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1 Department of Exercise Science and 2 Free Radical and Radiation Biology Program, University of Iowa, Iowa City, Iowa 52242
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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This work tested the hypotheses that
splanchnic oxidant generation is important in determining heat
tolerance and that inappropriate ·NO production may be involved in
circulatory dysfunction with heat stroke. We monitored colonic
temperature (Tc), heart rate, mean arterial pressure, and
splanchnic blood flow (SBF) in anesthetized rats exposed to 40°C
ambient temperature. Heating rate, heating time, and thermal load
determined heat tolerance. Portal blood was regularly collected for
determination of radical and endotoxin content. Elevating
Tc from 37 to 41.5°C reduced SBF by 40% and stimulated
production of the radicals ceruloplasmin, semiquinone, and
penta-coordinate iron(II) nitrosyl-heme (heme-·NO). Portal endotoxin
concentration rose from 28 to 59 pg/ml (P < 0.05).
Compared with heat stress alone, heat plus treatment with the nitric
oxide synthase (NOS) antagonist
N
-nitro-L-arginine methyl
ester (L-NAME) dose dependently depressed heme-·NO production and increased ceruloplasmin and semiquinone levels. L-NAME also significantly reduced lowered SBF,
increased portal endotoxin concentration, and reduced heat tolerance
(P < 0.05). The NOS II and diamine oxidase antagonist
aminoguanidine, the superoxide anion scavenger superoxide dismutase,
and the xanthine oxidase antagonist allopurinol slowed the rates of
heme-·NO production, decreased ceruloplasmin and semiquinone levels,
and preserved SBF. However, only aminoguanidine and allopurinol
improved heat tolerance, and only allpourinol eliminated the rise in
portal endotoxin content. We conclude that hyperthermia stimulates
xanthine oxidase production of reactive oxygen species that activate
metals and limit heat tolerance by promoting circulatory and intestinal barrier dysfunction. In addition, intact NOS activity is required for
normal stress tolerance, whereas overproduction of ·NO may contribute
to the nonprogrammed splanchnic dilation that precedes vascular
collapse with heat stroke.
oxidative stress; nitrosative stress; free radicals; ischemia; heat stroke; electron paramagnetic resonance; endotoxemia
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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HEAT STROKE IS THE MOST CATASTROPHIC FORM of debilitating illness resulting from environmental heat stress. Described clinically as either classical (nonexertional) or exertional in nature, heat stroke is a systemic disorder that is characterized by multiorgan injury, severe hypotension, and multisystem organ failure with evidence of multiorgan involvement in precipitating cardiovascular shock (26, 43). The etiology of shock with heat stroke remains unclear, but work from Kregel et al. (28) has shown that the initial decline in peripheral resistance occurs within the splanchnic vascular bed and may precipitate the systemic hypotension that is characteristic of heat illness.
In the present study, we tested the hypotheses that splanchnic oxidant generation is an important determinant of heat tolerance and that inappropriate nitric oxide (·NO) production is involved in splanchnic vascular dysfunction with heat stroke. We developed these hypotheses based on previous work from our laboratory (18) demonstrating that hyperthermia stimulates splanchnic production of ·NO and biomarkers of cellular oxidative stress.
With the use of electron paramagnetic resonance (EPR) spectroscopy to directly detect radicals in vivo (18), we observed that hyperthermia progressively increased portal venous content of the following: semiquinone radical, a biomarker of mitochondrial reductive stress (52); ceruloplasmin, an acute phase antioxidant protein that acts to reduce metal-catalyzed oxidant production (29); and penta-coordinate iron(II) nitrosyl-heme (heme-·NO) (27). The respective characteristics of these radicals suggest that heat stress stimulates ·NO and reactive oxygen species (ROS) production within splanchnic viscera leading to transition metal activation and cellular oxidative stress. Indeed, our group (19) and others (36, 44) have proposed that metal-catalyzed oxidative stress is involved in hyperthermia-related pathology.
