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J Biol Chem, Vol. 275, Issue 4, 2342-2348, January 28, 2000
From the It has been reported that free hemoglobin (Hb)
reacts with NO at an extremely high rate (KHb
~107 M Despite the well documented importance of nitric oxide, the
transfer of NO from the producing cell to the target is poorly understood, because the free radical NO can be degraded in a variety of
reactions. In particular, NO reacts with deoxy- and oxyhemoglobin (deoxyHb and oxyHb,
respectively)1 at a very high
rate (1, 2) to form nitrosyl Hb (HbNO) and met hemoglobin (metHb),
respectively. If Hb in the RBC behaved like Hb in dilute solution, the
half-life of NO in the blood (which contains about 12-15
mM heme) would be only about 1 µs. For such a rapid
reaction, it would seem likely that a large portion of NO produced from
the endothelium would be scavenged by the blood.
Indeed, in vivo and in vitro evidence suggests
that free Hb is an effective NO scavenger that can deplete NO. For
example, infusion of free Hb solution into experimental animals or
human subjects results in hypertension (3, 4), most likely due to the reaction of NO with oxyHb in the circulation (3). Modeling
analyses (5-7) also showed that if endothelium-produced NO reacted as
rapidly with blood as it does with free Hb, the NO concentrations in
vascular smooth muscle would be too low to activate soluble guanylate
cyclase, the primary target of NO. Mathematical modeling based on
diffusion theory and in vitro measurements of kinetic
constants (5-8) have confirmed that Hb could effectively scavenge
endothelial produced NO and mitigate its effect. Because 3-10
µM free Hb can abolish NO-mediated vasodilation in
vitro (4, 9), it is unclear how NO can exercise its vasoregulatory function with 12-15 mM Hb concentration in the blood. This
discrepancy is described as the "NO-Hb paradox," and is one of the
most important questions regarding the physiological and pathological
functions of NO.
NO produced from the endothelium must go through four steps to react
with the RBC-enclosed Hb (Fig. 1): (a) diffusion through the
RBC-free (or depleted) region, created by intravascular flow (10), to
the bulk solution; (b) diffusion from the bulk solution to
the RBC surface; (c) diffusion across RBC membrane; and
(d) diffusion and reaction inside RBC cytosol. The first two
are affected by extracellular factors, whereas the last two steps are
affected by intracellular components intrinsic to RBC itself. In
discussing the diffusion flow of molecules, it is customary to define
the diffusional resistance as a measure of the diffusional barrier. Resistance is defined as driving force divided by flux; where, for
diffusion, the driving force is the concentration difference, and the
flux is the molar flow/unit area. Although this definition can be made
precise, the term diffusional resistance is often most useful as a
concept. The diffusional resistance, if significant, results in the
formation of layer around the RBC, within which the NO concentration is
much smaller (e.g. less than 10%) than the bulk of the
solution (Fig. 1b). This layer is termed the diffusion (or
unstirred) layer, and the thickness of this layer is proportional to
the diffusional resistance. In reality, the layer has no actual boundary, and the concentration profile is continuous. However, the use
of the diffusion layer in discussion is conceptually convenient.
It is now recognized that the NO consumption rate by RBCs is much
slower than that expected based on the in vitro reaction rate of NO with free Hb (9, 11). The in vivo quenching of NO
by RBC is first reduced by the RBC-free zone (Fig. 1a)
created by the flow field (7, 9, 12). However, even without the RBC-free zone, the NO consumption by RBC is still about 500-1000 times
slower than the NO reaction rate with free Hb (9, 11). This low
reaction rate has been attributed to diffusion resistance from the bulk
solution to the surface of RBC (11). It has been suggested that the
diffusional resistance is mainly associated with a diffusion or
"unstirred" layer around each RBC (Fig. 1b).
Unfortunately, experimental difficulties have prevented distinguishing
the contribution of the extracellular diffusion resistance (Fig. 1b) from transmembrane (Fig. 1c)
or intracellular (Fig. 1d)
resistance. For in vitro experiments measuring the uptake of
NO (11, 13) or oxygen (14-16) by dilute erythrocyte suspensions, it
has been impossible to separate the effect of the extracellular from
transmembrane or intracellular diffusion resistances. Therefore, it has
been reasonable to make the simplest assumption and assign all
differences between RBCs and the equivalent solution of free Hb to
extracellular diffusional resistance (11, 15). In the dilute RBC
suspensions that are accessible experimentally, the extracellular
diffusional barrier around the cell is likely to be quite significant
and perhaps dominant whether or not additional cellular factors are
involved. Nonetheless, the RBC may possess a specific intrinsic
(transmembrane or intracellular) barrier that limits the NO consumption
rate.
