|
| |
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online June 15, 2007
Journal of Experimental Biology 210, 2290-2299 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.002824
Oxygen-sensitive regulatory volume increase and Na transport in red blood cells from the cane toad, Bufo marinus
1 Department of Zoophysiology, Aarhus University, Denmark
2 School of Biological Sciences, The Biosciences Building, Crown Street,
Liverpool L69 7ZB, UK
* Author for correspondence (e-mail: piak{at}liv.ac.uk)
Accepted 24 April 2007
 |
Summary |
|---|
 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
5.5 kPa. This activation was completely abolished by the Na/H
exchanger (NHE) inhibitor EIPA (10–4 mol l-1).
Hyperosmotic shrinkage is particularly interesting in B. marinus as
it withstands considerable elevation in extracellular osmolarity following
dehydration. Parallel studies showed that deoxygenated B. marinus
RBCs had a much faster regulatory volume increase (RVI) response than
air-equilibrated RBCs, reflecting the difference in magnitude of Na influxes
at the two PO2 values. The extent of RVI
(60%) after 90 min, however, was similar under the two conditions,
reflecting a more prolonged elevation of the shrinkage-induced Na influx in
air-equilibrated RBCs. There were no significant differences in the ability to
perform RVI between whole blood cells at a PCO2
of 1 and 3 kPa or washed RBCs, and 10–4 mol l-1
amiloride reduced the RVI under all conditions, whereas 10–5
mol l-1 bumetanide had no effect. Isoproterenol
(10–5 mol l-1) induced a significant and prolonged
increase in an EIPA-sensitive and bumetanide-insensitive Na influx at low
PO2 under iso-osmotic conditions, whilst there
was no stimulation by isoproterenol for up to 45 min in air-equilibrated RBCs.
The prolonged ß-adrenergic activation of the Na influx at low
PO2 is distinctly different from the rapid and
transient stimulation in teleost RBCs, suggesting significant differences in
the signal transduction pathways leading to transporter activation between
vertebrate groups.
Key words: Bufo marinus, oxygen-dependent ion transport, erythrocyte, NHE
|
Introduction |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
; Gibson et al.,
2000). The regulatory volume increase (RVI) in RBCs from mammals,
most teleosts and amphibians is mediated by increased NHE activity, resulting
in a net gain of inorganic osmolytes followed by osmotically obliged water
(Cala, 1980;
Parker et al., 1991;
Orlov et al., 1994;
Romero et al., 1996;
Gusev et al., 1997;
Weaver et al., 1999) whereas
bird RBCs activate the NKCC (Kregenow,
1981; Muzyamba et al.,
1999). In addition to shrinkage, the activity of the NHE and NKCC
and other RBC ion transporters is modulated by oxygen, catecholamines or
acidification [NHE (Motais et al.,
1987; Salama and Nikinmaa,
1988; Nielsen,
1997; Virkki et al.,
1998) and NKCC (Palfrey and
Greengard, 1981; Muzyamba et
al., 1999; Flatman,
2005; Berenbrink et al.,
2006)]. Thus, in various vertebrates, the NHE, NKCC and/or RVI
response are inhibited in air-equilibrated cells compared with cells at low
oxygen partial pressure (PO2)
(Gibson et al., 2000;
Brauner et al., 2002;
Jensen et al., 2002;
Drew et al., 2004).
Plasma osmolality increases naturally in animals during dehydration,
physical exercise and upon movement from freshwater to seawater
(Shoemaker, 1964;
Maxime et al., 1991;
Madsen et al., 1996;
McKenna et al., 1997;
Hyndman et al., 2003;
Peterson and Greenshields,
2001). Because of their water-permeable skin, amphibians are prone
to dehydration, but the dependence on water differs among species. Terrestrial
anurans, such as Bufo, tend to be rather tolerant to dehydration
(Thorson, 1955), and the cane
toad, Bufo marinus, tolerates severe dehydration with 30% loss of
body mass (Shoemaker, 1964;
Shoemaker, 1965). However,
little is known about the RVI response from terrestrial amphibians.
Furthermore, the cardiac shunts of the amphibian circulatory system cause
desaturation of arterial blood (Johansen
and Ditadi, 1966; Wang et al.,
1998) and it is of interest, therefore, to examine how RBC volume
regulation is affected by oxygen in these animals.
Catecholamines are potent activators of the NKCC and NHE in many vertebrate
RBCs (Palfrey et al., 1981;
Motais et al., 1987;
Nikinmaa, 1992;
Weaver et al., 1999;
Pedersen and Cala, 2004;
Berenbrink et al., 2005) with
maximal stimulation at low PO2 levels
(Motais et al., 1987;
Salama and Nikinmaa, 1988).
The influence of adrenergic stimulation on isotonic volume responses and ion
fluxes has been examined in some amphibian RBCs, but responses were only
significant in the presence of phosphodiesterase inhibitors; thus, the
physiological significance of the responses remains unknown
(Rudolph and Greengard, 1980;
Tufts et al., 1987a;
Tufts et al., 1987b;
Kaloyianni et al., 1997).
The present work examines the PO2 dependency of the Na transport mechanism induced by hyperosmotic shrinkage or ß-adrenergic stimulation in washed B. marinus RBCs using ouabain-insensitive unidirectional 22Na influx. In parallel experiments, the influence of oxygenation on the ability to restore cell volume after hyperosmotic shrinkage is studied in both whole blood cells and washed RBCs. The Na influx and RVI mechanisms are characterized pharmacologically.
|
Materials and methods |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Blood sampling
Animals were anaesthetized by immersion in a solution (1 g l-1)
of benzocaine (ethyl p-amino benzoate). When all reflexes had
disappeared, an incision was made in the hind limb and a blood sample
(3–4 ml) was obtained immediately after inserting a heparinised cannula
(PE-50) into the femoral artery. The cannula was removed and the incision was
closed using sutures, whereupon the animal was placed under running tapwater
until it restored spontaneous ventilation. Andersen and Wang reported no
change in plasma lactate concentration immediately after similar surgical
procedure in B. marinus (Andersen
and Wang, 2002) and this sampling procedure should therefore not
affect whole blood in vitro studies.
