红罗非鱼Na~+-K~+-ATPase α基因的克隆及其在不同盐度条件下mRNA表达差异

为获知红罗非鱼(Oreochromis sp.)Na+-K+-ATPaseα基因的全长分子结构及其在不同盐度条件下的表达情况,采用同源克隆及cDNA末端快速扩增(RACE-PCR)方法,首次在红罗非鱼鳃组织中克隆到了全长为3 379 bp的Na+-K+-ATPaseα基因全长cDNA序列,该序列包含3 072 bp的开放阅读框(ORF),143 bp的5'末端非编码区(UTR)和164 bp的3'末端非编码区(UTR),编码1023个氨基酸,预测分子量为112.5 kD,理论等电点为5.26。BLAST分析显示红罗非鱼Na+-K+-ATPaseα基因编码的氨基酸序列与其它已知物种相应基因编码的氨基酸序列的同源性达到97%-99%;系统进化分析显示,红罗非鱼Na+-K+-ATPaseα亚基与萨罗罗非鱼(Sarotherodon melanotheron)和莫桑比克罗非鱼(Oreochromis mossambicus)亲缘关系较近。应用Real-time PCR技术,以β-actin基因为内参,对不同盐度(0、15、25、32 g/L)条件下鳃组织中Na+-K+-ATPaseα基因的表达情况进行了比较分析,结果表明,当盐度为25 g/L时,Na+-K+-ATPaseα基因的表达量达到峰值,而盐度升至32 g/L时,其表达量呈下降趋势;各盐度处理组中,养殖12 h后Na+-K+-ATPaseα基因的mRNA表达量显著上升(P0.05)并达到最高;随着养殖时间的延长,24 h后该基因mRNA表达量开始降低,但仍然显著高于对照组的表达量值(P0.05)。     

盐度对大菱鲆幼鱼鳃Na -K -ATPase活力、血清离子浓度 和激素水平的影响

采用饲养试验方法,研究了平均体质量为(7.16±0.07)g的大菱鲆(Scophthalmus maximus)幼鱼分别在盐度12、18、24、30和36下饲养60 d后,其鳃Na+-K+-ATPase活力、血清离子浓度、生长激素(GH)、皮质醇激素(COR)、特定生长率(SGR)和饲料效率(FCE)的变化。结果表明:幼鱼鳃Na+-K+-ATPase活力、血清Na+浓度均随盐度的升高而上升,分别在3.48~8.30 U/mg和169.99~180.00 mmol/L之间,其中12盐度组最低,36盐度组最高。幼鱼血清中K+和Cl-浓度分别在2.20~3.47 mmol/L和136.67~142.00 mmol/L之间,各盐度组之间差异不显著。幼鱼血清中GH和COR浓度分别在0.41~1.66 ng/ml和35.33~76.41 ng/ml之间;其中GH在36盐度组最高,12盐度组最低,而COR在12盐度组最高,36盐度组最低。幼鱼SGR和FCE分别在(1.45~2.00)%/d和1.12%~1.38%之间,与盐度的相关性不显著,两者均为12盐度组最低。由此可见,盐度变化显著影响大菱鲆幼鱼鳃Na+-K+-ATPase活力、血清Na+浓度和激素含量。本研究对大菱鲆养殖生理生态条件分析具有重要参考意义,研究结果可为大菱鲆养殖的盐度选择提供理论依据。     

盐度对日本囊对虾仔虾生长发育和Na+-K+-ATPase活力的影响

研究了盐度对日本囊对虾仔虾(Marsupenaeus Japonicus)的生长发育和Na -K -ATPase活力的影响。结果表明,盐度对日本囊对虾仔虾存活率、增重率和Na -K -ATPase活力的影响显著(P<0.05)。随着向低盐度变化的增加,仔虾的存活率和增重率明显下降,而向高盐度变化无显著差异;在盐度变化48h内,随着盐度变化的增加仔虾Na -K -ATPase活力变化增大,各处理组仔虾Na -K -ATPase活力随着取样时间的增加呈峰值变化,至24h时低盐度处理组酶活力达到最大值,而高盐度处理组达到最小值:至48h~72h时,不同盐度下仔虾Na -K -ATPase活力趋于稳定,而且盐度越低酶活力越大。由此表明日本囊对虾仔虾在低盐度地下卤水中的适宜盐度为20‰—24‰。     

