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First published online September 14, 2007
Journal of Experimental Biology 210, 3451-3460 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.008524
In vivo red blood cell sickling and mechanism of recovery in whiting, Merlangius merlangus
School of Biological Sciences, University of Liverpool, Crown Street, Liverpool L69 7ZB, UK
* Author for correspondence (e-mail: piak{at}liv.ac.uk)
Accepted 17 July 2007
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Key words: Merlangius merlangus, sickle cell disease, Na/H exchanger, red blood cell, haemoglobin, Hb polymerisation, fish
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). However, a single mutation in human sickle Hb (HbS)
replaces a polar glutamic acid on the surface of the protein by a non-polar
valine, which greatly reduces the solubility of the protein in the
deoxygenated state and causes Hb polymerisation
(Ingram, 1957;
Perutz and Mitchison, 1950).
The HbS polymers form an extremely viscous, solid-like gel, which causes
peculiar cell deformations, the most obvious of which is the sickle-shape
(Eaton and Hofrichter, 1987).
Sickled RBCs show increased fragility and reduced deformability, causing
severe anaemia and vasoocclusion. The latter can cause severe pain, organ
failure and death (Serjeant,
2001).
Hb mutations leading to a reduced solubility apparently occurred in several
other vertebrate groups. This is suggested by early reports of intracellular
crystal formation or paracrystalline arrangements of fibres with associated
RBC deformations in white-tailed deer
(Undritz et al., 1960),
annulated gecko (Mattei et al.,
1985), common mudpuppy
(Dawson, 1930) and several
groups of fishes (e.g. Yoffey,
1929; Dawson,
1932; Thomas,
1971) [for reference to even earlier literature see Perutz and
Mitchison (Perutz and Mitchison,
1950)]. However, most of these reports were chance observations of
cells under the microscope and it is not clear to what extent observations are
artefacts of RBC preparation or under which physiological conditions sickling
may occur in vivo.
In a more recent in vitro study on sickling in RBCs of several
fish species, Hárosi and co-workers
(Hárosi et al., 1998),
using polarised light microscopy techniques, were able to correlate RBC
morphology with oxygenation state, spectral absorbance, linear dichroism and
linear birefringence. They concluded that the large aggregates in Atlantic cod
and oyster toadfish RBCs indeed consisted of Hb and that their
physico-chemical characteristics were remarkably similar to those in human
sickle RBCs (Hárosi et al.,
1998). However, the method to induce in vitro sickling in
these fish RBCs involved complete anoxia at very low pH and a temperature at
the upper physiological limit.
Here, we report extensive in vivo sickling of whiting RBCs after capture stress without any apparent haemolysis and its subsequent recovery. We describe the in vitro effects of saline pH and PO2 on sickling and show that low pH is most likely to elicit sickling in vivo. We further demonstrate the ameliorating effect of ß-adrenergic activation of the RBC Na/H exchanger (ßNHE) on sickling induced by low pH in vitro and discuss the possible physiological role of the phenomenon.
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Blood sampling, fixation and light microscopy
The fish were killed according to a British Home Office Schedule 1 method
by a sharp blow to the head and destruction of the brain, before blood
sampling from the caudal blood vessels into a heparinised syringe. In eight of
the fish, a 50-µl sample was fixed as described below immediately after
sampling. The remainder of the RBCs were washed three times in an isotonic
saline (125.5 mmol l–1 NaCl, 3 mmol l–1 KCl,
1.5 mmol l–1 MgCl2, 1.5 mmol l–1
CaCl2, 5 mmol l–1 D-glucose and 20 mmol
l–1 Hepes; 299 mOsm kg–1; pH adjusted to
7.97 at 15°C), each time removing the buffy coat. The cells were finally
suspended at a haematocrit (Hct) of 15–20%, oxygenated by contact with
air and stored at 4°C for 16–24 h. Before experimentation, the RBCs
were washed an additional two times and resuspended in fresh saline.
