(Hypertension. 2001;38:142.)
© 2001 American Heart Association, Inc.
Scientific Contributions |
Potentiation of Kinin Analogues by Ramiprilat Is Exclusively Related to Their Degradation
From Medical University of Lübeck, Institute of Experimental and Clinical Pharmacology and Toxicology (A.D., S.W., W.R., P.D.), Lübeck; and Friedrich-Schiller-University Jena, Institute of Biochemistry and Biophysics (S.R.), Jena, Germany.
Correspondence to Dr A. Dendorfer, Medical University of Lübeck, Institute of Experimental and Clinical Pharmacology and Toxicology, Ratzeburger Allee 160, D-23538 Lübeck, Germany. E-mail dendorfe{at}medinf.mu-luebeck.de
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Abstract |
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Abstract— The potentiation of kinin actions represents a cardioprotective property of ACE inhibitors. Although a clear contribution to this effect is related to the inhibition of bradykinin (BK) breakdown, the high efficacy of potentiation and the ability of ACE inhibitors to provoke a B2-receptor-mediated response even after receptor desensitization has also triggered hypotheses concerning additional mechanisms of kinin potentiation. The application of kinin analogues with enhanced metabolic stability for the demonstration of degradation-independent mechanisms of potentiation, however, has yielded inconsistent results. Therefore, the relation between the susceptibility of B2-agonists to ACE and the potentiation of their actions by ACE inhibitors was investigated with the use of minimally modified kinin derivatives that varied in their degree of ACE resistance. The B2-agonists BK, D-Arg-[Hyp3]-BK, [Hyp,3 Tyr(Me)8]-BK, [
Phe5]-BK, [D-NMF7]-BK, and [Phe8
(CH2-NH)Arg9]-BK were tested for degradation by purified rabbit ACE and for their potency in contracting the endothelium-denuded rabbit jugular vein in the absence and presence of ramiprilat. Purified ACE degraded D-Arg-[Hyp3]-BK and [Hyp,3 Tyr(Me)8]-BK at 81% and 71% of BK degradation activity, respectively, whereas other peptides were highly ([Phe5]-BK) or completely ([D-NMF7]-BK, [Phe8(CH2-NH)Arg9]-BK) resistant. The EC50 of BK-induced venoconstriction (1.15±0.2 nmol/L) was reduced by a factor of 5.7 in the presence of ramiprilat. Likewise, D-Arg-[Hyp3]-BK and [Hyp,3 Tyr(Me)8]-BK were both significantly potentiated by a factor of 4.4, whereas the activities of the other agonists were not affected. Ramiprilat exerted no influence on the maximum contraction induced by any of the agonists. It is concluded that the potentiation of kinin analogues during ACE inhibition correlates quantitatively with the susceptibility of each substance to degradation by ACE. As such, no evidence of degradation-independent potentiating actions of ACE inhibitors could be obtained.
Key Words: bradykinin • angiotensin-converting enzyme • receptors, bradykinin • kinins • rabbits
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Introduction |
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Potentiation of kinin actions is a well-known property of angiotensin I-converting enzyme inhibitors, an effect attributed to a protection of kinins against ACE enzymatic degradation. This mechanism contributes to the therapeutic spectrum of ACE inhibitors in vivo. A variety of experimental studies have demonstrated that enhancement of kinin effectiveness is responsible for beneficial influences of ACE inhibitors on the cardiovascular system, such as reduction of infarct size, inhibition of myocardial hypertrophy and fibrosis, and protection against the development of hypertension (reviewed by Linz et al1). The contribution of endogenous kinins to the vasodilatory effect of an ACE inhibitor has recently been demonstrated in humans as well.2 Such kinin-mediated effects can arise because of the high extent to which ACE inhibitors potentiate the effectiveness of kinins. In volunteers, ACE inhibitor treatment sensitized the blood pressure response to injected bradykinin (BK) by a factor of
40.3 However, attempts to correlate this intriguing effect with BK accumulation have failed because (1) plasma kinin levels increase only modestly after ACE inhibition,4 and (2) kinin potentiation by ACE inhibitors is also observed in experimental models in which kinin degradation should be negligible (eg, in superfused vessels5). This fact, and the observation that after desensitization by prolonged kinin exposure an ACE inhibitor can reestablish a B2-receptor-mediated response in the continued presence of the desensitizing kinin concentration (a phenomenon addressed as "receptor resensitization"),6,7 has triggered hypotheses of degradation-independent mechanisms by which ACE inhibitors might potentiate the actions of BK. Such proposed mechanisms include stabilization of B2 receptors in a high-affinity state,8 attenuation of receptor sequestration and internalization,8,9 and enhancement of the spontaneous activity of unoccupied B2-receptors.5
The key approach for demonstrating such degradation-independent mechanisms of kinin potentiation and for excluding the consequences of reduced kinin cleavage is to use degradation-resistant kinin analogues. In fact, both potentiation and resensitization to ACE inhibitors have been observed after application of some modified B2-agonists,5,8 but this has not been confirmed for structurally different kinin analogues.10,11 This inconsistency might be related to the various experimental conditions used or to differences between the agonists considering their individual metabolic stabilities or their particular abilities to enable potentiating mechanisms of ACE inhibitors.
