Recently two bradykinin-receptor subtypes (B1 and B2) have been defined, based on their pharmacological properties (1,2). Activation of B2 receptors causes pronounced vasodilation, pain, bronchoconstriction, and inflammation (3,4), whereas B1 receptors appear to be involved in the pathophysiology of chronic inflammation (5,6). The well-known vasodilator activity of bradykinin, mediated by vascular bradykinin receptors of the B2 subtype, is clearly endothelium dependent. Activation of the B2 receptors may enhance the release of nitric oxide or prostacyclin, two major processes underlying the vasodilatation (7).
In addition to its vasodilator activity, bradykinin may also modulate the function of the heart, in particular under pathologic conditions such as hemorrhagic shock or severe myocardial ischemia (8). It may also counteract ventricular arrhythmias associated with reperfusion, as well as tissue damage during ischemia (9,10). Bradykinin is assumed to modulate heart rate predominantly in the sense of activation. A direct chronotropic effect of bradykinin was observed in guinea pig isolated atria, thus reflecting stimulation of the sinoatrial node (11). However, when it was injected into the sinus node artery in autonomically blocked anesthetized dogs, heart rate was found to be reduced by bradykinin and mediated by bradykinin B2 receptors (12).
The subtype of bradykinin receptors in the chronotropic effects of bradykinin in rat atria remains unclear, although a direct positive chronotropic effect has been suggested to be involved in the bradykinin-induced cardiac-output increase in anesthetized rats (13). A previous study in the pithed normotensive rat by our group demonstrated a positive chronotropic activity of bradykinin, which is mediated principally by the release of noradrenaline from peripheral sympathetic nerve terminals (14). We therefore studied the chronotropic effect of bradykinin in some detail in spontaneously beating rat isolated atria. The kinin-receptor subtypes involved in mediating the effect of bradykinin were characterized by using Lys[Leu8]Des-Arg9-bradykinin, a selective bradykinin B1-receptor antagonist (15), and Hoe 140, a selective bradykinin B2-receptor antagonist (16), respectively.
The effects of bradykinin can be modulated by inhibitors of angiotensin-converting enzyme (ACE), which is identical with kininase II and as such largely responsible for the degradation of bradykinin (17-19). The effects of bradykinin were therefore studied in preparations pretreated or not pretreated with the ACE-inhibitor ramipril. The possible roles of nitric oxide and cyclooxygenase products also were investigated by using a nitric oxide synthesis inhibitor and cyclooxygenase inhibitors.
Rat atrial preparation
Male, normotensive Wistar rats (240-280 g) were stunned by a blow on the skull and then exsanguinated. The heart was carefully dissected and placed in a Krebs-Henseleit solution containing (in mM): NaCl, 118.0; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.2; NaHCO3, 24.9; KH2PO4, 1.2; and glucose, 11.1; pH 7.4. Sutures were tied to the upper and lower tips of the right atrium, and the atrium was then dissected from the heart. The spontaneously beating atrium was suspended between a fixed end and a force transducer (UFL Co. Morro Bay, CA, U.S.A.) for the measurement of contractile force. Beating rates of the atria were derived from the contractile signal thus obtained by means of a rate meter in a MacLab/8-computer system (A.D. Instruments Ltd, London, U.K.). The atria were placed in a 10-ml organ bath filled with Krebs-Henseleit solution, kept at 37°C and gassed with 95% O2 and 5% CO2. The resting tension was set at 0.5 g during a 1-h equilibration period. Contractile force and beating rates of the spontaneously beating atria were recorded and continuously displayed with a MacLab/8-computer system (A.D. Instruments).
Cumulative concentration-response curves for the positive chronotropic effect of bradykinin were determined 30 min after the atrial preparation had been incubated with saline or one of the following antagonists: propranolol (1 μM), prazosin (1 μM), indomethacin (3 μM), meclofenamate (10 μM), Nω-nitro-L-arginine methyl ester (L-NAME; 30 μM), ramipril (30 μM), Hoe 140 (1, 3, and 10 μM) and Lys-[Leu8]Des-Arg9-bradykinin (1 μM), respectively. The beating rate was assessed 2 min after the addition of each successive half-log concentration of bradykinin. Under these conditions, tachyphylaxis to bradykinin did not occur.
In separate experiments, the effect of cumulative additions of Des-Arg9-bradykinin (1 nM-10 μM), a bradykinin B1-receptor agonist, was investigated in the right atria.
