小肠毒蕈碱型胆碱受体的多样性

从小鼠小肠收缩实验测得,乙酰胆碱引起纵,环肌收缩的50%有效浓度各为7～10^-5M和10^-4～10^-2M,阿托品的PA₂约为9和7。实验结果提示小肠上至少存在两种不同亲和性的毒蕈碱型胆碱受体,分别与纵、环肌收缩有关。又用Yamamura法将小肠全平滑肌层、纵肌层匀浆与³H-ONB做结合实验,测得解离常数KD值约为1 nm和15 nm的两种特异结合部位。     

小肠毒蕈碱型胆碱受体的多样性

从小鼠小肠收缩实验测得,乙酰胆碱引起纵,环肌收缩的50%有效浓度各为7～10~(-5)M和10~(-4)～10~(-2)M,阿托品的PA_2约为9和7。实验结果提示小肠上至少存在两种不同亲和性的毒蕈碱型胆碱受体,分别与纵、环肌收缩有关。又用Yamamura法将小肠全平滑肌层、纵肌层匀浆与~3H-ONB做结合实验,测得解离常数KD值约为1 nm和15 nm的两种特异结合部位。     

P2Y受体介导大鼠胃体环行肌收缩反应的药理学特点

观察大鼠离体胃体环行肌和胃底环行肌不同的药理学特征，分析核苷及核苷酸类物质诱发胃体环行肌收缩反应的作用特点和受体机制。制备大鼠离体胃体环行肌和胃底环行肌标本，利用受体药理学技术观察药物诱发的收缩反应。在胃体环行肌KCl所致收缩反应与胃底环行肌无显著性差别；但是，CCh收缩胃体环行肌的EC₅₀值 [(0.45 ± 0.15) μmol·L⁻¹] 显著高于胃底环行肌 [(0.20 ± 0.09) μmol·L⁻¹, P < 0.01]。5-HT和His收缩两种标本的EC₅₀值无显著差异 (P > 0.05); 但是, 在胃体环行肌5-HT和His产生收缩反应的E_max值 [(0.81 ± 0.26) 和 (0.88 ± 0.27) g] 显著小于胃底环行肌 [(2.67 ± 0.61) 和 (1.90 ± 0.68) g, P < 0.01]。在预收缩胃体环行肌，ATP (0.1～3 000 μmol·L⁻¹) 诱发浓度依赖性收缩反应，未见舒张反应；在预收缩胃底环行肌标本，同浓度ATP诱发先舒张后收缩的双相反应，并呈浓度依赖性。ATP、UTP、ADP、2-MeSATP和α, β-MeATP浓度依赖性诱发大鼠胃体环行肌收缩反应，2-MeSATP的EC₅₀值为 (7.2 ± 5.2) nmol·L⁻¹比Ach [(3.47 ± 1.20) μmol·L⁻¹] 低500倍；各药物产生收缩反应的效价序列为：2-MeSATP>>ADP>ATP=UTP>α, β-MeATP>>腺苷。酚妥拉明、普萘洛尔、阿托品及河豚毒素不影响ATP和UTP诱发的胃体环行肌收缩反应。研究结果表明, 大鼠胃体环行肌的药理学特征明显不同于胃底环行肌; 核苷酸类物质通过某种特殊的P2Y受体介导胃体环行肌收缩反应，是调节胃体环行肌收缩功能的重要介质。
     

