Department of Veterinary Preclinical Sciences, University of Liverpool, Liverpool L69 7ZJ, United Kingdom
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ABSTRACT |
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The effect of oxidants on
K+-Cl cotransport (KCC) was
investigated in equine red blood cells. Carbon monoxide
mimicked O2. The substituted benzaldehyde, 12C79 (5 mM),
markedly increased O2 affinity. In N2, however,
O2 saturation was low (<10%) but KCC remained active.
Nitrite (NO2
) oxidized heme to methemoglobin (metHb).
High concentrations of NO2
(1 and 5 mM vs. 0.5 mM)
increased KCC activity above control levels; it became O2
independent but remained sensitive to other stimuli.
1-Chloro-2,4-dinitrobenzene (1-3 mM) depleted reduced glutathione
(GSH). Prolonged exposure (60-120 min, 1 mM) or high concentrations (3 mM) stimulated an O2-independent KCC
activity; short exposures and low concentrations (30 min, 0.5 or 1 mM)
did not. The effect of these manipulations was correlated with changes in GSH and metHb concentrations. An oxy conformation of Hb was necessary for KCC activation. An increase in its activity over the
level found in oxygenated control cells required both accumulation of
metHb and depletion of GSH. Findings are relevant to understanding the
physiology and pathology of regulation of KCC.
oxygen; nitrite; 1-chloro-2,4-dinitrobenzene; erythrocytes
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INTRODUCTION |
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POTASSIUM-CHLORIDE
COTRANSPORTERS (KCCs) are present in a variety of tissues,
including red blood cells (19). A number have been cloned
and share homology with the family of electroneutral cation-Cl cotransporters, whose members include the
Na+-K+-Cl
cotransporter (NKCC)
and the Na+-Cl
cotransporter
(30). Four KCCs have been sequenced to date from human and
other tissues; that of red blood cells is probably KCC1 (15, 18,
31, 35).
KCC in vertebrate red blood cells responds to a number of potential
physiological stimuli, including cell swelling, H+, and
urea (19, 27). In normal high-K+-containing
red blood cells, which have an outwardly directed chemical gradient for
the transported ions, the activated cotransporter will mediate net KCl
efflux, with water following osmotically. In some cells, it contributes
to cell shrinkage following swelling and has therefore been implicated
in regulatory volume decrease (19). In addition,
inappropriate activity of KCC will result in excessive KCl loss, red
blood cell shrinkage, elevation of Hb concentration, and also
cytoplasmic viscosity (38). Such events will eventually
cause deleterious rheological effects, including increased vascular
resistance. They may also elevate plasma K+ concentration.
KCC activity is inappropriately elevated in certain hemoglobinopathies
[notably, in cells containing hemoglobin S (HbS) and in
-thalassaemics; Refs. 23 and 34] and certain enzyme deficiencies
and treatment with oxidants such as nitrite (NO2
;
Refs. 1 and 33), diamide (27), and hydrogen peroxide (H2O2; Refs. 4 and 33). Understanding
regulation of KCC in red blood cells is therefore important both
physiologically and pathologically.
Recently, it has become apparent that physiological O2
tension (PO2) represents an important regulator
of KCC (9, 12). In red blood cells from many vertebrate
species, KCC is inhibited at low PO2 values and
then becomes largely refractory to other stimuli such as cell swelling
and low pH (5, 7, 13, 32), although species differences
are apparent (see Ref. 12 for a review). A widely held hypothesis
suggests that the cotransporter is controlled by the conformation of
Hb, being activated by the oxy or relaxed form assumed on combination
with O2 (5). A similar explanation has been
proposed for the effect of carbon monoxide (CO) and
NO2, which support KCC activity in deoxygenated fish
red blood cells, because CO-Hb and methemoglobin (metHb), respectively,
assume the same conformation as oxyhemoglobin (oxyHb) (22,
21).