To probe the existence of such mechanisms, the external diffusion
resistance must be distinguished from any intrinsic barrier that
reduces the NO consumption rate. To accomplish this, we designed an
experiment that exploited the competition for NO between RBC and
extracellular Hb. This design allowed us to use high concentrations of
RBC and, most importantly, avoid much of the extracellular diffusional
resistance and concentration limitations of traditional kinetic
measurements. In studies of oxygen uptake, about half of the resistance
is associated with oxygen transfer from the bulk solution to the
surface of RBC, and the other half is attributed to the diffusion
resistance from oxygenated hemoglobin within the RBC (17). With our
technique, the extracellular diffusional resistance can be eliminated,
because NO is generated uniformly throughout the continuous phase and a
high hematocrit can be used to reduce the distance that NO needs to
travel. In addition, only a small quantity of NO is used, and thus the
intracellular diffusional resistance through previously reacted Hb is
minimized. The NO consumption in the extracellular phase is monitored
directly by measuring the NO/Hb reaction product; NO oxidizes oxyHb to
metHb. The uptake rate of NO by RBCs is calculated by the use of a
kinetic equation derived for this experimental condition from the
extracellular metHb and oxyHb concentrations with or without RBCs present.
Chemicals--
The NO donor, spermine NONOate
(N-[4[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl]-1,3-propanediamine)
was purchased from Alexis Corp. (San Diego, CA). Some tests used SIN-1
(3-morpholinosydnonimine HCl) purchased from Alexis Corp. or DPTA
NONOate (3-3'-(hydroxynitrosohydrazono) bis 1-propanamine) purchased
from Cayman Chemical (Ann Arbor, MI). Dulbecco's phosphate-buffered
saline, Sephadex G-25 (Amersham Pharmacia Biotech), DEAE-Sephadex A-50
(Amersham Pharmacia Biotech), Drabkin's solution, and Tris base were
purchased from Sigma. The mixed bed ion exchange resin AG 501-X8 was
purchased from Bio-Rad.
Preparation of RBCs--
Bovine blood was collected in
heparinized (5 IU/ml) tubes. The plasma and the buffy coat were removed
following centrifugation at 800 × g for 20 min. The
cells were resuspended and immediately washed four times in Dulbecco's
phosphate-buffered saline (0.122 molar NaCl, 0.030 molar
KH2PO4 + Na2HPO4, 2 mg/ml glucose, pH 7.4, 290 mosmol/kg). After each wash, the cells were
centrifuged at 800 × g for 10 min to separate the RBCs
from the supernatant. The erythrocytes were purified by filtration
through a mixture of microcrystalline cellulose and Preparation of Oxyhemoglobin--
OxyHb solution was prepared
from bovine RBCs using the modification of Riggs' procedure (19).
Purified bovine RBCs were centrifuged (800 × g, 10 min), and the supernatant discarded. The cells were lysed by diluting
with 2 volumes of ice-cold deionized water, freezing in liquid
nitrogen, and then thawing at room temperature. Cell debris was removed
by centrifugation at 15,000 × g for 30 min in a
refrigerated centrifuge. Salts were removed by passing through a bed of
AG 501-X8 resin and then eluting at 4 °C through a column of
Sephadex G-25 that had been pre-equilibrated with 20 mM
Tris acetate + 0.5 mM EDTA, pH 8.3. The Hb was stored on ice and used within three days.
NO Donor--
Spermine NONOate was prepared as a stock solution
of 1-2 mM. Approximately 0.4 mg of spermine NONOate was
measured into a 1.5-ml micro-centrifuge tube. The donor was diluted in
1 ml of ice-cold, isotonic NaCl solution containing 0.001 N NaOH. The concentration was verified by diluting the concentrate in 0.01 N NaOH
and measuring the absorbance at 250 nm (extinction coefficient 7500 cm The Competition Experiment--
Each test consisted of four
solutions, run simultaneously: oxyHb in buffer, oxyHb with NO donor in
buffer, free oxyHb in a suspension of RBCs, and free oxyHb in a
suspension of RBCs with NO donor. A solution of oxyHb was prepared by
adding oxyHb concentrate to Dulbecco's phosphate-buffered saline to
produce the desired Hb concentration (normally 10 µM).
This solution was used without further dilution for the buffer samples.
RBC suspensions were produced by centrifuging purified RBCs (800 × g, 10 min), removing the supernatant, and diluting the
packed cells with the oxyHb solution to produce the desired hematocrit
(normally 15%). The samples were equilibrated (1 h) at 25 °C, and
then 15 ml of each solution were loaded into a 20-ml syringe
(Becton-Dickenson, Inc.). For the samples containing NO donor, spermine
NONOate was added immediately before loading into the syringe. The NO
donor concentration was computed based on the total volume of solution.
Immediately after the solutions were added to the syringes, the initial
1-ml sample was taken, and the syringes were placed on a rocking mixer (Clay Adams, Fisher Scientific) to keep the cells uniformly dispersed. The initial sample was centrifuged (20 s at 10,000 × g) to separate the RBCs, and the supernatant was assayed for
oxyHb and metHb using a Beckman DU 640 spectrophotometer. Subsequent
1-ml samples taken at time intervals were assayed in the same manner.
Hemoglobin Assay--
The concentrations of metHb and oxyHb were
determined spectrophotometrically by fitting a set of "basis
spectra" to the measured spectra by means of linear regression. All
samples and basis spectra were scanned from 380 to 700 nm using a
Beckman DU 640 spectrophotometer. To obtain the pure components for the
basis spectra, six samples of oxyHb were obtained as above using
freshly drawn bovine blood. Residual metHb was removed by loading the
Hb onto a DEAE-Sephadex A-50 column (2.5 by 50 cm), maintained at
4 °C and previously equilibrated with 50 mM Tris
acetate, pH 7.6/0.5 mM EDTA (19). Hb was eluted with this
buffer, and the concentration of the eluent was determined using
Drabkin's solution to convert the Hb to cyano-metHb (19). Each sample
was serially diluted to 10, 5, 2.5, and 1.25 µM and
scanned, and the digitized spectra were stored on computer. The
averaged spectrum was obtained by use of linear regression. These same
oxyHb samples were converted to metHb (without change in concentration)
by adding a slight excess of NO gas to each sample, and the average
spectrum was obtained in the same way.
