Department of Biology, McMaster University, Hamilton, ON L8S 4K1, Canada; Instituto de Ciências Biológicas, Universidade Federal do Rio Grande-FURG, 96203-900 Rio Grande, Brazil.
Department of Biology, McMaster University, Hamilton, ON L8S 4K1, Canada; Department of Zoology, University of British Columbia, Vancouver, BC V6T 1Z4, Canada; University of Miami Rosenstiel, School of Marine, Atmospheric, and Earth Science, Miami, FL 33149, USA.
Comp Biochem Physiol A Mol Integr Physiol. 2024 Nov;297:111715. doi: 10.1016/j.cbpa.2024.111715. Epub 2024 Jul 31.
There is a consensus that electroneutral Na/H exchangers (NHEs) are important in branchial Na uptake in freshwater fish. There is also widespread belief, based on mammalian data, that EIPA [5-(N-ethyl-N-isopropyl)-amiloride]], and HMA [5-(N,N-hexamethylene)-amiloride)] are more potent and specific in blocking Na uptake than amiloride. We evaluated this idea by testing the three drugs at 10 to 10 M, i.e. 0.1 to 100 μM in two model species, rainbow trout (Oncorhynchus mykiss) and goldfish (Carassius auratus), using Na to measure unidirectional Na influx and efflux rates. In both species, the potency order for inhibiting unidirectional Na influx was HMA > amiloride > EIPA (IC values in the 10-70 μM range), very different from in mammals. At 100 μM, all three drugs inhibited Na influx by >90% in both species, except for amiloride in goldfish (65%). However, at 60-100 μM, all three drugs also stimulated unidirectional Na efflux rates, indicating non-specific effects. In trout, HMA and EIPA caused significant increases (2.1- to 2.3-fold) in efflux rates, whereas in goldfish, significant efflux elevations were greater (3.1- to 7.2-fold) with all three drugs. We conclude that the inhibitory potency profile established in mammals does not apply to the NHEs in fish gills, that non-specific effects on Na efflux rates are a serious concern, and that EIPA and HMA offer no clear benefits in terms of potency or specificity. Considering its much lower cost, we recommend amiloride as the drug of choice for in vivo experiments on freshwater fishes.
人们普遍认为，电中性的 Na/H 交换器（NHEs）在淡水鱼类的鳃部 Na 吸收中很重要。基于哺乳动物的数据，人们还普遍认为，EIPA[5-（N-乙基-N-异丙基）-阿米洛利]和 HMA[5-（N，N-六亚甲基）-阿米洛利]在阻断 Na 吸收方面比阿米洛利更有效和更特异。我们通过在两种模式物种，虹鳟（Oncorhynchus mykiss）和金鱼（Carassius auratus）中，在 10 至 10-7 M （即 0.1 至 100 μM）的浓度下测试这三种药物，评估了这一观点，使用 Na 来测量单向 Na 内流和外排率。在这两个物种中，抑制单向 Na 内流的效价顺序为 HMA>阿米洛利>EIPA（IC 值在 10-70 μM 范围内），与哺乳动物非常不同。在 100 μM 时，除了金鱼中的阿米洛利（65%）之外，所有三种药物都使两种物种的 Na 内流抑制了>90%。然而，在 60-100 μM 时，所有三种药物也刺激了单向 Na 外排率，表明存在非特异性效应。在鳟鱼中，HMA 和 EIPA 导致外排率显著增加（2.1-2.3 倍），而在金鱼中，所有三种药物都引起更大的外排率显著升高（3.1-7.2 倍）。我们得出结论，在哺乳动物中建立的抑制效价谱不适用于鱼类鳃部的 NHEs，对 Na 外排率的非特异性影响是一个严重的问题，并且 EIPA 和 HMA 在效价或特异性方面没有明显的优势。考虑到其成本低得多，我们建议在淡水鱼类的体内实验中选择阿米洛利作为药物。
