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Departments of 1Pharmacology and 2Integrative Physiology, The University of Tokushima School of Medicine, Tokushima 770-8503, Japan
Submitted 10 December 2002 ; accepted in final form 19 March 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-nitro-L-arginine methyl ester
(L-NAME; 120 mg · kg–1 ·
day–1) for 1 wk to obtain HbNO-depleted blood.
When this method was applied to the analysis of untreated fresh whole blood,
the five-coordinate state of HbNO was observed. HbNO concentration in
pentobarbital-anesthetized rats was augmented (change in [HbNO] =
1.6–5.5 µM) by infusion of L-arginine (0.2–0.6 g/kg)
but not D-arginine. Using this method, we attempted to evaluate the
effects of temocapril on HbNO dynamics in an L-NAME-induced rat
endothelial dysfunction model. The oral administration of L-NAME
for 2 wk induced a serious hypertension, and the HbNO concentration was
reduced (change in [HbNO] = 5.7 µM). Coadministration of temocapril dose
dependently improved both changes in blood pressure and the systemic HbNO
concentration. In this study, we succeeded in measuring the blood HbNO level
as an index of NO by the EPR HbNO signal subtraction method. We also
demonstrated that temocapril improves abnormalities of NO dynamics in
L-NAME-induced endothelial dysfunction rats using the EPR HbNO
signal subtraction method.
nitric oxide; hemoglobin-nitric oxide adduct; electron paramagnetic resonance; temocapril
Because of increasing interest in the effect of NO, an accurate method is required for the measurement of this radical. Several methods for the quantitation of NO such as chemiluminescence (46), methemoglobin formation (23), and electron paramagnetic resonance (EPR) spectroscopy of nitrosyl-metal complexes (15) have been developed (3). The Griess and cGMP methods have been widely used to measure NO production in vivo (35, 62) and in vitro (34, 61), but these values do not always reflect the concentration of NO, because of their poor specificities for blood NO. EPR spectroscopy is a technique used for the detection and measurement of free radical species. EPR methods using endogenous and exogenous spin-trapping agents, which stabilize NO as a polyatomic spin adduct, have been developed (1, 4, 29, 41, 72).
In the circulation, NO exists as relatively stable hemoglobin (Hb)-NO adduct (HbNO) (48), which means that the amount of HbNO may reflect the blood NO concentration.
Hb is known to bind to NO, resulting in the formation of HbNO, which has a characteristic EPR spectrum, and hence is used as a spin-trapping agent for NO in lipopolysaccharide (LPS)-treated (67, 69) and NO donor-administered (10, 28) animals.
In addition, HbNO signal has been reported to be successfully detected in untreated animals and human blood using the EPR method (39, 51, 55, 68). However, there were still some difficulties in obtaining a fine HbNO signal, because of existing of paramagnetic compounds that give a strong EPR signal overlapping the same region to HbNO (5, 7, 10, 13, 19, 42). In the present study, we developed an improved method to detect this HbNO signal in whole blood by EPR spectroscopy. To overcome the difficulties mentioned above, we introduced the EPR signal subtraction method, which is based on the subtraction of the EPR spectrum of HbNO-depleted whole blood from that of each sample. In addition, changes of blood NO levels induced by angiotensin-converting enzyme inhibitor (ACEI) treatment were assessed to confirm the relevance of this method.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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L-Arginine hydrochloride (L-arginine) and
D-arginine hydrochloride (D-arginine) were purchased
from Wako Pure Chemical Industries (Tokyo, Japan).
N-nitro-L-arginine methyl ester
(L-NAME) was from Nacalai Tesque (Kyoto, Japan). Temocapril was
kindly provided by Sankyo (Tokyo, Japan). Other reagents were of the highest
grade available from Wako Pure Chemical Industries. NO gas was obtained
commercially from Sumitomo Seika Chemicals (Osaka, Japan), and higher oxides
such as NO2 and NO3 (NOx) were removed by
passing through a trap containing 1 M KOH. An NO-saturated aqueous solution
was prepared by bubbling NO gas for 15 min through water that had been
previously deoxygenated by bubbling with purified argon gas for 30 min
(32). Temocapril (0.5 or 5
mg/ml) was suspended in carboxyl methyl cellulose (0.5%) and orally given to
the animals.