Another group of whiting (mean mass 200.0±107.0 g and SL 25.5±4.5 cm; N=5), rainbow trout (Oncorhynchus mykiss Walbaum; mean mass 913±132 g and SL 37.2±1.8 cm; N=8) and common carp (Cyprinus carpio L.; mean mass 1378±463 g and SL 36.8±4.6 cm; N=5) were killed as above immediately after being caught on hook and line. Reeling in whiting, rainbow trout and common carp usually took 2–4 min irrespective of species. The trout and carp were caught at the Pennine Trout Farm and Fishery, Lancashire, UK, and Llyn y gors, Anglesey, UK, respectively, during February 2007. A blood sample was taken from the caudal vessels into a heparinised syringe and fixed as described below. Another group of whiting (mean mass 132.8±26.1 g and SL 23.6±1.3 cm; N=4) were transported to the university and allowed to recover in the holding tank for 24 h, whereupon the animals were netted individually with a minimum of disturbance and quickly killed. Blood was sampled and fixed as described below.
All blood samples for light microscopy and transmission electron microscopy (TEM) were fixed in iso-osmotic glutaraldehyde immediately after sampling, but on several occasions freshly sampled blood was studied directly under the light microscope and confirmed the presence of sickled RBCs in unfixed samples. The glutaraldehyde fixation solution was made from a 25% stock solution (Grade I; Sigma-Aldrich Company Ltd, Gillingham, UK), diluted with distilled water to obtain an osmolality of 300 mOsm kg–1 and further diluted with saline (see above) to obtain a 2% glutaraldehyde solution. Samples were fixed in an equal volume of this iso-osmotic solution to yield a final glutaraldehyde concentration of 1%. They were stored at 4°C until counting or further treatment for TEM. The samples were counted on an Axiovert 135 TV microscope (Carl Zeiss Microlmaging GmbH, Göttingen, Germany; using a 100x oil immersion lens and direct interference contrast) fitted with a video camera (3-CCD Color Video Camera, KY-F55B, JVC; Alrad Instruments Ltd, Newbury, UK) and viewed with Scion Image software (Scion Corporation, Frederick, MD, USA).
Effects of pH, oxygen tension and hyperosmotic shrinkage on RBC sickling and recovery
350-µl aliquots (70 µl washed RBCs with an Hct of 
20% and 280
µl experimental saline with pH values ranging from 6.75 to 7.97) were
incubated at 15°C in rotating Eschweiler tonometers (Eschweiler GmbH,
Engelsdorf, Germany). Saline pH was measured with a combined pH electrode and
a PHM 85 Precision pH Meter (Radiometer, Copenhagen, Denmark). Final pH values
of the mixed salines were assumed to be identical to extracellular pH due to
the low final Hct value and ranged from 7.03 to 7.97. The samples were gassed
with humidified N2, air or O2, and samples were taken
and fixed after 45 min.
Recovery from in vitro sickling by an increase in extracellular pH
was studied in air-equilibrated RBCs. After 45 min, a sub-sample of RBCs
suspended in saline pH 7.03 was taken and fixed, whereupon the remainder of
the sample was briefly centrifuged (10 s at 9000 g;
Eppendorf model 5415 D; Fisher Scientific, Loughborough, UK) and had the
supernatant replaced with pH 7.97 saline. The resuspended cells were incubated
as before and further samples were taken and fixed at different time points at
the new, elevated saline pH.
Recovery from in vitro sickling by an increase in oxygen tension was studied at pH 7.61 in RBC suspensions that had been gassed with humidified nitrogen for 45 min. After a sub-sample was taken and fixed (time zero), the gas was changed to humidified oxygen. Further samples were taken and fixed after 5, 15, 30 and 45 min.
To assess the effects of intracellular Hb concentration, washed RBCs were pre-equilibrated to air in pH 7.97 saline for 45 min. Sub-samples were taken and fixed at 5 and 45 min. Appropriate volumes of sucrose stock solution (sucrose in pH 7.97 saline; osmolality of 2.5 Osm kg–1) were added to change the extracellular osmolality to 350, 400, 450 or 600 mOsm kg–1, causing cell shrinkage and calculated 17, 33, 50 and 100% increases in intracellular [Hb], respectively. Further samples were taken and fixed at 5, 15, 30, 60 and 120 min after shrinkage.
