601 Hydroxyl Radical Mediates the Endothel ium-Dependent Relaxation Produced by Bradykinin in Mouse Cerebral Arterioles William I. Rosenblum Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017 Bradykinin relaxes arterioles on the brain's surface. This response is endothelium-dependent. The data presented here confirm the hypothesis that hydroxyl free radical mediates this response and may be the endothelium-dependent relaxing factor for bradykinin in this microvascular bed. The response to a locally applied bolus of bradykinin (80 ng/mY) was monitored by intravital TV microscopy. The response was significantly inhibited or totally blocked by the presence of superoxide dismutase 60U/ml, catalase 46U/ml, or deferoxamine 0.1 or 0.2 mM. The superoxide dismutase scavenges superoxide radical, which is known to enter the subarachnoid space as a consequence of cyclooxygenase activation. Cyclooxygenase is activated by bradykinin. The superoxide can form H2O2, scavenged by catalase, and the two together generate hydroxyl. The formation of hydroxyl radical is catalyzed by iron. Deferoxamine O.lmM scavenges the iron, blocking the generation of hydroxyl. Deferoxamine 0.2mM also directly scavenges the hydroxyl. None of the pharmacologic probes had an effect on arteriolar diameter when locally applied without bradykinin. Since the dilation produced by bradykinin was inhibited or totally blocked by probes that prevented hydroxyl formation or directly scavenged hydroxyl radical, that radical is either an essential mediator of the arteriolar relaxation, or is the endothelium-dependent relaxing factor for bradykinin in pial arterioles. (Circulation Research 1987;61:601-603) ecently,12 endothelium-dependent relaxation has been demonstrated in the microcirculation in vivo. This demonstration depended on the selective elimination of the relaxation produced by acetylcholine and bradykinin, following injury of endothelium in situ, and the vessels were arterioles on the surface of the brain (pial arterioles). In pial arterioles, the relaxations produced by acetylcholine and bradykinin have thus far been shown to have different mediators. The response to acetylcholine is eliminated by an oxygen-centered free radical and restored by radical scavengers. In contrast, the response to bradykinin is eliminated by catalase and superoxide dismutase. w The inhibitory effect of scavengers on the response to bradykinin, suggested that the hydroxyl radical was either the endothelium-dependent relaxing factor (EDRF) for bradykinin or that the production or action of that EDRF was dependent on the hydroxyl radical.3 The suggestion that free radicals modulate endothelium-dependent relaxation or other physiologic vascular responses is relatively new and important.36'7 Oxygen-centered radicals are generally considered to be destructive rather than essential for the action of vasoactive agents. Consequently, it is essential to replicate the report that an oxygen-centered radical is involved in the response to bradykinin.3 The following R From the Department of Pathology (Neuropathology), Medical College of Virginia, Virginia Commonwealth University, Richmond, Va. Supported by National Institutes of Health gTant HL 35935. Address for correspondence and reprints: William I. Rosenblum, MD, Neuropathology, Medical College of Virginia, Box 17, MCV Station, Richmond, VA 23298 Received October 27, 1986; accepted May 8, 1987. data confirm that report by extending the findings to a different species and uses an additional pharmacologic probe that implicates the hydroxyl radical as the essential mediator. Materials and Methods Male mice (ICR strain, Dominion Labs, Va.), were anesthetized with urethane and the pial vessels exposed by craniotomy as previously described.8"10 Body temperature was maintained at 37° C. The cerebral surface was continuously suffused at 1 ml/minute with artificial cerebrospinal fluid at pH 7 . 