The action of various reagents on KCC activity has also been correlated
with their redox potential. NO2 is a powerful oxidant
that will oxidize Hb to metHb and deplete the red blood cells of
reduced glutathione (GSH). Other oxidants (e.g., acetylphenylhydrazine
or H2O2; Refs. 4 and 33) and reagents such as
1-chloro-2,4-dinitrobenzene (CDNB), which removes GSH nonoxidatively
(26), also stimulate KCC. The high-KCC activity and
abnormal O2 dependence, which is characteristic of sickle cells, may be due to their low content of GSH (1, 25, 33). Because GSH represents a major component of the antioxidant mechanisms of the red blood cell, its depletion represents another form of oxidative stress and will allow the accumulation of metHb secondarily. How these oxidative stresses affect the O2 dependency of
the cotransporter has not been established.
In this paper, we examine the responses of the equine red blood cell
KCC. This species was chosen because equine red blood cells have a high
capacity for KCC and minimal K+ fluxes through other
pathways and because their O2 dependence has been
characterized in detail (6, 20, 37). We have compared the
effects of several agents that cause Hb to assume an oxy shape, O2 per se, CO, and 12C79, a left-shift reagent acting at a
site away from the heme group (2), together with
NO2, which directly generates metHb, and CDNB, an
agent that removes GSH and thereby indirectly results in formation of
metHb. The effect of these manipulations on the activity and
O2 dependence of KCC was examined and correlated with
changes in GSH and metHb. We found that oxy conformation of Hb was a
prerequisite for KCC activity and that oxidative stress at an
unidentified site amplified this activity. Findings are discussed in
relation to the pathology of oxidant toxicity, red blood cell defects
affecting GSH metabolism (e.g., glucose 6-phosphate dehydrogenase
deficiency and sickle cell disease), and the mechanism by which
O2 regulates membrane transporters.
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METHODS |
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Chemicals. CDNB, MOPS, N-ethylmaleimide (NEM), ouabain, salts, and staurosporine were purchased from Sigma Chemical (Poole, Dorset, UK). Calyculin A was purchased from Calbiochem (Nottingham, UK), 86Rb was from DuPont-NEN (Stevenage, UK), and CO and N2 were obtained from BOC (Guilford, UK). 12C79 was a gift from Glaxo Wellcome (Stevenage, UK).
Solutions.
The standard saline solution was composed of (in mM) 145 NaCl, 5 glucose, and 10 MOPS (pH 7.4 at 37°C; 290 ± 5 mosmol/kgH2O). For experiments in which
Cl dependence of K+ influx was examined,
Cl
was substituted with NO3
. To
investigate the effects of anisotonic saline, osmolality was adjusted
by addition of distilled water or hypertonic sucrose; when required, pH
was altered by addition of HNO3 or NaOH. Stock solutions of
ouabain (10 mM) were prepared in distilled water and used at a final
concentration of 100 µM. Stock solutions of NEM (100 mM) were
prepared daily in distilled water; those of calyculin A and
staurosporine were prepared in DMSO and frozen until required. Finally,
CDNB was dissolved in methanol (100 mM). In all cases, controls and
cells treated with inhibitors or other reagents were exposed to the
same concentrations of solvents (methanol or DMSO, whose final
concentrations did not exceed 0.5%).
Sample collection and handling. Blood samples were obtained from horses kept at the Department of Veterinary Clinical Sciences and Animal Husbandry (Leahurst, UK) by jugular venepuncture into heparinized vacutainers and prepared as previously described (37).
Tonometry. Before influx or O2 saturation measurements were made, red blood cell suspensions were incubated at about 40% hematocrit in glass tonometers (Eschweiler, Kiel, Germany) flushed with gas mixtures of the appropriate O2 tension (air replaced with N2 using a Wösthoff gas mixing pump), warmed to 37°C, and fully humidified through three humidifiers before delivery. For experiments with CO, cells were treated with CO for 5 min (after which O2 saturation was reduced to <1%) before incubation in the tonometers.
metHb, GSH, and O2 saturation. metHb content was determined colorimetrically by following the method of Hegesh et al. (17) and expressed as a percentage of total Hb. GSH (expressed in mM) was assayed following the procedure described by Beutler (3). O2 saturation was determined using the method of Tucker (39).
12C79. Stock solutions of 12C79 (282 mM) were made daily in Tris base (500 mM) and diluted in the appropriate saline to give a final concentration of 5 mM. Cell samples at 40% hematocrit were incubated with 12C79 (5 mM) in air for 15 min before they were placed in tonometers to adjust PO2. Measurement of both O2 saturation and K+ influx were made in the presence of 12C79 (5 mM). Control samples and those with 12C79 had the same extracellular and intracellular pH.