Effects of ß-adrenergic stimulation on RBC sickling
Aliquots of 350 µl washed RBCs with an Hct of 10–20% were
pre-equilibrated to humidified air in rotating Eschweiler tonometers kept at
15°C in a thermostatically controlled water bath. Samples were taken and
fixed at 5 and 45 min, whereupon sub-samples of the RBC suspension were
transferred to test tubes, containing four volumes of experimental saline
equilibrated with air. The cells were exposed to (1) pH 7.03 saline, (2) pH
7.03 saline and the ß-adrenergic agonist isoproterenol (final
concentration of 10–5 mol l–1; saline
vehicle) or (3) pH 7.03 saline and 10–5 mol
l–1 isoproterenol simultaneously with the Na/H exchanger
inhibitor amiloride (final concentration of 10–4 mol
l–1; 0.5% v/v DMSO vehicle). Parallel control samples were
kept in the pH 7.97 saline. Sub-samples were fixed at 5, 15, 30 and 45 min
after transfer to the test tubes.
Na influx studies
Aliquots of 350–700 µl washed RBCs with an Hct of 10–20%
were pre-equilibrated to humidified air as described above. Ouabain (final
concentration of 10–4 mol l–1; 0.5% v/v DMSO
vehicle) was added after 40 min, and duplicate samples for Hct were taken (3
min centrifugation at 10 000 g; Micro-haematocrit
centrifuge; Hawksley, Lancing, UK). Sub-samples were then transferred to test
tubes (defined as time zero), containing four volumes of pH 6.75 (final saline
pH 7.03) or 7.97 saline equilibrated with air, 22Na (16.7 kBq
ml–1) and ouabain (final concentration of
10–4 mol l–1). In addition, at both pH
values, some test tubes contained isoproterenol (final concentration of
10–5 mol l–1) and/or amiloride (final
concentration of 10–4 mol l–1). Triplicate
200-µl samples were taken from each test tube at predetermined time points.
To terminate Na uptake, the RBCs were immediately washed three times in
ice-cold isotonic, Hepes-containing (20 mmol l–1)
MgCl2 solution (adjusted to pH 7.97) by centrifugation (7–8 s
at 6000 g; Eppendorf Model 5410; Eppendorf GmbH,
Engelsdorf, Germany) and re-suspension. The RBC pellets and extracellular
samples were lysed in 0.5 ml 0.05% Triton X-100 solution and deproteinised by
subsequent addition of 0.5 ml 5% trichloroacetic acid. After each experiment,
a 20-µl sample was also treated with Triton X-100 and trichloroacetic acid
to count the total 22Na activity, which was assumed to correspond
to the extracellular 22Na activity at the beginning of the uptake
experiment. The samples were centrifuged for 2 min (6000
g; Eppendorf Model 5410; Eppendorf GmbH, Engelsdorf, Germany),
and 0.8 ml of the supernatant was counted (Tri-Carb 2100 TR; Packard
Instruments Ltd, Caversham, UK) in 3 ml scintillation cocktail
(Pico-Fluor 40; Perkin-Elmer Ltd, Beaconsfield, UK).
Na uptake (mmol Na l–1 RBCs) was calculated as:
 | (1) |
 | (2) |
c.p.m. denotes
the difference in triplicate 22Na c.p.m. between two time points,
and t is the time (in min) between samplings.
Transmission electron microscopy
Fixed RBC samples were washed three times in pH 7.97 saline, embedded in 2%
agarose and stained for 1 h in 1% OsO4. The embedded cells were
washed in 30% ethanol for 10 min and incubated for 1 h in 0.5% uranyl acetate
(in 30% ethanol) followed by 10 min in each of 30, 60, 70, 80, 90 and 100%
ethanol and two washes in 100% acetone to dehydrate samples. The samples were
then incubated in acetone:resin (Araldite; Sil-Mid Ltd, Coleshill, UK) for 30
min in a 1:1 ratio followed by 30 min pure resin, before the final incubation
in moulds for 24–48 h at 60°C in the oven. Sections were cut on an
ultramicrotome (Reichert Ultracut, Leica UK Ltd, Milton Keynes, UK) and
stained for 5 min each with 5% uranyl acetate and 2% Reynold's lead citrate
(Sigma-Aldrich Co. Ltd, Gillingham, UK). The samples were viewed on a Tecnai
electron microscope (FEI 120 kV Tecnai G 2 Spirit BioTWIN; FEI UK Ltd,
Cambridge, UK; equipped with a SIS Megaview III camera; Olympus Soft Imaging
Solutions Ltd, Helperby, UK). The pictures were analysed using AnalySIS Pro
[SIS] software (Olympus UK Ltd, London, UK).
