Regulation of the Brain’s Vascular Responses to Oxygen
The mechanism of oxygen-induced cerebral vasoconstriction has been sought for more than a century. Using genetically altered mice to enhance or disrupt extracellular superoxide dismutase (EC-SOD, SOD3), we tested the hypothesis that this enzyme plays a critical role in the physiological response to oxygen in the brain by regulating nitric oxide (NO·) availability. Cerebral blood flow responses in these genetically altered mice to changes in Po2 demonstrate that SOD3 regulates equilibrium between superoxide (·O2−) and NO·, thereby controlling vascular tone and reactivity in the brain. That SOD3 opposes inactivation of NO·is shown by absence of vasoconstriction in response to Po2 in the hyperbaric range in SOD3+/+ mice, whereas NO-dependent relaxation is attenuated in SOD3−/− mutants. Thus, EC-SOD promotes NO·vasodilation by scavenging ·O2− while hyperoxia opposes NO·and promotes constriction by enhancing endogenous ·O2− generation and decreasing basal vasodilator effects of NO·.
Aphysiological role for extracellular superoxide dismutase (EC-SOD, SOD3), which catalyzes superoxide (·O2−) dismutation to hydrogen peroxide and oxygen (O2) extracellularly, has never been demonstrated experimentally.1–5⇓⇓⇓⇓ Because of relatively low content in most tissues, SOD3 was initially presumed not to be an efficient antioxidant enzyme.6 Actually, SOD3 is highly expressed in blood vessels where it constitutes up to 70% of aortic SOD activity.7 SOD3 activity in arterial wall is approximately 10-fold higher than in other tissues and present at roughly the same activity as SOD1.8,9⇓ The SOD3 tetramer contains Cu, Zn, and a positively charged heparin domain linking it to extracellular matrix and cell surfaces.4,8,10⇓⇓ It is located strategically between endothelium and smooth muscle where ·O2− is generated11,12⇓ and dilatory NO·is present.
Endothelial cells release ·O2−,13 which is converted to H2O2 or reacts with NO·to generate the strong oxidant, peroxynitrite (ONOO−).14 By reacting rapidly with NO·, extracellular ·O2− should decrease biologically available NO·, which diffuses from endothelium, erythrocytes, and vascular nerves to smooth muscle. Catalytic removal of ·O2− from this domain should prevent inactivation of NO·by ·O2− and increase its availability for vasodilation. Hence, we have proposed a primary function for EC-SOD in blood vessels to minimize microenvironment concentrations of ·O2− and prevent inactivation of NO·.15 However, the validity of this hypothesis and the actual importance of SOD3 to NO·-mediated control of vascular tone are not known. In vitro studies have reported antagonism of NO·-dependent vasorelaxation after exposure to exogenous ·O2− or chemical inhibition of CuZn-SOD,16–20⇓⇓⇓⇓ but no physiological studies have yet linked SOD3 function to NO·-regulated blood flow in vivo. Genetically altered animals with increased or absent expression of SOD have made it possible to investigate this hypothesis in the brain.
Using mice that overexpress (+/+) or lack (−/−) SOD3, we performed experiments to determine whether the enzyme regulates the balance between ·O2− and NO·in the brain, and how the two radicals affect basal tone and vasoconstrictor responses to increasing O2 concentrations. SOD3+/+ and SOD3−/− mice were compared for blood flow changes in specific brain regions (rCBF) in response to Po2 changes. rCBF was correlated by cerebral microdialysis with NO·release (NOx), and a biochemical marker, 3-nitrotyrosine, to assess ONOO− as a product of SOD3-controlled reactions between ·O2− and NO.
Materials and Methods
The SOD3 mutant strains have been described and characterized.15,21⇓ SOD3+/+ mice were generated by microinjecting nuclei of fertilized oocytes from (C57BL/6) (C3H) F1 female mice with cDNA of human SOD3 containing a β-actin promoter. Mice carrying the transgene were identified by polymerase chain reaction (PCR) amplification of the human SOD3 gene and backcrossed into (C57BL/6×C3H) F1 mice. SOD3 transgenic mice were out-bred to improve fertility. Thus, the background of the SOD3+/+ was not identical to the BL/6 WT strain for null mice (see blood flow methods). Overexpression of EC-SOD in the brains and aortas of the transgenic mice was verified by activity analysis using xanthine/xanthine oxidase and cytochrome c. SOD3+/+ mice had a 5-fold increase in brain and 2-fold in aortic EC-SOD activity. Other antioxidant enzyme levels were not changed. Activity measurements were not done on isolated mouse brain vessels because tissue was insufficient for assay.
