大鼠腮腺M受体亚型分布的实验研究

EXPERIMENTAL STUDY ON THE DISTRIBUTION OF MUSCARINIC RECEPTOR SUBTYPES IN RAT PAROTID GLAND

目的：检测大鼠腮腺组织中Ｍ受体亚型的分布。方法：取ＳＤ大鼠腮腺制备膜蛋白，应用３Ｈ－ＱＮＢ测定特异性饱和曲线；选择Ｍ１、Ｍ２、Ｍ３的选择性拮抗剂Ｐｉｒｅｎｚｅｐｉｎｅ，Ｇａｌａｍｉｎｅ，４－ＤＡＭＰ进行竟争性结合实验。结果：饱和曲线示平衡解离常数Ｋｄ为０．８４±０．０８ｎＭ，最大结合量Ｂｍａｘ为１００．２±７．７ｆｍｏｌ／ｍｇ蛋白，Ｈｉｌ系数为０．８８，这说明３Ｈ－ＱＮＢ与Ｍ受体为单一结合位点；竟争性结合实验示三种拮抗剂的半数最大抑制浓度ＩＣ５０分别为１．９８×１０－９（Ｍ１），７．８×１０－８（Ｍ２），４．７６×１０－１０ｍｏｌ／Ｌ。结论：以上结果表明腮腺Ｍ受体以Ｍ３最多，Ｍ１次之，Ｍ２最少，这对阐明唾液腺的分泌机制、Ｍ受体与疾病状态均有重要的意义。

Objectives: The main receptors controlling salivary secretion are muscarinic receptors,which have five subtypes(m1 m5).There are differrnt distribution and quantity of the muscarinic receptor subtypes in different tissues.In order to determine the distribution of muscarinic receptor subtypes in rat parotid glands,radioactive ligand receptor binding experiment was performed. Methods: Parotid glands from SD rats were excised and the crude membranes were prepared.Saturation curves were generated by incubating membranes with 3H QNB.Dissociation constant Kd and maximum binding number (Bmax) were calculated by scatchard formula and Hill coefficient (nH) by Hill formula.According to saturation curves,we selected proper 3H QNB concentration and m1 m2 m3 selective antagonist (Pirenzepine)(M 1) Gallamine(M 2) 4 DAMP(M 3)) for the competition studies,using pharm software to calculate IC 50 . Results: For saturation curves,The dissociation constant Kd was 0.84±0.08nM,maximum binding number (Bmax) 100.2±7.7fmol/mg protein and Hill coefficient (nH) 0.88. 3H QNB labeled a single class of highaffinity binding sites in membranes of parotid glands.For competition studies,the IC 50 value of each antagonist was 1.98×10 -9 M (Prienzepine) 7.8×10 -8 M(Gallamime) and 4.76×10 -10 M (4 DAMP),respectively. Conclusion: The result showed that M 3 receptor was predominant,M 1 was the second and M 2 was minimum in rat parotid.It is significant for us to clarify secretive mechanism of salivary gland and the relation between muscarinic receptor and salivary diseases.