| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Emergency Medicine and 2 Institute for Environmental Medicine, Department of Biochemistry and 5 Biophysics and 6 Department of Physiology, University of Pennsylvania Medical Center, Philadelphia 19104; 3 Philadelphia Biomedical Research Institute, King of Prussia, Pennsylvania 19406; and 4 Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES
|
|---|
We hypothesized that elevated partial pressures of O2 would increase perivascular nitric oxide (·NO) synthesis. Rodents with O2- and ·NO-specific microelectrodes implanted adjacent to the abdominal aorta were exposed to O2 at partial pressures from 0.2 to 2.8 atmospheres absolute (ATA). Exposures to 2.0 and 2.8 ATA O2 stimulated neuronal (type I) NO synthase (nNOS) and significantly increased steady-state ·NO concentration, but the mechanism for enzyme activation differed at each partial pressure. At both pressures, elevations in ·NO concentration were inhibited by the nNOS inhibitor 7-nitroindazole and the calcium channel blocker nimodipine. Enzyme activation at 2.0 ATA O2 appeared to be due to an altered cellular redox state. Exposure to 2.8 ATA O2, but not 2.0 ATA O2, increased nNOS activity by enhancing nNOS association with calmodulin, and an inhibitory effect of geldanamycin indicated that the association was facilitated by heat shock protein 90. Infusion of superoxide dismutase inhibited ·NO elevation at 2.8 but not 2.0 ATA O2. Hyperoxia increased the concentration of ·NO associated with hemoglobin. These findings highlight the complexity of oxidative stress responses and may help explain some of the dose responses associated with therapeutic applications of hyperbaric oxygen.
neuronal nitric oxide synthase; heat shock protein 90; calmodulin; hyperbaric oxygen
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES
|
|---|
THE PURPOSE of this investigation was to determine the effect of hyperoxia in vivo on perivascular nitric oxide (·NO) synthesis by using ·NO- and O2-sensitive microelectrodes. The impact of elevated O2 tension on ·NO synthesis has not been clearly established by studies with cell cultures and isolated enzymes. We considered that inconsistencies in the literature may be due to reliance on indirect measurements of ·NO production, such as nitrite and nitrate, and because the incubation conditions used in model systems may not accurately reflect those that exist in vivo.
Among the three NO synthase (NOS) enzymes, the activity of inducible (type II) NOS (iNOS) is controlled at the level of gene transcription, whereas the activities of neuronal (type I) NOS (nNOS) and endothelial (type III) NOS (eNOS) are controlled by intracellular calcium/calmodulin, several different phosphorylation mechanisms, and by binding of the molecular chaperone heat shock protein 90 (HSP90) (5, 12, 13, 16, 36). Studies with all three NOS enzymes in vitro have shown that enzyme activity is influenced by the redox state and specifically by O2 tension. During NOS-mediated arginine catalysis, some of the self-generated ·NO reduces ferric heme in the active site to the ferrous form. Ferrous-state NOS has ~10% activity compared with ferric NOS (2, 3, 20). Elevated O2 tension influences NOS activity by hastening conversion of ferrous heme back to the native ferric conformation. Conversion of ferric heme to the ferrous state is influenced by the rate of ·NO synthesis. Therefore, this process appears less important with eNOS, which exhibits catalytic activity between four and eight times lower than the other two NOS isoforms (1). The effect of O2 on catalytic activity is reflected by values for the apparent Michaelis-Menten constant (Km) for O2 of each enzyme. Purified nNOS exhibits an apparent Km of ~400 µM and saturation at 800 µM (2), values far greater than the Km for binding O2. This contrasts sharply with the value for eNOS, 4 µM (1). iNOS has catalytic activity similar to that of nNOS, and its apparent Km for O2 is ~190 µM (11). Incubation conditions can alter these findings; however, as Hurshman and Marletta (20) reported that under reducing conditions, there was little enhancement of iNOS activity by super-normal O2 concentrations.
Hypoxic conditions diminish synthesis of ·NO in cells from both pulmonary and systemic circulations, likely because of the enzyme O2 requirement (8, 11, 30, 31, 39, 46, 48). Elevated O2 tensions above ambient will increase ·NO production by pulmonary endothelial cells and intact lungs (8, 11, 30-32, 38). In contrast, O2 tensions above ~55 mmHg were reported to have little effect on ·NO production by cells obtained from the systemic circulatory system (31, 48). In the central nervous system, elevated partial pressures of O2 increase the steady-state concentration of ·NO by stimulating nNOS activity (42). This action is mediated by binding HSP90 and is an oxidative stress response inhibitable by geldanamycin and infusion of superoxide dismutase (SOD) (42).
This study examined ·NO production in the vicinity of the abdominal great vessels. We avoided studies of the microvasculature because ·NO synthesis in this region is influenced by variations in blood flow controlled by proximal resistance vessels and by diffusion of an array of chemical mediators (6, 7, 19, 30, 38). We examined the dose response between elevated partial pressures of O2 and the steady-state concentrations of ·NO, the mechanism for NOS activation, and changes in concentration of ·NO-carrying substances in the blood. The majority of experiments were conducted in rats, with a complementary series carried out using "knockout" mice lacking functional genes for eNOS or nNOS.