The EC-SOD gene was disrupted in Neor-generated SOD3-deficient mice (SOD3−/−). Null mice were bred into WT C57BL/6 mice to yield heterozygotes, which were crossed to produce homozygous knockout and WT breeding pairs. Control mice were derived from WT pairs, whereas SOD3 deficient mice were derived from knockout breeding pairs. The genetic identity of breeders was identified by PCR using unique primers for SOD3 and Neor. Homozygote SOD3 mice have no brain SOD3 but normal activities of other SOD isozymes. SOD3−/− mice have a normal phenotype.
The Institutional Animal Care and Use Committee approved the protocol. At 10 to 12 weeks of age, mice were anesthetized with urethane (800 mg/kg, IP) followed by continuous infusion of 1/4 initial dose/hour by IP pump. Anesthetized mice underwent tracheotomy and ventilation (tidal volume=0.4 mL, rate 110/min) with 30% O2 (balance N2) and paralysis (pancuronium 0.1 mg/kg, IV). The head was fixed in a stereotaxic frame, and hydrogen/oxygen-sensitive microelectrodes inserted through a burr hole into substantia nigra (SN), caudate putamen (CP), hippocampus (HC), and/or parietal cortex (PC). Electrodes were made of platinum wire of 100-μm diameter insulated with epoxy except 1.0 mm at the pointed tip (15 to 20 μm) covered by Nafion to prevent fouling. In some mice, a microdialysis probe (CMA/10: 2-mm membrane, 0.24-mm OD, CMA/AB, Sweden) was placed into CP and perfused with artificial CSF at 2 μL/min using a microinjection pump. For EEG recording, stainless steel screws were inserted into the skull symmetrically over parietal cortex.
Regional Cerebral Blood Flow and Brain Po2
Regional cerebral blood flow (rCBF) was measured by a H2 clearance method.22 Platinum electrodes were inserted into brain and reference Ag-AgCl electrodes attached to the base of the tail. H2 (2.5%) was added via the respirator for 60 seconds then switched to the base breathing gas and the H2-washout curve recorded. Absolute values of CBF (mL · g−1 · min−1) were calculated by initial slope using a WINDAQ data system (DATAQ Inst) and Mathematica 3.0 software (Wolfrom Research, Inc) with minor modification.22 CBF data from the two WT strains, C57/BL6 (n=6) and C57BL6/C3H F1 (n=9), were compared for baseline flow and responses to HBO2, CO2, or L-NAME. There were no significant differences in the mean values; therefore, nontransgenic C57BL6/C3H F1 littermates were used as WT unless indicated otherwise.
Brain Po2 during HBO2 exposures was measured continuously in CP of rats using the same microelectrodes as for rCBF measurements. Po2 electrode calibration was performed with 100% N2 and 20% O2 at 1 ATA and 100% O2 at 1 ATA and 5 ATA. Absolute values for brain Po2 were calculated from a calibration curve for each electrode.
Measurement of Interstitial NO·and Peroxynitrite
Extracellular NO·was estimated from the microdialysis yield of nitrite and nitrate (NOx).23 After system equilibration, dialysate was collected every 15 minutes before and during HBO2. Samples were assayed for NOx after catalytic reduction to NO gas and chemiluminescence detection. Nitrite and nitrate signals, quantitatively linear between 25 and 500 pmol/25 μL, were used for calibrations. Recovery efficiency for the dialysis probes were 31±6% and 29±8% for NO2 and NO3, respectively. The data were expressed as [μmol/L] nitrite and nitrate (NOx). The amount of 3-nitrotyrosine (3-NTyr) in dialysate was measured relative to tyrosine by HPLC and electrochemical detection in timed samples and retention time compared with authentic standards. 3-NTyr values were expressed as 3-NTyr/Tyr ×100.
Hyperbaric O2 Exposures
Hyperbaric O2 (HBO2) exposures was used to maximize O2-induced vasoconstriction in the brain, which in mice occurs at 4 to 5 atmospheres absolute (ATA). Mice were instrumented and placed in a chamber containing the respirator, EEG amplifier, pressure transducer and amplifier, heating pad, and microdialysis system. After placing electrodes in the brain, the animals were recovered for 60 minutes. For microdialysis studies, the recovery period was 2 hours. Once the mice were stable and blood gases and temperature in the physiological range, three control H2 clearance curves were recorded and three dialysate samples collected. The respirator was connected to 100% O2 and the chamber compressed to a maximum pressure of 5 ATA at 0.6 ATA per min. During HBO2, rCBF was measured every 15 minutes using a remotely operated pneumatic system for H2 delivery. Brain Po2, EEG and blood pressure were monitored continuously. Body temperature was maintained between 36° and 37°C. Dialysate was collected every 15 minutes and NOx and 3-NTyr measured later. Arterial blood was taken at 1 ATA to determine Pao2, Paco2, and pH while the mice breathed 100% O2.
