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Am J Physiol Heart Circ Physiol 280: H2023-H2029, 2001;
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Vol. 280, Issue 5, H2023-H2029, May 2001

COX-2-dependent delayed dilatation of cerebral arterioles in response to bradykinin

Johnny E. Brian Jr.1, Frank M. Faraci2
Steven A. Moore3
(With the Technical Assistance of Paula Ludwig)

Departments of 1 Anesthesia, 2 Internal Medicine and Pharmacology, and 3 Pathology, University of Iowa College of Medicine, Iowa City, Iowa 52242


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Bradykinin (BK) is released in the brain during injury and inflammation. Activation of endothelial BK receptors produces acute dilatation of cerebral arterioles that is mediated by reactive oxygen species (ROS). ROS can also modulate gene expression, including expression of the inducible isoform of cyclooxygenase (COX-2). We hypothesized that exposure of the brain to BK would produce acute dilatation, which would be followed by a delayed dilatation mediated by COX-2. To test this hypothesis in anesthetized rats, BK was placed twice in cranial windows for 7 min, after which the windows were flushed to remove residual BK. The two BK exposures were separated by 30 min. Each BK exposure produced acute dilatation of cerebral arterioles, after which diameter rapidly returned to baseline. Over the subsequent 4.5 h after the second BK exposure, arterioles dilated 48 ± 8%. Treatment of the cranial window with NS-398, a selective COX-2 inhibitor, or dexamethasone, significantly attenuated the delayed dilatation. Aminoguanidine, a selective inhibitor of inducible nitric oxide synthase, did not alter the delayed dilatation. Cotreatment of cranial windows with BK, superoxide dismutase, and catalase also prevented the delayed dilatation. In separate experiments, exposure of the cortical surface to BK upregulated leptomeningeal expression of COX-2 mRNA. Our results suggest that acute, time-limited exposure of the brain to BK produces delayed dilatation of cerebral arterioles dependent on expression and activity of COX-2.

inflammation; NS-398; reactive oxygen species; cyclooxygenase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

BRADYKININ , a member of the kinin family of inflammatory mediators, is produced by cleavage from a precursor peptide kininogen. Bradykinin produces acute, reversible dilatation of peripheral and cerebral blood vessels. In the peripheral circulation, bradykinin produces vascular dilation by endothelial-dependent release of nitric oxide (NO), a hyperpolarizing factor, and/or prostaglandins (25). In cerebral arterioles, bradykinin produces acute, reversible dilatation dependent on activation of bradykinin type 2 (B2) receptors (29, 43). In contrast to the peripheral circulation, bradykinin produces acute endothelial-dependent dilatation of pial arterioles mediated by reactive oxygen species (ROS) (20, 21). Various ROS species have been suggested to mediate this vascular response, including superoxide (20), H2O2 (38), and hydroxyl radical (21).

Bradykinin, kininogen, and related compounds are expressed in the brain, where bradykinin is present in some nerve fibers (12, 44). During brain injury, bradykinin is produced and contributes to cerebrovasodilatation. After brain freeze lesion, interstitial kinin concentration increases to between 10-7 and 10-6 M (42). Spinal cord kinin concentration remains elevated for several hours after spinal cord trauma (46). Blockade of bradykinin receptors attenuates dilatation of cerebral arterioles during meningitis (26) and after fluid percussion brain injury (15).

Cyclooxygenase (COX) converts arachidonic acid to prostaglandin H2, which is metabolized by subsequent enzymes to produce prostaglandins and thromboxane. COX is expressed as two isoforms, COX-1, which is regarded as the constitutive isoform, and COX-2, the inducible isoform. Bradykinin receptors can activate phospholipase A2, which provides substrate for COX (44). COX-1 most likely produces the ROS responsible for acute bradykinin-induced dilatation of adult cerebral arterioles, because the cerebral endothelium does not express COX-2 under basal conditions (9) and acute bradykinin-induced dilatation is intact in COX-2 knockout mice (32). Although only a subpopulation of neurons express COX-2 under basal conditions, astrocytes, neurons, microglia, vascular, and leptomeningeal cells can upregulate COX-2 expression during inflammatory conditions (5, 6, 8, 31, 33). Increased COX-2 expression is associated with dilatation of cerebral arterioles and increased cerebral blood flow (8, 34).

