| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CELLULAR |
1 School of Biological Sciences, University of Liverpool, Biosciences Building, Crown Street, Liverpool L69 7ZB, UK
2 Department of Animal Physiology, Humboldt Universität zu Berlin, Philippstrasse 13, Abderhaldenhaus, D-10115 Berlin, Germany
3 Centre of Excellence in Evolutionary Genetics and Physiology, Department of Biology, University of Turku, FI-20014 Turku, Finland
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
(Received 3 May 2006;
accepted after revision 4 June 2006;
first published online 8 June 2006)
Corresponding author M. Berenbrink: School of Biological Sciences, The University of Liverpool, Biosciences Building, Crown Street, Liverpool L69 7ZB, UK. Email: michaelb{at}liv.ac.uk
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Red blood cells (RBCs) of certain non-mammalian vertebrates that express high activities of either of the two transporters have become important model systems for studying the functional properties, regulation and pharmacology of these widely expressed ion transport systems (Gibson et al. 1998). Indeed, turkey and rainbow trout RBCs were among the first model systems in which the powerful effects of molecular O2 on the activity of NKCC and KCC, respectively, were described (Palfrey & Greengard, 1981; Borgese et al. 1991; Nielsen et al. 1992). In all species studied so far, including man, O2 has opposing effects on the two RBC transport systems, activating KCC and deactivating NKCC (Gibson et al. 2000). Because the effects of O2 are rapid, they appear to depend on constitutive signal transduction pathways, rather than on hypoxia-induced changes in gene expression (López-Barneo et al. 2001). However, despite continuous efforts, the nature of the O2 sensor(s) and the transduction pathway(s) modulating RBC ion transport are still unknown.
Studies on rainbow trout RBCs – which appear to lack the NKCC but express high activities of KCC and a ß-adrenergically activated Na+–H+ exchange system (NHE) – have suggested that haemoglobin (Hb) is a component of the O2 sensor transducing the effects of molecular O2 on NHE and KCC activity (Motais et al. 1987; Borgese et al. 1991; Nielsen et al. 1992). Subsequently, Hb has also been discussed as the O2 sensor modulating NKCC and KCC in mammalian and avian RBCs, where the O2 affinities for KCC activation or NKCC deactivation approximately match the O2 affinity of Hb inside RBCs (Speake et al. 1997; Muzyamba et al. 1999; Flatman, 2005). However, O2 affinity of KCC in rainbow trout RBCs is much lower than that of Hb, casting doubt on a direct role of bulk Hb oxygenation in modulating KCC in this species (Berenbrink et al. 2000). It is presently not known whether the occurrence of O2 sensors with Hb-like and non-Hb-like (low) O2 affinity is species-specific, or whether they may even occur simultaneously in RBCs of a single species.
Here we use crucian carp RBCs as a model that expresses significant levels of both KCC and NKCC, and dissect their O2 affinities. RBCs from hypoxia-tolerant carp species are especially suited for distinguishing between Hb-like and non-Hb-like (low) affinity O2 sensors, because of an unusually high Hb O2 affinity, which is coupled with approximately threefold lower normoxic resting arterial PO2 values as compared with rainbow trout, birds and mammals (PO2 for half-maximal blood O2 saturation and for normoxic arterial blood at rest in carp species is typically 0.4–0.7 and 3.2–4.0 kPa, respectively; Prosser, 1950; Burggren, 1982; Knudsen & Jensen, 1998). Our results suggest that crucian carp RBCs simultaneously express two different O2 sensors, one with Hb-like O2 affinity that governs NKCC, as found in mammalian and avian RBCs, and another with significantly lower O2 affinity that governs KCC, as found in rainbow trout RBCs. These experiments characterize a model system for the coordinated regulation of differentially O2-sensitive ion transport systems in a single cell type, and are relevant for understanding pathologically altered O2 sensitivities of RBC ion transporters in human sickle cell disease, and 
and ß thalassaemia (Olivieri et al. 1994; Gibson et al. 1998; Drew et al. 2004).
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Crucian carp (Carassius carassius) were obtained either from a pond in the area of Turku, Finland (mass 0.7–1.8 kg, total length 31–40 cm, n = 20), or from a pond near Ipswich, UK (20–123 g, 12.1–18.3 cm, n = 25). Rainbow trout (Oncorhynchus mykiss, 130–214 g, 24.5–28.0 cm, n = 4) were purchased from a commercial fish farm near Turku, Finland. Both species were kept indoors at 15°C in running, dechlorinated tap water for at least 1 week prior to experimentation at the fish holding facilities of Åbo Akademi University, Turku, Finland, or of the School of Biological Sciences, University of Liverpool, UK. Experiments were performed from July to early December to minimize seasonal variations in the magnitude of RBC ion-transport pathways (Berenbrink & Bridges, 1994). As no differences in K+ (Rb+) influx were apparent between the RBCs of the two crucian carp stocks, despite marked difference in their size, results were combined.