Kinetic Modeling and Data Interpretation--
The solutions that
do not contain NO donor ensured that the Hb autooxidation and RBC lysis
were negligible. The ratio of the reaction rates between the NO-Hb and
the NO-RBC reactions is determined by comparing the NO production rate
with the metHb production rate in the oxyHb plus RBC suspension with NO
donor. The rate of NO production can be determined from the control
experiment, free oxyHb plus NO donor in buffer. Because only free oxyHb
is present (in excess) in this control experiment, the total amount of
metHb produced is the total amount of NO generated. Because the
NO-oxyHb reaction rate is known, the ratio can be used to compute the
NO-RBC reaction rate. For each solution, the oxyHb consumed and the
metHb produced in the extra erythrocyte space are measured at various
time points.
The NO-RBC reaction rate constant is determined from kinetic equations
describing NO uptake by oxyHb and RBCs. These equations assume that the
extracellular diffusional resistance is negligible, and thus the NO
concentration in the solution is homogeneous. Therefore, deviations
from the model may suggest that the NO concentration is nonhomogeneous,
which in turn indicates the significance of extracellular diffusional
resistance. By using the pseudo-steady-state approximation,
d[NO]/dt
If the extracellular diffusional resistance is negligible (which is the
model assumption), Equation 1 will fully describe the experimental
data, and the measured KRBC will be independent of hematocrit, extracellular Hb, and NO donor concentration. Otherwise, the measured KRBC will decrease as hematocrit
decreases, as extracellular Hb increases, and as NO donor concentration decreases.
The Competition Experiment Allows the Measurements Of
KRBC at High Hematocrit--
As stated above, previous
experimental techniques for measuring KRBC were
limited to very dilute RBC suspension and could not distinguish
external diffusion limitation from intrinsic barrier in the RBC. To
avoid these limitations, we designed the following competition
approach. In this set of experiments, known concentrations of
RBC-contained oxyHb and free oxyHb competed for a limiting amount of NO
generated by NO donor in solution. If oxyHb in the RBC reacted with NO
as fast as free oxyHb in the solution, then NO would be consumed by the
RBC and free oxyHb at the same specific rate. On the other hand, if
free oxyHb consumed NO faster than the RBC-enclosed oxyHb, then NO
would be consumed by the two species in a ratio determined by their
reaction rates. This ratio was determined by measuring the metHb and
oxyHb in the extra erythrocyte space. The metHb in the RBC was
continuously regenerated by metHb reductase systems and could not be
used to indicate NO consumption by RBC. However, the NO consumed by the
RBCs could be calculated from mass balance using the total amount of NO
generated and the amount consumed by the free Hb in the solution. The
rate of NO generated was determined from a control experiment, where
free oxyHb was present (without RBC) in excess to the NO donor. Kinetic equations were formulated to describe the experimental system, and the
solution is shown in Equation 1.
The results from a typical Hb-RBC competition experiment are shown in
Fig. 2. In this experiment, the sample
contained 10 µM spermine NONOate as the NO donor, 7.5 µM extracellular oxyHb (heme concentration), and 7.8%
hematocrit (1.5 mM RBC heme). Fig. 2a depicts
the time course of metHb production in the extra erythrocyte space.
Fig. 2b shows the K plot of this and three additional
experiments using the same sample of blood. The formation of the
straight line in this plot indicates that the result was consistent
with the kinetic model. This straight line also indicates that the reaction rate of the RBCs was constant throughout the test, suggesting that no internal diffusion layer formed during the test to slow NO
uptake. The ratio
KRBC/KHb = 0.00089 ± 0.00005 (mean ± S.E., n = 4) is determined
from slope of this line. The result indicates that RBC reaction with NO
is about three orders of magnitude lower than free Hb, consistent with
other evidence (9, 11).
The Competition Experiment Allows the Measurement of
KRBC Without External Diffusion Limitation--
The
generation of NO from a homogeneous NO source, soluble NO donor,
reduces the barrier of external diffusion. However, it cannot eliminate
the barrier of external diffusion, particularly at low concentrations
of RBC suspension. Because RBCs are in the particulate form, they
compete less favorably with the free Hb, which is in the homogeneous
phase. This disadvantage is amplified at low hematocrit, where the
external diffusion barrier is larger. Therefore, we predicted that at
low hematocrit the KRBC measured is not an
intrinsic quantity of the NO-RBC reaction rate. Rather, it is reduced
by external diffusion. Hence, the apparent KRBC measured will decrease as the hematocrit decreases. Conversely, the
apparent KRBC will increase as the external
diffusion limitation is reduced by increasing hematocrit, until it is
eliminated. In that region, the KRBC measured is
independent of hematocrit, and it represents the intrinsic rate
constant of the NO-RBC reaction. In this high hematocrit region, the
NO-RBC reaction is limited by intrinsic factors such as RBC membrane
permeability or intra-erythrocytic conditions.
