title: Characterisation of the membrane transport of pilocarpine in cell suspension cultures of Pilocarpus microphyllus.
abstract: Pilocarpine is an alkaloid obtained from the leaves of Pilocarpus genus, with important pharmaceutical applications. Previous reports have investigated the production of pilocarpine by Pilocarpus microphyllus cell cultures and tried to establish the alkaloid biosynthetic route. However, the site of pilocarpine accumulation inside of the cell and its exchange to the medium culture is still unknown. Therefore, the aim of this study was to determine the intracellular accumulation of pilocarpine and characterise its transport across membranes in cell suspension cultures of P. microphyllus. Histochemical analysis and toxicity assays indicated that pilocarpine is most likely stored in the vacuoles probably to avoid cell toxicity. Assays with exogenous pilocarpine supplementation to the culture medium showed that the alkaloid is promptly uptaken but it is rapidly metabolised. Treatment with specific ABC protein transporter inhibitors and substances that disturb the activity of secondary active transporters suppressed pilocarpine uptake and release suggesting that both proteins may participate in the traffic of pilocarpine to inside and outside of the cells. As bafilomicin A1, a specific V-type ATPase inhibitor, had little effect and NH4Cl (induces membrane proton gradient dissipation) had moderate effect, while cyclosporin A and nifedipine (ABC proteins inhibitors) strongly inhibited the transport of pilocarpine, it is believed that ABC proteins play a major role in the alkaloid transport across membranes but it is not the exclusive one. Kinetic studies supported these results.
authors.affiliation: Departamento de Biologia Vegetal, Instituto de Biologia, CP 6109, Universidade Estadual de Campinas, 13083-970 Campinas, SP, Brazil.
authors.forename: Nathalia Luiza
authors.initials: NL
authors.lastname: Andreazza
authors.affiliation: Departamento de Biologia Vegetal, Instituto de Biologia, CP 6109, Universidade Estadual de Campinas, 13083-970 Campinas, SP, Brazil.
authors.forename: Ilka Nacif
authors.initials: IN
authors.lastname: Abreu
authors.affiliation: Departamento de Biologia Vegetal, Instituto de Biologia, CP 6109, Universidade Estadual de Campinas, 13083-970 Campinas, SP, Brazil.
authors.forename: Alexandra Christine Helena Frankland
authors.initials: AC
authors.lastname: Sawaya
authors.affiliation: Departamento de Biologia Vegetal, Instituto de Biologia, CP 6109, Universidade Estadual de Campinas, 13083-970 Campinas, SP, Brazil. Electronic address: pmazza@unicamp.br.
authors.forename: Paulo
authors.initials: P
authors.lastname: Mazzafera
mesh_terms: D001692:Biological Transport; D018929:Cell Culture Techniques; D040503:Coffea / Q000187:drug effects; D003470:Culture Media; D016572:Cyclosporine / Q000494:pharmacology; D007700:Kinetics; D009543:Nifedipine / Q000494:pharmacology; D010862:Pilocarpine / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity; D032103:Pilocarpus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D031701:Piper / Q000187:drug effects; D018515:Plant Leaves / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D003470:Culture Media; D010940:Plant Proteins; D010862:Pilocarpine; D016572:Cyclosporine; D025262:Vacuolar Proton-Translocating ATPases; D009543:Nifedipine
keywords: ABC proteins; Pilocarpine; Subcellular localisation; Transport