温度与盐度对吉富品系尼罗罗非鱼幼鱼生长和肝脏抗氧化酶活力的协同影响

采用中心复合试验设计(CCD)和响应曲面方法(response surface methodology, RSM),探讨了温度(16～37 ℃)和盐度(0～18)对吉富罗非鱼幼鱼生长和肝脏抗氧化酶活力的协同影响.结果表明： 温度和盐度的一次与二次效应对吉富罗非鱼幼鱼特定生长率（SGR）有显著影响(P<0.05),随着温度或盐度的上升,其特定生长率呈先升后降的变化.温度与盐度间存在互作效应(P<0.05),温度为16～20 ℃时,幼鱼的特定生长率在盐度为9～10时较高;在温度27～32 ℃、盐度3～5时较高;高温环境下（35～37 ℃）,淡水环境中的幼鱼生长较快.温度和盐度分别为28～30 ℃和6～8时,超氧化物歧化酶（SOD）和过氧化氢酶（CAT）的活力较高,温度的一次效应与盐度的二次效应对两种酶活力均有显著影响(P<0.05),温度与盐度对CAT活力有互作效应,高温与高盐环境会抑制SOD和CAT活力的表达.SGR、SOD和CAT因子与响应值间二次多项回归方程的决定系数分别达到0.954、0.831和0.942(P<0.05),可用于预测;温度效应对吉富罗非鱼幼鱼生长和抗氧化酶活力的影响较盐度明显.在罗非鱼的养殖过程中应合理安排养殖环境,降低氧化胁迫,以促进罗非鱼的生长与抗病力的提高.     

温度、盐度和pH对尼罗罗非鱼性别分化的影响

采用Box-Behnken设计及响应曲面法研究了温度(20～36℃)、盐度(0 ～16)和pH(5.5 ～8.5)3个主要环境因子对吉富品系尼罗罗非鱼性别分化的影响.结果表明:温度的一次和二次效应分别对吉富罗非鱼性别分化的影响显著.盐度和pH的一次和二次效应分别对吉富罗非鱼性别分化的影响不显著,3个环境因子中任意2个因子之间的互作效应都不显著.采用响应曲面法分析发现,随着温度的升高,吉富罗非鱼的雄性率呈逐渐上升的趋势;温度为36℃、盐度为8、pH为8.5时,吉富罗非鱼最大雄性率可达80％.建立雄性率与温度、盐度和pH三者之间关系的模型方程,并剔除相关不显著的因子后,得到雄性率与温度之间的模型方程,可用于预测吉富罗非鱼雄性率的变化.     

温度、盐度和pH对尼罗罗非鱼性别分化的影响

采用Box Behnken设计及响应曲面法研究了温度(20～36 ℃)、盐度(0～16)和pH(5.5～8.5)3个主要环境因子对吉富品系尼罗罗非鱼性别分化的影响.结果表明： 温度的一次和二次效应分别对吉富罗非鱼性别分化的影响显著.盐度和pH的一次和二次效应分别对吉富罗非鱼性别分化的影响不显著,3个环境因子中任意2个因子之间的互作效应都不显著.采用响应曲面法分析发现,随着温度的升高,吉富罗非鱼的雄性率呈逐渐上升的趋势;温度为36 ℃、盐度为8、pH为8.5时,吉富罗非鱼最大雄性率可达80%.建立雄性率与温度、盐度和pH三者之间关系的模型方程,并剔除相关不显著的因子后,得到雄性率与温度之间的模型方程,可用于预测吉富罗非鱼雄性率的变化.     