Although the acute effect of bradykinin on brain blood vessels has been studied, little is known about the delayed effect of bradykinin on the cerebral circulation. In noncerebral cells, superoxide and H2O2 induce expression of COX-2 (16). ROS have been linked to activation of nuclear factor-kappa B (NF-kappa B), which can promote expression of COX-2 (2, 3, 16). Thus because acute exposure of brain to bradykinin produces ROS, we hypothesized that acute, short term exposure of brain to bradykinin would cause COX-2 expression and delayed dilatation of cerebral arterioles hours later in the absence of bradykinin.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cranial windows. The University of Iowa Animal Care and Use Committee approved all experiments. Male Sprague-Dawley rats (n = 51, 342 ± 4 g) were anesthetized with pentobarbital sodium (50 mg/kg ip), a tracheotomy was performed and ventilation was maintained with a small animal ventilator. Arterial PCO2 was adjusted to ~40 mmHg by altering minute ventilation and arterial PO2 maintained >100 mmHg by supplementing room air with oxygen. Anesthesia was supplemented by administration of additional pentobarbital sodium (5-15 mg · kg-1 · h-1) via the femoral vein. Rectal temperature was measured and maintained at 37 ± 0.5°C with a heating pad.

A closed cranial window was prepared as previously described (8). The scalp muscle and periostium overlying the parietal area of the skull were reflected and bleeding was controlled with ferric chloride solution. A craniotomy (~3 × 4 mm) was made in the parietal bone with the use of an air-cooled drill and bone bleeding was controlled with bone wax. The dura overlying an arteriole was incised. Two blunt needles were affixed to a dam of bone wax surrounding the craniotomy, and a circular glass cover slip (12 mm) was fused to the wax. The window was reinforced with dental acrylic. An outlet tube was affixed to one needle and set to maintain intracranial pressure at 10 cmH2O. A stopcock was attached to the other needle, and the cranial window was filled with artificial cerebrospinal fluid (aCSF) warmed to 37°C and equilibrated with 90% N2-5% O2-5% CO2 (pH, 7.34 ± 0.01; PO2, 73 ± 1 mmHg; and PCO2, 41 ± 0.2 mmHg). Cerebral arterioles were observed by using a microscope equipped with a video camera and images recorded on videotape. Arteriolar diameter was measured with a calibrated video micrometer. Only one arteriole is measured per preparation. The preparation was allowed to equilibrate for 30 min, during which time the window was flushed with 2 ml of aCSF every 15 min. The use of aCSF to flush the cranial window did not alter the diameter of cerebral arterioles.

After the equilibration period, arteriolar diameter was measured under control conditions, and in response to topical ADP (10-5 and 10-4 M), was an activator of endothelial NO synthase in rat pial arterioles (28). Responses to ADP were examined to test responsiveness of the preparation. The cranial window was then flushed with aCSF several times, and the preparation was allowed to recover for 30 min. After a second measurement of baseline vessel diameter, the cranial windows were filled with aCFS containing bradykinin 10-5 M, and the response of the cerebral arterioles was measured. Bradykinin produced acute dilatation of arterioles that rapidly dissipated over several minutes and arteriolar diameter returned to baseline. Seven minutes after bradykinin application, the windows were flushed with aCSF to remove any residual bradykinin. Thirty minutes after the first bradykinin application, treatment with bradykinin 10-5 M for 7 min was repeated. Arteriole diameter was observed for 4 h beginning 30 min after the second bradykinin exposure. The total time of the experimental protocol was 5 h; 30 min each for the two bradykinin exposures, followed by 4 h of observation. In the subsequent data presentation, time 0 is the time of the first bradykinin treatment. Diameter of arterioles was measured at 1, 1.5, and 2-5 h. Changes in arteriolar diameter are expressed as percent change in diameter compared with baseline. Arterial blood pressure was continuously monitored, and arterial blood gases were measured at regular intervals.

