|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 333-340
Nitric Oxide-Mediated Augmentation of Polymorphonuclear Free Radical
Generation After Hypoxia-Reoxygenation
By
Sonia Sethi,
Mahendra Pratap Singh, and
Madhu Dikshit
From the Pharmacology Division, Central Drug Research Institute,
Lucknow, India.
 |
ABSTRACT |
Polymorphonuclear leukocytes (PMNLs), nitric oxide (NO), calcium,
and free radicals play an important role in hypoxia/ischemia and
reoxygenation injury. In the present study, NO donors, sodium nitroprusside (SNP), and diethylamine-NO
(DEA-NO) at low concentrations (10 and 100 nmol/L)
potentiated, while higher (10 µmol/L to 10 mmol/L) concentrations
inhibited free radical generation response in the rat PMNLs. Free
radical generation response was found to be significantly augmented
when hypoxic PMNLs were reoxygenated (hypoxia-reoxygenation
[H-R]). This increase in free radical generation after
reoxygenation or SNP (10 nmol/L) was blocked in the absence of
extracellular calcium. SNP (10 nmol/L) or H-R-mediated increases in
the free radical generation were prevented by the pretreatment of PMNLs
with NO scavenger (hemoglobin), the polyadenine diphosphate (ADP)-ribosylation synthase inhibitor (benzamide) or the calcium channel antagonist (felodipine). A significant augmentation in the
nitrite and intracellular calcium levels was observed during hypoxia.
Hemoglobin pretreatment also blocked the increase in intracellular
calcium levels due to SNP (10 nmol/L) or hypoxia. Thus, increased
availability of NO during SNP treatment or H-R, may have led to an
ADP-ribosylation-mediated increase in intracellular calcium, thereby
increasing the free radical generation from the rat PMNLs.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
THE ROLE OF polymorphonuclear leukocytes
(PMNLs) in ischemia/hypoxia-reperfusion damage has been demonstrated in
a variety of organs such as the heart, lungs, kidneys, brain, liver, and in striated muscles and the gastrointestinal tract.1-3
Migration of PMNLs into the ischemic tissue as early as 10 minutes
after ischemia and increases with time and reperfusion have been
reported.4,5 Augmentation of reactive oxygen species (ROS)
generation, chemotaxis, adherence, release of other cytotoxic
substances, and upregulation of opsonic receptors in the activated
PMNLs after reoxygenation has also been demonstrated.3,6
PMNLs, which play an important role in ischemic reoxygenation injury,
synthesize nitric oxide (NO) in addition to ROS from L-arginine by the
enzyme nitric oxide synthase (NOS).7 Peroxynitrite production in PMNLs has also been found to mediate
cytotoxicity.7 By contrast, the involvement of NO in the
inhibition and scavenging of free radicals has also been
demonstrated.8 Recently, it has been shown that the low
concentrations (nmol/L) of NO enhanced the free radical generation in
PMNLs, while higher concentrations (µmol/L) inhibited
it.9-11 NO in higher concentrations inhibits the
nicotinamide adenine dinucleotide phosphate (NADPH)
oxidase activity and scavenges free radicals.8-11 However,
the mechanisms of the increase in ROS generation induced by NO are not
clearly understood. Most of the biologic functions of NO are mediated by modification of the heme group, thiol groups, or by poly- adenine diphosphate (ADP)-ribosylation.12,13 NO in PMNLs has also
been shown to cause poly ADP-ribosylation of various proteins and thus modulate enzyme activities.9
Protection against ischemia and reperfusion injury has been reported
after pretreatment with NOS inhibitors, blockade of neutrophil adherence, or inhibition of free radicals.3,14,15 Recently Hewett et al16 have shown that the inducible NOS-mediated
NO production in astrocytes potentiates N-methyl-D-aspartate
(NMDA) receptor-dependent neuronal death after a
hypoxic/ischemic insult. However, some reports also indicate a
deleterious or negligible effect of NOS inhibitors.17 It is
likely that the increased concentration of NO in ischemia/hypoxia due
to decreased degradation18 or increased
synthesis16 might modulate ROS generation from PMNLs during
ischemia and reperfusion injury. Therefore, we investigated the
involvement of NO in ROS generation from the normal and reoxygenated PMNLs after hypoxia.
| |
MATERIALS AND METHODS |
Materials.
Aminoguanidine, arachidonic acid (AA), catalase, dextran T-500, flavin
adenine dinucleotide (FAD), hemoglobin (Hb), lactate dehydrogenase
(LDH), luminol, lucigenin, Fura-2 AM,
formyl-methionyl-leucyl-phenylalanine (FMLP),  NADPH-reduced form,
N-(1 naphthyl) ethylene diamine, nitrate reductase, o-dianisidine,
phorbol esters (phorbol myristate acetate [PMA]), phosphoric acid,
sodium nitroprusside (SNP), sodium pyruvate, sulfanilamide, superoxide
dismutase (SOD), zymosan particles, and Histopaque (1077 and 1119) were
purchased from Sigma Chemical Co (St Louis, MO). Diethylamine nitric
oxide complex sodium salt (DEA-NO), 7-nitroindazole, and benzamide were
procured from Research Biochemicals International (Natick,
MA). Felodipine was a gift from Astra IDL (Banglore,
India). Sodium nitrite was obtained from Glaxo India
(Bombay, India). All other chemicals used in the present study were of
analytical grade (SRL, Bombay, India).