The hypothesis was tested first by evaluating differences in basal rCBF and responses to pharmacological agents known to alter brain NO·and ·O2− availability. These studies were conducted in mice breathing 30% O2 (balance N2) at sea level. Then, time-dependent measurements of rCBF, EEG, and physiological variables were made in mice exposed to HBO2 at 3 and 5 ATA. Timed accumulation of NOx and 3-NTyr was correlated with rCBF responses and EEG activity.
Group values are given as mean±SE. Group sizes for mutant strains were 6 to 9 animals each. Changes in rCBF and physiological variables were compared using ANOVA followed by post hoc comparison (Fisher’s exact test) or unpaired t test using computer software. A value of P<0.05 was accepted as significant.
Modulation of Basal CBF by EC-SOD
Using hydrogen clearance, rCBF was measured in WT, SOD3−/−, or SOD3+/+ mice before and after interventions designed to unbalance the equilibrium between ·O2− and NO·. Resting rCBF in WT and mutant strains were similar in the same brain regions, but lower absolute values were observed in SOD3−/− than SOD3+/+ mice in CP and HC (Table 1). CBF responses were statistically identical in the inbred and outbred WT mice (data not shown); therefore, these values were pooled into one control group of 14 animals. To determine whether SOD3 modulates NO-mediated basal tone, we measured rCBF after treating mice with NOS inhibitor, L-NAME to block endothelial and neuronal NO synthesis. rCBF was reduced significantly in an l-arginine–dependent manner in all groups of L-NAME–treated mice, but SOD3+/+ mice showed larger decreases in rCBF in selected brain regions (Table 1). These data indicate that basal tone in brain vessels depends on both NO availability and SOD3 activity.
To confirm this assertion we measured changes in rCBF after infusing mice intravenously with a high concentration of SOD1 (10 000 U/kg body weight) to reduce [·O2−] at the endothelial surface. Preliminary studies showed that active but not inactive SOD1 produced dose- and time-dependent increases in rCBF in WT mice: rCBF responses were reproducible and statistically significant after SOD1 infusion and reached maximal amplitude within 10 minutes (Figures 1a and 1b). This SOD1 dose and time was used for rCBF measurement in mutant mice. In mice treated with SOD1, rCBF in SN, CP, and PC increased significantly in WT, SOD3−/−, and SOD3+/+ mice, but significantly less in SOD3+/+ than SOD3−/− mice (Figure 1c). Because these increases in rCBF after SOD1 treatment could reflect either an increase in NO·availability in vessels due to ·O2− removal or a dilator effect of the enzyme, we tested these possibilities by treating the mice with L-NAME to inhibit endothelial and neuronal NOS. Infusion of SOD1 into WT and mutant mice pretreated with L-NAME had no effect on rCBF (Figure 1d), suggesting SOD1 altered rCBF by preventing ·O2−-mediated inactivation of NO·. SOD3−/− mice have higher extracellular levels of ·O2− compared with SOD3+/+ mice, thus SOD1 infusion has less influence on the equilibrium between ·O2− and NO·in SOD3+/+ animals.
Functional Implications of EC-SOD in Endothelium-Derived Relaxation
Endothelium-derived relaxation is a physiological response of vessels to stimuli that activate NO·signals.24,25⇓ If ·O2− opposes endothelium-derived relaxation, then NO-mediated cerebrovascular responses will depend critically on vascular SOD3 activity. To determine the extent to which SOD3 contributes to endothelium-derived vasodilation, we compared rCBF responses in SOD3+/+ and SOD3−/− mice to an NO donor and to other stimuli that operate via NO-dependent mechanisms. Representative data are shown in Figure 2.
The NO donor PAPA NONOate (at ≥0.1 mg/kg) increased rCBF in WT and both genetic strains of mice, but rCBF responses in SOD3+/+ mutant were more pronounced and significantly greater than that of SOD3−/− mice (representative data in Figure 2a). At higher NONOate concentrations, SOD−/− mice also were found to have attenuated CBF responses relative to the WT, but these infusions caused hypotension in SOD+/+ mice (data not shown).