Animals
Male Sprague-Dawley rats (12 wk old) were obtained from Japan SLC (Shizuoka, Japan) and kept in plastic cages at a controlled temperature (25°C) under controlled lighting condition (12:12-h light-dark cycle). The animals were fed a commercial diet and had access to tap water ad libitum until the day of the experiments. All animal care and treatments were conducted in accordance with the guidelines of the animal use and care committee of the University of Tokushima.
Animal Experiments
Protocol 1: preparation of HbNO for calibration curves. Rats were anesthetized with pentobarbital sodium (40 mg/kg body wt ip). Venous blood was collected from the vena cava, and aliquots (1 ml) were mixed with various amounts of the NO-saturated solution at 0°C. Immediately, the sample was sucked into a disposable 1-ml plastic syringe and then immersed and stored in liquid nitrogen until the EPR measurement.
Protocol 2: arginine infusion. Rats were anesthetized with pentobarbital sodium as in protocol 1. The femoral vein and artery were cannulated with polyethylene catheters for administration of the drug and the recording of systolic blood pressure (SBP), respectively. SBP was measured using an Amplifier Case 7746 (NEC San-ei Instruments; Tokyo, Japan) equipped with a pressure transducer and recorded using a WT-6856 (Nihon Koden; Tokyo, Japan). After surgery, rats were kept for 30 min to stabilize the hemodynamic parameters, and the drug infusion was then started using a syringe pump (model 100, KD Scientific) from the femoral vein. Rats were divided into five groups, and one group (n = 4) each received saline, 0.2 g/kg L-arginine, 0.2 g/kg D-arginine, 0.6 g/kg L-arginine, or 0.6 g/kg D-arginine at a rate of 0.1 ml/min for 10 min. During experiments, rats were kept at 37°C by a water jacket. One minute after cessation of the infusion, venous blood was collected from the vena cava with a 1-ml plastic syringe and stored in liquid nitrogen until use.
Protocol 3: L-NAME-induced endothelial dysfunction
model. Rats were divided into four groups [group 1, control;
group 2, L-NAME; group 3, low-dose temocapril (an
ACEI); and group 4, high-dose temocapril;
Fig. 1]. Each group had 12 rats
except for the control group (n = 8), and the animals received tap
water containing distilled water (group 1) or L-NAME (1
g/l, groups 2–4) from day 0 to day 13. Water
consumption was 
35 ml · rat–1 ·
day–1 in group 1 and 25 ml ·
rat–1 · day–1
in the L-NAME-treated groups (groups 2–4), and these
values were constant throughout the experiment. On the basis of the drug
solution intake, the effective daily dose of L-NAME was estimated
to be 65 mg · kg–1 ·
day–1. Temocapril was administered twice a day by
oral gavage (group 3, 2 mg · kg–1
· day–1; group 4, 20 mg ·
kg–1 · day–1)
from day 7 to day 15. Groups 1 and 2 were given
vehicle instead of temocapril. Body weight, SBP, and heart rate (HR) were
measured at days 0, 7, 14, and 16. SBP and HR were measured
by the tail-cuff method using a BP-98A (Softron; Tokyo, Japan). To evaluate
the endothelial function for maintenance of blood NO levels, we stopped the
L-NAME treatment 2 days before the experiment. Rats were
anesthetized with pentobarbital sodium, and venous blood was obtained on
day 16 (see Protocol 2: arginine infusion).
