Departments of 1 Anesthesiology, 2 Biochemistry and Molecular Genetics, and 3 Pharmacology and 4 The Center for Free Radical Biology, The University of Alabama at Birmingham, Birmingham, Alabama 35233-4234
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ventilator strategies allowing for
increases in carbon dioxide (CO2) tensions (hypercapnia)
are being emphasized to ameliorate the consequences of
inflammatory-mediated lung injury. Inflammatory responses lead to the
generation of reactive species including superoxide
(O2), nitric oxide (·NO), and their product
peroxynitrite (ONOO
). The reaction of CO2 and
ONOO
can yield the nitrosoperoxocarbonate adduct
ONOOCO2
, a more potent nitrating species than
ONOO
. Based on these premises, monolayers of fetal rat
alveolar epithelial cells were utilized to investigate whether
hypercapnia would modify pathways of ·NO production and reactivity
that impact pulmonary metabolism and function. Stimulated cells exposed
to 15% CO2 (hypercapnia) revealed a significant increase
in ·NO production and nitric oxide synthase (NOS) activity. Cell
3-nitrotyrosine content as measured by both HPLC and immunofluorescence
staining also increased when exposed to these same conditions.
Hypercapnia significantly enhanced cell injury as evidenced by
impairment of monolayer barrier function and increased induction of
apoptosis. These results were attenuated by the NOS inhibitor
N-monomethyl-L-arginine. Our studies reveal that
hypercapnia modifies ·NO-dependent pathways to amplify cell injury.
These results affirm the underlying role of ·NO in tissue inflammatory reactions and reveal the impact of hypercapnia on inflammatory reactions and its potential detrimental influences.
carbon dioxide; nitration; inflammation; superoxide; free radical; peroxynitrite
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
DESPITE SIGNIFICANT ADVANCES in understanding the pathophysiology of acute respiratory distress syndrome (ARDS), mortality remains at 40-60%, a moderate change since its description over 30 years ago (18). Injury to the lung by orchestrated inflammatory responses has devastating clinical consequences, including impairment of oxygenation and ventilation, as well as perturbation of nonrespiratory function, eventually necessitating mechanical ventilatory support. Several issues regarding causation, supportive measures, and treatment of ARDS patients are still receiving special attention. First, the genesis of lung dysfunction is multifactorial but, once established, involves the complex interplay of many of the pro- and anti-inflammatory mediators present in systemic inflammatory responses (10). Second, mechanical ventilation has now been accepted as a contributor to lung injury (ventilator-induced lung injury) resulting from a myriad of interactions between the mechanical ventilator, inspired gases, inflammatory mediators, and the lung itself (9, 43, 49). Finally, although pharmacological agents for the treatment of established lung injury are still in evolution, additional supportive and "protective" clinical strategies are being explored to reduce undesirable side effects (34). One promising approach is to allow carbon dioxide (CO2) tensions to increase in concert with decreasing tidal volume and minute ventilation, a practice referred to as "permissive hypercapnia" (5, 45). This concept represents a major departure from traditional practice because for many years, mechanical ventilation for ARDS equated to escalation in tidal volume, inspiratory pressure, end-expiratory pressure, or inspiratory time. Permissive hypercapnia or variants thereof allow for decreases in overall ventilation to take place at a time when alveolar-capillary integrity is particularly vulnerable, resulting in permeability pulmonary edema. The respiratory acidosis that ensues from hypercapnia is well tolerated by most patients (20, 21). This trade-off seems intuitive in that repeated ventilatory cycles during the time of alveolar vulnerability predisposes the primary injured lung (i.e., sepsis) to a secondary injury (i.e., ventilator-induced lung injury). Recent clinical trials utilizing varying degrees of hypercapnia have resulted in improved survival in patients suffering from ARDS (2, 46).
Oxidant stress or the increased rate of production of reactive species
including superoxide (O2), hydrogen peroxide
(H2O2), hypochlorous acid (HOCl), nitric oxide
(·NO), and peroxynitrite (ONOO
) occurs in pulmonary
tissues of animal models of ARDS and in ARDS patients, with these and
other secondary biomolecules being proposed as contributing to the
induction and maintenance of lung injury (3, 27). Markers
of oxidative reactions are increased, and intrinsic host antioxidant
defense mechanisms are impaired as well. In patients with ARDS, plasma
ascorbate,
-tocopherol,
-carotene, and selenium were decreased
along with lung glutathione (GSH) content (24, 30).
Biochemical evidence of oxidant injury was also indicated by increases
in lipid peroxidation by-products (35).
Nitric oxide is an endogenously synthesized free radical species that
contributes to both oxidative and antioxidant reactions and acts to
maintain cellular homeostasis by way of multiple regulatory pathways
(29). Nitric oxide is principally known as a mediator of
signal transduction via the stimulation of guanylate cyclase-mediated cGMP synthesis. It is now also accepted that ·NO plays a pivotal and
diverse role in inflammatory processes as well (36).
Numerous pulmonary cell types including vascular endothelium, smooth
muscle cells, macrophages, neutrophils, platelets, and type 2 alveolar epithelial cells produce ·NO. Nitric oxide biosynthesis is enhanced during pulmonary inflammation via inducible nitric oxide synthase (iNOS) induction (19). A reaction of particular
significance during inflammatory responses occurs between ·NO and
O2, yielding the potent oxidant
ONOO
, providing an alternative pathway for oxidative
injury previously ascribed to individual reactions of ·NO,
O2
, and ·OH (4, 31, 32, 39).