This reasoning is supported by experimental results. In Fig.
3, we varied the hematocrit in the
competition experiments and showed the effect of external diffusion
limitation exactly as predicted. When the hematocrit was greater than
10%, KRBC calculated from the kinetic model
(Equation 1) is constant, suggesting that the RBC consumption rate is
fully described by the kinetic model that does not include any
extracellular diffusion resistance. On the other hand, when hematocrit
was smaller than 5%, KRBC calculated from the
kinetic model decreased as hematocrit decreased. This decrease in the
rate constant suggests that extracellular diffusional resistance, which
is not considered in the model, becomes significant.
These experiments concluded that when the hematocrit is higher than
about 10%, the KRBC measured is essentially
free of external diffusion limitation. Under this condition, the
thickness of the diffusion layer (Fig. 1b) surrounding the
RBC is very small, much smaller than the intercellular distance.
Therefore, external diffusion is no longer the limiting process. When
the hematocrit is lower than 10%, the diffusion layer thickens, and
thus external diffusion (Fig. 1b) is more important compared
with other processes (Fig. 1, c and d). Note that
this conclusion does not necessarily imply that external diffusion is
unimportant under physiological conditions. The competition experiment
uses a homogeneous NO generating system (soluble NO donor), where
external diffusion can be eliminated at high hematocrit. However, in
blood vessels, NO is generated from endothelium, which has a very
different mass transfer barrier. It is interesting to note that under
high hematocrit (without external diffusion limitation), the
KRBC is still about 800 times lower than
KHb, indicating that the NO-RBC reaction rate is
intrinsically lower than the NO-free Hb reaction rate. Therefore, the
limitation is attributed to intrinsic factors in RBCs, such as
transmembrane or intracellular limitations.
Varying the hematocrit provides a sensitive probe of the near RBC
space. In a very dilute RBC suspension, much of the extracellular Hb
and NO production is far from the cell. As the hematocrit increases, the average spacing between cells decreases. If a diffusion layer exists, the proportion of extracellular volume associated with the
diffusion layer increases as the hematocrit increases. The average NO
concentration in the diffusion layer is low so less metHb will be
produced. Thus, the apparent rate would depend on hematocrit. On the
other hand, if NO uptake is controlled by factors intrinsic to the RBC,
then the NO concentration in the extracellular space will be nearly
uniform. Then there would be little change in apparent rate as
hematocrit increases. This behavior is reflected in Fig. 3,
a and b, where, as the hematocrit increases
beyond 7.5%, there is little change in the apparent reaction rate coefficient.
If the apparent RBC reaction rate is independent of RBC concentration,
as it would be if the resistance of the diffusion layer were
negligible, then the plot of
KRBC/KHb × Hct
versus Hct will be a straight line with slope
KRBC/KHb. This is indeed
the behavior seen in Fig. 3c. Here the presence of a
non-zero intercept indicates that the apparent reaction rate depends on
Hct in dilute suspensions, where the diffusion layer becomes important
(11).
Extracellular Hb or NO Donor Concentrations Do Not Affect the Value
of KRBC/KHb--
It is expected that
increasing extracellular Hb or NO donor concentrations will increase
the metHb formation in the extracellular space. However, the
extracellular NO consumption will follow the kinetic model (Equation 1)
only if the system is homogeneous, namely, no extracellular diffusion
resistance, as the kinetic model assumes. In this case,
KRBC derived from the kinetic equation will be
independent of extracellular Hb or NO donor concentration, as the model states.
On the other hand, if the diffusion layer outside of RBC becomes
significant, the system is nonhomogeneous, which means that the NO
concentration in the layer is much smaller than the bulk solution, and
the homogeneous kinetic model fails. In this case, the
KRBC derived from Equation 1 will decrease when
the extracellular Hb increases or NO donor decreases, because the RBC
competes much less favorably than the model assumes in this case.
Fig. 4 displays a competition experiment
in which the concentration of the extracellular Hb is varied from 5 to
40 µM with Hct held constant at 15.6%. The time course
of metHb formation varies significantly as seen in Fig. 4a.
However, using our K plot analysis, Fig. 4b, the data fall
on the same straight line yielding
KRBC/KHb = 0.00116 ± 0.00006. Similarly, Fig. 5 shows a
competition experiment in which the NO donor varied from 5 to 15 µM. Again, even though the time course of reaction
product changes significantly (Fig. 5a), the K plot analysis
yielded the same reaction rate
(KRBC/KHb = 0.00126 ± 0.00007). Because the reaction rate ratio does not depend on
extracellular Hb or NO donor concentrations over these ranges, we
conclude that the effect of any diffusion layer present under these
experimental conditions was small.
The above experiments determined the NO consumption by RBCs
without the influence of an external diffusion barrier. Interestingly, under this experimental condition, the NO consumption by RBCs is still
much slower than by an equivalent concentration of free Hb. This result
suggests that either the transmembrane diffusion or intracellular
diffusion/reaction is also rate-limiting. Because these factors are
intracellular components, the data suggest the existence of intrinsic
factors in the erythrocytes that control the overall NO consumption rate.