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Protocol 4: preparation for NO-depleted whole blood. To deplete NO
in blood, eight rats were administered tap water containing L-NAME
(2 g/l) for 1 wk. The daily dose of L-NAME was 120 mg ·
kg–1 · day–1,
and it was constant throughout the experiment. Rats were euthanized, and
venous blood was taken (see Protocol 2: arginine
infusion).
EPR Measurement and Data Processing
All EPR measurements were carried out in liquid nitrogen. The frozen sample was directly transferred into a liquid nitrogen-filled quartz finger dewar, which was placed in the cavity of the EPR measurement device. A JES TE 300 ESR spectrometer (JEOL; Tokyo, Japan) with an ES-UCX2 cavity (JEOL) was utilized to collect EPR spectra at the X band (9.5 GHz). Each sample was measured four times and normalized using ESPRIT 432 software (JEOL) to improve the signal-to-noise ratio. Typical EPR conditions were as follows: power, 20 mW; frequency, 9.045 GHz; field, 3,200 ± 250 gauss; mod width, 6.3 gauss; sweep time, 60 min; time constant, 1 s; and amplitude, 250. Spectra were stored on an IBM personal computer for analysis.
The HbNO signal was obtained by subtracting the EPR spectrum of HbNO-depleted whole blood from that of each sample (14, 32). EPRMAIN computer software obtained from the National Institute of the Environmental Health Sciences (http://epr.niehs.nih.gov/pest.html) was used to accomplish this calculation.
Statistical Analysis
All data are expressed as means ± SE. Data were analyzed by a two-way ANOVA, followed by the Bonferroni test for comparisons between groups. P < 0.05 was accepted as statistically significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Typical EPR spectra obtained from venous whole blood of untreated rats and
L-NAME-treated NO-depleted rats are shown in
Fig. 2, A and
B, respectively. Most of the broad EPR signal in
Fig. 2A consists of
serum ceruloplasmin (g = 2.06, where g is the spectroscopic
splitting factor of HbNO). The EPR signal of HbNO consists of the components
of the gX, gY, and
gZ factors as shown in
Fig. 3 (where X, Y,
and Z indicate the direction of magnetic field and the
Z-axis is parallel with it), and it overlaps with same field as
ceruloplasmin. Therefore, the principal of the EPR signal subtraction method
of HbNO is to bring the EPR signal of HbNO into prominence
(14) by subtraction of the
NO-depleted EPR signal from objective one. This manipulation is accomplished
using special EPRMAIN software. The EPR signal of the Z-factor of
HbNO gives a specific triplet EPR signal [gZ =
2.01 and Z-factor hyperfine coupling constant
= 17.5 gauss], and we used this
region for qualification of HbNO and measured the signal height for
quantitation of HbNO (Fig.
2F). Figure 2,
C and D, shows the differential spectra obtained
by subtracting the spectrum of Fig.
2A from that of Fig.
2B using EPRMAIN.
Figure 2D shows the
signal of Fig. 2C
magnified by 10 times. It shows a weak but distinct triplet hyperfine
structure (gZ = 2.01) with a coupling constant of
17.5 gauss, and this EPR signal was assigned to HbNO
(13,
19).
Figure 2E shows a
typical EPR signal of HbNO observed when NO (6.3 µM) was added to the
venous blood.
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Preparation of Calibration Curve
Figure 3 shows the EPR
spectra and calibration curve. The reaction mixture contained venous whole
blood of untreated rats and the NO-saturated solution (final concentration:
0–152 µM). Each spectrum was obtained by subtracting the EPR spectrum
of untreated whole blood from that of the NO-added sample, and the intensities
of the EPR signals were determined by measuring the height of the left line of
the five-coordinate 
-nitrosyl heme-derived triplet signal
(Fig. 2F). The
intensity of the EPR signal linearly increased according to the NO
concentration (32) with the
correlation coefficient (r2) = 0.995.
