BIOCHEMICAL
Vol. 184, No. 2, 1992 April 30, 1992
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1119-1124
DETECTION OF NITRIC OXIDE PRODUCTION IN LIPOPOLYSACCBARIDE-TREATED BY ESR USING CARBON MONOXKIE HEMOGLOBIN
RATS
Hiroaki Kosaka, Manabu Watanabe, Harumasa Yoshihara, Noboru Harada, and Takeshi Shiga Department
of Physiology, 2-2
Yamadaoka,
Medical
School,
Osaka
Uniuersity,
Osaka 565, JAPAN
Suita,
Received March 26, 1992
SUMMARY Release of nitric oxide (NO), from macrophages activated with E. lipopolysaccharide (LPS) and endothelial cells, has been proposed using chemiluminescence and spectrophotometry. However these methods can not distinguish NO from NO?. The present study was aimed to prove in uiuo production of NO, by ESR using CO-hemoglobin (HbCO) as a trapping agent of NO in the peritoneal cavity of rats treated with LPS. We detected a broad signal in the recovered HbCO solution. Inositol hexaphosphate induced a three-line hyperfine structure, characteristic of NO-hemoglobin (HbNO). In the arterial blood, ESR signal of HbNO with faint hyperfine structure was detected. NG-Monomethyl-L-arginine inhibited the formation of HbNO. HbNO was not detected in the peritoneal cavity of the LPS-untreated rat given i.p. both NO; and HbCO. HbNO was, therefore, derived from NO, not from NO,. These results show that free NO is produced in viuo by the stimulation of LPS. IO1992Academic Press,Inc. coli
Nitric regulator,
oxide (NO) is endothelial
mediates a variety muscle, inhibition
one
of
derived
of cell
the
relaxing
functions
of platelet
candidates factor
for
On the other hand, Escherichia in vitro
and neurotransmission
We showed that administration
lipopolysaccharide
coli
and in viuo
after
ABBREVIATIONS:
smooth
(4). The
(LPS)
a time delay of 6 h
macrophage cell
NO
was reported (2).
of LPS to rats caused nitrosation
(8, 9). The release of NO from LPS-activated was also measured by TEA (10).
The
of vascular
release of NO from stimulated endothelial cells in culture using thermal energy analyzer (TEA) (1) or spectrophotometry NO&NO, generation
physiological
(EDRF) (l-3).
such as relaxation
aggregation,
the
of
caused (5-7). amines
line RAW264.7
ESR, electron spin resonance; Hb, hemoglobin; HbCO, COhemoglobin; HbOz, oxyhemoglobin, HbNO, NO-hemoglobin; IHP, inositol hexaphosphate; RBC, red blood cell; EDRF. endothelial derived relaxing factor; TEA, thermal energy analyzer: LPS, Escheric hia coli lipopolysaccharide; L-NMMA, NG-Monomethyl-L-arginine.
Vol.
184,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
On the contrary, EDRF is speculated as nitrosocystein nitrosated protein (11). EDRF could not be identified as free using
deoxyhemoglobin-agarose
converted
to NO&NO,
responses
also
may also
react
with
to
of NO by other
there
in the
(13, 14). However,
from
endothelial
into
red blood
direct
cells, cell
because
and specific blood
of
NO, also
gives
(RBC) (15). NO binds
by ESR using peritoneal
study
thus
concerns
CO-hemoglobin
(HbCO)
cavity
We simultaneously
of rats detected
treated
direct a
the
HbNO in the arterial
LPS
formation
HbNO after
an
of
agent cell-wall
of
the entry
with
evidence
trapping
bacterial
needed.
with
ESR signal (16-18). by the reaction with
with with
treated
to hemoglobin
as
is thus
of NO generation.
rats
does not indicate
high affinity, and HbNO gives characteristic HbNO is rapidly oxidized to methemoglobin The present
methods
in uiuo evidence
venous this
similar
nitroso
yield
has been no direct
HbNO formation reported
NOi
ESR is
result as NO. TEA compounds. Deoxyhemoglobin NO-hemoglobin (HbNO) slowly. The
to NO, (1) or to labile
identification Further,
NO, causes
other
NO by by OZ to NO,, which
(12). NO is oxidized
in solution.
or
NO of NO,
extremely However, oxygen.
NO of
was
production NO in
product,
the
LPS.
blood.