With the aim of correlating these two properties of B2-agonists in a single model, we compared (1) the ability of an ACE inhibitor to alter the potencies of BK and several of its analogues in constricting the rabbit jugular vein with (2) the ability of that ACE inhibitor to inhibit breakdown of bradykinin and the same analogues by purified rabbit ACE. As well as B2-agonists that are known to be potentiated by ACE inhibitors, we also used recently described kinin analogues with only single modifications of the BK structure to minimize the risk of losing the intrinsic suitability of BK for potentiation.
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Materials and Methods |
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Organ Bath Experiments
Male New Zealand White rabbits (weight, 3 to 3.5 kg; Charles River, Sulzfeld, Germany) were anesthetized with ketamine (15 mg/kg) and xylazine (6 mg/kg). Both jugular veins were removed and denuded of endothelium by means of a coarse steel rod. The organ bath incubation conditions were adopted from previous studies on kinin potentiation by ACE inhibitors.5 In brief, vessel rings were incubated in Krebs-Henseleit solution containing diclofenac (1 µmol/L) at a resting tension of 3 mN. Experiments with and without ACE inhibitor treatment (250 nmol/L ramiprilat, 30 minutes of preincubation) were performed independently. Venoconstriction was induced by cumulative 3-fold increases in peptide concentrations starting at 1 pmol/L and applied at 4-minute intervals. The experiments were performed in accordance with rules set by the state of Schleswig-Holstein.
ACE Activity of Rabbit Jugular Vein
ACE activity was determined in 8 endothelium-intact and endothelium-denuded segments of the jugular vein obtained from 2 rabbits, following an established protocol.12 In brief, vessel rings were homogenized in 40 parts (vol/wt) buffer containing 0.01 (vol/vol) Triton X-100, aprotinin (43 µmol/L), and soybean trypsin inhibitor (2.5 µmol/L). The extract was incubated at 37°C for 60 minutes with Abz-Gly-p-nitro-Phe-Pro (83 µmol/L, pH 7.4) in the absence or presence of ramiprilat (250 nmol/L). The reaction was stopped with perchloric acid (0.3 mol/L final concentration), and the product (o-aminobenzoylglycyl) was quantified by high-performance liquid chromatography and flourimetric detection (320/412 nm). The ramiprilat-sensitive reaction was considered to represent the ACE activity. Protein content of the samples was determined by the method of Lowry et al.13
Kinin Degradation by Purified ACE
BK and kinin analogues (1 µmol/L) were incubated at 37°C with purified rabbit ACE (0.1 IU, as determined with 83 µmol/L Abz-Gly-p-nitro-Phe-Pro) in 1 mL HEPES-buffered Earle’s salt solution (pH 7.4). Aliquots were drawn at 0, 5, 10, 20, and 40 minutes and stabilized by addition of trifluoroacetic acid (120 mmol/L final concentration). Intact peptides were determined by high-performance liquid chromatography as described earlier.14 Specificity of the assay was confirmed by inclusion of ramiprilat (250 nmol/L) in separate control experiments.
Substances
Bradykinin, purified rabbit ACE, and HEPES were purchased from Sigma-Aldrich. Abz-Gly-p-nitro-Phe-Pro was obtained from Bachem. [Hyp,3 Tyr(Me)8]-BK and [Phe8(CH2-NH)Arg9]-BK were bought from Calbiochem. The BK analogues D-Arg-[Hyp3]-BK, [D-NMF7]-BK, and [Phe5]-BK were synthesized by a solid-phase method by means of the Boc-strategy.15 The specific activation of B2-receptors by these kinin analogues has been reported.11,16,17 Ramiprilat was kindly donated by Hoechst Marion Roussel. All other chemicals, of the highest analytical grade available, were obtained from Merck or Sigma-Aldrich.
