title: Selective antagonism activity of alkaloids from bulbs Fritillariae at muscarinic receptors: functional studies.
abstract: 15 alkaloids were isolated from five Fritillariae species and 6 derivatives were synthesized. Alkaloids having anticholinergic effect on guinea-pig tracheal smooth muscle were screened out and their mechanism was further studied on the cAMP formation in Chinese hamster ovary cells stably expressing human muscarinic M2 receptor (CHO-hM2 cells) and intracellular calcium ([Ca(2+)](i)) transient in Chinese hamster ovary cells stably expressing human muscarinic M3 receptor (CHO-hM3 cells). In normal Krebs-Henseleit (KH) solution, imperialine (15), 3beta-acetylimperialine (16) and sinpeinine A (17) concentration-dependently relaxed 1 microM carbachol-induced contraction of guinea-pig tracheal rings with EC(50) of 4.19, 1.71 and 4.67 microM, respectively. In Ca(2+)-free KH solution, 10 microM 3beta-acetylimperialine (16), imperialine (15) and sinpeinnine A (17) caused 97.42%, 5.45% and 6.55% inhibition, respectively, which indicated that the three components might inhibit muscarinic receptor in different mechanism. Results of muscarinic M2 receptor-inhibited cAMP formation in CHO-hM2 cells showed that imperialine (15) and sinpeinine A (17) could potently elevate the cAMP formation whereas 3beta-acetylimperialine (16) only had weak effect on antagonism of cAMP inhibition. Furthermore, the investigations of muscarinic M3 receptor-stimulated [Ca(2+)](i) transient in CHO-hM3 cells revealed that imperialine (15) and sinpeinine A (17) could not antagonize [Ca(2+)](i) transient, but 3beta-acetylimperialine (16) significantly inhibited [Ca(2+)](i) peak elevation with an IC(50) of 5.26 microM. The functional studies suggest that the mechanism of relaxant action of imperialine (15) and sinpeinine A (17) is due to their selective inhibitory effects on muscarinic M2 receptors and the mechanism of 3beta-acetylimperialine (16) originates from its selective muscarinic M3 receptors antagonism.
authors.affiliation: Key Laboratory of Modern Chinese Medicines, Ministry of Education of PRC and Department of Pharmacognosy, China Pharmaceutical University, 24 Tongjia Xiang, Nanjing 210009, PR China.
authors.forename: Bao-Qin
authors.initials: BQ
authors.lastname: Lin
authors.forename: Hui
authors.initials: H
authors.lastname: Ji
authors.forename: Ping
authors.initials: P
authors.lastname: Li
authors.forename: Yan
authors.initials: Y
authors.lastname: Jiang
authors.forename: Wei
authors.initials: W
authors.lastname: Fang
mesh_terms: D000470:Alkaloids / Q000138:chemical synthesis / Q000302:isolation & purification / Q000494:pharmacology*; D000818:Animals; D018927:Anti-Asthmatic Agents / Q000494:pharmacology; D001285:Atropine / Q000494:pharmacology; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002595:Cevanes / Q000494:pharmacology; D018679:Cholinergic Agonists / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D031410:Fritillaria / Q000737:chemistry*; D006168:Guinea Pigs; D006801:Humans; D018727:Muscarinic Antagonists / Q000138:chemical synthesis / Q000302:isolation & purification / Q000494:pharmacology*; D009126:Muscle Relaxation / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects; D018517:Plant Roots; D043585:Receptor, Muscarinic M2 / Q000187:drug effects; D043587:Receptor, Muscarinic M3 / Q000187:drug effects; D011976:Receptors, Muscarinic / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014132:Trachea / Q000187:drug effects* / Q000378:metabolism; D014162:Transfection
chemical_list: D000470:Alkaloids; D018927:Anti-Asthmatic Agents; D002595:Cevanes; D018679:Cholinergic Agonists; D018727:Muscarinic Antagonists; D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; D001285:Atropine; D002217:Carbachol; D000242:Cyclic AMP; C047331:peiminine; D002118:Calcium