The concentration of bradykinin placed in cranial windows (10-5 M) was selected on the basis of bradykinin concentration in brain after injury (42). A single exposure of cranial windows to bradykinin (10-5 M) did not produce a delayed dilatation of cerebral arterioles (data not shown). This could be related to the transient half-life of bradykinin in cerebrospinal fluid (seconds) (19). To prolong the exposure of brain to bradykinin, we used a second application of bradykinin. We selected intermittent application because it is simpler than continuous perfusion and allows more control over environmental lipopolysaccharide (LPS), which could synergistically alter the response to bradykinin. To verify that continuous and intermittent exposure produced a similar effect, the cranial windows of separate animals were continuously perfused with bradykinin (10-5 M) for 30 min, which produced a similar delayed dilatation (n = 5, 38 ± 2%).

To identify possible mediators in delayed dilatation occurring after bradykinin exposure, some groups of animals had cranial windows treated with various inhibitors during or after bradykinin exposure. The cranial windows were flushed with inhibitors every 30 min during the observation period. The animals were randomly allocated to one of the following five groups: 1) bradykinin treatment, followed by flushing the cranial windows with aCSF (n = 9), 2) bradykinin treatment, followed by treatment of the cranial windows with NS-398 (100 µM, n = 6), a relatively selective inhibitor of COX-2, 3) bradykinin treatment, followed by flushing cranial windows with aminoguanidine (AG; 300 µM, n = 4), a relatively selective inhibitor of inducible NO synthase, 4) cotreatment with bradykinin and dexamethasone (Dex; 1 µM, n = 5), followed by flushing windows with Dex, and 5) bradykinin treatment with superoxide dismutase (SOD; 100 U/ml) and catalase (Cat; 400 U/ml, n = 6), followed by flushing windows with aCSF. Because Dex and SOD/Cat would potentially suppress COX-2 expression and not enzymatic activity, these compounds were coapplied with bradykinin. In group 4, Dex was present in the windows during the study. In group 5, the antioxidant enzymes were present in the window only when bradykinin was placed in the window. Three control groups were studied. In the first control group (n = 3), windows were flushed only with aCSF for the entire course of the experiment to serve as time controls and demonstrate stability of the preparation. The second control group had an initial dilatation to bradykinin (10-5 M; n = 6), after which the windows were treated with the nonselective COX inhibitor ibuprofen (10-5 M) for 15 min. Dilatation to bradykinin was then remeasured in the presence of ibuprofen. The third control group (n = 6) had ADP-induced dilatation (10-5, 10-4 M) measured under baseline conditions. The windows were then treated with SOD (100 U/ml) and Cat (400 U/ml) for 30 min. Dilatation to ADP (10-5, 10-4 M) was then remeasured in the presence of SOD/Cat.

Intracisternal injection. Initial studies attempted to document bradykinin-induced expression of COX-2 mRNA from brain tissue exposed to bradykinin in cranial windows. However, the amount of available tissue proved insufficient. To expose a larger cortical area to bradykinin and allow sufficient tissue for analysis, separate animals underwent intracisternal injection of bradykinin, LPS, or vehicle (aCSF). For intracisternal injection, rats were anesthetized with pentobarbital sodium (50 mg/kg ip) and treated with atropine (15 µg/kg ip) to inhibit respiratory secretions. During the procedure, the pentobarbital sodium was supplemented as needed (5-15 mg · kg-1 · h-1) to maintain an adequate level of anesthesia. The animals were placed in a stereotactic head frame and the atlantooccipital membrane was exposed through a small incision. The atlantooccipital membrane was punctured with a 27-gauge needle in a stereotactic arm and was confirmed by aspiration of CSF. After slow aspiration of 100 µl of native CSF, 100 µl of aCSF alone (n = 2) or with bradykinin 4 × 10-5 M (n = 2) were injected over 15 min. The dose of bradykinin was chosen to yield an estimated in vivo bradykinin CSF concentration of ~10-5 M, as was used in cranial window experiments. This is on the basis of an estimated rat CSF volume of 400 µl. Thirty minutes later, 100 µl of CSF were withdrawn and a second dose of BK or vehicle injected. After the second injection, animals were maintained under anesthesia for 4.5 h to mimic the time course of the cranial window experiments. Separate animals (n = 2) underwent two intracisternal injections of LPS (40 ng) in 100 µl of aCSF to serve as positive controls. Four and one-half hours after the second injection, the animals were euthanized with an overdose of pentobarbital sodium, the brain was rapidly removed, and leptomeningeal tissue containing pial blood vessels was separated from the cortex by microdissection.

