Effect of Lead on Ion Transport in Fathead Gill

描述(申请人提供)：这项建议的目的是评估慢性铅暴露(水中二价铅)对Pimephales proelas(愚头鱼)鳃中钠钾ATP酶(Na K-ATPase，钠泵)活性的影响，以及这种酶活性如何受到铅诱导的鳃脂肪过氧化的影响。我们的工作假设是，铅通过自由基介导的机制促进类前列腺素脂肪酸衍生物异前列腺素(IPS)的形成，从而影响Na K-ATPase。我们的初步研究，使用分离的鳃制剂，表明铅浓度接近1ppm时，会明显刺激Na K-ATPase活性。将优先获得这种体外效应的剂量和时间依赖性的更完整的图景。随后，我们将评估刺激效应对蛋白质磷酸化的可能依赖性。体内铅对鳃Na-K-ATPase活性的影响将在水族馆研究中进行，在水族馆研究中，成年笨头鱼暴露在选定浓度的铅(作为硝酸铅)中长达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)以及在鳃中积累的铅与钠泵活动之间的时间关系。此外，当局亦会进行类似的实验，以追踪鱼缸水中铅暴露的鱼体释放鱼鳃iPS的时间进程。鳃类脂将被提取，并通过LC/MS-电喷雾进行分析，结构确认与真实的和氚的同源标准。在一些实验中，在对照鱼的体外制剂(分离的鳃和膜部分)中添加铅后，将测量IP的释放。在体内和体外研究中，我们将测定富含钠泵活性的鳃膜组分的铅浓度。将对数据进行分析，以确定膜铅水平是否与Na K-ATPase活性和/或IP生成相关。在稍后的项目中，我们将评估慢性铅暴露可能导致鳃中还原型谷胱甘肽(GSH)水平补偿性上升的假说。此外，在过去的一年里，将从饮用水中摄入铅的大鼠身上分离出肾近端小管。将孵育肾小管悬液以评估IP生物合成、钠泵活性和GSH状态。将对结果进行分析，以确定铅是否会导致近端肾单位和鳃上皮类似的IP依赖性变化。

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.