A variety of experimental and theoretical studies of gas uptake
kinetics by intact RBCs has been published since the original measurements by Hartridge and Roughton (16). These studies have greatly
increased our understanding of the kinetics of fast biological processes. However, accounting for the transport resistance provided by
a time-varying diffusion layer is still a formidable challenge. This
has been the fundamental problem for studies of dilute RBC suspensions.
As stated by Merchuck et al. (15), " ... measurements made on diluted red cell suspensions cannot provide a good model for
oxygen transport in normal blood, where high hematocrits tend to
diminish boundary layer effects." It is likely that the same statement can be made for NO uptake.
Recent studies (17, 20) have tried to account for this diffusion
resistance using mathematical models of the RBC, which include
hydrodynamic effects contributing to the external diffusion layer.
Using this technique, they were able to obtain good agreement with
rapid mixing, stopped-flow data. However, the thickness of diffusional
layer estimated for these stopped-flow experiments, 13 µm for human
RBCs at 50% saturation, could not apply under physiological
conditions, because this layer is much thicker than the average
intracellular distance between RBCs.
The competition experiment overcomes many of the experimental
difficulties in measuring the uptake kinetics of RBCs. The primary cause of the diffusion layer is the depletion of NO immediately adjacent to the cell from insufficient NO transport from the bulk fluid. The competition experiment minimizes this diffusion layer, because NO is continually produced from the NO donor. Furthermore, under physiological conditions, close proximity of RBCs ensures that
the concentration gradient associated with one RBC affects the
surrounding RBCs (21). Because a relatively high hematocrit is used in
the competition experiment (15%, which is 30-40% of the normal
physiological value) this effect is taken into account. Note that the
NO donor used should not enter the RBCs. If the NO donor is transported
into RBC, then the NO consumption rate by the RBC measured by the
competition experiment would be higher than the true value. In this
study, we used spermine NONOate as an NO donor, which does not enter
RBC at an appreciable rate (22). The amount of polyamines that might
enter RBC within the time scale of our experiment is too small to
affect our results. Indeed, use of other NO donors, such as SIN-1 and
DPTA NONOate, gave the same results, suggesting that uptake of the NO
donor by RBC is insignificant.
Although this work has focused on overcoming the influence of the
external diffusion layer, the technique presented here should also
minimize the influence of an internal diffusion layer formed from
reacted intracellular Hb. In rapid-mixing experiments where the RBC
oxygen saturation is increased over a large range, this internal
diffusion layer can contribute about half of the resistance to oxygen
uptake (17). With our technique, very little metHb is formed in the
cells, typically, 1-5 µM or 0.004-0.02% of cytosolic Hb is oxidized, an amount readily reduced by the metHb reductase system
in the time scale of the experiment. We have verified experimentally that there is little effect from the internal diffusion layer. If a
diffusion layer forms over the course of the experiment, its presence
would cause KRBC/KHb to
decrease as the cells uptake NO. Consequently, the K plot line would
flatten with time. However, Figs. 2b, 3a,
4b, and 5b show that the K plot line is straight, indicating a constant uptake rate. The resistance of the internal diffusion layer would also depend on NO donor concentration, because more metHb would be produced in the RBC at higher donor concentration. However, Fig. 5b shows that
KRBC/KHb is independent
of NO donor concentration. Furthermore, we have performed competition
experiments using RBCs in which 10-88% of the cytosolic Hb has been
oxidized to metHb by pretreatment with NO donor or phenylhydrazine. We found that the value for
KRBC/KHb was not
significantly reduced (data not shown), suggesting that the uptake rate
was essentially independent of internal metHb. The same result was
reported previously (11).
The low NO consumption rate by the RBC may be viewed as a result of
isolation of the bulk of Hb from exposure to NO. However, such an
effect cannot be achieved simply by forming a concentrated Hb solution
within an NO-permeable membrane. In this case, the only possible, but
unlikely, diffusional resistance must come from the intracellular
space. It can be argued that one scenario for diffusional resistance is
the formation of a metHb shell near the membrane. As Hb is oxidized to
metHb by NO, this metHb shell increases, which in turn reduces the NO
consumption rate. However, we and others (11) have shown that NO
consumption rate by RBC is independent of metHb concentration in the
cell. It may also be argued that the metHb formed in RBC quickly
establishes a metHb layer with constant thickness and maintains a
constant steady-state effect on the rate of NO consumption. In this
case, specific biochemical mechanisms, such as the binding of metHb to
the membrane, must be involved. However, even if such a mechanism
exist, it is highly unlikely that a metHb shell can reduce the NO
consumption to any significant degree.