The pharmacologic agents used were bradykinin and Des-Arg9-bradykinin (Saxon Biochemicals, Hannover, Germany), Lys-[Leu8]Des-Arg9-bradykinin (Bachem, Bubendorf, Switzerland), Hoe 140 [D-Arg-(Hyp3, Thi5,D-Tic7, Oic8) bradykinin] and ramipril (Hoechst, Frankfurt, Germany), (−)-propranolol hydrochloride (Imperial Chemical Industries, Macclesfield, U.K.), indomethacin, prazosin, and Nω-nitro-L-arginine methyl ester hydrochloride (Sigma, St. Louis, MO, U.S.A.), and meclofenamate (ICN, Aurora, IL, U.S.A.). Unless otherwise specified, drugs were dissolved in distilled water. Indomethacin and prazosin were dissolved in 2% Na2CO3 and 0.5% glucose, respectively.
Changes in the spontaneous rate of beating were expressed as mean ± SEM for n preparations. The concentration-response curves for bradykinin were analyzed by means of a computer program (GraphPad Prism; GraphPad, San Diego, CA, U.S.A.), and the concentrations that produced 50% of the maximal effect (EC50) as well as the maximal effect (Emax) were thus obtained. The pKB values were calculated by using Shild regressions: log(r − 1) = log[B] − logKB. The concentration ratio (r) represents the EC50 value of the agonist in the presence of the antagonist divided by the EC50 value of the agonist in the absence of the antagonist. [B] is the concentration of the antagonist. The statistical significance of differences was analyzed by means of Student's t test, and p values of <0.05 were considered significant.
In isolated rat right atrial preparations, the basal beating rate and amplitude of contraction after equilibration amounted to 258 ± 2 beats/min and 0.54 ± 0.06 g (n = 42), respectively. The cumulative additions of bradykinin (0.3-100 nM) caused a concentration-dependent increase in the spontaneous beating rate of the right atria without altering the contractile force (Fig. 1). Bradykinin-induced maximal increase in frequency was 24.8 ± 0.8% of that induced by isoprenaline (n = 7). In contrast, Des-Arg9-bradykinin (0.1-10 μM), a bradykinin B1-receptor agonist, had no effect on the spontaneous beating rate of the atria (data not shown).
The combination of β- and α1-adrenoceptor antagonists, propranolol (1 μM) and prazosin (1 μM), slightly reduced the basal spontaneously beating rate by 7.9 ± 0.6% (p < 0.05; n = 8) but had no significant effect on the bradykinin-induced increases in frequency (Fig. 1; p > 0.05; n = 7). By contrast, the same concentration of propranolol was sufficient to block the 0.3-300 nM isoprenaline-induced positive chronotropic effect (EC50, 4.5 ± 0.3 nM and 3.8 ± 0.4 μM, in the absence and presence of propranolol, respectively; p < 0.01; n = 4).
Hoe 140 (1, 3, and 10 nM), a selective bradykinin B2-receptor antagonist, shifted the concentration-response curve for bradykinin to the right in a parallel manner without altering the maximal responses (Fig. 2). The pKB value for Hoe 140 was 9.94. The slope of the Schild plot was not significantly different from unity (1.38 ± 0.35; p > 0.05; n = 6-8). In contrast, Lys-[Leu8]Des-Arg9-bradykinin (1 μM), a selective bradykinin B1-receptor antagonist, did not significantly alter the bradykinin-induced positive chronotropic effect (Fig. 2; p > 0.05; n = 8).
The cyclooxygenase inhibitor indomethacin (3 μM) did not affect the basal rate of beating, whereas 30-min incubation with meclofenamate (10 μM) slightly reduced the basal frequency by 7.3 ± 0.4% (p < 0.05; n = 6). Both inhibitors completely blocked the bradykinin-induced positive chronotropic effect (Fig. 3).
Neither the basal spontaneously beating rate nor the bradykinin-induced tachycardic response were affected by the presence of nitric oxide synthase inhibitor L-NAME (30 μM; Fig. 3; p > 0.05; n = 6).
The inhibition of ACE/kininase II by ramipril (30 μM) did not cause significant changes of the basal spontaneously beating rate (p > 0.05; n = 8) but significantly shifted the bradykinin curves to the left (p < 0.05; n = 8) without altering the maximal responses (Fig. 4).
Early in 1963, Montague et al. (13) suggested that a direct positive chronotropic effect may be involved in the bradykinin-induced cardiac output increase observed in anesthetized rats. However, so far little knowledge has been gained on the chronotropic effect of bradykinin, although its vasodilator effect has been widely studied in detail. A previous study we performed in pithed normotensive rats showed that bradykinin elicits a positive chronotropic effect mainly by stimulating noradrenaline release from peripheral sympathetic nerve endings (14).
In this study, we demonstrated that bradykinin exhibits a direct positive chronotropic effect in rat isolated spontaneously beating atria. This chronotropic effect after bradykinin stimulation was not caused by the release of endogenous catecholamines because treatment of the rat atria with the adrenoceptor antagonists propranolol and prazosin did not affect the bradykinin-induced chronotropic effect. Several publications reported an enhanced catecholamine release induced by bradykinin (20-22). This effect appears to occur only in electrically stimulated preparations, but not with respect to the spontaneous noradrenaline release in spontaneously beating right atria.