MATERlAM AND MEYEIODS. Male Wistar rats (about 100 g) were treated intraperitoneally (i.p.) with E. coli LPS (type 0127:B8; Sigma, St Louis, MO). After 6 h, human HbCO solution (5 mM, 3 ml) was injected into the peritoneal cavity. A few hours later after the administration of HbCO peritoneal cavity, solution, 0.5 ml each was aspirated from the transferred to ESR tube, and frozen immediately under liquid nitrogen. The rats were anaesthesized by pentobarbital (50 mg/kg b. w., i.p.) ten minutes prior to the sampling. Hemolysate from fresh human blood was adjusted to 5 mM Hb and added superoxide dismutase (250 units/ml). CO gas was exposed to the Hb solution in the under stirring. After conversion of Hb to HbCO, CO gas dissolved solution was washed out with nitrogen gas. Deoxyhemoglobin was prepared HbO. solution under stirring for
by exposing 1 h.
ultrapure
nitrogen
gas to
NO-Monomethyl-L-arginine (L-NMMA) was purchased from Calbiochem. Jolla, CA. L-NMMA was administered i.p. 50 mg/kg b. w. at 0, 2. 4, 6 h, respectively, after LPS. A Hitachi 320L spectrophotometer reaction between deoxyhemoglobin phosphate buffer (pH 6.2).
the La
was used for in vitro anaerobic (0.3 mM) and NO, (0.4 mM) in 0.05 M
ESR spectra were recorded with a Varian E-12 spectrometer at 110 K. ESR spectrometer settings were as follows; incident microwave power, 10 mW; modulation frequency, 100 kHz; modulation amplitude, 0.5 mT; response time, 1 s; and sweep rate, 12.5 mT/min. 1120
Vol.
184,
No.
In viuo experiment
RESULTS.
later
BIOCHEMICAL
2, 1992
HbCO Lp.. Peritoneal
Hb concentration hexaphosphate
AND
BIOPHYSICAL
using EibCO. Rats fluid
RESEARCH
received
COMMUNICATIONS
LPS,
then
6 h
showed a broad ESR signal (Fig. 1A). The
in the recovered (IHP) was mixed
sample was not
with
peritoneal
diluted. fluid
When inositol
before
freezing,
three-line hyperfine structure, characteristic to HbNO, appeared in the ESR signal (Fig. 1B). The HbNO concentration increased with time. In spite of variation
of the LPS concentration
similar, indicating After
saturation
aspiration
(l-7.5 mg/kg), the yield
of HbNO was
at the concentration.
of the last sample from the peritoneal
sample was taken from abdominal aorta.
cavity,
blood
An ESR signal of HbNO was detected.
The concentration the peritoneal structure
of HbNO from arterial blood was greater than that from three-line hyperfine cavity (Fig. 2). There was faint
in the ESR signal of HbNO from the arterial
contrast
to the marked hyperfine
(13). With LPS-untreated recovered
rat, no
3 h 45 min later
structure
ESR signal
from peritoneal
HbCO (5 mM. 3 ml), nor in the arterial
reported
blood (Fig. 2C), in by Westenberger et al.
was detected cavity
of the
in rat
the
sample
given
i.p.
blood.
L-arginine analog, L-NMMA was administered i.p. 50 m&kg b. w. at 0, 2, 4, 6 h, respectively, after LPS injection to rats. LEffect
of L-NMMA.
NMMA inhibited cavity
the formation
and in the arterial
Zn uiuo
experiment.
of ESR spectra of HbNO both in the peritoneal
blood (Fig. 2).
using
deoxyhemoglobin.
When deoxyhemoglobin
given i.p. instead of HbCO, an ESR signal of HbNO was detected, distinct
three-line
hyperfine
structure,
and it had
compared with that from HbCO.
1. Formation of ESR signals of HhNOfrom HbCOin the peritoneal cavity of rats treated with LPS. 6 h after administration of LPS (7.5 mg/kg b. w.), HbCOsolution (5 mM,3 ml) was i.p. injected. A; 2 h 45 min later, 0.5 ml of the peritoneal fluid was recovered by aspiration. B; 3 h 45 min later, peritoneal fluid recovered from the samerat was mixed with IHP solution (10 mM)under N1 and was thus diluted to l.2-fold. Span of arrow corresponds to 5 mT. Figure
1121
was
Vol.
