Department of Biology, University of Ottawa, Ottawa, Ontario, Canada.
Am J Physiol Regul Integr Comp Physiol. 2011 Nov;301(5):R1517-28. doi: 10.1152/ajpregu.00282.2011. Epub 2011 Aug 10.
The involvement of a Na(+)/H(+) exchanger (NHE) in mediating Na(+) uptake by freshwater fish is currently debated. Although supported indirectly by empirical molecular and pharmacological data, theoretically its operation should be constrained thermodynamically, owing to unfavorable chemical gradients. Recently, there has been an increasing focus on ammonia channels (Rh proteins) as potentially contributing to Na(+) uptake across the freshwater fish gill. In this study, we tested the hypothesis that Rhcg1, a specific apical isoform of Rh protein, is critically important in facilitating Na(+) uptake in zebrafish larvae via its interaction with NHE. Treating larvae (4 days postfertilization) with 5-(N-ethyl-N-isopropyl) amiloride (EIPA), an inhibitor of NHE, caused a significant reduction in Na(+) uptake in fish reared in acidic water (pH ∼ 4.0). A role for NHE in Na(+) uptake was further confirmed by translational knockdown of NHE3b, an isoform of NHE thought to be responsible for Na(+)/H(+) exchange in zebrafish larvae. Exposing the larvae reared in acidic water to 5 mM external ammonium sulfate or increasing the buffering capacity of the water with 10 mM HEPES caused concurrent reductions in ammonia excretion and Na(+) uptake. Furthermore, translational knockdown of Rhcg1 significantly reduced ammonia excretion and Na(+) uptake in larvae chronically (4 days) or acutely (24 h) exposed to acidic water. Unlike in sham-injected larvae, EIPA did not affect Na(+) uptake in fish experiencing Rhcg1 knockdown. Additionally, exposure of larvae to bafilomycin A1 (an inhibitor of H(+)-ATPase) significantly reduced Na(+) uptake in fish reared in acidic water. These observations suggest the existence of multiple mechanisms of Na(+) uptake in larval zebrafish in acidic water: one in which Na(+) uptake via NHE3b is linked to ammonia excretion via Rhcg1, and another facilitated by H(+)-ATPase.
Na(+)/H(+) 交换器（NHE）在介导淡水鱼摄取 Na(+)中的作用目前存在争议。尽管有间接的经验分子和药理学数据支持，但由于化学梯度不利，其理论上的运作应该受到热力学的限制。最近，人们越来越关注氨通道（Rh 蛋白）作为跨淡水鱼鳃摄取 Na(+)的潜在贡献者。在这项研究中，我们测试了一个假设，即 Rh 蛋白的特定顶端同工型 Rhcg1 通过与 NHE 的相互作用对于斑马鱼幼虫中 Na(+)摄取至关重要。用 5-（N-乙基-N-异丙基）阿米洛利（EIPA）处理（受精后 4 天）幼虫，一种 NHE 的抑制剂，在酸性水（pH ∼ 4.0）中饲养的鱼类中，Na(+)摄取显著减少。NHE 在 Na(+)摄取中的作用进一步通过 NHE3b 的翻译敲低得到证实，NHE3b 是一种被认为负责斑马鱼幼虫中 Na(+)/H(+)交换的 NHE 同工型。将在酸性水中饲养的幼虫暴露于 5 mM 外源性硫酸铵或用 10 mM HEPES 增加水的缓冲能力，会导致氨排泄和 Na(+)摄取同时减少。此外，在慢性（4 天）或急性（24 小时）暴露于酸性水的幼虫中，Rhcg1 的翻译敲低显著降低了氨排泄和 Na(+)摄取。与在假注射幼虫中不同，EIPA 不影响经历 Rhcg1 敲低的鱼类中的 Na(+)摄取。此外，暴露于巴弗洛霉素 A1（一种 H(+)-ATP 酶抑制剂）显著降低了在酸性水中饲养的鱼类中的 Na(+)摄取。这些观察结果表明，在酸性水中，幼鱼存在多种摄取 Na(+)的机制：一种是通过 NHE3b 摄取 Na(+)与 Rhcg1 介导的氨排泄有关，另一种是由 H(+)-ATP 酶促进的。