Ribonuclease protection assay. Total RNA from leptomeningeal tissue was isolated with RNA-STAT 60 according to the manufacturer's instructions (Tel-Test). The probes for the ribonuclease (RNase) protection assay were gifts from Iain Campbell of Scripps Research Institute (La Jolla, CA). A murine COX-2 cDNA fragment (nt-205-505; GenBank accession no. M88242) was synthesized by RT-PCR by using mouse brain RNA as a template and was cloned into pGEM4; a fragment of the RPL32-4A gene (L32) was also cloned into pGEM4 and served as an internal loading control (14). The RNase protection assay was performed as previously described (39). For the synthesis of a 32P-radiolabeled anti-sense RNA probe, equimolar mixtures of the linearized COX-2 and L32 templates were used. Hybridization reactions were performed overnight at 56°C. After RNase digestion, the RNA duplexes were isolated by electrophoresis in a standard 7.5% acrylamide, 12 M urea, and 0.5% Tris-based EDTA sequencing gel. Dried gels were placed on BioMax Maximum Resolution film and were exposed at -70°C. Quantitation was performed by densitometry.

Statistics. Data are means ± SE. Data between groups were compared by ANOVA and Duncan's post hoc test. Data within groups were analyzed by repeated measures ANOVA and post hoc comparison by means contrast. P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Physiological variables. Baseline arteriolar diameter was not different between groups and averaged 58 ± 2 µm. Initial dilatations to ADP (10-5, 10-4 M) did not differ between groups (P > 0.05) and averaged 7 ± 1 and 19 ± 1%, respectively. Mean arterial pressure did not vary between groups or across time, averaging 127 ± 1 mmHg (n = 240; P > 0.05). Arterial pH, PO2, and PCO2 also did not vary between groups or across time, averaging 7.37 ± 0.01, 186 ± 5 mmHg, and 39 ± 1 mmHg, respectively (n = 60; P > 0.05).

Acute response to bradykinin. The two acute bradykinin (10-5 M) dilatations did not differ between groups and averaged 88 ± 7 and 68 ± 7%, respectively. After the two bradykinin-induced dilatations, arteriolar diameter returned to values that were not different from baseline (56 ± 2 and 56 ± 2 µm, respectively; P > 0.05).

In a separate group of animals, acute dilatation to bradykinin (10-5 M; 91 ± 10%) was not different from the above groups. Treatment with ibuprofen (10-5 M) significantly attenuated bradykinin-induced dilatation (Fig. 1; P < 0.05).


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  		Fig. 1.   Acute dilatation to bradykinin (BK; 10-5 M) was greatly attenuated by treatment of cranial windows with ibuprofen (IBU; 10 µM). Values are means ± SE. *P < 0.05 compared with BK alone.

Delayed response to bradykinin. When cranial windows were flushed with aCSF after bradykinin exposure, cerebral arterioles progressively dilated, reaching 48 ± 8% by the end of the study (Fig. 2A). In windows treated only with aCSF (no bradykinin exposure), there was no significant change in arteriolar diameter over the course of observation. The delayed increase in arteriolar diameter after bradykinin was significantly different from the aCSF group at hours 3, 4, and 5 (P < 0.05; Fig. 2A). Treatment with NS-398, a relatively selective inhibitor of COX-2, after bradykinin exposure abolished the delayed dilatation (see Fig. 2A). Change in arteriolar diameter in the group treated with NS-398 after bradykinin was not different from aCSF control animals (P > 0.05; Fig. 2A). Treatment with AG, a relatively selective inhibitor of inducible NO synthase, after bradykinin did not attenuate the delayed dilatation (Fig. 2B). The increase in diameter in the group treated with AG was significantly different from the aCSF group at hours 4 and 5 (P < 0.05). Treatment of windows with Dex, which suppresses induction of COX-2 during and after bradykinin exposure, abolished the delayed dilatation (Fig. 2C). Change in arteriolar diameter over time in the Dex group was not different from the aCSF control group (P > 0.05). Treatment of windows with SOD and Cat only during bradykinin exposure also abolished the delayed dilatation (Fig. 2D). Change in arteriolar diameter over time was not different from the aCSF control group (P > 0.05). Treatment of windows with SOD and Cat did not alter dilatation to ADP (Fig. 3; P > 0.05).