蜂斗菜不同提取物对豚鼠离体回肠收缩的影响

目的 比较蜂斗菜不同提取物对豚鼠离体回肠收缩的影响。方法 采用豚鼠离体回肠实验,以组胺、乙酰胆碱不同激动剂为研究模型,比较蜂斗菜CO₂超临界提取物、80%乙醇提取物、80%乙醇提取物的不同极性提取物及乙酸乙酯提取物经硅胶柱层析分离获得的不同馏分对豚鼠离体回肠的收缩作用,计算各个提取物对豚鼠回肠收缩幅度的抑制率。结果 蜂斗菜不同提取物对组胺、乙酰胆碱所致的豚鼠离体回肠收缩有显著拮抗作用,其中乙酸乙酯提取物硅胶柱色谱的馏分抑制作用最强。结论 蜂斗菜不同提取物均能抑制组胺、乙酰胆碱引起的豚鼠离体回肠收缩作用,脂溶性成分抑制作用最强,其机制可能与M、H₁受体的抑制有关。     

盐酸小檗碱对毒蕈碱性受体的激动作用

用大鼠离体胸主动脉环和豚鼠离体气管条功能实验及放射配基受体结合实验研究盐酸小檗碱（Ber）对组织毒蕈碱受体（Ｍ受体）的作用. 结果表明较高浓度的Ber（≥20 μmol·L^-1）对有内皮的动脉环具直接舒张作用;在去内皮或阻断Ｍ受体后,其舒张作用被完全抑制. Ber使豚鼠离体气管条产生浓度依赖性收缩,此作用可被阿托品拮抗,其pA2值为9.8. 放射配基受体结合实验显示,Ber对大鼠的唾液腺, 大脑皮质和心脏Ｍ受体均有中度亲和力,且使［³H］-二苯羟乙酸奎宁酯（QNB）与3种组织Ｍ受体结合的Ｋ_d值增大,最大结合量不变. 其Ｋ_i值分别为(7.6±1.3), (1.6±0.9)和(0.58±0.07) μmol·L^-1. 结果提示,Ber有Ｍ受体激动作用.     

美西律衍生物对α1肾上腺素受体的作用

为寻找新的α₁受体阻断剂,用[³H]-WB4101配体测定法测定了18种美西律衍生物。结果表明,其中6种化合物对大鼠脑皮层α₁受体有不同程度的亲和力。凡具有手性碳结构的化合物亲和力都较高。化合物M-85001的亲和力较妥拉唑林(tolazoline)高一个数量级并能抑制苯肾上腺素引起的大鼠肛尾肌收缩,其pA₂(6.86)与pK_i(6.51)相近。结果提示,美西律衍生物对α₁-受体的亲和力可能与手性碳结构有关,从美西律衍生物中研制新的α₁-受体阻断剂是有前途的。     

蚯蚓中平喘蛋白组分的提取分离及其作用机制的初探

目的初步研究蚯蚓平喘蛋白组分(Eisenia foetidaprotein,EP₂)及其平喘活性。方法以新鲜赤子爱胜蚓为原料,通过蛋白质的硫酸铵沉淀和离子交换色谱法,分离得到一组平喘活性蛋白成分(EP₂)。采用经典的肺阻力(R_L)和动态肺顺应性(C_dyn)指标评价EP₂对致敏豚鼠抗原攻击前后的肺功能变化;采用体外试验观察EP₂拮抗白三烯D₄引起豚鼠离体气管平滑肌的收缩反应;同时应用双向凝胶电泳-质谱技术对蛋白组分EP₂进行了初步分析鉴定。结果在整体试验中,EP₂16.8mg·kg^-1口含吞服预处理能明显抑制致敏豚鼠抗原攻击后引起的RL增加和C_dyn下降,作用稍弱于阳性对照药孟鲁司特;在离体试验中,EP₂能明显拮抗白三烯D₄收缩气管平滑肌的作用,作用强度与孟鲁司特接近;双向凝胶电泳-质谱技术分析证明蛋白组分EP₂中含有蚯蚓种属中的蛋白F-I-0、F-I-1以及纤溶酶组分A,均为丝氨酸蛋白酶类胰蛋白酶家族成员。结论EP₂具有平喘作用,作用靶点可能通过拮抗白三烯受体,提示EP₂的有效成分可能是白三烯拮抗剂。     