There are a number of differences between this experimental design and
those using bolus-dissolved NO solutions (11, 15, 20). Perhaps most
importantly, NO is released slowly into the solution similar to the
endothelial release of NO. The NO concentration to which the RBCs in
the competition experiment are exposed is quite low. In contrast, Liu
et al. (11) added a bolus of 1 µM NO to a
dilute RBC suspension that contains about 1-2 µM heme. Kinetic modeling computations (results not shown) suggests that the NO
concentration is typically much less than 1 nM. The effect of NO on the erythrocytes is small; less than 0.5% of the
intracellular Hb will react with the NO. This means that there is no
intracellular resistance caused by diffusion through reacted Hb. In the
competition experiment, NO is released continuously at low
concentrations over a time scale of minutes to hours, which is more
physiological than sudden exposure to a high NO concentration. In the
competition experiment, RBCs encounter much milder conditions than in
the stopped-flow experiment. Therefore, transient disruptions or
alterations of membrane structure, which might be caused by high shear
during rapid mixing, are avoided. Furthermore, as stated above, the
hematocrit used in this experiment (15%) is close to the physiological
value (35-50%), in contrast to the very dilute (typically,
0.01-0.15% Hct) RBC suspension used previously (11, 20). The low
concentration of RBC suspension may artificially amplify external
diffusion resistance that may not be as significant under physiological conditions. Our data are remarkably close to that obtained by Liu
et al. (11). We reason that their data may also represent an
intrinsic limitation in RBC consumption of NO. Although they used a low
hematocrit ( Despite the complications of the diffusion layer, there appear to be no
significant inherent resistances for oxygen uptake in the RBCs; most of
resistance can be assigned to purely diffusional processes inside and
outside the RBC. This has been confirmed through modeling experimental
oxygen uptake data (15, 17, 20) and from experiments using packed RBCs
(23). Apparently, this is not the case for NO, which is surprising,
because NO and O2 are similar in size, shape, and solubility.
Why should NO and O2 behave differently? There are
fundamental differences in the physical treatment of RBCs in the
competition experiment as compared with other methods of measuring
uptake rate. The competition experiment is performed under mild
circumstances in which the RBC membrane is not greatly disturbed. In
rapid-mixing experiments, RBCs are subjected to intense shear and
turbulent stresses, which could alter membrane and membrane skeletal
structure, as well as Hb-membrane interactions. Membrane perturbation
may also occur in preparing thin layers of packed cells, where intimate cell-cell contact could result in membrane stretching and shear, even
under gentle handling. Furthermore, there are significant differences
in the chemical treatment of the cells. To the best of our knowledge,
the gas uptake rate for all previous experiments has been measured by
monitoring the conversion of cytosolic Hb. This means that a
significant portion of the cytosolic Hb must react with the gas during
a short time period. For oxygenation, this is physiological, but for NO
uptake, it is not. Except under pathological conditions such as sepsis,
the reaction products between NO and cytosolic Hb are too low to
measure. Therefore, the uptake rate for very low NO concentration is
important, but previously inaccessible, so there are no directly
comparable data. Even for oxygen uptake, it may not be that the
membrane is so permeable, but that membrane resistance is comparable to
intracellular and extracellular resistance (23). The comparable
situation, membrane resistance to very small concentrations of oxygen
for completely deoxygenated cells, is not known.
There are at least two possible mechanisms that would decrease the
uptake of NO by RBCs: lower than expected membrane permeability or
reduced reaction rate between NO and the cytosolic Hb. The latter is
ruled out by the observation that NO consumption rate by RBC is
independent of intracellular oxyHb concentration
(11).2 The diffusion
coefficient of NO in lipid membranes seems too high (24) for the RBC
membrane to provide significant resistance. However, the measured
diffusion coefficient appears to depend on the position within the
membrane, decreasing away from the center of the membrane (24).
Therefore, how rapidly NO passes through the membrane from an aqueous
phase is still unclear. Although we do not expect the RBC membrane to
be impermeable to NO, the transmembrane diffusion rate of NO may be
relatively slow compared with the very fast reaction rate between NO
and the Hb inside of RBC. How the intra-erythrocyte environment affects
the NO reaction with Hb is also an open question.
S-nitrosylation of In summary, there appear to be multiple mechanisms that reduce the NO
consumption rate in vessels under physiological conditions. The first
is the presence of flow, which forms a RBC-free zone (Fig.
1a) near the vessel wall and increases the diffusion barrier (7, 9, 12). This phenomenon has recently been verified experimentally
(9). The second is the diffusion layer (Fig. 1b) surrounding
the RBC (11), although its role under physiological conditions remains
to be determined. The third, suggested by this work, is an intrinsic
factor of RBCs that reduces the NO consumption rate, such as
transmembrane diffusion (Fig. 1c) or intracellular limitation (Fig. 1d). In view of the fact that NO
consumption of NO is independent of oxyHb concentration (11), the
limitation due to intracellular reactions is highly unlikely.
Obviously, more work remains.
*
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.
2
M. W. Vaughn, K.-T. Huang, L. Kuo, and
J. C. Liao, unpublished results.
The abbreviations used are:
Hb, hemoglobin;
RBC, red blood cell;
Hct, hematocrit.
Erythrocytes Possess an Intrinsic Barrier to Nitric Oxide
Consumption*
,
Department of Chemical Engineering,
University of California, Los Angeles, California 90095, the
§ Department of Chemical Engineering, and the
¶ Department of Medical Physiology, Texas A&M University,
College Station, Texas 77843
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 s1) and
that the red blood cell (RBC) membrane is highly permeable to NO. RBCs,
however, react with NO 500-1000 times slower. This reduction of NO
reaction rate by RBCs has been attributed to the extracellular
diffusion limitation. To test whether additional limitations are also
important, we designed a competition test, which allows the
extracellular diffusion limitation to be distinguished from
transmembrane or intracellular resistance. This test exploited the
competition between free Hb and RBCs for NO generated in a homogenous
phase by an NO donor. If the extracellular diffusion resistance is
negligible, then the results would follow a kinetic model that assumes
homogenous reaction without extracellular diffusion limitation. In this
case, the measured effective reaction rate constant,
KRBC, would remain invariant of the hematocrit,
extracellular-free Hb concentration, and NO donor concentration.
