长效支气管扩张剂作用机制探讨

Inquiry into the Mechanism of Long-acting Bronchodilators

目的旨在比较长效支气管扩张剂福莫特罗(FOR)、沙美特罗(SLM)与短效支气管扩张剂芬诺特罗(FEN)、沙丁胺醇(SAL)及异丙肾上腺素(ISO)的内在活性(intrinsic activity,IA),受体亲和力及其在细胞膜的信号放大作用。方法采用酶解法制备牛支气管平滑肌细胞膜,以^(32)P-ATP为底物,测定各激动剂作用于β_2受体,激活腺苷酸环化酶生成cAMP的IA及其生成半数最大量cAMP所需浓度(EC_(50)),并计算出各激动剂的相对效应pEC_(50);以放射性标记配体法测定各激动剂与β_2受体亲和力。结果 ISO、FOR、SLM、FEN和SAL的IA分别为1.00、1.00、0.76、0.81和0.83,上述各激动剂的亲和力分别为6.69、8.02、8.43、5.84和5.84 mol/L,pEC_(50)分别为7.68、9.10、8.70、6.79和6.78 mol/L。结论 FOR与ISO一样,为高效激动剂,其亲和力比ISO高约20倍,其他激动剂为部分激动剂。各激动剂作用于细胞膜均可产生信号放大作用,以FOR为著。FOR的高IA和高亲和力及其在细胞膜的信号放大作用可能是其长效作用的机制之一。

Objective To compare the intrinsic activity and the affinity of long-(formoterol and salmeterol) and short-acting (fenoterol and salbutamol) bronchodilators with isoproterenol in membranes of calf tracheal smooth muscle. Methods Membranes were prepared after isolation of intact cells using a mixture of collagenase and pronase. Affinities were measured with radioligand binding. Intrinsic activities (IA) of agonists and effective concentrations Iof agonists used for half maximum stimulation of adenylate cyclase (AC) (EC_(50) were determined as stimulants of β_2-adrenoceptor coupled AC activity using α-^(32)P-ATP as substrate. Results The results showed that IA estimated as fraction of the maximum effect by isoproterenol (1.00) were 1.00, 0.76, 0. 81 and 0.83 for formoterol, salmeterol, fenoterol and salbutamol respectively. Affinities measured as dissociation constants (pK_D) exhibited higher affinities for long-acting (formoterol. 8.02, salmeterol: 8.43) than for short-acting agonists (fenoterol: 5.84, salbutamol. 5.84). Relative effects of agonists (pEC_(50) were much higher for formoterol (9.10) and salmeterol (8.70) than for fenoterol (6.69) and salbutamol (6.78). Conclusions The results indicated that formoterol was characterized as a full ago- nist with a 20-fold higher affinity than isoproterenol whereas the other agonists acted as partial agonists. Significant amplifications of receptor signalling quantified as difference of values between pEC_(50) and pKD was found for the agonists in the tested receptor membranes. It is suggested that high IA, affinity and the signal amplification of formoterol might play a role in its long-acting effect.