This study also examined whether mechanisms for perturbing NOS activity
differed with O2 partial pressure. There is an increasing interest in the use of hyperbaric O2 therapy for a variety
of disorders such as refractory wounds, radiation injury, and
decompression sickness (18). Dosing protocols have been
based on anecdotal experience because underlying mechanisms for benefit
have not been elucidated. Hyperbaric O2 has been shown to
cause angiogenesis and to inhibit neutrophil 
2-integrin
adhesion, two potentially beneficial actions that may be influenced by
changes in steady-state ·NO concentration (4, 18, 26, 34, 40,
41).
| |
METHODS AND MATERIALS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Materials.
Wistar male rats (Charles River Laboratories) weighing 220-240 g
were fed a standard diet and water ad libitum. House mice (Mus
musculus) of the C57B6J strain were raised at the animal facilities of the University of Pennsylvania. Mice used in the study
were either wild type or lacked a functional nNOS gene (nNOS
/). Mice lacking a functional eNOS gene (eNOS/), used in several studies, were graciously provided by Dr. Paul Huang from the
Massachusetts General Hospital. All aspects of this investigation were
reviewed and approved by the Institutional Animal Care and Use Committee.
80°C.
Microelectrode fabrication. Studies performed with dual electrodes selective for ·NO and for O2 were fabricated by Dr. Buerk's laboratory according to published methods (42). The majority of studies were performed with ·NO-selective microelectrodes obtained commercially from World Precision Instruments (Sarasota, FL). Studies in rats were done with the ISO-NOP200 electrode (sensor surface area 200 × 2,000 µm in length), and studies in mice were done with the ISO-NOP3020 (sensor surface area 30 × 2,000 µm).
Electrode studies. The standard procedure was to anesthetize both rats and mice with intraperitoneal ketamine (83 mg/kg) and xylazine (11 mg/kg). After the animals were anesthetized, the abdomen was opened and the peritoneum was reflected to allow placement of the electrode between aorta and vena cava. Abdominal contents were then replaced, and a sterile saline-soaked piece of gauze was placed over the abdominal incision. A second dose of ketamine-xylazine amounting to three-fourths of the initial dose was given just before the animal was placed in the hyperbaric chamber. The hyperbaric chamber used in this study was rated for a maximum pressure of 3.0 atmospheres absolute (ATA) and has been described in a prior publication (43). Once in the closed chamber, animals were monitored for ~30 min until electrode recordings became stable. During this time, air was flowed through the chamber to remove exhaled gases, but no additional pressure was applied. Where specified, rats were injected with 7-nitroindazole (12 mg/kg ip), NG-nitro-L-arginine methyl ester (L-NAME, 40 mg/kg ip), nimodipine (1 mg/kg ip), aminoguanidine (100 mg/kg ip), or geldanamycin (0.3 mg/kg ip) 30 min before pressurization in the hyperbaric chamber or with N-3-(aminomethyl)benzyl acetamine (1400-W, 1 mg/kg ip) 2 h before pressurization. Others received bovine erythrocyte (copper-zinc) SOD concentration (25,000 U/kg) intravenously immediately before the pressurization.
Tissue preparation for immunochemical and biochemical assays.
Rats were anesthetized with intraperitoneal ketamine (83 mg/kg) and
xylazine (11 mg/kg), and abdominal aortas were removed. Tissue was
immediately homogenized in 25 mM Tris · HCl, pH
7.5, containing 100 mM NaCl, 100 µM diethylene triaminepentaacetic acid, 40 µM PMSF, 0.5 µg/ml leupeptin, and 0.01% butylated
hydroxytoluene. For each 1 g of aorta, 2 ml buffer were added,
and tissue was homogenized with two 30-s pulses in a Polytron and then
two strokes with a Teflon plunger. Homogenates were centrifuged (12,000 g for 5 min), and supernatants were frozen at 80°C until
used in subsequent assays.
Immunoprecipitation.
Supernatants containing 250 µg protein were suspended in 500 µl of
precipitation buffer [20 mM MES (pH 7.6) containing 100 mM NaCl, 0.1 mM dithiothreitol, 0.1 mM EDTA, 10% glycerol, 0.4% Triton X-100, 1 mM
PMSF, 10 mM sodium fluoride, 10 µg/ml aprotinin, 1 mM sodium
orthovanadate, 10 mM sodium fluoride, 10 µM tetrahydrobiopterin, 1 mM
arginine, and 20 mM sodium molybdate]. Suspensions were all precleared
by incubation for 2 h at 4°C with 30 µl 20% (wt/vol) protein
G-Sepharose and then centrifuged at 8,200 g for 1 min. The
supernatant was saved, antibodies (10 µg of anti-HSP90, anti-nNOS, or
anti-eNOS) were added, and the suspension was incubated overnight at
4°C. The next day, 30 µl of 20% (wt/vol) protein G-Sepharose were
added to the suspensions, incubated 1.5 h at 4°C, and then centrifuged at 8,200 g for 1 min. The immune pellets were
washed twice with wash buffer (10 mM MES, pH 7.6 containing 50 mM NaCl, 20 mM sodium molybdate, 10% glycerol, and 0.4% Triton X-100), pellets
were suspended with 40 µl of sample buffer (100 mM sodium phosphate,
pH 7.4, containing 2% SDS, 10% glycerol, 5% -mercaptoethanol, and
0.00125% bromophenol), and the suspension was heated to 95°C for 10 min. After centrifugation at 8,200 g for 1 min, aliquots (30 µl) of the supernatant were electrophoresed by using a 12% SDS-polyacrylamide gel. Proteins were transferred to Immobilon-P membranes and probed with 1:1,000 dilutions of antibody (anti-HSP90, eNOS, nNOS, or calmodulin). Where indicated, aortic homogenate preparations containing 250 µg protein were incubated for 30 min at
30°C with 50 µl of phenyl-Sepharose in 10 mM
Tris · HCl, pH 7.5, containing 50 mM KCl, 5 mM
MgCl2, and 1 mM dithiothreitol. The phenyl-Sepharose was
washed three times with Tris · HCl, pH 7.5, plus
1 mM EDTA, suspended in 40 µl sample buffer, and subjected to
electrophoresis as described above.
