Ivanina Anna V, Sokolova Inna M
Biology Department, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC 28223, USA.
Aquat Toxicol. 2008 Jun 2;88(1):19-28. doi: 10.1016/j.aquatox.2008.02.014. Epub 2008 Feb 29.
Heavy metal pollution is a worldwide problem, and cadmium (Cd) is one of the most noxious pollutants in aquatic environments. We studied P-glycoprotein (P-gp) expression and function in control and Cd exposed (50microgL(-1) Cd, 30-40 days) oysters Crassostrea virginica as a possible mechanism of cell protection against Cd. Our data show that P-gp is expressed on cell membrane and in mitochondria of oyster gills and hepatopancreas. Inhibitor studies with verapamil, cyclosporine A and JS-2190 suggest that in the gills, mitochondrial P-gp pumps substrates from cytosol into the mitochondria, while cell membrane P-gp pumps substrates from cytosol out of the cell. Cd exposure resulted in a 2-2.5-fold increase in P-gp protein expression in cell membranes and a 3.5-7-fold increase in transport activity measured as the inhibitor-sensitive rhodamine B extrusion rate. In contrast, p-gp mRNA levels were similar in control and Cd-exposed oysters. No difference in P-gp protein expression was observed between mitochondria of control and Cd-exposed oysters but the apparent transport activity was higher in mitochondria from Cd-exposed oysters. Overall, a stronger increase in substrate transport activity in Cd-exposed oysters compared to a relatively weaker change in P-gp protein levels suggests that P-gp activity is post-translationally regulated. Our data show that direct determination of P-gp transport activity may be the best measure of the xenobiotic-resistant phenotype, whereas p-gp mRNA levels are not a good marker due to the likely involvement of multiple post-transcriptional regulatory steps. Cd exposure resulted in a significantly elevated rate of oxygen consumption of isolated oyster gills by 46%. Specific inhibitors of ATPase function of P-gp (cyclosporine A and JS-2190) had no significant effect on tissue oxygen consumption indicating that P-gp contribution to energy budget is negligible and supporting indirect estimates based on the ATP stoichiometry of substrate transport that also suggest low energy demand for P-gp function.
重金属污染是一个全球性问题，镉（Cd）是水生环境中最有害的污染物之一。我们研究了对照和镉暴露（50μg/L镉，30 - 40天）的弗吉尼亚牡蛎中P-糖蛋白（P-gp）的表达和功能，以此作为细胞抵御镉的一种可能机制。我们的数据表明，P-gp在牡蛎鳃和肝胰腺的细胞膜及线粒体中表达。用维拉帕米、环孢素A和JS - 2190进行的抑制剂研究表明，在鳃中，线粒体P-gp将底物从细胞质泵入线粒体，而细胞膜P-gp将底物从细胞质泵出细胞。镉暴露导致细胞膜中P-gp蛋白表达增加2 - 2.5倍，以抑制剂敏感的罗丹明B外排率衡量的转运活性增加3.5 - 7倍。相比之下，对照和镉暴露的牡蛎中p-gp mRNA水平相似。对照和镉暴露的牡蛎线粒体之间未观察到P-gp蛋白表达差异，但镉暴露牡蛎的线粒体中表观转运活性较高。总体而言，与P-gp蛋白水平相对较弱的变化相比，镉暴露牡蛎中底物转运活性有更强的增加，这表明P-gp活性受到翻译后调控。我们的数据表明，直接测定P-gp转运活性可能是抗外源性物质表型的最佳衡量指标，而p-gp mRNA水平不是一个好的标志物，因为可能涉及多个转录后调控步骤。镉暴露导致分离的牡蛎鳃的耗氧率显著升高46%。P-gp的ATP酶功能的特异性抑制剂（环孢素A和JS - 2190）对组织耗氧没有显著影响，这表明P-gp对能量预算的贡献可忽略不计，并支持基于底物转运的ATP化学计量学的间接估计，该估计也表明P-gp功能的能量需求较低。
