Rainbow Trout (Oncorhynchus mykiss) Na+/H+ Exchangers tNhe3a and tNhe3b Display Unique Inhibitory Profiles Dissimilar from Mammalian NHE Isoforms

Abstract

Freshwater fishes maintain an internal osmolality of ~300 mOsm, while living in dilute environments ranging from 0 to 50 mOsm. This osmotic challenge is met at least partially, by Na⁺/H⁺ exchangers (NHE) of fish gill and kidney. In this study, we cloned, expressed, and pharmacologically characterized fish-specific Nhes of the commercially important species Oncorhynchus mykiss. Trout (t) Nhe3a and Nhe3b isoforms from gill and kidney were expressed and characterized in an NHE-deficient cell line. Western blotting and immunocytochemistry confirmed stable expression of the tagged trout tNhe proteins. To measure NHE activity, a transient acid load was induced in trout tNhe expressing cells and intracellular pH was measured. Both isoforms demonstrated significant activity and recovered from an acute acid load. The effect of the NHE transport inhibitors amiloride, EIPA (5-(N-ethyl-N-isopropyl)-amiloride), phenamil, and DAPI was examined. tNhe3a was inhibited in a dose-dependent manner by amiloride and EIPA and tNhe3a was more sensitive to amiloride than EIPA, unlike mammalian NHE1. tNhe3b was inhibited by high concentrations of amiloride, while even in the presence of high concentrations of EIPA (500 µM), some activity of tNhe3b remained. Phenamil and DAPI were ineffective at inhibiting tNhe activity of either isoform. The current study aids in understanding the pharmacology of fish ion transporters. Both isoforms display inhibitory profiles uniquely different from mammalian NHEs and show resistance to inhibition. Our study allows for more direct interpretation of past, present, and future fish-specific sodium transport studies, with less reliance on mammalian NHE data for interpretation.

Keywords: AP-1; fish physiology; ion-regulation; pH regulation; pharmacology.

Figure 1

RT-PCR analysis of gene expression in trout tissues. Total RNA was extracted from adult rainbow trout gill and kidney tissue and analyzed with RT-PCR using gene specific primers for nhe3a nhe3b, and ef1α (elongation factor 1 alpha).

Figure 2

Western blot analysis of expression tNhe3 proteins. Western blot of whole cell lysates of stable cell lines expressing tNhe3a or tNhe3b proteins. Moreover, 100 μg of total protein was loaded in each lane. The sample was immunoblotted with anti-GFP tag antibody. AP-1 is a cell lysate from mock transfected AP-1 cells.

Figure 3

Confocal imaging of expression tNhe3 proteins. Confocal fluorescent imaging (63 × objective lens) of cell preparations of stable AP-1 cell lines either non-transfected or expressing tNhe3a or tNhe3b proteins. (A) Non-transfected AP-1 cell stained with DAPI. (B) GFP tagged-tNhe3a expressing cells. (C) GFP tagged-tNhe3b expressing cells. Intracellular and cell membrane protein expression is present in each of the stably expressing tNhe cell lines. Scale bar represents 20 μm.

Figure 4

Summary of activity of tNhe3 proteins in stably transfected AP-1 cells. Activity was measured after ammonium chloride pre-pulse as described in the “Materials and Methods”. Results are ∆ intracellular pH/s. Data are presented mean ± SE, while dissimilar letters indicate statistical significance between groups as demonstrated by one-way ANOVA, with Tukey’s multiple comparisons test (n ≥ 6, p < 0.05, ANOVA).

Figure 5

Effect of varying concentrations of amiloride on activity of tNhe3a. tNhe3a containing cells were subjected to a two pulse Na⁺/H⁺ exchanger activity assay and the activity (ROR, rate of recovery) of the exchanger in the second pulse was compared to that of the first pulse. The second pulse was in the presence of the indicated inhibitor. A control was in the presence of equal amounts of vehicle (DMSO). Amiloride concentrations were from 1 to 1000 µM. IC50 was estimated at 9.3 uM as described earlier [28]. Raw data presented in Figure S2. Asterisk * indicates significantly different from the control at p < 0.05, one-way ANOVA, with Tukey’s multiple comparison’s test. Data are presented as mean +/− SE. Each data point is mean of 8–11 measurements.

Figure 6

Effect of varying concentrations of EIPA on activity of tNhe3a. tNhe3a containing cells were subjected to a two pulse Na⁺/H⁺ exchanger activity assay and measured as described in Figure 5. EIPA concentrations were from 1 to 500 µM. IC50 estimated at 44 µM. Raw data presented in Figure S3. Asterisk * indicates significantly different from the control at p < 0.05, one-way ANOVA, with Tukey’s multiple comparison’s test. Data are presented as mean +/− SE and each experimental point is 8–10 measurements, control n = 18.

Figure 7

Effect of phenamil, or DAPI on tNhe3b activity. The cells were subjected to a two pulse Na⁺/H⁺ exchanger activity assay and measured as described in Figure 5. The control treatment was in the presence of equal amounts of vehicle (DMSO). Na⁺/H⁺ exchanger activity presented as % of control AP1 cells. Asterisk ^* indicates significance from the control as demonstrated by one-way ANOVA at p < 0.05. Results are mean +/− SE of at least eight independent experiments.

Figure 8

Effect of Amiloride on tNhe3b activity. The cells were subjected to a two pulse Na⁺/H⁺ exchanger activity assay and measured as described in Figure 5. A control was in the presence of equal amounts of vehicle (DMSO). Asterisk * indicates significantly different from the control at p < 0.05, one-way ANOVA, with Tukey’s multiple comparison’s test. IC50 relatively incalculable, but approximately 335 µM. Each experimental point is mean +/− SE of 7–11 measurements, control n = 17.

Figure 9

Effect of varying concentrations of EIPA on activity of tNhe3b. tNhe3b containing cells were subjected to a two pulse Na⁺/H⁺ exchanger activity assay and the activity was measured as described in Figure 5. EIPA concentrations were from 1 to 500 uM. IC50 cannot be calculated. Results are mean +/− SE of 8–16 independent experiments with no significant differences (p < 0.05, ANOVA).