The role of EC-SOD in vascular CO2 reactivity was examined by measuring the rCBF responses in WT and mutant mice to inhaled CO2. The use of 5% CO2 produced rCBF increases on the flat part of the CO2 response curve and relatively rapid reversal of the CBF effect allowed multiple interventions per animal (Figure 2b). WT and both genetically altered mouse strains showed rCBF increases of 17% to 87% to 5% CO2 relative to baseline, but the greatest increases (in striatum) were observed in SOD3+/+ mice. This response was greater than that of SOD3−/− mutants (Figure 2b). Increases in rCBF to inhaled CO2 were attenuated significantly and to approximately the same extent in WT and both mutant strains pretreated with L-NAME (data not shown). In mice given acetylcholine, arterial blood pressure decreased slightly and transiently for ≈5 minutes followed by a peak rCBF response within 10 minutes. The peak response was augmented significantly when a constant dose of acetylcholine (0.05 mg/kg) was administered to SOD3+/+ mice, whereas it was attenuated in SOD3−/− mice (Figure 2c). WT controls had similar responses to acetylcholine (not shown). NOS inhibitor, L-NAME completely blocked the rCBF responses to acetylcholine (Figure 2d). The data of Figure 2 collectively support the interpretation that EC-SOD in mouse brain modulates NO-dependent vasodilation via ·O2−-dependent regulation of NO availability.
Role of EC-SOD in Regulating Cerebrovascular Responses to Hyperoxia
We assessed regulation of the vasoconstrictor response to O2 by SOD3 by exposing mice to O2 during HBO2 to increase brain ·O2− production.26 We report here rCBF in WT and mutant mice exposed to HBO2 that showed stable, normal arterial BP, Pco2, and pH values for 60 minutes at 3 and 5 atmospheres absolute (ATA) (Tables 1 and 2⇓).
HBO2 altered rCBF in an O2 dose- and time-dependent manner unrelated to the change in hydrostatic pressure per se (data not shown). Mean blood pressure in mice at 3 or 5 ATA rose during the first few minutes of HBO2 exposure and remained elevated until decompression. At 3 ATA, EEG discharges of HBO2 toxicity were not apparent. At 5 ATA, HBO2 produced marked differences in EEG pattern in all strains consisting of paroxysmal EEG discharges of single or multiple spiking activity of amplitude >100 μV. These changes were observed after HBO2 exposures of 40±4 minutes in SOD3+/+ mice, 49±5 minutes in SOD3−/− mice, and 56±6 minutes in WT mice consistent with the enhanced susceptibility reported for both mutant strains (Figure 3a).
At 3 ATA, the rCBF flow in cortex and deep brain structures decreased significantly over 60 minutes of HBO2 in WT and SOD3−/− mice. At 30 to 45 minutes, vasoconstriction reached maximum where it was maintained until decompression (Figure 3b). In contrast, rCBF responses in SOD3+/+ mice to HBO2 were minimal and not statistically different from preexposure levels (Figure 3b). At 5 ATA, HBO2 decreased rCBF in WT and SOD3−/− mice over the initial 30 minutes, but then rCBF gradually rose. By 45 minutes, rCBF had returned to preexposure levels and over the next 15 minutes increased above baseline (Figure 3c). This secondary increase in rCBF always preceded abnormal EEG discharges. SOD3+/+ mice did not show significant cerebral vasoconstriction at 5 ATA of HBO2, and within 30 minutes rCBF had increased significantly. After 60 minutes of HBO2, rCBF had increased 57% above control values (Figure 3c).
Po2 values in the striatum of WT mice and SOD3 mutant strains breathing 30% O2 at 1 ATA ranged from 24 to 45 mm Hg. During compression, Po2 progressively rose and after one minute at 5 ATA ranged from 497 to 738 mm Hg. During HBO2 exposure, Po2 increased gradually and immediately before EEG spikes reached values of 1276 to 2197 mm Hg. No significant differences in Po2 responses to HBO2 were observed among strains of mice.
The rCBF responses to HBO2 in these mice collectively are consistent with the SOD3 mechanism of ·O2−-dependent regulation of NO availability. This was confirmed by measuring the effects of HBO2 on CBF after treating mice with L-NAME. L-NAME decreased rCBF equally in all strains of mice breathing air (data not shown). During exposure to HBO2 at 5 ATA, rCBF remained at or near the control level for air-breathing mice given L-NAME (Figure 3d) throughout 60 minutes exposures. Abnormal EEG discharges were not observed in L-NAME–treated mice exposed to HBO2 at 5 ATA for 60 minutes.