As a nonselective agonist, bradykinin stimulates both bradykinin B1 and B2 receptors (1,23). To characterize the receptor subtype involved in the positive chronotropic effect of bradykinin, the effects of selective bradykinin B1-and B2-receptor antagonists on bradykinin-induced responses were investigated. We observed that Lys-[Leu8]Des-Arg9-bradykinin, a selective bradykinin B1-receptor antagonist (15), did not influence the effect of bradykinin, whereas Hoe 140, a selective bradykinin B2-receptor antagonist (16), concentration-dependently inhibited bradykinin-induced positive chronotropic effects, in a competitive manner. The affinity of Hoe 140 (pKB 9.94) estimated in our study is within the range of the pKB or pA2 values (8.5-11.4) obtained in other preparations from the rat (1,16,24). These results indicate that the positive chronotropic effects of bradykinin in rat right atrial preparations are mediated by bradykinin B2 receptors, whereas B1 receptors are not involved. This interpretation is further substantiated by the fact that the selective bradykinin B1-receptor agonist Des-Arg9-bradykinin (23) did not cause any change in the beating rate.
Interestingly, the positive chronotropic effect of bradykinin in rat atria can be completely abolished by the cyclooxygenase inhibitors indomethacin and meclofenamate, indicating the involvement of the arachidonic acid cascade (25). Activation of this cascade appears to be mediated by bradykinin B2 receptors. This pattern of action for bradykinin is different from that observed in isolated guinea-pig atria, in which the bradykinin B2 receptor-mediated chronotropic effect is not mediated by cyclooxygenase products (26). In electrically stimulated human and rat atrial slices (22), Rump et al. observed that indomethacin reduced bradykinin-induced facilitation of noradrenaline release, and the effect of bradykinin was mimicked by prostaglandin F2α and thromboxane but not by prostaglandin I2. However, it is unlikely that in our study, the chronotropic effect of bradykinin was attributed to the facilitatory effects of prostaglandins on noradrenaline release, because propranolol and prazosin had no effect on bradykinin-induced tachycardia. Whether prostaglandins acted on the sinus node in a direct manner and which specific prostaglandin may be involved in the chronotropic effect in rat right atria remains to be elucidated.
In rat and guinea-pig, bradykinin stimulates capsaicinsensitive sensory neurons, which leads to the release of neuropeptides such as substance P and calcitonin gene-related peptides (27,28). Whether a concurrent release of neuropeptides or galanin may contribute to the bradykinin-induced chronotropic effects should be studied in future investigations. However, calcitonin gene-related peptides cause positive chronotropic and inotropic effects in rat spontaneously beating right atria, which occurs by activation of cyclic adenosine monophosphate (cAMP) responses but is not mediated by catecholamine and prostaglandin release (29). In our study, the bradykinin-induced tachycardic responses could be abolished by the inhibition of prostaglandin synthesis, suggesting that the release of calcitonin gene-related peptides is unlikely to be involved in the bradykinin effects.
An increase in nitric oxide release was described as a potential mediator in the cardioprotective and vasodilator actions of bradykinin (10). In isolated rat atria, the lack of effect of L-NAME, a nitric oxide synthesis inhibitor, suggests that nitric oxide plays no role in the positive chronotropic effect of bradykinin.
Two major enzymes responsible for the biodegradation of bradykinin in the circulation and in the tissues are kininase I and kininase II, respectively (1). Kininase II is known to be identical to ACE (17). Many studies have reported that the effects of bradykinin were potentiated by ACE inhibitors, which break down the rapid degradation of bradykinin (21,22,26). Similarly, in our study, an increased potency of bradykinin was observed in the presence of ramipril, an inhibitor of ACE/kininase II. However, ramipril did not influence the basal beating rate, suggesting that the basal release of bradykinin is not involved in the regulation of sinus node activity.
In conclusion, in rat isolated right atria, exogenous bradykinin induces a positive chronotropic effect, which occurs independent of adrenoceptors. The bradykinin-induced chronotropic effect is mediated by bradykinin B2 receptors, whereas B1 receptors do not play a role. Prostaglandins but not nitric oxide appear to be involved in the bradykinin-induced positive chronotropic effect.
In this study, the threshold concentration of bradykinin used to stimulate the sinoatrial node appears to be 20-50 times higher than the physiological levels. Accordingly, it seems unlikely that bradykinin increases heart rate under physiological conditions. However, high plasma concentrations of bradykinin may be observed under pathologic circumstances such as severe myocardial ischemia, and the local generation at the level of the sinoatrial node may also be enhanced. Under such conditions, bradykinin may then be expected to contribute to the frequently occurring increase in heart rate.
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