ONOO
oxidizes a variety of biomolecules, with its
reactions profoundly influenced both kinetically and mechanistically by
CO2 (13). ONOO
that is produced
in the presence of the almost ubiquitous biomolecule CO2
readily gives rise to the nitrosoperoxocarbonate adduct
ONOOCO2
(25). This adduct can undergo
both heterolytic and homolytic cleavage to reactive products that
possess enhanced nitrating and reduced oxidizing potential. Based on
chemically oriented studies, it has been postulated that the unique
reactivities of ONOOCO2
will either increase or
decrease the probability of ONOO
-mediated cell injury,
but these precepts have not been tested in cell or animal models
(23). Although nitration of pulmonary cell protein
tyrosine residues readily occurs in respiratory distress syndromes,
inferring both nitration and oxidation reactions of target molecules,
neither mechanisms nor consequences of ·NO-mediated lung cell protein
tyrosine nitration are well defined (23, 48). This
posttranslational protein modification can influence diverse aspects of
cell structure and function, including cytoskeletal structure and the
catalytic activity of antioxidant enzymes (14, 26, 47).
We hypothesized that hypercapnia would enhance cytokine plus
lipopolysaccharide (LPS)-mediated inflammatory lung cell injury by
amplifying ONOO-mediated protein nitration reactions. To
test this hypothesis, fetal rat type 2 alveolar epithelial cell
monolayers were exposed to either 5 or 15% CO2 in the
presence and absence of cytokines plus Escherichia coli LPS.
Our results revealed that in the setting of cytokine-stimulated
inflammatory responses and hypercapnia, which is currently being
advocated clinically, potentially deleterious reactions that can impair
cell and organ function ensue.
![]() |
EXPERIMENTAL METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials.
Minimum essential medium (MEM) and fetal bovine serum (FBS) were from
HyClone Laboratories (Logan, UT). Hanks' balanced salt solution (HBSS)
and antibiotic-antimycotic solution were from GIBCO BRL (Life
Technologies, Grand Island, NY). Recombinant murine interleukin
(IL)-1, interferon (IFN)-
, and tumor necrosis factor (TNF)-
were from R&D Systems (Minneapolis, MN). E. coli was from American Type Culture Collection (Manassas, VA). LPS (serotype 0111:B4) and N-monomethyl-L-arginine
(L-NMMA) were from Sigma (St. Louis, MO). Timed-pregnant
female Sprague-Dawley rats at 16 days gestation were from Charles River
Laboratories (Wilmington, MA) or Harlan Laboratories (Indianapolis, IN).
Cell culture of type 2 alveolar cells. Fetal rat lung type 2 alveolar epithelial (RFLE) cells were isolated and cultured as previously described (7). Briefly, 19- or 20-day-gestation fetuses were removed aseptically; the lungs were dissected, minced, and resuspended in cold HBSS (calcium and phosphate free). The minced tissue was trypsinized for 10 min, filtered, and centrifuged. After several differential adherence steps, a 95-98% pure suspension of epithelial cells was obtained as determined by cytokeratin and vimentin staining (7). The cells were cultured in MEM plus 10% heat-inactivated FBS plus antibiotic-antimycotic solution (GIBCO BRL); then, on cell confluence (24-36 h), the monolayers were washed twice with HBSS and exposed to various experimental conditions in MEM containing 10% heat-inactivated FBS.
Cell treatments.
The cells were preincubated with cytokines [a combination of IFN-
(100 U/ml), TNF-
(500 U/ml), and IL-1
(300 pM)] and LPS. In some
cases, L-NMMA (1 mM) was added, and the cells were
subsequently exposed to either 5 or 15% CO2 (hypercapnia)
in room air. Exposure times varied from 3 to 48 h depending on the
analysis being performed. The cells were exposed in either 6- or
12-well plates (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ),
with the plate covers loosened during the incubation. The 5%
CO2 group contained 26.1 mM NaHCO3, resulting
in a mean pH of 7.28 and mean partial pressure of CO2 of 41 mmHg. Cells exposed to 15% CO2 contained 59.5 mM
NaHCO3, resulting in a mean pH of 7.28 and mean partial pressure of CO2 of 93.5 mmHg.
NOS activity.
NOS activity was determined by the conversion of
[14C]arginine to [14C]citrulline
(6). Cell lysates (100 µl) were added to an equal volume
of reaction buffer containing 10 mM NADPH, 0.5 M Tris, 0.4 mM FAD, 0.4 mM flavin mononucleotide, 100 mM citrulline, 4 mM tetrahydrobiopterin,
1 mM cold arginine, and 1 µCi/ml of [14C]arginine at pH
7.5. After 30 min, the assays were terminated by the addition of 2 ml
of 20 mM HEPES, 2 mM EDTA, and 2 mM EGTA at pH 5.5. Samples were
applied to 1-ml Dowex AG50WX-8 (Tris form) columns and eluted with 2 ml
of 20 mM HEPES buffer. Eluant containing [14C]citrulline
was quantified by liquid scintillation counting of 100-µl aliquots of
medium from the cell incubation that were assayed for the oxidation
products ·NO, NO2, and
NO3
. The samples were first incubated with
E. coli nitrate reductase to convert NO3
to NO2
for subsequent analysis by the Griess reaction
(37). Standard solutions of NaNO3 were
prepared in medium to calculate the extent of NO3
reduction to NO2
and total NO2
yield.