Data analysis and statistics
For each sample, 250–300 RBCs were studied under the light microscope
and assigned as normal (Fig.
2A), sickled (Fig.
1A, Fig.
2C–F) or globular (Fig.
7A). Sickle cells were plotted as a fraction of the total cells.
The saline pH value at which 50% of the RBCs were sickled
(pKapp) was determined by non-linear curve fitting for
each individual, using:
 | (3) |
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;
Perutz and Mitchison, 1950).
This conclusion was based on single-cell polarized light microscopy
techniques, including spectral absorbance scans in the 350–650 nm range
of individual intracellular bars, which corresponded to the typical spectrum
of deoxygenated Hb (Hárosi et al.,
1998). Accepting that the bars consist of polymerised Hb, we refer
to these cells as sickle cells. By contrast, similar samples taken from
rainbow trout and common carp after capture on hook and line contained
95.9±0.03 and 97.0±0.03% RBCs with the flattened, ellipsoidal
shape and smooth surface typical for nucleated RBCs of other vertebrates (e.g.
Nikinmaa, 1990)
(Fig. 1B). After 24 h recovery
in the holding tank, the proportion of whiting sickle cells had significantly
declined to 63.3±5.3%, and after more than 1 week merely
10.6±15.5% of whiting RBCs were sickled
(Fig. 1B). The presence of
sickle RBCs even after more than one week's recovery is likely due to the
stress associated with netting of the fish before blood sampling and could be
avoided in catheterized fish in future studies. When, immediately after
capture, whiting RBCs were washed and incubated in physiological saline for
16–24 h, virtually all cells attained a normal shape similar to that
seen in trout and carp (Fig.
1B). Whiting did not seem to be adversely affected by sickling
during capture stress, as they behaved as other fishes under similar
conditions and resumed feeding after a few days in the holding tank, where
they could be kept in apparently healthy conditions for several months. In
addition, we did not observe any signs of haemolysis after blood sampling, as
confirmed by clear RBC supernatants.
Exhaustive exercise in teleost fishes, such as encountered during capture
stress, is associated with severe blood acidosis (pH 7.3–7.5)
(Holeton et al., 1983;
Jensen et al., 1983;
Nikinmaa and Jensen, 1986;
Milligan and Wood, 1987;
Wood and Milligan, 1987;
Knudsen and Jensen, 1998).
Fig. 2A shows that sickling was
absent in washed whiting RBCs incubated in air-equilibrated saline at 15°C
and pH 7.97, a typical value for normoxic resting animals at that temperature
(Butler et al., 1989;
Perry et al., 1991;
Ultsch and Jackson, 1996;
Larsen et al., 1997;
Knudsen and Jensen, 1998). The
cytosol and nucleoplasm of these RBCs appeared uniform in the TEM images
(Fig. 2B). At lower saline pH
values, however, the appearance of the cells changed dramatically to various
polygonal shapes, triangular and diamond shapes being most abundant, often
with visible bars (Fig.
2C–E), and they looked very similar to whiting RBCs taken
immediately after capture on hook and line
(Fig. 1A). Other cells at low
pH retained the usual ellipsoidal shape but acquired a granular texture
(Fig. 2F). In all these cells,
prominent bundles of filaments in both the longitudinal direction and in cross
section were clearly visible throughout the cytosol and nucleoplasm by TEM
(Fig. 2G). Similar filaments
have been described in RBCs of the Baltic subspecies of Atlantic cod,
Gadus morhua callarias (Thomas,
1971), and in human RBCs from sickle cell patients, where the
filaments are known to consist of polymerised HbS
(Döbler and Bertles,
1968). Sickling in whiting did not appear to increase RBC
fragility as no haemolysis was observed throughout the experiments.