Chemicals and solutions
Inorganic salts, and dimethyl sulfoxide (DMSO), D-glucose, imidazole, ouabain and perchloric acid (PCA) were obtained from Merck, Darmstadt, Germany. Bumetanide, ethyl-m-aminobenzoate (MS 222), Hepes, methanesulphonic acid, N-methyl-D-glucamine (NMDG), sodium heparin and Tris were purchased from Sigma-Aldrich Chemical Company, while Triton X-100 was from Serva, Heidelberg, Germany. The radioactive tracer 86Rb+ (as RbCl) was obtained from NEN Life Science Products, Belgium, and the KCC inhibitor DIOA (dihydroindenyloxyalkanoic acid) was from Research Biochemicals, Natick, MA, USA. Stock solutions (10 mM) of DIOA and ouabain were prepared in ethanol and DMSO, respectively, and the chemicals were used at a final concentration of 0.1 mM. Bumetanide (10 mM) was prepared in ethanol and used at final concentrations of 1, 10 and 100 µM. The final volume of the respective solvents did not exceed 1% of the volume of RBC suspensions. Standard fish saline for RBCs of crucian carp and rainbow trout consisted of (mM): 125.5 NaCl, 3 KCl, 1.5 MgCl2, 1.5 CaCl2, 5 D-glucose and 20 Hepes, adjusted with NaOH to pH 7.97 at 15°C (Berenbrink et al. 2000; Völkel et al. 2001). The pH of standard saline was varied between 6.9 and 8.4 by adding NaOH or HCl. In Cl–-free salines, Cl– salts were replaced either by the respective nitrate salts or, alternatively, by the respective cation hydroxides. In the latter case, pH was adjusted using methanesulphonic acid, creating a Cl–-free saline with methanesulphonate as the principal anion. Na+-free saline was prepared by equimolar replacement of NaCl with NMDG. In this case pH was adjusted with HCl.
Blood sampling and preparation of RBCs
Fishes were normally killed by a sharp blow on the head and immediate exsanguination by caudal venipuncture using heparinized hypodermic syringes. Large crucian carp were immersed in an overdose of anaesthetic (1 g l–1 MS 222, neutralized with Tris salt) until all movement ceased, before exsanguination. Procedures were carried out in accordance with national ethical committee guidelines. RBCs were washed three times in 3–5 vols of ice-cold standard saline, each time removing the buffy coat. The resulting RBC suspensions were adjusted to half the original blood haematocrit value, oxygenated by contact with air and stored at 5°C for at least 16 h to allow for stabilization of cell volume and cellular ion content.
Experimental procedure
Immediately before experimentation, RBCs were washed in ice-cold standard saline. In the case of small crucian carp, RBCs from three or four animals were pooled to obtain a sufficiently large volume. For ion replacements, RBCs were washed three times with a 10-fold excess of Cl–- or Na+-free saline, allowing 5 min for ion equilibration at room temperature after each wash. RBCs were then resuspended at the original blood haematocrit value in the respective saline, and subjected to 45 min standard pre-equilibration at 15°C in shaking glass tonometers (Eschweiler, Kiel, Germany) with a water-vapour-saturated gas mixture of 5% air–95% N2, provided by mass flow controllers (Gf-3MP Cameron Instruments, Port Aransas, TX, USA) or a precision gas-mixing pump (Wösthoff KG, Bochum, Germany). Standard pre-equilibration at the resulting PO2 of 1 kPa is frequently used to deactivate O2-dependent KCC in fish RBCs with minimal effects on cellular ATP levels (Nielsen et al. 1992; Berenbrink et al. 1997, 2000; Völkel et al. 2001). After 45 min, the haematocrit value was determined by microcentrifugation (Micro-Compur M110, Compur Elektronik, München, Germany), and equilibration continued in humidified gases at PO2 values of 0, 1, 4, 21 or 100 kPa. After 10 min of experimental equilibration, RBC suspensions were diluted with 9 vols pre-equilibrated standard saline adjusted to the desired pH range, for determination of unidirectional K+ (86Rb+) fluxes, Hb O2 saturation and final pH.
