© 1996 American Heart Association, Inc.
Articles |
Role of Poly-ADP Ribosyltransferase Activation in the Vascular Contractile and Energetic Failure Elicited by Exogenous and Endogenous Nitric Oxide and Peroxynitrite
From the Children's Hospital Medical Center, Division of Critical Care, Cincinnati, Ohio.
Correspondence to Dr Csaba Szabó, Children's Hospital Medical Center, Division of Critical Care, 3333 Burnet Ave, Cincinnati, OH 45229.
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Abstract Stimulation of vascular smooth muscle with bacterial lipopolysaccharide (LPS) and proinflammatory cytokines induces the expression of a distinct isoform of NO synthase (inducible NOS [iNOS]) contributing to the suppression of vascular contractility. We have obtained evidence of the involvement of an indirect pathway triggered by NO and its reaction product peroxynitrite (ONOO-) through the activation of the nuclear enzyme poly-ADP ribosyltransferase (PARS) in the pathogenesis of cellular energetic and contractile failure in vascular smooth muscle. Exposure of vascular smooth muscle cells caused DNA strand breaks, activation of PARS, depletion of NAD+, and inhibition of mitochondrial respiration. The NAD+ depletion and inhibition of mitochondrial respiration were reduced by pharmacological inhibition of PARS. Stimulation of vascular smooth muscle cells with LPS and interferon gamma (IFN-
) triggered the production of superoxide anion
over 3 to 48 hours and NO and ONOO- over 24 to 48
hours and resulted in significant DNA strand breakage. The decrease in
mitochondrial respiration in response to LPS and IFN- stimulation
was inhibited by the ONOO- scavenger uric acid (100
µmol/L) and by inhibitors of iNOS. The PARS
inhibitors 3-aminobenzamide (1 mmol/L), nicotinamide
(1 mmol/L), and PD 128763 (100 µmol/L) inhibited the reduction in
cellular NAD+ and ATP and the suppression of mitochondrial
respiration in response to LPS and IFN- stimulation.
Administration of 3-aminobenzamide also reduced PARS activation and
vascular hyporeactivity of rat thoracic aortas exposed to
ONOO- (300 µmol/L to 1.5 mmol/L) in vitro.
3-Aminobenzamide (10 mg/kg IP) preserved the ex vivo
contractility of aortas obtained from endotoxic rats
and improved survival in lethal murine endotoxic shock. These data
suggest that PARS activation due to iNOS induction (1) is involved in
the energetic depletion of vascular smooth muscle cells that express
iNOS and (2) contributes to the pathogenesis of vascular energetic and
contractile failure in endotoxic shock. Inhibition of PARS may be a
novel concept of therapeutic potential in shock.
Key Words: septic shock • contraction • inflammation • endotoxin • hyporeactivity
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Introduction |
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Proinflammatory cytokines and bacterial LPS induce the expression of a distinct inducible isoform of NOS (iNOS) in vascular smooth muscle cells and other cell types (see References 11 to 32 3 ). Production of large amounts of NO by iNOS in the vascular smooth muscle has been associated with the suppression of mitochondrial respiration4 5 and the reduction of the contractile ability of various arteries.6 7 8 Inasmuch as these effects are reversed by the inhibition of NOS, an independent role for NO has been proposed to account for the loss of cellular respiration and the development of vascular failure (vascular decompensation and vascular hyporeactivity to vasoconstrictor agents) in various forms of circulatory shock.9 10 11 12
Stimulation with proinflammatory cytokines, however, invokes a pleiotropic cellular response, including the stimulation of oxygen-centered free radicals, such as superoxide anion.13 14 Simultaneous generation of both oxygen- and nitrogen-centered free radicals favors the development of an even more toxic reaction product, the oxidant peroxynitrite (ONOO-),15 16 17 as has been recently demonstrated in inflammation and endotoxic shock.18 19 20 Both NO and ONOO- have been shown to exert cytotoxic effects by inhibiting mitochondrial respiration.4 5 21 22 23 24 Moreover, recent data indicate that ONOO- is a more potent suppressor of the mitochondrial respiration than is NO per se.23 24
NO and ONOO- directly inhibit mitochondrial respiration in cells expressing iNOS by covalent modification of iron-containing enzymes in the mitochondrial respiratory chain, such as NADH/ubiquinone oxidoreductase, NADH/succinate oxidoreductase, and cis-aconitase.1 4 23 24 Recently, an indirect mechanism has been identified in pancreatic islet cells25 26 and in neurons27 : NO-mediated DNA strand breakage triggers a futile cycle by the nuclear enzyme PARS.25 26 27 Activation of the PARS pathway is known to result in massive depletion of NAD+ and ATP and eventually induces irreversible cytotoxicity and cell death.28 29 30 Recent data show that ONOO-, when given to DNA or to intact cells, is an extremely potent activator of DNA strand breaks.31 32 33 34 We have recently demonstrated in murine macrophages that DNA strand breaks caused by ONOO- potently activate PARS, resulting in the exhaustion of cellular NAD+ and ATP stores and the suppression of mitochondrial respiration.34
The aim of the present study was to investigate whether activation
of PARS (triggered by endogenous NO and
ONOO-) is involved in the pathogenesis of energetic
failure and vascular collapse in shock. To this end, we have used
pharmacological inhibitors of PARS of various structural
classes.35 36 Our data establish that
endogenous ONOO- suppresses mitochondrial
respiration and vascular contractility in vascular
smooth muscle cells after stimulation with LPS and IFN- via the PARS
pathway, suggesting that this mechanism plays a key role in the
development of vascular contractile dysfunction in endotoxic shock.
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Materials and Methods |
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Cell Culture
RASM cells were cultured in RPMI medium, supplemented with L-glutamine (3.5 mmol/L) and 10% fetal calf serum, as described previously.5 Cells were cultured in 96-well plates (200 µL medium per well) or in 12-well plates (3 mL medium per well) until confluence. To induce iNOS, fresh culture medium containing Escherichia coli LPS (011:B4, 10 µg/mL) and murine IFN-
(50 U/mL) were added in the presence or absence of NOS or PARS
inhibitors. Moreover, cells were exposed to NO donor,
O2- generator, or ONOO-
scavenger compounds.