184,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Figure 2. Inhibition of L-NMMA on the generation of HbNO in the peritoneal cavity and in the arterial blood of rats treated with LPS. 6 h after administration of LPS (3 mg/kg b. w.), HbCO solution was i.p. injected. 3 h later, peritoneal fluid was recovered. A; peritoneal fluid of rat A, B; + L-NMMA, peritoneal fluid of rat B, C; arterial blood of rat A, D; + LNMMA, arterial blood of rat B. L-NMMA was administered i.p. 50 mg/kg b. w. at 0, 2, 4, 6 h. respectively, after LPS. Span of arrow corresponds to 5 mT.
reaction
Zn vitro
deoxyhemoglobin generation detected
and
mixed
methemoglobin
spectrophotometrically.
hyperfine
deoxyhemoglobin
NO; were
of HbNO and
three-line reaction
between
structure.
The
and
under with
a
ESR signal
The hyperfine
NO,
anaerobic molar of
the
structure
under
Np.
condition,
ratio HbNO
of
When slow
1:l
showed
was the
disappeared at the
end.
The reaction
between WC0 and NO,. If LPS was not given, HbNO was not
detected in the sample recovered 3 h 45 min later from peritoneal cavity of the rat given Lp. NO, (1 mM) and HbCO (5 mM). When HbCO and NO, were mixed in uifro,
DISCUSSION.
no spectrophotometric
change of HbCO was observed.
The present study first demonstrated in viuo production cavity of rats administered LPS, by ESR using
NO in the peritoneal The
ESR signal of HbNO showed no three-line 1122
hyperfine
structure,
of
HbCO. because
Vol.
184,
No.
2, 1992
all
hemes have their
hyperfine
ligands,
structure,
quaternary state)
BIOCHEMICAL
AND
RESEARCH
i.e., CO or NO. Addition
indicating
structure
BIOPHYSICAL
(R state)
conversion
of IHP induced the high affinity the
from
to the low affinity
(17). When deoxyhemoglobin
quaternary
The inhibition
by L-NMMA of HbNO formation
that NO is produced via the pathway from L-arginine HbNO was not detected
from the peritoneal
and HbCO but untreated
structure
was i.p. given, the three-line
structure was present in the absence of IHP, because of NO on the T state of deoxyhemoglobin (18). L-NMMA.
COMMUNICATIONS
fluid
with LPS. Therefore,
(T
hyperfine
subsaturation in
(10).
rats
of
indicates
ESR signal
of
of the rat given i.p.
the NO derives from
NO,
arginine,
not from NO,. The
three-line
hyperfine
et al. (13) detected structure
in
hyperfine
structure
structure
in ESR signal
ESR signal of HbNO with marked
venous
blood. was faint
But,
the
present
in arterial
of WNO.
Westenberger
three-line
hyperfine
study
showed that
blood. Because intensity
the
of
the
hyperfine structure increased when our sample was exposed to air, origin of the hyperfine signal is a valency hybrid of Hb with nitrosylated ferrous (r -subunits and ferric /3 -subunit (( ~1‘*NO)/? “‘), (19). Origin
of WNO
in arterial
blood.
Intravenous
injection
of NO, to
rats
(15) or to RBC (19) gives HbNO. In contrast, the reaction between HbOZ and NO, does not produce HbNO, but generates methemoglobin and NO, through the peroxidatic
activity
of H,O,-methemoglobin
by macrophages and liver,
complex (20-25). NO, generated
changes to NO,/NO,
by the reaction
blood. NO, easily enters into RBC and is reduced to system inside of RBC. A part of NO may directly i-p.
is
more
superior
conversion Further,
reaction
than
the
of deoxyhemoglobin
of
to Hb02 is
in
reducing
enter RBC to produce HbNO.
technique,
injection
the
0,
the injection
of HbCO since the
deoxyhemoglobin,
rapid
upon
contact
with
air.
preparation
deoxyhemoglobin. ligated
As an experimental
Experiments.
NO by
with
of HbCO is faster and easier than that of Since NO binds with Hb 1000 times stronger than CO, CO
to Hb is easily replaced with NO. Moreover, between deoxyhemoglobin
in
and NO: generating
contrast
to
the
HbNO, HbCO does not
react with NO,. The present study first demonstrated in niuo formation of peritoneal cavity of rats treated with LPS, by ESR using trapping
agent of NO. NO and NO,
dissolved
in
plasma
is
NO in
the
HbCO as a converted to
NO&NO,. Then, NO, produces HbNO in RBC.