Determination of K+ fluxes
Unidirectional K+ fluxes were determined in the presence of 0.1 mM ouabain using 86Rb+ as a tracer, substituting for K+, as previously described (Berenbrink et al. 2000). Briefly, for influx measurements, 11.1–18.5 kBq ml–1
86Rb+ was added to the diluted RBC suspensions (standard salines with or without ion replacements, 2–3% haematocrit) and after predefined time points 200 µl aliquots of the suspension were removed. RBCs were immediately washed three times by centrifugation and resuspension in ice-cold wash solution (100 mM MgCl2, 10 mM imidazole or Hepes, pH 7.97 at 15°C). After final centrifugation, the supernatant was removed and the RBC pellet lysed (0.5 ml 0.05 vol% Triton X-100) and deproteinized (0.5 ml 0.6 M PCA). Cellular 86Rb+ activity was determined by Cerenkov radiation. K+ (Rb+) influx was calculated by linear regression from the rate of increase in cellular 86Rb+ activity with time and the extracellular 86Rb+ activity per extracellular K+ concentration (3 mM). Influx is expressed in millimoles of K+ (Rb+) per hour and per litre of cells. RBC volume was determined from haematocrit measurements at the end of the pre-equilibration period. For efflux measurements, RBCs at high haematocrit (30–40%) were loaded with 86Rb+ for 
3.5 h at 20°C (37 kBq ml–1 suspension). After standard pre-equilibration, PO2 was changed to the experimental value and 10 min later RBCs were diluted 20-fold in saline pre-equilibrated with the same gas mixture. The accumulation of 86Rb+ activity in the extracellular medium (standard saline) was followed for 15 min by centrifugation of aliquots at predefined time points and processing the supernatant as described above. As in our previous study (Berenbrink et al. 2000), 86Rb+ release appeared linear with time and K+ (Rb+) efflux was determined from the initial 86Rb+ release rate and the initial cellular 86Rb+ activity per cellular K+ concentration. The latter was 102.2 ± 1.3 mmol per litre of RBCs (mean value ±
S.E.M., n
= 6 animals) in cells processed the same way as for K+ (Rb+) efflux determinations and measured by atomic absorption spectrometry (Perkin-Elmer 2380). Efflux is expressed in mmol K+ (Rb+) per hour and per litre of cells.
Hb O2 saturation and pH measurements
Hb O2 saturation was determined according to the method of Tucker (1967) in dilute RBC suspensions under the same experimental conditions as for K+ (Rb+) flux measurements. Experimental procedures and calculations were identical to those used in our previous study on rainbow trout RBCs (Berenbrink et al. 2000). The pH of final RBC suspensions used for Hb O2 binding studies and K+ (Rb+) flux measurements was checked using a thermostatted (14.9–15.1°C) capillary glass electrode with calomel reference (Radiometer BMS 3 Mk 2) and a pH meter (Radiometer PHM 72). The pH electrode assembly was calibrated at the experimental temperature with precision buffers (Radiometer). The final pH of RBC suspensions diluted with four different pH-adjusted salines was 6.942 ± 0.014, 7.387 ± 0.005, 7.931 ± 0.017 and 8.375 ± 0.036 (means ± S.E.M., n = 3). For readability, experiments at these pH values are referred to as pH 6.9, 7.4, 7.9 and 8.4 experiments. Oxygenation-induced pH changes of the extracellular medium amounted to maximally 0.07 pH units, as estimated by comparing the pH difference between O2- and N2-equilibrated RBCs in the four different salines.
Intracellular ion concentrations and net free energy for cotransport
Intracellular Na+, K+ and Cl– concentrations ([Na+]i, [K+]i and [Cl–]i) were measured in RBCs that had been pre-equilibrated for 45 min under standard conditions and subsequently exposed to a PO2 of 21 kPa for 10 min. Hence values refer to the time point where K+ (Rb+) flux determinations were started. RBC ion and water content were determined as described before (Berenbrink & Bridges, 1994). Briefly, RBCs were separated from the extracellular medium by rapid centrifugation in narrow 400 µl capacity Eppendorf tubes and quickly frozen in liquid nitrogen. Frozen tubes were cut with a razor blade 2 mm below the boundary between supernatant and pellet. The remaining pellet was weighed and deproteinized by addition of 200 µl 0.6 M PCA. After centrifugation (5 min, 10 000 g), ion concentrations in the supernatant were determined, Na+ and K+ by atomic absorption spectrometry (Perkin-Elmer 2380), and Cl– coulometrically using a chloride titrator (CMT 10, Radiometer, Copenhagen, Denmark). In parallel samples, pellets were preweighed and then dried to constant weight ( 40 h at 80°C) for determination of cellular water content. This allowed the intracellular ion concentrations to be expressed in millimoles per litre cell water for calculation of the net free energy in transmembrane ion gradients.
The combined net free energy change, 
G, for the transport of 1 mol Na+, 1 mol K+ and 2 mol Cl– into the cell was calculated according to Russell (2000):
|
| (1) |
|
| (2) |
G indicate that ion gradients favour a net outward direction of transport. Data analysis and representation
Values are expressed as means ± S.E.M. of n experiments on RBCs of separate animals, or on RBCs pooled from separate groups of animals. In contrast to classical model organisms, animals were not from inbred laboratory lines and K+ (Rb+) influx and efflux values were somewhat variable between individuals. A similar degree of variability in ion transport activity is also evident in RBCs from other species, including humans (Ellory et al. 1985). In yet other studies, interindividual variation may be obscured by the common presentation of mean values and error bars of multiple measurements in a single, representative, experiment.