Unidirectional Na influx studies on washed RBCs
A sub-sample of the blood was centrifuged at 5°C for 3 min at 1700
g (Sigma-3MK, Osterode, Germany) to remove plasma and buffy
coat. The cells were then washed three times in 10 volumes of ice-cold
isotonic saline and kept oxygenated by contact with air at 5°C overnight
for use in the experiments on washed blood cells. The standard isotonic saline
contained: 105 mmol l-1 NaCl, 6 mmol l-1 KCl, 1 mmol
l-1 MgSO4, 5 mmol l-1
CaCl2.2H2O, 5 mmol l-1 D-glucose
and 10 mmol l-1 Hepes. It was matched to the plasma osmolality of
B. marinus of 240 mOsm kg-1 and adjusted to pH 7.83 at
25°C (Andersen and Wang,
2003).
After overnight storage at 5°C, the RBCs were washed twice in the
isotonic saline and adjusted to a haematocrit (Hct) of 10–20%. For each
experiment, 800 µl of RBC suspension was equilibrated with humidified air
in a rotating Eschweiler tonometer (obtained from the Dept of Chemistry,
Aarhus University, Denmark) at 25°C for 45 min before ouabain was added to
a final concentration of 10–4 mol l-1. Hct was
determined in duplicate and, after an additional 10 min (time zero in the
experiment), aliquots of RBC suspension were diluted 10 times by transfer to
test tubes containing air-equilibrated isotonic saline, 22Na
(16.7 kBq ml-1), ouabain (10–4 mol
l-1) and other transport modifiers (specified below). When studying
effects of a change in PO2, the tonometer and
test tubes were gassed with the experimental gas mixture for 10 min before
transfer of RBCs. The blood cells were subjected to
PO2 values ranging from 0 (pure N2)
to 20.5 kPa (air) under isotonic conditions or a calculated 25% shrinkage with
or without 10–4 mol l-1 EIPA
[5-(N-ethyl-N-isopropyl)amiloride]. In shrunken RBCs
incubated under nitrogen, a dose–response curve for EIPA was constructed
using final concentrations between 10–7 and
10–5 mol l-1. The effect of 10–5
mol l-1 isoproterenol with or without 10–4 mol
l-1 EIPA (to inhibit the NHE) or 10–5 mol
l-1 bumetanide (to inhibit the NKCC) was studied in cells incubated
in isotonic saline under humidified nitrogen. Finally, the effect of
extracellular acidification to pH 7.28 by addition of appropriate volumes of a
pH 7.2 saline and of 10–5 mol l-1 isoproterenol
was studied in cells suspended in isotonic saline under air.
Triplicate samples (200 µl) were taken from the test tubes and
centrifuged at 6700 g (Sigma-113) for 10 s before removing the
supernatant by aspiration. The RBCs were immediately washed three times in
ice-cold isotonic MgCl2 solution (84 mmol l-1
MgCl2 and 20 mmol l-1 Hepes; adjusted to pH 7.83 at
25°C) before addition of 500 µl of 0.05% Triton X-100 solution and 500
µl of 5% trichloroacetic acid (TCA) to the pellet to lyse and deproteinate
the cells. At the end of each experiment, a sample (20 µl) was treated with
Triton X-100 and TCA to count the total 22Na activity, which was
assumed to correspond to the extracellular 22Na activity at the
beginning of the uptake experiment. All samples were centrifuged for 2 min
(6700 g), and 800 µl of supernatant was transferred to 5 ml
plastic vials or insert vials. Radioactivity was measured directly using a
gamma-counter (auto-gamma 5650; Packard Instruments, Greve, Denmark) or after
addition of 3–4 ml scintillation cocktail (Pico-Flour 40; Perkin-Elmer,
Caversham, England) using a beta-counter (Tri-Carb 2100 TR; Packard). The Na
influx (mmol Na l-1RBCs h-1) was calculated as:
 | (1) |
c.p.m.
denotes the difference in triplicate 22Na c.p.m. between two time
points, VRBC is the volume of RBCs (litres), determined
from the Hct samples taken after 45 min of pre-equilibration, and
t is the time in minutes between samplings.
Cell volume regulation
Whole blood
A sub-sample of 2 ml freshly drawn blood was kept on ice for 1–2
h until used for whole-blood RVI studies. Approximately 0.5 ml (or 0.7 ml if
pH was measured; see below) was incubated in each of four rotating Eschweiler
tonometers. The tonometers were immersed in a water bath kept at 25°C and
supplied with a humidified gas mixture of 1% or 3% CO2, balanced
with air, through a Wösthoff gas mixing pump (Bochum, Germany) to achieve
physiologically relevant PCO2 and pH values for
resting and active animals, respectively
(Andersen and Wang, 2003).
After a 30 min equilibration period, samples were taken for duplicate pH, Hct
and haemoglobin concentration ([Hb]) determinations and, five minutes later
(time zero in the experiment), the blood cells were subjected to the following
manipulations: (1) osmotic shrinkage alone by addition of appropriate volumes
of a 2.3 Osm kg-1 stock solution (sucrose-containing standard
saline) to increase plasma osmolarity from 240 to 320 mOsm
l-1 (calculated to yield 25% cell shrinkage) or with (2)
simultaneous addition of amiloride to a final concentration of
10–4 mol l-1 to inhibit the NHE or (3)
simultaneous addition of bumetanide to a final concentration of
10–5 mol l-1 to inhibit the NKCC. In addition,
parallel control experiments with no osmotic disturbance were performed to
determine the influence of incubation alone on the RBCs.
