European Journal of Pharmacology, 216 (1992) 357-362

357

© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00

EJP 52493

Comparative study on the mechanism of bradykinin potentiation induced by bradykinin-potentiating peptide 9a, enalaprilat and kinin-potentiating peptide M o z a r t S. R o d r i g u e s , R o n y S c h a f f e l a n d J a m i l A s s r e u y Departamento de Farmacologia Basica e Clinica, ICB / CCS, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

Received 6 March 1992, accepted 24 March 1992

The action of a kinin-potentiating peptide (KPP) obtained from tryptic digestion of human serum proteins was compared with that of bradykinin-potentiating peptide 9a (BPPga; obtained from snake venom) and enalaprilat (a synthetic inhibitor of angiotensin-converting enzyme; ACE) as a means of understanding the mechanism of action of KPP on smooth muscle. KPP potentiated bradykinin-induced contractile effects in guinea-pig ileum and rat uterus, but not the bradykinin-induced relaxation of pre-contracted ileum, whereas BPP9a and enalaprilat potentiated both bradykinin effects. The receptor mediating both the contraction and the relaxation elicited by bradykinin in the ileum was found to be of the B 2 type. KPP retained its potentiating effect in the presence of enalaprilat in the guinea-pig ileum and rat uterus, whereas the potentiation evoked by BPPga was abolished. Enalaprilat inhibited the activity of purified ACE, whereas KPP was completely devoid of such an effect. The potentiating effect of KPP, but not that of BPPga or enalaprilat, was blocked by compounds that inhibit phospholipase A 2 and lipoxygenase activity but not by inhibitors of cyclo-oxygenase or phosphodiesterases. The results suggest that the potentiating effect of KPP (i) does not involve inhibition of ACE; (ii) is not due to an increased affinity of the receptor for bradykinin, and (iii) probably involves post-receptor events linked to phospholipase A 2 and to the lipoxygenase pathway. Bradykinin; Angiotensin-converting enzyme; Smooth muscle; Phospholipase A2; Lipoxygenase; Cyclic nucleotides

I. Introduction

Kinins are potent peptides released from inactive plasma protein substrates and participate in several pathophysiological conditions (for a review see Marceau et al., 1983). As a result of interaction with specific m e m b r a n e receptors, kinins trigger several post-receptor transduction steps that involve activation of phospholipases, stimulation of polyphosphoinositide turnover, opening of ion channels and activation of cyclic nucleotide synthesis. These events mediate the biological responses (see Burch et al., 1990 for a review). Kinins have a very short half-life in plasma and in tissues, in the order of seconds (Ferreira and Vane, 1967), due to inactivation by enzymatic hydrolysis. The main enzyme responsible is kininase II, a peptidyl-dipeptidase, also known as angiotensin-converting enzyme (ACE) since it also cleaves angiotensin I to angiontensin II (reviewed by Erd6s, 1979). Various classes of substances, mainly peptides, can potentiate

Correspondence to: J. Assreuy, Department of Pharmacology, ICB/CCS, UFRJ, P.O. Box 68013, 21944 - Rio de Janeiro, Brazil. Tel. 55.21.270 8588, fax 55.21.270 8647.

the biological actions of kinins. The potentiating effects of these peptides have been attributed to their ability to inhibit A C E (for a review see Stewart, 1979). Of these peptides, the best studied are those derived from snake venoms, including bradykinin-potentiating peptide 9a (BPP9a; Stewart, 1979). The study of A C E inhibition by this peptide and its analogues has led to the design and synthesis of inhibitory drugs such as captopril and enalapril, now widely used in antihypertensive therapy (Ondetti, 1988). Besides A C E inhibition, other mechanisms have been postulated for the kinin potentiation induced by some peptides. A m o n g other methods, same potentiating peptides have been suggested to increase the affinity of bradykinin (BK) receptors (Ufkes et al., 1977). We have recently described the partial purification of a kinin-potentiating peptide (KPP) derived from trypsin-digested human serum proteins. In vitro, this peptide potentiates kinin-induced effects such as contraction of guinea-pig ileum and rat uterus (Assreuy et al., 1989). In vivo, it potentiates rat paw oedema (Fernandes et al., 1991). It was observed that KPP potentiated only kinins and not other agonists, such as antiotensin II, substance P, histamine, acetylcholine and barium chloride. It was postulated that KPP po-

358 tentiation of BK-induced contraction of guinea-pig ileum might involve some other mechanism(s) besides ACE inhibition, although this effect was not completely ruled out (Assreuy et al., 1989). The present study examines some putative mechanisms for the potentiation caused by KPP, using BPP9a and enalaprilat for comparison. The results indicate that the KPP-potentiating effect is not dependent on (i) inhibition of ACE or (ii) increases in BK receptor affinity. Most importantly, the results suggest that KPP activates one or more post-receptor pathways to cause its potentiating effect.

