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Prazosin attenuates hydroxyl radical generation in the rat myocardium
European Journal of Pharmacology 379 Ž1999. 161–166 www.elsevier.nlrlocaterejphar
Prazosin attenuates hydroxyl radical generation in the rat myocardium Toshio Obata ) , Yasumitsu Yamanaka Department of Pharmacology, Oita Medical UniÕersity, Hasama-machi, Oita 879-5593, Japan Received 22 March 1999; received in revised form 7 July 1999; accepted 9 July 1999
Abstract The present study examined whether tyramine-induced hydroxyl radical ŽPOH. generation via noradrenaline release was attenuated by prazosin. A flexibly mounted microdialysis technique was used to detect the generation of POH in in vivo rat hearts. The microdialysis probe was implanted in the left ventricular myocardium of anaesthetized rats and Ringer’s solution was used. To measure the level of POH, sodium salicylate in Ringer’s solution Ž0.5 nmolrmlrmin. was infused directly through a microdialysis probe to detect the generation of POH as reflected by the nonenzymatic formation of 2,3-dihydroxybenzoic acid ŽDHBA.. Tyramine Ž0.1, 0.5 and 1.0 mM. increased the level of 2,3-DHBA in a concentration-dependent manner. However, in the presence of prazosin Ž10 mM., the effect of tyramine was abolished. To confirm the generation of POH by a Fenton type reaction, iron ŽII. was infused through a microdialysis probe. A positive linear correlation between iron ŽII. and the formation of 2,3-DHBA Ž R 2 s 0.982. was observed. To examine the effect of prazosin on ischemicrreperfused rat myocardium, the heart was subjected to myocardial ischemia for 15 min by occlusion of the left anterior descending coronary artery. When the heart was reperfused, a marked elevation of the level of 2,3-DHBA was observed. However, in the presence of prazosin Ž10 mM., the elevation of 2,3-DHBA was not observed in ischemicrreperfused rat heart. Prazosin was shown to have a POH scavenging effect. These results suggest that tyramine-induced noradrenaline causes POH generation, an effect which is inhibited by prazosin as Naq channel blocker, but not through its a 1-adrenoceptor antagonistic action of prazosin. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Prazosin; Naq channel blocker; Tyramine; Noradrenaline; Hydroxyl radical; Microdialysis
1. Introduction Prazosin has been demonstrated to have a protective effect on myocardial ischemic injury ŽNayler et al., 1985; Billman, 1994.. Recent publications indicate ŽGrand et al., 1993; Perez ` et al., 1994. that prazosin has a Naq channel blocking action as well as the a 1-adrenoceptor blocking action. Reactive oxygen causes excessive Naq entry through the fast Ca2q channel, leading to intracellular Ca2q overload through the Naq–Ca2q exchange system, and hence, myocardial damage ŽDonck and Borgers, 1991.. Ca2q overload is then considered to ensue via activation of the Naq–Ca2q exchange, due to increased cytosolic Naq content. Naq channel blocking drugs have been demonstrated to provide protection in the ischemiarreperfusion model ŽDuff et al., 1989; Tosaki et al., 1990.. During
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reperfusion after ischemia, reactive oxygen species, such . Ž . as superoxide anion ŽOy 2 , hydroxyl radical POH and Ž . hydrogen peroxide H 2 O 2 , are generated intra- and extracellularly in the myocardium ŽObata et al., 1994; Pietri et al., 1997; Vaage et al., 1997.. Although the role of catecholamines in myocardial cellular injury is unclear, noradrenaline was found to be induced by ischemiarreperfusion ŽCarlsson and Abrahamsson, 1988; Schomig et al., ¨ 1992; Toombs et al., 1993; Obata et al., 1994.. The noradrenaline released is thought to play a significant role in the etiology of various cardiac pathophysiological disorders ŽRandall et al., 1976; Inoue and Zipes, 1987; Akiyama et al., 1991.. Tyramine, a sympathomimetic amine, induces noradrenaline release by displacing noradrenaline from storage vesicles; the noradrenaline subsequently exits the nerve ending via carrier-mediated outward transport ŽTrendelenburg et al., 1987.. To confirm the mechanism of endogenous noradrenaline-induced POH generation, we examined whether tyramine-induced POH generation via noradrenaline release was attenuated by prazosin, a Naq channel blocker, in the rat heart.
0014-2999r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 1 4 - 2 9 9 9 Ž 9 9 . 0 0 5 0 4 - X
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T. Obata, Y. Yamanakar European Journal of Pharmacology 379 (1999) 161–166
To achieve this goal, we measured POH formation in in vivo hearts, with a flexibly mounted microdialysis technique that we developed ŽObata et al., 1994.. The POH generated, when captured as the hydroxylated derivative of salicylic acid, was measured with a high-performance liquid chromatography–electrochemical ŽHPLC–EC. procedure. 2. Materials and methods 2.1. Animal preparation Adult male Wistar rats weighing 300–400 g were kept in an environmentally controlled room Ž20–238C, 50–60% humidity, illuminated from 0700 to 1900 h. with food and water ad libitum. The animals were anaesthetized with chloral hydrate Ž400 mgrkg, i.p.. and the level of anaesthesia was maintained by intraperitoneal injection of chloral hydrate Ž20 mgrkg.. Artificial ventilation was maintained by constant-volume respiration with room air mixed with oxygen. The heart rate, arterial blood pressure, and electrocardiogram ŽECG. were monitored and recorded continuously. This study was approved by the Ethical Committee for Animal Experiments, Medical University of Oita. 2.2. Drugs used Prazosin, tyramineP HCl, ferrous ammonium sulfate, sodium salicylate, and its hydroxylate metabolites were purchased from Sigma. Prazosin was dissolved in distilled water as a 10-mM stock solution. An appropriate volume of these stock solutions was added to Ringer’s solution consisting of 147 mM NaCl, 2.3 mM CaCl 2 , and 4 mM KCl ŽpH 7.4. immediately before use, as indicated in Section 3. The drugs were dissolved in Ringer’s solution. 2.3. In ÕiÕo trapping of POH by salicylate When a perfusion flow of 1 mlrmin was used, the relative recovery of a l0y6 M standard solution of noradrenaline was l7.0 " 0.7%. For trapping POH radicals ŽFloyd et al., 1984; Halliwell et al., 1991. in the myocardium, sodium salicylate in Ringer’s solution Ž0.5 nmolrmlrmin. was perfused by a micro-injection pump ŽCarnegie Medicine, CMAr100 Stockholm, Sweden. and the basal level of 2,3-dihydroxybenzoic acid ŽDHBA. during a definite period of time was determined. Tyramine added in sodium salicylate solution was infused directly through a microdialysis probe in the rat heart. Samples Ž1 mlrmin. were collected after 15 min into small tubes containing 15 ml of 0.1N HClO4 . 2.4. Experimental protocol Details of the flexibly mounted microdialysis technique and its application to measure biological substances in the
