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Mechanisms of bradykinin-induced catecholamine release in pithed spontaneously hypertensive rats
Immunopharmacology 44 Ž1999. 99–104 www.elsevier.comrlocaterimmpharm
Mechanisms of bradykinin-induced catecholamine release in pithed spontaneously hypertensive rats Andreas Dendorfer ) , Meike Fitschen, Walter Raasch, Klaus Tempel, Peter Dominiak Institute of Pharmacology, Medical UniÕersity of Lubeck, Ratzeburger Allee 160, D-23538 Lubeck, Germany ¨ ¨ Accepted 3 May 1999
Abstract Kinins have the potential to modulate the sympathetic system. However, the kinin receptor subtypes and secondary mediators involved in vivo are not fully characterized. Earlier studies failed to show complete inhibition by B1- or B 2-antagonists of bradykinin-induced catecholamine release, and were impeded by direct stimulatory actions of those substances. Such effects may arise from the involvement of histamine, the release of which is known to be stimulated by bradykinin and kinin-receptor antagonists. The present study was designed to evaluate the significance of B 2-receptors and histamine in the bradykinin-induced enhancement of plasma catecholamines in pithed spontaneously hypertensive rats. The effects of bradykinin, the B 2-receptor antagonist HOE 140, histamine and the H 1-receptor antagonist mepyramine were tested. Administration of histamine dose-dependently increased catecholamine release whereby a marked preference for adrenaline over noradrenaline was seen. The H 1-receptor antagonist mepyramine Ž0.3 mgrkg. prevented this effect. Bradykinin Ž7.2 mgrkg. enhanced plasma adrenaline and noradrenaline. In doses G 10 mgrkg, HOE 140 completely suppressed the bradykinin-induced increase in plasma noradrenaline, while a slight stimulation of adrenaline that even persisted after a high dose of HOE 140 Ž100 mgrkg., was only abolished by additional administration of mepyramine Ž0.3 mgrkg.. The H 1-receptor antagonist at this dose did not influence the effectivity of bradykinin. It is concluded that the bradykinin-induced enhancement of catecholamine release during electrical stimulation is completely Žnoradrenaline. or predominantly Žadrenaline. mediated by B 2-receptors. A minor stimulating effect of bradykinin on plasma adrenaline is provoked independently of B 2-receptors via histamine acting on H 1-receptors. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Bradykinin; Histamine; Catecholamines; B 2-receptor; H 1 -receptor; Pithed rat
1. Introduction Since it became known that the effectivity of endogenous and exogenous bradykinin is enhanced AbbreÕiations: ACE s angiotensin I-converting enzyme; HPLC s high performance liquid chromatography; PE s polyethylene; SHR sspontaneously hypertensive rat ) Corresponding author. Tel.: q49-451-500-2684r2680; fax: q49-451-500-3327; e-mail: [email protected]
during ACE-inhibitor therapy, evidence has appeared confirming that kinins can modulate the activity of the sympathetic nervous system and the extent of catecholamine release ŽDominiak, 1993.. The functional aspects of the bradykinin-dependent alterations, however, are still a subject of controversial discussion. It has been clearly demonstrated in neuronal cells and isolated organ preparations that bradykinin stimulates catecholamine release by acting on presynaptic B 2-receptors ŽChulak et al., 1995;
0162-3109r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 2 - 3 1 0 9 Ž 9 9 . 0 0 1 1 4 - 9
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Dendorfer and Dominiak, 1995; Warashina, 1997.. In more complex systems, however, bradykinin can enhance sympathetic activity indirectly via its hypotensive or nociceptive effects ŽKuo and Keeton, 1991., while other bradykinin-induced mediators such as NO or prostacyclin ŽPGI 2 . have been shown to attenuate catecholamine release ŽStarke et al., 1977; Schwieler et al., 1993.. Especially studies in whole animal models which may disclose interactions between various regulatory mechanisms, are impeded by non-specific properties or insufficient potencies of kinin receptor antagonists. In the pithed rat, which provides a model of an intact peripheral sympathetic nervous system, the bradykinin-induced stimulation of catecholamine release was found to be attenuated but not completely abolished by the B 2-receptor antagonist HOE 140 ŽDendorfer et al., 1996., thus indicating the possible existence of additional stimulatory pathways. However, even an involvement of the B 1-receptor subtype could not be clearly determined, since the respective antagonist, desArg 9-wLeu8 x-bradykinin, exerted stimulatory effects on plasma adrenaline on its own, an effect that was likewise observed for some B 2antagonists ŽDominiak et al., 1992; Dendorfer and Dominiak, 1995.. Since bradykinin and bradykinin antagonists are known to release histamine from mast cells ŽBueb et al., 1990; Vietinghoff et al., 1996., and histamine itself is a potent catecholamine-releasing substance ŽStaszewska-Barczak and Vane, 1965; Borges, 1994; Ries and Fuder, 1994; Choi et al., 1995., an involvement of histamine in the bradykinin-induced sympathetic activation was hypothesized. In the present study, we therefore characterized the sympathofacilitatory effect of histamine in spontaneously hypertensive rats ŽSHR. and tried to assess whether histamine contributes to the B 2-dependent and the possibly B 2-independent mechanisms by which bradykinin stimulates catecholamine release in pithed SHR.