Measurement of Nitrite Production
Nitrite production, an indicator of NO synthesis, was
measured in the supernatant, as previously described,7 by
adding 100 µL of Griess reagent (1% sulfanilamide and 0.1%
naphthylethylenediamide in 5% phosphoric acid) to 100-µL samples
of conditioned medium. OD550 was measured using a
Spectramax 250 microplate reader (Molecular Devices). Nitrite
concentrations were calculated by comparison with
OD550 of standard solutions of sodium nitrite prepared in
culture medium.
Measurement of Superoxide Production
Assay for superoxide release into the supernatant was carried
out by measuring superoxide dismutase–inhibitable reduction of
ferricytochrome c as described previously.37
Cells were rinsed with PBS and then replaced by a reaction buffer
containing (mmol/L) glucose 2, CaCl2 1, MgCl2
1.3, KCl 4, NaCl 100, and phosphate buffer 10. Ferricytochrome
c (final concentration, 70 µmol/L), in the absence or
presence of superoxide dismutase (350 U/mL), was then added. After a
60-minute incubation at 37°C, the reduction of ferricytochrome
c of the supernatant was measured in 180-µL aliquots at
550 nm by the Spectramax 250 microplate reader. The amount of
superoxide release was calculated by using the extinction coefficient
for the change of ferricytochrome c to ferrocytochrome
c (E550nm=21.0
mmol/L-1·cm-1).
For the time-course measurements, at various time points after
incubation with LPS and IFN-, the supernatant was replaced with the
reaction buffer, and reduction of ferricytochrome c was
measured as described.
Measurement of ONOO- Production
The formation of ONOO- was measured by the
ONOO--dependent oxidation of dihydrorhodamine 123 to
rhodamine 123; this measurement was based on the principles of the
method described by Kooy et al.38 Cells were rinsed with
PBS and then replaced with PBS containing 5 µmol/L dihydrorhodamine.
After a 60-minute incubation at 37°C, the fluorescence of
rhodamine 123 was measured using a Perkin-Elmer LS50B fluorimeter, at
an excitation wavelength of 500 nm (slit width, 5 nm), emission
wavelength of 536 nm (slit width, 5 nm), and an integration time of 10
seconds. The quantity of ONOO- formed was calculated
using a standard curve obtained using 5 µmol/L dihydrorhodamine 123
and authentic ONOO-.
Measurement of Mitochondrial Respiration
Cell respiration was assessed by the mitochondrial-dependent
reduction of MTT to formazan.10 Cells in 96-well plates
were incubated at 37°C with MTT (0.2 mg/mL) for 1 hour. Culture
medium was removed by aspiration, and the cells were solubilized in
dimethyl sulfoxide (100 µL). The extent of reduction of MTT to
formazan within cells was quantified by the measurement of
OD550.
Determination of DNA Single-Strand Breaks
The formation of strand breaks in double-stranded DNA was
determined by the alkaline unwinding method as previously
described.28 34 Cells in 12-well plates were scraped into
0.2 mL of solution A buffer containing (mmol/L) myoinositol 250,
NaH2PO3 10, and MgCl2 1, pH 7.2.
The cell lysate was then transferred into plastic tubes designated T
(maximum fluorescence), P (fluorescence in sample used
to estimate extent of DNA unwinding), or B (background
fluorescence). To each tube, 0.2 mL of solution B (alkaline
lysis solution containing 10 mmol/L NaOH, 9 mol/L urea, 2.5 mmol/L
EDTA, and 0.1% SDS) was added and incubated at 4°C for 10 minutes to
allow cell lysis and chromatin disruption. Solutions C (0.45 vol
solution B in 0.2N NaOH) and D (0.4 vol solution B in 0.2N NaOH) were
then added (0.1 mL each) to the P and B tubes. Solution E at 0.1 mL
(neutralizing solution containing 1 mol/L glucose and 14 mmol/L
mercaptoethanol) was added to the T tubes before solutions C and D were
added. From this point, incubations were carried out in the dark. A
30-minute incubation period at 0°C was then allowed during which the
alkali diffused into the viscous lysate. Since the neutralizing
solution, solution E, was added to the T tubes before addition of the
alkaline solutions C and D, the DNA in the T tubes was never exposed to
a denaturing pH. At the end of the 30-minute incubation, the contents
of the B tubes were sonicated for 30 seconds to ensure rapid
denaturation of DNA in the alkaline solution. All tubes were then
incubated at 15°C for 10 minutes. Denaturation was stopped by
chilling to 0°C and adding 0.4 mL of solution E to the P and B tubes.
Solution F at 1.5 mL (6.7 µg/mL ethidium bromide in 13.3 mmol/L NaOH)
was added to all the tubes, and fluorescence (excitation, 520
nm; emission, 590 nm) was measured by a Perkin-Elmer fluorimeter. Under
the conditions used, in which ethidium bromide binds preferentially to
double-stranded DNA, the percentage of double-stranded DNA (D)
may be determined using the following equation:
%D=100x[F(P)-F(B)]/[F(T)-F (B)], where F(P) is the
fluorescence of the sample, F(B) is the background
fluorescence (ie, fluorescence due to all cell
components other than double-stranded DNA), and F(T) is the maximum
fluorescence.
Measurement of Cellular PARS Activity
The culture medium in 12-well plates was replaced with 0.5 mL of
56 mmol/L HEPES buffer, pH 7.5, containing 28 mmol/L KCl, 28 mmol/L
NaCl, 2 mmol/L MgCl2, 0.01% digitonin, and 125 nmol
NAD+ spiked with 0.25 µCi
[3H]NAD+. PARS activity was then measured as
described previously.28 Digitonin was used to
permeabilize plasma membranes.28 The
permeabilized cells were incubated for 5 minutes at
37°C, and the protein that was ribosylated with
[3H]NAD+ was precipitated with 200 µL of
50% TCA. After two washes with TCA, the protein pellet was solubilized
in 2% SDS in 0.1 mol/L NaOH and incubated at 37°C overnight, and the
radioactivity was determined by a 1450 Microbeta Plus scintillation
counter (Wallac).
