S2057 Diabetes-Induced Changes in the Expression of Proteins Involved in the Regulation of Sustained Mlc 20 Phosphorylation and cGMP Levels in Gastrointestinal Smooth Muscle

Abstract

The gastrointestinal complications of diabetes mellitus include nausea, vomiting, increase in intragastric pressure and delay in gastric emptying. Several studies showed that the changes in gastric emptying in diabetes are associated with the loss of enteric neurons and interstitial cells of Cajal, and dysfunction of neuronal nitric oxide synthase activity. We have previously shown that in gastrointestinal smooth muscle sustained MLC20 phosphorylation and contraction are dependent on inhibition of MLC phosphatase, which is mediated by RhoA-dependent pathway involving phosphorylation of MYPT1 (at Thr696) by Rho kinase and CPI-17 (at Thr38) by PKC. Smooth muscle relaxation is dependent on stimulation of cAMP and cGMP levels and activation of cAMPand cGMP-dependent protein kinases. The levels of cAMP and cGMP are regulated mainly by phosphodiesterase (PDE) 4D5 and PDE5, respectively. We hypothesized that diabetes induces changes in the expression of proteins involved in the maintenance of smooth muscle tone and regulation of cGMP levels. Aim. To determine the expression of PDE5, CPI-17 and Rho kinase as well as agonist-induced Rho kinase activity in gastric smooth muscle from control and diabetic animal models.Methods. Gastric smooth muscle was isolated from spontaneously type II diabetic ob/ob mice and type I diabetic rats. Type I diabetes was induced in rats by intravenous injection of streptozotocin. The rats were kept for 9 weeks and induction of diabetes was ascertained by the determination of blood glucose concentrations. Expression of CPI-17, Rho kinase and PDE5 was measured by immunoblot analysis and acetylcholine-induced Rho kinase activity was measured by immunokinase assay. Results. Expression of CPI-17, Rho kinase and PDE5 are significantly higher in gastric smooth muscle from both models of diabetic animals than from control animals. Consistent with the increased expression of Rho kinase, acetylcholine-induced Rho kinase activity was also significantly higher in smooth muscle from both models of diabetic animals than from control animals. Conclusion. This study identified mechanisms for increased smooth muscle tone and decreased smooth muscle relaxation in diabetes that are directly attributable to changes in smooth muscle physiology. The mechanisms involved increased expression of proteins involved in the regulation of sustained MLC20 phosphorylation (CPI-17 and Rho kinase) and cGMP levels (PDE5). Upregulation CPI-17 and Rho kinase could contribute to increased intragastric pressure, whereas upregulation of PDE5 could contribute to rapid hydrolysis of cGMP and decreased relaxation leading to delay in gastric emptying.