2. Material and methods 2.1. Experimental protocol Male SHR ŽCharles River, Sulzfeld, Germany. weighing 200–250 g were pithed under ether anes-
thesia using a steel rod Ž1.5 mm diameter. coated with enamel except for the length of the thoracolumbar spinal cord ŽTh4–Th12 segment. as described by Gillespie and Muir Ž1967.. Both vagus nerves were cut at the neck and neuromuscular junctions were blocked by D-tubocurarine Ž3 mgrkg.. Drugs were infused through a PE10 catheter placed into a femoral vein and blood pressure was monitored in the right carotid artery. Rats were pre-treated with ramiprilat Ž0.1 mgrkg. and the neutral endopeptidase inhibitor phosphoramidon Ž4 mgrkg. to prevent bradykinin degradation, and with the up-take1 inhibitor desipramine Ž0.5 mgrkg. and the a 2-adrenoreceptor blocker rauwolscine Ž1 mgrkg. to enhance plasma catecholamine levels. After an equilibration period, drugs Žbradykinin, histamine or NaCl. were infused over 6 min and the sympathetic outflow was induced by preganglionic electrical stimulation Ž10 V, 0.5 Hz, 1 ms. during the last 3 min of infusion. At the end of each stimulation period, 500 ml blood samples were obtained from a catheter in the left carotid artery and stabilized with 20 ml of a solution containing 0.2 M reduced glutathione and 0.25 M EDTA. Plasma catecholamines as an index of sympathetic outflow were measured by HPLC and electrochemical detection ŽEriksson and Persson, 1982.. Prior to any stimulation, rats were pre-treated with the appropriate antagonists ŽHOE 140 andror mepyramine in 0.9% NaCl.. An initial control stimulation was performed followed by a second stimulation in the presence of the respective activator of catecholamine release Žbradykinin or histamine in 0.9% NaCl.. Only one dose of antagonist was tested in each experiment. For determination of the dose– response relationship, histamine was applied in increasing doses during five sequential stimulations. 2.2. Substances The B 2-receptor antagonist HOE 140 and the ACE inhibitor ramiprilat were generous gifts from Hoechst Marion Roussell ŽFrankfurt, Germany.. Bradykinin, histamine, D-tubocurarine, desipramine, mepyramine and phosphoramidon were obtained from Sigma ŽDeisenhofen, Germany., and rauwolscine was purchased from Carl Roth ŽKarlsruhe, Germany..
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2.3. Statistics Data are given as means " SEM calculated from 12 independent experiments. Differences between consecutive stimulations were evaluated by using the Student’s t-test for paired data at a significance level of p - 0.05. The effects of mepyramine on bradykinin-stimulated plasma catecholamines in different treatment groups were tested by ANOVA in combination with Dunnet’s post-hoc test.
3. Results 3.1. Effect of histamine on plasma catecholamines during preganglionic electrical stimulation Consecutive doses of histamine from 10 to 1000 mgrkg or corresponding volumes of 0.9% saline were administered to pithed SHR. Histamine at a dose of 1 mg kgy1 6 miny1 resulted in a 5.5-fold
Fig. 1. Effect of histamine on catecholamine release during preganglionic electrical stimulation Ž10 V, 1 ms, 0.5 Hz, 3 min.. Plasma concentrations of adrenaline ŽA. and noradrenaline ŽB. after consecutive applications of histamine Ž`. or saline Žv . are depicted. Data shown are means"SEM. U p- 0.05 vs. control Ž ns12..
Fig. 2. Inhibition of histamine-induced catecholamine release by the H 1 -antagonist mepyramine. Plasma concentrations of adrenaline ŽA. and noradrenaline ŽB. during infusion of histamine Ž1 mg kgy1 6 miny1 . and preganglionic electric stimulation Ž10 V, 1 ms, 0.5 Hz, 3 min. are depicted. Rats were pre-treated with mepyramine Ž0.3 mgrkg, filled columns. or saline Žhatched columns.. Data shown are means"SEM. U p- 0.05 vs. control Ž ns12..
increase in adrenaline and a 1.4-fold increase in noradrenaline levels ŽFig. 1.. A higher dose of histamine Ž3333 mg kgy1 6 miny1 . led to arrhythmias and irreversible hypotension Ždata not shown.. A bolus of the H 1-receptor antagonist mepyramine prior to each infusion of histamine Ž1 mg kgy1 6 miny 1 . significantly reduced plasma catecholamine concentrations during preganglionic electrical stimulation ŽFig. 2.. Progressively increasing doses of mepyramine were tested and 0.3 mgrkg was the most effective dose Ždata not shown..