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100% sickle cells upon lowering
saline pH occurred within a rather narrow interval of 0.2 pH units in
cells incubated under N2, air and O2
(Fig. 3). This is reflected in
high apparent cooperativity constants for extracellular protons, which
amounted to at least 4.7, 12.3 and 5.8 in N2, air and O2
equilibrated RBCs, respectively (Table
1). The pH value at half-maximal sickling, or
pKapp, was 7.64±0.07 (mean ± s.d.) in
N2-equilibrated RBCs but decreased significantly to
7.53±0.01 and 7.28±0.02 in air- and O2-equilibrated
RBCs, respectively (Fig. 3; see
Table 1 for individual data).
Thus, at intermediate pH values corresponding to blood pH under severe
exercise, high PO2 values partly protected
against RBC sickling by shifting its occurrence towards lower pH values.
However, at still lower saline pH values, the protective effect of
O2 was lost and all RBCs sickled. Low pH appeared to be a necessary
condition for sickling, as at high saline pH close to resting arterial pH
values deoxygenation per se did not induce sickling
(Fig. 3). Similarly,
hyperosmotic shrinkage of air-equilibrated RBCs at pH 7.97, resulting in a
calculated increase of intracellular Hb concentration of up to 100%, did not
induce sickling, neither shortly after shrinkage (5 min) nor after prolonged
incubation for up to 2 h (not shown).
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100 kPa resulted in a
gradual decline of the sickle cell fraction towards the low values found in
cells incubated under O2 throughout the experiment at that pH
(Fig. 4B).
Exhaustive exercise in teleosts leads to elevated plasma adrenalin and
noradrenalin levels (Primmett et al.,
1986; Berenbrink and Bridges,
1994b; Perry et al.,
1996). Many teleosts possess a ßNHE
(Berenbrink et al., 2005),
whose stimulation causes an increase in intracellular pH
(Nikinmaa, 1982;
Cossins and Richardson, 1985;
Berenbrink and Bridges, 1994b)
and thus may protect against pH-induced sickling in vivo. At pH 7.97,
the ß-adrenergic agonist isoproterenol increased the ouabain-insensitive
Na influx of air-equilibrated whiting RBCs approximately 8–10-fold
relative to basal influx, reaching values of around 400 mmol Na
l–1 RBCs h–1
(Fig. 5A), which is by far the
highest value measured in an analysis of 38 vertebrate species
(Berenbrink et al., 2005). The
isoproterenol-stimulated Na influx was significantly inhibited by the NHE
inhibitor amiloride, which had no effect on basal Na influx. Decreasing saline
pH to 7.03 did not activate any amiloride-sensitive Na influx; however, at the
same pH, isoproterenol induced an amiloride-sensitive Na influx of equal
magnitude as observed at pH 7.97 (Fig.
5A,B).
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0.7 after 5
min, and after 30–45 min practically no sickle cells were present
(Fig. 6A). The cells under
these conditions rather obtained a globular shape and to some extent still
contained aligned Hb filaments (Fig.
7A–C). However, with time, an increasing proportion of these
cells contained ordered filaments only in the nucleus, whereas the cytosolic
filaments were clearly fragmented and irregular
(Fig. 7D,E), showing that
recovery from Hb polymerisation and sickling had taken place. The change from
sickle to globular cells induced by isoproterenol was significantly inhibited
by the NHE inhibitor amiloride (Fig.
6A).
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Indeed, parallel experiments showed that the ouabain-insensitive Na uptake in isoproterenol-stimulated RBCs was an approximate mirror image of the reduction in sickle cell fraction in similarly treated RBCs (Fig. 6A,B). Thus, within the first 15 min, isoproterenol stimulated a profound, linear increase in intracellular Na, whereupon a distinct plateau was reached. In the absence of isoproterenol, Na uptake was significantly reduced throughout the experiment. The ß-adrenergically stimulated Na uptake was significantly inhibited by amiloride (Fig. 6B).
