Claiborne James B, Choe Keith P, Morrison-Shetlar Alison I, Weakley Jill C, Havird Justin, Freiji Abe, Evans David H, Edwards Susan L
Department of Biology, Georgia Southern University, Statesboro, GA 30460, USA.
Am J Physiol Regul Integr Comp Physiol. 2008 Mar;294(3):R1092-102. doi: 10.1152/ajpregu.00718.2007. Epub 2007 Dec 19.
The dogfish (Squalus acanthias) can make rapid adjustments to gill acid-base transfers to compensate for internal acidosis/alkalosis. Branchial Na+/H+ exchange (NHE) has been postulated as one mechanism driving the excretion of H+ following acidosis. We have cloned gill cDNA that includes an open reading frame coding for a 770-residue protein most homologous (approximately 71%) to mammalian NHE2. RT-PCR revealed NHE2 transcripts predominantly in gill, stomach, rectal gland, intestine, and kidney. In situ hybridization with an antisense probe against NHE2 in gill sections revealed a strong mRNA signal from a subset of interlamellar and lamellae cells. We developed dogfish-specific polyclonal antibodies against NHE2 that detected a approximately 70-kDa protein in Western blots and immunologically recognized branchial cells having two patterns of protein expression. Cytoplasmic and apical NHE2 immunoreactivity were observed in cells coexpressing basolateral Na+-K+-ATPase. Other large ovoid cells more generally staining for NHE2 also were strongly positive for basolateral H+-ATPase. Gill mRNA levels for NHE2 and H+-ATPase did not change following systemic acidosis (as measured by quantitative PCR 2 h after a 1- or 2-meq/kg acid infusion). These data indicate that posttranslational adjustments of NHE2 and other transport systems (e.g., NHE3) following acidosis may be of importance in the short-term pH adjustment and net branchial H+ efflux observed in vivo. NHE2 may play multiple roles in the gills, involved with H+ efflux from acid-secreting cells, basolateral H+ reabsorption for pHi regulation, and in parallel with H+-ATPase for the generation of HCO3(-) in base-secreting cells.
角鲨（棘鲨）能够迅速调整鳃的酸碱转运，以补偿体内的酸中毒/碱中毒。鳃部的钠氢交换（NHE）被认为是酸中毒后驱动氢离子排泄的一种机制。我们克隆了鳃部的互补DNA（cDNA），其包含一个开放阅读框，编码一种由770个氨基酸残基组成的蛋白质，该蛋白质与哺乳动物的NHE2最为同源（约71%）。逆转录聚合酶链反应（RT-PCR）显示，NHE2转录本主要存在于鳃、胃、直肠腺、肠道和肾脏中。用针对鳃切片中NHE2的反义探针进行原位杂交，结果显示在板间细胞和薄片细胞的一个亚群中有强烈的信使核糖核酸（mRNA）信号。我们制备了针对角鲨NHE2的特异性多克隆抗体，该抗体在蛋白质免疫印迹中检测到一种约70千道尔顿的蛋白质，并在免疫上识别出具有两种蛋白质表达模式的鳃细胞。在共表达基底外侧钠钾ATP酶的细胞中观察到细胞质和顶端的NHE2免疫反应性。其他对NHE2进行更广泛染色的大卵形细胞对基底外侧氢离子ATP酶也呈强阳性。在全身酸中毒后（通过在注入1或2毫当量/千克酸后2小时进行定量聚合酶链反应测量），鳃中NHE2和氢离子ATP酶的信使核糖核酸水平没有变化。这些数据表明，酸中毒后NHE2和其他转运系统（如NHE3）的翻译后调节可能在体内观察到的短期酸碱度调节和鳃部净氢离子外流中起重要作用。NHE2可能在鳃中发挥多种作用，参与分泌酸细胞的氢离子外流、用于调节细胞内酸碱度的基底外侧氢离子重吸收，以及与氢离子ATP酶并行在分泌碱细胞中生成碳酸氢根离子。
