Wrzos Helena-F, Tandon Tarun, Ouyang Ann
Division of Gastroenterology and Hepatology, Department of Medicine, College of Medicine, Pennsylvania State University, PO Box 850, Hershey, PA 17033, USA.
World J Gastroenterol. 2004 Nov 15;10(22):3292-8. doi: 10.3748/wjg.v10.i22.3292.
To investigate the pathway (s) mediating rat antral circular smooth muscle contractile responses to the cholinomimetic agent, bethanechol and the subtypes of muscarinic receptors mediating the cholinergic contraction.
Circular smooth muscle strips from the antrum of Sprague-Dawley rats were mounted in muscle baths in Krebs buffer. Isometric tension was recorded. Cumulative concentration-response curves were obtained for (+)-cis-dioxolane (cD), a nonspecific muscarinic agonist, at 10(-8)-10(-4) mol/L, in the presence of tetrodotoxin (TTX, 10(-7) mol/L). Results were normalized to cross sectional area. A repeat concentration-response curve was obtained after incubation of the muscle for 90 min with antagonists for M1 (pirenzepine), M2 (methoctramine) and M3 (darifenacin) muscarinic receptor subtypes. The sensitivity to PTX was tested by the ip injection of 100 mg/kg of PTX 5 d before the experiment. The antral circular smooth muscles were removed from PTX-treated and non-treated rats as strips and dispersed smooth muscle cells to identify whether PTX-linked pathway mediated the contractility to bethanechol.
A dose-dependent contractile response observed with bethanechol, was not affected by TTX. The pretreatment of rats with pertussis toxin decreased the contraction induced by bethanechol. Lack of calcium as well as the presence of the L-type calcium channel blocker, nifedipine, also inhibited the cholinergic contraction, with a reduction in response from 2.5+/-0.4 g/mm2 to 1.2+/-0.4 g/mm(2) (P<0.05). The dose-response curves were shifted to the right by muscarinic antagonists in the following order of affinity: darifenacin (M(3))>methocramine (M(2)) >pirenzepine (M(1)).
The muscarinic receptors-dependent contraction of rat antral circular smooth muscles was linked to the signal transduction pathway(s) involving pertussis-toxin sensitive GTP-binding proteins and to extracellular calcium via L-type voltage gated calcium channels. The presence of the residual contractile response after the treatment with nifedipine, suggests that an additional pathway could mediate the cholinergic contraction. The involvement of more than one muscarinic receptor (functionally predominant type 3 over type 2) also suggests more than one pathway mediating the cholinergic contraction in rat antrum.
研究介导大鼠胃窦环形平滑肌对拟胆碱药氨甲酰甲胆碱收缩反应的途径以及介导胆碱能收缩的毒蕈碱受体亚型。
将来自Sprague-Dawley大鼠胃窦的环形平滑肌条置于含有Krebs缓冲液的肌肉浴槽中。记录等长张力。在存在河豚毒素（TTX，10⁻⁷mol/L）的情况下，获得10⁻⁸ - 10⁻⁴mol/L的非特异性毒蕈碱激动剂（+）-顺式二氧戊环（cD）的累积浓度-反应曲线。结果根据横截面积进行标准化。在用M1（哌仑西平）、M2（甲溴东莨菪碱）和M3（达非那新）毒蕈碱受体亚型拮抗剂孵育肌肉90分钟后，获得重复的浓度-反应曲线。在实验前5天腹腔注射100mg/kg的百日咳毒素（PTX）来测试对PTX的敏感性。从经PTX处理和未经处理的大鼠中取出胃窦环形平滑肌条，并分离平滑肌细胞，以确定PTX相关途径是否介导对氨甲酰甲胆碱的收缩性。
观察到氨甲酰甲胆碱引起剂量依赖性收缩反应，不受TTX影响。用百日咳毒素预处理大鼠可降低氨甲酰甲胆碱诱导的收缩。缺乏钙以及存在L型钙通道阻滞剂硝苯地平也抑制胆碱能收缩，反应从2.5±0.4g/mm²降至1.2±0.4g/mm²（P<0.05）。毒蕈碱拮抗剂使剂量-反应曲线右移，亲和力顺序如下：达非那新（M3）>甲溴东莨菪碱（M2）>哌仑西平（M1）。
大鼠胃窦环形平滑肌的毒蕈碱受体依赖性收缩与涉及百日咳毒素敏感的GTP结合蛋白的信号转导途径以及通过L型电压门控钙通道的细胞外钙有关。用硝苯地平处理后仍存在残余收缩反应，表明可能存在另一条途径介导胆碱能收缩。多种毒蕈碱受体的参与（功能上M3型比M2型占优势）也表明在大鼠胃窦中存在不止一条途径介导胆碱能收缩。