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  		Fig. 2.   A: flushing cranial window with artificial cerebrospinal fluid (aCSF) after BK (; n = 9) led to progressive dilatation that was significantly different from the group treated only with aCSF (black-triangle; n = 3) at hours 3-5. Treatment of window with NS-398 (100 µM, ; n = 6) after BK exposure abolished the delayed dilatation. B: treatment of windows with aminoguanidine (black-lozenge ; 300 µM; F; n = 4) after BK exposure did not reduce the delayed dilatation after BK. C: treatment of windows with dexamethasone (1 µM, ; n = 5) during and after BK exposure abolished the delayed dilatation. D: treatment of windows with superoxide dismutase (SOD; 100 U/ml) and catalase (400 U/ml, open circle ; n = 6) only during bradykinin exposure abolished the delayed dilatation. Arrows refer to two BK treatments. The aCSF group is shown in each panel for comparison. Data are means ± SE. *P < 0.05 compared with the aCSF group.



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  		Fig. 3.   Dilatation of cerebral arterioles to ADP, an activator of endothelial nitric oxide (NO) synthase, was not different (P > 0.05) under control conditions (left) or after treatment with SOD (100 U/ml) and catalase (400 U/ml). Data are means ± SE.

Bradykinin-induced COX-2 expression. Two intracisternal injections of bradykinin (estimated in vivo CSF concentration 10-5 M), LPS (estimated in vivo CSF concentration 10 ng/ml), or vehicle (aCSF) were performed 5 and 4.5 h before dissection of leptomeningeal tissue from the surface of brains. Total RNA from the leptomeninges was analyzed by RNase protection assay by using probes for COX-2 (Fig. 4A) and L32 (not shown). Intracisternal injection of bradykinin or LPS increased expression of COX-2 mRNA. Bradykinin increased COX-2 expression by three- to fourfold relative to control, whereas LPS increased COX-2 mRNA by 40- to 50-fold (Fig. 4B).


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  		Fig. 4.   A: autoradiogram of ribonuclease (RNase) protection assay for inducible isoform cyclooxygenase (COX-2) expression in leptomeningeal tissue after intracisternal injection of BK, lipopolysaccharide (LPS), or aCSF. RPL32-4A gene (L32) was used as a control for loading (not shown). B: integrated optical density data expressed as the ratio of COX-2 to L32; units for control and BK are on the left, whereas units for LPS are on the right.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The principal new finding of this study is that acute exposure of brain to bradykinin induces expression of COX-2 mRNA in leptomeningeal tissue, as well as delayed dilatation of cerebral arterioles mediated by COX-2 activity. The delayed dilatation occurred hours after bradykinin exposure and in the absence of bradykinin. Production of ROS during the initial bradykinin exposure appears to be an essential step leading to delayed expression and functional effects of COX-2. These findings support the concept that bradykinin produced during acute brain injury may cause prolonged vascular dysfunction via expression and activity of COX-2.