鲻鱼鳃组织14-3-3a、NKCCla、Apo-14和Na~+-K~+-ATPaseβ基因表达对盐度变化的响应

探讨了鲻鱼(Mugil cephalus)鳃组织14-3-3a、NKCCla、Apo-14和Na+-K+-ATPaseβ基因对盐度变化的响应表达特性。结果表明:不同盐度处理组对14-3-3a、NKCC1a、Apo-14和Na+-K+-ATPaseβ等4种基因mRNA的表达量与对照组(盐度20)间存在一定的差异性,且各类基因与盐度适应相关性的差异性也较为显著;其中Na+-K+-ATPaseβ和NKCC1a在低盐环境下被显著诱导,表达量上升明显(P0.01),而14-3-3a与Apo-14在低盐环境下表达量均有明显的下调(P0.05);不同盐度处理组与对照组表达量的差异、各基因表达量与盐度的回归关系以及4种基因生物整合标志物响应值的不同,表明Na+-K+-ATPaseβ和NKCC1a相对于14-3-3a和Apo-14更能赋予生物在受到低盐度应激刺激后,产生渗透调节机制,提高生物抵抗环境的胁迫能力;可考虑Na+-K+-ATPaseβ和NKCC1a适宜作为鱼类鳃组织在低盐环境胁迫下调控渗透基因的潜在分子生物标志物。     

盐度变化对克氏原螯虾渗透调节影响机制的初步研究

本文研究了克氏原螯虾在盐度变化下血淋巴渗透压、鳃丝Na+-K+-ATPase活力和生物胺的变化过程和特征。结果显示：盐度变化（0－20‰）对克氏原螯虾血淋巴渗透压、鳃丝Na+-K+-ATPase活力和生物胺含量影响显著(p<0.05),而对照组无明显变化。在实验时间内各处理组血淋巴渗透压随盐度变化增大而升高,1－2d后趋于稳定,鳃丝Na+-K+-ATPase活力逐渐降低,6d后保持稳定,且渗透压正相关性,酶活力与盐度呈负相关性;各处理组在实验时间内血淋巴和鳃丝多巴胺、去甲肾上腺素、5-羟色胺含量均呈峰值变化,血淋巴中多巴胺、5-羟色胺含量分别在1d和3d时达到最小值和最大值,且变化过程分别与盐度变化值呈负和正相关性,并分别在6d和9d后趋于稳定,而各处理组的NE含量（除盐度为4的处理组）在0.5d内迅速升高,0.5－3d内略有波动,然后呈下降趋势,至15d时保持稳定;各处理组鳃丝多巴胺、去甲肾上腺素和5-羟色胺含量均于2d时达到最大值,且变化过程均与盐度变化值呈正相关性,均在9d后趋于稳定;稳定后各处理组血淋巴和鳃丝生物胺含量均与对照组无显著性差异。这些结果表明,在外界盐度变化下生物胺作为一种神经内分泌因子,在甲壳动物的渗透压调节过程中发挥重要作用,可引发甲壳动物产生的渗透调节过程,如激活鳃丝已有的Na+-K+-ATPase活力、调节血淋巴渗透压效应物含量以适应外界环境的变化。     

温度、pH和盐度对克氏原螯虾鳃 Na+-K+-ATPase活性的影响

采用钼蓝法测定克氏原螯虾(Procambarus clarkii)鳃Na~ -K~ -ATPase活性,探讨温度、pH、盐度3个环境因子在环境驯化和突变状态下对Na -K -ATPase活性的影响。实验结果表明,Na~ -K~ -ATPase的活性与环境因子密切相关。酶活性在温度、盐度驯化实验中都表现为正相关关系,不同pH驯化中则表现为中性pH活性最高。在突变状况下表现出明显的应激性,应激响应在2～8h之间,之后逐渐缓和,最终结果与驯化结果相同。     

温度和盐度对褐牙鲆幼鱼渗透生理及抗氧化水平的影响简

采用双因素析因实验设计方法,研究了温度(20℃、24℃、28℃)和盐度(10‰、30‰)急性应激对褐牙鲆(Paralichthys olivaceus)幼鱼渗透生理和抗氧化水平的影响。结果表明:盐度和温度变化对各实验组1d和6d时褐牙鲆幼鱼血浆皮质醇含量均无显著性差异。在高温低盐(28℃、10‰)环境中1d时渗透压显著高于其他各实验组,6d时无显著性差异。牙鲆幼鱼在28℃环境中1d时鳃Na+-K+-ATP酶活性显著高于20℃和24℃;6d时,温度和盐度对牙鲆幼鱼鳃Na+-K+-ATP酶活性有显著交互影响作用。1d时,随着温度的升高或盐度的降低牙鲆幼鱼肝脏超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性呈现上升趋势,并且高温低盐(28℃、盐度10‰)组褐牙鲆幼鱼肝脏丙二醛(MDA)含量显著高于其他各组;在3个实验温度下,10‰环境中牙鲆幼鱼肝脏脂质过氧化物(LPO)的含量高于30‰。在6d时,各实验组间肝脏SOD、CAT活性及MDA含量无显著性差异。因此,褐牙鲆能够耐受温度20—28℃和低至盐度10‰的环境条件,应激早期温度和盐度的变化可引起褐牙鲆幼鱼渗透生理和抗氧化水平的变化,高温低盐对褐牙鲆幼鱼抗氧化水平的影响最大,至6d可基本恢复稳定。     