Arginine Infusion and HbNO Formation
To confirm that the HbNO level reflects the enzymatic formation of NO, we infused L- or D-arginine (0.2 or 0.6 g/kg) into rats and determined the HbNO levels in blood. When saline was infused, SBP was unchanged and the HbNO concentration was 9.23 ± 0.80 µM (Fig. 4). Although L-arginine did not alter SBP, the HbNO concentration increased in a dose-dependent manner. In contrast, D-arginine altered neither the HbNO concentration nor SBP even at the maximum dose.
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L-NAME-Induced Hypertension and HbNO Concentration
Body weight increased continuously in all groups during the experimental
period (Table. 1). The increase
was slightly attenuated in the L-NAME-treated groups. When the
experiment was started, basal SBP was 110 mmHg in all groups
(Fig. 5). In the control
(group 1), SBP and HR did not alter throughout the experiment.
Administration of L-NAME induced hypertension and bradycardia,
consistent with previous findings
(26,
58,
59). Temocapril treatment dose
dependently lowered SBP in the L-NAME-treated rats (groups
3 and 4; Fig. 5).
After the washout period (days 14 and 15), both SBP and HR
in the L-NAME-induced hypertensive rats were slightly improved.
Figure 6 shows representative
EPR spectra and HbNO concentration for each group. In the control group, the
HbNO concentration was 10.12 ± 2.03 µM, which was similar to that
for normal animals (9.23 ± 0.80 µM). Treatment with
L-NAME alone significantly reduced the blood HbNO concentration
(4.39 ± 0.65 µM), and temocapril restored the
L-NAME-induced HbNO reduction in a dose-dependent manner (2 mg
· kg–1 ·
day–1, 5.71 ± 0.53 µM; 20 mg ·
kg–1 · day–1,
7.02 ± 0.82 µM). When we administered temocapril (20 mg ·
kg–1 · day–1)
to untreated rats (n = 6) for 9 days, the HbNO concentration slightly
but not significantly increased (data not shown).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Therefore, an accurate method is required for the measurement of systemic NO dynamics under physiological and pathophysiological conditions. To date, the plasma concentration and urinary excretion of NOx and cGMP have been employed as indexes of systemic NO levels in vivo (35, 62). However, NOx and cGMP are regulated by factors other than NO. The NOx level is affected not only by endogenous NO production but also by dietary NOx and metabolism (8, 20, 53). The formation of cGMP is also altered by carbon monoxide and atrial natriuretic factor via the activation of guanylate cyclase (27, 64, 66, 71). Therefore, a specific and reliable method of detection is necessary to understand the physiological and pathophysiological role of NO.
Recently, it has been reported that endothelium-derived NO diffuses into blood, permeates the erythrocyte membrane, and then binds to Hb to form relatively stable HbNO in erythrocytes (48). In the present study, we tried to develop a method to detect HbNO as an index of blood NO by the EPR technique. The EPR method is more specific for NO compared with other methods, such as the Griess and cGMP methods, and offers a characteristic EPR spectrum of HbNO (22, 30).
Previous studies have already shown that LPS and NO donor treatment increase the HbNO concentration in blood, and clear HbNO spectra have been successfully obtained using the EPR method without any modifications (10, 28, 31, 67, 69). However, we and others failed to detect the distinct EPR signal of HbNO in the blood of normal humans or animals.
There are several difficulties in detecting the distinct EPR signal for HbNO in whole blood. Other EPR signals, such as for ceruloplasmin (g = 2.06) and semiquinone radical (g = 2.00), overlap the magnetic field of HbNO (gZ = 2.01) (5, 7, 10, 13, 19, 42). Because the basal HbNO level is quite low (µM range) (11, 25), and its EPR signal overlaps with the strong EPR signal of ceruloplasmin (Fig. 2A), the EPR spectrum of HbNO cannot be observed in most cases. To solve these problems, we introduced and modified an EPR signal subtraction method (14, 32).