Determination of 3-nitrotyrosine. Protein-bound nitrotyrosine from hydrolyzed cell lysates was analyzed by HPLC (5 µM Waters C18 column, 4.6 × 300 mm). Briefly, 100 µg of protein hydrolysate were separated at a flow rate of 0.75 ml/min with an isocratic elution profile consisting of 95% eluant A (50 mM acetic acid adjusted to pH 4.7 with a concentrated solution of semiconductor grade sodium hydroxide) plus 5% eluant B (HPLC-grade methanol) over 35 min (11). Tyrosine and 3-nitrotyrosine were identified by electrochemical detection with a 12-channel electrochemical detector (Coulachem, ESA, Chelmsford, MA). Both products were quantified by comparison with external standards. For immunocytochemical analysis, cell monolayers were fixed for 30 min at room temperature with 4% paraformaldehyde. After being washed three times with PBS, the cells were incubated in 50 µM lysine and 0.1% Triton X-100 in PBS (pH 7.4) for 15 min followed by blocking with 10% goat serum and PBS for 1 h. The cells were then incubated overnight with anti- 3-nitrotyrosine polyclonal antibodies in blocking solution (1:500), rinsed three times with PBS, and then incubated for an additional 30 min with 1:100 indocarbocyanine-linked goat anti-rabbit IgG (red; Jackson ImmunoResearch) at 25°C. After three washes with PBS, the cells were postfixed in 4% paraformaldehyde for 5 min, rinsed three times with distilled water, and finally incubated for 15 min with 1 µg/ml of 4',6-diamidino-2-phenylindole. After three washes with distilled water, the slides were mounted in ProLong antifade (Molecular Probes, Eugene, OR). Fluorescence was visualized with an Olympus IX-70 epifluorescence microscope and an OlymPix cooled digital camera (resolution 1,328 × 1,024 pixels), and ESPRIT software was used for imaging.
Cell injury assessment. Cell monolayer barrier integrity was assessed by measuring the permeability to 125 I-albumin. Transwell plates (Costar, Cambridge, MA) consist of an upper well separated from a lower well by a micropore filter (2.4-cm diameter, 4.5-cm2 surface area, 0.4-µm pore size). The cells were plated in the upper well and grown to confluence (36-48 h) in MEM plus 10% FBS. On confluence (36- 48 h), the monolayers were washed twice with HBSS and exposed to experimental conditions in MEM plus 10% FBS. The lower well contained 2.65 ml of medium, and the upper well contained 1.5 ml of medium. Subsequently, 125I-albumin [1 × 106 counts/min (cpm)] and cytokines plus LPS with and without L-NMMA were added to the lower well. The addition of 125I-albumin (1 × 106 cpm) and cytokines plus LPS with and without L-NMMA represented time 0, and, subsequently, 10-µl samples were obtained from the upper well at 3, 24, and 48 h. Radioactivity was assessed by gamma counting (LKB-Wallac 1275 MiniGamma).
Western blot analysis for iNOS.
Expression of iNOS protein was determined from the cell protein lysates
after incubation for 12 h. The cells were lysed by sonication in
40 mM Tris · HCl (pH 7.9), 10% glycerol, 1% Igepal, 5 µg/ml
of aprotinin, 1 µg/ml of pepstatin, 1 µM leupeptin, 0.1 mM
phenylmethylsulfonyl fluoride, 4 µM FAD, 4 µM flavin
mononucleotide, 4 µM tetrahydrobiopterin, and 3 mM dithiothreitol;
heated to 100°C for 5 min; and then stored at 20°C. Proteins were
fractionated on 10% sodium dodecyl sulfate-polyacrylamide gels,
transferred to nitrocellulose, and incubated with a 1:2,000 dilution of
anti-mouse macrophage iNOS. The blots were then treated with a 1:50,000
dilution of peroxidase-linked anti-rabbit IgG and developed with a
maximum sensitivity chemiluminescence substrate kit (Pierce, Rockford, IL).
Apoptosis quantification. Type 2 alveolar cells were exposed for 48 h as in Cell treatments and then terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling staining was performed with the Apoptosis Detection System (Promega, Madison, WI). Each experimental condition was replicated in four wells, and six fields per well were recorded. Fluorescence was visualized with an Olympus IX-70 epifluorescence microscope and an OlymPix cooled digital camera (resolution of 1,328 × 1,024 pixels), and ESPRIT software was used for imaging.
Determination of total cell thiol content.
Net cell sulfhydryl content was quantified as previously described
(40). Briefly, a 0.05-ml sample was mixed with 1 ml of phosphate-buffered solution, pH 8.2, and added before transfer to a
cuvette containing 0.02 ml of 5,5'-dithio-bis(2-nitrobenzoic acid) in a
0.01 M phosphate-buffered solution. The formation of the
5-thio-2-nitrobenzoate ion was measured at 412 nm (molar extinction coefficient = 1.36 × 104
M1 · cm
1) after a 15-min
incubation at 25°C.
pH assessment. With combined pH and blood gas analysis monitoring (IL 1306 Instrumentation Laboratories), NaHCO3 was added to the cell culture medium (MEM) equilibrated with either 5% CO2 (26.1 mM NaHCO3) or 15% CO2 (59.5 g/l of NaHCO3), and the pH was titrated to 7.40. At pH 7.4, the 5% CO2 condition was determined to have 35-40 mmHg and 15% CO2 corresponded to 75-90 mmHg CO2 partial pressure. Type 2 alveolar epithelial cell intracellular pH measurements were made with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (Molecular Probes, Eugene, OR) 24 h after cytokines plus LPS and CO2 exposure as previously described (44).
Statistical analyses. Statistical analyses were performed with ANOVA with Tukey's post hoc analysis on Systat 7.0 software (SPSS, Chicago, IL). Results with P < 0.05 were considered significant. All values are means ± SD.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hypercapnia enhances type 2 alveolar epithelial cell ·NO
production.