The purpose of the present study was to investigate mechanisms of ·NO
and ROS production in vivo during environmental heat stress. Figure
1 details a working model that we propose
for the etiology of heat illness. We (17) have previously
shown that hyperthermia produces cellular hypoxia and metabolic stress
within the liver and intestine. We propose that subsequent cellular
biochemical events secondary to hypoxia (e.g., increased cytosolic
Ca2+) promote mitochondrial ROS production and stimulate
cellular oxidase and constitutive nitric oxide synthase (NOS) I and III enzymatic activities (Fig. 1B). We further propose that
constituitive synthesis of ·NO is protective, buffering the rise in
splanchnic vasoconstrictor activity (14) and cellular
oxidative stress. If heat stress continues (Fig. 1C),
intestinal barrier function may be compromised, increasing
translocation of gut contents into the splanchnic circulation where
endotoxins and local inflammatory mediators such as cytokines
(7) would upregulate NOS II enzymatic activity. The higher
cellular flux of ·NO produced by NOS II would promote reactive
nitrogen species generation and nonprogrammed splanchnic dilation.
Unresolved questions in this model include identification of the
principle source(s) of ROS and ·NO during heat stress.
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To test these ideas, in vivo experiments were designed to examine the effects of 1) antagonizing NOS enzymatic activity, 2) increasing NOS substrate availability, 3) limiting superoxide anion bioavailability with polyethyleneglycol (PEG)-conjugated copper and zinc-containing superoxide dismutase (PEG-SOD), and 4) antagonizing the heat-sensitive ROS-producing enzyme xanthine oxidase with allopurinol.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals
Male Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing 300-350 g served as subjects. Rats were purchased in groups of 15 and housed in the University of Iowa Animal Care Facility at an ambient temperature of 22-24°C on a 12:12-h light-dark cycle. Animals were handled and familiarized with laboratory procedures for 3-5 days before use as experimental subjects. Food and water were provided ad libitum.Rats were randomly assigned to the following groups: 1)
sham-operated normothermic control (n = 4),
2) saline-treated normothermic control (n = 4), 3) noninjected hyperthermic control (n = 4), 4) saline-treated hyperthermic control
(n = 10), 5)
N-nitro-L-argine methyl
ester (L-NAME)-treated (25, 50, and 125 µM) heat exposed
(n = 24), 6) aminoguanidine-treated (25 and
50 µM) heat exposed (n = 16), 7)
L-arginine-treated (100, 200, and 300 µM) heat exposed
(n = 24), 8) PEG-SOD-treated (10 and 30 mg/kg body wt) heat exposed (n = 16), 9)
PEG-SOD-treated normothermic control (n = 4), and
10) allopurinol-treated (10, 20, and 30 mg/kg body wt) heat
exposed (n = 24).
Experimental Design
Experiments were conducted between 800 and 1300 h. After surgical preparation, anesthetized rats were exposed to 40°C ambient temperature (Ta) in a temperature-controlled environmental chamber while they received intravenous injections of the designated pharmacological agent. Thermocouples placed around the animal continuously monitored Ta. Preliminary experiments established that enzyme blockade was not maintained for the duration of an experiment if a single bolus injection of a reagent was used. Therefore, rats received injections (10 µl/100 g body wt iv) at time 0 (beginning of heat exposure) and at 15-min intervals until termination of an experiment (~90 min). Because of its long vascular half-life (41), PEG-SOD was given as two injections within the first 15 min of heating.Colonic temperature (Tc), heating time, mean arterial
pressure (MAP), heart rate (HR), and superior mesenteric artery (SMA) blood flow were continually monitored. From these data, thermal load
and splanchnic resistance were calculated. Portal venous and femoral
artery blood samples were collected for analyses of radical and
endotoxin content at four time points: 1) immediately before
heat exposure (Tc 
37°C), 2) at
Tc = 41.5°C, 3) immediately after SMA
flow increased, and 4) after MAP fell below 100 mmHg. Heat
exposure was terminated when MAP fell below 100 mmHg. Experiments were
terminated when MAP fell below 60 mmHg.
Surgical Preparation
Animals were anesthetized with pentobarbital sodium (Nembutal; 50 mg/kg ip), injected with atropine (100 µl), and tracheotomized to ensure a patent airway. An incision was made in the inguinal region of the right hindlimb. The femoral artery was isolated for ~1 cm of its length and fitted with a catheter [polyethylene (PE)-50; Clay Adams, Parsippany, NJ] filled with heparinized saline (100 U/ml) to monitor HR and MAP and to sample blood. The leg incision was closed, and a midline laparotomy was performed. The portal vein was isolated ~2 mm from the liver, and a second catheter for sampling blood and delivering pharmacological agents was placed in the portal vein through a tributary vessel. Tributary vessels were chosen as cannulation sites to avoid impeding portal blood flow. Catheter placement was confirmed at the conclusion of each experiment.The superior mesenteric artery was next isolated for ~1 cm of its length, and a miniaturized Transonic Doppler flow probe was positioned around the vessel to monitor SMA blood flow. The abdominal incision was closed, and a colonic temperature (Tc) probe (36-gauge copper-constantan wire in PE-100 tubing) was inserted 7-8 cm past the anal sphincter to monitor Tc.