Duplicate Hct determinations were made at time zero as well as 15, 30, 60, 90 and 120 min after treatment, and duplicate samples for [Hb] determinations were taken at 0, 60 and 120 min. Haematocrit was determined after centrifuging heparinized micro-haematocrit tubes at 16 000 g for 3 min in a haemofuge (Heraeus Sepatech, Usingen, Germany). After conversion of Hb to cyanomethaemoglobin by Drabkin's reagent {11.9 mmol l-1 NaHCO3, 0.61 mmol l-1 K3[Fe(CN)6], 0.77 mmol l-1 KCN and 0.05% v/v Triton X-100}, [Hb] was determined at a wavelength of 540 nm (Ultrospec 2; LKB Biochrom, Cambridge, UK), applying a millimolar extinction coefficient of 11.0. The [Hb] was constant after time zero, so an average value was calculated for the time 0, 60 and 120 min measurements and used to calculate mean cellular haemoglobin concentration (MCHC) as [Hb]/Hct at all sampling points after treatment. MCHC was used as an indicator of red cell volume changes. pH was measured with a Radiometer pH electrode (BMS2, Copenhagen, Denmark) at 25°C.
Washed red blood cells
Washed RBCs, as above, were washed twice in the isotonic saline and
adjusted to an Hct value of 20–25% (22.2±0.9%, N=16).
The RBC suspension was then transferred to a rotating Eschweiler tonometer
kept in a water bath at 25°C and equilibrated to humidified air. After 20
min, ouabain was added to a final concentration of 10–4 mol
l-1, and 5 min later samples were taken for duplicate
determinations of Hct and [Hb]. After an additional 5 min (time zero in the
experiment), the suspension was distributed into open test tubes and subjected
to the same manipulations and sampling procedures as described above for whole
blood. The test tubes were kept at 25°C throughout the experiment and
gently swirled regularly to ensure oxygenation and avoid sedimentation. In
addition, identical volume recovery experiments on deoxygenated washed RBCs
were performed. To achieve deoxygenated conditions, humidified N2
replaced air in the tonometer 10 min before the suspension was transferred to
test tubes (at time zero in the experiment), which were also gassed with
humidified N2.
Data analysis and statistics
Differences in the mean MCHC and Na influx between treatments were analysed
by a two-way analysis of variance (ANOVA) test for repeated measures.
Differences in MCHC between washed cells and whole blood cells were analysed
by a three-way ANOVA test for repeated measures. ANOVA tests were followed by
a multiple comparison Bonferroni test. Data were transformed according to
x'=x
+(x+1)
if needed to fulfil the requirement of normal distribution
(Zar, 1984). All values shown
are means ± 1 s.e.m., and statistical significance was accepted at
P<0.05. The PO2 at which the
shrinkage-stimulated Na influx was half-maximal, P50, was
estimated by curve fitting, using:
 | (2) |
|
Results |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
50 mmol Na l-1 RBCs
h-1 in deoxygenated cells (0 kPa
PO2) (Fig.
1A). The kinetics of the shrinkage-induced Na influx also depended
on PO2. The Na influx in deoxygenated cells
remained high throughout the initial 30 min
(Fig. 1A,B), whereupon it
returned to basal levels between 45 and 60 min
(Fig. 1D). However, while the
Na influx at 4.1 kPa PO2 also remained high
(36–42 mmol Na l-1 RBCs h-1) during the first 30
min, the influx declined but remained significantly above basal levels
throughout the experiment (Fig.
1). At higher PO2 values, there
appeared to be a 15-min lag before maximal Na influx values of 30 mmol Na
l-1 RBCs h-1 were reached between 15 and 45 min
(Fig. 1B,C), whereupon the
influx declined but remained significantly elevated at 20–25 mmol Na
l-1 RBCs h-1 (Fig.
1D).
|
The PO2 dependency of the shrinkage-induced Na influx was maximal during the first 5–15 min (Fig. 1A), decreased between 15 and 30 min (Fig. 1B), and between 30 and 45 min there was no significant PO2 dependency of the shrinkage-induced Na influx (Fig. 1C). In the 45–60 min interval, the PO2 dependency even reversed, with maximal shrinkage-induced Na influxes at 8.2 and 20.5 kPa PO2 (Fig. 1D). In the 5–15 min interval, a P50 value of 5.5±1.0 kPa (N=4) was determined for shrinkage-induced Na influx.
The shrinkage-induced Na influx was completely abolished by
10–4 mol l-1 EIPA, a selective inhibitor of the
NHE, at all PO2 values
(Fig. 1). A dose–response
curve for EIPA is shown in Fig.
2, where the effect of EIPA was studied in deoxygenated RBCs
exposed to a calculated 25% shrinkage. An IC50 of
10–6 mol l-1 EIPA can be estimated from the
dose–response curve.
|
23 and 70 min,
respectively, under the two conditions
(Fig. 3B). The faster RVI
response of deoxygenated RBCs (Fig.
3B) reflects that the Na influx was already maximal in the initial
two time intervals under those conditions
(Fig. 1A,B), whereas maximal Na
influx values were not attained until after 15–45 min in oxygenated
cells (Fig. 1B,C), delaying the
RVI response (Fig. 3B). Maximal
volume recovery of deoxygenated cells was attained after 30 min
(Fig. 3B), after which the Na
influx was reduced and reached basal levels after 45–60 min
(Fig. 1C,D). The volume
recovery was slower in oxygenated cells, but after 90 min the extent of volume
recovery (60%) was similar to that attained in deoxygenated cells
(Fig. 3B), reflecting the more
sustained Na influx in cells kept at 20.5 kPa
PO2 (Fig.
1B–D). The RVI response was substantially reduced by the NHE
inhibitor amiloride (10–4 mol l-1), whereas the
NKCC inhibitor bumetanide (10–5 mol l-1) had no
effect (Fig. 3C).
|
The above experiments were carried out on RBCs suspended in a physiological
saline to allow comparisons with the Na influx data. However, RVI experiments
on whole blood were also carried out under similar conditions of pH and
temperature. Fig. 4 depicts the
time courses in MCHC of whole blood at 19.9–20.3 kPa
PO2 after different treatments at 1%
CO2 (PCO2 1 kPa) and 3%
CO2 (PCO2 3 kPa), corresponding
to pH 7.86±0.05 (N=7) and 7.55±0.03 (N=4),
respectively, as measured immediately before shrinkage. These are typical
PCO2 and pH values for resting and active or
burrowing animals, respectively (Boutilier
et al., 1979). MCHC of untreated control cells at both
PCO2 values did not change significantly during
the experiment (Fig. 4A,B). At
pH 7.86, the RVI response after a calculated 25% shrinkage of the cells was
statistically significant from 30 min onwards, and after 120 min volume
recovery had attained 89% (Fig.