Measurement of Cellular NAD+ Levels
Cells in 12-well plates were extracted in 0.25 mL of 0.5N
HClO4, scraped, neutralized with 3 mol/L KOH, and
centrifuged for 2 minutes at 10 000g. The
supernatant was assayed for NAD+ using a modification of
the colorimetric method,39 in which NADH,
produced by enzymatic cycling with alcohol dehydrogenase, reduces MTT
to formazan through the intermediation of phenazine methosulfate. The
rate of MTT reduction is proportional to the concentration of the
coenzyme. The reaction mixture consisted of 10 µL of a solution of
2.5 mg/mL MTT, 20 µL of a solution of 4 mg/mL phenazine methosulfate,
10 µL of a solution of 0.6 mg/mL alcohol dehydrogenase (300 U/mg),
and 190 µL of a 0.065 mol/L glycyl-glycine buffer (pH 7.4)
containing 0.1 mol/L nicotinamide and 0.5 mol/L ethanol. The mixture
was warmed to 37°C for 10 minutes, and the reaction was started by
the addition of 20 µL of the sample. The rate of increase in the
absorbance was read immediately after addition of the NAD+
samples and after 10 and 20 minutes of incubation at 37°C against a
blank at 560 nm in the Spectramax spectrophotometer.
Measurement of Cellular ATP Levels
ATP was extracted with 10% TCA containing 0.15% EDTA. The cell
suspension was sonicated for 10 seconds and centrifuged for 10
minutes at 720g at 4°C. The supernatant was stored at
-70°C before measurements. ATP was measured as described
previously40 with an ATP detection kit using firefly
luciferase in a luminometer (model 9020, Analytical Luminescence
Laboratory).
Measurement of Cellular Glutathione Levels
Cellular glutathione was measured as described
previously.41 Briefly, cells were incubated in the
presence of 2.5% sulfosalicylic acid in 0.2% Triton X-100 for 30
minutes at 37°C. Aliquots of the cell lysates were incubated using a
96-well plate assay in triplicate 10-µL samples after the addition of
200 µL phosphate buffer containing 0.15 mmol/L
5,5'-dithio-bis(2-nitrobenzoic acid), 0.2 mmol/L NADPH, and 0.06 U
glutathione reductase, pH 7.2. The difference in the optical density at
414 nm was measured on the Spectramax reader, and three time
points (0, 10, and 20 minutes) were used for quantification.
Measurement of LDH Release
The amounts of LDH released into the cell culture medium were
measured using the Sigma LD-L kit according to the manufacturer's
instructions. Optical density was measured at 340 nm using the
Spectramax reader, and three time points (0, 30, and 60 seconds) were
used for quantification. Aliquots of cells scraped and sonicated for 2
minutes served as positive controls (100% lysis).
Endotoxic Shock Model
Male Wistar rats (Charles River Laboratories, Wilmington, Mass)
were anesthetized with sodium pentathol (120 mg/kg IP) and
instrumented as described previously.42 The trachea was
cannulated to facilitate respiration, and temperature was maintained at
37°C using a homeothermic blanket. The right carotid artery was
cannulated and connected to a pressure transducer for the measurement
of phasic blood pressure, MAP, and heart rate, which were digitized
using a Maclab A/D converter (AD Instruments) and stored and displayed
on a Macintosh personal computer. The left femoral vein was cannulated
for the administration of drugs. Upon completion of the surgical
procedure, cardiovascular parameters were
allowed to stabilize for 10 minutes. After recording of
baseline hemodynamic parameters, endotoxic
shock was induced by injection of E coli LPS (15 mg/kg IV)
at time 0. Rats were injected with intravenous saline (0.2
mL/kg) or 3-aminobenzamide (10 mg/kg IV in 0.2 mL/kg saline) at 90
minutes and killed at 180 minutes after LPS injection. This protocol
was chosen in order to avoid potential interference of the PARS
inhibitor with the process of iNOS induction, which starts
at 60 to 90 minutes after LPS injection in this experimental
model.12 42 The control groups of rats were injected with
vehicle (saline, 0.1 mL/kg IV) at time 0. At 90 minutes, the rats were
injected with 3-aminobenzamide (10 mg/kg IV in 0.1 mL/kg saline) and
observed for a further 90 minutes.
Measurement of Isometric Force in Vascular Rings
Thoracic aortas from rats were cleared of adhering
periadventitial fat and cut into rings 3 to 4 mm in width.
Endothelium was removed from some of the rings by
gently rubbing the intimal surface. Lack of an
acetylcholine-induced relaxation was taken as evidence that the
endothelial cells had been removed. The rings were
mounted in organ baths (5 mL) filled with warmed (37°C),
oxygenated (95% O2/5% CO2)
Krebs' solution (pH 7.4) consisting of (mmol/L) NaCl 118, KCl 4.7,
KH2PO4 1.2, MgSO4 1.2,
CaCl2 2.5, NaHCO3 25, and glucose 11.7, in the
presence of indomethacin (10 µmol/L). Isometric force
was measured with isometric transducers (Kent Scientific Corp),
digitized using a Maclab A/D converter (AD Instruments), and stored and
displayed on a Macintosh personal computer. A tension of 1g
was applied, and the rings were equilibrated for 60 minutes. Fresh
Krebs' solution was provided at 15-minute intervals.
Concentration-response curves to noradrenaline (10-9 to 10-5 mol/L) were obtained in endothelium-denuded aortic rings from control and endotoxic rats obtained after 180 minutes of study. In these studies, LPS was administered intravenously at a dose of 15 mg/kg, in the presence or absence of 3-aminobenzamide treatment.
In a separate set of studies, endothelium-denuded rings in Krebs' solution were exposed to ONOO- (300 µmol/L to 1.5 mmol/L) or vehicle control, in the presence or absence of 1 mmol/L 3-aminobenzamide. After a 30-minute incubation, rings were analyzed for PARS activity or isometric contractility.
Measurement of PARS Activity in Vascular Tissue
Vascular rings were placed in 0.5 mL of 56 mmol/L HEPES buffer
(pH 7.5) containing 28 mmol/L KCl, 28 mmol/L NaCl, 2 mmol/L
MgCl2, 0.01% digitonin (to
permeabilize plasma membranes), and 125 nmol/L
NAD+ spiked with 0.25 µCi
[3H]NAD+. PARS activity was then measured
using a previously described method28 adapted for vascular
tissue. Vascular tissues were incubated with ONOO- in
the presence or absence of 3-aminobenzamide (1 mmol/L) for 10 minutes
at 37°C, and ADP-ribosylated protein was precipitated in 200 µL of
50% TCA. Aortic rings were washed twice and incubated overnight in 2N
HCl, followed by neutralization in NaOH. The protein pellet was
solubilized in 2% SDS in 0.1 mol/L NaOH, and the
[3H]NAD+ incorporation was determined by
scintillation counting. The 3-aminobenzamide–inhibitable component
of the [3H]NAD+ incorporation into proteins
was considered as a specific indicator of PARS activity.