In the previous reports, prepared Hb was directly injected into animal body
to trap endogenous NO (Hb2+ + NO 
HbNO)
(32). However, this method is
inappropriate for general use in NO detection in living systems due to the
prooxidative activity of Hb2+. In addition, although
Glover et al. (14) first
reported the definitive EPR spectra of HbNO in the systemic circulation of
humans receiving hydroxyurea, they did not determined the absolute value.
Therefore, in this study, we adopted a whole blood as an NO trap agent and tried to quantitate the HbNO concentration with the EPR subtraction method.
First of all, we tried to produce NO-depleted venous blood. L-NAME is a nonselective NO synthase (NOS) inhibitor (12, 17, 50) commonly used both in vivo (9, 38) and in vitro (33, 49), and chronic L-NAME (0.5–1 g/l)-treated rats have been widely used as a model of NO deficiency (6, 26, 56). To make NO-depleted rats, we administered a high dose of L-NAME for 1 wk. When we subtracted the EPR signal of blood of L-NAME (2 g/l)-treated rats from that of untreated rats, the apparent EPR signal of HbNO was observed (Fig. 2, C and D), whereas no HbNO EPR signal appeared when we subtracted the signal of blood of L-NAME (2 g/l)-treated rats from that of L-NAME (1 g/l)-treated rats (data not shown). Although we cannot confirm the completely depletion of NO in the L-NAME (2 g/l)-treated rats, these results suggest that 1-wk L-NAME treatment (2 g/l) is sufficient for the preparation of HbNO-depleted blood, and hence this treatment was employed to the prepare NO-depleted rats in the present study.
As expected, the EPR signal of whole blood of chronic
L-NAME-treated rats is identical to that of untreated rats except
for the HbNO signal in the field of 3,200 ± 250 gauss
(Fig. 2, A and
B). Therefore, we subtracted the EPR signal of
L-NAME-induced NO-depleted rats as a background to obtain a pure
HbNO signal. HbNO can exist as a mixture of five-coordinate and six-coordinate
hemes dependent on the environmental oxygen tension
(54,
73). The differential signal
showed a resolved triplet hyperfine structure (gZ
= 2.01) with a coupling constant of 17.5 gauss, which was assigned to a
five-coordinate -nitrosyl heme (Fig.
2, C and D). The signal intensity of HbNO
increased with the NO concentration (Fig.
3), which means that NO specifically binds to Hb in red blood
cells and forms HbNO (32).
L-Arginine is a physiological precursor of NO production. NO is synthesized from the terminal guanidine group of L-arginine by NOS (45, 52). To determine whether the EPR signal of HbNO is derived from L-arginine-dependent NO or not, we infused arginine into rats via the femoral vein and measured the EPR signal of HbNO. L-Arginine infusion (0.2 or 0.6 g/kg) significantly increased the HbNO concentration, but SBP was not affected by L-arginine at any dose. When D-arginine was infused, neither an increase in HbNO concentration nor a change of SBP was observed at the same dose as L-arginine. These results demonstrated that the EPR signal for HbNO reflects the endogenous changes in NO dynamics.
It has been reported that endothelium-derived vasodilation is impaired in hypertensive animals and patients compared with normotensive subjects (36, 44, 47, 63). This endothelial dysfunction may result in the progression of vascular structural change, cardiac hypertrophy, and renal failure, which may be associated with a decreased bioavailability of NO (16). ACEIs have been shown to significantly improve the endothelial dysfunction and structural changes of some organs in experimental and clinical studies (2, 21, 36, 37, 57, 65). It seems conceivable that the improvement of endothelial function caused by ACEIs may result from the restoration of blood NO levels. Therefore, we used chronic L-NAME-treated rats as an animal model of endothelial dysfunction and examined the effect of temocapril (24, 43) on the level of NO in blood using the HbNO signal subtraction method.
The oral administration of L-NAME (1 g/l) for 2 wk induced a
time-dependent hypertension, and the HbNO concentration was reduced to
40% of the control (Fig.