Fig. 3. Absence of effect of the B 2 -antagonist HOE 140 on catecholamine release during preganglionic electrical stimulation Ž10 V, 1 ms, 0.5 Hz, 3 min.. Plasma concentrations of adrenaline ŽA. and noradrenaline ŽB. after pre-treatment with either HOE 140 Ž1 mgrkg, filled columns. or saline Žhatched columns. are depicted. No significant differences were found between the two treatment groups. Data shown are means"SEM Ž ns12..
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miny1 . increased the plasma levels of adrenaline Ž200-fold. and noradrenaline Ž5-fold. ŽFig. 4.. Compared with histamine, bradykinin was a much more effective catecholamine liberator. A single bolus of HOE 140 given prior to the infusion of bradykinin, dose-dependently suppressed the bradykinin-induced stimulation of plasma noradrenaline ŽFig. 4.. At doses of 10 mgrkg or higher, HOE 140 abolished the effect of bradykinin on noradrenaline release completely. On the other hand, bradykinin-induced adrenaline plasma levels were not completely suppressed by HOE 140, even at a dose of 100 mgrkg. However, additional administration of mepyramine at a dose sufficient to antagonize exogenously applied histamine Ž0.3 mgrkg. did result in a complete attenuation of the bradykinin-induced effect on plasma adrenaline levels.
Fig. 4. Dose-dependent inhibition of bradykinin-induced catecholamine release by the B 2 -antagonist HOE 140 and the H 1 antagonist mepyramine. Plasma concentrations of adrenaline ŽA. and noradrenaline ŽB. during preganglionic electrical stimulation Ž10 V, 1 ms, 0.5 Hz, 3 min. before Žhatched columns. and after Žfilled columns. administration of bradykinin Ž7.2 mg kgy1 6 miny1 . are depicted. Rats were pre-treated with the indicated doses of HOE 140 or 100 mgrkg HOE 140 in addition to 0.3 mgrkg mepyramine Ž100qM.. Doses G10 mgrkg HOE 140 completely suppressed the bradykinin-induced release of noradrenaline. A stimulatory effect of bradykinin on plasma adrenaline which persisted even after application of 100 mgrkg HOE 140 was abolished by mepyramine. Data shown are means"SEM. U p- 0.05 vs. control Ž ns12 for each inhibitor condition..
3.2. Influence of HOE 140 on bradykinin-induced catecholamine release In the absence of exogenous bradykinin, the specific and highly selective B 2-receptor antagonist HOE 140 Ž1 mgrkg. did not influence stimulation-dependent plasma catecholamine concentrations as compared to an independent control group ŽFig. 3.. Intravenous administration of bradykinin Ž7.2 mg kgy1 6
Fig. 5. Influence of the H 1-receptor antagonist mepyramine on bradykinin-induced catecholamine release. Plasma concentrations of adrenaline ŽA. and noradrenaline ŽB. during preganglionic electrical stimulation Ž10 V, 1 ms, 0.5 Hz, 3 min. before Žhatched column. and after Žfilled column. administration of bradykinin Ž7.2 mg kgy1 6 miny1 . are depicted. Rats were pre-treated with the indicated doses of mepyramine. Data shown are means"SEM. U p- 0.05 vs. individual control, apF 0.05 vs. control treatment group Ž ns12 for each treatment group..
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3.3. Influence of mepyramine on basal and bradykinin-induced catecholamine release With the aim of determining whether histamine might also participate in the B 2-mediated stimulation of catecholamine release, basal and bradykininstimulated catecholamine levels were measured after pre-treatment with the H 1-antagonist mepyramine at increasing doses ŽFig. 5.. A 0.3-mgrkg dose of mepyramine did neither reduce basal nor bradykininstimulated catecholamine release. Only a substantially higher dose of mepyramine Ž3 mgrkg. could lower the bradykinin-induced plasma catecholamines. This effect, however, was non-specific, since mepyramine on its own also affected basal catecholamine release during electrical stimulation.