EC-SOD Regulates Interstitial NO· and ·O2− Equilibrium in the Brain
Intracerebral microdialysis experiments revealed dynamic changes in equilibrium between NO·and ·O2− in brain extracellular fluid as a function of EC-SOD expression. We measured interstitial NOx to assess NO·release and 3-NTyr to assess ONOO− formation in mice at 5 ATA. Basal interstitial NOx in the striatum of WT mice was stable at 3.9±0.4 μmol/L, in SOD3−/− and SOD+/+ mice these values were 3.8±0.5 μmol/L and 4.3±0.6 μmol/L, respectively. Within 30 minutes at 5 ATA, brain interstitial NOx decreased significantly. Thereafter, NOx gradually increased to above control levels (Figure 4a). The recovery of NOx in brain interstitial fluid at 5 ATA was remarkably different in SOD3+/+ and SOD3−/− mice. In SOD+/+ mice, NOx levels gradually rose during 60 minutes of HBO2 (Figure 4a). In SOD3−/− mice, NOx decreased significantly over 30 minutes at 5 ATA and then stabilized (Figure 4a) indicating HBO2-related differences in NO varied with EC-SOD expression in the different strains.
Brain interstitial 3-NTyr content, unlike NOx, accumulated steadily at different rates during HBO2 in WT and SOD3 mutant mice exposed to 5 ATA (Figure 4b). Marked increases in 3-NTyr were detected in WT and SOD3−/− mice at 5 ATA consistent with an NO-·O2−-dependent mechanism of ONOO− formation. This was confirmed by less accumulation of 3-NTyr in striatum of mice pretreated before HBO2 with L-NAME. Thus, HBO2-mediated increases in ONOO− production at 5 ATA were blocked by NOS inhibition to a similar degree in both mutants (Figure 4c).
These physiological measurements in genetically altered mice provide definite evidence that SOD3 regulates rCBF in vivo by controlling the equilibrium between NO and ·O2− in the extracellular brain space. Furthermore, SOD3 regulates both basal tone and NO-mediated rCBF responses during increases in Po2. The different rCBF responses to raising the Po2 of mice that overexpress or lack SOD3 supports the central hypothesis of the study: oxygen alters the equilibrium between ·O2− and NO·, which regulates vessel tone and blood flow. Enhanced ·O2− scavenging by SOD3 augments the relaxing effect of NO·on vascular smooth muscle. Because NO·is a central regulator of vascular tone, extracellular ·O2− production functionally reduces the influence of eNOS on guanylate cyclase in smooth muscle. As a major vascular sink for extracellular ·O2−, SOD3 activity regulates the vasodilator effect of NO on cerebral vessels. Evidence for this premise derives from differences in basal rCBF lacking in WT, SOD3+/+, and SOD−/− mice brought forth by interventions to alter the equilibrium between NO·and ·O2−. These interventions yield the predicted differences in rCBF response in the mutants. SOD3+/+ mice treated with an NO donor show more pronounced rCBF responses than in SOD3−/− mutants, which is consistent with less ·O2− bioactivity in cerebral vessels of SOD3+/+ and a lower tendency for NO· inactivation. Thus, we surmise that SOD3−/− mice have higher steady-state vascular ·O2− levels, which allow a more substantial increase in the NO·effect when SOD1 is administered.
One difference between this study and several earlier ones is that basal rCBF in these mice responded to active and not inactivated SOD1. This response could be related in part to blood brain barrier (BBB) damage by electrode insertion, which allowed SOD1 entry into parenchyma. However, ·O2− and NO·flux are regulated on both sides of a thin endothelium, and a large sink for ·O2− within vessels might well increase NO·flux on the other side of the barrier. NO released by RBCs also might have a greater dilator effect if ·O2− scavenging were increased. Notably, rCBF responses to SOD1 were abolished completely in mice pretreated with L-NAME, suggesting the vasoactivity of SOD1 was NO-dependent and did not require BBB damage. In any event, rCBF response to SOD1 infusion in SOD3−/− mice was significantly greater than in SOD3+/+ animals.