To determine whether cytokine-induced ·NO production was altered by
hypercapnia, cell NO2 and NO3
production was determined. Type 2 alveolar epithelial cells were exposed to 5 or 15% CO2 in the absence and presence of
cytokines plus LPS for 48 h. Cell NO2
and
NO3
production in the absence of cytokines plus LPS
was enhanced by exposure to 15% CO2 (Fig.
1). Although the increase in 5%
CO2-exposed cell NO2
and
NO3
production in the presence of cytokines plus LPS
was significant (P < 0.05), the greatest extent of
NO2
and NO3
production was induced
by exposure of cytokine-activated cells to 15% CO2
(P < 0.05). In this group, L-NMMA
inhibited NO2
and NO3
production
90% (P < 0.05).
|
NOS activity and protein expression were enhanced by hypercapnia.
The rate of cell lysate arginine-to-citrulline conversion indicated
changes in net NOS activity after cell exposure to hypercapnia and
cytokines plus LPS for 24 h. Cytokines plus LPS enhanced NOS activity in the presence of 5% CO2 (21.6 ± 3.2 nmol · min1 · mg protein
1;
P < 0.05), with an even greater increase induced in
cytokines plus LPS-treated cells in the presence of 15%
CO2 (40.3 ± 3.5 nmol · min
1 · mg protein
1;
P < 0.05; Fig.
2). The NOS activity of both
cytokines plus LPS-treated cell groups in 5 and 15% CO2
exposure conditions was significantly inhibited by
L-NMMA (1.5 ± 0.30 and 1.6 ± 0.45 nmol · min
1 · mg protein
1,
respectively; P < 0.05). Western blot analysis
confirmed that during exposure to 15% CO2 in the presence
of cytokines plus LPS, the level of expression of iNOS protein was
maximal. Compared with 5% CO2 with cytokines plus LPS,
iNOS protein expression was increased 70% by hypercapnia (Fig.
3). No detectable iNOS protein expression
was observed during exposure of non-cytokine-activated cells to 5 and
15% CO2 alone.
|
|
Evidence for formation of ·NO-derived nitrating species.
Cell protein 3-nitrotyrosine content was significantly increased by
hypercapnic exposure of cytokines plus LPS-activated cells. 3-Nitrotyrosine was quantified after 48 h both
immunohistochemically and by HPLC separation of cell protein
hydrolysates with a diode array detection. Hypercapnia and cytokines
plus LPS-activated type 2 alveolar epithelial cells displayed the
greatest 3-nitrotyrosine immunoreactivity, a response inhibitable by
L-NMMA (Fig. 4). Cell immunoreactivity for 3-nitrotyrosine was not observed in 5%
CO2-exposed cells, and only minimally increased
3-nitrotyrosine immunoreactivity was observed in activated cells
exposed to 5% CO2. Cells exposed to 15% CO2
also displayed only minimal 3-nitrotyrosine immunoreactivity compared
with the more intense 3-nitrotyrosine immunoreactivity of cells exposed
to 15% CO2 and cytokines plus LPS.
|
|
Transmonolayer albumin flux increased significantly in the presence
of 15% CO2 with cytokines plus LPS.
The transmonolayer flux of 125I-albumin from the
basolateral to the apical side of type 2 alveolar epithelial cell
monolayers was greatest in the cells exposed to 15% CO2
and cytokines plus LPS (P < 0.05) compared with that
in hypercapnia alone (Fig. 5). The
addition of L-NMMA to the hypercapnia-activated cells
reduced albumin flux 56% (P < 0.05) but increased the
rate of albumin flux in cytokines plus LPS-activated cells exposed to
5% CO2.
|
Hypercapnia enhances apoptosis of cytokines plus LPS-activated type
2 alveolar epithelial cells.
The occurrence of apoptotic nuclei was minimal in cells exposed to 5%
CO2 alone (4.4 ± 0.8%), with cell cytokines plus LPS treatment with and without L-NMMA also inducing no
significant difference (Fig. 6). In the
presence of 15% CO2, a significant increase in apoptotic
nuclei was observed in cells exposed to cytokines plus LPS (14.6 ± 1.0%) compared with that in 15% CO2-alone exposure
conditions (1.1 ± 0.3%; P < 0.05). The addition
of L-NMMA decreased cytokines plus LPS-activated
hypercapnic cell apoptotic nuclei by 63% (14.6 ± 1 vs. 5.5 ± 1.1%).
|
Hypercapnia decreases cell thiol content.
The total cell thiol (RSH) content (protein plus nonprotein thiols) was
not significantly influenced by hypercapnia (104 ± 3 nmol RSH/mg
protein in 5% CO2 and 138 ± 12 nmol RSH/mg
protein in 15% CO2; Fig.
7). Activation of cells by cytokines plus
LPS caused a moderate increase in thiol content in cells maintained in
5% CO2 (138 ± 12 nmol/mg), with exposure to 15%
CO2 alone decreasing total cell thiols 34% to 91 ± 5 nmol/mg protein (P < 0.05).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It was observed that hypercapnia modulates cell inflammatory
responses via reactions that were predominantly detrimental to cell
function. Cells exposed to hypercapnic conditions produced more ·NO
as inferred by increased iNOS protein expression and the extent of
L-NMMA-inhibitable cell NO3 and
NO2
production. Both HPLC and immunohistochemical
analysis showed cell protein tyrosine nitration was increased by
hypercapnia, inferring increased formation of the reactive nitrating
intermediate ONOOCO2
. Apoptotic cells were also
present in significantly larger quantities during hypercapnia with
cytokines plus LPS. Of functional significance was the loss of
epithelial barrier integrity when activated cells were exposed to a
proinflammatory hypercapnic environment.