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;
Bunn, 1997;
Christoph et al., 2005),
formation of long, aligned intracellular filaments in whiting RBCs
(Fig. 2G) results in a variety
of cell shapes. These include triangular, diamond-shaped or polygonal cells
with three, four or more bars, respectively
(Fig. 1A,
Fig. 2C–E). Yet other
RBCs retain their normal oval shape but acquire a granular appearance, very
similar to rapidly polymerising human sickle RBCs that contain numerous short
random fibres (Fig. 2F)
(Eaton and Hofrichter, 1987;
Bunn, 1997). In contrast to
human sickle cell disease (Lange et al.,
1951), these deformations are not associated with increased cell
fragility, as haemolysis of sickled whiting RBCs was not observed in
vitro, nor in vivo even immediately after capture. One factor
that could reduce haemolysis and alleviate other ill effects of sickling such
as vasoocclusion would be an increased capillary diameter relative to RBC
size. In goldfish and rainbow trout muscle, the capillary diameter of
3–4 µm is comparable to that in mammals
(Egginton, 2002;
Egginton et al., 2000;
Vink and Duling, 1996),
despite the generally larger size of fish relative to mammalian RBCs
(Prosser, 1950). However,
capillary diameters in the choroid and swimbladder retia mirabilia of various
teleosts are at least 2–3-fold larger than the above values for muscle
(Krogh, 1922;
Scholander, 1954;
Wittenberg and Wittenberg,
1974). Unfortunately, comparable data on fishes where RBC sickling
has been established are missing.
The primary cause of sickling in whiting RBCs appears to be low pH as
deoxygenation alone did not induce sickling of whiting RBCs at pH values
typical for arterial and venous blood in resting fishes (7.8–7.9 at
10–15°C; see above), which contrasts with the situation in human
sickle RBCs (Lange et al.,
1951; Bookchin et al.,
1976; Ueda and Bookchin,
1984). At the same pH, an increase in cellular Hb concentration in
air-equilibrated whiting RBCs by hyperosmotic shrinkage equally failed to
induce sickling, which again differs from human sickle RBCs, where Hb
polymerisation and sickling are extremely dependent on intracellular [Hb]
(Hofrichter et al., 1974;
Bookchin et al., 1976) and
where the solubility of HbS is reduced 100-fold upon deoxygenation, compared
with a 2-fold reduction in deoxygenated normal human HbA
(Perutz and Mitchison, 1950).
It is currently not known whether high pH also prevents shrinkage-induced
sickling in deoxygenated whiting RBCs.
Low PO2 can, however, induce sickling in
whiting RBCs under slightly more acidic conditions than in blood of resting
fishes (less than or equal to approximately pH 7.7). Our in vitro
results suggest that at these pH values, which may be regularly encountered
during routine activity, PO2 values close to
zero in poorly perfused muscles or even mixed venous
PO2 values may be sufficient to induce sickling
(Fig. 3), as already seen in
human sickle RBCs at normal resting pH
(Bookchin et al., 1976).
Dramatic changes in PO2 and pH are
experienced in blood perfusing the swimbladder and choroid retia mirabilia,
where oxygen is delivered by diffusion to the swimbladder and poorly
vascularised retina, respectively. Thus, capillary pH in the swimbladder rete
mirabile of the European eel may drop to 6.5–7.0
(Steen, 1963;
Kobayashi et al., 1990) (for a
review, see Berenbrink, 2007),
whereas capillary PO2 may rise to 120 kPa or
above in the swimbladder rete of the cod, as calculated on the basis of the
values for gas composition in the swimbladder given by Bohr
(Bohr, 1894). In the choroid
rete mirabile of the Atlantic cod, PO2 values
of up to 157 kPa have been measured
(Wittenberg and Wittenberg,
1962). In our study, 100 kPa O2 did not protect from
RBC sickling at pH values close to those measured in the swimbladder rete
mirabile, which suggests that RBC sickling can take place in these
capillaries. However, this may depend on the transit time of RBCs in the rete
mirabile and whether a lag time before sickling is present in whiting RBCs. In
human sickle RBCs, a sufficient delay time before polymer formation prevents a
large fraction of cells from sickling during the time spent in the capillaries
and venous system, where they are most likely to experience conditions of low
PO2 that induce sickling
(Mozzarelli et al., 1987;
Christoph et al., 2005).