A consistent finding in mice exposed to HBO2 is that seizure onset is accelerated in SOD3+/+ mutants,15 indicated here by abnormal EEG activity. Although this conflicts with predictions that antioxidant enzymes protect against O2 toxicity, SOD3−/− mice are also more sensitive to CNS O2 toxicity than WT animals. When inactivation of NO by ·O2− is circumvented in SOD3+/+ mice, rCBF is higher at high Po2, which appears to contribute to sensitivity to O2 toxicity. HBO2 also alters recovery of NOx from the brain in accordance with “escape” of rCBF from vasoconstriction before the EEG discharges. This escape is prevented by NOS inhibition in the different mice, as well as in rats in an l-arginine–dependent manner.22 The genetic changes in EC-SOD expression in the mice provide evidence that O2 initially alters brain NO in accordance with this concept as opposed to stimulating its production by providing substrate. Indeed, WT and SOD3−/− mice first show less interstitial NOx in HBO2 at 5 ATA followed by an increase in NOx to above control. By comparison, SOD3+/+ mice consistently show elevated brain NOx levels in HBO2 at 5 ATA. The directional changes in NO metabolites are appropriate to the O2-dependent rCBF responses, and these measurements are the first in vivo evidence that O2 causes cerebral vasoconstriction by interfering with NO·.
Oxygen may inactivate NO·because, in addition to heme and thiol interactions, NO residence time shows dependence on Po2 and rate of ·O2− generation.27–29⇓⇓ At the onset of HBO2, hemoglobin and thiol pools are stable while brain Po2 and ·O2− production rate increase in proportion to Po2.26 If [O2] greatly exceeds [NO·], NO2 and NO3 are produced by autoxidation of NO·.30 If autoxidation had been a major factor in NOx formation, high instead of low NOx would have been expected. On the other hand, the transient decrease in NOx is most readily explained by a decrease in extracellular NO·from rapid inactivation by ·O2−. This effect is also qualitatively independent of any effect of CBF on recovery of the compounds by dialysis because decreases in CBF tend to increase NOx recovery and vice versa.
NO·and ·O2− also produce ONOO− in a nearly diffusion limited reaction, and at steady ·O2− flux, ONOO− is formed almost as rapidly as NO·is released.31 This chemistry predicts NO activity is regulated directly by ·O2− rather than by independent and opposite effects of NO and ·O2− on tone. The lack of difference in O2-dependent vasoconstriction between WT and EC-SOD−/− mice and the lack of a greater fall in rCBF in HBO2 after L-NAME in EC-SOD−/− mice support this concept because HBO2 generates ·O2−. It is not possible to assess the reaction of NO·and ·O2− directly in brain, but the initial decline in NOx with HBO2 suggests NO·inactivation by increased ·O2− production.26 HBO2-mediated decreases in rCBF were not observed in SOD3+/+ mice, which together with increased NOx and decreased 3-NT are compatible with SOD3 opposition of NO·inactivation.
In WT mice at 5 ATA, the initial decrease in interstitial NOx was followed by an increase to above control in concert with increases in rCBF. This observation suggests that prolonged HBO2 exposure promotes NO·production, which augments CBF.22 Because NOS requires O2 it is possible that [O2] is limiting,32 and HBO2 stimulates NOS activity by increasing cerebral Po2. HBO2 also elevates brain l-arginine, the substrate for NOS.33 l-arginine synthesis is stimulated by O2-dependent catecholamine deamination, which produces H2O2 and ammonia. Ammonia is detoxified by transamination of glutamate to glutamine, which generates l-arginine.33 Finally, high Po2 increases sympathetic and metabolic activity in the preconvulsive period34 and calcium/calmodulin-dependent neuronal NOS is stimulated by postsynaptic receptor activation. These data, however, do not discriminate among these possibilities.
In summary, O2 vasoconstriction defines a physiological role for SOD3 in regulating CBF in vivo. SOD3 is present in high concentration in vessels where NO·is important for vascular relaxation. Inactivation by ·O2− interferes with NO-dependent basal tone and vasorelaxation. Scavenging of ·O2− by EC-SOD plays a critical role in NO·CBF function as shown by predictable differences in rCBF response to high Po2 in mice with varying enzyme expression. These findings have implications in vascular diseases where extracellular ·O2− production exceeds SOD3 function, diminishing responsiveness to NO·, and allowing production of oxidative/nitrosative stress. The strategic location of vascular SOD3 in the brain between endothelium and smooth muscle preserves and regulates the endogenous dilator function of NO·.
This work was supported by grants from NHLBI and the Office of Naval Research.
Original received April 17, 2002; revision received October 15, 2002; accepted October 15, 2002.
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