A cultured fetal rat type 2 alveolar epithelial cell model was chosen
based on its ability to reflect lung epithelial responses to
inflammatory stimuli and because the lung epithelium is a key target of
inflammatory injury. A combination of cytokines (TNF-, IL-1
, and
IFN-
) plus LPS was administered on cell confluence to stimulate
inflammatory responses, including oxidative and ·NO-mediated stress.
Cells were exposed for up to 48 h in 5 (35-40 mmHg) or 15%
(75-90 mmHg) CO2. Normally, tissues exposed to
hypercapnia would become more acidic, secondary to bicarbonate
dissociation and carbonic anhydrase-catalyzed increases in
H+ concentration. Because we focused on testing whether
CO2 reacted directly with ·NO-dependent oxidative
pathways to then modify susceptible target molecules, we normalized the
values for the ancillary actions of CO2 (i.e., acidosis).
This involved additional buffering of the medium and documentation of
similar extracellular and intracellular acid-base environments for all
CO2 exposure conditions. Although there was relative ease
in maintaining and monitoring comparable extracellular pH levels, the
use of a fluorescent intracellular pH indicator probe was necessary to
confirm that intracellular pH was consistent with the extracellular
milieu. For all experimental conditions, it was confirmed that
intracellular and extracellular pH values were within 0.08 pH units of
each other after 24 h of exposure (5% CO2 mean
intracellular pH of 7.31; 15% CO2 mean intracellular pH of
7.33). Thus the results herein were reflective of the chemical
reactivity of CO2 and are not due to indirect responses to acidosis.
The hypercapnia-induced increased formation of 3-nitrotyrosine was used
to indicate CO2-stimulated nitration reactions of ONOO (e.g., ONOOCO2
). Because the
activity of iNOS was increased significantly by cell activation with a
combination of cytokines and LPS, especially during hypercapnic
exposures (Figs. 1-3), it remains possible that increased cell
·NO production also contributes in part to CO2-stimulated nitration reactions.
The reaction between ONOO and CO2 has a
facile rate constant (k = 5.8 × 104
M
1 · s
1) (8) and is
highly favored in biological milieus, especially during periods of
metabolic stress when inflammatory and hemodynamic events impact on
O2
and ·NO production and CO2 removal.
The net effect of ONOOCO2
formation on cell and organ
function could be either injurious or protective because
ONOOCO2
decays in a neutral aqueous solution more
rapidly than ONOO
(e.g., a half-life of 1 µs to 1 ms
for ONOOCO2
compared with a half-life for
ONOO
of 0.1-1.6 s depending on the molecular
composition of the microenvironment). Importantly,
ONOOCO2
and its secondary products also manifest an
altered reactivity relative to ONOO
, displaying an
increased propensity for nitration reactions and less potential as an
oxidant (25). Because of these characteristics, CO2-mediated formation of ONOOCO2
might
alternatively protect cells from cytotoxicity due to redirection of
ONOO
to less oxidizing and kinetically faster decay
mechanisms, thus decreasing cell exposure time to reactive species.
This is in contrast to a scenario where the influence of
CO2 yields not only ONOO
but also
ONOOCO2
that would react with a host of susceptible
target molecules (membrane, cytoskeletal, nuclear, or metabolic
components) and defense mechanisms (i.e., ascorbate, tocopherols,
thiols) to both oxidize and extensively nitrate targets in a manner
detrimental for cell viability. For example, recent data
(42) convincingly supports the concept that
ONOO
-mediated biological nitration reactions are
predominantly CO2 dependent and ONOOCO2
mediated.
Based on thermodynamic measurements (free energy of
activation = 7.3 ± 5.5 kcal/mol), reaction A
would be the predominant pathway of further ONOOCO2
reaction, with the other decay reactions, reactions
B-D, being either less thermodynamically feasible or
such that the reactants possess half-lives much shorter in magnitude
than ONOOCO2
(38)
![]() |
(A) |
![]() |
(B) |
![]() |
(C) |
![]() |
(D) |
The potent protection lent by L-NMMA to the loss of monolayer barrier function in 15% CO2-exposed, cytokine-activated cells affirms that hypercapnia deleteriously influences ·NO-mediated oxidative and nitration pathways, a pattern also observed in cells stained for the occurrence of apoptosis. This finding also lends support to the hypothesis that ·NO-derived species mediate, in part, cell responses to oxidant stress and hypercapnia.
Multiple response variables revealed a difference in basal
(nonactivated) cell responses to 5 and 15% CO2. For
instance, there was an 87% increase in cell ·NO production as
indicated by the increase in medium NO2 plus
NO3
concentrations on type 2 cell exposure to 15%
CO2. Impaired monolayer barrier function was also observed,
with increased transmonolayer 125I-albumin flux occurring
in the 15% (2,198 ± 321 cpm) and 5% (1,455 ± 98 cpm)
CO2 groups. Immunofluorescent staining of 3-nitrotyrosine was apparent in the 15% CO2-alone group, whereas no
staining was observed in the control 5% CO2 group.
CO2 has historically been considered a relatively innocuous
molecule during normocapnia, with limited data on the effects of
CO2 in varying concentrations on cellular and humoral
functions. When elevated to concentrations of 15% or greater,
stimulation of the autonomic and central nervous systems, increased
pulmonary vascular resistance, and elevated cardiac output have been
recognized (50). Other regional vascular beds, including
those of the renal-glomerular axis and splanchnic circulation, have
been reported to have hypercapnia-induced increases in blood flow
(12, 17). The predominant explanation for the
physiological consequences of hypercapnia has been indirect effects
resulting from the action of CO2 as a weak acid. Herein, pH
was controlled for by additional buffering, with intracellular and
extracellular acid-base environments documented to be similar in both
normocapnic and hypercapnic conditions. Thus it is proposed that
hypercapnia alone can alter normal cell signaling and that elevated
CO2 levels serve as a catalyst for directing
ONOO-mediated reaction pathways, leading to impairment of
cell structure and function.