4. Discussion Recent studies suggest that bradykinin, at least during ACE-inhibitor therapy, contributes to the modulation of catecholamine release ŽDominiak, 1993.. Investigations on the intact sympathetic system of the pithed SHR have revealed that bradykinin and desArg 9-bradykinin, as well as B 1- and B 2-receptor antagonists, can exert stimulatory effects on catecholamine release ŽDominiak et al., 1992.. Thus, the receptor subtypes and secondary mediators involved in the stimulatory actions of bradykinin have not been clearly delineated and a contribution of non-specific mechanisms has been suspected. The present study addressed the question as to the involvement of B 2-receptors and the secondary mediator histamine in the bradykinin-induced stimulation of the peripheral sympathetic system in the pithed SHR. Under the conditions of effective kininase inhibition, exogenous bradykinin potentiates the release of catecholamines during electrical preganglionic stimulation. Bradykinin is more effective compared to histamine, whereas both substances preferentially release adrenaline. Doses G 10 mgrkg of the B 2-receptor antagonist HOE 140 are able to completely abolish the bradykinin-induced increase in plasma noradrenaline, and to inhibit, although incompletely, the increase in plasma adrenaline. Therefore, exogenous bradykinin induces sympathetic activation in the pithed SHR essentially via
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B 2-receptors which are most likely located on peripheral neurons and chromaffin cells. In view of the effective inhibitory actions of HOE 140, a significant involvement of the B 1-receptor subtype can be excluded. The failure of HOE 140 to influence plasma catecholamine levels during electrical stimulation alone also demonstrates that endogenous kinins do not exist in sufficient amounts to stimulate catecholamine release. Furthermore our data confirm the earlier observation of an incomplete suppression of bradykinin-stimulated catecholamine release by 1 mgrkg HOE 140 ŽDendorfer et al., 1996., and also show that this dose is insufficient to provoke an optimum inhibition of presynaptic kinin actions. The hypothesis that a liberation of histamine might be involved in the B 2-independent activation of adrenaline was derived from observations on the stimulating activities of some B 1- and B 2-antagonists ŽdesArg 9-wLeu8 x-BK, HOE 890307, wThi 5,8 , D-Phe7 xBK. on catecholamine release ŽDominiak et al., 1992; Dendorfer and Dominiak, 1995.. Since these compounds are known to be strong releasers of histamine from rat mast cells, this effect provided a rational explanation. Compared to these antagonists, the histamine releasing activity of bradykinin is even stronger and not mediated by B 2-receptors, whereas HOE 140 only has a slight effect on histamine liberation ŽVietinghoff et al., 1996.. Indeed, the failure of bradykinin to increase plasma catecholamines during combined blockade of the B 2- and H 1-receptors with mepyramine and HOE 140 indicated that such non-specific mechanisms contribute to the stimulatory effect of bradykinin on adrenaline release. We have shown that histamine is able to increase plasma catecholamines in the pithed rat via the H 1receptor. Since it is not impossible that bradykinin could also stimulate histamine release via a B 2-dependent mechanism, we also wondered whether such an effect could contribute to the overall B 2-mediated activation of catecholamine release which is of predominant importance. Such a mechanism, however, could be excluded since mepyramine, at a dose sufficient to block the effect of exogenous histamine completely, did not influence the bradykinin-induced stimulation of catecholamine release. In summary, exogenous bradykinin stimulates catecholamine release in the pithed SHR mainly via B 2-receptors. A minor component of the bradyki-
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nin-stimulated release of adrenaline becomes apparent after complete B 2-receptor blockade and is mediated via H 1-receptors, probably involving a nonspecific, bradykinin-induced liberation of histamine. Histamine, on the other hand, does not take part in the B 2-mediated sympathoadrenal activation provoked by bradykinin. Acknowledgements The authors would like to thank Dr. J.P. Keogh for his editing and critical review of the manuscript. References Borges, R., 1994. Histamine H 1 receptor activation mediates the preferential release of adrenaline in the rat adrenal gland. Life Sci. 54, 631–640. Bueb, J.L., Mousli, M., Bronner, C., Rouot, B., Landry, Y., 1990. Activation of Gi-like proteins, a receptor-independent effect of kinins in mast cells. Mol. Pharmacol. 38, 816–822. Choi, A.Y., Fukui, H., Perlmann, R.L., 1995. Glucocorticoids enhance histamine-evoked catecholamine secretion from bovine chromaffin cells. J. Neurochem. 64, 206–212. Chulak, C., Couture, R., Foucart, S., 1995. Modulatory effect of bradykinin on the release of noradrenaline from rat isolated atria. Br. J. Pharmacol. 115, 330–334. Dendorfer, A., Dominiak, P., 1995. Characterisation of bradykinin receptors mediating catecholamine release in PC12 cells. Naunyn-Schmiedeberg’s Arch. Pharmacol. 351, 274–281. Dendorfer, A., Hauser, W., Falias, D., Dominiak, P., 1996. ¨ Bradykinin increases catecholamine release via B 2 receptors. Pflugers Arch.–Eur. J. Physiol. 432, 99–106.