4A). Amiloride (10–4 mol l-1)
significantly reduced RVI, whereas bumetanide (10–5 mol
l-1) did not have any effect on the extent of RVI
(Fig. 4A). Whole blood, at pH
and PCO2 values more typical for an active or
burrowing animal, showed an overall lowering of the MCHC from 4.7 to
3.9 mmol Hb4 l-1 RBCs and, thereby, had a new
increased steady-state RBC volume (Fig.
4B). The time course and extent of volume recovery after shrinkage
were similar to those at 1% CO2 but, due to larger differences in
MCHC values between individual experiments, there was no difference between
MCHC values of control and shrunken cells from 60 min onwards
(Fig. 4B).
|
Effects of deoxygenation per se and extracellular acidification on Na influx
In the 15–30 min interval, reduction of
PO2 to 4.1 and 0 kPa induced a 7- and 16-fold
increase in the ouabain-insensitive basal flux, respectively, relative to that
measured in fully oxygenated cells (Fig.
1B). In cells exposed to a PO2 of 0
kPa, a 10-fold increase in basal flux relative to that of oxygenated cells was
still present in the 30–45 min interval
(Fig. 1C). Deoxygenation
per se increased cell volume, as illustrated by significantly lowered
MCHC values compared with oxygenated RBCs from 60 min onwards
(Fig. 3A), corresponding well
with the increased Na influx in deoxygenated RBCs in the 15–45 min
interval (Fig. 1B,C).
Extracellular acidification (to pH 7.28) of oxygenated RBCs suspended in isotonic saline did not change the Na influx compared with basal values. Thus, basal Na influxes in oxygenated RBCs in the 5–15, 15–30 and 30–45 min intervals were 3.9±2.1, 0.4±1.3 and 2.2±1.2 mmol l-1 RBCs h-1 (N=6) whereas the Na influxes in acidified RBCs were 1.5±2.9, 3.5±2.8 and 1.3±1.0 mmol l-1 RBCs h-1 (N=6).
Oxygen dependence of isoproterenol-induced Na influx
The ß-adrenergic agonist isoproterenol significantly increased the
ouabain-insensitive Na influx in deoxygenated RBCs from 6 and 11
mmol Na l-1 RBCs h-1 to 25 and 35 mmol Na
l-1 RBCs h-1, respectively, within the first two time
intervals (Fig. 5A). The effect
of isoproterenol on Na influx was statistically significant for up to 45 min,
whereupon the influx was reduced to basal levels
(Fig. 5A). Bumetanide did not
significantly reduce the isoproterenol-stimulated Na influx at any time,
whereas EIPA completely blocked the effects of isoproterenol on Na influx
(Fig. 5A). In air-equilibrated
RBCs, on the other hand, isoproterenol had no effect on Na influx within 45
min of stimulation (Fig.
5B).
|
|
Discussion |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
;
Nielsen et al., 1992;
Gibson et al., 1998;
Virkki et al., 1998;
Muzyamba et al., 1999;
Berenbrink et al., 2000;
Berenbrink et al., 2006;
Flatman, 2005) (reviewed by
Gibson et al., 2000). This
also applies to the transporters involved in volume regulation
(Jensen, 1995;
Nielsen, 1997;
Gibson et al., 2000;
Brauner et al., 2002;
Drew et al., 2004). In
Bufo marinus, there is a strong PO2
dependency of the shrinkage-induced Na influx, with maximal stimulation at low
PO2 during the initial 30 min
(Fig. 1). The shrinkage-induced
Na influx was completely abolished by the specific NHE inhibitor EIPA
(10–4 mol l-1), showing that the Na uptake was
entirely due to activation of the NHE (Fig.
1). The P50 value of 5.5 kPa for half-maximal
shrinkage-induced Na influx in the first time interval is similar to the
in vitro Hb P50 value of 5.3 kPa that can be
estimated for B. marinus at a similar pH and temperature [based on
data in table 1 of Andersen et al.
(Andersen et al., 2001)].
Therefore, with matching oxygen affinities of NHE activation and Hb, it is
conceivable that Hb is involved in oxygen sensing and modulation of NHE
activation in B. marinus RBCs. This role of Hb has been suggested for
modulation of NHE and KCC (K/Cl co-transporter) activity in rainbow trout RBCs
(Motais et al., 1987;
Nielsen et al., 1992).
However, other studies on rainbow trout and crucian carp RBCs indicate that
the P50 of KCC activation is significantly different from
the P50 of Hb, implying that the effect of oxygen is not
regulated by bulk Hb oxygen saturation
(Berenbrink et al., 2000;
Berenbrink et al., 2006). In
rainbow trout, the effect of oxygen on transporter activity seems to be
mediated through hydroxyl radicals, which stimulate KCC activity, whilst they
inhibit the ßNHE at high PO2
(Bogdanova and Nikinmaa, 2001;
Nikinmaa et al., 2003). The
oxygen dependency of NKCC activity in RBCs of some tetrapods such as turkey
and ferret and in crucian carp, however, is consistent with modulation by the
Hb oxygenation state (Muzyamba et al.,
1999; Flatman,
2005; Berenbrink et al.,
2006).
Shrinkage of B. marinus RBCs was attended by a pronounced
amiloride-sensitive RVI response (Fig.
3C, Fig. 4C) that
was much faster under deoxygenated conditions
(Fig. 3B), reflecting the time
course of activation and the magnitude of the Na influxes under the two levels
of PO2 (Fig.