小儿健脾膏整方及其拆方对豚鼠离体胃肌、回肠平滑肌的作用比较

目的 观察比较小儿健脾膏整方及其拆方对豚鼠离体胃、回肠平滑肌的作用,进一步辨识方中促胃肠动力药效物质及其与整方的关系。方法 运用生物信号采集系统记录小儿健脾膏整方及其拆方对正常豚鼠离体胃、回肠平滑肌收缩张力的影响。结果 整方能显著促进胃、回肠平滑肌收缩。拆方中丁香、吴茱萸和山楂对胃肌收缩有促进作用,肉桂、白胡椒作用不显著。丁香和山楂对回肠平滑肌收缩有促进作用,且丁香 > 山楂 > 整方,吴茱萸则呈抑制作用,肉桂、白胡椒作用不显著。两两药物配伍对胃肌收缩有促进作用的组合为：丁香+肉桂、丁香+白胡椒、吴茱萸+肉桂、吴茱萸+白胡椒、山楂+肉桂。对回肠平滑肌收缩有促进作用的组合为：丁香+肉桂、丁香+白胡椒、山楂+肉桂;而吴茱萸+肉桂组、吴茱萸+白胡椒组则呈显著抑制作用。结论 小儿健脾膏整方能显著促进豚鼠胃、回肠平滑肌收缩,其中,丁香和山楂为主要促胃肠动力单味药。     

二苯氰胂对毒蕈碱性受体的抑制作用

目的研究了日本在我国遗弃的化学武器———二苯氰胂(DC)对离体豚鼠回肠纵肌和毒蕈碱性受体的作用机制,为评估DC的毒理学作用及其对环境的影响提供依据。方法取豚鼠8只,分离豚鼠回肠纵肌16条,分4组,用累积给药法,从低到高分别给予不同浓度的DC,记录离体豚鼠回肠纵肌收缩的幅度,求出DC作用于离体回肠纵肌的EC50值,并通过DC与乙酰胆碱(AChR)的相互作用,研究DC的作用机制。在放射性配体-受体结合实验中,从10只大鼠大脑中提取受体,通过DC与[3H]QNB的竞争结合作用,研究了DC对毒蕈碱性受体的作用。结果DC可抑制离体豚鼠回肠纵肌的收缩作用,其对离体豚鼠回肠纵肌的EC50值为(7.335±2.377)×10-7mol/L,AChR可收缩离体豚鼠回肠,并可反转DC引起离体豚鼠回肠纵肌的舒张作用。在放射性配体-受体结合实验中,DC与毒蕈碱性受体结合的Ki值为(9.19±1.52)nmol/L,IC50值为(16.00±2.65)nmol/L。结论DC可作用于mAChR受体上,初步断定为AChR的拮抗剂。     

蚯蚓总蛋白对豚鼠哮喘模型肺功能的影响

目的研究蚯蚓总蛋白(Eisenia foetida protein,EP)对豚鼠哮喘模型支气管平滑肌收缩功能的影响,初步分析其作用机制。方法以新鲜赤子爱胜蚓为原料,经硫酸铵沉淀得到EP。采用经典的肺阻力(R_L)和动态肺顺应性(C_dyn)指标评价EP对致敏豚鼠抗原攻击前后的肺功能变化;采用体外试验观察EP对豚鼠的离体气管平滑肌静息张力以及拮抗组胺、氨甲酰胆碱和白三烯引起的收缩反应。结果在整体试验中,EP10g·kg^-1(生药量,po)或EP5g·kg^-1(生药量,im)预处理能明显抑制致敏豚鼠抗原攻击后引起的R_L增加和C_dyn下降;在离体试验中,EP对离体豚鼠气管平滑肌静息张力无显著影响,对组胺或氨甲酰胆碱引起的气管平滑肌收缩反应无明显拮抗作用,但对白三烯D₄引起的收缩反应呈现非常显著的拮抗作用(P<0.01)。结论EP抑制豚鼠哮喘模型肺功能下降可能与抗白三烯作用有关。     