Rats were allowed to stabilize for 30 min after surgery. Baseline HR, MAP, and SMA blood flow data were collected for an additional 30 min. During this 60-min period, Tc was maintained at 37.0 ± 0.2°C with a heating pad. At the end of the control period, baseline portal venous and femoral artery blood samples were collected, and the Ta of the environmental chamber was elevated to 40°C with a relative humidity of 30%.
Sample Collection and Handling
Cardiovascular data. MAP was determined by connecting the femoral artery catheter to a Gould P23 ID pressure transducer that electronically averages the pulsatile signal. HR was determined from the number of electronic pulsations sensed by a cardiotachometer (Beckman Instruments). SMA blood flow was determined using a pulsed Transonics Doppler flowmeter (University of Iowa Bioengineering Resource Facility). MAP, HR, and SMA blood flow were continuously monitored, and the mean value over each 60-s period was recorded.
Radicals and serum endotoxin.
Portal venous and femoral artery blood samples (350 µl each) were
collected in sterile, 1-ml Monoject syringes (2). Whole blood (200 µl) was immediately delivered into a quartz EPR tube (inner diameter 3 mm) and frozen at 77 K pending EPR analyses as
previously described (18). The remaining 150 µl of blood was processed for quantitation of gram-negative bacterial endotoxin concentration per manufacturer's instructions [limulus amebocyte lysate (LAL), quantitative chromogenic LAL, BioWhittaker, Walkersville, MD]. Briefly, cell and serum fractions were isolated by centrifugation in a refrigerated clinical microcentrifuge. The serum was extracted and
delivered to pyrogen-free storage tubes (BioWhittaker), diluted 1:10
with pyrogen-free water, and heated for 10 min at 70°C to remove
nonspecific LAL inhibitors present in blood products (8). Processed serum was stored at 
80°C pending endotoxin assay. Serum from all samples was tested in triplicate. All materials coming into
contact with blood or test materials not purchased as pyrogen-free from
BioWhittaker were rendered pyrogen-free by heating at 200°C for
4 h.
EPR Conditions
EPR spectra were recorded with a Bruker ESP 300 EPR spectrometer (Bruker Instruments, Karlsruhe, Germany) equipped with an ER035M gaussmeter, an ER4111VT variable-temperature unit, and an EIP-625A microwave-frequency counter. Signal averaging (multiple scans of the same sample) was used to improve the signal-to-noise ratio. Sample volume and geometry were kept constant to allow for comparisons of radical concentration between samples. All spectra were collected at 100 K, with data reported as the normalized average of 20 scans. EPR conditions were the following: receiver gain 5.00 × 105, modulation frequency 100 kHz, modulation amplitude 4.0 Gauss (G), microwave frequency 9.43 GHz, microwave power 10 mW, and scan rate 6.0 G/s.Calculations
Thermal stress was quantified by determining thermal load, a product of Tc and time spent above 40.4°C (22). Tc was recorded at 1-min intervals, and thermal load (°C × min) was calculated as the following: the sum of the time interval (in min) × [(Tc above 40.4°C 40.4°C)]. Heating rate (in °C/min) was calculated as the following: (maximum Tc attained during heat
exposure baseline Tc)/total heating time (in min).
Splanchnic vascular resistance is equal to pressure (in mmHg) divided
by SMA flow (kHz shift). The percent change in splanchnic vascular
resistance was calculated using the following formula: [(Rt Rc)/Rc] × 100, where Rc is baseline MAP divided by the mean SMA flow value
in the control period and Rt equals MAP divided by the SMA
flow value in the test period (20).
Statistical Analyses
Thermal responses, endotoxin values, and cardiovascular data were analyzed by one-way analysis of variance. Groups were categorized according to Tukey's multiple comparisons procedure. Significant differences were identified with Dunnet's test. Differences were considered significant at the P < 0.05 level.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Normal Heat Stress Responses
Our initial set of experiments was designed to characterize the typical radical, hemodynamic, and splanchnic endotoxin responses of the heat-stressed rat. To examine splanchnic hemodynamics and splanchnic radical production in parallel, Fig. 2 presents SMA blood flow and portal EPR spectra versus Tc. There were no differences in the responses of noninjected hyperthermic rats and saline-treated hyperthermic animals; therefore, data from these two groups were pooled and are referred to as hyperthermic controls.
