Andreazza Nathalia Luiza, Abreu Ilka Nacif, Sawaya Alexandra Christine Helena Frankland, Mazzafera Paulo
Departamento de Biologia Vegetal, Instituto de Biologia, CP 6109, Universidade Estadual de Campinas, 13083-970 Campinas, SP, Brazil.
Departamento de Biologia Vegetal, Instituto de Biologia, CP 6109, Universidade Estadual de Campinas, 13083-970 Campinas, SP, Brazil.
J Plant Physiol. 2015 Mar 1;175:37-47. doi: 10.1016/j.jplph.2014.11.002. Epub 2014 Nov 20.
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.
毛果芸香碱是一种从毛果芸香属植物叶子中提取的生物碱，具有重要的药用价值。先前的报道研究了小叶毛果芸香细胞培养物中毛果芸香碱的产生，并试图建立生物碱的生物合成途径。然而，细胞内毛果芸香碱的积累部位及其向培养基中的交换情况仍然未知。因此，本研究的目的是确定毛果芸香碱在小叶毛果芸香细胞悬浮培养物中的细胞内积累情况，并表征其跨膜转运特性。组织化学分析和毒性试验表明，毛果芸香碱很可能储存在液泡中，可能是为了避免细胞毒性。向培养基中添加外源性毛果芸香碱的试验表明，该生物碱能迅速被吸收，但会迅速代谢。用特异性ABC蛋白转运体抑制剂和干扰次级主动转运体活性的物质处理后，毛果芸香碱的吸收和释放受到抑制，这表明这两种蛋白可能都参与了毛果芸香碱进出细胞的运输过程。由于特异性V型ATP酶抑制剂巴弗洛霉素A1作用较小，氯化铵（诱导膜质子梯度消散）作用中等，而环孢素A和硝苯地平（ABC蛋白抑制剂）强烈抑制毛果芸香碱的转运，因此认为ABC蛋白在生物碱跨膜转运中起主要作用，但不是唯一作用。动力学研究支持了这些结果。