Measurement of ·NO-containing substances in blood. Heparinized blood was obtained by puncturing the abdominal aorta of anesthetized rats. Where indicated, rats were exposed to 2.8 ATA O2 for 45 min immediately before anesthesia. Assays were performed exactly as described by Sonoda et al. (35).
Electron paramagnetic resonance measurements.
Deionized water used for stock solutions was bubbled with nitrogen to
remove dissolved O2, and stock solutions of 1 M
cysteine-HCl and 0.45 M
FeSO4 · 7H2O were bubbled
a second time before storage at 80°C. A solution of MGD-Fe-cysteine
was prepared by dissolving 3.4 g MGD in 3.4 ml H2O, to
which 0.13 ml FeSO4 stock solution was added. After several
minutes to allow equilibration, 0.6 ml cysteine stock solution was
added, and the solution was kept under an argon flow until aliquots of
0.12 ml were distributed to Eppendorf tubes and stored at 80°C.
Data analysis. Statistical significance was determined by ANOVA followed by Scheffé's test. The level of significance was taken as P < 0.05. Results are expressed as means ± SE.
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Temporal changes in tissue O2 and ·NO elevations.
A dual-barrel electrode placed adjacent to the wall of the abdominal
aorta was used to examine the relationship between elevation of tissue
O2 tension and steady-state ·NO concentration. As shown in a representative experiment (Fig. 1),
tissue O2 tension rose rapidly from a value between 10 and
40 Torr while air was breathed to a mean value of 1,900 Torr ± 100 (n = 4) when rats were pressurized to 2.8 ATA while
breathing pure O2. The steady-state ·NO concentration also increased rapidly, and the mean concentration while O2
was breathed at 1, 1.5, 2.0, and 2.8 ATA is shown in Fig.
2. The concentration at 2.0 and 2.8 ATA
O2 were both greater than control, and 2.8 ATA
O2 had a significantly greater effect than 2.0 ATA
O2 (P < 0.05, ANOVA). The markedly higher
number of samples for air and 2.8 ATA O2 studies occurred
because the results in Fig. 2 included studies in which only the
effects of O2 were examined and studies related to
pharmacological inhibitors (see Pharmacological inhibitors of NOS
activation in rats).
|
|
|
|
NOS enzymes and regulatory proteins present in aorta.
Protein amounts, normalized to actin present on Western blots, were
measured for the three NOS isoforms and for calmodulin and HSP90.
Homogenates of the abdominal aorta were made from control animals, and
rats were killed immediately after a 45-min exposure to 2.8 ATA
O2. A representative group of images from Western
blots is shown in Fig. 5. The
concentrations of proteins were not significantly different between
control and 2.8 ATA O2 groups (Table
1). No iNOS was detected on the blots.
|
|
Pharmacological inhibitors of NOS activation in rats.
Pharmacological manipulations were performed in rats to gain insight
into the mechanism by which O2 elevated the perivascular steady-state ·NO concentration. Injection with the nonspecific NOS
inhibitor L-NAME or with the 7-nitroindazole, a
relatively specific inhibitor of nNOS, significantly reduced ·NO
production due to exposure to 2.8 ATA O2 (Fig.
6). In contrast, there was no significant
inhibition when rats were treated with aminoguanidine or 1400-W, two
inhibitors of iNOS. Significant inhibition was observed with infusions
of geldanamycin, an inhibitor of HSP90, with nimodipine, a calcium
channel blocker, and with SOD. Inhibition due to SOD was reversible. In
three trials, the experiment was repeated 1 h after SOD infusion,
and the response to hyperoxia returned to 102.3 ± 9% of the
response observed before SOD infusion. SOD did not change the temporal
pattern of elevations in O2 tension and steady-state ·NO
concentration (as shown in Fig. 3, A and B).
Times to achieve half-maximal O2 tension and ·NO
concentration were within 3 ± 2% (n = 4) of
those observed with exposure to 2.8 ATA O2 on trials
conducted before SOD infusion.
|
Investigation with knockout mice.
Production of ·NO in mice in response to elevations in O2
partial pressure is shown in Fig. 7. No
significant difference was observed in the periaortic ·NO
concentration between wild-type and eNOS knockout mice exposed to 2.8 ATA O2. There were significantly lower steady-state ·NO
concentrations in wild-type mice exposed to 1.5 and 2.0 ATA
O2 and in nNOS knockout mice exposed to 1.5, 2.0, and 2.8 ATA O2. We conclude that nNOS was the predominant isoform
activated in response to hyperoxia.
|
Protein associations and NOS phosphorylation.