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EPR spectra of portal blood collected before heat stress displayed a weak composite signal, suggesting the presence of multiple radicals. The narrow g = 2.005 feature (peak-to-peak line width ~10 G) represents semiquinone radical (Fig. 2A) (18). The broad deflection at g = 2.06, indicative of a transition metal, represents ceruloplasmin (Fig. 2B) (4).
Elevating Tc from 37 to 41.5°C stimulated a 127 ± 7% increase in splanchnic resistance (Table
1), a 40 ± 2% reduction in splanchnic blood flow (SBF) (Fig. 2 and Table 1), and increased circulating ceruloplasmin and semiquinone radical levels (Fig. 2B). The decline in SBF was nonlinear in nature, with a
sharp decrease occurring at Tc > 40°C (Fig. 2).
Heart rate and MAP (Fig. 3, control)
progressively increased with rising Tc, peaking at 589 ± 16 beats/min and 170 ± 2 mmHg, respectively (Table 1).
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At Tc of 41.5°C, we observed the evolution of a triplet EPR signal centered at g = 2.012 (splitting constant ~17.5 G) (Fig. 2, B and C) that represents heme-·NO (18, 27). EPR evidence of heme-·NO was not observed in arterial blood (data not shown), suggesting that this was a local phenomenon within the splanchnic region. Splanchnic resistance fell and SBF rapidly increased at Tc of 41.8 ± 0.2°C. EPR of portal blood collected immediately after the initial rise in SBF shows a strong heme-·NO signal (Fig. 2C), suggesting that overproduction of ·NO may contribute to declining splanchnic resistance. Only the ceruloplasmin signal was observed in arterial blood samples (data not shown), again suggesting that the rise in heme-·NO levels was a local event within the splanchnic circulation. HR and MAP (Fig. 3, control) declined at Tc of 42.0 ± 0.2°C ~5-7 min after rising SBF. Total heating time for hyperthermic controls was 77 ± 4 min, and thermal load was 34.4 ± 3.2°C × min (Table 1). We interpret these results as evidence that heat stress increases metal activation and ·NO production within splanchnic tissues.
L-NAME
These experiments tested the hypothesis that globally antagonizing NO synthase (NOS) with L-NAME (37) would lower heme-·NO production and prevent splanchnic dilation. However, because of the antioxidant actions of ·NO (21, 25, 50, 51), we further hypothesized that antagonizing NOS would enhance cellular oxidative stress and lower heat tolerance.Compared with hyperthermic controls, treatment with 25 µM
L-NAME reduced SBF by >20% and rapidly increased
ceruloplasmin and semiquinone radical levels (Fig.
4, B vs. A).
Low-dose L-NAME also reduced, but did not eliminate,
splanchnic heme-·NO production (Fig. 4C vs. Fig.
2C) and marginally elevated the Tc at which splanchnic dilation was observed (Fig. 4). The net effect of 25 µM
L-NAME treatment was a decreased rate of splanchnic
heme-·NO production and maintenance of splanchnic constriction but an
increase in biomarkers of oxidative stress. Peak MAP and heat
tolerance, as determined by total heating time and thermal load, were
not altered (Table 1).
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In contrast, EPR-detectable heme-·NO was not observed with higher
concentrations of L-NAME (Fig.
5, B and C).
Compared with hyperthermic controls, both 50 and 125 µM
L-NAME significantly increased resistance (Table 1) and
reduced SBF (Fig. 5). Both 50 and 125 µM L-NAME increased
ceruloplasmin and semiquinone radical levels (Fig. 5B vs.
Fig. 4B), suggesting that antagonizing NOS increased
cellular oxidative stress and metal activation. L-NAME treatment also eliminated the rise in SBF at high Tc (Fig.
5), suggesting that activation of NOS contributes to the observed splanchnic dilation. In addition, 50 and 125 µM L-NAME
dose dependently reduced peak HR (Fig. 3 and Table 1) and lowered heat
tolerance (Table 1).