This research was supported in part by Grants-in-Aid from of Education, Science and Culture and the Ministry of Health
ACKNOWLEDGMENTS.
the Ministry
and Welfare of Japan. 1123
Vol.
184,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
REFERENCES
1. Palmer, R. M. J, Ferrige, A. G., Moncada, S. (1987) Nature 327, 524526. 2. Ignarro, L. J., Buga, G. M., Wood, K. S., Byrns, R. E., and Chaudhuri, G. (1987) Proc. Natl. Acad. Sci. USA 84, 9265-9269. 3. Garthwaite, J., Charles, S. L., and Chess-Williams, R. (1988) Nature 336, 385-388. 4. Moncada, S. and Palmer, R. M. J. (1990) Nitric Oxide from L-arginins: A Bioregulatory System (S. Moncada, and E. A. Higgs, eds.) pp. 19-34, Elsevier Science Publishers B. V., Amsterdam. 5. Wagner, D. A., Young, V. R., and Tannenbaum, S. R,, (1983) Proc. Natl. Acad. Sci. USA 80, 4518-4521. 6. Stuehr, D. J. and Marletta, M. A. (1985) Proc. Natl. Acad. Sci. USA 82, 7738-7742. 7. Hibbs, J. B., Taitor, R. E., and Vavrin, Z. (1987) Science 235, 473476. 8. Kosaka, H., Tsuda, M., Kurashima, Y., Esumi, H., Terada, N., Ito, Y., and Uozumi, M. (1990) Carcinogenesis 11, 1887-1889. 9. Leaf, C. D., Wishnok, J. S., and Tannenbaum, S. R. (1991) Carcinogenesis 12, 537-539. 10. Marletta, M. A., Yoon, P. S., Iyengar. R., Leaf, C. D., and Wishnok, J. S. (1988) Biochemisty 27, 8706-8711. 11. Myers, P. R., Minor Jr, R. L., Guerra Jr, R., Bates, J. N., and Harrison, D. G. (1990) Nature 345, 161-163. 12. Greenberg, S. S., Wilcox, D. E., and Rubanyi, G. M. (1990) Circ. Res. 67. 1446-1452. 13. Westenberger, U.. Thanner, S., Ruf, H. H., Gersonde, K., Sutter, G., Trenz, 0. (1990) Free Rad. Res. Comms. 11, 167-178. 14. Wang, Q., Jacobs, J., Deleo, J., Kruszyna, H., Kruszyna, R., Smith, R. P, and Wilcox, D. (1991) Life Sci. Pharmacol Lett. 49, 55-60. 15. Imaizumi, K., Tyuma, I., Imai, K., Kosaka, H., and Ueda, Y. (1980) Int. Arch. Occup. Environ. Health 45, 97-104. 16. Shiga, T.. Hwang, K.-J., and Tyuma, I. (1969) Biochemistry 8, 378-383. 17. Rein, H., Ristau, O., and Scheler, W. (1972) FEBS Lett. 24, 24-26. 18. Hille, R. Olson, J. S., and Palmer, G. (1979) J. Biol. Chem. 254, 12110-12120.
19. Kruszyna, R., Kruszyna, H., Smith, R. P., Thron, C. D. and Wilcox, D. E. (1987) J. Pharmacol. Exp. Ther. 241, 307-313. 20. Kosaka, H., Imaizumi, K., Imai, K., and Tyuma, I. (1979) Biochim. Biophys. Acta 581, 184-188. 21. Kosaka, H., Imaizumi, K.. and Tyuma, I. (1982) Biochim. Biophys. Acta 702, 237-241. 22. Kosaka, H. and Tyuma, I. (1982) Biochim. Biophys. Acta 709, 187-193. 23. Kosaka, H., Uozumi, M. (1986) Biochim. Biophys. Acta 871, 14-18. 24. Kosaka, H., Uozumi, M., and Tyuma, I. (1989) Free Radical Biol. Med. 7, 653-658. 25. Shiga, T. and Imaizumi, K. (1975) Arch. Biochem. Biophys. 167, 469479.
