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Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 333-340
By
From the Pharmacology Division, Central Drug Research Institute,
Lucknow, India.
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.
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.
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), 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 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 × 10 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 × 10 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.
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%).
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).
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).
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).
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).
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 × 10 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.
We are grateful to S.K. Mandal for statistical analysis of the data.
Submitted February 4, 1998;
accepted August 14, 1998.
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.
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This article has been cited by other articles:
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Top
Abstract
Introduction
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).
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.
-5
i protein in bovine ciliary body is stimulated by NO.
Arch Biochem Biophys
235:482, 1997