Muscarinic receptor heterogeneity in guinea pig intestinal smooth muscle: binding studies with AF-DX 116

Muscarinic receptor subtypes in longitudinal and circular smooth muscles of the guinea pig ileum were characterized with the use of the cardioselective antagonist AF-DX 116 in binding competition experiments against 0.3 nM [3H] N-methylscopolamine [( 3H]NMS). This compound recognized a heterogeneous receptor population in both smooth muscles, revealing the existence of different percentages of the cardiac (KD = 92-110 nM) and the glandular (KD = 1150-2541 nM) muscarinic receptor subtypes. These results, together with the low potency displayed by AF-DX 116 to inhibit the agonist-stimulated smooth muscle contraction and salivary secretion allow the suggestion that the glandular muscarinic receptor subtype, showing a low affinity for AF-DX 116, is involved in smooth muscle contraction.     

Calcium reversal, relaxation by calcium ion of various smooth muscles contracted by carbachol, norepinephrine or a phorbol ester in calcium-ion free medium

1. Ca reversal was reported on the longitudinal smooth muscle of rat uterus and the fundic part of the circular smooth muscle of guinea pig stomach; that is, addition of Ca ion to the Ca-free bathing solution in which the muscle is contracting to oxytocin or carbachol, respectively, relaxes the muscle. 2. We tested whether this inhibitory action of Ca ion is seen in other smooth muscles including vascular, air way, gastric and genital smooth muscles. 3. We found that Ca reversal was observed in the smooth muscle of rat thoracic aorta contracted by norepinephrine or by a phorbol ester, TPA, guinea pig tracheal smooth muscle contracted to carbachol, fundic smooth muscle of rat stomach to carbachol, corporal and antral smooth muscle of guinea pig stomach to carbachol and rat vas deferens to norepinephrine. 4. Ca reversal was observed not only when the muscle contracted by an agonist of the surface receptor such as oxytocin, norepinephrine or carbachol but also by a phorbol ester that is believed to cause contraction in the cell by activating phosphorylation. 5. Thus, we conclude that Ca reversal is a universal phenomenon in smooth muscles.     

Smooth muscle,neurons and interstitial cells of guinea pig ileum: are there tachykinin neurokinin 1 receptor subtypes?

Binding and autoradiographic studies were carried out in guinea pig ileum to examine neurokinin (NK) 1 receptor location and subtypes using the NK-1-selective radioligand [(125)I]Bolton-Hunter[Sar(9),Met(O(2))(11)]SP. Two membrane preparations were made: (1) longitudinal muscle containing the myenteric plexus and (2) circular muscle containing the interstitial cells of Cajal. In saturation binding studies, the K(D) was estimated as 1.3 and 1.0 nmol/l in each preparation, respectively. In competition binding, the rank order of potency was similar in both membrane preparations: SR140333 approximately CP99994 > or = [Sar(9),Met(O(2))(11)]SP approximately physalaemin approximately CP96345 (pIC(50) 9.5-8.7) > neuropeptide gamma > or = septide (pIC(50) 7.8-7.4). Similarly, scyliorhinin I displayed equal affinity in both preparations, although binding was at two sites, of high affinity (pIC(50) 9.1, 30%) and low affinity (pIC(50) 7.2-6.6, 70%). The only competitor to bind differently in the two muscle preparations was scyliorhinin II, which bound to one site with low potency in the circular muscle (pIC(50) 6.9) but to high-affinity (pIC(50) 9.0, 17%) and low-affinity (pIC(50) 6.7, 83%) sites in the longitudinal muscle. In autoradiographic studies, dense specific binding was associated with the myenteric plexus and the inner circular muscle containing the interstitial cells of Cajal, with minimal specific binding to longitudinal and circular smooth muscle. These results suggest that the NK-1 receptor on the interstitial cells and the myenteric plexus is similar. The apparently low numbers of binding sites on intestinal smooth muscle may be due to a low expression of the NK-1 receptor. Alternatively, the radioligand may not recognize the guinea pig ileum muscle NK-1 receptors due to possible minor differences in their sequence or glycosylation.     

