Effect of Lead on Ion Transport in Fathead Gill

描述（由申请人提供）：本提案的目的是评估慢性铅暴露（水生二价铅）对pmephales promelas（鱼头鲦鱼）鳃中钠、钾atp酶（Na+K+- atp酶，钠泵）活性的影响，以及这种酶活性如何受到铅诱导的鳃脂过氧化的影响。我们的工作假设是，铅通过自由基介导的机制促进鳃膜内异前列腺素（iPs）（前列腺素样脂肪酸衍生物）的形成，从而影响Na+K+- atp酶。我们最初的研究，使用分离的鳃制剂，表明接近1ppm的铅浓度会引起Na+K+- atp酶活性的明显刺激。将优先考虑更全面地了解这种体外效应的剂量和时间依赖性。随后，我们将评估刺激作用对蛋白质磷酸化的可能依赖性。铅对鳃Na+K+- atp酶活性的体内影响将在水族馆研究中进行检验，其中成年黑头鲦鱼暴露于选定浓度的铅（如硝酸铅）长达3个月。作为这项工作的一部分，将确定铅的组织分布（例如，鳃，皮肤和肌肉，内脏，骨骼）以及鳃中铅积累与钠泵活性之间的时间关系。将进行类似的实验，以跟踪接触水族馆水中铅的鱼鳃释放iPs的时间过程。鳃脂将被提取，并通过LC/MS-Electrospray进行分析，用正品和氘化的同源标准进行结构确认。在一些实验中，将铅添加到对照鱼的体外制剂（分离的鳃和膜部分）后，将测量iP释放。在体内和体外研究中，我们将确定富含钠泵活性的鳃膜组分的铅浓度。将分析数据以确定膜铅水平是否与Na+K+- atp酶活性和/或iP生成相关。在项目的后期，我们将评估慢性铅暴露可能导致鳃内还原型谷胱甘肽（GSH）水平代偿性上升的假设。此外，在去年，肾近端小管将从饮用铅水的老鼠身上分离出来。微管悬液将被孵育以评估iP生物合成、钠泵活性和谷胱甘肽状态。结果将分析，以确定是否铅引起类似的ip依赖改变近端肾细胞和鳃上皮。

DESCRIPTION (provided by applicant): The objective of this proposal is to evaluate the effect of chronic lead exposure (water-borne divalent lead) on sodium, potassium ATP-ase (Na+K+-ATPase, sodium pump) activity in the gill of Pimephales promelas (fathead minnow) and how this enzymatic activity is affected by lead-induced peroxidation of gill lipids. Our working hypothesis is that lead affects Na+K+-ATPase by promoting formation of isoprostanes (iPs), prostaglandin-like fatty acid derivatives, within gill membranes by a free radical-mediated mechanism. Our initial studies, using isolated gill preparations, indicate that concentrations of lead near 1 ppm cause pronounced stimulation of Na+K+-ATPase activity. Priority will be given to gaining a more complete picture of the dose and time-dependence of this in vitro effect. Subsequently, we will evaluate the possible dependence of the stimulatory effect on protein phosphorylation. The in vivo influence of lead on gill Na+K+-ATPase activity will be examined in aquarium studies wherein adult fathead minnows are exposed to selected concentrations of lead (as lead nitrate) for up to 3 months. As part of this work, the tissue distribution of lead will be determined (e.g., gills, skin and muscle, viscera, skeleton) as well as the temporal relationships between accumulation of lead in the gill and sodium pump activity. Similar experiments will be carried out to follow the time course for the release of gill iPs in fish exposed to lead in the aquarium water. Gill lipids will be extracted, and analyzed by LC/MS-Electrospray, with structural confirmation with authentic and deuterated homologous standards. In some experiments iP release will be measured after adding lead to in vitro preparations (isolated gills and membrane fractions) from control fish. In both the in vivo and in vitro studies, we will determine the lead concentration of gill membrane fractions enriched in sodium pump activity. Data will be analyzed to determine if membrane lead levels correlate with Na+K+-ATPase activity and/or iP generation. Later in the project, we will evaluate the hypothesis that chronic lead exposure may result in a compensatory rise in levels of reduced glutathione (GSH) within the gill. Also, in the last year, renal proximal tubules will be isolated from rats administered lead in the drinking water. Suspensions of tubules will be incubated to assess iP biosynthesis, sodium pump activity, and GSH status. Results will be analyzed to determine if lead causes analogous iP-dependent changes in proximal nephron and gill epithelium.