Aortic tissue homogenates were subjected to immunoprecipitation to
examine the associations among proteins. Samples from rats exposed to
2.8 ATA O2, but not 2.0 ATA O2, had twice as
much calmodulin associated with immunoprecipitated nNOS compared with
control, air-breathing rats (Fig. 8). A
representative group of images from Western blots is shown in Fig.
9. The elevation of calmodulin-nNOS association was inhibited by treatment with geldanamycin but not by
SOD. There was no significant increase in the association between HSP90
and nNOS among samples from rats exposed to 2.0 or 2.8 ATA O2 (Figs. 9 and 10).
|
|
|
, nor
association with PIN, played a role in aortic nNOS inactivation.
Alterations were not found in the associations between eNOS and HSP90
or eNOS and calmodulin after exposures to 2.8 ATA O2 in
aortic homogenates immunoprecipitated with anti-eNOS (Table 2). A representative group of images from
Western blots is shown in Fig. 11. Akt
protein kinase-dependent phosphorylation will activate eNOS by
phosphorylating serine-1177 (10, 13). There was no difference in the ratio of phosphorylated eNOS to total eNOS on Western
blots of aortic homogenates probed with an antibody that recognizes
serine-1177-phosphorylated eNOS (Table 2 and Fig. 11). Blots were also
probed for Akt and its activated form phospho-Akt. The ratio of
phosphorylated Akt to total Akt was 0.5 ± 0.2 (n = 4) for aortic homogenates from control rats and 0.48 ± 0.2 (n = 4) for samples from rats exposed to 2.8 ATA
O2 (no significant difference).
|
|
HSP90 binding characteristics. Because of the inhibitory effects of geldanamycin on O2-mediated ·NO production, we examined whether some binding functions attributed to HSP90 may be altered by exposure to 2.8 ATA O2. Geldanamycin binds at the amino-terminal portion of HSP90 where ATP binding occurs (17). ATP binding regulates the HSP90 conformational change required for binding p23, a cochaperone that confers stability to protein heterocomplexes (22, 28). Aortic homogenates were subjected to immunoprecipitation using anti-HSP90 and Western blots probed for p23 that coprecipitated with HSP90. The band density ratios of p23 to HSP90 on the blots were 0.91 ± 0.23 (n = 4) for control samples and 0.99 ± 0.41 (n = 4, no significant difference) for samples obtained from rats exposed to 2.8 ATA O2 for 45 min.
The ADP-bound form of HSP90 exhibits an elevated affinity for binding to phenyl-Sepharose (17, 40). The mean band density of HSP90 was measured on Western blots prepared from aortic homogenate samples eluted from phenyl-Sepharose, and this was compared with the total amount of HSP90 in the homogenate expressed as the band density from lanes loaded with samples that had not been eluted from Sepharose. The band density ratio of control samples was 0.18 ± 0.02 (n = 4), and for samples obtained from rats exposed to 2.8 ATA O2 for 45 min it was 0.19 ± 0.03 (n = 4, no significant difference).Elevations of ·NO-carrying substances in blood.
The concentration of ·NO-carrying substances in the blood by a
fluorometric method was 4.7 ± 0.7 µM (n = 6)
for control rats and 4.4 ± 0.4 (n = 6, no
significant difference versus control) after 2.8 ATA O2.
The limit of detection for this assay was ~0.5 µM, so a more
sensitive technique was sought. We developed an EPR method by using
erythrocyte lysates from which ·NO was extracted using the spin trap
MGD (see MATERIALS AND METHODS). Examples of ·NO-MGD EPR
spectra obtained from control rats and rats exposed to 2.8 ATA using
either pure O2 or hypoxic gas (7.46% O2) are shown in Fig. 12. Where indicated, rats
were pretreated with L-NAME. Approximately a 50% increase
in spin concentration occurred with exposure to 2.8 ATA O2
but not by exposure to normoxic gas at 2.8 ATA or in rats infused with
L-NAME before exposure to 2.8 ATA O2 (Table
3). The ·NO concentration in
erythrocyte lysates was estimated by determining the amount of ·NO
required to cause a ~50% increase in spin concentration in standard
curves. The concentration following exposure to 2.8 ATA O2
was estimated to be 910 ± 105 nM (means ± SE,
n = 4), whereas the concentration in control,
air-breathing rats was 590 ± 66 nM (means ± SE,
n = 4).
|
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES
|
|---|
This investigation was focused on extending the understanding of how ·NO synthesis in vivo is altered by elevated partial pressures of O2. The array of inhibitor studies in rats (Fig. 6) and results in knockout mice (Fig. 7) indicate that nNOS is the isoform responsible for elevations of steady-state ·NO concentration due to increases in tissue oxygenation. We exclude iNOS activity based on a lack of effect of specific iNOS inhibitors. The inhibitory effect of nimodipine on O2-mediated stimulation of ·NO synthesis is consistent with the role of calcium/calmodulin in nNOS activation (5, 39).
Geldanamycin prevented the 2.8 ATA O2-mediated elevation in steady-state ·NO concentration (Fig. 6) and inhibited the O2-mediated increase in association between nNOS and calmodulin (Fig. 8). This indicates that HSP90 plays a role in nNOS activation. HSP90 is a molecular chaperone that interacts with its substrate proteins by forming transient multiprotein complexes. Two mechanisms have been identified for how HSP90 can activate nNOS. HSP90 can lower the dissociation constant (Kd) of calmodulin binding and sustain the opening of the heme-binding cleft to allow substrate entry (5, 36, 37).
