptor agonists, and RGS2, which regulates Gi activity;(ii) G?13 and G?12/Rho-GEF (AKAP-Lbc), which regulate RhoA activity and sustained muscle contraction;and (iii) the MLC phosphatase activators, telokin and p116Rip, which promote MLC20 dephosphorylation (Specific Aim I). The strength, duration, and specificity of cAMP/PKA signaling are modulated by caveolin-1, AKAP79, and AKAP-Lbc (Specific Aim II). Preliminary studies show that caveolin-1 dampens the cAMP/PKA signal by inhibiting AC V/VI and accelerating internalization of VPAC2 receptors, whereas AKAPs dampen the cAMP/PKA signal by promoting activation of PDE4D5 and inhibition of AC V/VI;in addition, AKAP79 and AKAP-Lbc facilitate the targeting and inactivation of PLC-?3, G?13, Rho-GEF, and RhoA by PKA leading to muscle relaxation. The strength, duration, and specificity of cGMP/PKG signaling are modulated by caveolin-1, Hsp90, and the IP3R-I binding protein, PRIP-2 (Specific Aim III). Preliminary studies show that caveolin-1 binds PDE5 and inhibits cGMP degradation, whereas Hsp90 stabilizes sGC and enhances cGMP synthesis;PRIP-2 facilitates targeting and inactivation of IP3- RI by PKG-I?. The mechanisms involving RGS2, p116Rip, PRIP-2, caveolin-1/PDE5, AKAP79/PLC-?3, AKAP-Lbc/RhoA represent new discoveries resulting from our preliminary studies. The functional significance of these molecular mechanisms was confirmed in preliminary studies on muscle relaxation in integrated tissues using muscle strips and whole segments and in caveolin-1-/- mice. Analysis of these mechanisms should lead to new insights for the development of therapeutic agents that act on smooth muscle of the gut and other regions (e.g., airway and vascular smooth muscle).

Public Health Relevance

The objective of this proposal is to characterize the signal transduction pathways that mediate gastrointestinal smooth muscle relaxation. The project involves analysis of the molecular targets of PKA and PKG: PLC-b3, Ga13, telokin, p116Rip, and their ability to modulate muscle relaxation, and define the role of scaffolding proteins in regulating cAMP/PKA and cGMP/PKG signaling.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK028300-33
Application #
8468678
Study Section
Special Emphasis Panel (ZRG1-DIG-D (02))
Program Officer
Grey, Michael J
Project Start
1984-04-01
Project End
2014-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
33
Fiscal Year
2013
Total Cost
$301,876
Indirect Cost
$96,166
Name
Virginia Commonwealth University
Department
Physiology
Type
Schools of Medicine
DUNS #
105300446
City
Richmond
State
VA
Country
United States
Zip Code
23298
Kumar, Divya P; Asgharpour, Amon; Mirshahi, Faridoddin et al. (2016) Activation of Transmembrane Bile Acid Receptor TGR5 Modulates Pancreatic Islet α Cells to Promote Glucose Homeostasis. J Biol Chem 291:6626-40
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Liu, Miao; Shen, Shanwei; Kendig, Derek M et al. (2015) Inhibition of NMDAR reduces bladder hypertrophy and improves bladder function in cyclophosphamide induced cystitis. J Urol 193:1676-83
Anderson Jr, Charles D; Kendig, Derek M; Al-Qudah, Mohammad et al. (2014) Role of various kinases in muscarinic M3 receptor-mediated contraction of longitudinal muscle of rat colon. J Smooth Muscle Res 50:103-19
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Bala, Vanitha; Rajagopal, Senthilkumar; Kumar, Divya P et al. (2014) Release of GLP-1 and PYY in response to the activation of G protein-coupled bile acid receptor TGR5 is mediated by Epac/PLC-ε pathway and modulated by endogenous H2S. Front Physiol 5:420
Huang, Jiean; Nalli, Ancy D; Mahavadi, Sunila et al. (2014) Inhibition of Gαi activity by Gβγ is mediated by PI 3-kinase-γ- and cSrc-dependent tyrosine phosphorylation of Gαi and recruitment of RGS12. Am J Physiol Gastrointest Liver Physiol 306:G802-10
Nalli, Ancy D; Kumar, Divya P; Al-Shboul, Othman et al. (2014) Regulation of Gβγi-dependent PLC-β3 activity in smooth muscle: inhibitory phosphorylation of PLC-β3 by PKA and PKG and stimulatory phosphorylation of Gαi-GTPase-activating protein RGS2 by PKG. Cell Biochem Biophys 70:867-80

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