3 5 . ' " An arteriole 30-50 fj.m in diameter was arbitrarily selected for observation through a Leitz microscope. Whenever possible, a segment exactly 35 fxm in diameter was used because it simplified calibration of the chart paper in the recorder used to monitor the diameters. This recorder was linked to a TV image splitter, which in turn sheared the image of the vessel on a TV camera as described by Baez.12 The drugs used were administered topically at pH 7.35 in a bolus of artificial cerebrospinal fluid. The duration of the bolus was 60 seconds. The diameter was monitored continuously before, during, and for 5 minutes after the bolus was delivered. The maximal change in diameter produced by the bolus was used to calculate a drug's effect. This change was expressed as a percent of baseline diameter. Thus a 10% dilation was expressed as 110% of baseline. The drugs used were bradykinin triacetate (Sigma Chemical Co., St. Louis, Mo.); acetylcholine chloride (Sigma); superoxide dismutase (SOD), 2,000 U/mg (Sigma); catalase 11,500 U/mg, thymol free (Sigma); deferoxamine mesylate (Desferal, Ciba, Medfield, Mass.). All stock solutions were freshly made in deionized water on the day of use and kept on ice. Final 602 Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017 dilution was made by adding no more than 5 /LX.1 to 1 ml of artificial CSF. Preliminary experiments confirmed the observation3 that heat inactivated SOD and catalase had no effect on the response to acetylcholine or bradykinin. Thereafter, experiments were performed as described below. Four separate experiments were performed with bradykinin triacetate, 80 /ig/ml. This dose was selected after experiments showing dose dependent reduction of responses at lower doses. In each experiment responses to bradykinin triacetate plus scavenger vehicle (i.e., 5 /xl or less of deionized water) were compared with responses to bradykinin triacetate plus scavenger, mixed together in the 1 ml bolus of artificial CSF. The responses were obtained 15 minutes apart to allow for complete washoff of the bolus and restoration of baseline diameter. Thus, each mouse served as its own control, permitting analysis of the results by paired t test after log transformation of the data to normalize the distribution. Fifteen minutes after the final test of bradykinin's action, the scavenger in question was tested by itself. The first experiment tested the effect of 60 U/ml SOD on the response to bradykinin in 10 mice. SOD is a scavenger of superoxide radical, 3 and the dose chosen was used by Kontos et al to inhibit bradykinin. The second study tested the effect of 46 U/ml catalase in 10 mice. Catalase destroys hydrogen peroxide and the dose was similar to that used by Kontos et al to inhibit bradykinin. Superoxide plus hydrogen peroxide can generate the hydroxyl radical.13 This reaction is catalyzed by free iron in amounts known to exist in the cerebrospinal fluid bathing the pial vessels. Therefore, a third set of experiments was performed on 10 mice, using deferoxamine, a chelator of this free iron. High concentrations of deferoxamine not only inhibit formation of hydroxyl radical but also directly scavenge hydroxyl. Therefore, the fourth experiment employed a higher concentration of deferoxamine in 5 mice. After these experiments were completed the effect of SOD on the relaxation produced by acetylcholine chloride (80 /Ag/ml) was also tested. At the end of each experiment blood gas determinations were made from 100 /AI of blood from the carotid artery. These values were similar from study to study. The mean values ( ± SD) were as follows and will not be referred to again: CO2, 36 ± 3 mm Hg; O2, 106± 10 mm Hg; pH 7.34±0.04. Results The results are shown in Table 1. Bradykinin triacetate, 80 /xg/ml, produced relaxation that was significantly inhibited by each of the tested drugs, SOD, catalase, deferoxamine (at 2 dose levels). The inhibition was most complete by SOD or 2 mM deferoxamine. Indeed, some small constrictions were seen when bradykinin plus SOD were applied. In each experiment, scavengers by themselves failed to change the diameter. This data is not shown