There was no significant increase in the association between nNOS and HSP90 in aortic preparations from rats exposed to 2.8 ATA O2 (Fig. 10). This is different from observations in the brain, where exposure to 2.8 ATA O2 has been shown to elevate ·NO synthesis by stimulating nNOS activity. In the brain, the association between HSP90 and nNOS was increased by 2.8 ATA O2, but there was no significant increase in the association between calmodulin and nNOS (42). This suggests that the mechanism for O2-mediated activation of nNOS may differ between these two locations. In the aorta, nNOS activation may arise due to augmented calmodulin binding, whereas in the brain, nNOS activation may be related to sustained opening of the heme-binding cleft.
Infusion of SOD inhibited ·NO elevation due to 2.8 ATA O2
(Fig. 6). This observation is similar to findings in the brain
(42) but counter to findings with aortic rings perfused
with air-equilibrated buffer (27). Our findings with the
aorta and those in the brain differ from an expectation that
O
The steady-state concentration of ·NO due to exposure to 2.0 ATA O2 was significantly greater than the control, but also significantly less than that caused by 2.8 ATA O2 (Fig. 2). At 2.0 ATA O2, there was no elevation in the nNOS-calmodulin association (Fig. 8), and neither geldanamycin nor SOD prevented the elevation in ·NO concentration. These data suggest that the mechanism for nNOS activation was not as complex as with 2.8 ATA O2 exposure. Better oxygenation, and thus maintaining a higher percentage of enzyme in the more active ferric conformation, may be the predominant mechanism at 2.0 ATA O2 (2, 3, 20).
Whereas the inhibitory effect of geldanamycin indicates that HSP90 binding to nNOS is important for responses to 2.8 ATA O2, there were no apparent alterations in HSP90-binding characteristics. Hyperbaric O2 did not increase HSP90 binding to eNOS or nNOS (Figs. 10 and 11, and Table 2). In addition, interactions between HSP90 and p23 and HSP90 and phenyl-Sepharose were not altered by hyperoxia. The small protein p23 confers stability to many HSP90 heterocomplexes, and p23 is thought to bind specifically to the ATP-bound state of HSP90 (23, 29). When in the ADP-bound state, HSP90 binds to phenyl-Sepharose (17, 40). Therefore, the data indicate that hyperoxia does not perturb the HSP90 structure. This suggests that nNOS, itself, or some additional factor, is altered by hyperoxia and leads to the HSP90-mediated enhancement in the calmodulin-nNOS association.
It is well established that low-molecular-weight thiols, albumin and hemoglobin, can carry ·NO in the blood stream (14, 15, 22). We did not find an elevation in the steady-state concentration of total ·NO-containing substances using a fluorescence technique, but an elevation in hemoglobin-associated ·NO by hyperoxia was identified with a sensitive EPR technique. We have previously used diethyldithiocarbamate (DETC) for ·NO trapping in animal tissues (28, 45). However, DETC is water insoluble and not suitable for blood experiments. Kotake (25) used water-soluble MGD for detecting the serum concentration of ·NO. This method is limited because of the concentration of MGD that can be injected without causing side effects, and we found this method was not adequately sensitive to detect a small change of blood ·NO level during hyperbaric O2. We also tried spin trapping ·NO by using blood hemoglobin but found no difference with hyperoxia, perhaps because the amount of ·NO was too small compared with the heme concentration in the red blood cell (20 mM as heme concentration). Therefore, we devised a method by which a small amount of hemoglobin-trapped ·NO was extracted ex vivo by MGD at a high concentration (~0.5 M, which cannot be reached in vivo). This method provided us with sufficient sensitivity to detect differences cause by hyperbaric O2. It must be recognized that estimating the actual ·NO concentration by standard curve has several assumptions. It was assumed that ·NO was efficiently trapped with hemoglobin and extracted completely by MGD. The efficiency of adduct formation in the presence of red blood cells is also assumed to be the same as that in the pure buffer solution used for calibration. Therefore, the results are best viewed as an approximation. The ·NO concentration measured with the EPR technique was approximately twice the concentration measured by microelectrode, which may also have underestimated the concentration achieved by hyperoxia because the electrode measured values from outside of the blood vessel.
The studies with inhibitors and knock-out mice offer a consistent impression of the relative importance of nNOS. However, ·NO from eNOS associated with vascular endothelium may not have been detected as readily, given that most of the ·NO would diffuse to the vascular lumen away from the electrodes on the abluminal surface of blood vessels. Therefore, our methodology could have underestimated the apparent contribution of eNOS to ·NO synthesis in response to hyperoxia.
The physiological relevance of elevations in ·NO concentration due to
hyperoxia require added investigation. These changes may contribute to
augmentation of angiogenesis and inhibition of neutrophil
2-integrin function that have been reported with hyperbaric O2 (33, 41, 44). Differences in the
mechanism for ·NO production at 2.0 versus 2.8 ATA O2, as
well as differences in magnitude of ·NO synthesis, may offer insight
into dose-response phenomena. Our findings also indicate the complex
influence that O
| |
ACKNOWLEDGEMENTS |
|---|
This work was supported by National Institutes of Health Grants AT-00428 (to S. R. Thom), ES-05211 (to S. R. Thom), CA-82506 (to Y. Kotake), and GM-30736 (to T. Ohnishi).
| |
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: S. R. Thom, Institute for Environmental Medicine, Univ. of Pennsylvania, 1 John Morgan Bldg., 3620 Hamilton Walk, Philadelphia, PA 19104-6068 (E-mail: sthom{at}mail.med.upenn.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.
