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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Characterisation of the membrane transport of pilocarpine in cell suspension cultures of Pilocarpus microphyllus

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Andreazza, Nathalia Luiza [1] ; Abreu, Ilka Nacif [1] ; Helena Frankland Sawaya, Alexandra Christine [1] ; Mazzafera, Paulo [1]
Total Authors: 4
[1] Univ Estadual Campinas, Inst Biol, Dept Biol Vegetal, BR-13083970 Campinas, SP - Brazil
Total Affiliations: 1
Document type: Journal article
Source: Journal of Plant Physiology; v. 175, p. 37-47, MAR 1 2015.
Web of Science Citations: 2

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. (C) 2014 Elsevier GmbH. All rights reserved. (AU)