Both protein and nonprotein thiols (e.g., GSH, which represents >90%
of nonprotein thiols in cells) are ubiquitous in vivo and serve as
important intrinsic antioxidants. ONOO readily reacts
with GSH, a reaction that is inhibited by its more facile reaction with
CO2 (13, 33). Cytokines plus LPS exposure
consistently enhanced cell thiol content, a not unexpected observation
because the rate-limiting enzyme of GSH synthesis,
-glutamylcysteine
synthetase, is induced by ·NO and redox-activated transcription
factors (28). Net cell thiol content decreased after
exposure to hypercapnia, a significant response when
cytokine-stimulated cells maintained in 5% CO2 were
compared with 15% CO2-exposed cytokine-stimulated cells
(P < 0.05). Potential explanations for these findings
include 1) the possibility that in cell systems, hypercapnia
may stimulate the formation of secondary propagating radical species
(e.g., HCO3
) that depletes thiols; 2)
integrated metabolic reactions responsible for maintaining thiol
reduction are impaired by hypercapnia; and 3) hypercapnia
may also accelerate cellular production of partially reduced species of
oxygen. This emphasizes the widespread metabolic and oxidative effects
of hypercapnia and is in contrast to observations of decreased
ONOO
-mediated oxidation of GSH occurring in the presence
of increasing concentrations of CO2 (51).
Recent clinical studies have confirmed the efficacy of protective ventilatory strategies in decreasing the expression of proinflammatory mediators (34) and increased survival in patients with ARDS (2, 21, 46). A consequence in several of these studies has been varying degrees of hypercapnia that occurred compared with that in patients treated with conventional strategies. Importantly, pulmonary cell iNOS protein expression and ·NO production will be elevated in the proinflammatory milieu of ARDS lungs (9, 19). This, combined with the clinical use of inhaled ·NO, hyperoxia, and the induction of hypercapnia, infers that a complex set of oxidative and free radical reactions can be occurring within lung cells. Last, low tidal volume strategies and accompanying normocapnia have been associated with improved survival in patients with ARDS (41). The consequences of the increased respiratory rates (versus increased tidal volume) required to maintain these traditional targeted levels of CO2 are not fully known, thus opening up an additional area for investigation.
In summary, these data reveal the detrimental cellular effects of hypercapnia in an inflammatory environment that includes amplification of the production of ·NO and secondary nitrating species. These findings are of particular clinical significance and timing, showing a novel reactivity for CO2 in the context of emerging ventilator strategies that allow hypercapnia to occur in patients suffering from inflammatory-mediated lung injury. The current literature pertaining to hypercapnia in the setting of critical illness is both limited and in conflict (22). The present results thus encourage clinical studies prospectively evaluating the contribution of oxidative inflammatory reactions and aberrant lung cell ·NO-dependent signal transduction during the evaluation of protective ventilation modes that result in varying degrees of hypercapnia.
![]() |
ACKNOWLEDGEMENTS |
---|
This study was supported by a Foundation for Anesthesia and Education Research Grant (New Investigator Award); Arrow International (J. D. Lang); and National Heart, Lung, and Blood Institute Grants HL-58418, HL-58115, and HL-51245 (B. A. Freeman).
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: J. D. Lang, Jr., Dept. of Anesthesiology, The Univ. of Alabama at Birmingham, 845M Jefferson Tower, 619 19th St., Birmingham, AL 35233-6810 (E-mail: john.lang{at}ccc.uab.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 18 April 2000; accepted in final form 20 July 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Adams, GE.
Free radicals in biology: the pulse radiolysis approach.
In: Free Radicals in Biology, edited by Pryor WA.. New York: Academic, 1977, p. 53-91.
2.
Amato, MBP,
Barbas CSV,
Medeiros DM,
Magaldi RB,
Schettino GP,
Lorenzo-Filho G,
Kairalla RA,
Deheinzelin D,
Munoz C,
Oliveira R,
Takagaki TY,
and
Carvalho CR.
Effect of a protective ventilation strategy on mortality in the acute respiratory distress syndrome.
N Engl J Med
338:
347-354,
1998
3.
Baldwin, SR,
Grum CM,
Boxer LA,
Simon RH,
Ketai L,
and
Devall LJ.
Oxidant activity in expired breath of patients with adult respiratory distress syndrome.
Lancet
1:
11-14,
1986[ISI][Medline].
4.
Beckman, JS,
Beckman TW,
Chen J,
Marshall PA,
and
Freeman BA.
Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide.
Proc Natl Acad Sci USA
87:
1620-1624,
1990[Abstract].
5.
Bidani, A,
Tzouanakis AE,
Cardenas VJ,
and
Zwischenberger JB.
Permissive hypercapnia in acute respiratory failure.
JAMA
272:
957-962,
1994[Abstract].
6.
Bredt, DS,
and
Schmidt HHW
The citrulline assay.
In: Methods in Nitric Oxide Research, edited by Feelisch M,
and Stamler J.. Chichester, UK: Wiley, 1996, p. 249-255.
7.
Caniggia, I,
Tseu I,
Han RN,
Smith BT,
Tanswell K,
and
Post M.
Spatial and temporal differences in fibroblast behavior in fetal rat lung.
Am J Physiol Lung Cell Mol Physiol
261:
L424-L433,
1991
8.
Chen, SN,
and
Hoffman MZ.
Rate constants for the reaction of the carbonate radical with compounds of biochemical interest in neutral aqueous solution.