First published December 27, 2002;10.1152/ajpheart.01043.2002
Received 17 December 2002; accepted in final form 19 December 2002.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Abu-Soud, HM,
Ichimori K,
Presta A,
and
Stuehr DJ.
Electron transfer, oxygen binding, and nitric oxide feedback inhibition in endothelial nitric oxide synthase.
J Biol Chem
275:
17349-17357,
2000
2.
Abu-Soud, HM,
Rousseau DL,
and
Stuehr DJ.
Nitric oxide binding to the heme of neuronal nitric-oxide synthase links its activity to changes in oxygen tension.
J Biol Chem
271:
32515-32518,
1996
3.
Abu-Soud, HM,
Wangs J,
Rousseau DL,
Fukuto JM,
Ignarro LJ,
and
Stuehr DJ.
Neuronal nitric oxide synthase self-inactivates by forming a ferrous-nitrosyl complex during aerobic catalysis.
J Biol Chem
270:
22997-23006,
1995
4.
Banick, PD,
Chen Q,
Xu YA,
and
Thom SR.
Nitric oxide inhibits neutrophil B2 integrin function by inhibiting membrane-associated cyclic GMP synthesis.
J Cell Physiol
172:
12-24,
1997[Web of Science][Medline].
5.
Bender, AT,
Silverstein AM,
Demady DR,
Kanelakis KC,
Noguchi S,
Pratt WB,
and
Osawa Y.
Neuronal nitric-oxide synthase is regulated by the hsp90-based chaperone system in vivo.
J Biol Chem
274:
1472-1478,
1999
6.
Bohlen, HG.
Mechanism of increased vessel wall nitric oxide concentrations during intestinal absorption.
Am J Physiol Heart Circ Physiol
275:
H542-H550,
1998
7.
Bohlen, HG,
and
Nase GP.
Dependence of intestinal arteriolar regulation on flow-mediated nitric oxid eformation.
Am J Physiol Heart Circ Physiol
279:
H2249-H2258,
2000
8.
Cornfield, DN,
Reeve HL,
Tolarova S,
Weir EK,
and
Archer S.
Oxygen causes fetal pulmonary vasodilation through activation of a calcium-dependent potassium channel.
Proc Natl Acad Sci USA
93:
8089-8094,
1996
9.
Demchenko, IT,
Boso AE,
Bennett PB,
Whorton AR,
and
Piantadosi CA.
Hyperbaric oxygen reduces cerebral blood flow by inactivating nitric oxide.
Nitric Oxide
4:
597-608,
2000[Web of Science][Medline].
10.
Dimmeler, S,
Fleming I,
Fisslthaler B,
Hermann C,
Busse R,
and
Zeiher AM.
Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation.
Nature
399:
601-605,
1999[Medline].
11.
Dweik, RA,
Laskowski D,
Abu-Soud HM,
Kaneko FT,
Hutte R,
Stuehr DJ,
and
Erzurum SC.
Nitric oxide synthesis in the lung.
J Clin Invest
101:
660-666,
1998[Web of Science][Medline].
12.
Fontana, J,
Fulton D,
Chen Y,
Fairchild TA,
McCabe TJ,
Fujita N,
Tsuruo T,
and
Sessa WC.
Domain mapping studies reveal that the M domain of hsp90 serves as a molecular scaffold to regulate Akt-dependent phosphorylation of endothelial nitric oxide synthase and NO release.
Circ Res
90:
866-873,
2002
13.
Garcia-Cardone, G,
Fan R,
Shah V,
Sorrentino R,
Cirino G,
Papapetropoulos A,
and
Sessa WC.
Dynamic activation of endothelial nitric oxide synthase by Hsp 90.
Nature
392:
821-824,
1998[Medline].
14.
Gow, AJ,
Luchsinger BP,
Pawloski JR,
Singel DJ,
and
Stamler JS.
The oxyhemoglobin reaction of nitric oxide.
Proc Natl Acad Sci USA
96:
9027-9032,
1999
15.
Gow, AJ,
and
Stamler JS.
Reactions between nitric oxide and haemoglobin under physiological conditions.
Nature
391:
169-173,
1998[Medline].
16.
Gratton, JP,
Fontana J,
O'Connor DS,
Garcia-Cardena G,
McCabe T,
and
Sessa WC.
Reconstitution of an endothelial nitric oxide synthase (eNOS), hsp90, and caveolin-1 complex in vitro.
J Biol Chem
275:
22268-22272,
2000
17.
Grenert, JP,
Sullivan WP,
Fadden P,
Haystead AJ,
Clark J,
Mimnaugh E,
Krutzsch H,
Ochel HJ,
Schulte TW,
Sausville E,
Neckers LM,
and
Toft DO.
The amino-terminal domain of heat shock protein 90 (hsp90) that binds geldanamycin is an ATP/ADP switch domain that regulates hsp90 conformation.
J Biol Chem
272:
23843-23850,
1997
18.
Hampson, N.
Hyperbaric Oxygen Therapy: 1999 Committee Report. Kensington, MD: Undersea and Hyperbaric Medical Society, 1999.
19.
Hester, RL.
