MRP (ABCC) transporters-mediated efflux of anti-HIV drugs, saquinavir and zidovudine, from human endothelial cells
MRP (ABCC) transporters-mediated efflux of anti-HIV drugs, saquinavir and zidovudine, from human endothelial cells
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DOI：
10.3181/0802-rm-59
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发表时间：
2008-09-01
影响因子：
3.2
通讯作者：
Mondal, Debasis
中科院分区：
文献类型：
--
作者：
Eilers, Mark;Roy, Upal;Mondal, Debasis
高效抗逆转录病毒疗法（HAART）的成分包括HIV-1蛋白酶抑制剂（HPI）和核苷逆转录酶抑制剂（NRTI）。内皮细胞（EC）屏障，尤其是血脑屏障（BBB）抑制HAART药物进入内皮下HIV-1储存库。 ATP 结合盒 (ABC) 转运蛋白家族成员、多药耐药 1 (MDR-1) 和多药耐药相关蛋白 (MRP) 可以从细胞内区室中流出 HPIs 和 NRTI。使用非人类来源的脑源性 EC，之前的研究表明 MDR-1 在 HAART 从 BBB 流出中起主导作用。然而，由于 ABC 转运蛋白表达的物种差异，需要研究使用人脑 EC 的药物流出功能。此外，需要研究 ABC 转运蛋白在药物从全身 EC 屏障流出中的作用。我们监测了从大脑（HBMVEC）、主动脉（HAEC）、肺动脉（HPAEC）、真皮微血管（HDMVEC）和脐静脉（HUVEC）获得的原代人EC中ABC转运蛋白的表达。通过逆转录聚合酶链式反应 (RT-PCR) 分析 MDR-1 和 MRP（MRP-1 至 MRP-5）的基因表达。通过钙黄绿素保留测定来确定药物流出功能。还监测了 HAEC 和 HBMVEC 中 H-3-沙奎那韦（HPI）和 H-3-齐多夫定（NRTI）的细胞内积累。两种测定均在MDR-1和MRP的维拉帕米(20-60μM)或MK-571(12.5-50μM)抑制剂存在下进行，分别在维拉帕米或MK-571存在下进行。 HBMVEC 表达的 MRP 水平高于 MDR-1，并且只有 MK-571 显着 (P < 0.01) 抑制这些细胞的钙黄绿素外流。然而，HAEC 和 HPAEC 均显示 MDR-1 和 MRP 表达，并且维拉帕米和 MK-571 均抑制钙黄绿素外流。两种抑制剂均抑制 H-3-沙奎那韦从 HAEC 流出，但只有 MK-571 抑制沙奎那韦从 HBMVEC 流出。在两个 EC 中，H-3-齐多夫定外流仅被 MK-571 抑制。因此，原代人EC，尤其是脑源性EC，主要表达MRP，其特异性抑制可能增强HAART在内皮下HIV-1储存库中的功效。
The constituents of highly active anti-retroviral therapy (HAART) include HIV-1 protease inhibitors (HPIs) and nucleoside reverse transcriptase inhibitors (NRTIs). Endothelial cell (EC) barriers, especially the blood-brain-barrier (BBB) suppresses the entry of HAART drugs to subendothelial HIV-1 reservoirs. The ATP binding cassette (ABC) transporter family members, multidrug resistant-1 (MDR-1) and multidrug resistance-associated proteins (MRPs) can efflux both HPIs and NRTIs from intracellular compartments. Using brain derived ECs from non-human sources, previous studies suggested a dominant role for MDR-1 in HAART efflux from the BBB. However, due to species variations in ABC-transporter expression, drug-efflux functions using human brain ECs need to be investigated. Furthermore, roles of ABC-transporters in drug-efflux from systemic EC barriers need to be studied. We monitored the expression of ABC-transporters in primary human ECs obtained from brain (HBMVECs), aorta (HAECs), pulmonary-artery (HPAECs), dermal-microvessel (HDMVECs) and umbilical vein (HUVECs). Gene expression for MDR-1 and MRPs (MRP-1 to MRP-5) were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). Drug efflux functions were determined by calcein retention assays. Intracellular accumulation of both H-3-saquinavir (an HPI) and H-3-zidovudine (an NRTI) were also monitored in HAECs and HBMVECs. Both assays were carried out in presence of verapamil (20-60 mu M) or MK-571 (12.5-50 mu M) inhibitors of MDR-1 and MRPs, respectively in presence of verapamil or MK-571. The HBMVECs expressed higher levels of MRPs than MDR-1 and only MK-571 significantly (P < 0.01) suppressed calcein efflux from these cells. However, both HAECs and HPAECs showed MDR-1 and MRP expression and calcein efflux was inhibited by both verapamil and MK-571. Both inhibitors suppressed H-3-saquinavir efflux from HAECs, but only MK-571 suppressed saquinavir efflux from HBMVECs. In both ECs H-3-zidovudine efflux was only suppressed by MK-571. Thus, primary human ECs, especially brain derived ECs, predominantly express MRPs and their specific inhibition may enhance HAART efficacy in subendothelial HIV-1 reservoirs.