Dominiak, P., 1993. Modulation of sympathetic control by ACE inhibitors. Eur. Heart J. 14, 169–172, Suppl. I. Dominiak, P., Simon, M., Blochl, A., Brenner, P., 1992. Modula¨ tion of presynaptic sympathetic activity by kinins and related compounds: influence of converting enzyme inhibition. Agents Actions Suppl. 38 ŽIII., 52–61. Eriksson, B.M., Persson, B.A., 1982. Determination of catecholamines in rat heart tissue and plasma samples by liquid chromatography with electrochemical detection. J. Chromatogr. 228, 143–154. Gillespie, J.S., Muir, T.C., 1967. A method of stimulating the complete sympathetic outflow from the spinal cord to blood vessels in the pithed rat. Br. J. Pharmacol. Chemother. 30, 78–87. Kuo, Y.J.J., Keeton, T.K., 1991. Captopril increases norepinephrine spillover rate in conscious spontaneously hypertensive rats. J. Pharmacol. Exp. Ther. 258, 223–231. Ries, P., Fuder, H., 1994. Differential effects on sympathetic neurotransmission of mast cell degranulation by compound 48r80 or antigen in the rat isolated perfused heart. Methods Find. Exp. Clin. Pharmacol. 16, 419–435. Schwieler, J.H., Kahan, T., Nussberger, J., Hjemdahl, P., 1993. Converting enzyme inhibition modulates sympathetic neurotransmission in vivo via multiple mechanisms. Am. J. Physiol. 264, 631–637. Starke, K., Peskar, B.A., Schumacher, P.A., Taube, H.D., 1977. Bradykinin and postganglionic sympathetic transmission. Naunyn-Schmiedeberg’s Arch. Pharmacol. 299, 23–32. Staszewska-Barczak, J., Vane, J.R., 1965. The release of catecholamines from the adrenal medulla by histamine. Br. J. Pharmacol. 25, 728–742. Vietinghoff, G., Paegelow, I., Reissmann, S., 1996. Induction of histamine release from rat mast cells by bradykinin analogues. Peptides 17, 1467–1470. Warashina, A., 1997. Potentiation by indomethacin of receptormediated catecholamine secretion in rat adrenal medulla. Jpn. J. Pharmacol. 73, 197–205.
Mechanisms of bradykinin-induced catecholamine release in pithed spontaneously hypertensive rats Andreas Dendorfer ) , Meike Fitschen, Walter Raasch, Klaus Tempel, Peter Dominiak Institute of Pharmacology, Medical UniÕersity of Lubeck, Ratzeburger Allee 160, D-23538 Lubeck, Germany ¨ ¨ Accepted 3 May 1999
Abstract Kinins have the potential to modulate the sympathetic system. However, the kinin receptor subtypes and secondary mediators involved in vivo are not fully characterized. Earlier studies failed to show complete inhibition by B1- or B 2-antagonists of bradykinin-induced catecholamine release, and were impeded by direct stimulatory actions of those substances. Such effects may arise from the involvement of histamine, the release of which is known to be stimulated by bradykinin and kinin-receptor antagonists. The present study was designed to evaluate the significance of B 2-receptors and histamine in the bradykinin-induced enhancement of plasma catecholamines in pithed spontaneously hypertensive rats. The effects of bradykinin, the B 2-receptor antagonist HOE 140, histamine and the H 1-receptor antagonist mepyramine were tested. Administration of histamine dose-dependently increased catecholamine release whereby a marked preference for adrenaline over noradrenaline was seen. The H 1-receptor antagonist mepyramine Ž0.3 mgrkg. prevented this effect. Bradykinin Ž7.2 mgrkg. enhanced plasma adrenaline and noradrenaline. In doses G 10 mgrkg, HOE 140 completely suppressed the bradykinin-induced increase in plasma noradrenaline, while a slight stimulation of adrenaline that even persisted after a high dose of HOE 140 Ž100 mgrkg., was only abolished by additional administration of mepyramine Ž0.3 mgrkg.. The H 1-receptor antagonist at this dose did not influence the effectivity of bradykinin. It is concluded that the bradykinin-induced enhancement of catecholamine release during electrical stimulation is completely Žnoradrenaline. or predominantly Žadrenaline. mediated by B 2-receptors. A minor stimulating effect of bradykinin on plasma adrenaline is provoked independently of B 2-receptors via histamine acting on H 1-receptors. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Bradykinin; Histamine; Catecholamines; B 2-receptor; H 1 -receptor; Pithed rat
1. Introduction Since it became known that the effectivity of endogenous and exogenous bradykinin is enhanced AbbreÕiations: ACE s angiotensin I-converting enzyme; HPLC s high performance liquid chromatography; PE s polyethylene; SHR sspontaneously hypertensive rat ) Corresponding author. Tel.: q49-451-500-2684r2680; fax: q49-451-500-3327; e-mail: [email protected]
during ACE-inhibitor therapy, evidence has appeared confirming that kinins can modulate the activity of the sympathetic nervous system and the extent of catecholamine release ŽDominiak, 1993.. The functional aspects of the bradykinin-dependent alterations, however, are still a subject of controversial discussion. It has been clearly demonstrated in neuronal cells and isolated organ preparations that bradykinin stimulates catecholamine release by acting on presynaptic B 2-receptors ŽChulak et al., 1995;
0162-3109r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 2 - 3 1 0 9 Ž 9 9 . 0 0 1 1 4 - 9
100
A. Dendorfer et al.r Immunopharmacology 44 (1999) 99–104
Dendorfer and Dominiak, 1995; Warashina, 1997.. In more complex systems, however, bradykinin can enhance sympathetic activity indirectly via its hypotensive or nociceptive effects ŽKuo and Keeton, 1991., while other bradykinin-induced mediators such as NO or prostacyclin ŽPGI 2 . have been shown to attenuate catecholamine release ŽStarke et al., 1977; Schwieler et al., 1993.. Especially studies in whole animal models which may disclose interactions between various regulatory mechanisms, are impeded by non-specific properties or insufficient potencies of kinin receptor antagonists. In the pithed rat, which provides a model of an intact peripheral sympathetic nervous system, the bradykinin-induced stimulation of catecholamine release was found to be attenuated but not completely abolished by the B 2-receptor antagonist HOE 140 ŽDendorfer et al., 1996., thus indicating the possible existence of additional stimulatory pathways. However, even an involvement of the B 1-receptor subtype could not be clearly determined, since the respective antagonist, desArg 9-wLeu8 x-bradykinin, exerted stimulatory effects on plasma adrenaline on its own, an effect that was likewise observed for some B 2antagonists ŽDominiak et al., 1992; Dendorfer and Dominiak, 1995.. Since bradykinin and bradykinin antagonists are known to release histamine from mast cells ŽBueb et al., 1990; Vietinghoff et al., 1996., and histamine itself is a potent catecholamine-releasing substance ŽStaszewska-Barczak and Vane, 1965; Borges, 1994; Ries and Fuder, 1994; Choi et al., 1995., an involvement of histamine in the bradykinin-induced sympathetic activation was hypothesized. In the present study, we therefore characterized the sympathofacilitatory effect of histamine in spontaneously hypertensive rats ŽSHR. and tried to assess whether histamine contributes to the B 2-dependent and the possibly B 2-independent mechanisms by which bradykinin stimulates catecholamine release in pithed SHR.