Statistical differences between treatments were assessed using one-way or two-way analyses of variance, as appropriate, followed by Tukey's test for pairwise comparisons (SigmaStat version 2.03). Data deviating from a normal distribution were transformed before further analysis according to x' = log(x + 1) or x' = 1/(x + 1) (Sachs, 1988). Statistical significance was accepted at P < 0.05.
PO2 values for half-maximal K+ (Rb+) influx (P50) were calculated by non-linear curve fitting (SigmaPlot version 8) using equations for simple saturation curves of the form
|
| (3) |
|
| (4) |
The P50 for Hb O2 binding was calculated from non-linear curve fits according to a simple saturation curve
|
| (5) |
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
|
Replacing external Na+ by NMDG yielded somewhat reduced K+ (Rb+) influx values at low PO2 values, although the effect was statistically not significant (Fig. 1B). Independence from extracellular Na+ has frequently been used as supporting evidence for excluding NKCC as the mechanism for K+ tracer fluxes (e.g. Gillen et al. 1996; Muzyamba et al. 1999; Mercado et al. 2000). Importantly, however, this cannot be taken as evidence against the involvement of NKCC in Cl–-dependent K+ (Rb+) tracer fluxes because in the absence of external Na+, the NKCC can perform a partial reaction and operate in a K+ (Rb+) self-exchange mode in many systems, provided some internal Na+ is present (Lauf et al. 1987; Lytle et al. 1998).
In most cell types, the combined net free energy in the physiological transmembrane ion gradients greatly favours net efflux of cotransported ions via the KCC (Lauf et al. 1992). In contrast, the NKCC in RBCs is close to equilibrium and may be operating in a net inward or outward direction, depending on physiological plasma K+ concentrations (Duhm & Göbel, 1984). Unidirectional K+ (Rb+) efflux showed a similar O2 dependence as K+ (Rb+) influx, but was about 4–5 times higher over the whole PO2 range, predicting a net outward direction of ouabain-insensitive K+ transport pathways at low and high PO2 (Fig. 1C). Figure 2 shows that at PO2 21 kPa the net free energy of the measured ion gradients across the crucian carp red cell membrane was compatible with a net efflux via both NKCC and KCC.
|
In mammalian RBCs, NKCC activity is strongly stimulated by alkaline pH (Flatman, 1991), whereas KCC is stimulated by acidification (e.g. Speake et al. 1997). In contrast, KCC in fish RBCs is strongly activated by alkaline pH (Berenbrink et al. 2000; Völkel et al. 2001). K+ (Rb+) influxes in crucian carp RBCs at low and high PO2 fundamentally differed in their pH dependence (Fig. 3A). Between PO2 values from 0 to 4 kPa, pH did not significantly affect K+ (Rb+) influx. However, at PO2 values of 21 and 100 kPa, K+ (Rb+) influx was significantly reduced by acidification below pH 8.4 and reached close to baseline levels at pH 7.4 and 6.9. This was similar to the inhibition of KCC by acidification in rainbow trout RBCs measured under the same experimental conditions (Fig. 3B). In rainbow trout, K+ (Rb+) influx was minimal at PO2 values of 1 and 0 kPa, independent of pH.
|
|
To test the idea of two different K+ (Rb+) transport pathways, which are differentially modulated by O2, further experiments were carried out using bumetanide at pH 8.4, where K+ (Rb+) influx was maximally activated at both high and low PO2. At low concentrations, bumetanide specifically inhibits NKCC in various vertebrate and invertebrate tissues with a 50% inhibitory concentration (IC50) of about 0.1 µM (Russell, 2000). At higher concentrations bumetanide also inhibits KCC, albeit with a more than 1000-fold higher IC50 (
180 µM for KCC1 and 900 µM for KCC4; Mercado et al. 2000). Figure 5A shows that K+ (Rb+) influx at low PO2 was already significantly inhibited by more than 50% in the presence of 1 µM bumetanide. Increasing the concentration to 10 µM led to significant further reductions, and 100 µM essentially abolished K+ (Rb+) influx at low PO2. With IC50 values greater than 100 µM, any KCC isoform should be less than 50% inhibited at this bumetanide concentration. Therefore the near elimination of K+ (Rb+) influx by 100 µM bumetanide suggests that the flux at PO2 0–1 kPa is almost entirely due to NKCC. At an intermediate PO2 of 4 kPa, significant inhibition of K+ (Rb+) influx required 10 µM bumetanide. At 21 and 100 kPa PO2, only 100 µM bumetanide caused a significant reduction of K+ (Rb+) influx. Thus, at higher PO2, an increasing fraction of K+ (Rb+) influx was carried by a more bumetanide-resistant pathway such as KCC. However, the moderate, but consistent reductions of K+ (Rb+) influx by low bumetanide concentrations (1 and 10 µM) even at high PO2 values suggest that NKCC was still partially active at 21 and 100 kPa.
