The Role Of Pyk2-mediated RhoA/ROCK Signaling Pathway In The Contraction Process Of Colonic Smooth Muscle

The mechanisms of contraction of smooth muscles include"Ca²⁺-dependent"and"Ca²⁺-independent"pathways.These two pathways cooperate well to make a constant and steady smooth muscle contraction.It has been reported that Pyk2 can be activated and then accelerate the process of differentiation,proliferation and contraction of vascular smooth muscle cells.However,whether Pyk2 plays a similar role in the contraction of gastrointestinal?GI?smooth muscle cells has not been reported by far.In addition,Pyk2 and RhoA/ROCK signaling pathway which blongs to"Ca²⁺-independent"pathway show abnormality in many vascular diseases recently.We wondered if GI motility disorder disease is also related to the abnormal expression of Pyk2 and RhoA/ROCK signaling pathway.But few studies have been reported about this area.Consequently,we firstly studied the relationship between Pyk2 and colonic smooth muscle contraction in normal mouse.Secondly,we explored if the change of Pyk2 signaling pathway mediated contraction in the colonic tissue of Hirschsprung's disease?HD?.The isometric tension measurement method was used to explore the contraction of colonic smooth muscle,the western blotting method was used to observe the protein expression which related to contraction,and the HE staining was used to compare the difference among the structure of the wall in HD patients.The results showed that Pyk2 protein was expressed in colonic smooth muscle tissue of normal mouse.When Pyk2was inhibited,the amplitudes of colonic smooth muscle contraction were significantly reduced,and the MLCK protein expression which related to“Ca²⁺-dependent”pathway was not affected,however,the protein expressions involved in RhoA/ROCK signaling pathway were decreased.On the other hand,the level of P?Tyr402?-Pyk2 and the protein expressions involved in RhoA/ROCK signaling pathway were both increased in Ach-induced smooth muscle contraction.Next,in the colon of HD children,we found that the contraction in narrow segment colon was stronger compared with the normal part,and the muscular layer and submucosa in narrow segment were also thicker compared with normal colon.In addition,the expression of Pyk2 was decreased in both narrow and dilated segments compared with the normal part,and the protein expressions involved in RhoA/ROCK signaling pathway in narrow segment were reduced compared with the normal part,but between the dilated and the normal parts there was no significant difference in the protein expressions.Combined with the above results,we concluded that Pyk2 is expressed in the colonic tissue of normal mouse,which can participate in the colonic smooth muscle contraction via RhoA/ROCK signaling pathway.Moreover,the contraction in narrow segment is stronger than that in the normal part in HD patients,which may result from the decrease of Ca²⁺-independent pathway and thus the compensatory role of Ca²⁺dependent pathway.