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Aminoguanidine
These experiments were designed to test the tenet that NOS II activation may be involved in splanchnic dilation during heat stress. We hypothesized that aminoguanidine treatment would slow the rate of heme-·NO accumulation, delay splanchnic dilation, and improve heat tolerance. We chose concentrations of 25 and 50 µM aminoguanidine based on the work of Grisham et al. (16) and Corbett et al. (6), who demonstrated in vivo the relative specificity of aminoguanidine for NOS II over NOS I and III. Vascular radical profile was similar between 25 and 50 µM aminoguanidine treatments; therefore, EPR spectra from the 25 µM-treated group are presented.Compared with hyperthermic controls, aminoguanidine treatment increased
ceruloplasmin and semiquinone radical levels and significantly increased the magnitude of rise in Tc required to produce
portal heme-·NO accretion and splanchnic dilation (42.7 vs. 41.5°C
for hyperthermic controls, P < 0.05) (Fig.
6 vs. Fig. 2). Aminoguanidine (25 µM)
also shifted the blood flow curve rightward (Fig. 6), significantly
slowing the rate of reduction in SBF. The net effect was a slower rate
of splanchnic heme-·NO production (Fig. 6, B and
C) and maintenance of splanchnic constriction but a rise in the levels of biomarkers of cellular oxidative stress. Aminoguanidine (25 µM) also reduced the magnitude of rise in SBF on splanchnic dilation (Fig. 6), increased peak MAP and HR, and improved heat tolerance more than any other treatment (Table 1). In contrast, 50 µM
aminoguanidine significantly reduced heat tolerance (Table 1).
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L-Arginine
L-Arginine is a critical substrate for NOS. These experiments tested the hypothesis that L-arginine supplementation would increase hyperthermia-induced ·NO production, blunt the rise in splanchnic resistance, and preserve SBF. Contrary to our hypothesis, L-arginine dose dependently increased splanchnic resistance and MAP and lowered SBF (Table 1); events that occurred independent of alterations in splanchnic heme-·NO production (data not shown). We speculate that the increase in resistance and MAP may be due to arginine-stimulated splanchnic vasoconstrictor release (32).PEG-SOD
SOD scavenges O2
·, directly blocking
O2·-mediated chemistry and indirectly increasing
the bioavailability of ·NO. PEG-SOD is a large molecule with a
vascular half-life of 18-24 h (41). With the use of
PEG-CuZnSOD, these experiments tested the hypothesis that heat stress
stimulates O2· production, which is involved in
regulating splanchnic hemodynamics and in cellular events leading to
semiquinone radical production and ceruloplasmin release. Vascular
radical profile was similar between 10 and 30 mg/kg PEG-SOD treatments;
therefore, spectra from 10 mg/kg-treated animals are presented.
Treatment with PEG-SOD markedly reduced splanchnic
constriction (Table 1) and maintained SBF at or near euthermic control levels throughout heat exposure (Fig. 7).
PEG-SOD also blunted the rise in ceruloplasmin and semiquinone radical
levels (Fig. 7, B vs. A) and
significantly increased the magnitude of rise in Tc
required to elicit heme-·NO accumulation and splanchnic dilation
(42.5 vs. 41.5°C for hyperthermic controls, P < 0.05) (Fig. 7 vs. Fig. 2). Once resistance declined in this group, SBF rose to 44 ± 8% above euthermic baseline. Peak HR and MAP were significantly increased (Fig. 8), and HR
was preserved at or near peak levels until termination of an experiment
(Tc = 43.3 ± 0.3°C) (Fig. 8).
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Both 10 and 30 mg/kg PEG-SOD significantly increased thermal load (Table 1). However, neither dose increased total heating time, and both the rate of rise in Tc and the maximum Tc attained before shock were greater than that of hyperthermic controls (Table 1), suggesting that lowering splanchnic constriction negatively impacted heat-dissipating capacity in this group.
Allopurinol
Allopurinol is a competitive antagonist of xanthine oxidase, an enzyme that directly produces O2· and hydrogen
peroxide (H2O2). The intestine and liver are
rich in xanthine oxidase protein, which is localized primarily to
hepatic sinusoidal endothelial cells and intestinal epithelial cells
(23). These experiments tested the hypothesis that heat
stress can stimulate xanthine oxidase production of ROS that influence
splanchnic hemodynamics and participate in cellular events leading to
semiquinone radical production and ceruloplasmin release. Vascular
radical profile was similar between 10 and 30 mg/kg allopurinol
treatments; therefore, spectra from 10 mg/kg-treated animals are presented.
