Phosphorylation state of the Na+-K+-Cl- cotransporter (NKCC1) in the gills of Atlantic killifish (Fundulus heteroclitus) during acclimation to water of varying salinity

Abstract

Euryhaline teleosts such as Atlantic killifish (Fundulus heteroclitus) are able to acclimate to changing environmental salinity by tightly regulating NaCl absorption and secretion across their gills. Many studies have examined the mechanisms responsible for long-term (days) salinity acclimation; however, much remains unknown about the mechanisms of acute (hours) salinity acclimation. In this study, we tested the hypotheses that phosphorylation of the Na(+)-K(+)-Cl(-) cotransporter (NKCC1) located in the basolateral membrane of the gill plays a role in acute salinity acclimation and that changes in NKCC1 phosphorylation are mediated by a cAMP-protein kinase A (cAMP-PKA) pathway. Using a phospho-specific antibody, we determined the time course of changes in total and phosphorylated NKCC1 protein during acclimation to water of various salinities. Long-term (>or=14 days) acclimation of killifish to seawater (SW) and 2x SW resulted in 4- to 6-fold and 5- to 8-fold increases, respectively, in total gill NKCC1 protein relative to fish maintained in freshwater (FW). NKCC1 was found to be between 20% and 70% activated in fish, with lower average activation in fish acclimated to SW and 2x SW compared with FW fish. Increases and decreases in the fractional level of NKCC1 phosphorylation were seen within 1 h of transfer of fish to water of higher and lower salinity, respectively, consistent with a regulatory role of phosphorylation prior to an increase in the biosynthesis of NKCC1; large changes in protein expression of NKCC1 were observed over periods of hours to days. We found that NKCC1 phosphorylation is acutely regulated in the killifish gill in response to changing environmental salinity and that phosphorylation in excised gills increases in response to forskolin stimulation of the cAMP-PKA pathway. The role of phosphorylation is further underscored by the observation that mRNA expression of sterile 20 (Ste20)-related proline-alanine-rich kinase (SPAK) changes with salinity acclimation, being 2.7-fold greater in SW-acclimated killifish relative to FW fish. Overall, these results demonstrate an important role of NKCC1 phosphorylation in the gill of Atlantic killifish during acute salinity acclimation.

Fig. 1.

Representative western blot of gills taken from Atlantic killifish acclimated for ≥14 days to freshwater (FW), seawater (SW) or 2× SW. Gills were rapidly excised from fish, briefly rinsed in teleost Ringer solution, and incubated without (A) or with (B) 10 μmol l⁻¹ forskolin for 15 min at room temperature. Each lane represents gill samples pooled from 6 individual fish. Equal amounts (30 μg) of SDS-solubilized protein were loaded in each lane and probed with anti-phospho Na⁺–K⁺–Cl⁻ cotransporter antibody R5. The R5 antibody recognized a strongly stained band centered at ~170 kDa.

Fig. 2.

Time course of NKCC1 cotransporter phosphorylation in excised killifish gills stimulated with forskolin. Gills (three pieces per fish) were excised from fish maintained in SW and incubated in teleost Ringer solution containing 10 μmol l⁻¹ forskolin at room temperature. p-NKCC was analyzed on dot blots using the anti-p-NKCC antibody R5 and normalized for total protein. Data are means ± s.e.m. of 2–6 fish at a given time point. One-way ANOVA determined a significant effect (P<0.001) of time on p-NKCC. Asterisks indicate a significant difference (P<0.05) from time zero.

Fig. 3.

Representative immunofluorescence images showing Na⁺,K⁺-ATPase (α5, left column) and p-NKCC (R5, center column) immunoreactivity in the gills of Atlantic killifish (N=3–4) acclimated to FW (A), SW (B), and 2× SW (C) both with and without 10 μmol l⁻¹ forskolin for 15 min at room temperature as indicated. The scale bar represents 50 μm.

Fig. 4.

‘Steady-state’ protein expression and fractional level of phosphorylation of NKCC1 in gills isolated from fish acclimated to FW, SW or 2× SW. NKCC1 was measured on dot blots probed with anti-phospho NKCC1 (R5) and normalized to protein content. (A) Total NKCC1, measured with anti-phospho NKCC1 in gill samples that were incubated with 10 μmol l⁻¹ forskolin for 15 min in order to maximally stimulate NKCC1. Each bar represents the mean ± s.e.m. of 6 individual fish in a cohort of fish studied in one experiment. Results are shown relative to standards for four series of experiments with different cohorts of killifish. (B) p-NKCC in freshly excised gills as a ratio to the total level for the paired forskolin-stimulated gills shown in A. One-way ANOVA determined that salinity had a significant effect (P<0.007) on total NKCC1 in all experiments. One-way ANOVA determined that salinity had a significant effect (P<0.02) on p-NKCC in two of the four experiments. Asterisks indicate a significant difference (P<0.05) from FW within an experiment.

Fig. 5.

Time course of total NKCC1 protein expression (A) and NKCC1 phosphorylation (B) after transition of fish from SW to FW. Each point represents the mean ± s.e.m. of 6 individual fish. One-way ANOVA determined a significant effect of time on both total NKCC1 (P<0.001) and p-NKCC (P=0.004). Asterisks indicate a significant difference (P<0.05) from pre-treatment control.

Fig. 6.

Time course of total NKCC1 protein expression (A) and NKCC1 phosphorylation (B) after transition of fish from FW to SW. Each point represents the mean ± s.e.m. of 6 individual fish in a cohort of fish studied in one experiment. Different symbols represent different experiments. One-way ANOVA determined a significant effect of time on total NKCC1 in all three experiments (P<0.002), and a significant effect of time on p-NKCC in one experiment (P=0.023; open circles). Asterisks indicate a significant difference (P<0.05) from pre-treatment control within an experiment.

Fig. 7.

Time course of total NKCC1 protein expression (A) and NKCC1 phosphorylation (B) after transition of fish from SW to 2× SW. Each point represents the mean ± s.e.m. of 6 individual fish in a cohort of fish studied in one experiment. Different symbols represent different experiments. One-way ANOVA determined a significant effect of time on both total NKCC1 (P=0.003) and p-NKCC (P=0.004). Asterisks indicate a significant difference (P<0.05) from pre-treatment control within an experiment.

Fig. 8.

Short-term changes in total NKCC1 protein expression (A–C) and NKCC1 phosphorylation (D–F) after transition of fish from SW to 2× SW (A,D), 2× SW to SW (B,E), and 2× SW to 3× SW (C,F). Each point represents the mean ± s.e.m. of 6 individual fish in a cohort of fish studied in one experiment. Different symbols represent different experiments. One-way ANOVA determined a significant effect of time on p-NKCC in all three transfer experiments (P<0.001). Asterisks indicate a significant difference (P<0.05) from pre-treatment control within an experiment. There was no effect of time on total NKCC1 protein in any of the three experiments (P>0.191).