2. Materials and methods

BK was purchased from the Department of Biophysics, Escola Paulista de Medicina, S~o Paulo, Brazil. Angiotensin II, [D-Arg °, Hyp 3, Thi 5'8, D-Phe7]BK (Hyp = hydroxyproline; Thi = thienylalanine), diethylstilbestrol, mepacrine, quercetin, acetylsalicylic acid, nordihydroguaiaretic acid (NDGA), theophylline, atropine sulphate, angiotensin-converting enzyme (ACE; EC 3.4.15.1; from rabbit lung; 3 U / m g protein) and N-(3-[2-furyl]acryloyl)Phe-Gly-Gly (FAPGG) were purchased from Sigma, USA. Naproxen was from Syntex, Brazil. BPP9a was a gift from Prof. S.H. Ferreira (Departamento de Farmacologia, Universidade de S~o Paulo, Brazil). Enalaprilat was a gift from Merck, Sharp and Dohme (USA). Acetylsalicylic acid, quercetin and N D G A were dissolved in a few drops of N a O H (5 N) and neutralized with Tris buffer. Indothemacin was dissolved in Tris buffer (0.2 M, pH 9.0). The other drugs or peptides were dissolved in Tyrode solution.

2.1. lsolated smooth muscle preparations The guinea-pig ileum preparation was set up as described previously (Webster and Prado, 1970). A 3-4 cm length of terminal ileum was removed from either male or female animals (180-220 g) killed by a blow on the head. The preparation was mounted in a 15-ml bath using Tyrode solution containing atropine sulphate, 1.0 /zg/ml, bubbled with air at 37°C. The composition of the Tyrode solution was (in mM): NaC1 138; KCI 2.7; MgCI 2 1.05; NaH2PO 2 0.42; NaHCO 3 11.9; glucose 5.55 and CaCI 2 1.8. The ileum was attached to a lever that provided an amplification of 6- to 7-fold, and subjected to a load of 1 g. After an equilibration period of 1 h, the isotonic contractions elicited by BK were recorded on a smoked drum. For BK-induced relaxations, the preparation was set up as described above and pre-contracted with sub-maximal concentrations of angiotensin II (1 nM). At the peak of the contraction, BK was added and the relaxing effect was recorded as above.

Rat uterus (3-4 cm long) was obtained from female Wistar animals that had been treated with diethylstilbestrol in vegetable oil (100 /~g/kg s.c.) 18-20 h before they were killed. Both uterine horns were mounted as described above, except that the temperature was 30 °C and de Jalon's solution containing 1.0 /zg/ml atropine sulphate was used. The composition of the Jalon's solution was (in raM): NaCI 154; KCI 5.63; CaCI 2 0.54; NaHCO 3 5.95 and glucose 2.8.

2.2. KPP The initial steps of the KPP purification were performed as described previously (Assreuy et al., 1989; Fernandes et al., 1990). After ion-exchange chromatography on SP-Sephadex C-25 at pH 3.5, additional purification was obtained by high-performance liquid chromatography, using 3 passes through a reverse-phase C18 column. Only one potentiating peptide was found. The complete purification scheme and the sequence of the peptide will be published elsewhere. The molecular weight was estimated as 1200 by gel-filtration chromatography on Sephadex G-15.

2.3. A C E inhibition 2.3.1. Smooth muscle preparations In pilot experiments, it was found that 300 nM enalaprilat abolished the contraction induced by 2.6 nM angiotensin I (a nearly maximal concentration) which is converted by the guinea-pig ileum to angiotensin II. This concentration of enalaprilat was therefore used and a 10-fold excess of angiotensin I was added to all preparations to ensure that the ACE activity was totally inhibited; it elicited no response. For rat uterus, 30 nM enalaprilat blocked the conversion of angiotensin I. 2.3.2. Spectrophotometric assay The enzyme assay was carried out as described (Holmquist et al., 1979). Briefly, ACE (2 /zg in 20 /zl buffer) was added to a spectrophotometer cuvette containing warm (37 ° C) substrate solution (FAPGG 100 /zM in Tris. HCI 50 m M / N a C I 300 mM, pH 7.5). The decrease in absorbance at 328 nm was followed for 2 min. For inhibition assays, enalaprilat or KPP was added to the cuvette immediately before the enzyme. Results are expressed as percentage of residual enzyme activity. 2.4. Potentiating effect in the presence of drugs To examine the influence of different drugs on the BK-induced contraction of guinea-pig ileum, a doseresponse curve for BK was obtained and the curve was repeated afger a drug had been added to the bath

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fluid. The BK-potentiating effect of one of the agents (KPP, BPP9a and enalaprilat) was obtained before and after addition of a given drug to the bath fluid. Only one drug and only one potentiating agent were used per preparation.

2. 5. Potentiating factor The potentiating factor is defined as: PF---(BK equivalent response obtained in the presence of the potentiating agent)/(actual dose of BK added). The BK equivalent response was obtained by interpolation of the height of the contraction (in mm) on a previously obtained BK dose-response curve. When drugs were used, the interpolation was performed with a BK dose-response curve obtained in the presence of the suitable drug.

2.6. Statistical analysis Results are presented as means ± S.D. The significance of the results was determined by anlaysis of variance (ANOVA), followed by Bonferroni's t-test or by Student's t-test for paired samples. The difference was considered significant when P < 0.05.

3. Results

3.1. Potentiation of BK-relaxing effect in guinea-pig ileum We have previously shown that KPP potentiates BKoinduced contractions in a dose-dependent manner, shifting the BK dose-response curve to the left (Assreuy et al., 1989). Since it has been described that a

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Comparative study on the mechanism of bradykinin potentiation induced by bradykinin-potentiating peptide 9a, enalaprilat and kinin-potentiating peptide.

The action of a kinin-potentiating peptide (KPP) obtained from tryptic digestion of human serum proteins was compared with that of bradykinin-potentia...
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