Preparation of the reagents.
Stock solutions of FMLP (100 µmol/L), PMA (1.625 mmol/L), felodipine
(0.5 mmol/L), Fura-2 AM (5 mol/L) and luminol (100 mmol/L) were made in
dimethyl sulfoxide (DMSO), while a stock solution of AA (5 × 10 2 mol/L) was prepared in alcohol and sodium
carbonate; further dilutions were made in normal saline. Solutions of
lucigenin (50 mmol/L) and aminoguanidine (0.5 mol/L) were prepared in
water. Benzamide (0.2 mol/L) was dissolved in methanol. Opsonized
zymosan (OZ) was made by treating washed zymosan particles with fresh autologous serum for 30 minutes at 37°C, then removing the serum by
centrifugation. The particles were resuspended in Hanks' Balanced Salt
Solution (HBSS) and a stock solution of 12.5 mg/mL was stored in
100-µL aliquots at  70°C. Hemoglobin (0.5 mmol/L) solution was prepared in normal saline.
PMNLs isolation.
Rat blood PMNLs were obtained from male Sprague Dawley rats (130 to 150 g) by the Boyum method.19 Blood was collected by cardiac
puncture in sodium citrate (0.129 mol/L, pH 6.5, 9:1, vol/vol) under
ether anesthesia from normal animals. Platelet-rich plasma was removed
by centrifugation at 250g for 20 minutes at 20°C (Sigma
centrifuge, Osterode, Germany) and the buffy coat was
subjected to dextran sedimentation as described
previously.20 PMNLs were further purified on
Histopaque-density gradient at 700g for 30 minutes at 25°C.
The PMNL-rich layer was recovered at the interface of Histopaque
1119/1077 and was washed three times with HBSS (sodium chloride 138 mmol/L, potassium chloride 2.7 mmol/L, disodium hydrogen phosphate 8.1 mmol/L, potassium dihydrogen phosphate 1.5 mmol/L, magnesium chloride
0.6 mmol/L, calcium chloride 1.0 mmol/L, glucose 10 mmol/L, pH 7.4).
The viability of the cells, tested by trypan blue exclusion, was never
less than 95%. Before experiments, PMNLs were kept at 4°C for no
more than 2 to 3 hours.
Estimation of LDH activity.
LDH activity was measured8 in the supernatant and in the
PMNLs lysate after the disruption of PMNLs by sonication. The amount
(%) of the enzyme released in each set was calculated relative to the
total activity present in the supernatant and in the lysate.
Measurement of chemiluminescence response.
Free radical generation from PMNLs stimulated with various inducers
such as AA (1 to 5 × 105 mol/L), FMLP (1 µmol/L), OZ (1.25 µg) or PMA (3 × 108
mol/L) was measured at 37°C using a dual channel lumiaggregometer (Model 560; Chronolog Corp, Havertown, PA) with constant stirring at
900 rpm. Chemiluminescence is reported as units calculated from the
maximum output (obtained from stimulated PMNLs) divided by the "gain
setting" of the instrument.8 An assay mixture (1,000 µL) contained 1 to 5 × 106 PMNLs, 10 µmol/L
luminol (LCL), or 50 µmol/L of lucigenin (LUCDCL), the test substance
and FMLP (1 × 106 mol/L), AA (1 × 105 mol/L), OZ (1.25 µg) or PMA (3 × 108 mol/L).
The effect on the AA (1 × 105 mol/L)-induced
chemiluminescence response of pretreatment with different
concentrations of sodium nitroprusside (SNP, 10 nmol/L to 10 mmol/L),
DEA-NO (10 nmol/L to 1 mmol/L), poly-ADP-ribosylation inhibitor
(benzamide, 2 × 103 mol/L), hemoglobin (Hb, 5 or 10 µmol/L), calcium channel antagonist (felodipine, 5 × 106 mol/L), and NOS inhibitor (aminoguanidine, 5 × 103 mol/L) was studied. Chemiluminescence
responses were also studied in the absence of extracellular calcium (a
calcium-free medium).
Preparation of hypoxic and hypoxic-reoxygenated cells.