|
In the presence of 100 µM bumetanide, NKCC is expected to be completely inhibited, and this allowed the determination of the P50 value for the O2-activated, bumetanide-resistant flux mechanism (KCC). In addition, calculation of the 1 µM-bumetanide-sensitive flux in Fig. 5B allowed the determination of the P50 value for the activation of NKCC by low PO2. Figure 4 shows that the P50 for the relative bumetanide-resistant K+ (Rb+) influx pathway (KCC) was significantly higher than the P50 values for NKCC and Hb. In contrast, P50 values for Hb and NKCC were not significantly different.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Identification of two separate O2-dependent K+ (Rb+) transport pathways
We propose that the first K+ (Rb+) flux pathway, whose activity positively correlated with the fraction of deoxyHb in crucian carp RBCs, is carried by NKCC. At low PO2, where this mechanism was maximally activated, K+ (Rb+) influx was virtually abolished by replacement of extracellular Cl– with nitrate or methanesulphonate. The mechanism was highly sensitive to the specific NKCC inhibitor bumetanide, with 1 µM of the drug causing more than 50% inhibition, and 100 µM bumetanide almost abolishing the flux. The mechanism was progressively activated by decreasing PO2, similar to NKCC in turkey, chicken, human and ferret RBCs (Muzyamba et al. 1999; Drew et al. 2004; Flatman, 2005).
The second K+ (Rb+) influx mechanism was maximally activated by high PO2 and showed the characteristics of KCC. It was abolished by replacement of Cl– with nitrate or methanesulphonate, or by 100 µM DIOA. In contrast to NKCC, it showed low bumetanide sensitivity, with 50% inhibition requiring 100 µM bumetanide or more, like the KCC in other systems (Ellory et al. 1985; Mercado et al. 2000). Similar to KCC in normal human, horse and rainbow trout RBCs, the mechanism was virtually silent at a PO2 of 1 kPa or lower (Nielsen et al. 1992; Speake et al. 1997; Berenbrink et al. 1997; Gibson et al. 1998). Contrary to KCC in mammalian RBCs (e.g. Speake et al. 1997), the K+ transport mechanism at high PO2 was also strongly activated by alkaline extracellular pH, similar to KCC in rainbow trout RBCs (Nielsen et al. 1992; Berenbrink et al. 2000).
The partial inhibition of K+ (Rb+) fluxes at low PO2 by the KCC inhibitor DIOA was unexpected, because the high bumetanide sensitivity of the flux strongly suggests that it is carried by NKCC. However, partial inhibition of the NKCC by 100 µM DIOA has previously been noted in human RBCs (18 ± 6% inhibition, mean value ± S.E.M, n = 3; Culliford et al. 2003), calling for a cautionary interpretation of inhibitory effects of DIOA (Berenbrink et al. 2000).
Ultimate proof of both KCC and NKCC requires demonstration that concentration changes of each of the respective cotransported ions influence the transport of the other ions in the predicted way. This has been reported only in a few cases, partly because of the high anion exchange activity and anion conductance of RBCs, which presents a difficulty in varying Cl– independently on both sides of the membrane (Lauf, 1985; Russell, 2000). In the absence of such evidence, we follow previous studies and use Cl– dependence and low bumetanide sensitivity provisionally to ascribe the O2-activated K+ (Rb+) flux pathway in crucian carp RBCs to a KCC mechanism.
Differential O2 sensitivity of KCC and NKCC
At pH 8.4, where both NKCC and KCC were maximally activated, titration of K+ (Rb+) influxes with bumetanide allowed dissection of the O2 affinities of these two transport systems. P50 for NKCC under these conditions was 1.4 kPa. At pH values of 7.9 and 6.9, P50 of total K+ (Rb+) flux was 2.5 and 2.4 kPa, respectively. Figure 3A and comparable data on rainbow trout RBCs (Fig. 3B) suggest that KCC was largely deactivated at these PO2 and pH values, and hence the majority of the flux was likely to be due to NKCC. These P50 values were not significantly different from the P50 of crucian carp Hb under the same conditions, although they are considerably lower than P50 values for NKCC in turkey and ferret RBCs (5.5 and 3.2 kPa, respectively; Muzyamba et al. 1999; Flatman, 2005).