1124
Vol. 184, No. 2, 1992 April 30, 1992
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1119-1124
DETECTION OF NITRIC OXIDE PRODUCTION IN LIPOPOLYSACCBARIDE-TREATED BY ESR USING CARBON MONOXKIE HEMOGLOBIN
RATS
Hiroaki Kosaka, Manabu Watanabe, Harumasa Yoshihara, Noboru Harada, and Takeshi Shiga Department
of Physiology, 2-2
Yamadaoka,
Medical
School,
Osaka
Uniuersity,
Osaka 565, JAPAN
Suita,
Received March 26, 1992
SUMMARY Release of nitric oxide (NO), from macrophages activated with E. lipopolysaccharide (LPS) and endothelial cells, has been proposed using chemiluminescence and spectrophotometry. However these methods can not distinguish NO from NO?. The present study was aimed to prove in uiuo production of NO, by ESR using CO-hemoglobin (HbCO) as a trapping agent of NO in the peritoneal cavity of rats treated with LPS. We detected a broad signal in the recovered HbCO solution. Inositol hexaphosphate induced a three-line hyperfine structure, characteristic of NO-hemoglobin (HbNO). In the arterial blood, ESR signal of HbNO with faint hyperfine structure was detected. NG-Monomethyl-L-arginine inhibited the formation of HbNO. HbNO was not detected in the peritoneal cavity of the LPS-untreated rat given i.p. both NO; and HbCO. HbNO was, therefore, derived from NO, not from NO,. These results show that free NO is produced in viuo by the stimulation of LPS. IO1992Academic Press,Inc. coli
Nitric regulator,
oxide (NO) is endothelial
mediates a variety muscle, inhibition
one
of
derived
of cell
the
relaxing
functions
of platelet
candidates factor
for
On the other hand, Escherichia in vitro
and neurotransmission
We showed that administration
lipopolysaccharide
coli
and in viuo
after
ABBREVIATIONS:
smooth
(4). The
(LPS)
a time delay of 6 h
macrophage cell
NO
was reported (2).
of LPS to rats caused nitrosation
(8, 9). The release of NO from LPS-activated was also measured by TEA (10).
The
of vascular
release of NO from stimulated endothelial cells in culture using thermal energy analyzer (TEA) (1) or spectrophotometry NO&NO, generation
physiological
(EDRF) (l-3).
such as relaxation
aggregation,
the
of
caused (5-7). amines
line RAW264.7
ESR, electron spin resonance; Hb, hemoglobin; HbCO, COhemoglobin; HbOz, oxyhemoglobin, HbNO, NO-hemoglobin; IHP, inositol hexaphosphate; RBC, red blood cell; EDRF. endothelial derived relaxing factor; TEA, thermal energy analyzer: LPS, Escheric hia coli lipopolysaccharide; L-NMMA, NG-Monomethyl-L-arginine.
Vol.
184,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
On the contrary, EDRF is speculated as nitrosocystein nitrosated protein (11). EDRF could not be identified as free using
deoxyhemoglobin-agarose
converted
to NO&NO,
responses
also
may also
react
with
to
of NO by other
there
in the
(13, 14). However,
from
endothelial
into
red blood
direct
cells, cell
because
and specific blood
of
NO, also
gives
(RBC) (15). NO binds
by ESR using peritoneal
study
thus
concerns
CO-hemoglobin
(HbCO)
cavity
We simultaneously
of rats detected
treated
direct a
the
HbNO in the arterial
LPS
formation
HbNO after
an
of
agent cell-wall
of
the entry
with
evidence
trapping
bacterial
needed.
with
ESR signal (16-18). by the reaction with
with with
treated
to hemoglobin
as
is thus
of NO generation.
rats
does not indicate
high affinity, and HbNO gives characteristic HbNO is rapidly oxidized to methemoglobin The present
methods
in uiuo evidence
venous this
similar
nitroso
yield
has been no direct
HbNO formation reported
NOi
ESR is
result as NO. TEA compounds. Deoxyhemoglobin NO-hemoglobin (HbNO) slowly. The
to NO, (1) or to labile
identification Further,
NO, causes
other
NO by by OZ to NO,, which
(12). NO is oxidized
in solution.
or
NO of NO,
extremely However, oxygen.
NO of
was
production NO in
product,
the
LPS.
blood.