Calculations and Statistics
Dose-response curves were constructed from the increases in isometric tension induced by each of the B2-agonists. Maximum tension and EC50 values were derived by nonlinear regression (Prism, GraphPad Software). For calculating degradation rates, the complete kinetics were fitted by means of a monoexponential function (BK(t)=BK0 · e-k · t, k=degradation rate). All quantitative data are given as mean±SEM. For each peptide, the parameters of venoconstriction were compared between groups with and without ramiprilat treatment with the Student’s t test. Differences were considered statistically significant at an error level of P<0.05.
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Results |
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Bradykinin and all investigated analogues induced a dose-dependent constriction of the jugular vein, the quantitative parameters of which are summarized in the Table. As exemplified in Figure 1, the potency of BK was increased by a factor of 5.7 after pretreatment with ramiprilat. A significant shift in potency by a factor of 4.4 during ACE inhibition was also observed for D-Arg-[Hyp3]-BK and [Hyp,3 Tyr(Me)8]-BK. On the other hand, the activities of the kinin analogues [
Phe5]-BK, [D-NMF7]-BK, and [Phe8(CH2-NH)Arg9]-BK were not affected, a fact that is exemplarily depicted for [D-NMF7]-BK in Figure 1. Ramiprilat exerted no influence on the maximum efficacy of any of the B2-agonists (Table).
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) significantly shifted EC50 of BK (upper graph) by factor of 5.7 compared with control stimulations (•), whereas potency of [D-NMF7]-BK was not affected (lower graph). Ramiprilat did not influence maximum contraction in response to any agonist. Data represent mean±SEM of 6 to 7 independent determinations.The intact rabbit jugular vein contained ACE at an activity of 8.4±1.4 nmol/min per gram of protein. The majority of this enzyme activity (7.7±1.9 nmol/min per gram of protein, corresponding to 92% of the intact vein) was still present in the endothelium-denuded preparation.
During in vitro incubation with purified rabbit ACE, BK was rapidly degraded, as were the peptides D-Arg-[Hyp3]-BK and [Hyp,3 Tyr(Me)8]-BK (Figure 2). A low rate of degradation was also observed for [Phe5]-BK, whereas [D-NMF7]-BK and [Phe8(CH2-NH)Arg9]-BK were completely resistant to ACE (Table). Degradation was completely blocked by inclusion of ramiprilat in the incubation assay (data not shown).
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) or [Hyp,3 Tyr(Me)8]-BK(
) nearly abolished cleavage by ACE (9% degradation compared with BK). Data represent mean±SEM of 3 independent determinations. |
Discussion |
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This study has shown that the ACE inhibitor-induced potentiation of kinin effects in the rabbit jugular vein is quantitatively correlated to the susceptibility of the individual agonist to degradation by ACE; it is completely abolished in the cases of 3 substances that are virtually or completely resistant to ACE. The rabbit jugular vein was proved to be a suitable model for the investigation of kinin potentiation by ACE inhibitors, as demonstrated in a previous study that confirmed that ramiprilat was a highly effective substance in this respect.5 In this model and in the rabbit isolated heart, a potentiation by ramiprilat not only of BK but of the B2-agonist D-Arg-[Hyp3]-BK had been observed earlier.5,6 Because of the rapid degradation of D-Arg-[Hyp3]-BK by rabbit ACE, however, this effect may not be considered as a degradation-independent action of the ACE inhibitor. Rather, a quantitative relation exists for D-Arg-[Hyp3]-BK and BK between their ACE-mediated degradation and their functional potentiation by the ACE inhibitor, which means that both phenomena are equally attenuated (by 19% and 22%, respectively) when D-Arg-[Hyp3]-BK is administered instead of BK (Figure 3). A similar relation was also demonstrated for [Hyp,3 Tyr(Me)8]-BK, indicating that the ability of a modified B2-agonist to become potentiated by an ACE inhibitor does not represent a structure-related property but is causally linked to its degradation by ACE.
