Phosphorylation state of the Na+-K+-Cl- cotransporter (NKCC1) in the gills of Atlantic killifish (Fundulus heteroclitus) during acclimation to water of varying salinity.
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.
Flemmer
Andreas W
AW
Department of Cellular and Molecular Physiology, Yale University School of Medicine, PO Box 208026, New Haven, CT 06520, USA.
Monette
Michelle Y
MY
Djurisic
Maja
M
Dowd
Brian
B
Darman
Rachel
R
Gimenez
Ignacio
I
Forbush
Biff
B
D000064:Acclimatization; D000818:Animals; D015536:Down-Regulation; D023781:Fundulidae / Q000378:metabolism / Q000502:physiology*; D005880:Gills / Q000378:metabolism*; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics; D054712:Salinity; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
D012333:RNA, Messenger; D028021:Sodium-Potassium-Chloride Symporters
J Clin Invest. 1991 Oct;88(4):1422-31
1717515
Nature. 1991 Aug 15;352(6336):628-31
1714039
J Membr Biol. 1995 Feb;143(3):207-17
7769606
J Biol Chem. 2002 Oct 4;277(40):37551-8
12145305
J Biol Chem. 1992 Dec 15;267(35):25438-43
1334094
Am J Physiol. 1996 Feb;270(2 Pt 1):C437-48
8779905
Biochem J. 2008 Jan 15;409(2):321-31
18092945
J Biol Chem. 2003 Jul 18;278(29):26946-51
12732642
Comp Biochem Physiol B Biochem Mol Biol. 2001 Jul;129(4):821-9
11435136
J Exp Zool. 2002 Jul 1;293(2):106-18
12115907
J Exp Biol. 2005 Mar;208(Pt 6):1063-77
15767308
Acta Physiol (Oxf). 2006 May-Jun;187(1-2):103-13
16734747
J Exp Biol. 2008 Aug;211(Pt 16):2584-99
18689412
Comp Biochem Physiol A Mol Integr Physiol. 2007 Sep;148(1):29-43
17289411
Science. 1982 Jan 8;215(4529):164-6
7053566
Curr Opin Nephrol Hypertens. 2006 Sep;15(5):517-23
16914965
J Exp Biol. 2005 Jul;208(Pt 14):2719-29
16000541
Physiology (Bethesda). 2006 Oct;21:326-35
16990453
Am J Physiol Cell Physiol. 2004 Aug;287(2):C300-9
15044150
Am J Physiol. 1999 Dec;277(6):C1184-93
10600770
Annu Rev Physiol. 2005;67:445-69
15709965
Physiol Rev. 2005 Apr;85(2):423-93
15788703
J Exp Biol. 1999 Jun;202 (Pt 11):1535-44
10229699
Am J Physiol. 1988 Apr;254(4 Pt 1):C535-47
3354651
J Exp Zool A Comp Exp Biol. 2004 Dec 1;301(12):979-91
15562450
Biochem J. 2005 Oct 1;391(Pt 1):17-24
16083423
J Biol Chem. 2001 Sep 14;276(37):34359-62
11466303
J Biol Chem. 2002 Oct 4;277(40):37542-50
12145304
Acta Physiol Scand. 1985 Sep;125(1):55-66
2864785
Am J Physiol. 1992 Dec;263(6 Pt 1):G823-37
1476190
Comp Biochem Physiol A Mol Integr Physiol. 2007 Nov;148(3):479-97
17689996
J Exp Zool. 2000 Sep 1;287(4):263-74
10951386
Physiol Rev. 2005 Jan;85(1):97-177
15618479
Gen Comp Endocrinol. 1998 Aug;111(2):103-12
9679082
Comp Biochem Physiol A Mol Integr Physiol. 1998 Jan;119(1):97-106
11253824
J Biol Chem. 2003 Jul 25;278(30):27347-53
12740379
J Exp Biol. 1994 Nov;196:405-18
7529818
Gen Comp Endocrinol. 2001 Nov;124(2):134-43
11703079