in the table. SOD failed to alter the dilation produced by acetylcholine (baseline diameter 3 6 ± 2 fim, n= 10, di- Circulation Research Vol 61, No 4, October 1987 Table 1. Inhibition of Bradykinin Relaxation by Agents Interfering With Production of Hydroxyl Radical Study No. 1 (n=10) BK + 60 U/ml SOD BK alone Resting diameter (/xm, mean ± SD) Response to bradykinin (80 ng/ml) (Diameter as % (mean + SD) resting diameter) 34±3 96 + 7* 113 + 7 BK + 46 U/ml catalase BK alone 3 (n=10) BK + 0.1 mM deferoxamine BK alone 34±1 106 + 2* 110 + 3 34±4 107 + 3* 113 + 5 BK + 0.2 mM deferoxamine BK alone 31+5 101+2* 113+6 •Dilation significantly impaired compared to bradykinin alone; p<0.01, paired t test after log transformation to normalize the distribution. ameter increased to 110 ± 3% (mean ± SD) of baseline in presence of 60 U/ml SOD, and to 1 1 2 ± 3 % of baseline in absence of SOD; p>0.2, paired t test.) Discussion Our data confirm the inhibitory effects of SOD and catalase on the response of pial arterioles to bradykinin.3 Kontos et al3 suggested that these data implicated the hydroxyl radical that is formed when superoxide and H2O2 interact in an iron catalyzed reaction.1314 We extended the research design testing a role for hydroxyl radical by using deferoxamine, which chelates iron and would, therefore, inhibit hydroxyl production.14 A dose of 0.1 mM significantly inhibited bradykinin, and a dose of 0.2 mM had an even greater effect. The higher dose not only chelates iron but also begins to have significant ability to directly scavenge hydroxyl.1617 None of the inhibitors had any effect on the diameter of pial arterioles when used alone. Thus, inhibition of the response to bradykinin was not caused by an opposing constricting influence. The selectivity of the effect of SOD was indicated by failure of this scavenger to significantly impair dilation produced by acetylcholine. This agrees with the findings of Kontos et al3 who failed, also, to find an action of catalase on acetylcholine. Our data establishes in mice what the data of Kontos et al suggested in cats. Mutually confirmatory data coming from two species is extremely important because others have reported no effect of SOD on the response to bradykinin in rat cremaster.18 The negative report using rats left open the possibility of species differences. Such differences are common when endo- Rosenblum Hydroxyl Radical and Endothelium Dependent Dilation Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017 thelium-dependent relaxations, like that to bradykinin, are investigated." 20 However, our data makes less likely interspecies differences as an explanation for failure of others to inhibit bradykinin with SOD. Perhaps there are different mechanisms for bradykinin's action in different vascular beds. For example, since the production of a dilating prostaglandin2' occurs as a result of cyclooxygenase stimulation by bradykinin, in some beds this prostaglandin could conceivably dilate even in the absence of a concomitantly produced oxygen-centered radical. The mechanism by which bradykinin may initiate production of superoxide has been discussed by Kontos et al. 3 They point out that bradykinin is known to stimulate prostaglandin synthesis by blood vessels. They have shown that cyclooxygenase, the enzyme responsible for prostaglandin synthesis, generates superoxide radical from pial arterioles during prostaglandin synthesis and that the radical is released into the subarachnoid cerebrospinal fluid.22 Left unresolved is the question of whether hydroxyl radical is a sufficient, or merely a necessary, mediator of the relaxation of pial arterioles produced by bradykinin. In other words, is the hydroxyl radical the EDRF for bradykinin in these vessels or merely necessary for its action? This question will not be resolved unless another EDRF for bradykinin's action on pial arterioles can be identified and isolated. If so, one might attempt to block its action with the scavengers and the chelator used here. That would demonstrate the