6). However, the administration of temocapril dose dependently
improved the HbNO concentration and SBP (Figs.
5 and
6). Because the increase in the
HbNO level was accompanied by a reduction in SBP
(Fig. 5), the SBP-lowering
effect of temocapril may be, at least in part, associated with an improvement
in systemic NO levels (60).
Although the exact mechanism behind the effect of temocapril in improving NO
dynamics is still unclear, we confirmed using our EPR HbNO signal subtraction
method that temocapril reversed the decrease of blood NO in
L-NAME-treated rats.
In the present study, we succeeded in measuring the systemic HbNO levels as an index of NO by the EPR HbNO signal subtraction method. Our method is specific for NO and does not require any pretreatment, and hence it seems simple and relevant as a method for measurement of the NO concentration in blood. As the balance between production and quenching of NO determines the level of bioactive NO, the determination of blood HbNO levels by our method may be useful in investigating a potential role of NO in physiological and pathophysiological functions. We recognize that there are some limitations with our method, and we will attempt to correct for these in successive experiments. The detection limit of HbNO was in the micromolar range (Fig. 3) with the present equipment. Further improvements will be required to apply this technique in clinical use.
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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excretion as an indicator
of nitric oxide formation in vivo during oral administration of
L-arginine or L-NAME in rats. Clin Exp
Pharmacol Physiol 23:
11–15, 1996.[Web of Science][Medline]
-stimulated NO and
cGMP via V1 receptor in cultured rat mesangial cells. Am
J Physiol Renal Physiol 276:
F433–F441, 1999.This article has been cited by other articles:
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  Y. Kanematsu, K. Yamaguchi, H. Ohnishi, Y. Motobayashi, K. Ishizawa, Y. Izawa, K. Kawazoe, S. Kondo, S. Kagami, S. Tomita, et al. Dietary doses of nitrite restore circulating nitric oxide level and improve renal injury in L-NAME-induced hypertensive rats Am J Physiol Renal Physiol, November 1, 2008; 295(5): F1457 - F1462. [Abstract] [Full Text] [PDF]
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 B. Derakhshan, G. Hao, and S. S. Gross Balancing reactivity against selectivity: The evolution of protein S-nitrosylation as an effector of cell signaling by nitric oxide Cardiovasc Res, July 15, 2007; 75(2): 210 - 219. [Abstract] [Full Text] [PDF]
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 K. Tsuchiya, Y. Kanematsu, M. Yoshizumi, H. Ohnishi, K. Kirima, Y. Izawa, M. Shikishima, T. Ishida, S. Kondo, S. Kagami, et al. Nitrite is an alternative source of NO in vivo Am J Physiol Heart Circ Physiol, May 1, 2005; 288(5): H2163 - H2170. [Abstract] [Full Text] [PDF]
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M. Okamoto, K. Tsuchiya, Y. Kanematsu, Y. Izawa, M. Yoshizumi, S. Kagawa, and T. Tamaki Nitrite-derived nitric oxide formation following ischemia-reperfusion injury in kidney Am J Physiol Renal Physiol, January 1, 2005; 288(1): F182 - F187. [Abstract] [Full Text] [PDF]
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 M. W. Foster, J. R. Pawloski, D. J. Singel, and J. S. Stamler Role of Circulating S-Nitrosothiols in Control of Blood Pressure Hypertension, January 1, 2005; 45(1): 15 - 17. [Full Text] [PDF]
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 J. S. Stamler S-Nitrosothiols in the Blood: Roles, Amounts, and Methods of Analysis Circ. Res., March 5, 2004; 94(4): 414 - 417. [Full Text] [PDF]
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is the
Z-factor hyperfine coupling constant.
, Control; 
,
L-NAME; 
, low-dose temocapril; 
, high-dose temocapril.
Values are expressed as means ± SE. Each group had 12 rats except for
the control group (n = 8).