Venular-arteriolar diffusion of adenosine in hamster cremaster microcirculation.
Am J Physiol Heart Circ Physiol
258:
H1918-H1924,
1990
20.
Hurshman, AR,
and
Marletta MA.
Nitric oxide complexes of inducible nitric oxide synthase: spectral characterization and effect on catalytic activity.
Biochemistry
34:
5627-5634,
1995[Medline].
21.
Jaffrey, SR,
and
Snyder SH.
PIN: an associated protein inhibitor of neuronal nitric oxide synthase.
Science
274:
774-777,
1996
22.
Jia, L,
Bonaventura C,
Bonaventura J,
and
Stamler SJ.
S-nitrosohaemoglobin: a dynamic activity of blood involved in vascular control.
Nature
380:
221-226,
1996[Medline].
23.
Johnson, JL,
and
Toft DO.
Binding of p23 and hsp90 during the assembly with the progesterone receptor.
Mol Endocrinol
9:
670-678,
1995
24.
Komeima, K,
Hayashi Y,
Naito Y,
and
Watanabe Y.
Inhibition of neuronal nitric-oxide synthase by calcium/calmodulin-dependent protein kinase II through Ser847 phosphorylation in NG108-15 neuronal cells.
J Biol Chem
275:
28139-28143,
2000
25.
Kotake, Y.
Continuous and quantitative monitoring of rate of cellular Nitric oxide generation.
Methods Enzymol
268:
222-229,
1996[Web of Science][Medline].
26.
Lee, RH,
Efron D,
Tantry U,
and
Barbul A.
Nitric oxide in the healing wound: a time-course study.
J Surg Res
101:
104-108,
2001[Web of Science][Medline].
27.
Mian, KB,
and
Martin W.
Differential sensitivity of basal and acetylcholine-stimulated activity of nitric oxide to destruction by superoxide anion in rat aorta.
Br J Pharmacol
115:
993-1000,
1995[Web of Science][Medline].
28.
Ohnishi, ST.
Measurement of nitric oxide using electron paramagnetic resonance.
In: Methods in Molecular Biology, edited by Titheradge MA.. Totowa, NJ: Humana, 1998, vol. 100, p. 129-153.
29.
Richter, K,
and
Buchner J.
Hsp90: chaperoning signal transduction.
J Cell Physiol
188:
281-290,
2001[Web of Science][Medline].
30.
Saito, Y,
Eraslan A,
and
Hester RL.
Importance of venular flow in control of arteriolar diameter in hamster cremaster muscle.
Am J Physiol Heart Circ Physiol
265:
H1294-H1300,
1993
31.
Schmetterer, L,
Findl O,
Strenn K,
Graselli U,
Kastner J,
Eichler HG,
and
Wolzt M.
Role of NO in the O2 and CO2 responsiveness of cerebral and ocular circulation in humans.
Am J Physiol Regul Integr Comp Physiol
273:
R2005-R2012,
1997
32.
Shaul, PW,
and
Wells LB.
Oxygen modulates nitric oxide production selectively in fetal pulmonary endothelial cells.
Am J Respir Cell Mol Biol
11:
432-438,
1994[Abstract].
33.
Sheikh, AY,
Gibson JJ,
Rollins MD,
Hopf HW,
Hussain Z,
and
Hunt TK.
Effect of hyperoxia on vascular endothelial growth factor levels in a wound model.
Arch Surg
135:
1293-1297,
2000
34.
Shinobu, LA,
Jones SG,
and
Jones MM.
Sodium N-methyl-D-glucamine dithiocarbamate and cadmium intoxication.
Acta Pharmacol Toxicol (Copenh)
25:
171-191,
1984.
35.
Sonoda, M,
Kobayashi J,
Takezawa M,
Miyazaki T,
Nakajima T,
Shimomura H,
Koike K,
Satomi A,
Ogino H,
Omoto R,
and
Komoda T.
An assay method for nitric oxide-related compounds in whole blood.
Anal Biochem
247:
417-427,
1997[Web of Science][Medline].
36.
Song, Y,
Zweir JL,
and
Xia Y.
Heat shock protein 90 augments neuronal nitric oxide synthase activity by enhancing Ca+2/calmodulin binding.
Biochem J
355:
357-360,
2001[Web of Science][Medline].
37.
Song, Y,
Zweir JL,
and
Xia Y.
Determination of the enhancing action of HSP90 on neuronal nitric oxide synthase by EPR spectroscopy.
Am J Physiol Cell Physiol
281:
C1819-C1824,
2001
38.
Steenbergen, JM,
Bohlen HG,
and
Lash JM.
Role of a lymphatic system in glucose absorption and the accompanying microvascular hyperemia.
Am J Physiol Gastrointest Liver Physiol
267:
G529-G535,
1994
39.
Su, Y,
and
Block ER.
Role of calpain in hypoxic inhibition of nitric oxide synthase activity in pulmonary endothelial cells.
Am J Physiol Lung Cell Mol Physiol
278:
L1204-L1212,
2000
40.
Sullivan, W,
Stensgard B,
Caucutt G,
Bartha B,
McMahon N,
Alnemri ES,
Litwack G,
and
Toft D.
Nucleotides and two functional states of hsp90.
J Biol Chem
272:
8007-8012,
1997
41.
Thom, SR.
Functional inhibition of leukocyte 2-integrins by hyperbaric oxygen in carbon monoxide-mediated brain injury in rats.