Rat PMNLs were subjected to hypoxic conditions by suspending the cells
in HBSS, which was sponged with nitrogen gas for 30 minutes, and the
partial pressure of oxygen was then measured to assess the oxygen
depletion. Partial pressures of oxygen (pO2) and pH (7.3 ± 0.007) were measured in the cells suspended in HBSS before and
after hypoxia (H) and reoxygenation (H-R) using a blood gas analyzer
(Eschweiler System C 2000; Keil, Germany). PMNLs were
incubated in the oxygen-depleted HBSS for 30 minutes at 37°C and
pO2 was measured at the end of the incubation time
(pO2 68.05 ± 3 mm Hg, n = 90). PMNLs were reoxygenated
by suspending the cells in normoxic HBSS (pO2 120.9 ± 2.6 mm Hg, n = 90) after centrifugation, and pO2 was again
measured (117.2 ± 4.2 mm Hg, n = 50). Viability of the cells was
measured at the end of the experiment by a trypan blue exclusion test
(96% ± 1.3%) and LDH release, which was 7.29% for normoxic
cells, and was unchanged after hypoxia and hypoxia-reoxygenation.
Measurement of the myeloperoxidase (MPO) activity.
Experiments were performed to determine the AA (1 × 105 mol/L)-induced MPO release from PMNLs granules
during H and H-R. Enzyme activity was estimated in the supernatant, and
the remaining activity was estimated in the lysed PMNLs preparations by
the methods described earlier.12 Briefly, 0.1 mL lysate or
supernatant was incubated in a 2.5-mL phosphate buffer (pH 6.2) with
0.05% hydrogen peroxide and O-dianisidine (250 µg) at 37°C for
30 minutes. Reaction was stopped by adding sodium azide, and the
absorbance of the solutions was read at 460 nm. The percentage of the
enzyme released was calculated relative to the total activity (obtained
in each set by adding the activities estimated in supernatant and in
the lysate).
Nitrite measurement.
Nitrite contents in the normal control, H, and H-R PMNLs were measured
by diazo formation.21 The cell suspensions (5 × 107 cells/mL) were incubated in normoxic, hypoxic, and
reoxygenated conditions in the presence of superoxide dismutase and
catalase (50 U/mL); the cells were then sonicated and supernatants were used for nitrite estimations. The supernatants were incubated with
NADPH (40 µmol/L), FAD (4 µmol/L), and nitrate reductase (0.1 U)
for 30 minutes at 37°C. The reaction mixture was then treated with
lactate dehydrogenase (2 U/mL) and sodium pyruvate (5 µmol/L) and
incubated for a further 20 minutes at 37°C. The color was developed
by using Griess reagent, and absorbance was recorded at 548 nm after a
30-minute incubation at 37°C. The amount of nitrite was calculated
from a standard curve of sodium nitrite.
Measurement of intracellular calcium levels.
The PMNLs suspended in calcium-free HBSS were loaded with Fura 2-AM (3 µmol/L) for 60 minutes at 4°C. After 60 minutes, the cells were
diluted twofold with calcium-free HBSS and centrifuged, resuspended in
normoxic or hypoxic HBSS, and incubated at 37°C for 30 minutes.
[Ca+2]i was estimated by using the method of
Grynkiewiez et al.22
Calculations and statistical analysis.
Each experimental group consisted of at least four or five sets of
experiments. Results were expressed as the mean ± standard error
(SE) for separate experiments, and comparisons were made by paired
Student's t-test or by one way analysis of variance for
multiple comparisons of three or more groups. When F was significant, the differences between individual groups were compared with
t-test results. The differences were considered to be
statistically significant when P value was less than .05.
| |
RESULTS |
Effect of hypoxia-reoxygenation on the rat PMNLs chemiluminescence
response induced by various agents.
PMNLs were subjected to hypoxia for variable time intervals (10, 20, and 30 minutes) and then reoxygenated for 30 minutes. There was an
increase in the AA-induced free radical generation after reoxygenation
and no significant difference between hypoxia for 20 and 30 minutes was
observed (Fig 1). In another set of experiments, cells were subjected to 10-, 20-, and 30-minute periods of
reoxygenation after 15 and 30 minutes of hypoxia (Fig 1). Although 10 minutes of hypoxia followed by 30 minutes of reoxygenation was
sufficient to induce an increase in free radical generation, we
observed a significant and consistent increase at 30 minutes of hypoxia
and 30 minutes of reoxygenation. Therefore, in all of the subsequent
studies, 30 minutes of hypoxia followed by 30 minutes of reoxygenation
was used (Fig 2A). A significant increase in the luminol, as well as the lucigenin-dependent chemiluminescence response induced by FMLP, OZ, or PMA, was also observed after reoxygenation (30 minutes) of the hypoxic (30 minutes) rat PMNLs (Table 1). AA-induced MPO release from the
normoxic cells was 24% ± 6%, which was not altered after hypoxia
(30% ± 5%) or H-R (31% ± 6%).
View larger version (32K):
[in this window]
[in a new window]
View larger version (18K):
[in this window]
[in a new window]
| Fig 1.