2. Material and methods 2.1. Experimental protocol Male SHR ŽCharles River, Sulzfeld, Germany. weighing 200–250 g were pithed under ether anes-
thesia using a steel rod Ž1.5 mm diameter. coated with enamel except for the length of the thoracolumbar spinal cord ŽTh4–Th12 segment. as described by Gillespie and Muir Ž1967.. Both vagus nerves were cut at the neck and neuromuscular junctions were blocked by D-tubocurarine Ž3 mgrkg.. Drugs were infused through a PE10 catheter placed into a femoral vein and blood pressure was monitored in the right carotid artery. Rats were pre-treated with ramiprilat Ž0.1 mgrkg. and the neutral endopeptidase inhibitor phosphoramidon Ž4 mgrkg. to prevent bradykinin degradation, and with the up-take1 inhibitor desipramine Ž0.5 mgrkg. and the a 2-adrenoreceptor blocker rauwolscine Ž1 mgrkg. to enhance plasma catecholamine levels. After an equilibration period, drugs Žbradykinin, histamine or NaCl. were infused over 6 min and the sympathetic outflow was induced by preganglionic electrical stimulation Ž10 V, 0.5 Hz, 1 ms. during the last 3 min of infusion. At the end of each stimulation period, 500 ml blood samples were obtained from a catheter in the left carotid artery and stabilized with 20 ml of a solution containing 0.2 M reduced glutathione and 0.25 M EDTA. Plasma catecholamines as an index of sympathetic outflow were measured by HPLC and electrochemical detection ŽEriksson and Persson, 1982.. Prior to any stimulation, rats were pre-treated with the appropriate antagonists ŽHOE 140 andror mepyramine in 0.9% NaCl.. An initial control stimulation was performed followed by a second stimulation in the presence of the respective activator of catecholamine release Žbradykinin or histamine in 0.9% NaCl.. Only one dose of antagonist was tested in each experiment. For determination of the dose– response relationship, histamine was applied in increasing doses during five sequential stimulations. 2.2. Substances The B 2-receptor antagonist HOE 140 and the ACE inhibitor ramiprilat were generous gifts from Hoechst Marion Roussell ŽFrankfurt, Germany.. Bradykinin, histamine, D-tubocurarine, desipramine, mepyramine and phosphoramidon were obtained from Sigma ŽDeisenhofen, Germany., and rauwolscine was purchased from Carl Roth ŽKarlsruhe, Germany..
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2.3. Statistics Data are given as means " SEM calculated from 12 independent experiments. Differences between consecutive stimulations were evaluated by using the Student’s t-test for paired data at a significance level of p - 0.05. The effects of mepyramine on bradykinin-stimulated plasma catecholamines in different treatment groups were tested by ANOVA in combination with Dunnet’s post-hoc test.
3. Results 3.1. Effect of histamine on plasma catecholamines during preganglionic electrical stimulation Consecutive doses of histamine from 10 to 1000 mgrkg or corresponding volumes of 0.9% saline were administered to pithed SHR. Histamine at a dose of 1 mg kgy1 6 miny1 resulted in a 5.5-fold
Fig. 1. Effect of histamine on catecholamine release during preganglionic electrical stimulation Ž10 V, 1 ms, 0.5 Hz, 3 min.. Plasma concentrations of adrenaline ŽA. and noradrenaline ŽB. after consecutive applications of histamine Ž`. or saline Žv . are depicted. Data shown are means"SEM. U p- 0.05 vs. control Ž ns12..