K+ influx.
K+ influx was measured at 37°C using 86Rb as
a tracer for K+ (11). 86Rb was
added in 150 mM KNO3 to give a final K+
concentration of 7.5 mM. Ouabain (100 µM) was present in all experiments, obviating any K+ influx through the
Na+-K+-ATPase. Hematocrit was measured either
by the cyanomethemoglobin method or by microhematocrit determination.
Influxes are expressed as millimoles of K+ per liter of
cells per hour. In horse red blood cells, in the presence of ouabain,
KCC represents the predominant K+ transport pathway;
however, in several experiments, Cl dependence
(substituted with NO3
) of K+ influx was
determined. Also, although in these experiments K+ influx
was measured because of the outwardly facing chemical gradient for
KCl, a net loss of ions will occur through this pathway (as
indicated by the cell volume measurements). Because this concept often
causes confusion, in much of the text influx has been replaced with transport.
Measurement of cell volume. Cell water content was determined by the wet weight-to-dry weight method of Borgese et al. (5) and expressed as milliliter per gram of dry cell solids.
Statistics. Data are presented as means ± SD for n replicates for single experiments representative of at least two others on samples from different animals or as means ± SE for n experiments.
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RESULTS |
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Effect of CO.
We have demonstrated previously that KCC in equine red blood cells is
O2 dependent: stimulation by swelling, H+, and
moderate (but not high) concentrations of urea only occurs if
PO2 is sufficiently high. This is confirmed in
the experiments shown in Figs. 1 and 2. Equine red blood cells swollen
anisosmotically by suspension in hypotonic saline (260 mosmol/kgH2O) had a high K+ transport in air
(Fig. 1) and shrank with time (Fig.
2); when equilibrated with
N2, K+ transport was minimal (<10% of that at
high PO2), and there was no decrease in cell
water content.
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Effect of 12C79.
12C79 is a substituted benzaldehyde developed to increase the
O2 affinity of human Hb, thereby protecting against
sickling. The effect of 5 mM 12C79 on O2 saturation of
equine red blood cells is shown in Fig.
3. The relationship of O2
saturation against PO2 was shifted to the left;
the PO2 required for half-maximal O2 saturation fell from 25 ± 2 to 8 ± 2 mmHg
(mean ± SD, n = 4). This marked increase in
O2 affinity was very similar to the effect of 12C79 on
human red blood cells (14). Notwithstanding the increase
in O2 affinity, at very low PO2
values, O2 saturation was low (< 10%) and cells were dark
in color, implying that Hb was in the deoxy form.
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Effect of nitrite.
In the next series of experiments, we examined the effect of
NO2 on K+ transport in equine red blood
cells. It has been suggested that NO2
acts by
oxidizing Hb to metHb, which has a conformation like that of oxyHb. If
this is the case, its action should be identical with that of CO.
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Effect of CDNB.
CDNB reacts with GSH in the presence of
glutathione-S-transferase, thereby depleting the red blood
cell of GSH. The effect of various concentrations of CDNB on
K+ transport in equine red blood cells is shown in Figs. 9
and 10. In Fig. 9, oxygenated or
deoxygenated cells were treated with 1 mM CDNB at a hematocrit of 40%
for 30 min and then K+ transport was measured in the
absence of CDNB. In these experiments, the magnitude of K+
transport was similar in control and CDNB-treated cells in both air and
in N2. After a 3-h exposure to 1 mM CDNB, however, KCC activity was stimulated over oxygenated control levels and only inhibited about 50% by deoxygenation (data not shown). In the second
series of experiments, cells at 40% hematocrit were treated with
progressively higher concentrations of CDNB (1, 2, and 3 mM) for 30 min, and transport was measured at one-tenth this hematocrit and CDNB
concentration. As the concentration of CDNB was increased, K+ transport was stimulated over oxygenated control levels
(Fig. 10). K+ transport
(expressed as a percentage) in N2 compared with that in air
increased from 7% in control cells to 19, 56, and 66% in cells
treated with 1, 2, and 3 mM CDNB, respectively, so that the transport
also became progressively less dependent on O2.