Bradykinin, precursor peptide, and related enzymes have been identified in brain (10, 12, 44). B2 receptors are located on the cerebral vascular endothelium, and activation of these receptors leads to acute cerebral vasodilatation (29, 43). B2 receptors are linked via G proteins to activation of phospholipase A2, which liberates arachidonic acid (44). Arachidonic acid is metabolized by COX, which under normal conditions in the adult cerebral vascular endothelium appears to be only COX-1 (9, 32). In the current study, acute bradykinin-induced dilatation was greatly attenuated by ibuprofen, a nonselective COX inhibitor, supporting the concept that acute bradykinin-induced dilatation is dependent on COX-1. Previous studies (30) also report that nonselective inhibition of COX will abolish acute bradykinin-induced dilatation of cerebral arterioles. Furthermore, acute bradykinin-induced dilatation of cerebral arterioles is intact in COX-2-deficient mice (32). These data suggest that in the adult cerebral circulation, acute bradykinin-induced dilatation depends on COX-1.

The mechanism of acute bradykinin-induced dilatation has been relatively well studied (21, 27, 38, 43). In large cerebral arteries and in extracerebral vessels, NO, prostaglandins, or an EDHF mediate bradykinin-induced vasodilatation (17, 25, 27). In cerebral arterioles, however, ROS are the mediators for bradykinin-induced vasodilatation (20, 21, 38). Various ROS have been identified in different models, including hydroxyl radical, superoxide, and H2O2 (20, 21, 38). Although bradykinin and related systems are present in the brain, they apparently do not contribute to regulation of basal cerebral vascular tone, because blockade of bradykinin receptors does not alter resting cerebral blood flow (29, 45).

In contrast to normal physiology, bradykinin does appear to contribute to cerebral vasodilatation during pathophysiology. In rabbits with meningitis, blockade of B2 receptors attenuates cerebral vasodilatation, suggesting that active bradykinin is released during meningeal inflammation (26). Fluid-percussion brain trauma in rats causes dilatation of pial arterioles and loss of response to hypocapnia (15). Bradykinin receptor blockade attenuates the vasodilatation and preserves the response to hypocapnia (15). Spinal cord trauma in rats increases tissue kinin concentration (peptides that include bradykinin) 40-fold (46), and the brain freeze lesion increases brain interstitial kinin concentration to between 10-7 and 10-6 M (42).

We found that treatment of cranial windows with NS-398 greatly attenuated the delayed dilatation after bradykinin exposure. NS-398 is a relatively selective inhibitor of COX-2. A study of isolated enzymes indicates that NS-398 (1-300 µM) inhibits COX-2 and not COX-1 (11). In the brain in vivo, NS-398 (10-300 µM) does not reduce the acute increase in cerebral blood flow secondary to activation of COX-1 by bradykinin (32). We reported (8) that several hours of exposure of the brain to NS-398 (100 µM) did not inhibit acute ADP- or bradykinin-induced dilatation of cerebral arterioles. These data suggest that NS-398 does not produce nonspecific vascular effects, because it did not reduce ADP-induced dilatation, which depends on endothelial NO production. These data suggest that NS-398 is specific for COX-2 and not COX-1 in our model, because acute bradykinin-induced dilatation was not attenuated.

COX is a bifunctional enzyme that converts arachidonic acid to prostaglandin G2 (PGG2) via COX activity and then PGG2 to prostaglandin H2 via peroxidase activity. The peroxidase reaction also produces ROS (24). In the adult brain in vivo, activation of constitutive COX, presumably COX-1, with bradykinin or arachidonic acid increases production of superoxide (20). COX-dependent production of superoxide also occurs in endothelial cells stimulated with bradykinin (18). Because H2O2 may be the mediator of acute bradykinin-induced dilatation in rat cerebral arterioles, it is possible that superoxide produced by COX is converted to H2O2 by Cat.

ROS have been implicated in expression of COX-2. In cultured noncerebral cells, H2O2 and superoxide induce expression of COX-2 (16). An important mechanism by which ROS can cause gene expression appears to be activation of NF-kappa B. Activation of NF-kappa B causes nuclear translocation and transcription of mRNA from genes with a NF-kappa B response element. The promoter region of the COX-2 gene contains NF-kappa B response elements (3). H2O2 and superoxide activate NF-kappa B in noncerebral cells in culture (1, 2). In cultured human fibroblasts, bradykinin activates NF-kappa B via G proteins (35), which is consistent with known receptor-effector mechanisms of bradykinin in cerebral blood vessels.