Survival Studies
Swiss albino mice (26 to 30 g, Charles River Laboratories,
Wilmington, Mass) were injected with E coli LPS (120 mg/kg
IP). At the time of injection and every 6 hours thereafter, animals
were treated with 3-aminobenzamide (5 mg/kg IP) or a vehicle
control.
Materials
RPMI and fetal calf serum were obtained from GIBCO. Perchloric
acid and S-methylisothiourea sulfate were obtained from
Aldrich. L-NMA monoacetate and SNAP were purchased from Calbiochem.
Murine IFN- was obtained from Genzyme. Diethylamine NONOate was
purchased from Cayman, and SIN-1 was obtained from Cassella AG.
Dihydrorhodamine 123 and rhodamine 123 were obtained from Molecular
Probes. [3H]NAD+ was purchased from
DuPont/NEN. Alcohol dehydrogenase and NAD+ were obtained
from Boehringer Mannheim. The ATP detection kit was obtained
from Analytical Luminescence Laboratory. Bacterial LPS (E
coli, serotype No. 0127:B8), superoxide dismutase (from bovine
erythrocytes), and all other chemicals were from Sigma Chemical Co.
ONOO- was synthesized and kindly provided by Dr H.
Ischiropoulos (University of Pennsylvania). The PARS
inhibitor PD 12876335 was obtained from
Parke-Davis Co.
Data Analysis
All values in the figures and text are expressed as mean±SEM of
n observations, where n represents the number of wells or
vascular rings studied (six to nine wells or rings from two or three
independent experiments). Data sets were examined by one- and
two-way ANOVA, and individual group means were then compared with
Student's unpaired t test. For comparisons in the survival
rate, the 
2 test was used. A value of
P<.05 was considered significant.
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Effect of Uric Acid on NO- and ONOO--Mediated Inhibition of Mitochondrial Respiration
In order to establish the relative inhibitory potencies of NO, superoxide, and ONOO- on cellular respiration, we exposed cultured vascular smooth muscle cell monolayers to varying concentrations of compounds spontaneously releasing oxygen- or nitrogen-centered free radicals. Cellular respiration was inhibited by exposure to the NO donor compounds diethylamine NONOate and SNAP, with EC50 values of 0.5 and 2 mmol/L, respectively (n=6 to 9). Exposure to SIN-1, a compound that releases NO and superoxide anion in equimolar amounts,43 inhibited cellular respiration, with an EC50 of 3 mmol/L (n=6). Exposure to superoxide anion, provided by treatment with pyrogallol (up to 1 mmol/L), only slightly inhibited mitochondrial respiration (Fig 1
). Simultaneous exposure to pyrogallol and
SNAP (100 µmol/L) exhibited a marked inhibitory effect on
cell respiration (Fig 1). We have observed a similar synergy between
pyrogallol and diethylamine NONOate (not shown).
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In order to specifically implicate the role of
ONOO-, and not the reactants NO and superoxide
anion, in the alterations in cellular energetics, we used uric acid, a
compound that is known to scavenge
ONOO-.44 45 46 We observed that uric acid
(100 µmol/L) prevents the decrease in respiration in response to (1)
the combination of SNAP and pyrogallol or (2) SIN-1 (which
spontaneously generates NO and superoxide anion) but that it confers no
protection against the decrease in respiration in response to
individual free radical formation by SNAP, pyrogallol, or diethylamine
NONOate (Fig 1
). Similar to uric acid, the putative
ONOO- scavengers cysteine (1 mmol/L), glutathione (1
mmol/L), and ascorbic acid (1 mmol/L) also significantly reduced the
SIN-1–induced suppression of mitochondrial respiration (not
shown).
Production of NO, Superoxide, and ONOO- in
RASM Cells Stimulated With LPS and IFN-
Similar to our recent findings in J774
macrophages,47 stimulation of RASM cells with LPS
(10 µg/mL) and IFN- (50 U/mL) resulted in a rapid and
sustained production of superoxide anion: superoxide
production increased from 104±2 to 134±2 and 208±5 pmoles
per million cells per minute at 3 and 24 hours after stimulation with
LPS and IFN- (n=6). Superoxide production remained constant
at this level over the next 24-hour period. The formation of nitrite
could not be detected until 24 hours after stimulation with LPS and
IFN- but significantly increased at 24 and 48 hours to 8±1 and
24±2 µmol/L (P<.01, n=9). Similarly,
ONOO- production increased from <0.2 to
5.4±0.5 pmoles per million cells per minute at 48 hours after LPS and
IFN- stimulation (n=6). There was an increase in the DNA
single-strand breaks in these cells at 24 to 48 hours after LPS and
IFN- stimulation (from 4±2% to 27±8% and 32±8% at 24 and 48
hours, respectively; n=6). The inhibition of mitochondrial respiration
and the development of DNA strand breaks coincided with the
production of NO and ONOO- starting at 24
hours after stimulation and became pronounced at 48 hours (Fig 2b). The NOS inhibitor L-NMA
dose-dependently inhibited the production of NO (Fig 2a)
and the formation of ONOO- (to 38±2% of
LPS/IFN-–stimulated control at 1 mmol/L L-NMA, n=6) and
completely reversed the depression of mitochondrial respiration (Fig 2b). Similarly, S-methylisothiourea, a potent
isoform-selective inhibitor of iNOS,10
caused a dose-dependent reversal of the mitochondrial respiration
and inhibition of nitrite and ONOO-
production (at 1 to 100 µmol/L, n=6; not shown).
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In order to identify the relative contribution of NO and
ONOO- to the loss of cellular energetics, we used a
scavenger of ONOO-, uric acid, which has no
effect on NOS activity or the half-life of NO (not shown). Uric
acid (100 µmol/L) protected against the suppression of the
mitochondrial respiration in cells exposed to LPS and IFN- (Fig 2b).
The effect of uric acid was unrelated to the induction of iNOS (similar
to L-NMA), as evidenced by its protective action when administered 24
hours after the exposure of the cells to LPS/IFN- (Fig 2). Similar
to uric acid, the putative ONOO- scavengers cysteine
(1 mmol/L), glutathione (1 mmol/L), and ascorbic acid (1 mmol/L)
significantly reduced the LPS/IFN-–induced suppression of
mitochondrial respiration (not shown).