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Determination of GSH and metHb.
Finally, we determined the effect of CO, NO2, 12C79,
and CDNB on levels of GSH and metHb under the conditions used for
K+ transport measurements. Results are given in Table
1. In air and N2, and after
treatment with CO and 12C79, metHb levels were low (<1%) and reduced
GSH concentrations were high (about 3 mM). After treatment with
NO2
, metHb levels increased progressively with
NO2
concentration. At low concentrations of
NO2
, the percentage of metHb was higher in air than
in N2; the reverse occurred at higher NO2
concentrations. GSH levels were reduced by NO2
but,
even at 5 mM, NO2
levels in deoxygenated cells (in
N2) remained at about 50% those in control cells.
NO2
therefore appeared to oxidize Hb before lowering
the GSH concentration. With CDNB, GSH levels were most sensitive, being
reduced by about 50% by 0.5 mM CDNB and reduced totally at higher
levels, whereas metHb only increased at the highest CDNB concentration
(15% in N2 with 3 mM CDNB). At 1 mM CDNB, metHb levels
were 1.7, 9.9, and 16.4% after 10 min, 1.5 h, and 3 h,
respectively, whereas at all time points GSH was depleted (<7%).
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DISCUSSION |
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In this study, we compared the effects of several manipulations
that affect KCC. First, we looked at reagents that bind to the
O2 site of Hb: O2 per se and CO. Second, we
looked at the substituted benzaldehyde 12C79, which interacts with
other sites in the Hb molecule but again increases O2
affinity and causes it to assume the oxy conformation. Third, we
examined an oxidant, NO2, which oxidizes the heme
from Fe2+ to Fe3+ to produce metHb. Fourth, we
looked at the reagent CDNB, which does not directly alter Hb but has
removal of GSH as its primary action, although loss of the antioxidant
capacity of the red blood cell will secondarily encourage the
accumulation of metHb. We correlated the effects of these manipulations
on the activity and O2 dependence of KCC and on GSH and
metHb levels. Our findings show that an oxy conformation of Hb was
required for KCC activation. An increase in KCC activity over control
levels in oxygenated cells, however, did not correlate with either
oxyHb conformation or depletion of GSH alone but rather required both
accumulation of metHb and depletion of GSH, implying that oxidative
damage was required at some as yet unidentified target. Finally, the lack of additivity between NO2
and staurosporine or
NEM, and the continued sensitivity to calyculin A, may imply a common
site of action on the regulatory phosphorylation cascade that controls
KCC upstream of the calyculin-sensitive phosphatase.
O2-dependent membrane transporters and oxidants.
A number of membrane transporters in vertebrate red blood cells respond
to PO2 (9, 12). The effect occurs
over physiological PO2 levels and is selective.
Most work has involved inorganic ion cotransporters and
countertransporters (9, 12), but ion channels and other
transport systems (e.g., amino acid transporters; Ref. 24) can also be
O2 dependent. As a general rule, regulatory volume decrease
pathways (e.g., KCC) are stimulated by O2 and regulatory
volume increase pathways [e.g., NKCC or Na+/H+
exchanger (NHE)] are stimulated by deoxygenation (9).
Although the effect is potentially significant both physiologically and pathologically (as reviewed in the introduction), little is known about
its mechanism. A role for Hb has been proposed (29). The differential binding of oxyHb and deoxyHb to the cytoplasmic tail of
band 3 (8) may modulate membrane permeability via a
signaling pathway involving other parts of the cytoskeleton or via
regulatory enzymes (glycolytic, kinases, phosphatases), which also
associate with this site (28). Most evidence in support of
this hypothesis comes from work with fish red blood cells. Thus, for
trout NHE and KCC, CO mimics O2 (5, 29);
alteration of Hb O2 affinity in carp red blood cells
through modulation of intracellular pH causes changes in KCC activity
that correlate with the conformation of Hb (21).
NO2 activates KCC in deoxygenated carp red blood
cells, which is explained because metHb has the oxy conformation
(21, 22). In addition, other oxidants (e.g.,
acetyl-phenylhydrazine and H2O2; Refs. 4 and
33) and nonoxidizing agents, such as CDNB (26), increase
KCC activity to levels greater than those in control cells. All are
associated with a decrease in GSH and sometimes elevation in metHb, but
their O2 dependence has not been investigated.