(Top) Effect of hypoxia for different time intervals (10, 20, and 30 minutes) and reoxygenation (30 minutes) on free radical
generation. (Bottom) Effect of variable time of reoxygenation (10, 20, and 30 minutes) on hypoxic cells. Cells were subjected to hypoxia for
15 (solid bars) or 30 (empty bars) minutes. Free radical generation
response was induced by AA (1 to 5 × 105 mol/L) after
H-R. *P < .01 in comparison with the control.
|
|
View larger version (12K):
[in this window]
[in a new window]
| Fig 2.
(A) AA-induced chemiluminescence response in normoxic
(C), hypoxic (H), and reoxygenated (H-R) PMNLs (5 × 106
cells). (B) AA-induced chemiluminescence response in SNP pretreated
cells. SNP was used at 10 nmol/L and 100 µmol/L concentrations.
|
|
Effect of NO donors on AA induced chemiluminescence response of rat
PMNLs.
Pretreatment of rat peripheral PMNLs with SNP or DEA-NO for 10 to 20 minutes increased the AA-induced chemiluminescence response at 10 and
100 nmol/L concentrations (Figs 2B and 3).
However, higher concentrations (10 µmol/L to 10 mmol/L) reduced the
LCL response significantly (Figs 2B and 3).
View larger version (25K):
[in this window]
[in a new window]
| Fig 3.
Histogram showing effect of SNP (open bars) and DEA-NO
(hatched bars) at different concentrations (10 nmol/L to 10 mmol/L) on
the AA-induced PMNLs chemiluminescence response. *P < .05, **P < .01, and ***P < .001 in comparison with the
control.
|
|
Modulation of chemiluminescence response in the SNP pretreated cells
by intracellular and extracellular calcium.
While AA-induced LCL response in the normoxic cells was not altered in
the absence of extracellular calcium, potentiation in the AA-induced
LCL response after H-R was not observed in the absence of extracellular
calcium (Fig 4). Interestingly,
augmentation in the AA-induced LCL response in the presence of the NO
donor SNP (10 nmol/L) was also blocked when extracellular calcium was absent (Fig 4).
View larger version (38K):
[in this window]
[in a new window]
View larger version (29K):
[in this window]
[in a new window]
| Fig 4.
(Top) AA (1 × 105 mol/L) stimulated
chemiluminescence response in normoxic, H-R, or SNP (10 nmol/L)
pretreated cells in the presence and absence of calcium. (Bottom)
Intracellular calcium concentration in the normoxic (C), SNP (S, 10 nmol/L) pretreated, hypoxic (H), and reoxygenated (H-R) PMNLs.
Alteration in the cell calcium levels was estimated in absence of Hb
(left set) and in the presence of Hb (10 µmol/L, right set).
*P < .05 and **P < .01 in comparison with the
control.
|
|
Effect of SNP pretreatment, hypoxia, or H-R on the intracellular
calcium levels.
Intracellular calcium levels were found to be augmented after the
addition of 10 nmol/L SNP to the rat PMNLs (Fig 4). A significant increase in intracellular calcium levels was also observed after hypoxia, and this increase did not revert back to the normal control level after reoxygenation (Fig 4). In the presence of hemoglobin (5 µmol/L) an increase in the intracellular calcium levels after SNP
pretreatment or hypoxia was blocked (Fig 4). However, Hb had no
significant effect on the basal calcium levels in the normoxic cells
(Fig 4).
Effect of hypoxia and H-R on the PMNLs nitrite content.
Nitrite content was measured in the normoxic, H, and H-R PMNLs cell
suspensions. PMNLs nitrite concentrations (1.15 ± 0.07 µmol/L/5 × 107 cells) after hypoxia were found to be
significantly augmented (42% ± 5%, P < .01) in
comparison to the control (Fig 5). While AA
stimulation also increased the nitrite content in the PMNLs, there was
no significant difference in normoxic versus hypoxic or H-R cells (data
not shown).
View larger version (50K):
[in this window]
[in a new window]
| Fig 5.
Nitrite content in normoxic (C), hypoxic (H), and
reoxygenated (H-R) cell suspensions. Nitrite was estimated in 5 × 107 cells after a 30-minute incubation at 37°C.
*P < .01 in comparison with the control.
|
|
Effect of hemoglobin, benzamide, and felodipine pretreatment on SNP
or H-R induced increases in the chemiluminescence response.
The H-R or SNP-induced potentiation of the AA-induced PMNLs free
radical generation response was prevented by the addition of Hb to the
hypoxic cells or by the pretreatment of cells with benzamide or
felodipine (Figs 6 and
7).
View larger version (15K):
[in this window]
[in a new window]
View larger version (15K):
[in this window]
[in a new window]
| Fig 6.
(Top) Effect of Hb (10 µmol/L) pretreatment on the
chemiluminescence response after H-R and SNP (10 nmol/L) pretreatment.
