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

SUMMARYEuryhaline 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 (≥14 days) acclimation of killifish to seawater (SW) and 2ϫ 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 2ϫ 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.

Branchial ionocyte organization and ion-transport protein expression in juvenile alewives acclimated to freshwater or seawater

“…Juvenile alewife salinity acclimation in freshwater-acclimated individuals. This might be an inactive nonphosphorylated pool of NKCC1 that could be quickly activated for an acute response upon exposure to elevated salinities (Flemmer et al, 2010). Notably, the proportional increase in NKCC1 abundance upon seawater acclimation was much greater than that of NKA, further suggesting that NKA isoforms have a role in ion absorption.…”

Inhibition of Na+-K+-2Cl–Cotransporter isoform 1 Accelerates Temozolomidemediated Apoptosis in Glioblastoma Cancer Cells

“…The rapid upregulation of the two proteins likely results from de novo protein synthesis by using an existing pool of mRNA. Similarly, rapid increase in NKCC1 protein has been observed in the gills of Atlantic killifish when transferred from fresh to seawater [< 6 hours, [43]]. Therefore, the fast upregulation of transporter proteins and their regulatory kinases will allow cells to quickly acclimate to an altered osmotic environment.…”

Expression of aquaporin 3 in gills of the Atlantic killifish (Fundulus heteroclitus): Effects of seawater acclimation

“…Therefore, killifish are a valuable model for elucidating the mechanisms of acclimation to osmotic stress, and the effects of environmental toxicants on acclimation (Burnett et al, 2007; Marshall et al, 2005; Scott et al, 2006; Shaw et al, 2010; 2007b; 2008; Stanton et al, 2006). Most of the studies examining acclimation to osmotic stress in killifish have focused on the regulation of ion transport, such as transport of Cl - and Na + by the gill and opercular membrane (Flemmer et al, 2010; Marshall et al, 2005; Scott et al, 2008; Shaw et al, 2007a; 2008; Wood and Marshall 1994; Zadunaisky et al, 1995). These studies have demonstrated that in seawater, marine teleosts drink copious amounts of water, thus they must excrete the NaCl that is absorbed by the gastrointestinal tract to maintain plasma concentrations of Na + and Cl - (i.e., plasma osmolality).…”