CO and 12C79. In the first series of experiments, we examined the effect of CO. This gas binds to heme with an affinity about 200-fold greater than O2, forming CO-Hb, which has the same conformation as oxyHb. As observed for fish, CO-treated equine red blood cells behaved like oxygenated ones with regard to K+ transport via KCC or the change in cell volume that it mediates, irrespective of PO2. Responses to a number of PP-PK inhibitors (NEM, staurosporine, calyculin A) were also unaffected. These observations are all consistent with CO mimicking O2 and agree well with previous work on fish red blood cells.
12C79 is one of a series of substituted benzaldehyde compounds, originally developed as antisickling agents (2). These compounds combine with Hb, forming Schiff bases with amino groups; those with the terminal amino groups of theNO2 and CDNB.
NO2
oxidizes Fe2+ to Fe3+,
forming metHb, which has an oxy conformation. If this is its only
significant effect, NO2
should behave like CO.
Indeed, like CO and 12C79, NO2
also stimulated KCC in
deoxygenated cells, making it O2 independent. Unlike either
of these, however, it increased KCC activity over the control levels
observed in untreated, oxygenated cells. Cells dehydrated faster when
treated with NO2
compared with oxygenated controls or
CO-treated cells, and this effect was fully Cl
dependent,
consistent with mediation via KCC. In addition to being stimulated by
NO2
, the transporter also remained sensitive to other
stimuli, such as H+, urea, and cell volume. These features
will potentially exacerbate the deleterious effects of
NO2
toxicity. MetHb, which cannot transport
O2, will cause tissue hypoxia, anaerobic metabolism, and
acidosis, further stimulating the cotransporter. These stimuli are
thought to act via the phosphorylation cascade that regulates KCC1.
Pharmacologically, enzymes of this cascade can be modulated by PK
inhibitors (e.g., staurosporine and NEM) to stimulate KCC1 activity or
by PP inhibitors (e.g., calyculin A) to inhibit it. The response to
such stimuli in cells treated with NO2
, the lack of
additivity between NO2
and staurosporine or NEM, and
the continued sensitivity to calyculin A may imply a common site of
action on the regulatory phosphorylation cascade that controls KCC
(10). If this is the case, NO2
must act
upstream of the calyculin A-sensitive phosphatase.
OxyHb, GSH, and control of KCC.
Table 2 summarizes the effects of the
various manipulations on transporter activity together with their
effects on metHb and GSH levels. KCC was activated in deoxygenated
cells by CO and 12C79 and by the higher concentrations of
NO2 and CDNB. CO and 12C79 directly cause adoption of
the oxyHb conformation either through effects on heme or elsewhere.
Nevertheless, in red blood cells treated with 12C79 and incubated in
N2, most Hb is in the deoxy form. Low levels of CDNB,
despite completely depleting cells of GSH, did not activate the
transporter in N2 nor did they increase its activity in
oxygenated red blood cells. At the higher levels of
NO2
and CDNB, when KCC became O2
independent, significant accumulations of metHb were present, again
indicative of Hb in the oxy form, and GSH was depleted. Higher
concentrations of CDNB and NO2
also increased KCC
activity above that observed in oxygenated controls. Similar results
were obtained with prolonged exposure (3 h) to 1 mM CDNB. In agreement
with our findings, for elevated KCC activity in LK sheep red blood
cells treated with NO2
, both accumulation of metHb
and depletion of GSH (1) are shown, whereas in human red
blood cells, activity of KCC in response to a number of oxidants did
not correlate with GSH depletion alone (33).
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ACKNOWLEDGEMENTS |
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We thank J. E. Cox for provision of equine blood samples.
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FOOTNOTES |
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This work was supported by the Wellcome Trust.
M. C. Muzyamba holds a Beit Fellowship and ORS award.
Address for reprint requests and other correspondence: J. S. Gibson, Dept. of Physiology, St. George's Hospital Medical School, Univ. of London, Cranmer Terrace, Tooting, London, SW17 0RE, UK (E-mail:jsgibson{at}sghms.ac.uk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 6 December 1999; accepted in final form 13 April 2000.
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