Few studies have measured the O2 affinity of Hb and of O2-dependent ion transport under strictly comparable experimental conditions. However, the decreasing P50 values of the NKCC from bird to mammalian and fish RBCs are closely matched by the respective P50 values for blood O2 binding in these groups (Prosser, 1950). These results are thus compatible with a role for Hb as the O2 sensor affecting NKCC in crucian carp RBCs, as previously suggested for several membrane ion transporters in other RBC systems (Motais et al. 1987; Gibson et al. 2000). Discussing NKCC in ferret RBCs, Flatman (2005) suggested that competitive binding of deoxyHb to band 3 in the RBC membrane may release a kinase into the cytoplasm, which activates neighbouring NKCC proteins via phosphorylation of the transporter or of another protein, which could then activate NKCC. Indeed, protein kinase inhibitors suppress activation of NKCC by deoxygenation in both turkey and ferret RBCs (Muzyamba et al. 1999; Flatman, 2005). Alternatively, binding of deoxyHb to band 3, which may be modulated by band 3 phosphorylation, could directly activate NKCC via the cytoskeleton or by direct protein–protein interactions (Flatman, 2005).
In contrast, activation of KCC by elevated PO2 in crucian carp RBCs was associated with a distinctly higher P50 (8.5 kPa at pH 8.4) than displayed by Hb O2 binding or the NKCC. Therefore in this case the mechanism of O2 sensing does not involve changes in bulk Hb O2 saturation, as previously also shown for the KCC in rainbow trout RBCs (Berenbrink et al. 2000).
In human sickle cell disease, and and ß thalassaemia, the RBC KCC also appears to be at least partially uncoupled from its normal, Hb-like O2 sensor. These human haemoglobinopathies are all associated with increased production of reactive O2 species (ROS), and an altered O2 dependency of KCC in their RBCs (Repka & Hebbel, 1991; Olivieri et al. 1994; Gibson et al. 1998). KCC activity in normal human RBCs is low but can be activated by combined cell swelling and acidification (Gibson et al. 1998). Decreasing PO2 inhibits the human KCC with a P50 of 4.8 kPa, a value that may be similar to the P50 of Hb under the acidic conditions used to stimulate KCC in that study (pH 7.0; Gibson et al. 1998). However, KCC in RBCs from homozygous sickle cell patients fails to become inactivated at low PO2, resulting in a biphasic O2 dependence (Gibson et al. 1998). Conceivably, an increased rate of ROS production in human sickle cells (Repka & Hebbel, 1991) bypasses or overrides an O2 sensor with Hb-like affinity that regulates KCC in RBCs of healthy subjects. It is tempting to speculate that this prevents KCC deactivation at low PO2 just as experimentally increased ROS levels prevent deactivation of KCC in rainbow trout RBCs at low PO2(Bogdanova & Nikinmaa, 2001). The mechanism behind abnormally elevated KCC activity in human sickle cells at low PO2 is of considerable clinical importance, since it may contribute to RBC dehydration and thereby increased propensity for sickling (Lew & Bookchin, 2005).
In summary, the present study on crucian carp RBCs provides the first evidence for more than one constitutively expressed O2 sensor in a single cell type. We confirm the presence of an O2-dependent KCC in fish RBCs with significantly lower O2 affinity than Hb. This has allowed its regulating O2 sensor to be distinguished from another, high affinity O2 sensor, which governs the activity of NKCC, described here for the first time in fish RBCs. Similar to NKCC in mammalian and avian RBCs, it is activated by low PO2 and shows a similar O2 affinity to that of Hb. These findings are relevant for the understanding of cellular O2 sensing in general and in particular for the altered O2 dependence of RBC ion transport in certain haemoglobinopathies.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Berenbrink M & Bridges CR (1994). Active Na+-, Cl–-, and HCO3–-dependent acid extrusion in Atlantic cod red blood cells in winter activated by hypercapnia. J Exp Biol 192, 239–252.[Abstract]
Berenbrink
M, Koldkjær
P, Kepp
O
&
Cossins
AR (2005). Evolution of oxygen secretion in fishes and the emergence of a complex physiological system. Science
307, 1752–1757.
Berenbrink
M, Völkel
S, Heisler
N
&
Nikinmaa
M (2000). O2-dependent K+ fluxes in trout red blood cells: the nature of O2 sensing revealed by the O2 affinity, cooperativity and pH dependence of transport. J Physiol
526, 69–80.
Berenbrink M, Weaver YR & Cossins AR (1997). Defining the volume dependence of multiple K+ flux pathways in trout red blood cells. Am J Physiol 272, C1099–C1111.[Medline]
Bogdanova
AY
&
Nikinmaa
M (2001). Reactive oxygen species regulate oxygen-sensitive potassium flux in rainbow trout erythrocytes. J Gen Physiol
117, 181–190.