Characterization of solubilized bradykinin B2 receptors from smooth muscle and mucosa of guinea pig ileum.

Bradykinin (BK) B2 receptors in guinea pig ileum were characterized in both membrane and soluble form. [3H]BK bound to a single class of sites with almost identical affinities in membranes prepared from the longitudinal muscle, circular muscle and mucosal layers of the ileum. The pharmacology of the binding in the distinct layers was indistinguishable. The detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS) maximally solubilized nearly 80% of membrane binding activity in a very stable conformation. In soluble preparations, [3H]BK labeled a single class of sites but with about 10-fold lower affinity. The affinities of BK analogs in competition studies were similarly reduced. There was no difference in the pharmacology of the binding in soluble receptors prepared from the different layers of the ileum. The results show that the ileum is a good source of solubilized B2 receptors and that the receptors in the smooth muscle and the mucosa are very similar.     

Receptor binding profiles of some selective muscarinic antagonists

The binding of hexahydrosiladifenidol, procyclidine, 4-DAMP (4-diphenylacetoxy-N-methylpiperidine) and AF-DX 116 to muscarinic receptors in the heart, ileum, urinary bladder, parotid gland and cerebral cortex from guinea pig was studied in competition experiments with (-)-[3H]QNB. The affinity of AF-DX 116 was higher in the heart than in the cortex and it was extremely low in the parotid gland. The affinities of hexahydrosiladefinidol, procyclidine and 4-DAMP were higher in the cortex and parotid gland than in the heart, bladder and ileum. Hexahydrosiladifenidol and 4-DAMP recognized two classes of muscarinic binding sites in the cortex. However, in contrast to functional data, binding results showed that 4-DAMP hexahydrosiladifenidol and procyclidine did not distinguish between the sites in the smooth muscles and those in the heart. Nevertheless, the present data support the view that the putative M2-receptors are heterogeneous, since the four drugs examined were found to distinguish between the muscarinic binding sites in the parotid gland and those in smooth muscles and heart.     

A difference in effects of physiological Ca2+ concentrations on activity of guanylate cyclase preparations obtained from the taenia caecum of guinea pig and from the longitudinal muscle of rat duodenum.

A cholinergic stimulant, butyltrimethylammonium bromide and serotonin increased the tissue levels of cyclic GMP in the taenia caecum of guinea pig but not those in the longitudinal muscle of rat duodenum. On the other hand, physiological Ca2+ concentrations enhanced the activity of a guanylate cyclase preparation obtained from the taenia caecum of guinea pig, while guanylate cyclase in the longitudinal muscle of rat duodenum was not influenced by Ca2+. The difference in the effects of the smooth muscle stimulants on the tissue levels of cyclic GMP in two different smooth muscles in attributed to differences in the properties of guanylate cyclase of smooth muscles.     

正丁基东莨菪碱与东莨菪碱药理作用的比较

本研究用放射配基结合法测定正丁基东莨菪碱和东莨菪碱对大鼠脑、豚鼠回肠纵长肌M胆碱受体以及大鼠脑GABA受体的亲和力。结果表明,正丁基东莨菪碱与中枢和外周M胆碱受体的亲和力均比东莨菪碱弱,而两药对GABA受体无特异性结合。正丁基东莨菪碱抗Ach收缩回肠效应也比东莨菪碱弱,表明生物效应的大小与其对受体亲和力的高低相关。用小鼠一次性训练的被动回避性反应法,观察两药对记忆获得的影响,所得资料表明,正丁基东莨菪碱阻抑记忆获得的用量至少比东莨菪碱大10～20倍。