1). The RVI response was, however, not complete under either of
the conditions within the 90–120 min studied here. It appears that the
fast RVI response in deoxygenated B. marinus RBCs is due to immediate
and large NHE activation, whereas the slower RVI in oxygenated cells reflects
the fact that the Na influx is not maximal until 15–45 min after
shrinkage activation (Fig. 1,
Fig. 3B). In European flounder,
the RVI is progressively faster with decreasing levels of Hb oxygen saturation
(Jensen et al., 2002). In
light of this, studies carried out at high PO2
values, as for example during air equilibration, may mistakenly underestimate
the ability of RBCs to perform RVI under conditions prevailing in the venous
circulation or, in animals with cardiac shunts, even in arterial blood.
The NHE also mediates RVI in Rana ridibunda RBCs, and a flux of
11.6±1.1 mmol Na l-1 RBCs h-1 under oxygenated
conditions was measured within the first 30 min after a calculated 31%
shrinkage (Gusev and Ivanova,
2003). This Na influx is lower than the influx between 0 and 30
min in B. marinus RBCs, which at PO2
20.5 kPa can be estimated to be 19.1±2.0 mmol Na l-1 RBCs
h-1 (N=4) and it suggests that, at least initially, the
NHE activity upon shrinkage is larger in the terrestrial toad than in the
semi-aquatic frog. These data, however, are too sparse to allow a realistic
comparison of the RVI ability of terrestrial and semi-aquatic amphibians
because, as shown here, the RVI responses depend on blood
PO2 and time frame.
The mechanism of RVI in B. marinus RBCs seems to be the same at
low and high PO2 as the response was
significantly reduced by amiloride but insensitive to bumetanide under both
conditions (Fig. 3C and
Fig. 4C, respectively). At a
concentration of 10–4 mol l-1, amiloride did not
completely block the RVI response, which was also found in shrunken Rana
temporaria RBCs (Jørgensen,
1995). Based on the Na influx experiments
(Fig. 1), it is likely that
10–4 mol l-1 EIPA would abolish the RVI
completely, but at present we cannot rule out that other, Na-independent,
mechanisms may be involved in the RVI response.
Many RVI studies are only carried out on cells suspended in defined,
artificial media containing Na+/K+-ATPase inhibitors
(Cala, 1977;
Siebens and Kregenow, 1985;
Romero et al., 1996;
Weaver et al., 1999). Our
study shows that the RVI response was not significantly different in whole
blood and washed RBCs from B. marinus
(Fig. 4). RVI did therefore not
depend on washing and storage procedures or activity of the
Na+/K+-ATPase. Cala also reported that RVI did not
depend on the Na+/K+-ATPase
(Cala, 1977), and Caldwell et
al. showed that effects of ß-adrenergic stimulation in trout RBCs taken
via caudal puncture and then washed and stored for up to 96 h were
not significantly different from those of whole blood taken via
cannulae 48 h after surgery (Caldwell et
al., 2006). Haemolysis was not detected in B. marinus
whole blood during the course of the experiment, making it appear less fragile
than the washed RBCs. However, the influence of, for instance, stress hormones
can be avoided when replacing the plasma and incubating the cells overnight
(Bourne and Cossins,
1982).
Effects of deoxygenation per se and extracellular acidification on Na influx
Volume changes due to the Haldane effect and associated shifts in the
Donnan equilibrium are very fast (Hladky
and Rink, 1977; Borgese et al.,
1991) and can therefore not explain the slow volume increase in
B. marinus RBCs caused by deoxygenation alone
(Fig. 3A). The same treatment
caused a significant increase in the basal Na influx
(Fig. 1B,C), which could
explain the volume increase. The P50 for half-maximal
deoxygenation-induced Na influx in the 15–30 min time interval is in the
same range as the values for Hb and shrinkage-induced Na influx (see above)
and, thus, support that Hb oxygenation could also modulate basal NHE activity.
The mechanism of the increased deoxygenation-induced Na influx was not
addressed specifically, but 10–4 mol l-1 EIPA
lowered the shrinkage-induced Na influx in deoxygenated cells to basal values
measured in oxygenated RBCs (Fig.
1B,C), so it likely involved the NHE. Weaver et al. showed that
deoxygenation per se activates the NHE in European flounder RBCs
(Weaver et al., 1999).
Elevation of PCO2 caused swelling of the
RBCs (Fig. 4B), but since
extracellular acidification in washed RBCs did not influence the Na influx
(see above), swelling is probably caused by a change in the Donnan equilibrium
across the membrane and not a pH regulatory Na transport. In rainbow trout
RBCs, extracellular acidification stimulates the NHE in the acidic pH range,
whereas intracellular acidification stimulates the NHE in the alkaline pH
range (Borgese et al., 1987).
Intracellular acidification also stimulates the NHE in Amphiuma
tridactylum and R. temporaria RBCs
(Cala and Maldonado, 1994;
Jørgensen, 1995). This
regulation of intracellular pH may help maintain the oxygen affinity of
haemoglobin. As at 1 kPa PCO2, bumetanide did
not affect RVI at 3 kPa PCO2, indicating also
that, under conditions typical for an active or burrowing animal, the NKCC did
not contribute to volume regulation.
Oxygen dependence of isoproterenol-induced Na influx
Stimulation by the ß-adrenergic agonist isoproterenol at 0 kPa
PO2 elicited a pronounced increase in the
EIPA-sensitive and bumetanide-insensitive Na influx
(Fig. 5A). At present, we do
not know how widespread ß-adrenergic NHE activation is within amphibians,
but it was not found in deoxygenated Xenopus laevis RBCs
(Berenbrink et al., 2005).
Adrenergic stimulation increased cAMP and caused swelling of deoxygenated RBCs
from R. ridibunda, but the mechanism(s) involved in the response were
not characterized (Kaloyianni and
Rasidaki, 1996). In preliminary experiments on B. marinus
RBCs there was a slower, but significant, increase in the
isoproterenol-stimulated Na influx at high PO2
and there also appeared to be a prolonged lag phase in the Na influx in
shrunken RBCs (data not shown). The reason for the variable lag time between
experiments is currently not understood.