Development of DNA Single-Strand Breaks and Activation of PARS in
Smooth Muscle Cells Exposed to Authentic ONOO- or
Compounds That Generate ONOO-
Similar to J774 cells34 and rat
thymocytes,31 RASM cells exposed to
ONOO- (1 mmol/L) produced a marked increase in DNA
strand breakage (Fig 3a). ONOO- also
caused the activation of PARS (Fig 3b), a decrease in the intracellular
NAD+ (Fig 3c) and glutathione (Fig 3d) levels, inhibition
of the mitochondrial respiration (Fig 3e), and release of LDH into the
culture medium (Fig 3f), indicating terminal cell injury. Inhibition of
PARS activity by 3-aminobenzamide (1 mmol/L) did not affect the extent
of DNA strand breakage but abolished ONOO--induced
PARS activation and provided a partial protection against the
NAD+ depletion, against the inhibition of mitochondrial
respiration, and against the LDH release (Fig 3a through 3e). The
glutathione depletion, on the other hand, was not affected by the PARS
inhibitor (Fig 3d).
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Although exposure of RASM cells to SNAP (1 mmol/L) or pyrogallol (100
µmol/L) did not cause a significant increase in DNA strand breakage,
the simultaneous administration of these two compounds
resulted in marked DNA strand breakage (Fig 4).
Similarly, and in accordance with recent observations,32
we have found that exposure of RASM cells to SIN-1 (1 mmol/L), a
compound that releases both NO and superoxide,43 resulted
in significant DNA strand breakage (Fig 4). These observations are in
line with our finding that there is an enhancement by pyrogallol of the
inhibition of the mitochondrial respiration in cells exposed to SNAP
(see Fig 1).
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Effect of PARS Inhibitors on the Suppression of
Mitochondrial Respiration in Smooth Muscle Cells Exposed to NO,
Superoxide, and ONOO- Donor Compounds
Inhibition of PARS activity by 3-aminobenzamide (1 mmol/L)
slightly blunted the reduction in mitochondrial respiration in cells
treated with SNAP (Fig 5a), completely prevented the
decrease in mitochondrial respiration in response to SIN-1 (Fig 5b),
and provided a modest protection against the decrease in respiration
due to diethylamine NONOate (Fig 5c). Similar results were obtained
with nicotinamide (1 mmol/L) and PD 128763 (100 µmol/L), PARS
inhibitors of different structural classes (not shown).
Moreover, similar to 3-aminobenzamide, nicotinamide and PD 128763 (data
not shown) inhibited the suppression of mitochondrial respiration by
exogenous ONOO-. The decrease in mitochondrial
respiration elicited by pyrogallol, however, was unaffected by these
PARS inhibitors (not shown).
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Effect of PARS Inhibitors on the Suppression of
Mitochondrial Respiration in Smooth Muscle Cells
Endogenously Producing NO, Superoxide, and
ONOO-
Stimulation of RASM cells with LPS and IFN- produced NO,
superoxide anion, and ONOO- and resulted in an
increase in the DNA single-strand breaks at 24 to 48 hours (see
above). In order to evaluate the role of subsequent PARS activation, we
treated vascular smooth muscle cells in the presence of LPS and IFN-
with 3-aminobenzamide (Fig 6a), PD 128763 (Fig 6b), or
nicotinamide (not shown). Similar to our finding in J774 cells
stimulated by LPS,44 these PARS inhibitor
agents potently reduced the suppression of mitochondrial respiration
but had no effect on the formation of NO (Fig 6c) or superoxide anion
(not shown).
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Inhibition of PARS by 3-aminobenzamide and inhibition of NOS by L-NMA
significantly blocked the LPS/IFN-–induced depletion of
intracellular NAD+ and ATP (Fig 7). The
protection was more pronounced with L-NMA than with 3-aminobenzamide,
suggesting that NO- or ONOO--dependent but
PARS-independent mechanisms may also contribute to the vascular
energetic failure in these cells. This is in line with our observation
that inhibition of PARS caused only a partial protection against the
cellular injury in RASM cells exposed to authentic
ONOO- (Fig 3). There was no detectable LDH release at
24 hours after stimulation by LPS and IFN-, and at 48 hours, a
marginal increase was observed (by 4±1% over the background, n=6),
which was unaffected by 3-aminobenzamide pretreatment. At 48 hours
after LPS/IFN- stimulation, there was a reduction in the
cellular-reduced glutathione content (by 34±2% of control), which
was not affected by inhibition of PARS with 3-aminobenzamide (n=6).
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Effect of PARS Inhibition on the Contractility of
Rat Aortic Rings Exposed to ONOO- In Vitro
In vitro exposure of rat aortic rings to ONOO-
caused a dose-dependent reduction in vascular
contractility (Fig 8). In addition, we
have observed a significant increase in PARS activity. The
incorporation of tritiated NAD+ into proteins (an indicator
of PARS activity) increased from 89±13 to 183±23 cpm
(P<.01, n=9) in vascular rings exposed to
ONOO- (900 µmol/L) for 30 minutes. When
3-aminobenzamide was included into the incubation mixture,
incorporation of tritiated NAD+ into proteins was to 69±11
cpm in vehicle-treated rings (n=9) and 71±13 cpm in rings treated
with ONOO- (n=9).
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) and the effects of
3-aminobenzamide (3AB, 1 mmol/L) (
). *P<.05,
indicating significant effect of 3AB in rings exposed to
ONOO-. Data are expressed as mean±SEM of n=7 to 9
vascular rings.
Although in vitro treatment with 3-aminobenzamide, at 1 mmol/L,
completely abolished the ONOO--induced increase in
PARS activity, its effect on the change in vascular reactivity was only
partial (Fig 8), suggesting the presence of parallel PARS-independent
mechanisms of depression of vascular contractility in
rings exposed to ONOO- in vitro.
Effect of In Vivo PARS Inhibition on Vascular
Contractility in Endotoxic Shock
In rats injected with LPS, there was a significant reduction at
180 minutes in (1) MAP (from 130±3 to 108±4 mm Hg, n=8,
P<.01) and (2) ex vivo contractility of
thoracic aortas. Intravenous administration of
3-aminobenzamide (10 mg/kg) completely prevented the fall in MAP in
endotoxic rats (control MAP, 123±5 mm Hg; MAP at 180 minutes, 115±4
mm Hg; n=8) and preserved vascular contractility (Fig 9). LPS caused no significant change in heart rate in
either group (not shown). In vivo treatment with 3-aminobenzamide for
90 minutes in the absence of LPS had no effect on blood pressure, heart
rate, or ex vivo vascular reactivity to norepinephrine (n=3
or 4; not shown).
|
). **P<.01,
indicating significant effect of endotoxic shock. ##P<.01,
indicating significant protective effect of 3AB. Data are expressed as
mean±SEM of n=8 or 9 vascular rings.
