Increased PDE5 activity and decreased Rho kinase and PKC activities in colonic muscle from caveolin-1^−/− mice impair the peristaltic reflex and propulsion

Abstract

Murthy KS. Increased PDE5 activity and decreased Rho kinase and PKC activities in colonic muscle from caveolin-1 Ϫ/Ϫ mice impair the peristaltic reflex and propulsion. Am J Physiol Gastrointest Liver Physiol 305: G964 -G974, 2013. First published October 24, 2013 doi:10.1152/ajpgi.00165.2013.-Caveolae are specialized regions of the plasma membrane that concentrate receptors and associated signaling molecules critical in regulation of cellular response to transmitters and hormones. We have determined the effects of caveolin-1 (Cav-1) deletion, caveolin-1 siRNA, and caveolar disruption in mice on the signaling pathways that mediate contraction and relaxation in colonic smooth muscle and on the components of the peristaltic reflex in isolated tissue and propulsion in intact colonic segments. In Cav-1 Ϫ/Ϫ mice, both relaxation and contraction were decreased in smooth muscle cells and muscle strips, as well as during both phases of the peristaltic reflex and colonic propulsion. The decrease in relaxation in response to the nitric oxide (NO) donor was accompanied by a decrease in cGMP levels and an increase in phosphodiesterase 5 (PDE5) activity. Relaxation by a PDE5-resistant cGMP analog was not affected in smooth muscle of Cav-1 Ϫ/Ϫ mice, suggesting that inhibition of relaxation was due to augmentation of PDE5 activity. Similar effects on relaxation, PDE5 and cGMP were obtained in muscle cells upon disruption of caveolae by methyl-␤-cyclodextrin or suppression of Cav-1. Sustained contraction mediated via inhibition of myosin light chain phosphatase (MLCP) activity is regulated by Rho kinase and PKC via phosphorylation of two endogenous inhibitors of MLCP: myosin phosphatasetargeting subunit (MYPT1) and 17-kDa PKC-potentiated protein phosphatase 1 inhibitor protein (CPI-17), respectively. The activity of both enzymes and phosphorylation of MYPT1 and CPI-17 were decreased in smooth muscle from Cav-1 Ϫ/Ϫ mice. We conclude that the integrity of caveolae is essential for contractile and relaxant activity in colonic smooth muscle and the maintenance of neuromuscular function at organ level.caveolin; phosphodiesterase; Rho kinase; smooth muscle; peristaltic reflex CAVEOLAE AND THEIR MAIN MEMBRANE PROTEINS, caveolin-1 (Cav-1), -2, and -3, form regular invaginations of the plasma membrane, which are sites that concentrate receptors, G proteins, effector enzymes, and associated signaling molecules in several cell types (reviewed in Refs. 2, 4, 22, 37, 50). As such, they play a critical role in regulating, either by augmenting or inhibiting, the physiological response of cells to transmitters and hormones. Most studies have concentrated on identifying their role in regulating the response to agonists at the cellular level; however, the translation of these cellular effects of caveolin to the organ level is less well understood although many roles have been postulated (1, 25, 43-45, 50, 58).In the gut, caveoli are present on many cell types, including smooth muscle cells, interstitial cells of Cajal (ICC), and endothelial cell...

Cytokine-Induced S-Nitrosylation of Soluble Guanylyl Cyclase and Expression of Phosphodiesterase 1A Contribute to Dysfunction of Longitudinal Smooth Muscle Relaxation

“…Colonic tissue from mice treated with TNBS exhibited typical histologic characteristics of colitis (Alkahtani et al, 2013;Al-Shboul et al, 2014;Mahavadi et al, 2014;Nalli et al, 2014). Distal colonic segments 2-3 cm long were obtained from control and TNBS-treated mice, and muscle cells from the longitudinal muscle layer were obtained as described previously (Murthy et al, 2002(Murthy et al, , 2003Mahavadi et al, 2013Mahavadi et al, , 2014. Briefly, colonic longitudinal muscle strips were dissected and incubated at 31°C for 30 minutes in HEPES medium containing 120 mM NaCl, 4 mM KCl, 2.6 mM KH 2 PO 4 , 0.6 mM MgCl 2 , 25 mM HEPES, 14 mM glucose, 2.1% Eagle's essential amino acid mixture, 0.1% collagenase, and 0.1% soybean trypsin inhibitor.…”

“…[ Assay for PDE Activity. PDE5 and PDE1A activity was measured in immunoprecipitates of PDE5 and PDE1A as described previously (Wyatt et al, 1998;Murthy, 2001;Mahavadi et al, 2013). One-milliliter aliquots (3 Â 10 6 cells/ml) of muscle cells were incubated with S-nitroso-N-acetylpenicillamine (SNAP) for 5 minutes.…”

“…Cyclic GMP production was measured by radioimmunoassay as described previously (Murthy, 2001;Mahavadi et al, 2013). In brief, muscle cells (3 Â 10 6 cells) were treated with SNAP for 5 minutes, and the reaction was terminated with 10% trichloroacetic acid.…”

“…Contraction in freshly dispersed colonic longitudinal muscle cells was determined by scanning micrometry (Murthy and Makhlouf, 1998;Murthy, 2001;Mahavadi et al, 2013Mahavadi et al, , 2014). An aliquot (0.4 ml) of cells containing approximately 10 4 cells/ml was treated with 100 ml of medium containing different concentrations of SNAP for 5 minutes followed by carbachol (1 mM) for 30 seconds, and then the reaction was terminated with 1% acrolein at a final concentration of 0.1%.…”

“…PDE5 activity is stimulated via binding of cGMP and cGMPdependent protein kinase (PKG)-mediated phosphorylation of PDE5 at a conserved serine residue in the N-terminal region (Francis et al, 2001;Rybalkin et al, 2003). PDE5 expression is abundant in smooth muscle and plays an important role in the regulation of cGMP levels and smooth muscle relaxation (Murthy, 2001;Mahavadi et al, 2013). PDE1 (PDE1A and PDE1B) hydrolyzes cGMP with higher affinity and is regulated by Ca 21 /calmodulin (Sharma et al, 2006).…”