interstitial space have been described previously ŽObata et al., 1994.. We created a suitable microdialysis probe. The probe was implanted from the epicardial surface into the left ventricular myocardium to a depth of 3 mm and perfused through the inlet tube. The synchronized movement of the tip of the microdialysis probe with the beating ventricle minimized the tissue injury that would otherwise be caused by friction between the probe and the muscle tissue. The drugs were dissolved in Ringer’s solution for perfusion Ž1 mlrmin. through a microdialysis probe into the myocardium. 2.5. Preparation of ischemic rats After microdialysis probe implantation in the ischemic zone, the left anterior descending coronary artery branch was clamped by a thread through a tube surrounding the coronary artery. The heart was subjected to regional ischemia for 15 min by the occlusion of the left anterior descending coronary artery followed by reperfusion for 60 min. 2.6. Analytical procedure The dialysate samples were immediately injected for analysis into an HPLC–EC system equipped with a glassy carbon working electrode ŽEicom, Kyoto, Japan. and an analytic reverse-phase column on an Eicompak MA-5ODS column Ž5 mm 4.6 = 150 mm; EICOM.. The working electrode was set at a detector potential of 0.75 V. Each liter of mobile phase contained 1.5 g 1-heptansulfonic acid sodium salt ŽSigma., 0.1 g Na 2 EDTA, 3 ml triethylamine ŽWako, Japan. and 125 ml acetonitrile ŽWako. dissolved in H 2 O. The pH of the solution was adjusted to 2.8 with 3 ml phosphoric acid ŽWako.. 2.7. Statistical analysis All values are presented as means " S.E.M. Differences between the time courses of the levels of 2,3-DHBA were evaluated with the Mann–Whitney U-test. All values are given as means " S.E.M. Analysis of variance ŽANOVA. combined with a Fisher’s post hoc test or Student’s t-test was used to determine significance. A P-value of less than 0.05 was regarded as statistically significant.
3. Results 3.1. Relationship between tyramine-induced noradrenaline and POH products of salicylate Using the in vivo perfusion system, time-dependent changes in the level of noradrenaline and the formation of 2,3-DHBA were monitored in the dialysates from rat heart after tyramine treatment. When tyramine Ž1 mM or
T. Obata, Y. Yamanakar European Journal of Pharmacology 379 (1999) 161–166
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1 nmolrmlrmin. was directly infused in the rat heart through a microdialysis probe, the noradrenaline level at 150–165 min Žor 15 min after administration of tyramine. increased significantly in relation to the level at 135–150 min after probe implantation ŽFig. 1A.. However, no increase in noradrenaline level in the absence of tyramine treatment was observed Ždata not shown.. The POH radical generated when captured as the hydroxylated derivative of salicylic acid was measured by HPLC. The authentic standards of 2,3-DHBA Žreaction products of salicylic acid and POH. had an identical retention time. The level of 2,3DHBA in the heart dialysate samples from control animals following infusion of salicylate Ž0.5 nmolrmlrmin. was 0.039 " 0.009 mM. When the level of noradrenaline after tyramine treatment increased, a marked elevation in the level of 2,3-DHBA was observed in the cardiac dialysates Fig. 2. Concentration-dependent effects of tyramine measured in the absence and presence of prazosin, an a 1-adrenoceptor antagonist, on the level of dialysate noradrenaline. Concentration of dialysate noradrenaline measured at 30–45 min after application of various concentrations of tyramine Žas indicated in abscissa. in the absence Žopen column. and presence Ždiagonally shaded column. of prazosin Ž10 mM. and given as a percentage of the value measured just before application of tyramine. Each column and vertical bar indicates means"S.E.M. for six animals; U P - 0.05 vs. 2,3-DHBA levels immediately before application of tyramine ŽStudent’s t-test.. §P - 0.05: significant difference ŽANOVA and Fisher’s test.. ns, nonsignificant.
ŽFig. 1B.. As summarized in Fig. 2, tyramine applied at a variety of concentrations Ž0.1, 0.5 and 1.0 mM. increased the level of 2,3-DHBA in a concentration-dependent manner. In the presence of tyramine, the application of pra-
Fig. 1. Relationship between noradrenaline and hydroxyl radical generation. After a 90-min washout with Ringer’s solution, the time course of dialysate noradrenaline ŽA. and in vivo trapping of highly reactive POH ŽB. in extracellular fluid of myocardium after tyramine was investigated by infusing sodium salicylate in Ringer’s solution Žopen bar; 0.5 nmolrmlrmin. through a myocardial microdialysis probe placed in the epicardial surface into the left ventricular myocardium and perfused with Ringer’s solution by a microinjection pump. When tyramine Ždiagonally shaded bar; 1 mM or 1 nmolrmlrmin. was infused directly through the microdialysis probe, dialysate samples were collected at 15-min intervals and immediately assayed for noradrenaline and 2,3-DHBA using an HPLC–EC procedure. Values are means"S.E.M. for six animals. Differences between the time courses of the levels of noradrenaline and 2,3-DHBA were evaluated by means of the Mann–Whitney U-test. U P - 0.05 vs. levels at 135–150 min. ŽAbscissa. after 90-min washout with Ringer’s solution, infusion of salicylic acid was started.
Fig. 3. Dose–response relationship between iron ŽII. and the formation of POH products of salicylate. Iron ŽII. and sodium salicylate Ž0.5 nmolrmlrmin. were infused through the dialysis probe. When iron ŽII. Ž0, 5, 25 and 50 mM. was administered, it was given directly through the dialysis probe. The levels of 2,3-DHBA in all three groups tyramine-only treated rats Ž^. and tyramine plus prazosin-pretreated rats ŽI. and nontreated rats Ž`.x were compared. Dialysate samples were collected and immediately assayed for 2,3-DHBA, using an HPLC–EC procedure. Values are means"S.E.M. for six animals.