Effect of Inhibition of PARS on Endotoxin-Induced Mortality
in Mice
Intraperitoneal injection of LPS (120 mg/kg)
resulted in 
90% mortality over 42 hours. In vivo administration of
3-aminobenzamide significantly reduced the LPS-induced mortality (Fig 10).
|
). *P<.05, indicating
significant improvement in survival by 3-AB. Data are expressed as
percentage of initial survival rate (100%). There were 18 animals in
the vehicle-treated group and 16 animals in the 3-AB–treated
group. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ONOO- is a recently identified toxic oxidant formed in situ by the reaction of NO and superoxide anion, free radicals generated by multiple cell types in response to stimulation.15 16 Accumulating evidence supports a role for ONOO- in the mechanism of cellular and tissue injury in a variety of inflammatory states.16 17 18 19 20 Because ONOO- has been noted in many cell types to impair cellular energetics,21 22 23 24 34 we hypothesized a role for this oxidant in the characteristic loss of cellular respiration and contractile function of the vascular smooth muscle in endotoxic shock. In the present study, we present data obtained from isolated cultured vascular smooth muscle cells, isolated vascular rings, and in vivo shock models suggesting that ONOO- impairs vascular function in circulatory shock. Moreover, because of recent data demonstrating the development of DNA injury31 32 33 34 and subsequent activation of a suicidal DNA repair pathway, governed by PARS, in cells exposed to ONOO-, we have investigated the contribution of PARS to the impairment of vascular function and obtained data suggesting that part of the ONOO--mediated injury is related to the activation of PARS.
ONOO- Is a Potent Suppressor of Mitochondrial
Respiration in Vascular Smooth Muscle Cells
Mitochondrial respiration was inhibited by the exogenous
addition of (1) authentic ONOO- or (2)
ONOO- (authentic or pharmacologically generated in
situ by the combination of pyrogallol, a drug that undergoes
auto-oxidation and releases superoxide, in combination with the NO
donor compounds SNAP or diethylamine NONOate). In the millimolar
concentration range used in the present study,
ONOO- has previously been noted to inhibit
respiration and sodium transport in alveolar type II
cells22 and macrophages.34 45
Moreover, in other cell types ONOO- has been observed
to be a relatively more potent inhibitor of cellular
respiration than NO.21 22 34 45 We did not observe a
marked difference, however, between the inhibitory potency
of the NO donors SNAP and diethylamine NONOate compared with SIN-1, a
compound that releases equimolar amounts of NO and superoxide and thus
may be considered a ONOO- donor.22 32 43
It is noteworthy in this context, however, that the above-mentioned
compounds have different half-lives, which may affect the rate and
durability of cellular injury. An additional complexity in the
interpretation of data derived from studies using SIN-1 is that the
ratio of NO to ONOO- appears to be an important
determinant of ONOO- toxicity. Excess NO inhibits the
effects of authentic ONOO- on lipid
peroxidation,48 oxidation of dihydrorhodamine
123,20 and the suppression of mitochondrial respiration in
cultured macrophages and smooth muscle cells (authors'
unpublished data, 1995).
Endogenous ONOO- Is Responsible for the
Depression of Mitochondrial Respiration in Cells Stimulated With LPS
and IFN-
In order to clarify the relative contribution of the individual
free radicals, NO and superoxide anion, and their reaction product,
ONOO-, we used a
ONOO--specific scavenger, uric acid. Treatment with
uric acid did not inhibit pyrogallol and diethylamine
NONOate–induced suppression of cellular respiration yet provided
full protection against the effect of SIN-1 or of the combined effect
of diethylamine NONOate and pyrogallol. Similar results were obtained
with a range of putative ONOO- scavenger agents.
These data strongly support the proposition that uric acid acts as a
scavenger of ONOO-38 44 45 46 in our in
vitro system and that, therefore, in the RASM cells, similar to the
J774 cells,47 endogenous
ONOO- is involved in the depression of mitochondrial
respiration. The proposition that ONOO- and NO are
the major endogenous cytotoxic factors is further
strengthened by the following findings: (1) The time course of the
suppression of mitochondrial respiration coincides with the time course
of NO and ONOO- formation but not with the formation
of superoxide. (2) The NOS inhibitor L-NMA also protects
against the depression of mitochondrial respiration, suggesting that NO
or a closely related species is involved in the process. (3) Uric acid
does not inhibit NO or superoxide formation. (4) Uric acid is still
effective when given 24 hours after LPS/IFN-, suggesting that it
does not interfere with the induction of iNOS in these cells. (5) The
cell-permeable superoxide dismutase analogue49 and
ONOO- scavenger50 (but not NO
scavenger50 ) MnIII tetrakis (4-benzoic acid) porphyrin
(300 µmol/L) and Trolox (1 mmol/L), a putative
ONOO- scavenger,31 which is known to
prevent the development of DNA single-strand breaks in cells
exposed to ONOO-,31 cause a full
prevention of the depression of mitochondrial respiration in
macrophages50 and smooth muscle cells (authors'
unpublished data, 1995) exposed to LPS and IFN- in vitro. Other
putative ONOO- scavenger agents, such as ascorbate,
L-cysteine, desferoxamine, and Trolox, when given in
millimolar concentrations, also provided protection of variable
extent against the decrease in mitochondrial respiration in RASM cells
exposed to SIN-1 or stimulated with LPS and IFN- (authors'
unpublished data, 1995).
PARS Activation Contributes to the Depression of Mitochondrial
Respiration in RASM Cells Stimulated With LPS and IFN-
We have previously demonstrated the activation of PARS and the
ensuing reduction of intracellular NAD+, ATP, and
mitochondrial respiration in macrophages exposed to authentic
ONOO- in vitro.34 In the present
study, we demonstrate that in RASM cells, authentic
ONOO- causes DNA strand breakage, PARS activation,
NAD depletion, inhibition of mitochondrial respiration, and LDH
release. All of the above changes, except for the development of DNA
strand breaks, are ameliorated by pharmacological inhibition of PARS.
