Mandal Goutam, Sarkar Avijit, Saha Piu, Singh Neeloo, Sundar Shyam, Chatterjee Mitali
Department of Pharmacology, Institute of Post Graduate Medical Education and Research, Kolkata, 244B Acharya JC Bose Road, Kolkata 700 020 India.
Indian J Biochem Biophys. 2009 Feb;46(1):86-92.
The recent upsurge of antimony (Sb) resistance is a major impediment to successful chemotherapy of visceral leishmaniasis (VL). Mechanisms involved in antimony resistance have demonstrated an upregulation of drug efflux pumps; however, the biological role drug efflux pumps in clinical isolates remains to be substantiated. Thus, in this study, the functionality of drug efflux pumps was measured in promastigotes and axenic amastigotes isolated from VL patients, who were either Sb-sensitive (AG83, 2001 and MC9) or resistant (NS2, 41 and GE1) using rhodamine123 as a substrate for multidrug resistant (MDR) pumps and calcein as a substrate for multidrug resistance-associated proteins (MRP) respectively; their specificity was confirmed using established blockers. Sb-resistant (Sb-R) isolates accumulated higher amounts of R123, as compared to Sb-sensitive (Sb-S) isolates. Verapamil, a MDR inhibitor failed to alter R123 accumulation, suggesting absence of classical MDR activity. In Sb-R isolates, both promastigotes and axenic amastigotes accumulated significantly lower amounts of calcein than Sb-S isolates and probenecid, an established pan MRP blocker, marginally increased calcein accumulation. Depletion of ATP dramatically increased calcein accumulation primarily in Sb-R isolates, indicating existence of a MRP-like pump, which was more active in Sb-R isolates. In conclusion, our data suggested that overfunctioning of a MRP-like pump contributed towards generation of Sb-R phenotype in L. donovani field isolates.
近期锑（Sb）耐药性的激增是内脏利什曼病（VL）化疗成功的主要障碍。与锑耐药相关的机制已表明药物外排泵上调；然而，药物外排泵在临床分离株中的生物学作用仍有待证实。因此，在本研究中，使用罗丹明123作为多药耐药（MDR）泵的底物，使用钙黄绿素分别作为多药耐药相关蛋白（MRP）的底物，测量了从VL患者分离的前鞭毛体和无菌无鞭毛体中药物外排泵的功能，这些患者要么对锑敏感（AG83、2001和MC9），要么耐药（NS2、41和GE1）；使用既定的阻滞剂证实了它们的特异性。与锑敏感（Sb-S）分离株相比，锑耐药（Sb-R）分离株积累了更高量的R123。MDR抑制剂维拉帕米未能改变R123的积累，表明不存在经典的MDR活性。在Sb-R分离株中，前鞭毛体和无菌无鞭毛体积累的钙黄绿素量均明显低于Sb-S分离株，而既定的泛MRP阻滞剂丙磺舒略微增加了钙黄绿素的积累。ATP的消耗主要在Sb-R分离株中显著增加了钙黄绿素的积累，表明存在一种类似MRP的泵，其在Sb-R分离株中更活跃。总之，我们的数据表明，类似MRP的泵功能亢进有助于杜氏利什曼原虫野外分离株中Sb-R表型的产生。