(Bottom) Histogram representing effect of benzamide (2 × 103 mol/L) on the AA-induced chemiluminescence response
of H-R and SNP pretreated PMNLs. *P < .01 in comparison with
the control.
|
|
Effect of NOS inhibitors on the chemiluminescence response after H-R.
Aminoguanidine pretreatment prevented the enhancing effect of H-R on
the AA-induced chemiluminescence response (Fig 7). Another NOS
inhibitor, 7-nitroindazole (1 × 103 mol/L),
was also used to assess the effect of NOS inhibition on H-R-mediated
augmentation of OZ-induced chemiluminescence response (Table 1).
OZ-induced LCL responses in normoxic and H-R PMNLs in the presence of
7-nitroindazole were 639.36 ± 93.47 and 671.56 ± 70.03 (n = 20)
chemiluminescence units, respectively.
| |
DISCUSSION |
The results obtained in the present investigation suggest that NO
donors had a biphasic effect on the rat PMNLs free radical generation
response. Low concentrations (10 and 100 nmol/L) potentiated, but
higher concentrations (10 µmol/L to 10 mmol/L) attenuated ROS
generation from the rat PMNLs. We postulate that H-R increases the NO
level causing ADP-ribosylation and increases the intracellular calcium
level, which potentiates PMNLs free radical generation.
Rubanyi et al23 have demonstrated the scavenging role of NO
on the PMNLs superoxide radicals. Other investigators have shown that
augmentation in the exogenous or endogenous NO inhibits NADPH-oxidase activity and scavenges free radicals.8,24,25 An increase in
the intracellular calcium levels in a guanosine 3 -5 cyclic monophosphate (cGMP) independent manner by SNP in
platelets, endothelial cells, and PMNLs has been
observed.11,26,27 Consistent with these results, we also
observed an increase in the intracellular calcium levels along with
augmented chemiluminescence response in the presence of SNP at 10 nmol/L concentration. In contrast, higher concentrations (10 µmol/L
to 10 mmol/L) inhibited free radical generation8 (Figs 2
and 3). We also observed that benzamide, an ADP ribosylation
inhibitor,12,28 and felodipine, an inhibitor of calcium
channels on the PMNLs,29 inhibited SNP-mediated
augmentation in the chemiluminescence response. SNP did not augment ROS
generation in the calcium-deficient medium. Bromo cGMP (a cell
permeable analogue of cGMP) did not modify the free radical generation
response (data not shown). Thus, it appears that NO donors (SNP and
DEA-NO), possibly by ADP-ribosylation-mediated mechanisms, raised the
intracellular calcium, which augments ROS generation in the rat PMNLs.
Studies on inflammation, ischemia, and reperfusion have recognized
PMNLs as the principal participant and effector cells in acute
inflammatory conditions.1,2 The availability of NO during
hypoxia/ischemia has been found to be augmented.16,18 To
delineate the biological importance and relevance of NO-mediated increases in free radical generation, we investigated the role of NO in
H-R-mediated increases in free radical generation response. Reoxygenation of PMNLs and endothelial cells in vitro after brief periods of hypoxia has been shown to augment free radical
generation,6,8 a result replicated in the present study. We
have found that nitrite contents in the hypoxic cell suspensions
paralleled the concentrations of NO donors used, which also potentiated
free radical generation response in the normal PMNLs. When the cells
were incubated in the presence of Hb, a NO scavenger,30
during hypoxia and reoxygenation, there was no increase in the free
radical generation, suggesting involvement of NO. During hypoxia there
was a significant increase in the intracellular calcium levels, which
did not revert back to the control levels after reoxygenation. In the
cerebellar granule cells, it was reported that hypoxia-induced
elevation of the intracellular calcium was greatly attenuated by NOS
inhibition.31 Interestingly, in the presence of the NO
scavenger, Hb, we observed no increase in the intracellular calcium in
the PMNLs subjected to H-R. Hb did not affect the basal calcium levels
in the control cells (Fig 4). H-R-mediated augmentation in ROS
generation was also totally prevented by pretreatment of the cells with
a calcium antagonist, felodipine. Furthermore, the basal free radical
generation response was found to be independent of the presence of
extracellular calcium in the normal PMNLs. However, H-R-mediated
potentiation in free radical generation response was totally dependent
on the presence of extracellular calcium (Fig 4), thereby suggesting
that NO mediates an increase in the intracellular calcium during H-R,
which augments the ROS generation.