Results show that the KRBC approaches a
constant only when the hematocrit is greater than 10%, suggesting that
at higher hematocrit, the extracellular diffusion resistance is
negligible. Under such a condition, the NO consumption by RBCs is still
500-1000 times slower than that by free Hb. This result suggests that
intrinsic RBC factors, such as transmembrane diffusion limitation or
intracellular mechanisms, exist to reduce the NO consumption by RBCs.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (27K):
[in a new window]
Fig. 1.
Four stages of NO diffusion from endothelium
to Hb in RBCs. a, diffusion from the endothelium to the
bulk of RBC suspension. The flow field tends to drive suspended
particles (RBCs) away from the vessel wall. Therefore, blood flow
creates an RBC-free (or depleted) region near the vessel wall (10),
which reduces NO scavenging by RBCs through increasing the distance
that NO has to diffuse (9). b, diffusion of NO from the bulk
of solution to the RBC membrane. Conceptually, the diffusional
resistance can be represented by a boundary layer (or undisturbed layer
(11)), where the concentration of NO is much smaller than the bulk
concentration. The thickness of the boundary layer depends heavily on
the hematocrit and the flow field. c, diffusion across the
RBC membrane. d, diffusion within the RBC cytoplasm to reach
the reactive Hb.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
cellulose
(18).
1 M1). The spermine NONOate
solution was prepared fresh daily and kept in the dark and on ice until
use. Some additional tests used DPTA NONOate and
3-morpholinosydnonimine HCl prepared in the same manner.
0, solution of these equations can he simplified to
the following equation,
in which square brackets denote concentration. Here Hct is
hematocrit, [metHb]c is the metHb
concentration in the cell-free control, [metHb]ex is the
metHb concentration in the extracellular space of the RBC suspension,
[oxyHb]ex and [totalHb]ex are the oxyHb and
initial total Hb concentrations in the extracellular space of the RBC
suspension, respectively. KRBC and
KHb are the rate constants
(M
![([<UP>metHb</UP>]<SUB>c</SUB>−(1−<UP>Hct</UP>)[<UP>metHb</UP>]<SUB><UP>ex</UP></SUB>)<UP>/</UP>[<UP>oxyHb</UP>]<SUB><UP>RBC</UP></SUB>=(K<SUB><UP>RBC</UP></SUB>/K<SUB><UP>Hb</UP></SUB>)<UP> ln </UP>([<UP>totalHb</UP>]<SUB><UP>ex</UP></SUB><UP>/</UP>[<UP>oxyHb</UP>]<SUB><UP>ex</UP></SUB>)](/content/vol275/issue4/fulltext/2342/img002.gif)
(Eq. 1)
1s1) of the NO-RBC and
NO-free Hb reactions, respectively. KRBC is defined based on the Hb concentration in the solution as if all the
RBCs are lysed. The Hb concentration (mM, heme) in the
solution can be calculated as 19.1 × Hct. By plotting the experimental data according to the above equation, we obtained a straight line of
slop eKRBC/KHb and an
intercept of zero. This plot (termed the Kplot) allowed the
verification of the experiment and the determination of the rate
constant. During the experiment, if RBC lysis, determined by an
increase in total Hb in extra-erythrocytespace, contributed more than
6% of the extraerythrocyte Hb concentration, then the data were discarded.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (20K):
[in a new window]
Fig. 2.
A competition experiment determines the RBC
reaction rate. a, the NO uptake rate of RBCs is deduced
from the time course of metHb production in the extracellular space and
metHb in a cell free sample. In this test, the extracellular Hb
concentration was 7.5 µM and the Hct was 7.8%. The error
bars for the RBC sample (n = 4, maximum S.E. ± 0.03)
are smaller than the symbols, and thus cannot be seen on the plot.
b, using the K plot analysis, we determined the ratio of
reaction rates, KRBC/KHb = 0.00089 ± 0.00005. The metHb production for these four
independent RBC-containing samples are representative of the 100+ tests
we have performed. The symbols represent four independent runs.
View larger version (17K):
[in a new window]
Fig. 3.
By varying hematocrit, the competition
experiment provides a probe of conditions near the erythrocyte. In
a dilute suspension of RBCs, NO produced far from the cell is consumed
by extracellular Hb, so NO uptake becomes diffusion-limited. As the
concentration of RBCs increases, the "true" reaction rate is
measured. a, K plot of the competition experiment data for
1.6-15.7% Hct. For 7.8 and 15% Hct, 9 µM extracellular
oxyHb and 10 µM NO donor were used. For 1.6 and 3.4% Hct
tests, extracellular oxyHb and NO donor concentration were scaled down
to 2 µM, so that the local NO concentration remain
roughly the same. The slope of the line is
KRBC/KHb. For more
concentrated RBC suspensions (7.8-15.7% Hct) the relative reaction
rate does not depend on Hct. This trend is seen clearly in
b, which shows
KRBC/KHb as a function of
Hct. These data were obtained from a plot (a) combined with
several additional experiments. Error bars indicate S.E.,
n = 10 for 7.8% and 15.7% Hct and n = 2 for 3.4% Hct. For 4.4% and 8.9% Hct, 9 µM
extracellular oxyHb and 10 µM NO donor were used,
n = 3. KRBC/KHb shows little
change at higher Hct indicating little diffusion layer resistance. The
graph (c) shows data from the graph in b
replotted. KRBC/KHb × Hct, a measure of the NO consumption by RBCs, is shown as a function of
Hct. The straight line indicates
KRBC/KHb does not depend
on Hct. This is expected if the NO uptake is controlled by a barrier
intrinsic to the erythrocytes rather than by a diffusion layer.