Treatment with allopurinol markedly decreased splanchnic
semiquinone radical and ceruloplasmin levels (Fig.
9). EPR spectra of portal blood collected
at 41.5°C (Fig. 9B) and 42.7°C (Fig. 9C) showed little change in ceruloplasmin or semiquinone
radical concentrations from 37°C levels. As with PEG-SOD, allopurinol also increased the Tc at which heme-·NO accumulation and
splanchnic dilation were observed (Tc > 42.5 vs.
41.5°C for controls, P < 0.05). Indeed, the vascular
radical profile from allopurinol-treated animals and the magnitude of
thermal stress required for radical generation were similar to those of
animals treated with PEG-SOD (compare Figs. 9 and 7). However, there is
a notable absence of EPR-detectable ceruloplasmin with allopurinol
treatment (Fig. 9, A-D).
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Allopurinol did not alter the magnitude of change in resistance or SBF (Fig. 9 and Table 1) but shifted the SBF curve rightward, significantly slowing the rate of rise in resistance and the rate of decline in SBF (Fig. 9). On splanchnic dilation, SBF increased to +20 ± 4% above euthermic baseline. Allopurinol also increased peak HR, MAP (Fig. 8), and thermal load (Table 1). HR was maintained at or near peak levels until termination of an experiment (Fig. 8).
Portal Endotoxin
Portal venous blood collected before heat exposure tested positive for gram-negative bacterial endotoxins at a concentration of 28 ± 7 pg/ml (Fig. 10). Arterial blood tested negative for endotoxin (data not shown), suggesting that the liver clears endotoxins under these conditions. Heat stress alone and heat stress plus saline treatment significantly increased portal endotoxin concentration to 59 ± 7 and 62 ± 4 pg/ml, respectively (P < 0.05) (Fig. 10). Arterial blood tested negative for endotoxin, suggesting that the liver capacity to clear endotoxins remains intact at Tc of 41.5°C. Compared with hyperthermic controls, neither PEG-SOD (58 ± 5 pg/ml), aminoguanidine (60 ± 5 pg/ml), nor L-arginine (59 ± 7 pg/ml) treatments altered 41.5°C portal endotoxin concentration, whereas L-NAME (78 ± 4 pg/ml) significantly increased portal endotoxin levels. Only allopurinol treatment significantly reduced 41.5°C endotoxin levels (29 ± 8 pg/ml, P < 0.05) (Fig. 10).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The purpose of the present study was to test the hypotheses that splanchnic oxidant generation is an important determinant of heat tolerance and that ·NO is involved in splanchnic vascular dysfunction with heat stroke. The major results of the present experiments support these proposals, suggesting that environmental heat stress can stimulate oxidase production of ROS that contribute to metal activation and circulatory and intestinal barrier dysfunction. In addition, the present data demonstrate that intact NOS activity is required for normal heat tolerance, whereas overproduction of ·NO may be responsible for the nonprogrammed loss of splanchnic resistance that precedes heat stroke. We speculate that cellular hypoxia and derangements in intracellular Ca2+ control may be central to the observed increase in ROS and ·NO production (Fig. 1).
EPR Results
EPR data from the present study demonstrate that environmental heat stress can increase circulating ceruloplasmin levels and stimulate splanchnic semiquinone radical production. Ceruloplasmin is an acute phase protein synthesized by the liver that exhibits robust ferroxidase activity, oxidizing loosely bound transition metals and decreasing their participation in cellular redox chemistry (24, 29). The involvement of catalytic metals in oxidant generation and cellular injury is a well-documented phenomenon (33). Accordingly, ceruloplasmin is viewed as a potent antioxidant protein. Hyperthermia-induced elevations in circulating ceruloplasmin and semiquinone radical levels suggest that metal-catalyzed oxidative stress may contribute to heat stress pathology. We speculate that hepatic tissues (46) and vascular endothelial cells (9) may be sources of catalytic metals under these conditions.Mechanisms by which hyperthermia causes metal activation are unclear,
but both O2· (9) and ·NO
(38) can reduce protein-bound metals, promoting their
participation in redox chemistry. While the current results suggest
that production of both O2· and ·NO are
stimulated by heat stress, the fact that PEG-SOD and allopurinol
treatments lowered ceruloplasmin concentrations, whereas
L-NAME increased ceruloplasmin, suggests that
O2· may be the effector molecule that activates
transition metals during hyperthermia. It is unlikely, given the large
size of the PEG-SOD complex and the relatively short duration of these
experiments, that PEG-SOD could be impacting mitochondrial
O2· production. Rather, these results imply that
extramitochondrial sources of O2·, such as cellular
oxidases, may be involved. Data from experiments using allopurinol as
an intervention support this conclusion. Collectively, we interpret
these results as evidence that heat stress stimulates oxidase
production of O2· and H2O2,
leading to transition metal activation within splanchnic tissues.