MATERlAM AND MEYEIODS. Male Wistar rats (about 100 g) were treated intraperitoneally (i.p.) with E. coli LPS (type 0127:B8; Sigma, St Louis, MO). After 6 h, human HbCO solution (5 mM, 3 ml) was injected into the peritoneal cavity. A few hours later after the administration of HbCO peritoneal cavity, solution, 0.5 ml each was aspirated from the transferred to ESR tube, and frozen immediately under liquid nitrogen. The rats were anaesthesized by pentobarbital (50 mg/kg b. w., i.p.) ten minutes prior to the sampling. Hemolysate from fresh human blood was adjusted to 5 mM Hb and added superoxide dismutase (250 units/ml). CO gas was exposed to the Hb solution in the under stirring. After conversion of Hb to HbCO, CO gas dissolved solution was washed out with nitrogen gas. Deoxyhemoglobin was prepared HbO. solution under stirring for
by exposing 1 h.
ultrapure
nitrogen
gas to
NO-Monomethyl-L-arginine (L-NMMA) was purchased from Calbiochem. Jolla, CA. L-NMMA was administered i.p. 50 mg/kg b. w. at 0, 2. 4, 6 h, respectively, after LPS. A Hitachi 320L spectrophotometer reaction between deoxyhemoglobin phosphate buffer (pH 6.2).
the La
was used for in vitro anaerobic (0.3 mM) and NO, (0.4 mM) in 0.05 M
ESR spectra were recorded with a Varian E-12 spectrometer at 110 K. ESR spectrometer settings were as follows; incident microwave power, 10 mW; modulation frequency, 100 kHz; modulation amplitude, 0.5 mT; response time, 1 s; and sweep rate, 12.5 mT/min. 1120
Vol.
184,
No.
In viuo experiment
RESULTS.
later
BIOCHEMICAL
2, 1992
HbCO Lp.. Peritoneal
Hb concentration hexaphosphate
AND
BIOPHYSICAL
using EibCO. Rats fluid
RESEARCH
received
COMMUNICATIONS
LPS,
then
6 h
showed a broad ESR signal (Fig. 1A). The
in the recovered (IHP) was mixed
sample was not
with
peritoneal
diluted. fluid
When inositol
before
freezing,
three-line hyperfine structure, characteristic to HbNO, appeared in the ESR signal (Fig. 1B). The HbNO concentration increased with time. In spite of variation
of the LPS concentration
similar, indicating After
saturation
aspiration
(l-7.5 mg/kg), the yield
of HbNO was
at the concentration.
of the last sample from the peritoneal
sample was taken from abdominal aorta.
cavity,
blood
An ESR signal of HbNO was detected.
The concentration the peritoneal structure
of HbNO from arterial blood was greater than that from three-line hyperfine cavity (Fig. 2). There was faint
in the ESR signal of HbNO from the arterial
contrast
to the marked hyperfine
(13). With LPS-untreated recovered
rat, no
3 h 45 min later
structure
ESR signal
from peritoneal
HbCO (5 mM. 3 ml), nor in the arterial
reported
blood (Fig. 2C), in by Westenberger et al.
was detected cavity
of the
in rat
the
sample
given
i.p.
blood.
L-arginine analog, L-NMMA was administered i.p. 50 m&kg b. w. at 0, 2, 4, 6 h, respectively, after LPS injection to rats. LEffect
of L-NMMA.
NMMA inhibited cavity
the formation
and in the arterial
Zn uiuo
experiment.
of ESR spectra of HbNO both in the peritoneal
blood (Fig. 2).
using
deoxyhemoglobin.
When deoxyhemoglobin
given i.p. instead of HbCO, an ESR signal of HbNO was detected, distinct
three-line
hyperfine
structure,
and it had
compared with that from HbCO.
1. Formation of ESR signals of HhNOfrom HbCOin the peritoneal cavity of rats treated with LPS. 6 h after administration of LPS (7.5 mg/kg b. w.), HbCOsolution (5 mM,3 ml) was i.p. injected. A; 2 h 45 min later, 0.5 ml of the peritoneal fluid was recovered by aspiration. B; 3 h 45 min later, peritoneal fluid recovered from the samerat was mixed with IHP solution (10 mM)under N1 and was thus diluted to l.2-fold. Span of arrow corresponds to 5 mT. Figure
1121
was
Vol.