A significant reduction in the cerebral infarct size in nNOS (neuronal
NOS) knock-out mice32 and augmentation in NO synthesis due
to the induction of NOS after hypoxia/ischemia has been
shown.18,33 It is interesting to observe that NOS
inhibition in the nNOS containing cells protects against
ischemia/hypoxia and reoxygenation injury.28,32 NOS in the
PMNLs resembles nNOS, as shown by specific antibodies and DNA
probes.34,35 However, it has not yet been demonstrated that
there is any interrelationship between NO and calcium in the
augmentation of free radical generation response after H-R in the
PMNLs. Recently Volk et al36 have shown in human
endothelial cells that the blockade of the receptor-mediated calcium
entry or prevention of intracellular calcium release inhibits
NO-induced interleukin-8 secretion, suggesting an interrelationship
between NO and calcium. Our work shows for the first time that the
NO-mediated increase in the intracellular calcium in the hypoxic cells
is responsible for the increase in free radical generation after reoxygenation.
Previous research in our laboratory demonstrated the nonspecific
interference of L-nitro arginine analogues with the chemiluminescence response.37 Chen and Mehta34 have also shown
that L-arginine analogues do not inhibit PMNLs NOS activity. Therefore,
we used 7-nitroindazole (an inhibitor of nNOS28) and
aminoguanidine (a NOS inhibitor38), which inhibited PMNLs
NOS (data not shown) and prevented H-R-mediated increases in the PMNLs
free radical generation. Thus, inhibition of NO synthesis in PMNLs was
protective against H-R-mediated increases in the free radical
generation, indicating the involvement of NO in this response.
In addition, the role of granular MPO18 release in the
augmentation of chemiluminescence response was also assessed. However, no significant change in MPO release after AA stimulation from normoxic
versus H-R cells was observed. This indicates that the increase in ROS
generation after H-R is not mediated by MPO. Furthermore, H-R-mediated
augmentation in ROS generation was also prevented by the pretreatment
of the cells with an ADP ribosylation inhibitor, benzamide.
Recently Thiemermann et al28 reported that ADP-ribosylation
inhibitors reduced the infarct size caused by ischemia and reperfusion of rabbit myocardium and skeletal muscles. In the skeletal muscles, inhibition of nNOS by 7-nitroindazole also decreased the infarct size.
However, they did not investigate the involvement of calcium in the
ADP-ribosylation-mediated injury.28 NO-mediated ADP
ribosylation of G protein has been demonstrated to regulate the
intracellular second messenger level in the bovine ciliary
body.39 Regulation of the mitochondrial calcium homeostasis
in the isolated hepatocyte has also been shown to be controlled by the
protein ADP-ribosylation.40,41 In the present study, we
have achieved the blockade of H-R-mediated increase in the ROS
generation by the calcium antagonist, NOS, and ADP-ribosylation
inhibitors. It, therefore, appears that during H-R, NO induces
ADP-ribosylation, which increased the intracellular calcium, which in
turn, increases the ROS generation. The role of these mechanisms in
hypoxia/ischemic injury to various organs needs further examination.
| |
ACKNOWLEDGMENT |
We are grateful to S.K. Mandal for statistical analysis of the data.
| |
FOOTNOTES |
Submitted February 4, 1998;
accepted August 14, 1998.
This is CDRI Communication No. 5742.
Supported by a financial grant to M.D. from the Department of Science
and Technology, New Delhi, India, and an award of research fellowship
to M.P.S. from the Council of Scientific and Industrial Research New
Delhi, India.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Madhu Dikshit, PhD,
Pharmacology Division, Central Drug Research Institute, Lucknow 226 001, India; e-mail: root{at}csdri.ren.nic.in.
| |
REFERENCES |
1.
Sheridan FM, Dauber IM, McMurty IF, Lesenfsky EJ, Horwitz LD:
The role of leukocytes in coronary vascular endothelial injury due to ischemia and reperfusion.
Circ Res
69:1566, 1991[Abstract/Free Full Text]
2.
Albertine KH, Weyrich AS, Ma X-L, Lefer DJ, Becker LC, Lefer AM:
Quantification of neutrophil migration following myocardial ischemia and reperfusion in cats and dogs.
J Leukoc Biol
55:557, 1994[Abstract]
3.
Sharar SR, Mihelcic DD, Han KT, Harlan JM, Winn RK:
Ischemia reperfusion injury in the rabbit ear is reduced by both immediate and delayed CD18 leukocyte adhesion blockade.
J Immunol
153:2234, 1994[Abstract]
4.
Lehr H-A, Menger M, Messmer K:
Impact of leukocyte adhesion on myocardial ischemia/reperfusion injury: Conceivable mechanisms and proven facts.
J Lab Clin Med
121:539, 1993[Medline]
[Order article via Infotrieve]
5.
Hansen PR:
The role of neutrophils in myocardial ischemia and reperfusion.
Circulation
91:1872, 1995[Abstract/Free Full Text]
6.
Simms H, D'Amico R, Garner C:
Polymorphonuclear leukocyte opsonic receptor expression after hypoxia/reoxygenation.
J Lab Clin Med
127:364, 1996[Medline]
[Order article via Infotrieve]
7.
Carreras MC, Pargament GA, Catz SD, Poderoso JJ, Boveris A:
Kinetics of nitric oxide and hydrogen peroxide production and formation of peroxynitrite during the respiratory burst of human neutrophils.