The finding that 3-aminobenzamide does not inhibit the development of
DNA strand breaks (the trigger of PARS activation) is in line with
previous data47 demonstrating that 3-aminobenzamide is not
a direct scavenger of ONOO-. Similar to the results
observed with authentic ONOO-, we found that
stimulation of RASM cells with LPS and IFN- or exposure of these
cells to conditions that generate ONOO- (also see
Reference 3232 ) resulted in DNA strand breakage. Moreover,
pharmacological inhibitors of PARS activity inhibited the
depression of respiration elicited by various NO and
ONOO- generators as well as by stimulation with LPS
and IFN-. We obtained similar results using three different
structurally unrelated PARS inhibitors,35 36
suggesting that the mechanism by which these agents exerted protective
effects is, indeed, via the inhibition of PARS activity. Thus, taken
together, the present data suggest that activation of PARS, caused
by DNA strand breakage due to endogenously produced
ONOO-, contributes to the cell damage in
response to stimulation with LPS and IFN-.
Initial reports have proposed that the decomposition of ONOO- may yield hydroxyl radical, which mediates its oxidant activity.15 In this respect, it is noteworthy that hydroxyl radical (produced by hydrogen peroxide) has been previously identified as a major trigger of DNA strand breakage and PARS activation.28 29 30 However, subsequent work suggests that the decomposition of ONOO- does not yield hydroxyl radical but peroxynitrous acid or its activated isomer, which mediates its oxidant actions. This appears to hold true, in general, for all oxidations mediated by ONOO-16 and also specifically for the DNA strand breaks induced by ONOO- or SIN-1 (see References 3131 to 3332 33 ).
Pretreatment with very high doses of PARS inhibitors (10 to 30 times higher than the ones used in the present study) has been noted to inhibit the induction of iNOS and subsequent production of nitrite in L929 cells,51 pancreatic islet cells,52 and macrophages,53 possibly because a basal activity of PARS in unstimulated cells is involved in the process of iNOS induction.53 In contrast to these observations, we found that PARS inhibitors, in the concentration range used in the present study, did not inhibit NO or superoxide formation in vascular smooth muscle cells, implying that these agents did not interfere with iNOS induction in this cell type. It is noteworthy that macrophages,34 53 but not cultured RASM cells (present study), have a significant "basal" PARS activity under unstimulated conditions.
Inhibition of PARS activity preserved mitochondrial respiration in
cells subjected to endogenous and exogenous sources of NO
and ONOO-. The degree of protection afforded by PARS
inhibition was greatest in the cells stimulated with LPS/IFN- and
cells treated by the ONOO- donor SIN-1, more modest
in SNAP-treated cells, and least efficacious in cells exposed to the
"pure" NO donor diethylamine NONOate. Treatment with the
ONOO- scavenger uric acid revealed a similar order of
potency, further suggesting that in smooth muscle cells exposed to LPS
and IFN-, as in J774 macrophages,45
ONOO- and not NO per se is likely to account for the
majority of the suppression of the respiration.
The measurement of reduction of MTT by the mitochondria is a useful assay for investigation of the effect of the general metabolic status of the cell on the mitochondrial respiration. Although the reduction of MTT appears to be mainly by the mitochondrial complexes I and II, it also may involve NADH- and NADPH-dependent energetic processes that occur outside the mitochondrial inner membrane.54 55 Thus, this method cannot be used to separate the effect of free radicals, oxidants, or other factors on the individual enzymes in the mitochondrial respiratory chain but is useful for monitoring changes in the general energetic status of the cells. There are data demonstrating direct effects of ONOO- on isolated mitochondria56 or on purified mitochondrial enzymes.23 24 Interestingly, it has been reported that although a decrease in the activity of NADH-COQ1 reductase, succinate-cytochrome c reductase, and cytochrome c oxidase was found in neurons exposed to ONOO-, in the same study, in isolated mitochondria exposed to ONOO-, only cytochrome c oxidase is affected.56 These data suggest that the presence of intracellular processes is required to detect a general inhibition by ONOO- on the mitochondrial chain.56 To determine the extent to which these intracellular processes are related to PARS will be the subject of follow-up studies.
Inhibition of PARS activity resulted in only partial restoration of
mitochondrial respiration and vascular reactivity when vascular smooth
muscle cells or aortic rings were exposed to high doses of authentic
ONOO- (Figs 6e and 9d). Moreover, inhibition of PARS
did not inhibit the depletion in cellular glutathione levels in cells
exposed to ONOO-. Thus, the present data support
the view that ONOO- also exerts direct effects on the
mitochondrial respiratory chain or inhibits other intracellular redox
processes (see also References 1, 4, and 211 4 21 -24). In line with this
proposal, we observed that NOS inhibition by L-NMA provided greater
protection than did 3-aminobenzamide against the LPS/IFN-– induced
decrease in intracellular NAD+ and ATP. Heller et
al57 have also reported that murine PARS/pancreatic islet
cells were resistant to cell lysis by intermediate but not high
doses of NO donors. These observations support the existence of NO-
and/or ONOO--related, but PARS-independent, cytotoxic
pathways in smooth muscle cells stimulated with LPS and IFN-. It is
conceivable that PARS-related pathways play a more important role in
the cellular energy depletion and cell damage in response to lower and
intermediate doses of NO and ONOO-,
particularly when cells are under conditions of chronic exposure. Our
data, demonstrating the protective effect of 3-aminobenzamide against
the cell lysis and LDH release in response to
ONOO-, support the view that PARS activation
is an important factor that leads to terminal and irreversible cell
injury. In agreement with our finding, the importance of PARS in the
cytotoxic process was underlined by previous work in
endothelial cells, where, using hydrogen peroxide as a
trigger, it has been demonstrated that PARS activation alone is
sufficient to terminally damage the cells so that acute cell lysis
occurs, which can be prevented by pharmacological
inhibitors of PARS.58 In contrast, chronic
exposure of cells to endogenous ONOO- (in
response to LPS and IFN- stimulation) did not cause substantial cell
lysis in our experiments. This observation is in line with current data
showing that acute exposure of cells to NO or ONOO-
causes cell necrosis, whereas chronic exposure or lower concentrations
result in apoptotic cell death.59 In this respect,
it is noteworthy that PARS is known to be involved in the
apoptotic process.60 61
PARS Inhibitors Prevent
Hypocontractility in Rat Aortic Rings Exposed to
ONOO- In Vitro and In Vivo
Recently, we reported histological evidence
of tyrosine nitration in the aortas of endotoxic rats,
consistent with endogenous ONOO-
formation in the vasculature.19 The functional
consequences of ONOO- exposure include a loss of
contractile ability and/or relaxation of various blood vessels exposed