Effect of water temperature,salinity, and their interaction on growth,plasma osmolality,and gill Na,K-ATPase activity in juvenile GIFT tilapia Oreochromis niloticus (L.)

We used a central composite rotatable experimental design and response surface methodology to evaluate the effects of temperature (18–37 °C), salinity (0–20‰), and their interaction on specific growth rate (SGR), feed efficiency (FE), plasma osmolality, and gill Na⁺, K⁺-ATPase activity in GIFT tilapia juveniles. The linear and quadratic effects of temperature and salinity on SGR, plasma osmolality, and gill Na⁺, K⁺-ATPase activity were statistically significant (P<0.05). The interactive effects of temperature and salinity on plasma osmolality were significant (P<0.05). In contrast, the interaction term was not significant for SGR, FE, and gill Na⁺, K⁺-ATPase activity ⁽P>0.05). The regression equations for SGR, FE, plasma osmolality, and gill Na⁺, K⁺-ATPase activity against the two factors of interest had coefficients of determination of 0.944, 0.984, 0.966, and 0.960, respectively (P<0.01). The optimal temperature/salinity combination was 28.9 °C/7.8‰ at which SGR (2.26% d¹) and FE (0.82) were highest. These values correspond to the optimal temperature/salinity combination (29.1 °C/7.5‰) and the lowest plasma osmolality (348.38 mOsmol kg⁻¹) and gill Na⁺, K⁺-ATPase activity (1.31 µmol Pi. h⁻¹ g⁻¹ protein), and resulted in an energy-saving effect on osmoregulation, which promoted growth.     

Decoupling the Na+-K+-ATPase in vivo: a possible new role in the gills of freshwater fishes

The literature suggests that when Na(+)-K(+)-ATPase has reduced access to its glycosphingolipid cofactor sulfogalactosyl ceramide (SGC), it is converted to a Na(+) uniporter. We recently showed that such segregation can occur within a single membrane when Na(+)-K(+)-ATPase is excluded from membrane microdomains or 'lipid rafts' enriched in SGC (D. Lingwood, G. Harauz, J.S. Ballantyne, J. Biol. Chem. 280, 36545-36550). Specifically we demonstrated that Na(+)-K(+)-ATPase localizes to SGC-enriched rafts in the gill basolateral membrane (BLM) of rainbow trout exposed to seawater (SW) but not freshwater (FW). We therefore proposed that since the freshwater gill Na(+)-K(+)-ATPase was separated from BLM SGC it should also transport Na(+) only, suggesting a new role for the pump in this epithelium. In this paper we discuss the biochemical evidence for SGC-based modulation of transport stoichiometry and highlight how a unique asparagine-lysine substitution in the FW pump isoform and FW gill transport energetics gear the Na(+)-K(+)-ATPase to perform Na(+) uniport.     

Impact of environmental DDT concentrations on gill adaptation to increased salinity in the tilapia Sarotherodon melanotheron