Fig. 2. Inhibition of histamine-induced catecholamine release by the H 1 -antagonist mepyramine. Plasma concentrations of adrenaline ŽA. and noradrenaline ŽB. during infusion of histamine Ž1 mg kgy1 6 miny1 . and preganglionic electric stimulation Ž10 V, 1 ms, 0.5 Hz, 3 min. are depicted. Rats were pre-treated with mepyramine Ž0.3 mgrkg, filled columns. or saline Žhatched columns.. Data shown are means"SEM. U p- 0.05 vs. control Ž ns12..
increase in adrenaline and a 1.4-fold increase in noradrenaline levels ŽFig. 1.. A higher dose of histamine Ž3333 mg kgy1 6 miny1 . led to arrhythmias and irreversible hypotension Ždata not shown.. A bolus of the H 1-receptor antagonist mepyramine prior to each infusion of histamine Ž1 mg kgy1 6 miny 1 . significantly reduced plasma catecholamine concentrations during preganglionic electrical stimulation ŽFig. 2.. Progressively increasing doses of mepyramine were tested and 0.3 mgrkg was the most effective dose Ždata not shown..
Fig. 3. Absence of effect of the B 2 -antagonist HOE 140 on catecholamine release during preganglionic electrical stimulation Ž10 V, 1 ms, 0.5 Hz, 3 min.. Plasma concentrations of adrenaline ŽA. and noradrenaline ŽB. after pre-treatment with either HOE 140 Ž1 mgrkg, filled columns. or saline Žhatched columns. are depicted. No significant differences were found between the two treatment groups. Data shown are means"SEM Ž ns12..
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miny1 . increased the plasma levels of adrenaline Ž200-fold. and noradrenaline Ž5-fold. ŽFig. 4.. Compared with histamine, bradykinin was a much more effective catecholamine liberator. A single bolus of HOE 140 given prior to the infusion of bradykinin, dose-dependently suppressed the bradykinin-induced stimulation of plasma noradrenaline ŽFig. 4.. At doses of 10 mgrkg or higher, HOE 140 abolished the effect of bradykinin on noradrenaline release completely. On the other hand, bradykinin-induced adrenaline plasma levels were not completely suppressed by HOE 140, even at a dose of 100 mgrkg. However, additional administration of mepyramine at a dose sufficient to antagonize exogenously applied histamine Ž0.3 mgrkg. did result in a complete attenuation of the bradykinin-induced effect on plasma adrenaline levels.
Fig. 4. Dose-dependent inhibition of bradykinin-induced catecholamine release by the B 2 -antagonist HOE 140 and the H 1 antagonist mepyramine. Plasma concentrations of adrenaline ŽA. and noradrenaline ŽB. during preganglionic electrical stimulation Ž10 V, 1 ms, 0.5 Hz, 3 min. before Žhatched columns. and after Žfilled columns. administration of bradykinin Ž7.2 mg kgy1 6 miny1 . are depicted. Rats were pre-treated with the indicated doses of HOE 140 or 100 mgrkg HOE 140 in addition to 0.3 mgrkg mepyramine Ž100qM.. Doses G10 mgrkg HOE 140 completely suppressed the bradykinin-induced release of noradrenaline. A stimulatory effect of bradykinin on plasma adrenaline which persisted even after application of 100 mgrkg HOE 140 was abolished by mepyramine. Data shown are means"SEM. U p- 0.05 vs. control Ž ns12 for each inhibitor condition..
3.2. Influence of HOE 140 on bradykinin-induced catecholamine release In the absence of exogenous bradykinin, the specific and highly selective B 2-receptor antagonist HOE 140 Ž1 mgrkg. did not influence stimulation-dependent plasma catecholamine concentrations as compared to an independent control group ŽFig. 3.. Intravenous administration of bradykinin Ž7.2 mg kgy1 6
Fig. 5. Influence of the H 1-receptor antagonist mepyramine on bradykinin-induced catecholamine release. Plasma concentrations of adrenaline ŽA. and noradrenaline ŽB. during preganglionic electrical stimulation Ž10 V, 1 ms, 0.5 Hz, 3 min. before Žhatched column. and after Žfilled column. administration of bradykinin Ž7.2 mg kgy1 6 miny1 . are depicted. Rats were pre-treated with the indicated doses of mepyramine. Data shown are means"SEM. U p- 0.05 vs. individual control, apF 0.05 vs. control treatment group Ž ns12 for each treatment group..
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3.3. Influence of mepyramine on basal and bradykinin-induced catecholamine release With the aim of determining whether histamine might also participate in the B 2-mediated stimulation of catecholamine release, basal and bradykininstimulated catecholamine levels were measured after pre-treatment with the H 1-antagonist mepyramine at increasing doses ŽFig. 5.. A 0.3-mgrkg dose of mepyramine did neither reduce basal nor bradykininstimulated catecholamine release. Only a substantially higher dose of mepyramine Ž3 mgrkg. could lower the bradykinin-induced plasma catecholamines. This effect, however, was non-specific, since mepyramine on its own also affected basal catecholamine release during electrical stimulation.