184,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Figure 2. Inhibition of L-NMMA on the generation of HbNO in the peritoneal cavity and in the arterial blood of rats treated with LPS. 6 h after administration of LPS (3 mg/kg b. w.), HbCO solution was i.p. injected. 3 h later, peritoneal fluid was recovered. A; peritoneal fluid of rat A, B; + L-NMMA, peritoneal fluid of rat B, C; arterial blood of rat A, D; + LNMMA, arterial blood of rat B. L-NMMA was administered i.p. 50 mg/kg b. w. at 0, 2, 4, 6 h. respectively, after LPS. Span of arrow corresponds to 5 mT.
reaction
Zn vitro
deoxyhemoglobin generation detected
and
mixed
methemoglobin
spectrophotometrically.
hyperfine
deoxyhemoglobin
NO; were
of HbNO and
three-line reaction
between
structure.
The
and
under with
a
ESR signal
The hyperfine
NO,
anaerobic molar of
the
structure
under
Np.
condition,
ratio HbNO
of
When slow
1:l
showed
was the
disappeared at the
end.
The reaction
between WC0 and NO,. If LPS was not given, HbNO was not
detected in the sample recovered 3 h 45 min later from peritoneal cavity of the rat given Lp. NO, (1 mM) and HbCO (5 mM). When HbCO and NO, were mixed in uifro,
DISCUSSION.
no spectrophotometric
change of HbCO was observed.
The present study first demonstrated in viuo production cavity of rats administered LPS, by ESR using
NO in the peritoneal The
ESR signal of HbNO showed no three-line 1122
hyperfine
structure,
of
HbCO. because
Vol.
184,
No.
2, 1992
all
hemes have their
hyperfine
ligands,
structure,
quaternary state)
BIOCHEMICAL
AND
RESEARCH
i.e., CO or NO. Addition
indicating
structure
BIOPHYSICAL
(R state)
conversion
of IHP induced the high affinity the
from
to the low affinity
(17). When deoxyhemoglobin
quaternary
The inhibition
by L-NMMA of HbNO formation
that NO is produced via the pathway from L-arginine HbNO was not detected
from the peritoneal
and HbCO but untreated
structure
was i.p. given, the three-line
structure was present in the absence of IHP, because of NO on the T state of deoxyhemoglobin (18). L-NMMA.
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fluid
with LPS. Therefore,
(T
hyperfine
subsaturation in
(10).
rats
of
indicates
ESR signal
of
of the rat given i.p.
the NO derives from
NO,
arginine,
not from NO,. The
three-line
hyperfine
et al. (13) detected structure
in
hyperfine
structure
structure
in ESR signal
ESR signal of HbNO with marked
venous
blood. was faint
But,
the
present
in arterial
of WNO.
Westenberger
three-line
hyperfine
study
showed that
blood. Because intensity
the
of
the
hyperfine structure increased when our sample was exposed to air, origin of the hyperfine signal is a valency hybrid of Hb with nitrosylated ferrous (r -subunits and ferric /3 -subunit (( ~1‘*NO)/? “‘), (19). Origin
of WNO
in arterial
blood.
Intravenous
injection
of NO, to
rats
(15) or to RBC (19) gives HbNO. In contrast, the reaction between HbOZ and NO, does not produce HbNO, but generates methemoglobin and NO, through the peroxidatic
activity
of H,O,-methemoglobin
by macrophages and liver,
complex (20-25). NO, generated
changes to NO,/NO,
by the reaction
blood. NO, easily enters into RBC and is reduced to system inside of RBC. A part of NO may directly i-p.
is
more
superior
conversion Further,
reaction
than
the
of deoxyhemoglobin
of
to Hb02 is
in
reducing
enter RBC to produce HbNO.
technique,
injection
the
0,
the injection
of HbCO since the
deoxyhemoglobin,
rapid
upon
contact
with
air.
preparation
deoxyhemoglobin. ligated
As an experimental
Experiments.
NO by
with
of HbCO is faster and easier than that of Since NO binds with Hb 1000 times stronger than CO, CO
to Hb is easily replaced with NO. Moreover, between deoxyhemoglobin
in
and NO: generating
contrast
to
the
HbNO, HbCO does not
react with NO,. The present study first demonstrated in niuo formation of peritoneal cavity of rats treated with LPS, by ESR using trapping
agent of NO. NO and NO,
dissolved
in
plasma
is
NO in
the
HbCO as a converted to
NO&NO,. Then, NO, produces HbNO in RBC.
This research was supported in part by Grants-in-Aid from of Education, Science and Culture and the Ministry of Health
ACKNOWLEDGMENTS.
the Ministry
and Welfare of Japan. 1123
Vol.
184,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
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REFERENCES
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