Radiat Res
56:
40-47,
1973[ISI][Medline].
9.
Chiumello, D,
Pristine G,
and
Slutsky A.
Mechanical ventilation affects local and systemic cytokines in an animal model of acute respiratory distress syndrome.
Am J Respir Crit Care Med
160:
109-116,
1999
10.
Chollet-Martin, S,
Jourdain B,
Gibert C,
Elbim C,
Chastre J,
and
Gougerot-Pocidalo MA.
Interactions between neutrophils and cytokines in blood and alveolar spaces during ARDS.
Am J Respir Crit Care Med
154:
594-601,
1996[Abstract].
11.
Crow, JP.
Measurement and significance of free and protein-bound 3-nitrotyrosine, 3-chlorotyrosine, and free 3-nitro-4-hydroxyphenylacetic acid in biologic samples: a high-performance liquid chromatography method using electrochemical detection.
Methods Enzymol
301:
151-160,
1999[ISI][Medline].
12.
Daugherty, RM,
Scott JB,
Dabney JM,
and
Haddy FJ.
Local effects of O2 and CO2 on limb, renal, and coronary vascular resistances.
Am J Physiol
213:
1102-1110,
1967[ISI][Medline].
13.
Denicola, A,
Freeman B,
Trujillo M,
and
Radi R.
Peroxynitrite reaction with carbon dioxide/bicarbonate: kinetics and influence on peroxynitrite-mediated oxidations.
Arch Biochem Biophys
333:
49-58,
1996[ISI][Medline].
14.
Eiserich, JP,
Estevez AG,
Bamberg TV,
Ye YZ,
Chumley PH,
Beckman JS,
and
Freeman BA.
Microtubule dysfunction by posttranslational nitrotyrosination of -tubulin: a nitric oxide-dependent mechanism of cellular injury.
Proc Natl Acad Sci USA
96:
6365-6370,
1999
15.
Eiserich, JP,
Hristova M,
Cross CE,
Jones AD,
Freeman BA,
Halliwell B,
and
van der Vliet A.
Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils.
Nature
391:
393-397,
1998[ISI][Medline].
16.
Frampton, MW,
Morrow PE,
Cox C,
Gibb FR,
Speers DM,
and
Utell MJ.
Effects of nitrogen dioxide exposure on pulmonary function and airway reactivity in normal humans.
Am Rev Respir Dis
143:
522-527,
1991[ISI][Medline].
17.
Fujita, Y,
Sakai T,
Ohsumi A,
and
Takaori M.
Effects of hypocapnia and hypercapnia on splanchnic circulation and hepatic function in the beagle.
Anesth Analg
69:
152-157,
1989[Abstract].
18.
Fulkerson, WJ,
MacIntyre N,
Stamler J,
and
Crapo JD.
Pathogenesis and treatment of the adult respiratory distress syndrome.
Arch Intern Med
156:
29-38,
1996[Abstract].
19.
Gutierrez, HH,
Pitt BR,
Schwarz M,
Watkins SC,
Lowenstein C,
Caniggia I,
Chumley P,
and
Freeman B.
Pulmonary alveolar epithelial inducible NO synthase gene expression: regulation by inflammatory mediators.
Am J Physiol Lung Cell Mol Physiol
268:
L501-L508,
1995
20.
Hickling, KG,
and
Joyce C.
Permissive hypercapnia in ARDS and its effects on tissue oxygenation.
Acta Anaesthesiol Scand Suppl
107:
201-208,
1995[Medline].
21.
Hickling, KG,
Walsh J,
Henderson S,
and
Jackson R.
Low mortality rate in adult respiratory distress syndrome using low-volume, pressure-limited ventilation with permissive hypercapnia: a prospective study.
Crit Care Med
22:
1568-1578,
1994[ISI][Medline].
22.
Laffey, JG,
Engelberts D,
and
Kavanagh BP.
Buffering hypercapnic acidosis worsens acute lung injury.
Am J Respir Crit Care Med
161:
141-146,
2000
23.
Lamb, NJ,
Quinlan GJ,
Westerman ST,
Gutteridge MD,
and
Evans TW.
Nitration of proteins in bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome receiving inhaled nitric oxide.
Am J Respir Crit Care Med
160:
1031-1034,
1999
24.
Leff, JA,
Parsons PE,
Day CE,
Taniguchi N,
Jochum M,
Fritz H,
Moore FA,
Moore EE,
McCord JM,
and
Repine JE.
Serum antioxidants as predictors of adult respiratory distress syndrome in patients with sepsis.
Lancet
341:
777-780,
1993[ISI][Medline].
25.
Lymar, SV,
and
Hurst JK.
Carbon dioxide: physiological catalyst for peroxynitrite-mediated cellular damage or cellular protectant?
Chem Res Toxicol
9:
845-850,
1996[ISI][Medline].
26.
MacMillan-Crow, LA,
Crow JP,
and
Thompson JA.
Peroxynitrite-mediated inactivation of manganese superoxide dismutase involves nitration and oxidation of critical tyrosine residues.
Biochemistry
37:
1613-1622,
1998[ISI][Medline].
27.
Mathru, M,
Rooney MR,
Dries DJ,
Hirsch LJ,
Barnes L,
and
Tobin MJ.
Urine hydrogen peroxide during adult respiratory distress syndrome in patients with and without sepsis.
Chest
105:
232-236,
1994[Abstract].
28.
Moellering, D,
McAndrew J,
Patel RP,
Forman HJ,
Mulcahy RT,
Jo H,
and
Darley-Usmar VD.
The induction of GSH synthesis by nanomolar concentrations of NO in endothelial cells: a role for -glutamylcysteine synthetase and
-glutamyl transpeptidase.