We found that cotreatment with Cat and SOD during bradykinin exposure prevented the delayed dilatation. In our model, SOD and Cat were applied only during the bradykinin exposure. We used both SOD and Cat to maximize scavenging of ROS. Our goal was not to identify specific ROS responsible for COX-2 expression, but rather to implicate a role for ROS in general. SOD and Cat are specific enzymatic scavengers of superoxide and H2O2, respectively, and did not attenuate dilatation to ADP in our model. This strongly suggests that ROS produced in response to the acute application of bradykinin were essential elements leading to the delayed dilatation dependent on COX-2.

In the current study, Dex treatment abolished the delayed dilatation after bradykinin exposure. We reported (8) that Dex suppressed LPS-induced COX-2 expression in leptomeningeal tissue, as well as COX-2-mediated dilatation of cerebral arterioles during LPS exposure. The COX-2 gene lacks a glucocorticoid response element by which Dex could suppress COX-2 expression (3). However, Dex can directly inhibit NF-kappa B and suppress COX-2 expression (4). Dex can also suppress COX-2 expression in cultured cerebral vascular smooth muscle and in cultured human airway smooth muscle (36, 37).

Dex appears to have minimal, if any, direct vascular effects. Brian and Faraci (7) reported that several hours of exposure of cerebral arterioles to Dex does not alter resting diameter or response of cerebral arterioles to ADP, an activator of endothelial NO synthase. Others (40, 41) have reported that Dex does not alter constrictor or dilator response of cerebral arterioles. More importantly, Dex does not alter the enzymatic activity of COX-1 (23). Together, these data suggest that Dex suppressed expression of COX-2 in the current study.

Other studies support our hypothesis that bradykinin causes delayed vascular dysfunction via COX-2. Exposure of cerebral arterioles to arachidonic acid for 15 min caused a progressive, delayed dilatation in vivo (22). The delayed dilatation could be prevented by treatment with indomethacin, a nonselective COX inhibitor (22). Kontos et al. (22) assumed that the delayed dilatation after arachidonic acid exposure was due to "damage" from ROS. Although not studied, their data are consistent with our hypothesis that bradykinin-induced release of arachidonic acid activates COX-1, producing ROS that induce expression of COX-2 with subsequent dilatation of cerebral arterioles. Our hypothesis is also consistent with data from a study of the newborn cerebral circulation, where ischemia-induced expression of COX-2 was inhibited by indomethacin (13). This suggests that activation of constitutive COX was necessary for COX-2 expression. Bradykinin-mediated activation of COX-1 with subsequent expression and activity of COX-2 has also been demonstrated in cultured airway smooth muscle cells (36). In these cells, bradykinin-induced expression of COX-2 could be blocked with Dex, indomethacin, and Hoechst-140 (a B2 receptor antagonist), which suggests that COX-2 expression was dependent on activation of B2 receptors and COX-1 (36).

Our data support the concept that acute, short-term exposure of cerebral arterioles to bradykinin leads to delayed vasodilatation. On the basis of current and previous data, we suggest that bradykinin activates cerebral vascular endothelial B2 receptors, which are linked to phospholipase A2. This liberates arachidonic acid, which serves as substrate for COX-1, producing ROS. ROS produce acute dilatation as well as activate NF-kappa B, leading to expression of COX-2 and delayed vasodilatation (Fig. 5). The concept that acute vasodilator mechanisms may lead to delayed effects on the cerebral circulation is a new concept with potentially important implications for pathophysiology in the cerebral circulation.


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  		Fig. 5.   Diagrammatic representation of the signaling cascade leading from BK to COX-2 expression and delayed dilatation of cerebral blood vessels.


    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants NS-24621 and HL-38901, by American Heart Association Grant-in-Aid 96-50661N (to S. A. Moore), and by research funds from the Department of Anesthesia.


    FOOTNOTES

Address for reprint requests and other correspondence: J. E. Brian Jr., Dept. of Anesthesia 6 JCP, Univ. of Iowa College of Medicine, Iowa City, IA 52242 (E-mail: eddie-brian{at}uiowa.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8 September 2000; accepted in final form 27 December 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 280(5):H2023-H2029
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