The magnitude of the ß-adrenergic response of B. marinus RBCs
is comparable to that measured in the initial 5 min after stimulation in some
teleosts such as chub (36±6 mmol Na l-1 RBCs h-1;
N=3) and striped bass (40±8 mmol Na l-1 RBCs
h-1; N=4), but much higher values of more than 200 mmol Na
l-1 RBCs h-1 have been measured in other teleost RBCs
(Berenbrink et al., 2005).
However, in teleosts, maximal activation of the ßNHE is observed within
1–2 min followed by a rapid desensitization
(Motais et al., 1992) (P.K.
and M.B., personal observations), whereas in B. marinus the Na influx
remained maximal for up to 30 min and was still elevated significantly above
basal level for up to 45 min (Fig.
5A).
Tufts et al. found elevated plasma catecholamine levels after forced
activity in B. marinus (Tufts et
al., 1987a), and it is tempting to speculate that, as in teleosts
(Nikinmaa, 1992), the
physiological role of ßNHE activation in deoxygenated RBCs is to
safeguard intracellular pH and oxygen binding under conditions of general
acidosis. Tufts et al., however, found no evidence of in vivo
ßNHE activation as cell volume remained unaltered after 30 min of
vigorous exercise (Tufts et al.,
1987a).
In rainbow trout RBCs, the ßNHE is partially inhibited at high
PO2 values
(Motais et al., 1987;
Nikinmaa et al., 2003). We did
not find ßNHE activation at 20.5 kPa PO2
for up to 45 min in B. marinus RBCs
(Fig. 5B). Tufts et al. were
similarly unable to show in vitro ß-adrenergic changes in
intracellular pH and water content of air-equilibrated B. marinus
RBCs kept under 5% CO2 (extracellular pH of 7.5)
(Tufts et al., 1987a) or of
air-equilibrated A. tridactylum RBCs kept under 4 or 8%
CO2 (extracellular pH of 7.65 or 7.44, respectively)
(Tufts et al., 1987b).
Air-equilibrated RBCs from R. ridibunda and R. pipiens,
nevertheless, increase cAMP levels and swell when stimulated by isoproterenol,
but these responses have only been demonstrated under non-physiological
conditions in the presence of the phosphodiesterase inhibitor EDTA
(Rudolph and Greengard, 1980;
Kaloyianni and Rasidaki,
1996). In air-equilibrated RBCs, therefore, it seems that
ßNHE stimulation is lacking within amphibians.
In conclusion, this study demonstrates a pronounced
PO2 sensitivity of the shrinkage-induced RVI
response and the underlying Na influxes mediated through the NHE in RBCs from
the terrestrial anuran Bufo marinus. Deoxygenation reveals
ß-adrenergic activation of the NHE mediated Na influx, which has been
missed in earlier studies on air-equilibrated amphibian RBCs. The regulation
of the RBC ßNHE seems to differ from that in teleosts, where rapid
desensitization of the transporter may be the rule, whereas the activation was
prolonged for up to 45 min in B. marinus. The RVI response in B.
marinus RBCs may protect the HbO2-affinity in vivo by
reducing intracellular concentrations of Hb and organic phosphates and it may
thus contribute to the high tolerance to elevated plasma osmolarity following
dehydration in this species (Shoemaker,
1964; Shoemaker,
1965). A description of the relationship between RBC volume and
HbO2-affinity in amphibians with different tolerance to dehydration
would help understand the role of RVI.
List of abbreviations and symbols
|
Acknowledgments |
|---|
|
References |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Andersen, J. B. and Wang, T. (2002). Effects of anaesthesia on blood gases, acid-base status and ions in the toad, Bufo marinus. Comp. Biochem. Physiol. 131A,639 -646.
Andersen, J. B. and Wang, T. (2003). Cardio-respiratory effects of forced activity and digestion in toads. Physiol. Biochem. Zool. 76,459 -470.[CrossRef][Medline]
Andersen, J. B., Jensen, F. B. and Wang, T. (2001). Effects of temperature and oxygen availability on circulating cateholamines in the toad Bufo marinus. Comp. Biochem. Physiol. 129A,473 -486.
Berenbrink, M., Völkel, S., Heisler, N. and 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., Koldkjær, P., Kepp, O. and Cossins, A.
R. (2005). Evolution of oxygen secretion in fishes and the
emergence of a complex physiological system. Science
307,1752
-1757.
Berenbrink, M., Völkel, S., Koldkjær, P., Heisler, N.
and Nikinmaa, M. (2006). Two different oxygen sensors
regulate oxygen-sensitive K+ transport in carp red blood cells.
J. Physiol. 575,37
-48.
Bogdanova, A. Y. and Nikinmaa, M. (2001).
Reactive oxygen species regulate oxygen-sensitive potassium flux in rainbow
trout erythrocytes. J. Gen. Physiol.
117,181
-190.
Borgese, F., Garcia-Romeu, F. and Motais, R.
(1987). Ion movements and volume changes induced by
catecholamines in erythrocytes of rainbow trout: effect of pH. J.
Physiol. 382,145
-157.
Borgese, F., Motais, R. and 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]
Bourne, P. K. and Cossins, A. R. (1982). On the
instability of K+ influx in erythrocytes of the rainbow trout,
Salmo gairdneri, and the role of catecholamine hormones in
maintaining in vivo influx activity. J. Exp.
Biol. 101,93
-104.
Boutilier, R. G., Randall, D. J., Shelton, G. and Toews, D.
P. (1979). Acid–base relationships in the blood of the
toad, Bufo marinus. J. Exp. Biol.
82,357
-365.
Brauner, C. J., Wang, T. and Jensen, F. B. (2002). Influence of hyperosmotic shrinkage and ß-adrenergic stimulation on red blood cell volume and oxygen binding properties in rainbow trout and carp. J. Comp. Physiol. B 172,251 -262.[CrossRef][Medline]
Cala, P. M. (1977). Volume regulation by
flounder red blood cells in anisotonic media. J. Gen.