Estuaries of tropical developing countries suffering from severe droughts induced by climate change are habitats to fish, which face drastic salinity variations and the contact with pollutants. The Western Africa tilapia Sarotherodon melanotheron is highly resistant to hypersalinity, but the effect of human-released xenobiotics on its adaptation is barely known. Controlled experiments were conducted to observe S. melanotheron gill adaptation to abrupt salinity variations in the presence of waterborne DDT, at concentrations detected in their natural habitat. The gills appeared as an important site of DDT conversion to DDD and/or depuration. A 12-days DDT exposure resulted in decreased gill epithelium thickness at all salinities (from fresh- to hypersaline-water), and the structure of gills from freshwater fish was particularly altered, relative to controls. No unbalance in tilapia blood osmolality was observed following DDT exposure, which however caused a decrease in branchial Na(+)-K(+)-ATPase (NKA) activity. Gill cellular NKA expression was reduced in salt-water, together with the expression of the CFTR chloride channel in hypersaline water. Although S. melanotheron seems very resistant (especially in seawater) to short-term waterborne DDT contamination, the resulting alterations of the gill tissue, cells and enzymes might affect longer term respiration, toxicant depuration and/or osmoregulation in highly fluctuating salinities.     

Increases in transepithelial vectorial Na+ transport facilitates Na+-dependent L-DOPA transport in renal OK cells

The present study evaluated the hypothesis of whether increases in vectorial Na+ transport translate into facilitation of Na+-dependent L-DOPA uptake in cultured renal epithelial tubular cells. Increases in vectorial Na+ transport were obtained in opossum kidney (OK) cells engineered to overexpress Na+-K+-ATPase after transfection of wild type OK cells with the rodent Na+-K+-ATPase alpha1 subunit. The most impressive differences between wild type and transfected OK cells are that the latter overexpressed Na+-K+-ATPase accompanied by an increased activity of the transporter. Non-linear analysis of the saturation curve for l-DOPA uptake revealed a Vmax value (in nmol mg protein/6 min) of 62 and 80 in wild type and transfected cells, respectively. The uptake of a non-saturating concentration (0.25 microM) of [14C]-L-DOPA in OK-WT cells was not affected by Na+ removal, whereas in OK-alpha1 cells accumulation of [14C]-L-DOPA was clearly dependent on the presence of extracellular Na+. When Na+ was replaced by choline, the inhibitory profile of neutral l-amino acids, but not of basic and acidic amino acids, upon [14C]-L-DOPA uptake in both cell types, was significantly greater than that observed in the presence of extracellular Na+. It is concluded that enhanced ability of OK cells overexpressing Na+-K+-ATPase to translocate Na+ from the apical to the basal cell side correlates positively with their ability to accumulate L-DOPA, which is in agreement with the role of Na+ in taking up the precursor of renal dopamine.     

Cyclic stretch translocates the alpha2-subunit of the Na pump to plasma membrane in skeletal muscle cells in vitro

The Na+-K+-ATPase and its regulation is important for maintaining membrane potential and transmembrane Na(+) gradient in all skeletal muscle cells and thus is essential for cell survival and function. In our previous study, cyclic stretch activated the Na pump in cultured skeletal muscle cells. Presently, we investigated whether this stimulation was the result of translocation of Na+-K+-ATPase from endosomes to the plasma membrane, and also evaluated the role of phosphatidylinositol 3-kinase (PI 3-kinase), the activation of which initiated vesicular trafficking and targeting of proteins to specific cell compartments. Skeletal muscle cells were stretched at 25% elongation continuous for 24h using the Flexercell Strain Unit. The plasma membrane and endosome fractions were isolated and Western blotted to localize the Na+-K+-ATPase alpha1- and alpha2-subunit protein. The results showed stretch increased Na+-K+-ATPase alpha1- and alpha2-subunit protein expression in plasma membrane fractions and decreased it in endosomes. The alpha2-subunit had a more dynamic response to mechanical stretch. PI 3-kinase inhibitors (LY294002) blocked the stretch-induced translocation of the Na+-K+-ATPase alpha2-subunit, while LY294002 had no effect on the transfer of alpha1-subunit. We concluded that cyclic stretch mainly stimulated the translocation of the alpha2-subunit of Na+-K+-ATPase from endosomes to the plasma membrane via a PI 3-kinase-dependent mechanism in cultured skeletal muscle cells in vitro, which in turn increased the activity of the Na pump.     