4. Discussion Recent studies suggest that bradykinin, at least during ACE-inhibitor therapy, contributes to the modulation of catecholamine release ŽDominiak, 1993.. Investigations on the intact sympathetic system of the pithed SHR have revealed that bradykinin and desArg 9-bradykinin, as well as B 1- and B 2-receptor antagonists, can exert stimulatory effects on catecholamine release ŽDominiak et al., 1992.. Thus, the receptor subtypes and secondary mediators involved in the stimulatory actions of bradykinin have not been clearly delineated and a contribution of non-specific mechanisms has been suspected. The present study addressed the question as to the involvement of B 2-receptors and the secondary mediator histamine in the bradykinin-induced stimulation of the peripheral sympathetic system in the pithed SHR. Under the conditions of effective kininase inhibition, exogenous bradykinin potentiates the release of catecholamines during electrical preganglionic stimulation. Bradykinin is more effective compared to histamine, whereas both substances preferentially release adrenaline. Doses G 10 mgrkg of the B 2-receptor antagonist HOE 140 are able to completely abolish the bradykinin-induced increase in plasma noradrenaline, and to inhibit, although incompletely, the increase in plasma adrenaline. Therefore, exogenous bradykinin induces sympathetic activation in the pithed SHR essentially via
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B 2-receptors which are most likely located on peripheral neurons and chromaffin cells. In view of the effective inhibitory actions of HOE 140, a significant involvement of the B 1-receptor subtype can be excluded. The failure of HOE 140 to influence plasma catecholamine levels during electrical stimulation alone also demonstrates that endogenous kinins do not exist in sufficient amounts to stimulate catecholamine release. Furthermore our data confirm the earlier observation of an incomplete suppression of bradykinin-stimulated catecholamine release by 1 mgrkg HOE 140 ŽDendorfer et al., 1996., and also show that this dose is insufficient to provoke an optimum inhibition of presynaptic kinin actions. The hypothesis that a liberation of histamine might be involved in the B 2-independent activation of adrenaline was derived from observations on the stimulating activities of some B 1- and B 2-antagonists ŽdesArg 9-wLeu8 x-BK, HOE 890307, wThi 5,8 , D-Phe7 xBK. on catecholamine release ŽDominiak et al., 1992; Dendorfer and Dominiak, 1995.. Since these compounds are known to be strong releasers of histamine from rat mast cells, this effect provided a rational explanation. Compared to these antagonists, the histamine releasing activity of bradykinin is even stronger and not mediated by B 2-receptors, whereas HOE 140 only has a slight effect on histamine liberation ŽVietinghoff et al., 1996.. Indeed, the failure of bradykinin to increase plasma catecholamines during combined blockade of the B 2- and H 1-receptors with mepyramine and HOE 140 indicated that such non-specific mechanisms contribute to the stimulatory effect of bradykinin on adrenaline release. We have shown that histamine is able to increase plasma catecholamines in the pithed rat via the H 1receptor. Since it is not impossible that bradykinin could also stimulate histamine release via a B 2-dependent mechanism, we also wondered whether such an effect could contribute to the overall B 2-mediated activation of catecholamine release which is of predominant importance. Such a mechanism, however, could be excluded since mepyramine, at a dose sufficient to block the effect of exogenous histamine completely, did not influence the bradykinin-induced stimulation of catecholamine release. In summary, exogenous bradykinin stimulates catecholamine release in the pithed SHR mainly via B 2-receptors. A minor component of the bradyki-
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nin-stimulated release of adrenaline becomes apparent after complete B 2-receptor blockade and is mediated via H 1-receptors, probably involving a nonspecific, bradykinin-induced liberation of histamine. Histamine, on the other hand, does not take part in the B 2-mediated sympathoadrenal activation provoked by bradykinin. Acknowledgements The authors would like to thank Dr. J.P. Keogh for his editing and critical review of the manuscript. References Borges, R., 1994. Histamine H 1 receptor activation mediates the preferential release of adrenaline in the rat adrenal gland. Life Sci. 54, 631–640. Bueb, J.L., Mousli, M., Bronner, C., Rouot, B., Landry, Y., 1990. Activation of Gi-like proteins, a receptor-independent effect of kinins in mast cells. Mol. Pharmacol. 38, 816–822. Choi, A.Y., Fukui, H., Perlmann, R.L., 1995. Glucocorticoids enhance histamine-evoked catecholamine secretion from bovine chromaffin cells. J. Neurochem. 64, 206–212. Chulak, C., Couture, R., Foucart, S., 1995. Modulatory effect of bradykinin on the release of noradrenaline from rat isolated atria. Br. J. Pharmacol. 115, 330–334. Dendorfer, A., Dominiak, P., 1995. Characterisation of bradykinin receptors mediating catecholamine release in PC12 cells. Naunyn-Schmiedeberg’s Arch. Pharmacol. 351, 274–281. Dendorfer, A., Hauser, W., Falias, D., Dominiak, P., 1996. ¨ Bradykinin increases catecholamine release via B 2 receptors. Pflugers Arch.–Eur. J. Physiol. 432, 99–106.
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