Adaptive responses include hepatic release of ceruloplasmin and local
·NO production.
The present study strongly suggests that constituitive synthesis of ·NO is essential for successful adaptation to acute heat stress, whereas NOS II activation may underlie hyperthermia-induced splanchnic dilation. Antagonizing NOS with L-NAME dose dependently lowered heme-·NO accumulation and SBF, increased semiquinone radical levels, and lowered heat tolerance. In contrast, aminoguanidine treatment slowed but did not eliminate heme-·NO accumulation, significantly delayed splanchnic dilation, and improved heat tolerance more than any other intervention. On the basis of previously cited in vivo work (6, 16), we speculate that aminoguanidine antagonized inducible but not constituitive ·NO synthesis in these experiments. We interpret these results as evidence that NOS II activation contributes to hyperthermia-related splanchnic dilation. Alternatively, because aminoguanidine also inhibits diamine oxidase, these results may indicate that antagonizing amine metabolism during heat stress lowers NOS activation. However, while the precise mechanism of action for aminoguanidine remains unclear, these results suggest that constituitive ·NO production protects normal heat tolerance, whereas NOS II activation may compromise thermoregulatory processes by lowering peripheral resistance.
The present work was not designed to evaluate mechanisms underlying the protective effects of ·NO, but low flux ·NO production has been shown to enhance cellular antioxidant capacity, acting as a primary antioxidant (51) and as a chain-breaking antioxidant (21, 25, 50, 51). During heat stress, ·NO may act to buffer the rise in intracellular ROS levels and subsequent generation of lipid peroxides. In addition, ·NO opposes splanchnic vasoconstrictor activity, thereby preserving SBF and potentially reducing tissue hypoxia and acidosis (17). Local hypoxia elicits vasoconstriction followed by relaxation in mesenteric arterioles (31), and acidosis is a key predictor of negative clinical outcomes from heat illness (35). Moreover, ·NO inhibits platelet aggregation (34), neutrophil adherence to activated endothelial cells (30), and leukocyte NADPH oxidase activity (5), all of which may contribute to heat stress pathology.
Interestingly, in addition to antagonizing NOS II, aminoguanidine also
slows vasoactive amine turnover by antagonizing enzymes that metabolize
polyamines (15). Cellular oxidases involved in amine
metabolism directly produce both O2· and
H2O2 as byproducts; therefore, this raises the
possibility that aminoguanidine may have exerted beneficial effects by
simultaneously antagonizing NOS II and by decreasing cellular oxidase activity.
Cardiovascular Results
The present data suggest that ·NO is directly involved in hyperthermia-induced splanchnic dilation. However, experiments using PEG-SOD and allopurinol as antioxidant interventions also support a role for ROS in this outcome. Dismuting O2· to
H2O2 with PEG-SOD lowered splanchnic
constriction more than any other intervention. EPR evidence suggests
that the profound decrease in splanchnic resistance occurred
independent of increased ·NO bioavailability. Conversely, reducing
both O2· and H2O2
production with allopurinol slowed the rate of constriction but did not
alter the magnitude of reduction in SBF. These results establish that
O2· is a powerful heat-induced constricting agent
within the splanchnic circulation (39, 42) and suggest
that H2O2 may have synergized with ·NO in
producing splanchnic dilation.
Support for this interpretation is provided by previous work from Rubanyi et al. (40) and Vanhoutte et al. (49), who reported that H2O2 can produce vasodilation by acting directly on vascular smooth muscle (40) or by stimulating guanylate cyclase activity and depolarizing K+ channels (49). Moreover, like ·NO, H2O2 can readily cross biological membranes; therefore, we speculate that both ROS and reactive nitrogen species may contribute to local circulatory dysfunction during heat stress.
