Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL 33149, USA.
J Exp Biol. 2012 Apr 1;215(Pt 7):1199-209. doi: 10.1242/jeb.067496.
The euryhaline fish Cyprinodon variegatus variegatus is capable of tolerating ambient salinities ranging from 0.3 to 160 PSU, but is incapable of long-term survival in freshwater (<2 mmol l(-1) Na(+)). A population isolated in several freshwater (0.4-1 mmol l(-1) Na(+)) lakes in central Florida is now designated as a subspecies (Cyprinodon variegatus hubbsi). We conducted a comparative study of Na(+) transport kinetics in these two populations when acclimated to different ambient Na(+) concentrations. Results reveal that the two subspecies have qualitatively similar low affinity Na(+) uptake kinetics (K(m)=7000-38,000 μmol l(-1)) when acclimated to 2 or 7 mmol l(-1) Na(+), but C. v. hubbsi switches to a high affinity system (K(m)=100-140 μmol l(-1)) in low-Na(+) freshwater (≤1 mmol l(-1) Na(+)). Inhibitor experiments indicate that Na(+) uptake in both subspecies is EIPA-sensitive, but sensitivity decreases with increasing external Na(+). EIPA induced a 95% inhibition of Na(+) influx in C. v. hubbsi acclimated to 0.1 mmol l(-1) Na(+), suggesting that this subspecies is utilizing a Na(+)/H(+) exchanger to take up Na(+) in low-Na(+) environments despite theoretical thermodynamic constraints. Na(+) uptake in C. v. hubbsi acclimated to 0.1 mmol l(-1) Na(+) is phenamil-sensitive but not bafilomycin-sensitive, leading to uncertainty about whether this subspecies also utilizes Na(+) channels for Na(+) uptake. Experiments with both subspecies acclimated to 7 mmol l(-1) Na(+) also indicate that a Cl(-)-dependent Na(+) uptake pathway is present. This pathway is not metolazone-sensitive (NCC inhibitor) in either species but is bumetanide-sensitive in C. v. variegatus but not C. v. hubbsi. This suggests that an apical NKCC is increasingly involved with Na(+) uptake for this subspecies as external Na(+) increases. Finally, characterization of mitochondria-rich cell (MRC) size and density in fish acclimated to different ambient Na(+) concentrations revealed significant increases in the number and size of emergent MRCs with decreasing ambient Na(+). A linear relationship between the fractional area of emergent MRCs and Na(+) uptake rate was observed for both subspecies. However, C. v. variegatus have lower Na(+) uptake rates at a given MRC fractional area compared with C. v. hubbsi, indicating that the enhanced Na(+) uptake by C. v. hubbsi at low ambient Na(+) concentrations is not strictly a result of increased MRC fractional area, and other variables, such as differential expression of proteins involved in Na(+) uptake, must provide C. v. hubbsi with the ability to osmoregulate in dilute freshwater.
杂色石斑鱼（Cyprinodon variegatus variegatus）能够耐受 0.3 至 160 PSU 的环境盐度，但不能在淡水中长期生存（<2 mmol l(-1) Na(+))。佛罗里达州中部的几个淡水（0.4-1 mmol l(-1) Na(+))湖泊中的一个种群现在被指定为亚种（Cyprinodon variegatus hubbsi）。我们对这两个种群在适应不同环境 Na(+)浓度时的 Na(+)转运动力学进行了比较研究。结果表明，当适应 2 或 7 mmol l(-1) Na(+)时，两个亚种的低亲和力 Na(+)摄取动力学（K(m)=7000-38,000 μmol l(-1))具有定性相似性，但 C. v. hubbsi 在低 Na(+)的淡水中（≤1 mmol l(-1) Na(+))切换为高亲和力系统（K(m)=100-140 μmol l(-1))。抑制剂实验表明，两种亚种的 Na(+)摄取均对 EIPA 敏感，但随着外 Na(+)的增加，敏感性降低。EIPA 诱导 C. v. hubbsi 在适应 0.1 mmol l(-1) Na(+)时对 Na(+)内流的 95%抑制，表明尽管存在理论热力学限制，该亚种仍在利用 Na(+)/H(+)交换器在低 Na(+)环境中摄取 Na(+)。在适应 0.1 mmol l(-1) Na(+)的 C. v. hubbsi 中，Na(+)摄取对苯氟胺敏感但对巴弗洛霉素不敏感，这导致对该亚种是否也利用 Na(+)通道摄取 Na(+)存在不确定性。对适应 7 mmol l(-1) Na(+)的两个亚种的实验也表明存在 Cl(-)依赖性 Na(+)摄取途径。该途径在两种物种中均不受 metolazone（NCC 抑制剂）影响，但在 C. v. variegatus 中受 bumetanide 影响，而在 C. v. hubbsi 中不受影响。这表明，随着外部 Na(+)的增加，一种顶端 NKCC 越来越参与该亚种的 Na(+)摄取。最后，对适应不同环境 Na(+)浓度的鱼类中富含线粒体的细胞（MRC）大小和密度的特征表明，随着环境 Na(+)的降低，出现的 MRC 的数量和大小显著增加。在两个亚种中，都观察到出现的 MRC 分数面积与 Na(+)摄取率之间存在线性关系。然而，与 C. v. hubbsi 相比，C. v. variegatus 在给定的 MRC 分数面积下具有较低的 Na(+)摄取率，这表明 C. v. hubbsi 在低环境 Na(+)浓度下增强的 Na(+)摄取并非仅仅是由于 MRC 分数面积的增加，其他变量，例如参与 Na(+)摄取的蛋白质的差异表达，必须为 C. v. hubbsi 在稀淡水中提供渗透调节能力。
