Tong L, Ao J-P, Lu H-L, Huang X, Zang J-Y, Liu S-H, Song N-N, Huang S-Q, Lu C, Chen J, Xu W-X
Department of Anatomy and Physiology, Shanghai Jiao Tong University School of Medicine, Shanghai, China,
Physiol Res. 2019 Mar 6;68(1):89-98. doi: 10.33549/physiolres.933857. Epub 2018 Oct 23.
The contraction of gastrointestinal (GI) smooth muscles is regulated by both Ca(2+)-dependent and Ca(2+) sensitization mechanisms. Proline-rich tyrosine kinase 2 (Pyk2) is involved in the depolarization-induced contraction of vascular smooth muscle via a Ca(2+) sensitization pathway. However, the role of Pyk2 in GI smooth muscle contraction is unclear. The spontaneous contraction of colonic smooth muscle was measured by using isometric force transducers. Protein and phosphorylation levels were determined by using western blotting. Pyk2 protein was expressed in colonic tissue, and spontaneous colonic contractions were inhibited by PF-431396, a Pyk2 inhibitor, in the presence of tetrodotoxin (TTX). In cultured colonic smooth muscle cells (CSMCs), PF-431396 decreased the levels of myosin light chain (MLC20) phosphorylated at Ser19 and ROCK2 protein expression, but myosin light chain kinase (MLCK) expression was not altered. However, Y-27632, a Rho kinase inhibitor, increased phosphorylation of Pyk2 at Tyr402 and concomitantly decreased ROCK2 levels; the expression of MLCK in CSMCs did not change. The expression of P(Tyr402)-Pyk2 and ROCK2 was increased when CSMCs were treated with Ach. Pyk2 is involved in the process of colonic smooth muscle contraction through the RhoA/ROCK pathway. These pathways may provide very important targets for investigating GI motility disorders.
胃肠道（GI）平滑肌的收缩受钙依赖性和钙敏化机制的调节。富含脯氨酸的酪氨酸激酶2（Pyk2）通过钙敏化途径参与血管平滑肌的去极化诱导收缩。然而，Pyk2在胃肠道平滑肌收缩中的作用尚不清楚。使用等长力传感器测量结肠平滑肌的自发收缩。通过蛋白质免疫印迹法测定蛋白质和磷酸化水平。Pyk2蛋白在结肠组织中表达，在存在河豚毒素（TTX）的情况下，Pyk2抑制剂PF-431396可抑制结肠的自发收缩。在培养的结肠平滑肌细胞（CSMCs）中，PF-431396降低了Ser19位点磷酸化的肌球蛋白轻链（MLC20）水平和ROCK2蛋白表达，但肌球蛋白轻链激酶（MLCK）的表达没有改变。然而，Rho激酶抑制剂Y-27632增加了Pyk2在Tyr402位点的磷酸化，并同时降低了ROCK2水平；CSMCs中MLCK的表达没有变化。当CSMCs用乙酰胆碱处理时，P（Tyr402）-Pyk2和ROCK2的表达增加。Pyk2通过RhoA/ROCK途径参与结肠平滑肌收缩过程。这些途径可能为研究胃肠动力障碍提供非常重要的靶点。