FEBS Lett
448:
292-296,
1999[ISI][Medline].
29.
Moncada, S,
and
Higgs A.
The L-arginine-nitric oxide pathway.
N Engl J Med
329:
2002-2012,
1993
30.
Pacht, ER,
Timerman AP,
Lykens MG,
and
Merola J.
Deficiency of alveolar glutathione in patients with sepsis and the adult respiratory distress syndrome.
Chest
100:
1397-1403,
1991[Abstract].
31.
Patel, RK,
McAndrew J,
Sellak H,
White CR,
Jo H,
Freeman BA,
and
Darley-Usmar V.
Biological aspects of reactive nitrogen species.
Biochim Biophys Acta
447:
1-16,
1999.
32.
Radi, R,
Beckman JS,
Bush KM,
and
Freeman BA.
Peroxynitrite oxidation of sulfhydryls: the cytotoxic potential of superoxide and nitric oxide.
J Biol Chem
266:
4244-4250,
1991
33.
Radi, R,
Denicola A,
and
Freeman BA.
Peroxynitrite reactions with carbon dioxide-bicarbonate.
Methods Enzymol
301:
353-367,
1999[ISI][Medline].
34.
Ranieri, VM,
Suter PM,
Tortorella C,
De Tullio R,
Dayer JM,
Brienza A,
Bruno F,
and
Slutsky AS.
Effect of mechanical ventilation on inflammatory mediators in patients with acute respiratory distress.
JAMA
282:
54-61,
1999
35.
Roumen, RM,
Hendriks T,
Man MD,
and
Goris RJ.
Serum lipofuscin as a prognostic indicator of adult respiratory distress syndrome and multiple organ failure.
Br J Surg
81:
1300-1305,
1994[ISI][Medline].
36.
Rubbo, H,
Darley-Usmar V,
and
Freeman B.
Nitric oxide regulation of tissue free radical injury.
Chem Res Toxicol
9:
809-820,
1996[ISI][Medline].
37.
Schmidt, HHW,
and
Kelm M.
Determination of nitrite and nitrate by the Griess reaction.
In: Methods in Nitric Oxide Research, edited by Freelisch M,
and Stamler J.. Chichester, UK: Wiley, 1996, p. 491-497.
38.
Schofield, K.
Aromatic Nitration. Cambridge, UK: Cambridge University Press, 1980.
39.
Squadrito, GL,
and
Pryor W.
Oxidative chemistry of nitric oxide: the roles of superoxide, peroxynitrite, and carbon dioxide.
Free Radic Biol Med
25:
392-403,
1998[ISI][Medline].
40.
Suzuki, Y,
Lyall V,
Biber TUL,
and
Ford GD.
A modified technique for the measurement of sulfhydryl groups oxidized by reactive oxygen intermediates.
Free Radic Biol Med
9:
479-484,
1990[ISI][Medline].
41.
The Acute Respiratory Distress Syndrome Network. Ventilation with lower tidal volumes compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome.
N Engl J Med
342:
1301-1308,
2000
42.
Tien, M,
Berlett BS,
Levine RL,
Chock PB,
and
Stadtman ER.
Peroxynitrite-mediated modification of proteins at physiological carbon dioxide concentration: pH dependence of carbonyl formation, tyrosine nitration, and methionine oxidation.
Proc Natl Acad Sci USA
96:
7809-7814,
1999
43.
Tremblay, L,
Valenza F,
Ribeiro SP,
Li J,
and
Slutsky AS.
Injurious ventilatory strategies increase cytokines and c-fos m-RNA expression in an isolated rat lung model.
J Clin Invest
99:
944-952,
1997
44.
Tsien, RY,
Pozzan T,
and
Kirk TJ.
Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator.
J Cell Biol
94:
325-334,
1982[Abstract].
45.
Tuxen, DV.
Permissive hypercapnic ventilation.
Am J Respir Crit Care Med
150:
870-874,
1994[ISI][Medline].
46.
Ullrich, R,
Lorber C,
Roder G,
Urak G,
Faryniak B,
Sladen RN,
and
Germann P.
Controlled airway pressure therapy, nitric oxide inhalation, prone position, and extracorporeal membrane oxygenation (ECMO) as compounds of an integrated approach to ARDS.
Anesthesiology
91:
1577-1586,
1999[ISI][Medline].
47.
Uppu, RM,
Squadrito GL,
and
Pryor WA.
Acceleration of peroxynitrite oxidations by carbon dioxide.
Arch Biochem Biophys
327:
335-343,
1996[ISI][Medline].
48.
Van der Vliet, A,
Eiserich JP,
Shigenaga MK,
and
Cross CE.
Reactive nitrogen species and tyrosine nitration in the respiratory tract. Epiphenomena or a pathobiologic mechanism of disease?
Am J Respir Crit Care Med
160:
1-9,
1999
49.
Von Bethmann, AN,
Brasch F,
Nusing R,
Vogt K,
Volk HD,
Muller KM,
Wendel A,
and
Uhlig S.
Hyperventilation induces release of cytokines from perfused mouse lung.
Am J Respir Crit Care Med
157:
263-272,
1998
50.
Walley, KR,
Lewis TH,
and
Wood LDH
Acute respiratory acidosis decreases left ventricular contractility but increases cardiac output in dogs.
Circ Res
67:
628-635,
1990[Abstract].
51.
Zhang, H,
Squadrito GL,
Uppu RM,
Lemercier J,
Cueto R,
and
Pryor WA.
Inhibition of peroxynitrite-mediated oxidation of glutathione by carbon dioxide.
Arch Biochem Biophys
339:
183-189,
1997[ISI][Medline].