Role Of Protein Kinase A And RhoA/ROCK Pathway In Diabetic Colon Dysmotility

Background Gastrointestinal(GI)motility disorder is one of the most common complications in diabetic patients.The pathogenesis of GI dysmotility in diabetes is still unclear and it is related with multiple factors,such as autonomic neuropathy,decreased number of interstitial cells of Cajal(ICCs),and lesions of smooth muscle cells(SMCs).Phosphorylation of myosin light chain(MLC)is one of the key factors in the process of SMCs contraction.The phosphorylation of MLC is regulated by myosin light chain kinase and myosin light chain phosphatase(MLCP).Therefore,any factor that can influence MLCP activity can affect SMCs contraction by affecting the phosphorylation of MLC.Evidence has shown that Rho A/ROCK pathway is the main way to regulate MLCP.This pathway is involved in many pathological processes,and recently,it has been concerned for its role in DM.Although many studies have done to evalue the association between this pathway and diabetic complications,there is little research on the association between diabetic GI dysmotility and Rho A/ROCK pathway.Protein kinase A is involved in regulation of many cellular signaling pathway.It is believed that PKA can inhibit Rho A signal;Meanwhlie,the activity of PKA alters in DM.But whether its activity is also changed in diabetic GI muscle tissues remains unknown.Advanced glycation end products(AGEs)are significantly increased in serum and GI tissue of diabetic patients.AGEs are closely related to diabetic complications.Based on these studies,this study is to assess whether the expression of PKA and Rho A/ROCK pathway alter in diabetic muscle tissues and SMCs,thus provide the basis for the research of diabetic GI dysmotility.Objectives 1.To investigate the expression of PKA and Rho A/ROCK pathway in diabetic colonic muscle tissues.2.To investigate the effect of AGEs on Rho A/ROCK pathway in colonic SMCs and its possible mechanisms.Methods 1.Tissues preparation: Male SD rats were used for the experiments.Diabetic rats were induced by a single intraperitoneal injection of streptozotocin and age-matched control rats received equal volumes of buffer.Diabetes was confirmed 1 wk later by measurement of tail vein blood glucose levels.Rats with final blood glucose levels > 16.7 mmol/L were included in the study.2.Isolation and culture of colonic SMCs: The colon was taken from male SD rats.Smooth muscle layer was removed from the serosa and mucosa and then was cut into pieces.SMCs were isolated by enzymatic digestion and cultured with DMEM.(1)Effect of AGEs on Rho A/ROCK signal pathway in SMCs: Colonic SMCs were treated with AGEs,the protein expression levels in Rho A/ROCK pathway were measured by western blot.(2)Effect of AGEs on PKA levels: Colonic SMCs were cultured with different concentrations of AGEs for different times.PKA activity was tested by PKA activity assay kit.(3)Effect of PKA inhibitor on AGEs-inhibited Rho A/ROCK pathway in SMCs: Colonic SMCs were treated with H89 for 2min before exposure to AGEs.Western blot were used to examine the protein expression levels in Rho A/ROCK pathway.Results(1)The expression level of PKA and Rho A/ROCK pathway in colonic muscle tissues: Compared with the control group,the expression of PKA in diabetic colonic muscle tissues was significant increased(1.73±0.28 vs 2.21±0.88,P<0.05);While the protein expression levels of Rho A/ROCK/MLC pathway including Rho A,ROCK,p-MYPT1 and p-MLC were decreased(0.64±0.02 vs 1.17±0.04,P<0.01;1.00±0.007 vs 1.03±0.01,P<0.05;0.80±0.01 vs 1.02±0.007,P<0.01;0.98±0.098 vs 1.61±0.123,P<0.05)(2)The effect of AGEs on PKA activity: AGEs at the concentration of 100μg/ml,150μg/ml or 200μg/ml significantly increased PKA activity compared with the control group(1.81±0.118、1.89±0.069,1.91±0.061 vs 1.22±0.097,P<0.01),while there was no significance at the concentration of 50μg/ml(1.50±0.092 vs 1.22±0.097,P>0.05).(3)The effect of AGEs on Rho A/ROCK pathway: Compared with the control group,AGEs can inhibit the protein expression of Rho A/ROCK pathway including Rho A,ROCK,p-MYPT1(0.81±0.06 vs 1.24±0.08,0.70±0.07 vs 1.02±0.10 and 0.85±0.03 vs 1.17±0.03,P<0.05).(4)The effect of PKA on AGEs-mediated inhibition of Rho A/ROCK pathway: Pretreatment with PKA inhibitor H89 significantly enhanced AGEs-mediated effect on the decrease of Rho A/ROCK signaling pathway.Conclusions(1)Expression of PKA in diabetic colonic muscle tissues is increased,while the expression of Rho A/ROCK pathway is reduced.(2)AGEs inhibit activity of Rho A/ROCK pathway in colonic SMCs,and the mechanism involves the increase of PKA activity.