Borgese F, Motais R & Garcia-Romeu F (1991). Regulation of Cl–-dependent K+ transport by oxy-deoxyhemoglobin transitions in trout red cells. Biochim Biophys Acta 1066, 252–256.[Medline]
Burggren W (1982). ‘Air gulping’ improves blood oxygen transport during aquatic hypoxia in the goldfish Carassius auratus. Physiol Zool 55, 327–334.
Culliford SJ, Ellory JC, Lang H-J, Englert H, Staines HM & Wilkens RJ (2003). Specificity of classical and putative Cl– transport inhibitors of membrane transport pathways in human erythrocytes. Cell Physiol Biochem 13, 181–188.[CrossRef][Medline]
Drew C, Ball V, Robinson H, Ellory JC & Gibson JS (2004). Oxygen sensitivity of red cell membrane transporters revisited. Bioelectrochemistry 62, 153–158.[CrossRef][Medline]
Duhm J & Göbel BO (1984). Role of the furosemide-sensitive Na+/K+ transport system in determining the steady-state Na+ and K+ content and volume of human erythrocytes in vitro and in vivo. J Membr Biol 77, 243–254.[CrossRef][Medline]
Ellory JC, Hall AC & Stewart GW (1985). Volume-sensitive cation fluxes in mammalian red cells. Mol Physiol 8, 235–246.
Flatman
PW (1991). The effects of metabolism on Na+–K+–Cl– co-transport in ferret red cells. J Physiol
437, 495–510.
Flatman
PW (2005). Activation of ferret erythrocyte Na+–K+–2Cl– cotransport by deoxygenation. J Physiol
563, 421–431.
Fuchs
DA
&
Albers
C (1988). Effect of adrenaline and blood gas conditions on red cell volume and intra-erythrocytic electrolytes in the carp, Cyprinus carpio. J Exp Biol
137, 457–477.
Garay RP, Nazaret C, Hannaert PA & Cragoe EJ Jr (1988). Demonstration of a [K+, Cl–]-cotransport system in human red cells by its sensitivity to [(dihydroindenyl)oxy]alkanoic acids: Regulation of cell swelling and distinction from the bumetanide-sensitive [Na+, K+, Cl–]-cotransport system. Mol Pharmacol 33, 696–701.[Abstract]
Gibson JS, Cossins AR & Ellory JC (2000). Oxygen-sensitive membrane transporters in vertebrate red cells. J Exp Biol 203, 1395–1407.[Abstract]
Gibson
JS, Speake
PF
&
Ellory
JC (1998). Differential oxygen sensitivity of the K+–Cl– cotransporter in normal and sickle human red blood cells. J Physiol
511, 225–234.
Gillen
CM, Brill
S, Payne
JA
&
Forbush
B
III (1996). Molecular cloning and functional expression of the K+–Cl– cotransporter from rabbit, rat, and human. J Biol Chem
271, 16237–16244.
Hladky SB & Rink TJ (1977). pH equilibrium across the red cell membrane. In Membrane Transport in Red Cells, ed. Ellory JC & Lew VL, pp. 115–135. Academic Press, London.
Hoffmann
EK
&
Simonsen
LO (1989). Membrane mechanisms in volume and pH regulation in vertebrate cells. Physiol Rev
69, 315–382.
Jensen FB & Brahm J (1995). Kinetics of chloride transport across fish red blood cell membranes. J Exp Biol 198, 2237–2244.[Medline]
Knudsen PK & Jensen FB (1998). Effects of exhausting exercise and catecholamines on K+ balance, acid–base status and blood respiratory properties in carp. Comp Biochem Physiol A 119, 301–307.[CrossRef][Medline]
Lauf PK (1985). K+:Cl– cotransport: Sulfhydryls, divalent cations, and the mechanism of volume activation in a red cell. J Membr Biol 88, 1–13.[CrossRef][Medline]
Lauf PK, Bauer J, Adragna NC, Fujise H, Zade-Oppen AMM, Ryu KH & Delpire E (1992). Erythrocyte K+–Cl– cotransport: properties and regulation. Am J Physiol 263, C917–C932.[Medline]
Lauf PK, McManus TJ, Haas M, Forbush B, Duhm J, Flatman PW, Saier MH & Russell JM (1987). Physiology and biophysics of chloride and cation cotransport across cell-membranes. Fed Proc 46, 2377–2394.[Medline]
Lew
VL
&
Bookchin
RM (2005). Ion transport pathology in the mechanism of sickle cell dehydration. Physiol Rev
85, 179–200.
