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).

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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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Nalli, Ancy D; Kumar, Divya P; Al-Shboul, Othman et al. (2014) Regulation of G??i-dependent PLC-?3 activity in smooth muscle: inhibitory phosphorylation of PLC-?3 by PKA and PKG and stimulatory phosphorylation of G?i-GTPase-activating protein RGS2 by PKG. Cell Biochem Biophys 70:867-80
Mahavadi, Sunila; Sriwai, Wimolpak; Huang, Jiean et al. (2014) Inhibitory signaling by CB1 receptors in smooth muscle mediated by GRK5/?-arrestin activation of ERK1/2 and Src kinase. Am J Physiol Gastrointest Liver Physiol 306:G535-45
Huang, Jiean; Nalli, Ancy D; Mahavadi, Sunila et al. (2014) Inhibition of G?i activity by G?? is mediated by PI 3-kinase-?- and cSrc-dependent tyrosine phosphorylation of G?i and recruitment of RGS12. Am J Physiol Gastrointest Liver Physiol 306:G802-10
Mahavadi, Sunila; Nalli, Ancydimpy; Al-Shboul, Othman et al. (2014) Inhibition of MLC20 phosphorylation downstream of Ca2+ and RhoA: A novel mechanism involving phosphorylation of myosin phosphatase interacting protein (M-RIP) by PKG and stimulation of MLC phosphatase activity. Cell Biochem Biophys 68:1-8
Rajagopal, Senthilkumar; Kumar, Divya P; Mahavadi, Sunila et al. (2013) Activation of G protein-coupled bile acid receptor, TGR5, induces smooth muscle relaxation via both Epac- and PKA-mediated inhibition of RhoA/Rho kinase pathway. Am J Physiol Gastrointest Liver Physiol 304:G527-35
Al-Shboul, Othman; Mahavadi, Sunila; Sriwai, Wimolpak et al. (2013) Differential expression of multidrug resistance protein 5 and phosphodiesterase 5 and regulation of cGMP levels in phasic and tonic smooth muscle. Am J Physiol Gastrointest Liver Physiol 305:G314-24
Alkahtani, Reem; Mahavadi, Sunila; Al-Shboul, Othman et al. (2013) Changes in the expression of smooth muscle contractile proteins in TNBS- and DSS-induced colitis in mice. Inflammation 36:1304-15
Mahavadi, Sunila; Bhattacharya, Sayak; Kim, Jennnifer et al. (2013) Caveolae-dependent internalization and homologous desensitization of VIP/PACAP receptor, VPACýýý, in gastrointestinal smooth muscle. Peptides 43:137-45
Bhattacharya, Sayak; Mahavadi, Sunila; Al-Shboul, Othman et al. (2013) Differential regulation of muscarinic M2 and M3 receptor signaling in gastrointestinal smooth muscle by caveolin-1. Am J Physiol Cell Physiol 305:C334-47
Ahmed, Rashad; Mahavadi, Sunila; Al-Shboul, Othman et al. (2013) Characterization of signaling pathways coupled to melatonin receptors in gastrointestinal smooth muscle. Regul Pept 184:96-103

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