FEBS Lett
341:65, 1994[Medline]
[Order article via Infotrieve]
8.
Seth P, Kumari R, Dikshit M, Srimal RC:
Modulation of rat peripheral polymorphonuclear leukocyte response by nitric oxide and arginine.
Blood
84:2741, 1994[Abstract/Free Full Text]
9.
Brune B, Lapetine EG:
Activation of cytosolic ADP-ribosyl transferase by nitric oxide generating agents.
J Biol Chem
269:8455, 1989[Abstract/Free Full Text]
10.
Piper GM, Clarke GA, Gross GJ:
Stimulatory and inhibitory action of nitric oxide donor agents vs. nitrovasodilators on reactive oxygen production by isolated polymorphonuclear leukocytes.
J Pharmacol Exp Ther
269:451, 1994[Abstract/Free Full Text]
11.
Morikawa M, Inoue M, Tokumaru S, Kogo H:
Enhancing and inhibitory effects of nitric oxide on superoxide anion generation in human polymorphonuclear leukocytes.
Br J Pharmacol
115:1302, 1995[Medline]
[Order article via Infotrieve]
12.
Zhang J, Dawson VL, Dawson TM, Snyder SH:
Nitric oxide activation of poly (ADP ribose) synthetase in neurotoxicity.
Science
263:687, 1994[Abstract/Free Full Text]
13.
Kanagy NL, Charpie JR, Webb RC:
Nitric oxide regulation of ADP-ribosylation of G proteins in hypertension.
Med Hypotheses
44:159, 1995[Medline]
[Order article via Infotrieve]
14.
Bolli R, Jeroudi MO, Patel BS:
Direct evidence that oxygen derived free radical contributes to post ischemic myocardial dysfunction in the intact dog.
Proc Natl Acad Sci USA
86:4695, 1989[Abstract/Free Full Text]
15.
Terada LS:
Hypoxia-reoxygenation increases O2 efflux which injures endothelial cells by an extracellular mechanism.
Am J Physiol
270:H945, 1996[Abstract/Free Full Text]
16.
Hewett SJ, Muir JK, Lobner D, Symons A, Choi DW:
Potentiation of oxygen-glucose deprivation induced neuronal death after induction of iNOS.
Stroke
27:1586, 1996[Abstract/Free Full Text]
17.
Dalkara T, Moskowitz MA:
The complex role of nitric oxide in the pathophysiology of focal cerebral ischemia.
Brain Pathol
4:49, 1994[Medline]
[Order article via Infotrieve]
18.
Archer SL, Freude KA, Shultz PJ:
Effect of graded hypoxia on the induction and function of inducible nitric oxide synthase in rat mesangial cells.
Circ Res
77:21, 1995[Abstract/Free Full Text]
19.
Boyum A:
Isolation of granulocyte cells and lymphocytes from human blood.
Scand J Lab Invest
21:77, 1968 (suppl 97)
20.
Dikshit M, Kumari R, Srimal RC:
Pulmonary thromboembolism induced alteration in nitric oxide release from rat circulating neutrophils.
J Pharmacol Exp Ther
265:1369, 1993[Abstract/Free Full Text]
21.
Granger DL, Taintor RR, Read R, Boockvar KS, Hibbs JB:
Measurement of nitrate and nitrite in biological samples using nitrate reductase and Griess reagent.
Methods Enzymol
268:142, 1996[Medline]
[Order article via Infotrieve]
22.
Grynkiewiez G, Poenia M, Tsien RY:
A new generation of calcium indicators with greatly improved fluorescence properties.
J Biol Chem
260:3440, 1985[Abstract/Free Full Text]
23.
Rubanyi GM, Ho EH, Canter EH, Lumma WC, Botelho LHP:
Cytoprotective function of nitric oxide: Inactivation of superoxide radicals produced by human leukocytes.
Biochem Biophys Res Commun
181:1392, 1991[Medline]
[Order article via Infotrieve]
24.
Clancy RM, Leszezynoka-Piziak J, Abramson SB:
Nitric oxide an endothelial cell relaxation factor, inhibits neutrophil superoxide anion production via a direct action on the NADPH oxidase.
J Clin Invest
90:116, 1992
25.
Forslund T, Sundqvist T:
Nitric oxide reduces hydrogen peroxide production from human polymorphonuclear neutrophils.
Eur J Clin Invest
25:9, 1995[Medline]
[Order article via Infotrieve]
26.
Sang K-HLQ, Lantoine F, Devynck M-A:
Influence of authentic nitric oxide on basal cytosolic [Ca2+] and Ca2+ release from internal stores in human platelets.
Br J Pharmacol
119:1361, 1996[Medline]
[Order article via Infotrieve]
27.
Volk T, Mading K, Hensel M, Kox WJ:
Nitric oxide induces transient Ca2+ changes in endothelial cells independent of cGMP.
