Gill Na+/K+ ATPase Capacity
0,00 0,25 0,50 0,75 1,00 1,25 1,50 1,75 2,00 2,25 2,50
mRNA Expression Protein Level ATPase Activity
*
*
*
*
* * *
* *
*
control 8 hours 24 hours 2 days 4 days 7 days 2 weeks 6 weeks
Na+/K+ ATPase levels relative to control values [AU] Na+/K+ ATPase regulati-
on follows a similar pat- tern on different regula- tion levels.
Higher functional capaci- ties are established by higher protein and mRNA levels.
Even though whole animal energy consumption is not affected by acute hypercapnic incubation, large re- arrangements in all investigated gill transporters at the molecular level could be shown.
Since the protein levels of Na+/H+ exchanger and Na+/K+ ATPase follow their respective mRNA patterns, similar increments of Na+/HCO3- cotransporter may be predicted according to the observed higher mRNA levels.
Elevated Na+/K+ ATPase capacities might influence the energy budget of the euythermal species.
Conclusion & Prospects
Adaptation of ion regulatory capacities in fish gills under hypercapnic acidosis
Katrin Deigweiher, Hans-O. Pörtner, Magnus Lucassen
Marine Animal Physiology, Alfred Wegener Institute for Polar and Marine Research, Bremerhaven, Germany.
Correspondence: katrin.deigweiher@awi.de
Literature
Evans, D.H., Piermarini, P.M. & Choe, K.P. (2005). "The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste." Physiological Reviews 85(1): 97-177.
Ishimatsu, A., Hayashi, M. & Lee, K.S. (2005). "Physiological effects on fishes in a high-CO2- world." Journal of Geophysical Research 110: C09S09
Pörtner, H.O., Langenbuch, M. & Reipschläger, A. (2004). "Biological impact of elevated CO2 concentrations: lessons from animal physiology and earth history?" Journal of Oceanography 60: 705-18.
Introduction
The currently rising CO2 levels in the atmos- phere and in marine surface waters combine with trends of increasing temperatures.
Simultaneous shifts in those factors will likely enhance sensitivity especially in stenothermal animals (reviewed in Pörtner et al. 2004).
The preservation of ion balance and the regu- lation of pH despite environmental fluctuati- ons play a special role in this context, and sup- port the maintenance of vital cellular functions.
Therefore, it comprises a significant fraction of the energy budget.
Here, we investigated the time course of accli- mation to hypercapnia (10,000 ppm) in the benthic eelpout Zoarces viviparus at optimum temperature.
Na+ Na+
H+
H+
H+
2K+ 2K+
3Na+ 3Na+
1 Na+
HCO3- 3 HCO3-
+ H2O CO2
HCO3- Cl- Cl-
HCO3-
2 Cl- 2 Cl-
Na+
Na+
K+ K+
N B C NAK V-
ATPase
NHE CA
AE
Water Intracellular Extracellular
CO2
N K C C Na+
Cl--ChannelCl- Cl-
Na+-Channe
l H+
NAK Na+/K+ ATPase NBCNa+/HCO3- cotransporter AECl-/HCO3- exchanger NHE Na+/H+ exchanger
NKCC Na+/K+/2Cl- cotransporter
The contribution of different trans- porters to ion and pH regulation in the gills were studied on functional, translational and transcriptional levels.
Acute exposure to Hypercapnia
Ion Transporter mRNA Expression
Overall, the expression of the „passive“ transporters is reduced during the early phase of acclimation, and is restored at the end.
The expression of the active Na+ pump remains high during the early phase and doubles until the end of acclimation.
During early phase the Na+/HCO3- cotransporter behaves like the other passive transporters, but expression is about threefold at the end of acclimation.
Na+/K+ ATPase Na+/HCO3- cotransporter Cl-/HCO3- exchanger Na+/H+ exchanger Isoform A Na+/H+ exchanger Isoform B
0 1,0 2,0 3,0
control
8 hours
24 hours
2 days
4 days
7 days
2 weeks
6 weeks
mRNA level relative to control value [AU]
*
*
*
*
**
*
Ion Transporter Protein Levels
# ) reference sample
Protein level relative to control value [AU]
control 8 hours 24 hours 2 days 4 days 7 days
2 weeks
6 weeks
1 2#34 5 6
1 2 3 # 4 5 6 7 sample no.
sample no.
*
*
*
*
0 0,5 1,0 1,5
control 8 hours 24 hours 2 days 4 days 7 days
2 weeks
6 weeks
0 0,5 1,0 1,5
Protein level relative to control value [AU]
12 3 # 4 5 6 7 sample no.
1 2 3 # 4 5 6 sample no.
Na+/K+ ATPase
Na+/H+ exchanger protein level shows a slight decrease within the first 2 days of hy- percapnia incubation, followed by recovery after 4 days and stabilization at control level until the end of the 6 weeks trial.
The protein levels corres- pond to the respecitve tran- script levels.
Na+/K+ ATPase protein level increases significantly after 2 days of hypercapnia incubation and stabilizes to 135% of control level at the end of the 6 weeks time course.
This pattern is paralleled by increased transcript and activity levels.
# ) reference sample
Na+/H+ exchanger
Zoarces viviparus shows a high capacity for pH and ion regulation, comparable to other studies on fish (reviewed in Ishimatsu et al. 2005).
Whole animal oxygen consumption remains con- stant during and after acute exposure to 10,000 ppm hypercapnic incubation and recovery (time course 3.5 - 4.0 - 3.0 days).
0,0 0,5 1,0 1,5 2,0 2,5
1 2 3 4 5 6 7 8 9 10 6,8
7 7,2 7,4 7,6 7,8 8 8,2 8,4
Time course [days]
Oxygen consumption [μmol O2 g-1 h-1] Seawater pH
Whole Animal Regulation Capacity
Long term exposure to hypercapnia
Individuals of Zoarces viviparus were kept at optimum tempe- rature (10°C) before and during exposure to 10,000 ppm hy- percapnic incubation (corresponding pH of 6,9).
Groups of 8 - 10 fishes were sampled for each point of the time course.
Gill tissue was dissected and frozen immediatly in liquid ni- trogen.
RNA was isolated and used for mRNA quantification of single ion transporters by Real-time PCR.
Tissue homogenates were prepared to determine Na+/K+ ATPase activity as well as Na+/H+ exchanger and Na+/K+ ATPase protein quantities by Western blotting.
Experimental Procedure
redrawn after Evans et al. 2005