Whereas the very low pH values in the retia mirabilia are localized and
only experienced by RBCs during their passage, the general blood acidosis down
to pH 7.3–7.5 caused by strenuous exercise in many teleosts may take
several hours to recover to normal resting pH values (e.g.
Holeton et al., 1983;
Milligan and Wood, 1987).
Under these conditions, a possible delay time before sickling in whiting RBC,
even if amounting to minutes, cannot protect from sickling.
However, strenuous exercise in vivo is associated with the release of adrenalin and noradrenalin into the plasma, which stimulates the RBC ßNHE and thereby maintains or even increases intracellular pH as well as cell volume (see above). We show that the sickle cell fraction in ß-adrenergically stimulated RBCs indeed decreases as a mirror image of ßNHE activation. The previously sickled cells attain a globular, swollen shape in which the cytosolic Hb filaments become very fragmented. It remains to be shown to what extent cell swelling and the increase in intracellular pH contribute to the break-up of Hb filaments and the recovery from sickling.
The mechanism of Hb polymerisation and consequent sickling in whiting RBCs
is presently unknown. The apparent cooperativity constants for low-pH-induced
sickling suggest highly cooperative proton binding to Hb during the sickling
process. In the absence of catecholamines, protons are usually passively
distributed across the RBC membrane, with changes in plasma pH causing
distinctly smaller changes in intracellular pH, as also seen in the close
relative of whiting, the Atlantic cod
(Berenbrink and Bridges, 1994a;
Berenbrink and Bridges, 1994b)
(for a review, see Brauner and Berenbrink,
2007). When sickling is more appropriately related to
intracellular pH changes in the vicinity of Hb, even higher cooperativity
constants can be calculated. For example, using the in vitro
relationship between extracellular and intracellular pH of air-equilibrated
Atlantic cod RBCs (Berenbrink and Bridges,
1994a), the value for the apparent cooperativity constant under
air increases from 12 (Table 1)
to 30 (not shown). This indicates that about 30 proton binding sites
interact during sickling and demonstrates the extreme pH-dependent nature of
the process. Using the same approach, an intracellular
pKapp value for sickling of air-equilibrated RBCs of
7.16±0.01 can be calculated. This suggests the involvement of protein
moieties with pK values in the physiological range, such as
N-terminal C-alpha amino groups or the imidazole ring of histidines
(Berenbrink, 2006).
Indeed, despite the general trend towards a reduction in histidine content
in teleost haemoglobins (Berenbrink et al.,
2005), one of the two different ß globin chains in Atlantic,
Arctic and Polar cod (Verde et al.,
2006) contains two extra histidine residues on the external
surface of the protein. These occur in position 7 of the A-helix (His A7) and
position 1 of the corner between the E and F helices (His EF1) and are absent
in other teleost ß chains (M.B., unpublished). In other teleosts, His A7
is usually substituted by a variety of non-ionisable amino acids and His EF1
is occupied by neutral asparagine. Interestingly, in all three species the
mutation to His EF1 is accompanied by a change of the preceding amino acid in
position E20 from positively charged lysine to negatively charged aspartic
acid (M.B., unpublished), which may facilitate proton binding by His EF1. It
is tempting to speculate that one or both of the above two histidine
substitutions, which are likely to change surface charge, are responsible for
the pH-dependent polymerisation of at least some Hb isoforms in codfishes.
Alternatively, or in addition, the globins of codfishes are also unusually
rich in cysteine, which has been implicated in the in vitro formation
of Hb polymers in some other teleosts
(Borgese et al., 1988;
Borgese et al., 1992;
Fago et al., 1993). Clearly,
the elucidation of the mechanism behind Hb polymerisation awaits detailed
molecular structure–function analyses, ideally on otherwise similar,
polymerising and non-polymerising Hbs.
