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-23, which mediates initial muscle contraction by Gi-coupled receptor agonists, and RGS2, which regulates Gi activity;(ii) G113 and G112/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-23, G113, 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-I1. The mechanisms involving RGS2, p116Rip, PRIP-2, caveolin-1/PDE5, AKAP79/PLC-23, 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).

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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.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Special Emphasis Panel (ZRG1-DIG-D (02))
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Grey, Michael J
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Virginia Commonwealth University
Schools of Medicine
United States
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Mahavadi, Sunila; Grider, John R; Murthy, Karnam S (2018) Muscarinic m2 receptor-mediated actin polymerization via PI3 kinase ? and integrin-linked kinase in gastric smooth muscle. Neurogastroenterol Motil :e13495
Mahavadi, Sunila; Nalli, Ancy D; Wang, Hongxia et al. (2018) Regulation of gastric smooth muscle contraction via Ca2+-dependent and Ca2+-independent actin polymerization. PLoS One 13:e0209359
May, Alexander T; Crowe, Molly S; Blakeney, Bryan A et al. (2018) Identification of expression and function of the glucagon-like peptide-1 receptor in colonic smooth muscle. Peptides 112:48-55
Blakeney, Bryan A; Crowe, Molly S; Mahavadi, Sunila et al. (2018) Branched Short-Chain Fatty Acid Isovaleric Acid Causes Colonic Smooth Muscle Relaxation via cAMP/PKA Pathway. Dig Dis Sci :
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Mahavadi, Sunila; Sriwai, Wimolpak; Manion, Olivia et al. (2017) Diabetes-induced oxidative stress mediates upregulation of RhoA/Rho kinase pathway and hypercontractility of gastric smooth muscle. PLoS One 12:e0178574
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Parikh, Jay; Zemljic-Harpf, Alice; Fu, Johnny et al. (2017) Altered Penile Caveolin Expression in Diabetes: Potential Role in Erectile Dysfunction. J Sex Med 14:1177-1186
Qian, Jie; Mummalaneni, Shobha K; Alkahtani, Reem M et al. (2016) Nicotine-Induced Effects on Nicotinic Acetylcholine Receptors (nAChRs), Ca2+ and Brain-Derived Neurotrophic Factor (BDNF) in STC-1 Cells. PLoS One 11:e0166565

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