Endotoxin Results
Results from the present study establish that severe heat stress can produce splanchnic endotoxemia. However, hepatic reticuloendothelial cell function remains intact up to 41.5°C because endotoxins were not detected in the systemic circulation. These effects were eliminated with allopurinol treatment and exacerbated with L-NAME. This suggests that ROS produced by xanthine oxidase may be responsible for the observed rise in splanchnic endotoxin levels, whereas ·NO may be a critical molecule that protects intestinal barrier function and/or contributes to reticuloendothelial cell endotoxin removal.The heat sensitivity and physiological significance of xanthine oxidase was first demonstrated by Skibba et al. (44, 45), who reported that stimulation of hepatic xanthine oxidase activity produced metal-catalyzed oxidative injury in the hyperthermic liver. Subsequent works by Tan et al. (47) and Terada et al. (48) have shown that an ischemic insult to the intestine and liver can stimulate cellular export of xanthine oxidase. Once in the vascular compartment, xanthine oxidase can be bound by endothelial cell glycosaminoglycans (47), localizing the enzyme next to endothelial cell surfaces.
In the current work, heat stress reduced SBF by ~40% as Tc rose from 37 to 41.5°C. We (17) have previously shown that heat stress of this magnitude produces significant cellular hypoxia in the liver and intestine. Hypoxia can stimulate conversion of xanthine dehydrogenase to xanthine oxidase and increase its cellular export (23). Although the current project did not directly measure xanthine oxidase activity, our data suggest that the combined effects of heat and cellular hypoxia may have stimulated cellular xanthine oxidase activity, leading to intestinal injury and splanchnic endotoxemia.
The role of endotoxins in heat illness remains to be established. The current view of the involvement of splanchnic organs in the pathogenesis of heat stroke is that ischemic and/or thermal stresses can damage the intestinal wall, allowing endotoxins to escape the intestinal lumen and enter the portal circulation (1, 13). Thermal injury to the liver would then allow endotoxins to spill over into the systemic circulation, stimulating a cascade of events similar to systemic inflammatory response syndrome. However, it generally requires much higher Tc to produce spillover; therefore, the role of endotoxins in the current work remains debatable. However, it is noteworthy that pretreating experimental animals with steroids, antiendotoxin antibodies, or with antibiotics to reduce gut flora reduces heat stress lethality (3, 10-12). On the basis of the present work, we suggest that the above-listed interventions may have acted by reducing NOS II activation.
In summary, interventions that increased systemic antioxidant capacity (aminoguanidine, PEG-SOD, or allopurinol) decreased production of radical biomarkers of metal-catalyzed oxidative stress. Agents that reduced NOS II or cellular oxidase activity (aminoguanidine and allopurinol) also significantly improved cardiovascular performance and heat tolerance. The mechanisms responsible for these results could involve decreased cellular ROS production and transition metal activation in combination with reduced flux of ·NO.
Additionally, these data establish that intact NOS activity is required for normal heat tolerance, whereas overproduction of ·NO and H2O2 may be responsible for local circulatory dysfunction with heat stroke. Xanthine oxidase may be a critical heat-sensitive cellular oxidase that contributes to intestinal barrier dysfunction and splanchnic dilation. On the basis of these data, we conclude that splanchnic oxidant generation is an important determinant of heat tolerance and that ·NO is involved in vascular dysfunction with heat stroke.
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ACKNOWLEDGEMENTS |
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The authors acknowledge the expert secretarial assistance of Joan Seye.
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FOOTNOTES |
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Deceased 3 June 2000.
This work was supported by NIH Grants CA-66081, CA-81090, and AG-12350 (to G. R. Buettner and L. W. Oberley), HL-61389 (to C. V. Gisolfi), and T32 AG-00214 (Interdisciplinary Research Training Program on Aging Fellowship; to D. M. Hall).
Address for reprint requests and other correspondence: D. M. Hall, Dept. of Exercise Science, Field House, Univ. of Iowa, Iowa City, IA 52242 (E-mail: dmhall{at}kirkwood.cc.ia.us).
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 4 April 2000; accepted in final form 23 August 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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39.0°C and rapidly increased semiquinone radical
and ceruloplasmin formation (A vs. B,
respectively), suggesting that blocking NOS activity increased metal
activation and cellular oxidative stress. L-NAME treatment
also blunted, but did not eliminate, the rise in heme-·NO and delayed
splanchnic dilation until Tc ~ 42°C. Note that a
strong heme-·NO signal was evident immediately before the rapid rise
in SBF at 41.8°C, consistent with ·NO involvement in splanchnic
dilation. *Significantly different from control, P < 0.05.