Hárosi and co-workers
(Hárosi et al., 1998)
reported that the intracellular aggregates in Atlantic cod RBCs consist of
deoxygenated Hb. Many fishes possess Hbs with a highly pH-sensitive oxygen
affinity, which can be deoxygenated by low pH even when equilibrated with 100
kPa O2. This so-called Root effect has been explained as a
stabilization of the low O2 affinity T-state conformation over the
high O2 affinity R-state of Hb by the binding of protons (see
Berenbrink, 2007). Our
observation that fully deoxygenated cells with Hb in the T-state only sickle
at low pH suggests that sickling in whiting does not depend on a
conformational transition from R- to T-state Hb. Further exploration of the
mechanism of Hb polymerisation awaits sequencing of whiting Hb and detailed
molecular structure–function studies.
In vitro, and possibly also in vivo, haemoglobin
polymerisation and RBC sickling appears to be widespread in codfishes,
occurring in whiting as well as in poor cod and pouting
(Yoffey, 1929), Atlantic cod
and haddock (Hárosi et al.,
1998) and pollack, saithe and five-bearded rockling (P.K. and
M.B., unpublished). Curiously, RBCs from all individuals within populations
appear to sickle (Hárosi et al.,
1998; present study), in contrast to the balanced polymorphism for
human sickle cell trait (Aluoch,
1997; Jones,
1997). In the latter case, the disadvantage to homozygote human
carriers of the disease is outweighed by the benefits of heterozygote carriers
against infection with malaria parasites. Our in vitro results
suggest that activation of the ßNHE in vivo may protect whiting
RBCs to a certain degree from sickling, at least at intermediate pH values.
Thus, the disadvantages of possessing a Hb with the potential of polymerising
and causing sickling appear reduced in whiting when compared with homozygous
human sickle cell patients. It is less clear whether RBC sickling in fishes
also offers an advantage like in human heterozygote carriers. Fishes are
infected by numerous parasites, including several protozoan species in the
blood stream as well as in the RBCs
(Hemmingsen and MacKenzie,
2001; Davies and Johnston,
2000). The intracellular RBC parasites include a virus causing
viral erythrocytic necrosis (VEN), which is widespread in Atlantic cod, chum
salmon and Atlantic herring but has adverse effects such as haemorrhage,
haemolysis, anaemia and increased mortality only in the latter two species
(Smail, 1982), whose RBCs (in
contrast to Atlantic cod) are not known to sickle. Even more interestingly,
VEN infection in Atlantic cod is associated with the occurrence of
intracellular fibrillar material, which looks similar to the Hb filaments
observed in the present work and was absent in uninfected RBCs
(Appy et al., 1976). It is
tempting to speculate that, in analogy to the human condition
(Ayi et al., 2004),
VEN-infected RBCs have a higher tendency to sickle, which may enable selective
removal from the circulation or impair intracellular virus propagation.
The present study on whiting is the first to suggest a physiological mechanism that protects against RBC sickling, which remains a lethal disease in humans. We can only speculate about the selective advantages that led to the fixation and spread of the initial mutation causing sickling through populations of the ancestral species. However, the disadvantages of this mutation seem to have been sufficiently suppressed by the presence of a ßNHE, such that the mutation persisted during several speciation events, which gave rise to the current diversity of codfishes. This mechanism offers a unique opportunity to study how the problem of RBC sickling has been managed successfully by nature through evolutionary time.
Abbreviations and symbols
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  L. Blackburn WHITING SICKLE CELLS J. Exp. Biol., October 1, 2007; 210(19): ii - ii. [Full Text] [PDF]
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indicates filaments cut in
longitudinal direction, and 
indicates filaments in cross section.
; N=4). Data points on the
right-hand side of the dotted line indicate values for cell suspensions kept
at pH 7.97 under air (•) or pH 7.61 at a
PO2 of 
); saline pH of 7.03 (•); saline pH
of 7.03 with 10–5 mol l–1 isoproterenol
alone (
indicates cells at pH 7.03 only that are significantly
different (P<0.05) from samples at saline pH 7.03 with
isoproterenol. Values are means ± s.d. N=8 (A) and 5 (B).
Error bars are in some cases smaller than the symbol.