Physiol. 69,537
-552.
Cala, P. M. (1980). Volume regulation by
Amphiuma red blood cells. J. Gen. Physiol.
76,683
-708.
Cala, P. M. and Maldonado, H. M. (1994). pH
regulatory Na/H exchange by Amphiuma red-blood-cells. J.
Gen. Physiol. 103,1035
-1053.
Caldwell, S., Rummer, J. L. and Brauner, C. J. (2006). Blood sampling techniques and storage duration: effects of the presence and magnitude of the red blood cell ß-adrenergic response in rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Physiol. 144A,188 -195.[CrossRef][Medline]
Cossins, A. R. and Gibson, J. S. (1997). Volume-sensitive transport systems and volume homeostasis in vertebrate red blood cells. J. Exp. Biol. 200,343 -352.[Abstract]
Drew, C., Ball, V., Robinson, H., Ellory, J. C. and Gibson, J. S. (2004). Oxygen sensitivity of red cell membrane transporters revisited. Bioelectrochemistry 62,153 -158.[CrossRef][Medline]
Flatman, P. W. (2005). Activation of ferret
erythrocyte Na+-K+-2Cl– cotransport by
deoxygenation. J. Physiol.
563,421
-431.
Gibson, J. S., Speake, P. F. and Ellory, J. C. (1998). Differential oxygen sensitivity of the K+-Cl– cotransporter in normal and sickle human red blood cells. J. Physiol. 551,225 -234.
Gibson, J. S., Cossins, A. R. and Ellory, J. C. (2000). Oxygen-sensitive membrane transporters in vertebrate red cells. J. Exp. Biol. 203,1395 -1407.[Abstract]
Gusev, G. P. and Ivanova, T. I. (2003). Activation of Na+/H+ exchange by protein phosphatase inhibitors in red blood cells of the frog Rana ridibunda. J. Comp. Physiol. B 173,429 -435.[CrossRef][Medline]
Gusev, G. P., Lapin, A. V. and Agulakova, N. I. (1997). Volume regulation in red blood cells of the frog Rana temporaria after osmotic shrinkage and swelling. Membr. Cell Biol. 11,305 -317.[Medline]
Hladky, S. B. and Rink, T. J. (1977). pH equilibrium across red cell membrane. In Membrane Transport in Red Cells (ed. J. C. Ellory and V. L. Lew), pp.115 -135. London: Academic Press.
Hyndman, C. A., Kieffer, J. D. and Benfey, T. J. (2003). Physiology and survival of triploid brook trout following exhaustive exercise in warm water. Aquaculture 221,629 -643.[CrossRef]
Jensen, F. B. (1995). Regulatory volume decrease in carp red blood cells: mechanisms and oxygenation-dependency of volume-activated potassium and amino acid transport. J. Exp. Biol. 198,155 -165.[Medline]
Jensen, F. B., Lecklin, T., Busk, M., Bury, N. R., Wilson, R. W., Wood, C. M. and Grosell, M. (2002). Physiological impact of salinity increase at organism and red blood cell levels in the European flounder (Platichthys flesus). J. Exp. Mar. Biol. Ecol. 274,159 -174.[CrossRef]
Johansen, K. and Ditadi, A. S. F. (1966). Double circulation in the giant toad, Bufo paracnemis. Physiol. Zool. 39,140 -150.
Jørgensen, N. C. (1995). Amiloride-sensitive Na+/H+ exchange stimulated by cellular acidification of shrinkage in red blood cells of the frog, Rana temporaria. J. Comp. Physiol. B 165,450 -457.
Kaloyianni, M. and Rasidaki, A. (1996). Adrenergic responses of R. ridibunda red cells. J. Exp. Zool. 276,175 -185.[CrossRef][Medline]
Kaloyianni, M., Giannisis, G., Gavriil, P. and Boukla, A. (1997). Metabolic effects and cellular volume responses induced by noradrenaline in nucleated erythrocytes. J. Exp. Zool. 279,337 -346.[CrossRef][Medline]
Kregenow, F. M. (1981). Osmoregulatory salt transporting mechanisms: control of cell volume in anisotonic media. Annu. Rev. Physiol. 43,493 -505.[CrossRef][Medline]
Madsen, S. S., Larsen, B. K. and Jensen, F. B. (1996). Effects of freshwater to seawater trasnfer on osmoregulation, acid-base balance and respiration in river migrating whitefish (Coregonus lavaretus). J. Comp. Physiol. B 166,101 -109.[CrossRef]
Maxime, V., Pennec, J. P. and Peyraud, C. (1991). Effects of direct transfer from freshwater to seawater on respiratory and circulatory variables and acid-base status in rainbow trout. J. Comp. Physiol. B 161,557 -568.[CrossRef]
McKenna, M. J., Heigenhauser, J. F., McKelvie, R. S., MacDougall, J. D. and Jones, N. L. (1997). Sprint training enhances ionic regulation during intense exercise in men. J. Physiol. 501,687 -702.[CrossRef][Medline]
Motais, R., Garcia-Romeu, F. and Borgese, F.
(1987). The control of Na+/H+ exchange by
molecular oxygen in trout erythrocytes. J. Gen.
Physiol. 90,197
-207.
Motais, R., Borgese, F., Fievet, B. and Garcia-Romeu, F. (1992). Regulation of Na+/H+ exchange and pH in erythrocytes of fish. Comp. Biochem. Physiol. 102A,597 -602.[Medline]
Muzyamba, M. C., Cossins, A. R. and Gibson, J. S. (1999). Regulation of Na+-K+-2Cl– cotransport in turkey red cells: the role of oxygen tension and protein phosphorylation. J. Physiol. 512,421 -429.[CrossRef]
Nielsen, O. B. (1997). Effects of haemoglobin O2 saturation on volume regulation in adrenergically stimulated red blood cells from the trout, Oncorhynchus mykiss. J. Comp. Physiol. B 167,159 -168.[CrossRef][Medline]
Nielsen, O. B., Lykkeboe, G. and Cossins, A. R. (1992). Oxygenation-activated K fluxes in trout red blood cells. Am. J. Physiol. 263,C1057 -C1064.[Medline]
Nikinmaa, M. (1992). Membrane transport and
control of hemoglobin-oxygen affinity in nucleated erythrocytes.