J Cell Physiol
172:296, 1997[Medline]
[Order article via Infotrieve]
28.
Thiemermann C, Bowes J, Myint FP, Vane JR:
Inhibition of the activity of poly (ADP ribose) synthetase reduces ischemia-reperfusion injury in the heart and skeletal muscle.
Proc Natl Acad Sci USA
94:679, 1997[Abstract/Free Full Text]
29.
Haga Y, Dumitrscu A, Zhang Y, Stain-Malmgren R, Sjoquist P-O:
Effects of calcium blockers on the cytosolic calcium, H2O2 production and elastase release in human neutrophils.
Pharmacol Toxicol
79:312, 1996[Medline]
[Order article via Infotrieve]
30.
Iha S, Orita K, Kanno T, Utsumi T, Sata EF, Inoue M, Utsumi K:
Oxygen-dependent inhibition of neutrophil respiratory burst by nitric oxide.
Free Radic Res
25:489, 1996[Medline]
[Order article via Infotrieve]
31.
Mei JM, Wei MC, Benjamin FT, Christine UE:
Involvement of nitric oxide in the deregulation of cytosolic calcium in cerebellar neurons during combined glucose and oxygen deprivation.
Mol Chem Neuropathol
27:155, 1996[Medline]
[Order article via Infotrieve]
32.
Huang Z, Huang PL, Panahian N, Dalkara T, Fishman MC, Moskowitz MA:
Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase.
Science
265:1883, 1994[Abstract/Free Full Text]
33.
Hampl V, Cornfield DN, Cowan NJ, Archer SL:
Hypoxia potentiates nitric oxide synthesis and transiently increases cytosolic calcium levels in pulmonary artery endothelial cells.
Eur Respir J
8:515, 1995[Abstract]
34.
Chen LY, Mehta JL:
Variable effects of L-arginine analogs on L-arginine-nitric oxide pathway in human neutrophils and platelet may relate to different nitric oxide synthase isoforms.
J Pharmacol Exp Ther
276:253, 1996[Abstract/Free Full Text]
35.
Wallerath T, Gath I, Aulitzky WE, Pollock JS, Kleinert H, Fostermann U:
Identification of NO synthase isoforms expressed in human neutrophil granulocytes, megakaryocytes and platelets.
Thromb Haemost
77:163, 1983
36.
Volk T, Hensel M, Mading K, Erger K, Kox WJ:
Intracellular Ca2+ dependence of nitric oxide mediated enhancement of interleukin-8 secretion in human endothelial cells.
FEBS Lett
415:169, 1997[Medline]
[Order article via Infotrieve]
37.
Dikshit M, Chari SS, Seth P, Kumari R:
Interaction of nitric oxide synthase inhibitors and their D-enantiomers with rat neutrophil luminol dependent chemiluminescence response.
Br J Pharmacol
119:578, 1996[Medline]
[Order article via Infotrieve]
38.
Tracey WR, Tse J, Carter G:
Lipopolysaccharide-induced changes in plasma nitrite and nitrate concentrations in rats and mice: Pharmacological evaluation of nitric oxide synthase inhibitors.
J Pharmacol Exp Ther
272:1011, 1995[Abstract/Free Full Text]
39.
Pozdnyakov N, Margulis A, Sitaramayya A:
Endogenous ADP-ribosylation of a G i protein in bovine ciliary body is stimulated by NO.
Arch Biochem Biophys
235:482, 1997
40.
Weis M, Kass GEN, Orrenius S, Moldeus P:
N-Acetyl-p-benzoquinone imine Ca2+ release from mitochondria by stimulating pyridine nucleotide hydrolysis.
J Biol Chem
267:804, 1992[Abstract/Free Full Text]
41.
Juedes MJ, Kass GEN, Orrenius S:
m-Iodobenzylguanidine increases the mitochondrial Ca2+ pool in isolated hepatocytes.
FEBS Lett
313:39, 1992[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
R. Saini, S. Patel, R. Saluja, A. A. Sahasrabuddhe, M. P. Singh, S. Habib, V. K. Bajpai, and M. Dikshit
Nitric oxide synthase localization in the rat neutrophils: immunocytochemical, molecular, and biochemical studies
J. Leukoc. Biol.,
March 1, 2006;
79(3):
519 - 528.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Tsukimori, K. Fukushima, A. Tsushima, and H. Nakano
Generation of Reactive Oxygen Species by Neutrophils and Endothelial Cell Injury in Normal and Preeclamptic Pregnancies
Hypertension,
October 1, 2005;
46(4):
696 - 700.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Sharma, S. A.V. Raghavan, R. Saini, and M. Dikshit
Ascorbate-mediated enhancement of reactive oxygen species generation from polymorphonuclear leukocytes: modulatory effect of nitric oxide
J. Leukoc. Biol.,
June 1, 2004;
75(6):
1070 - 1078.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction