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zosin Ž10 mM. significantly decreased the level of 2,3DHBA in the presence of a variety of tyramine concentrations; inhibition was complete at tyramine concentrations of 0.1, 0.5 and 1.0 mM. To confirm the POH generation by means of a Fenton reaction, iron ŽII. was infused through the microdialysis probe. When iron ŽII. Ž0, 5, 25 and 50 mM. was administered to the tyramine-pretreated animals, a marked elevation in 2,3-DHBA was obtained, as compared with iron ŽII. only-treated animals, showing a positive linear correlation between iron ŽII. and POH formed and trapped as 2,3-DHBA Ž R 2 s 0.982. in the dialysate. However, when corresponding experiments were performed with the prazosin Ž10 mM.-pretreated animals, small increases in the level of 2,3-DHBA products were observed ŽFig. 3.. In contrast, atenolol, a b 1-adrenoceptor antagonist, did not prevent the effect of tyramine Ždata not shown..
3.2. Effect of prazosin on POH formation in ischemiar reperfused rat heart The presence of POH was observed in ischemicrreperfused rat heart. Sodium salicylate Ž0.5 nmolrmlrmin. was infused for 60 min to trap POH, which was formed during ischemiarreperfusion of myocardium. After the dialysate probe was implanted in the left ventricular myocardium, the levels of 2,3-DHBA remained unchanged until reperfusion. When the heart was reperfused, a marked elevation of the levels of 2,3-DHBA was observed in the dialysate. This elevation of 2,3-DHBA was not observed outside the ischemic area. To confirm the ischemic zone, after reperfusion, the heart was removed and cannulated via the aorta. The ligature remaining around the left anterior descending coronary artery was retightened, and ethanol was injected into the heart. Then the heart was sliced. Discolored areas in the sliced sections of the heart showed nonischemic regions Žnot illustrated.. When corresponding experiments were performed with prazosin Ž10 mM.-pretreated animals, the elevation of 2,3-DHBA was not observed in ischemicrreperfused rat heart ŽFig. 4.. In contrast, atenolol did not prevent the increase in 2,3-DHBA on ischemicrreperfusion Ždata not shown..
4. Discussion
Fig. 4. Effect of prazosin in ischemicrreperfused rat heart on formation of POH products of salicylate. The level of dialysate noradrenaline ŽA. and in vivo trapping of highly reactive POH ŽB. were investigated by infusing sodium salicylate in Ringer’s solution Ž0.5 nmolrmlrmin. through a myocardial microdialysis probe placed in the epicardial surface into the left ventricular myocardium and perfused with Ringer’s solution by a microinjection pump. Prazosin Ž10 mM.-treated animals Žv . Ždiagonally shaded column. were compared with control animals Ž`.. Dialysate samples were collected at 15-min intervals and immediately assayed for the level of noradrenaline and the level of 2,3-DHBA using an HPLC–EC procedure. Values are means"S.E.M. for six animals. Differences between the time courses of the levels of noradrenaline and the level of 2,3-DHBA were evaluated by means of the Mann–Whitney U-test. U P - 0.05 vs. levels at 135–150 min.
Our results with the microdialysis technique demonstrated that tyramine-induced noradrenaline release caused POH generation. Details of the microdialysis technique required for use with a flexibly mounted microdialysis probe in in vivo rat hearts to measure biological substances in the interstitial space have already been described ŽObata et al., 1994.. With this technique, it is feasible to make stable and long-term measurements of interstitial noradrenaline and POH generation. Admittedly, the concentration profile of the administered compounds in the surrounding interstitial space is unknown; in general, the extracellular concentration of a compound given through the probe would never reach the concentration in the dialysis probe ŽBenveniste, 1989.. This is an unavoidable limitation of the microdialysis technique that should be kept in mind when interpreting the experimental data. We confirmed that tyramine increased the dialysate noradrenaline in a concentration-dependent manner ŽFig. 2.. Our results showed that the tyramine concentration for the half-maximal effect of noradrenaline to produce release ŽEC 50 . was approximately 1 mM. Therefore, we used a concentration of 1 mM tyramine in the experiments. Tyramine supplied from an inlet tube diffused out into the interstitial fluid through the dialysis membrane, and was converted to noradrenaline in the concentration found in the dialysate. The results indicated that the elevation in tyramine-induced noradrenaline may cause POH generation, as reflected by 2,3-DHBA levels in the cardiac
T. Obata, Y. Yamanakar European Journal of Pharmacology 379 (1999) 161–166
dialysate ŽFig. 1B.. This result suggests that accumulation of endogenous noradrenaline can lead to the formation of cytotoxic POH radicals. Although the mechanism of POH generation by tyramine is unclear, noradrenaline is considered to be autooxidized in the presence of oxygen and transition metal ŽGraham, 1984; Riederer et al., 1989; Obata and Yamanaka, 1996.. Radical scavengers or antioxidant substances have been shown to attenuate myocardial dysfunction ŽSlezak et al., 1995; Pietri et al., 1997; Vaage et al., 1997.. Theoretically, POH may be formed in vivo during nonenzymatic oxidation ŽGraham, 1984; Fornstedt et al., 1989; Ben-Shachar et al., 1991. andror enzymatic oxidation of noradrenaline. Our data indicate that the elevation in noradrenaline may cause POH generation, as reflected by 2,3-DHBA levels in the myocardial dialysate ŽFig. 1.. However, in the presence of prazosin Ž10 mM., a Naq channel blocker, the effect of tyramine was abolished ŽFig. 2.. Tyramine-induced POH formation was attenuated by prazosin. It is possible that Naq accumulation triggers the Naq–Ca2q exchange, leading to Ca2q overload. It is well known that Ca2q overload may involve the formation of free radicals ŽHoffmann et al., 1995; Obata and Yamanaka, 1997.. According to recent studies ŽGrand et al., 1993; Perez et al., 1994., prazosin has a Naq channel ` blocking action as well as the a 1-adrenoceptor blocking action. Su et al. Ž1995. have found that prazosin Ž1–10 mM. inhibits the inward Naq current in the rat, guinea-pig and human myocardium and that this action of prazosin is unrelated to the blockade of a 1-adrenoceptors. On the other hand, the reactive oxygen causes excessive Naq entry through the fast Naq channel, leading to intracellular Ca2q overload through the Naq–Ca2q exchange system, and hence, myocardial damage ŽVer Donck and Borgers, 1991.. The beneficial effect of prazosin on the H 2 O 2-induced derangements, therefore, may be due to its blocking effect on the Naq channel in the cardiac cells. This notion is in agreement with data from a recent study, in which the a 1-adrenoceptor antagonistic action of prazosin may not have contributed to its action to attenuate the free radicalinduced damage, although the possibility of a contribution of a 1-adrenoceptor blockade cannot be completely excluded ŽAkahira et al., 1998.. Intracellular Ca2q overload is considered to be the pathway leading to cell death. To determine whether prazosin has a radical-scavenging or antioxidant effect, we examined the effect of prazosin on POH generation, using a Fenton type reaction. When iron ŽII. was administered to tyramine-pretreated animals, a marked increase in the level of 2,3-DHBA was obtained, as compared with the iron ŽII.-only treated group, showing a positive linear correlation between iron ŽII. and POH formation trapped as 2,3-DHBA Ž R 2 s 0.982. in the dialysate. When corresponding experiments were performed with prazosin-pretreated animals, small increases in the levels of 2,3-DHBA products were observed ŽFig. 3.. Therefore, in the presence of prazosin Ž10 mM., tyramine failed to increase 2,3-DHBA formation in the
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dialysate. This result suggests that prazosin, a Naq channel blocker, may attenuate POH generation in the rat myocardium. Although the mechanism of POH generation in the ischemic heart is obscure, we previously found a concomitant increase of noradrenaline and POH generation on myocardiac injury ŽObata et al., 1994.. Numerous investigators have reported that myocardial ischemia also increases catecholamine levels ŽDart et al., 1984; Schomig et ¨ al., 1984; Rona, 1985; Schomig et al., 1987.. When the ¨ ischemic arrhythmia appeared, noradrenaline and free radicals were found in ischemiarreperfused rat heart ŽObata et al., 1994.. Noradrenaline release by ischemiarreperfusion of the myocardium may be associated with oxidant damage due to POH generation in the heart. These results suggest that noradrenaline release in the extracellular fluid may play a key role in the generation of POH radicals in the heart. Prazosin was shown to have a POH scavenging effect ŽAkahira et al., 1998.. Thus, these results of our observations using an in vivo microdialysis technique are consistent with the finding that prazosin attenuates the reactive oxygen species in isolated rat cardiomyocytes ŽAkahira et al., 1998.. Increases in intracellular Naq levels could be caused by a modified Naq channel function ŽVer Donck and Borgers, 1991; Wermelskirchen et al., 1991.. In the presence of prazosin Ž10 mM., or during ischemiarreperfusion, tyramine failed to increase 2,3-dihydroxylation of salicylate in the cardiac microdialysis perfusion ŽFig. 4.. The present results seem to indicate that ischemiarreperfusion of myocardium may significantly elevate the level of noradrenaline through oxidant damage during POH generation. The results of the present study demonstrated that prazosin attenuates the endogenous noradrenaline-induced POH generation, suggesting that it protects from ischemiarreperfusion-induced POH generation partly through the radical scavenging action or its antioxidant action. In fact, prazosin had a POH radical scavenging effect. The contribution of the Naq channel blocking activity of prazosin to attenuation of the ischemiarreperfusion-induced damage remains to be studied. During myocardial ischemia, reduced ATP production generates disturbances in intracellular ion homeostasis, which ultimately lead to cellular Ca2q overload and contractile failure. Ca2q overload is then considered to ensue via activation of the Naq–Ca2q exchange due to the increase in cytosolic Naq content under ischemic conditions. Intracellular Ca2q overload is then considered to lead to cell death under pathological conditions such as ischemiarreperfusion injury ŽFarber, 1982; Tani, 1990.. These results suggest that Naq-mediated Ca2q overload may be relevant to ischemiarreperfusion Ca2q overload ŽVer Donck and Borgers, 1991.. This is a new cardioprotective principle. Although free radicals are a part of normal metabolism, we found that a mechanism other than the improvement of the myocardium oxygen balance contributes to the protective effect of prazosin against ischemiarreperfusion dam-
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age. Microdialysis heart perfusion experiments using the hydroxylation of salicylate to detect POH generation may be useful to answer some of the fundamental questions concerning the relevance of oxidant damage in the pathogenesis of heart disorders such as infarction.
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protective effect of prazosin on the ischaemic and reperfused myocardium. J. Mol. Cell. Cardiol. 17, 685–699. Obata, T., Yamanaka, Y., 1996. Effect of iron ŽII. on the generation of hydroxyl free radicals in rat myocardium. Biochem. Pharmacol. 51, 1411–1413. Obata, T., Yamanaka, Y., 1997. Cardiac microdialysis of salicylic acid POH generation on nonenzymatic oxidation by norepinephrine in rat heart. Biochem. Pharmacol. 51, 1411–1413. Obata, T., Hosokawa, H., Yamanaka, Y., 1994. In vivo monitoring of norepinephrine and POH generation on myocardial ischemic injury by dialysis technique. Am. J. Physiol. 266, H903–H908. Perez, O., Valenzuela, C., Delpon, ` ´ E., Tamargo, J., 1994. Class I and III antiarrhythmic actions of prazosin in guinea-pig papillary muscles. Br. J. Pharmacol. 111, 717–722. Pietri, S., Maurelli, E., Drieu, K., Culcasi, M., 1997. Cardioprotective and anti-oxidant effects of the terpenoid constituents of Ginkgo biloba extract ŽEGb 761.. J. Mol. Cell. Cardiol. 29, 733–742. Randall, W.C., Keye, M.P., Hageman, G.R., Jacobs, H.K., Euler, D.E., Wehrmacher, W., 1976. Cardiac dysrhythmias in the conscious dog after surgically induced autonomic imbalance. Am. J. Cardiol. 38, 178–183. Riederer, P., Sofic, E., Rausch, W.-D., Schmidt, B., Reynolds, G.P., Jellinger, K., Youdim, M.B.H., 1989. Transition metals, ferritin, glutathione, and ascorbic acid in parkinsonian brains. J. Neurochem. 52, 515–520. Rona, G., 1985. Catecholamine cardiotoxicity. J. Mol. Cell. Cardiol. 17, 291–306. Schomig, A., Dart, A.M., Dietz, R., Mayer, E., Kubler, W., 1984. ¨ ¨ Release of endogenous catecholamines in the ischemic myocardium of the rat. Part A: Locally mediated release. Circ. Res. 55, 689–701. Schomig, A., Fischer, S., Kurz, T., Richardt, G., Schomig, E., 1987. ¨ ¨ Nonexocytotic release of endogenous noradrenaline in the ischemic and anoxic rat heart: mechanism and metabolic requirements. Circ. Res. 60, 194–205. Schomig, A., Rehmert, G., Kurz, T., Richardt, G., 1992. Calcium antago¨ nism and norepinephrine release in myocardial ischemia. J. Cardiovasc. Pharmacol. 7, S16–S20. Slezak, J., Tribulova, N., Pristacova, J., Uhrik, B., Thomas, T., Khaper, N., Kaul, N., Singal, P.K., 1995. Hydrogen peroxide changes in ischemic and reperfused heart. Cytochemistry and biochemical and X-ray microanalysis. Am. J. Pathol. 147, 772–781. Su, M.J., Chi, J.F., Chu, S.H., 1995. Effects of prazosin on rat, guinea pig and human cardiac tissues. Chin. J. Physiol. 38, 159–169. Tani, M., 1990. Mechanisms of Ca2q overload in reperfused ischemic myocardium. Annu. Rev. Physiol. 52, 543–559. Toombs, C.F., Wiltse, A.L., Shebuski, R.J., 1993. Ischemic preconditioning fails to limit infarct size in reserpinized rabbit myocardium. Implication of norepinephrine release in the preconditioning effect. Circulation 88, 2351–2358. Tosaki, A., Koltai, M., Paubert-Braquet, M., 1990. Effect of iloprost on reperfusion-induced arrhythmias and myocardial ion shifts in isolated rat hearts. Eur. J. Pharmacol. 191, 69–81. Trendelenburg, U., Langeloh, A., Bonisch, H., 1987. Mechanism of action of indirectly acting sympathomimetic amines. Blood Vessels 24, 261–270. Vaage, J., Antonelli, M., Bufi, M., Irtun, O., DeBlasi, R.A., Corbucci, G.G., Gasparetto, A., Semb, A.G., 1997. Exogenous reactive oxygen species deplete the isolated rat heart of antioxidants. Free Radical Biol. Med. 22, 85–92. Ver Donck, L., Borgers, M., 1991. Myocardial protection by R 56865: a new principle based on prevention of ion channel pathology. Am. J. Physiol. 266, H903–H908. Wermelskirchen, D., Wilffert, B., Nebel, U., Wirth, A., Peters, T., 1991. Veratridine-induced intoxication in the isolated left atrium of the rat: effects of some anti-ischemic compounds. Naunyn-Schmiedeberg’s Arch. Pharmacol. 344, 101–106.
Prazosin attenuates hydroxyl radical generation in the rat myocardium Toshio Obata ) , Yasumitsu Yamanaka Department of Pharmacology, Oita Medical UniÕersity, Hasama-machi, Oita 879-5593, Japan Received 22 March 1999; received in revised form 7 July 1999; accepted 9 July 1999
Abstract The present study examined whether tyramine-induced hydroxyl radical ŽPOH. generation via noradrenaline release was attenuated by prazosin. A flexibly mounted microdialysis technique was used to detect the generation of POH in in vivo rat hearts. The microdialysis probe was implanted in the left ventricular myocardium of anaesthetized rats and Ringer’s solution was used. To measure the level of POH, sodium salicylate in Ringer’s solution Ž0.5 nmolrmlrmin. was infused directly through a microdialysis probe to detect the generation of POH as reflected by the nonenzymatic formation of 2,3-dihydroxybenzoic acid ŽDHBA.. Tyramine Ž0.1, 0.5 and 1.0 mM. increased the level of 2,3-DHBA in a concentration-dependent manner. However, in the presence of prazosin Ž10 mM., the effect of tyramine was abolished. To confirm the generation of POH by a Fenton type reaction, iron ŽII. was infused through a microdialysis probe. A positive linear correlation between iron ŽII. and the formation of 2,3-DHBA Ž R 2 s 0.982. was observed. To examine the effect of prazosin on ischemicrreperfused rat myocardium, the heart was subjected to myocardial ischemia for 15 min by occlusion of the left anterior descending coronary artery. When the heart was reperfused, a marked elevation of the level of 2,3-DHBA was observed. However, in the presence of prazosin Ž10 mM., the elevation of 2,3-DHBA was not observed in ischemicrreperfused rat heart. Prazosin was shown to have a POH scavenging effect. These results suggest that tyramine-induced noradrenaline causes POH generation, an effect which is inhibited by prazosin as Naq channel blocker, but not through its a 1-adrenoceptor antagonistic action of prazosin. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Prazosin; Naq channel blocker; Tyramine; Noradrenaline; Hydroxyl radical; Microdialysis
1. Introduction Prazosin has been demonstrated to have a protective effect on myocardial ischemic injury ŽNayler et al., 1985; Billman, 1994.. Recent publications indicate ŽGrand et al., 1993; Perez ` et al., 1994. that prazosin has a Naq channel blocking action as well as the a 1-adrenoceptor blocking action. Reactive oxygen causes excessive Naq entry through the fast Ca2q channel, leading to intracellular Ca2q overload through the Naq–Ca2q exchange system, and hence, myocardial damage ŽDonck and Borgers, 1991.. Ca2q overload is then considered to ensue via activation of the Naq–Ca2q exchange, due to increased cytosolic Naq content. Naq channel blocking drugs have been demonstrated to provide protection in the ischemiarreperfusion model ŽDuff et al., 1989; Tosaki et al., 1990.. During
) Corresponding author. Tel.: q81-97-586-5724; fax: q81-97-5865729; E-mail: [email protected]
reperfusion after ischemia, reactive oxygen species, such . Ž . as superoxide anion ŽOy 2 , hydroxyl radical POH and Ž . hydrogen peroxide H 2 O 2 , are generated intra- and extracellularly in the myocardium ŽObata et al., 1994; Pietri et al., 1997; Vaage et al., 1997.. Although the role of catecholamines in myocardial cellular injury is unclear, noradrenaline was found to be induced by ischemiarreperfusion ŽCarlsson and Abrahamsson, 1988; Schomig et al., ¨ 1992; Toombs et al., 1993; Obata et al., 1994.. The noradrenaline released is thought to play a significant role in the etiology of various cardiac pathophysiological disorders ŽRandall et al., 1976; Inoue and Zipes, 1987; Akiyama et al., 1991.. Tyramine, a sympathomimetic amine, induces noradrenaline release by displacing noradrenaline from storage vesicles; the noradrenaline subsequently exits the nerve ending via carrier-mediated outward transport ŽTrendelenburg et al., 1987.. To confirm the mechanism of endogenous noradrenaline-induced POH generation, we examined whether tyramine-induced POH generation via noradrenaline release was attenuated by prazosin, a Naq channel blocker, in the rat heart.
0014-2999r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 1 4 - 2 9 9 9 Ž 9 9 . 0 0 5 0 4 - X
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To achieve this goal, we measured POH formation in in vivo hearts, with a flexibly mounted microdialysis technique that we developed ŽObata et al., 1994.. The POH generated, when captured as the hydroxylated derivative of salicylic acid, was measured with a high-performance liquid chromatography–electrochemical ŽHPLC–EC. procedure. 2. Materials and methods 2.1. Animal preparation Adult male Wistar rats weighing 300–400 g were kept in an environmentally controlled room Ž20–238C, 50–60% humidity, illuminated from 0700 to 1900 h. with food and water ad libitum. The animals were anaesthetized with chloral hydrate Ž400 mgrkg, i.p.. and the level of anaesthesia was maintained by intraperitoneal injection of chloral hydrate Ž20 mgrkg.. Artificial ventilation was maintained by constant-volume respiration with room air mixed with oxygen. The heart rate, arterial blood pressure, and electrocardiogram ŽECG. were monitored and recorded continuously. This study was approved by the Ethical Committee for Animal Experiments, Medical University of Oita. 2.2. Drugs used Prazosin, tyramineP HCl, ferrous ammonium sulfate, sodium salicylate, and its hydroxylate metabolites were purchased from Sigma. Prazosin was dissolved in distilled water as a 10-mM stock solution. An appropriate volume of these stock solutions was added to Ringer’s solution consisting of 147 mM NaCl, 2.3 mM CaCl 2 , and 4 mM KCl ŽpH 7.4. immediately before use, as indicated in Section 3. The drugs were dissolved in Ringer’s solution. 2.3. In ÕiÕo trapping of POH by salicylate When a perfusion flow of 1 mlrmin was used, the relative recovery of a l0y6 M standard solution of noradrenaline was l7.0 " 0.7%. For trapping POH radicals ŽFloyd et al., 1984; Halliwell et al., 1991. in the myocardium, sodium salicylate in Ringer’s solution Ž0.5 nmolrmlrmin. was perfused by a micro-injection pump ŽCarnegie Medicine, CMAr100 Stockholm, Sweden. and the basal level of 2,3-dihydroxybenzoic acid ŽDHBA. during a definite period of time was determined. Tyramine added in sodium salicylate solution was infused directly through a microdialysis probe in the rat heart. Samples Ž1 mlrmin. were collected after 15 min into small tubes containing 15 ml of 0.1N HClO4 . 2.4. Experimental protocol Details of the flexibly mounted microdialysis technique and its application to measure biological substances in the
interstitial space have been described previously ŽObata et al., 1994.. We created a suitable microdialysis probe. The probe was implanted from the epicardial surface into the left ventricular myocardium to a depth of 3 mm and perfused through the inlet tube. The synchronized movement of the tip of the microdialysis probe with the beating ventricle minimized the tissue injury that would otherwise be caused by friction between the probe and the muscle tissue. The drugs were dissolved in Ringer’s solution for perfusion Ž1 mlrmin. through a microdialysis probe into the myocardium. 2.5. Preparation of ischemic rats After microdialysis probe implantation in the ischemic zone, the left anterior descending coronary artery branch was clamped by a thread through a tube surrounding the coronary artery. The heart was subjected to regional ischemia for 15 min by the occlusion of the left anterior descending coronary artery followed by reperfusion for 60 min. 2.6. Analytical procedure The dialysate samples were immediately injected for analysis into an HPLC–EC system equipped with a glassy carbon working electrode ŽEicom, Kyoto, Japan. and an analytic reverse-phase column on an Eicompak MA-5ODS column Ž5 mm 4.6 = 150 mm; EICOM.. The working electrode was set at a detector potential of 0.75 V. Each liter of mobile phase contained 1.5 g 1-heptansulfonic acid sodium salt ŽSigma., 0.1 g Na 2 EDTA, 3 ml triethylamine ŽWako, Japan. and 125 ml acetonitrile ŽWako. dissolved in H 2 O. The pH of the solution was adjusted to 2.8 with 3 ml phosphoric acid ŽWako.. 2.7. Statistical analysis All values are presented as means " S.E.M. Differences between the time courses of the levels of 2,3-DHBA were evaluated with the Mann–Whitney U-test. All values are given as means " S.E.M. Analysis of variance ŽANOVA. combined with a Fisher’s post hoc test or Student’s t-test was used to determine significance. A P-value of less than 0.05 was regarded as statistically significant.
3. Results 3.1. Relationship between tyramine-induced noradrenaline and POH products of salicylate Using the in vivo perfusion system, time-dependent changes in the level of noradrenaline and the formation of 2,3-DHBA were monitored in the dialysates from rat heart after tyramine treatment. When tyramine Ž1 mM or
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1 nmolrmlrmin. was directly infused in the rat heart through a microdialysis probe, the noradrenaline level at 150–165 min Žor 15 min after administration of tyramine. increased significantly in relation to the level at 135–150 min after probe implantation ŽFig. 1A.. However, no increase in noradrenaline level in the absence of tyramine treatment was observed Ždata not shown.. The POH radical generated when captured as the hydroxylated derivative of salicylic acid was measured by HPLC. The authentic standards of 2,3-DHBA Žreaction products of salicylic acid and POH. had an identical retention time. The level of 2,3DHBA in the heart dialysate samples from control animals following infusion of salicylate Ž0.5 nmolrmlrmin. was 0.039 " 0.009 mM. When the level of noradrenaline after tyramine treatment increased, a marked elevation in the level of 2,3-DHBA was observed in the cardiac dialysates Fig. 2. Concentration-dependent effects of tyramine measured in the absence and presence of prazosin, an a 1-adrenoceptor antagonist, on the level of dialysate noradrenaline. Concentration of dialysate noradrenaline measured at 30–45 min after application of various concentrations of tyramine Žas indicated in abscissa. in the absence Žopen column. and presence Ždiagonally shaded column. of prazosin Ž10 mM. and given as a percentage of the value measured just before application of tyramine. Each column and vertical bar indicates means"S.E.M. for six animals; U P - 0.05 vs. 2,3-DHBA levels immediately before application of tyramine ŽStudent’s t-test.. §P - 0.05: significant difference ŽANOVA and Fisher’s test.. ns, nonsignificant.
ŽFig. 1B.. As summarized in Fig. 2, tyramine applied at a variety of concentrations Ž0.1, 0.5 and 1.0 mM. increased the level of 2,3-DHBA in a concentration-dependent manner. In the presence of tyramine, the application of pra-
Fig. 1. Relationship between noradrenaline and hydroxyl radical generation. After a 90-min washout with Ringer’s solution, the time course of dialysate noradrenaline ŽA. and in vivo trapping of highly reactive POH ŽB. in extracellular fluid of myocardium after tyramine was investigated by infusing sodium salicylate in Ringer’s solution Žopen bar; 0.5 nmolrmlrmin. through a myocardial microdialysis probe placed in the epicardial surface into the left ventricular myocardium and perfused with Ringer’s solution by a microinjection pump. When tyramine Ždiagonally shaded bar; 1 mM or 1 nmolrmlrmin. was infused directly through the microdialysis probe, dialysate samples were collected at 15-min intervals and immediately assayed for noradrenaline and 2,3-DHBA using an HPLC–EC procedure. Values are means"S.E.M. for six animals. Differences between the time courses of the levels of noradrenaline and 2,3-DHBA were evaluated by means of the Mann–Whitney U-test. U P - 0.05 vs. levels at 135–150 min. ŽAbscissa. after 90-min washout with Ringer’s solution, infusion of salicylic acid was started.
Fig. 3. Dose–response relationship between iron ŽII. and the formation of POH products of salicylate. Iron ŽII. and sodium salicylate Ž0.5 nmolrmlrmin. were infused through the dialysis probe. When iron ŽII. Ž0, 5, 25 and 50 mM. was administered, it was given directly through the dialysis probe. The levels of 2,3-DHBA in all three groups tyramine-only treated rats Ž^. and tyramine plus prazosin-pretreated rats ŽI. and nontreated rats Ž`.x were compared. Dialysate samples were collected and immediately assayed for 2,3-DHBA, using an HPLC–EC procedure. Values are means"S.E.M. for six animals.
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zosin Ž10 mM. significantly decreased the level of 2,3DHBA in the presence of a variety of tyramine concentrations; inhibition was complete at tyramine concentrations of 0.1, 0.5 and 1.0 mM. To confirm the POH generation by means of a Fenton reaction, iron ŽII. was infused through the microdialysis probe. When iron ŽII. Ž0, 5, 25 and 50 mM. was administered to the tyramine-pretreated animals, a marked elevation in 2,3-DHBA was obtained, as compared with iron ŽII. only-treated animals, showing a positive linear correlation between iron ŽII. and POH formed and trapped as 2,3-DHBA Ž R 2 s 0.982. in the dialysate. However, when corresponding experiments were performed with the prazosin Ž10 mM.-pretreated animals, small increases in the level of 2,3-DHBA products were observed ŽFig. 3.. In contrast, atenolol, a b 1-adrenoceptor antagonist, did not prevent the effect of tyramine Ždata not shown..
3.2. Effect of prazosin on POH formation in ischemiar reperfused rat heart The presence of POH was observed in ischemicrreperfused rat heart. Sodium salicylate Ž0.5 nmolrmlrmin. was infused for 60 min to trap POH, which was formed during ischemiarreperfusion of myocardium. After the dialysate probe was implanted in the left ventricular myocardium, the levels of 2,3-DHBA remained unchanged until reperfusion. When the heart was reperfused, a marked elevation of the levels of 2,3-DHBA was observed in the dialysate. This elevation of 2,3-DHBA was not observed outside the ischemic area. To confirm the ischemic zone, after reperfusion, the heart was removed and cannulated via the aorta. The ligature remaining around the left anterior descending coronary artery was retightened, and ethanol was injected into the heart. Then the heart was sliced. Discolored areas in the sliced sections of the heart showed nonischemic regions Žnot illustrated.. When corresponding experiments were performed with prazosin Ž10 mM.-pretreated animals, the elevation of 2,3-DHBA was not observed in ischemicrreperfused rat heart ŽFig. 4.. In contrast, atenolol did not prevent the increase in 2,3-DHBA on ischemicrreperfusion Ždata not shown..
4. Discussion
Fig. 4. Effect of prazosin in ischemicrreperfused rat heart on formation of POH products of salicylate. The level of dialysate noradrenaline ŽA. and in vivo trapping of highly reactive POH ŽB. were investigated by infusing sodium salicylate in Ringer’s solution Ž0.5 nmolrmlrmin. through a myocardial microdialysis probe placed in the epicardial surface into the left ventricular myocardium and perfused with Ringer’s solution by a microinjection pump. Prazosin Ž10 mM.-treated animals Žv . Ždiagonally shaded column. were compared with control animals Ž`.. Dialysate samples were collected at 15-min intervals and immediately assayed for the level of noradrenaline and the level of 2,3-DHBA using an HPLC–EC procedure. Values are means"S.E.M. for six animals. Differences between the time courses of the levels of noradrenaline and the level of 2,3-DHBA were evaluated by means of the Mann–Whitney U-test. U P - 0.05 vs. levels at 135–150 min.
Our results with the microdialysis technique demonstrated that tyramine-induced noradrenaline release caused POH generation. Details of the microdialysis technique required for use with a flexibly mounted microdialysis probe in in vivo rat hearts to measure biological substances in the interstitial space have already been described ŽObata et al., 1994.. With this technique, it is feasible to make stable and long-term measurements of interstitial noradrenaline and POH generation. Admittedly, the concentration profile of the administered compounds in the surrounding interstitial space is unknown; in general, the extracellular concentration of a compound given through the probe would never reach the concentration in the dialysis probe ŽBenveniste, 1989.. This is an unavoidable limitation of the microdialysis technique that should be kept in mind when interpreting the experimental data. We confirmed that tyramine increased the dialysate noradrenaline in a concentration-dependent manner ŽFig. 2.. Our results showed that the tyramine concentration for the half-maximal effect of noradrenaline to produce release ŽEC 50 . was approximately 1 mM. Therefore, we used a concentration of 1 mM tyramine in the experiments. Tyramine supplied from an inlet tube diffused out into the interstitial fluid through the dialysis membrane, and was converted to noradrenaline in the concentration found in the dialysate. The results indicated that the elevation in tyramine-induced noradrenaline may cause POH generation, as reflected by 2,3-DHBA levels in the cardiac
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dialysate ŽFig. 1B.. This result suggests that accumulation of endogenous noradrenaline can lead to the formation of cytotoxic POH radicals. Although the mechanism of POH generation by tyramine is unclear, noradrenaline is considered to be autooxidized in the presence of oxygen and transition metal ŽGraham, 1984; Riederer et al., 1989; Obata and Yamanaka, 1996.. Radical scavengers or antioxidant substances have been shown to attenuate myocardial dysfunction ŽSlezak et al., 1995; Pietri et al., 1997; Vaage et al., 1997.. Theoretically, POH may be formed in vivo during nonenzymatic oxidation ŽGraham, 1984; Fornstedt et al., 1989; Ben-Shachar et al., 1991. andror enzymatic oxidation of noradrenaline. Our data indicate that the elevation in noradrenaline may cause POH generation, as reflected by 2,3-DHBA levels in the myocardial dialysate ŽFig. 1.. However, in the presence of prazosin Ž10 mM., a Naq channel blocker, the effect of tyramine was abolished ŽFig. 2.. Tyramine-induced POH formation was attenuated by prazosin. It is possible that Naq accumulation triggers the Naq–Ca2q exchange, leading to Ca2q overload. It is well known that Ca2q overload may involve the formation of free radicals ŽHoffmann et al., 1995; Obata and Yamanaka, 1997.. According to recent studies ŽGrand et al., 1993; Perez et al., 1994., prazosin has a Naq channel ` blocking action as well as the a 1-adrenoceptor blocking action. Su et al. Ž1995. have found that prazosin Ž1–10 mM. inhibits the inward Naq current in the rat, guinea-pig and human myocardium and that this action of prazosin is unrelated to the blockade of a 1-adrenoceptors. On the other hand, the reactive oxygen causes excessive Naq entry through the fast Naq channel, leading to intracellular Ca2q overload through the Naq–Ca2q exchange system, and hence, myocardial damage ŽVer Donck and Borgers, 1991.. The beneficial effect of prazosin on the H 2 O 2-induced derangements, therefore, may be due to its blocking effect on the Naq channel in the cardiac cells. This notion is in agreement with data from a recent study, in which the a 1-adrenoceptor antagonistic action of prazosin may not have contributed to its action to attenuate the free radicalinduced damage, although the possibility of a contribution of a 1-adrenoceptor blockade cannot be completely excluded ŽAkahira et al., 1998.. Intracellular Ca2q overload is considered to be the pathway leading to cell death. To determine whether prazosin has a radical-scavenging or antioxidant effect, we examined the effect of prazosin on POH generation, using a Fenton type reaction. When iron ŽII. was administered to tyramine-pretreated animals, a marked increase in the level of 2,3-DHBA was obtained, as compared with the iron ŽII.-only treated group, showing a positive linear correlation between iron ŽII. and POH formation trapped as 2,3-DHBA Ž R 2 s 0.982. in the dialysate. When corresponding experiments were performed with prazosin-pretreated animals, small increases in the levels of 2,3-DHBA products were observed ŽFig. 3.. Therefore, in the presence of prazosin Ž10 mM., tyramine failed to increase 2,3-DHBA formation in the
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dialysate. This result suggests that prazosin, a Naq channel blocker, may attenuate POH generation in the rat myocardium. Although the mechanism of POH generation in the ischemic heart is obscure, we previously found a concomitant increase of noradrenaline and POH generation on myocardiac injury ŽObata et al., 1994.. Numerous investigators have reported that myocardial ischemia also increases catecholamine levels ŽDart et al., 1984; Schomig et ¨ al., 1984; Rona, 1985; Schomig et al., 1987.. When the ¨ ischemic arrhythmia appeared, noradrenaline and free radicals were found in ischemiarreperfused rat heart ŽObata et al., 1994.. Noradrenaline release by ischemiarreperfusion of the myocardium may be associated with oxidant damage due to POH generation in the heart. These results suggest that noradrenaline release in the extracellular fluid may play a key role in the generation of POH radicals in the heart. Prazosin was shown to have a POH scavenging effect ŽAkahira et al., 1998.. Thus, these results of our observations using an in vivo microdialysis technique are consistent with the finding that prazosin attenuates the reactive oxygen species in isolated rat cardiomyocytes ŽAkahira et al., 1998.. Increases in intracellular Naq levels could be caused by a modified Naq channel function ŽVer Donck and Borgers, 1991; Wermelskirchen et al., 1991.. In the presence of prazosin Ž10 mM., or during ischemiarreperfusion, tyramine failed to increase 2,3-dihydroxylation of salicylate in the cardiac microdialysis perfusion ŽFig. 4.. The present results seem to indicate that ischemiarreperfusion of myocardium may significantly elevate the level of noradrenaline through oxidant damage during POH generation. The results of the present study demonstrated that prazosin attenuates the endogenous noradrenaline-induced POH generation, suggesting that it protects from ischemiarreperfusion-induced POH generation partly through the radical scavenging action or its antioxidant action. In fact, prazosin had a POH radical scavenging effect. The contribution of the Naq channel blocking activity of prazosin to attenuation of the ischemiarreperfusion-induced damage remains to be studied. During myocardial ischemia, reduced ATP production generates disturbances in intracellular ion homeostasis, which ultimately lead to cellular Ca2q overload and contractile failure. Ca2q overload is then considered to ensue via activation of the Naq–Ca2q exchange due to the increase in cytosolic Naq content under ischemic conditions. Intracellular Ca2q overload is then considered to lead to cell death under pathological conditions such as ischemiarreperfusion injury ŽFarber, 1982; Tani, 1990.. These results suggest that Naq-mediated Ca2q overload may be relevant to ischemiarreperfusion Ca2q overload ŽVer Donck and Borgers, 1991.. This is a new cardioprotective principle. Although free radicals are a part of normal metabolism, we found that a mechanism other than the improvement of the myocardium oxygen balance contributes to the protective effect of prazosin against ischemiarreperfusion dam-
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age. Microdialysis heart perfusion experiments using the hydroxylation of salicylate to detect POH generation may be useful to answer some of the fundamental questions concerning the relevance of oxidant damage in the pathogenesis of heart disorders such as infarction.
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