Physiol. Rev. 72,301
-321.
Nikinmaa, M., Bogdanova, A. and Lecklin, T. (2003). Oxygen dependency of the adrenergic Na/H exchange in rainbow trout erythrocytes is diminished by a hydroxyl radical scavenger. Acta Physiol. Scand. 178,149 -154.[CrossRef][Medline]
Orlov, S. N., Kuznetsov, S. R., Kolosova, I. A. and Makarov, V. L. (1994). Na+/H+ and Na+/Na+ counter transport in human, rabbit, and rat erythrocytes: evidence for two independent ion transport systems. Biochemistry 59,467 -472.
Palfrey, H. C. and 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]
Palfrey, H. C., Feit, P. W. and Greengard, P. (1981). Specific inhibition by `loop' diuretics of an anion-dependent Na++K+ cotransport system in avian erythrocytes. Ann. N. Y. Acad. Sci. 341,134 -138.
Parker, J. C., Colclasure, G. C. and McManus, T. J.
(1991). Coordinated regulation of shrinkage-induced Na/H exchange
and swelling-induced [K-Cl] cotransport in dog red cells. J. Gen.
Physiol. 98,869
-880.
Pedersen, S. F. and Cala, P. M. (2004). Comparative biology of the ubiquitous Na+/H+ exchanger, NHE1: lessons from erythrocytes. J. Exp. Zool. A 301,569 -578.
Peterson, C. C. and Greenshields, D. (2001). Negative test for cloacal drinking in a semi-aquatic turtle (Trachemys scripta), with comments on the functions of cloacal bursae. J. Exp. Zool. 290,247 -254.[CrossRef][Medline]
Romero, M. G., Guizouarn, H., Pellissier, B., Garcia-Romeu, F. and Motais, R. (1996). The erythrocyte Na+/H+ exchanger in eel (Anguilla anguilla) and rainbow trout (Oncorhyncus mykiss): a comparative study. J. Exp. Biol. 136,405 -416.
Rudolph, S. A. and Greengard, P. (1980).
Effects of catecholamines and prostaglandin E1 on cyclic AMP,
cation fluxes, and protein phosphorylation in the frog erythrocyte.
J. Biol. Chem. 255,8534
-8540.
Salama, A. and Nikinmaa, M. (1988). The
adrenergic responses of carp (Cyprinus carpio) red cells: effects of
PO2 and pH. J. Exp.
Biol. 136,405
-416.
Shoemaker, V. H. (1964). The effects of dehydration on electrolyte concentrations in a toad, Bufo Marinus.Comp. Biochem. Physiol. 13,261 -271.[Medline]
Shoemaker, V. H. (1965). The stimulus for the water-balance response to dehydration in toads. Comp. Biochem. Physiol. 15,81 -88.[Medline]
Siebens, A. W. and Kregenow, F. M. (1985).
Volume-regulatory responses of Amphiuma red cells in anisotonic
media. J. Gen. Physiol.
86,527
-564.
Thorson, T. B. (1955). The relationship of water economy to terrestrialism in amphibians. Ecology 36,100 -116.[CrossRef]
Tufts, B. L., Mense, D. C. and Randall, D. J.
(1987a). The effects of forced activity on circulating
catecholamines and pH and water content of erythrocytes in the toad.
J. Exp. Biol. 128,411
-418.
Tufts, B. L., Nikinmaa, M., Steffensen, J. F. and Randall, D.
J. (1987b). Ion exchange mechanisms on the erythrocyte
membrane of the aquatic salamander, Amphiuma tridactylum. J. Exp.
Biol. 133,329
-338.
Virkki, L. V., Salama, A. and Nikinmaa, M. (1998). Regulation of ion transport across lamprey (Lampetra fluviatilis) erythrocyte membrane by oxygen. J. Exp. Biol. 201,1927 -1937.
Wang, T., Abe, A. S. and Glass, M. L. (1998). Temperature effects on lung and blood gases in Bufo paracnemis: consequences of bimodal gas exchange. Respir. Physiol. 113,231 -238.[CrossRef][Medline]
Weaver, Y. R., Kiessling, K. and Cossins, A. R. (1999). Responses of the Na+/H+ exchanger of European flounder red blood cells to hypertonic, ß-adrenergic and acidotic stimuli. J. Exp. Biol. 202, 21-32.[Abstract]
Zar, J. H. (1984). Data transformations. In Biostatistical Analysis, pp.236 -243. Upper Saddle River, NJ: Prentice-Hall.
| ||||||||||||||||||||||||||||||||||||||||||||||||
) and in cells
stimulated by a calculated 25% osmotic shrinkage alone (
) or in the
presence of 10–4 mol l-1 EIPA (•). The cell
suspensions were exposed to the experimental gas mixture 10 min before
shrinkage (time zero in the figures). (A) 5–15 min, (B) 15–30 min,
(C) 30–45 min and (D) 45–60 min. * indicates
statistical difference from basal influx within the same
PO2 and time interval; 
indicates statistical difference from air-equilibrated RBCs within the same
treatment and time interval; 
indicates statistical
difference from the same treatment and PO2
level in the 5–15 min time interval. N=4,
P<0.05.
) as well as deoxygenated
control (
) or with 10–5
mol l-1 bumetanide
(
). * indicates mean
values that are statistically different from the respective time zero value;
;
N=9, 4 and 11 for A, B and C, respectively) or with
10–5 mol l-1 bumetanide
(
; N=4, 4 and 6 in A, B
and C, respectively). * indicates mean values that are
statistically different from the respective control value at time zero;