Toxicol Appl Pharmacol
123:
248-256,
1993[Web of Science][Medline].
42.
Thom, SR,
Bhopale V,
Fisher D,
Manevich Y,
Huang PL,
and
Buerk D.
Stimulation of nitric oxide synthesis in cerebral cortex due to elevated partial pressures of oxygen: an oxidative stress response.
J Neurobiol
51:
85-100,
2002[Web of Science][Medline].
43.
Thom, SR,
and
Elbuken ME.
Oxygen-dependent antagonism of lipid peroxidation.
Free Radic Biol Med
10:
413-426,
1991[Web of Science][Medline].
44.
Thom, SR,
Mendiguren I,
Hardy KR,
Bolotin T,
Fisher D,
Nebolon M,
and
Kilpatrick L.
Inhibition of human neutrophil 2-integrin-dependent adherence by hyperbaric oxygen.
Am J Physiol Cell Physiol
272:
C770-C777,
1997
45.
Thom, SR,
Ohnishi ST,
Fisher D,
Xu YA,
and
Ischiropoulos H.
Pulmonary vascular stress from carbon monoxide.
Toxicol Appl Pharmacol
154:
12-19,
1999[Web of Science][Medline].
46.
Tiktinsky, MH,
and
Morin FC, III.
Increasing oxygen tension dilates fetal pulmonary circulation via endothelium-derived relaxing factor.
Am J Physiol Heart Circ Physiol
265:
H376-H380,
1993
47.
Turrens, JF,
Crapo JD,
and
Freeman BA.
Protection against oxygen toxicity by intravenous injection of liposome-entrapped catalase and superoxide dismutase.
J Clin Invest
73:
87-95,
1984[Web of Science][Medline].
48.
Whorton, AR,
Simonds DB,
and
Piantadosi CA.
Regulation of nitric oxide synthesis by oxygen in vascular endothelial cells.
Am J Physiol Lung Cell Mol Physiol
272:
L1161-L1166,
1997
This article has been cited by other articles:
|
|
 |
|
  R. T. Mahon, H. M. Dainer, M. G. Gibellato, and S. E. Soutiere Short oxygen prebreathe periods reduce or prevent severe decompression sickness in a 70-kg swine saturation model J Appl Physiol, April 1, 2009; 106(4): 1459 - 1463. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Rubinstein, Z. Abassi, F. Milman, E. Ovcharenko, R. Coleman, J. Winaver, and O. S. Better Hyperbaric oxygen treatment improves GFR in rats with ischaemia/reperfusion renal injury: a possible role for the antioxidant/oxidant balance in the ischaemic kidney Nephrol. Dial. Transplant., February 1, 2009; 24(2): 428 - 436. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. D. Bauser-Heaton, J. Song, and H. G. Bohlen Cerebral microvascular nNOS responds to lowered oxygen tension through a bumetanide-sensitive cotransporter and sodium-calcium exchanger Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H2166 - H2173. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Dainer, J. Nelson, K. Brass, E. Montcalm-Smith, and R. Mahon Short oxygen prebreathing and intravenous perfluorocarbon emulsion reduces morbidity and mortality in a swine saturation model of decompression sickness J Appl Physiol, March 1, 2007; 102(3): 1099 - 1104. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Hink, S. R. Thom, U. Simonsen, I. Rubin, and E. Jansen Vascular reactivity and endothelial NOS activity in rat thoracic aorta during and after hyperbaric oxygen exposure Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1988 - H1998. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. J. Goldstein, K. A. Gallagher, S. M. Bauer, R. J. Bauer, V. Baireddy, Z.-J. Liu, D. G. Buerk, S. R. Thom, and O. C. Velazquez Endothelial Progenitor Cell Release into Circulation Is Triggered by Hyperoxia-Induced Increases in Bone Marrow Nitric Oxide Stem Cells, October 1, 2006; 24(10): 2309 - 2318. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. P. Cabigas, J. Su, W. Hutchins, Y. Shi, R. B. Schaefer, R. F. Recinos, V. Nilakantan, E. Kindwall, J. A. Niezgoda, and J. E. Baker Hyperoxic and hyperbaric-induced cardioprotection: Role of nitric oxide synthase 3 Cardiovasc Res, October 1, 2006; 72(1): 143 - 151. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. R. Thom, V. M. Bhopale, O. C. Velazquez, L. J. Goldstein, L. H. Thom, and D. G. Buerk Stem cell mobilization by hyperbaric oxygen Am J Physiol Heart Circ Physiol, April 1, 2006; 290(4): H1378 - H1386. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Palm, D. G. Buerk, P.-O. Carlsson, P. Hansell, and P. Liss Reduced Nitric Oxide Concentration in the Renal Cortex of Streptozotocin-Induced Diabetic Rats: Effects on Renal Oxygenation and Microcirculation Diabetes, November 1, 2005; 54(11): 3282 - 3287. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. O Brubakk, D Duplancic, Z Valic, I Palada, A Obad, D Bakovic, U Wisloff, and Z Dujic A single air dive reduces arterial endothelial function in man J. Physiol., August 1, 2005; 566(3): 901 - 906. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. M. Denton, A. Shweta, R. L. Flower, and W. P. Anderson Predominant postglomerular vascular resistance response to reflex renal sympathetic nerve activation during ANG II clamp in rabbits Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2004; 287(4): R780 - R786. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