to ONOO-.62 63 In the present study,
we have shown that in vitro exposure of rat aortic rings to
ONOO- activates PARS and depresses
contractile function. It was not directly measured whether PARS
activation occurs in vivo after ONOO--mediated
vascular injury in severe endotoxic shock. However, our data
demonstrating beneficial effects of 3-aminobenzamide pretreatment in
endotoxic shock strongly imply that PARS-related mechanisms contribute
to the development of vascular hyporeactivity in endotoxemia. Similar
maintenance of the contractions was observed in endotoxemic
animals treated with MnIII tetrakis (4-benzoic acid) porphyrin in the
present model of endotoxin shock (authors' unpublished data,
1995), further pointing toward the involvement of
ONOO- in the development of vascular depression in
shock. It is noteworthy in this context that we have recently observed
that peritoneal macrophages obtained from animals subjected to
endotoxic shock maintain NAD+ and ATP levels in the
presence of 3-aminobenzamide pretreatment in a shock model similar to
the one used in the present study.47
The cell culture studies using RASM cells stimulated with LPS/IFN-
and the in vivo experiments in rats injected with LPS are very much
different in the time course of NO, superoxide, and
ONOO- production. Under the in vitro
conditions (macrophages or smooth muscle cells), the expression
of iNOS protein occurs after a lag phase of 6 to 24
hours,4 5 6 whereas in the in vivo model iNOS is detectable
at 1 hour and peaks at 3 to 6 hours after LPS
injection.7 10 12 However, in both experimental models,
the production of superoxide precedes the induction of
iNOS,19 20 and it is conceivable that there are many
similarities in the nature of the cytotoxic pathways that follow the
production of NO and ONOO-. A distinct
difference between the in vitro and in vivo model is that in the in
vivo model, early production of superoxide appears to produce
ONOO- before the induction of iNOS, by reacting with
NO derived from endothelial cell NOS.20
Since there is no constitutive NOS in the RASM cells, early
production of superoxide does not result in the early formation
of ONOO- in these cells.
The proposition that PARS activation and the resultant energetic depletion are responsible for the contractile deficit during shock may contradict the previous theory that the massive vasodilatation and vascular hyporeactivity in blood vessels that express iNOS are mediated by the NO-cGMP pathway.6 8 It is noteworthy in this context, however, that the evidence for the involvement of cGMP has been obtained by inhibitors of guanylyl cyclase, such as methylene blue and LY83583.6 8 Recent data, however, indicate that the action of these antagonists involves the inactivation of NO rather than inhibition of guanylyl cyclase.64 65 In fact, the hypothesis that the iNOS-mediated vascular contractile and energetic failure may be cGMP independent and related to the effects of NO on the mitochondrial respiratory chain or glycolysis has also been proposed by other investigators previously.4
Many of the agents used in the present study, such as pyrogallol, a compound that releases superoxide in aqueous solutions, the NO donor compounds used, or SIN-1, a compound that simultaneously releases NO and superoxide, are widely studied and well-established agents, without major notable side effects in the concentration range used in the present study. Our conclusions, implicating the role of PARS in the pathogenesis of shock, are based on data obtained with pharmacological inhibitors of the repair enzyme. Since thorough characterization of the potential additional pharmacological effects of the PARS inhibitor 3-aminobenzamide was not available, we have investigated the effects of this agent on NO-, superoxide-, and ONOO--related processes and have found that the drug does not scavenge NO or ONOO- and does not inhibit the production of superoxide or ONOO- production in macrophages stimulated with LPS.47 In addition, 3-aminobenzamide and nicotinamide (up to 3 mmol/L) do not inhibit the calcium-independent conversion of L-arginine to L-citrulline in homogenates of lungs from endotoxin-treated rats (authors' unpublished data, 1995), an experimental model useful for determining the potential direct effect of NOS inhibitors on iNOS activity.66 Furthermore, in the present study, similar to our previous investigation,34 we were able to reproduce the effects of 3-aminobenzamide by using several structurally unrelated PARS inhibitors. In addition, we demonstrate that 3-aminobenzamide, at 1 mmol/L, is sufficient to completely inhibit ONOO--induced PARS activation in RASM cells, and the same concentration of 3-aminobenzamide ameliorates against NAD+ depletion, inhibition of mitochondrial respiration, and the development of vascular hyporeactivity in vitro. Because 3-aminobenzamide is not a ONOO- scavenger, it is not surprising that the drug does not inhibit the development of DNA single-strand breaks, which is the trigger of PARS activation. Recently, a knockout animal has been produced that lacks a functional gene for PARS. The finding that pancreatic islet cells derived from a knockout mouse that lacks the gene for PARS are protected against damage in response to NO donors57 further supports the view that effects we observed with PARS inhibitor compounds are, indeed, due to inhibition of the activity of PARS. Future studies using these knockout animals should give important insights into the contribution of PARS in the pathogenesis of various pathophysiological conditions.
Current strategies aimed at limiting NO-mediated cell/organ injury
include agents that inhibit the induction of iNOS, NOS enzyme
inhibitors (preferably with selectivity for iNOS), various
agents that scavenge NO, and agents that limit substrate availability
for iNOS.9 12 Less attention has been directed to
strategies that inhibit the intracellular cytotoxic pathways initiated
by NO or its toxic derivatives. The present results, obtained from
studies of vascular smooth muscle cells, coupled with the in vitro and
ex vivo observations obtained from studies of vascular rings, are
further strengthened by the results of the survival study, in which
3-aminobenzamide dramatically limited endotoxin-mediated mortality
(Fig 10). Similar benefits have been observed after iNOS
inhibition10 11 or targeted disruption of the murine iNOS
gene.67 Taken together, the present data suggest that
pharmacological inactivation of PARS represents a novel
strategy to counteract the energy failure and vascular dysfunction
found in septic shock and in other
pathophysiological conditions associated with
iNOS induction. The viability of this potential therapeutic strategy is
also strengthened by recent observations demonstrating that the
presence of PARS is not obligatory for normal DNA repair68
because of the simultaneous existence of multiple efficient
DNA repair pathways. Thus, the PARS pathway may be an expendable and
potentially deleterious mechanism contributing to the vascular failure
in circulatory shock.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
The technical assistance of M. O'Connor and A. Denenberg and the assistance of J. Giles with the tissue cultures are appreciated.
Received October 20, 1995; accepted February 20, 1996.
|
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