Role of skeletal muscle Na+-K+ ATPase activity in increased lactate production in sub-acute sepsis

Bacterial sepsis is frequently accompanied by increased blood concentration of lactic acid, which traditionally is attributed to poor tissue perfusion, hypoxia and anaerobic glycolysis. Therapy aimed at improving oxygen delivery to tissues often does not correct the hyperlactatemia, suggesting that high blood lactate in sepsis is not due to hypoxia. Various tissues, including skeletal muscle, demonstrate increased lactate production under well-oxygenated conditions when the activity of the Na+-K+ ATPase is stimulated. Although both muscle Na+-K+ ATPase activity and muscle plasma membrane content of Na+, K+-ATPase subunits are increased in sepsis, no studies in vivo have demonstrated correlation between lactate production and changes in intracellular Na+ and K+ resulting from increased Na+-K+ pump activity in sepsis. Plasma concentrations of lactate and epinephrine, a known stimulator of the Na+-K+ pump, were increased in rats made septic by E. coli injection. Muscle lactate content was significantly increased in septic rats, although muscle ATP and phosphocreatine remained normal, suggesting oxygen delivery remained adequate for mitochondrial energy metabolism. In septic rats, muscle intracellular ratio of Na+:K+ was significantly reduced, indicating increased Na+-K+ pump activity. These data thus demonstrate that increased muscle lactate during sepsis correlates with evidence of elevated muscle Na+-K+ ATPase activity, but not with evidence of impaired oxidative metabolism. This study also further supports a role for epinephrine in this process.     

Acute changes in gill Na+-K+-ATPase and creatine kinase in response to salinity changes in the euryhaline teleost, tilapia (Oreochromis mossambicus)

Some freshwater (FW) teleosts are capable of acclimating to seawater (SW) when challenged; however, the related energetic and physiological consequences are still unclear. This study was conducted to examine the changes in expression of gill Na(+)-K(+)-ATPase and creatine kinase (CK) in tilapia (Oreochromis mossambicus) as the acute responses to transfer from FW to SW. After 24 h in 25 ppt SW, gill Na(+)-K(+)-ATPase activities were higher than those of fish in FW. Fish in 35 ppt SW did not increase gill Na(+)-K(+)-ATPase activities until 1.5 h after transfer, and then the activities were not significantly different from those of fish in 25 ppt SW. Compared to FW, the gill CK activities in 35 ppt SW declined within 1.5 h and afterward dramatically elevated at 2 h, as in 25 ppt SW, but the levels in 35 ppt SW were lower than those in 25 ppt SW. The Western blot of muscle-type CK (MM form) was in high association with the salinity change, showing a pattern of changes similar to that in CK activity; however, levels in 35 ppt SW were higher than those in 25 ppt SW. The activity of Na(+)-K(+)-ATPase highly correlated with that of CK in fish gill after transfer from FW to SW, suggesting that phosphocreatine acts as an energy source to meet the osmoregulatory demand during acute transfer.     

Gill Na+-K+-ATPase activity correlates with basolateral membrane lipid composition in seawater- but not freshwater-acclimated Arctic char (Salvelinus alpinus)

The successful migration of euryhaline teleost fish from freshwater to seawater requires the upregulation of gill Na+-K+-ATPase, an ion transport enzyme located in the basolateral membrane (BLM) of gill chloride cells. Following 39 days of seawater exposure, Arctic char had similar plasma sodium and chloride levels as individuals maintained in freshwater, indicating they had successfully acclimated to seawater. This acclimation was associated with an eightfold increase in gill Na+-K+-ATPase activity but only a threefold increase in gill Na+-K+-ATPase protein number, suggesting that other mechanisms may also modulate gill Na+-K+-ATPase activity. We therefore investigated the influence of membrane composition on Na+-K+-ATPase activity by examining the phospholipid, fatty acid, and cholesterol composition of the gill BLM from freshwater- and seawater-acclimated Arctic char. Mean gill BLM cholesterol content was significantly lower ( approximately 22%) in seawater-acclimated char. Gill Na+-K+-ATPase activity in individual seawater Arctic char was negatively correlated with BLM cholesterol content and positively correlated with %phosphatidylethanolamine and overall %18:2n6 (linoleic acid) content of the BLM, suggesting gill Na+-K+-ATPase activity of seawater-acclimated char may be modulated by the lipid composition of the BLM and may be especially sensitive to those parameters known to influence membrane fluidity. Na+-K+-ATPase activity of individual freshwater Arctic char was not correlated to any membrane lipid parameter measured, suggesting that different lipid-protein interactions may exist for char living in each environment.