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) and [Phe8
) resistant to ACE were not altered by ACE inhibitor.
These results might also influence current hypotheses concerning the potentiation of kinins by ACE inhibitors. The majority of studies that have addressed the mechanisms of degradation-independent kinin potentiation with modified B2-agonists have been performed with the substances D-Arg-[Hyp3]-BK or [Hyp,3 Tyr(Me)8]-BK,5–8,18–20 which are not resistant to rabbit ACE. Because the degradation stabilities of these substances and of [Phe8(CH2-NH)Arg9]-BK in a recent study21 have not been confirmed under the particular conditions of the respective assays, the observed effects of potentiation or resensitization by ACE inhibitors cannot be regarded as definitive proof for the involvement of degradation-independent mechanisms.
By investigating the influence of ramiprilat on ACE-resistant B2-agonists, the present study has exploited only one approach for demonstrating a degradation-independent potentiation of kinin actions. Previous investigations have identified an increased efficacy of B2 responses,5,7 a stabilization of B2-receptors in a high-affinity state,8 and a decreased internalization of B2-receptors8,9 as possible mechanisms by which ACE inhibitors in combination with ACE may enhance B2-receptor signaling. Those findings were not obtained with the use of stabilized kinin derivatives, and their interpretation is not affected by the present results. The failure of ramiprilat to potentiate stable kinins in the rabbit jugular vein only indicates that those postulated mechanisms may not have been present, properly activated, or sufficiently effective to provoke functional potentiation. Naturally, this conclusion can only be drawn for the specific kinin derivatives, ACE inhibitor, and experimental model used in our study.
Because of the lack of degradation-independent kinin potentiation in the rabbit jugular vein, the observed potentiation by ACE inhibitors can only be explained by a reduction of kinin degradation. When this line of reasoning is followed, a 5.7-fold increase in the potency of BK during ACE inhibition would reflect an equally effective increase in functional kinin concentrations at the B2-receptors. An alteration to that extent can only occur if the normal activity of ACE will reduce the availability of BK at the B2-receptors to <15% of the concentration administered. Clearly, the kinin concentration in the large volume of the organ bath cannot be lowered to this level by the ACE activity contained in the vessel preparation, as has already been confirmed from BK measurements in a similar setup.18 Therefore, it must be hypothesized that the functional B2-receptors are not in direct contact with the incubation medium but must be located behind a metabolic barrier, a situation that can also be described as a localization of B2-receptors in a compartment endowed with a highly active kinin metabolism. The actual existence of a distribution compartment featuring such properties has already been identified in rat myocardium by tracer transit studies that demonstrated a 3.1-fold increase of local BK concentrations after ACE inhibition.22 These significant local changes were not accompanied by major alterations of BK concentrations in the perfusion medium, a situation consistent with the constant overall peptide levels in the organ bath. In addition, our study has shown that the rabbit jugular vein, even in the absence of endothelium, contains sufficient ACE activity to allow substantial alterations of kinin concentrations to occur within the tissue.
Regarding an arrangement that would enable ACE to determine kinin availability at the B2-receptor site, a colocalization of both proteins may in fact exist in membrane compartments of individual cells. Properties shared by B2-receptors and ACE include a similar susceptibility of both proteins to solubilization23 and their presence within caveolae.9,24 A recent investigation has illustrated the functional significance of a colocalization of B2-receptors and ACE within the membrane.21 In that study, the association of ACE and B2-receptors was disrupted by directing ACE to cholesterol-rich domains through genetic modification of its membrane anchor; an alteration that abolished ramiprilat-induced B2-receptor resensitization. It may be hypothesized that such close proximity of ACE and B2-receptors within membrane domains, or even a direct association of the proteins,21 constitutes a minute compartment that enables an efficient kinin metabolism and allows a profound kinin potentiation to occur, although it might not be accessible for analysis of local kinin levels.
The finding that functional potentiation of B2-receptor-mediated venoconstriction is absent after stimulation with ACE-resistant kinin analogues and that it occurs as a graded phenomenon if stimulation is induced by partially stabilized peptides indicates that ramiprilat exerts no degradation-independent potentiation of kinin effects in the rabbit jugular vein.
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Acknowledgments |
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The authors thank Dr Julian P. Keogh for revising the English style of the manuscript.
Received November 30, 2000; first decision December 28, 2000; accepted January 8, 2001.
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References |
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