necessity of the radical for the function of the true EDRF. Until such a time, we can tentatively offer the hypothesis that the hydroxyl radical is the EDRF for bradykinin in pial arterioles. References 1. Rosenblum WI: Endothelial dependent relaxation demonstrated in vivo in cerebral arterioles. Stroke 1986; 17:494—497 2. Rosenblum WI, Nelson GH, Povlishock JT: Laser-induced endothelial damage inhibits endothelial-dependent relaxation in the cerebral microcirculation of the mouse. Circ Res 1987;60:169-176 3. Kontos HA, Wei EP, Povlishock JT, Christman CW: Oxygen radicals mediate the cerebral arteriolar dilation from arachidonate and bradykinin in cats. Circ Res 1984;55:295-303 4. Wei EP, Kontos HA, Christman CW, DeWitt DS, Povlishock JT: Superoxide generation and reversal of acetylcholine induced cerebral vasodilation after acute hypertension. Circ Res 1985;57:781-787 603 5. Kontos HA: Oxygen radicals in cerebral vascular injury. Circ Res 1985;57:508-516 6. Furchgott RF, Zawadzki JV, Cherry PD: Role of endothelium in the vasodilator response to acetylcholine, in Vanhoutte P, Leusen I (eds): Vasodilation. New York, Raven Press Publishers, 1981, p46 7. Rosenblum WI: Effects of free radical generation on mouse pial arterioles: Probable role of hydroxyl radicals. Am J Physiol 1983;245:H139-H142 8. Rosenblum WI, Zweifach BW: Cerebral microcirculation in the mouse brain. Arch Neurol 1963;9:414-423 9. Rosenblum WI: Constriction of pial arterioles by prostaglandin Fj,,. Stroke 1975 ;6:293-297 10. Rosenblum WI: Pial arteriolar responses in the mouse brain revisited. Stroke 1976;7:283-287 11. Elliott KAC, Jasper HH: Physiologic salt solutions for brain surgery. J Neurosurg 1949;6:140-152 12. Baez S: Recording of microvascular dimensions with an image splitter television microscope. J Appl Physiol 1966;21:299-301 13. Fridovich I: Oxygen radicals, hydrogen peroxide and oxygen toxicity, in Pryor WA (ed): Free Radicals in Biology. New York, Academic Press, Inc, 1976, pp 239-277 14. Gutteridge JMC, Rowley DA, Halliwell B: Superoxidedependent formation of hydroxyl radicals and lipid peroxidation in the presence of iron salts. Biochem J 1982;206:605-609 15. Gutteridge JMC, Richmond R, Halliwell B: Inhibition of the iron-catalyzed formation of hydroxyl radicals from superoxide and of lipid peroxidation by desferrioxamine. Biochem J 1979; 184:469-472 16. Hoe S, Rowley DA, Halliwell B: Reactions of ferrioxamine and desferrioxamine with the hydroxyl radical. Chem Biol Interactions 1982;41:75-81 17. Halliwell B, Gutteridge JMC: Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J 1984;219:1-14 18. Wolin MS, Rodenburg JM, Messina EJ, Kaley G: Reduced oxygen metabolites induce vasodilation of the rat cremasteric arterioles, but do not mediate the actions of bradykinin (BK) and arachidonic acid (AA) (abstract). Circulation 1985; 72:111-83 19. Furchgott RF: Role of endothelium in responses of vascular smooth muscle. Circ Res 1983;53:557-573 20. VanHoutte PM, Miller VM: Heterogeneity of endothelium dependent responses in mammalian blood vessels. J Cardiovasc Pharmacol 1985;7(suppl 3):S12-S23 21. Blumberg AL, Denny SE, Marshall GR, Needleman P: Blood vessel-hormone interactions: Angiotensin, bradykinin and prostaglandins. Am J Physiol 1977;232:H305-H310 22. Kontos HA, Wei EP, Ellis EF, Jenkins LW, Povlishock JT, Rowe GT, Hess ML: Appearance of superoxide anion radical in cerebral extracellular space during increased prostaglandin synthesis in cats. Circ Res 1985;57:142-151 KEY WORDS • bradykinin endothelium-dependent relaxation brain microcirculation Hydroxyl radical mediates the endothelium-dependent relaxation produced by bradykinin in mouse cerebral arterioles. W I Rosenblum Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017 Circ Res. 1987;61:601-603 doi: 10.1161/01.RES.61.4.601 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1987 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/61/4/601 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. 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