López-Barneo J, Pardal R & Ortega-Sáenz P (2001). Cellular mechanisms of oxygen sensing. Annu Rev Physiol 63, 259–287.[CrossRef][Medline]
Lytle C, McManus TJ & Haas M (1998). A model of Na+–K+–2Cl– cotransport based on ordered ion binding and glide symmetry. Am J Physiol 274, C299–C309.[Medline]
Mercado
A, Song
L, Vázquez
N, Mound
DB
&
Gamba
G (2000). Functional comparison of the K+–Cl– cotransporters KCC1 and KCC4. J Biol Chem
275, 30326–30334.
Motais
R, Garcia-Romeu
F
&
Borgese
F (1987). The control of Na+/H+ exchange by molecular oxygen in trout erythrocytes. J Gen Physiol
90, 197–207.
Muzyamba
MC, Cossins
AR
&
Gibson
JS (1999). Regulation of Na+–K+–2Cl– cotransport in turkey red cells: the role of oxygen tension and protein phosphorylation. J Physiol
517, 421–429.
Nielsen OB, Lykkeboe G & Cossins AR (1992). Oxygenation-activated K+ fluxes in trout red blood cells. Am J Physiol 263, C1057–C1064.[Medline]
Olivieri
O, De Franceschi
L, Capellini
MD, Girelli
D, Corrocher
R
&
Brugnara
C (1994). Oxidative damage and erythrocyte membrane transport abnormalities in thalassemias. Blood
84, 315–320.
Palfrey HC & Greengard P (1981). Hormone-sensitive ion transport systems in erythrocytes as models for epithelial ion pathways. Ann N Y Acad Sci 372, 291–308.[Medline]
Prosser CL (1950). Respiratory functions of body fluids. In Comparative Animal Physiology, ed. Prosser CL, pp. 290–340. Saunders, Philadelphia.
Repka
T
&
Hebbel
RP (1991). Hydroxyl radical formation by sickle erythrocyte membranes: role of pathologic iron deposits and cytoplasmic reducing agents. Blood
78, 2753–2758.
Russell
JM (2000). Sodium–potassium–chloride cotransport. Physiol Rev
80, 211–276.
Sachs L (1988). Statistische Methoden: Planung und Auswertung, 6th edn. Springer, Berlin.
Speake PF, Roberts CA & Gibson JS (1997). Effect of changes in respiratory blood parameters on equine red blood cell K+–Cl– cotransporter. Am J Physiol 273, 1811–1818.
Tucker
VA (1967). Method for oxygen content and dissociation curves on microliter blood samples. J Appl Physiol
23, 410–414.
Van den Thillart G & Van Waarde A (1990). pH changes in fish during environmental anoxia and recovery: the advantages of the ethanol pathway. In Physiological Strategies for Gas Exchange and Metabolism, ed. Woakes AJ, Grieshaber MK & Bridges CR, pp. 173–190. Cambridge University Press, Cambridge, UK.
Völkel S, Berenbrink M, Heisler N & Nikinmaa M (2001). Effects of sulfide on K+ flux pathways in red blood cells of crucian carp and rainbow trout. Fish Physiol Biochem 24, 213–223.[CrossRef]
|
Acknowledgements |
|---|
This article has been cited by other articles:
|
|
 |
|
  H. Chu, A. Breite, P. Ciraolo, R. S. Franco, and P. S. Low Characterization of the deoxyhemoglobin binding site on human erythrocyte band 3: implications for O2 regulation of erythrocyte properties Blood, January 15, 2008; 111(2): 932 - 938. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Kristensen, P. Koldkjaer, M. Berenbrink, and T. Wang Oxygen-sensitive regulatory volume increase and Na transport in red blood cells from the cane toad, Bufo marinus J. Exp. Biol., July 1, 2007; 210(13): 2290 - 2299. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||
, 0.1 mM dihydroindenyloxyalkanoic acid (DIOA) (n
= 4); 
and 
, medium chloride was replaced by nitrate and methanesulphonate, respectively (both n
= 3). B, 
, control; 
, sodium salts replaced by NMDG (both n
= 3). C, n
= 3. All values are means ±
S.E.M.
*Significantly different from corresponding control value at the same PO2 (A and B); 
significantly different from the corresponding control value and DIOA value at the same PO2 (A only). Note the logarithmic scale for PO2.
0.001). Thus, the effect of PO2 on K+ (Rb+) influx (n
= 3–10) depended on pH. aSignificantly different from values at 0 kPa at the same pH; bsignificantly different from values at pH 8.4 at the same PO2. B, comparable data on rainbow trout RBCs (n
= 3–4), partly taken from 
Significantly different from the value at PO2 0 kPa at the same bumetanide concentration. B, 1 µM bumetanide-sensitive K+ (Rb+) influx, calculated as the difference between fluxes in the absence and presence of 1 µM bumetanide. Mean values not labelled by the same letter differed significantly from each other. Note the logarithmic scale for PO2.