View larger version (25K):
[in a new window]
Fig. 4.
Altering the environment near the cell by
varying the extracellular Hb suggests that the NO uptake by
erythrocytes is dominated by factors intrinsic to the RBCs. The
time course of metHb production in the extracellular space is shown in
a. Here the extracellular Hb is varied from 5 to 40 µM for RBCs at 15.6% Hct. For 5 µM
extracellular Hb the NO donor concentration was 5 µM,
otherwise the NO donor concentration was 10 µM. The K
plot of these data is shown in b. The slope of the best fit
line is KRBC/KHb = 0.00116 ± 0.00006. The K plot analysis shows that the reaction
rate of the RBCs is independent of extracellular Hb, as expected if NO
uptake is limited by a barrier in the RBC.
View larger version (27K):
[in a new window]
Fig. 5.
The competition experiment is further
validated by varying the NO donor concentration. a, the
time course of metHb production in the extracellular space. Here the NO
donor concentration is varied from 5 to 15 µM for RBCs at
15.6% Hct with an extracellular Hb of 10 µM.
b, the K plot of these data. The slope of the best fit line
shows KRBC/KHb = 0.00126 ± 0.00007. This value is independent of NO donor, as
expected if NO uptake is limited by a barrier in the RBC.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
0.01%), their experimental setup allows very efficient
mixing, which reduces the external diffusion limitation. However, it
seems that the difference between the resistance offered by a diffusion
layer and that from an intrinsic barrier in the RBC would be hard to
discern in their experiments.
-93cys has been proposed to play an
important role in transferring of NO (25, 26). However,
S-nitrosylation does not affect the NO consumption rate and
cannot explain the results shown here. It has also been proposed that
low concentrations of NO in the blood under a partially oxygenated
condition can be effectively and rapidly sequestered by Hb through the
formation of (Fe-NO)2(Fe)2 (27). The
formation of an NO adduct has been suggested to be much faster than
previously reported when the NO to Hb ratio is low (28). However,
regardless of the nature of the NO consumption reaction by RBC, these
reactions cannot explain the results presented here.
FOOTNOTES
To whom correspondence should be addessed: Dept. of Chemical
Engineering, 5531 Boelter Hall, University of California, Los Angeles,
CA 90095-1592, Tel.: (310) 825-1656; Fax: (310) 206-4107; E-mail:
liaoj@ucla.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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T. H. Han, D. R. Hyduke, M. W. Vaughn, J. M. Fukuto, and J. C. Liao Nitric oxide reaction with red blood cells and hemoglobin under heterogeneous conditions PNAS, May 28, 2002; 99(11): 7763 - 7768. [Abstract] [Full Text] [PDF]
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K.-T. Huang, T. H. Han, D. R. Hyduke, M. W. Vaughn, H. Van Herle, T. W. Hein, C. Zhang, L. Kuo, and J. C. Liao Modulation of nitric oxide bioavailability by erythrocytes PNAS, September 25, 2001; 98(20): 11771 - 11776. [Abstract] [Full Text] [PDF]
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M. J. Coffey, R. Natarajan, P. H. Chumley, B. Coles, P.-R. Thimmalapura, M. Nowell, H. Kuhn, M. J. Lewis, B. A. Freeman, and V. B. O'Donnell Catalytic consumption of nitric oxide by 12/15- lipoxygenase: Inhibition of monocyte soluble guanylate cyclase activation PNAS, June 20, 2001; (2001) 141136098. [Abstract] [Full Text] [PDF]
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A. Bloodsworth, V. B. O'Donnell, and B. A. Freeman Nitric Oxide Regulation of Free Radical- and Enzyme-Mediated Lipid and Lipoprotein Oxidation Arterioscler. Thromb. Vasc. Biol., July 1, 2000; 20(7): 1707 - 1715. [Abstract] [Full Text] [PDF]
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M. J. Coffey, R. Natarajan, P. H. Chumley, B. Coles, P.-R. Thimmalapura, M. Nowell, H. Kuhn, M. J. Lewis, B. A. Freeman, and V. B. O'Donnell Catalytic consumption of nitric oxide by 12/15- lipoxygenase: Inhibition of monocyte soluble guanylate cyclase activation PNAS, July 3, 2001; 98(14): 8006 - 8011. [Abstract] [Full Text] [PDF]
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N. M. Tsoukias and A. S. Popel Erythrocyte consumption of nitric oxide in presence and absence of plasma-based hemoglobin Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H2265 - H2277. [Abstract] [Full Text] [PDF]
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