Cardiac-deleterious role of galectin-3 in chronic angiotensin II-induced hypertension.

Galectin-3 (Gal-3), a member of the ß-galactoside lectin family, has an important role in immune regulation. In hypertensive rats and heart failure patients, Gal-3 is considered a marker for an unfavorable prognosis. Nevertheless, the role and mechanism of Gal-3 action in hypertension-induced target organ damage are unknown. We hypothesized that, in angiotensin II (ANG II)-induced hypertension, genetic deletion of Gal-3 prevents left ventricular (LV) adverse remodeling and LV dysfunction by reducing the innate immune responses and myocardial fibrosis. To induce hypertension, male C57BL/6J and Gal-3 knockout (KO) mice were infused with ANG II (3 µg·min-1·kg-1 sc) for 8 wk. We assessed: 1) systolic blood pressure by plethysmography, 2) LV function and remodeling by echocardiography, 3) myocardial fibrosis by histology, 4) cardiac CD68+ macrophage infiltration by histology, 5) ICAM-1 and VCAM-1 expression by Western blotting, 6) plasma cytokines, including interleukin-6 (IL-6), by enzyme-linked immunosorbent assay, and 7) regulatory T (Treg) cells by flow cytometry as detected by their combined expression of CD4, CD25, and FOXP3. Systolic blood pressure and cardiac hypertrophy increased similarly in both mouse strains when infused with ANG II. However, hypertensive C57BL/6J mice suffered impaired ejection and shortening fractions. In these mice, the extent of myocardial fibrosis and macrophage infiltration was greater in histological sections, and cardiac ICAM-1, as well as plasma IL-6, expression was higher as assessed by Western blotting. However, all these parameters were blunted in Gal-3 KO mice. Hypertensive Gal-3 KO mice also had a higher number of splenic Treg lymphocytes. In conclusion, in ANG II-induced hypertension, genetic deletion of Gal-3 prevented LV dysfunction without affecting blood pressure or LV hypertrophy. This study indicates that the ANG II effects are, in part, mediated or triggered by Gal-3 together with the related intercellular signaling (ICAM-1 and IL-6), leading to cardiac inflammation and fibrosis.

Unveiling the vulnerability of C57BL/6J female mice to HFpEF and its related complications.

Introduction: The impact of female biological sex on the development of heart failure with preserved ejection fraction (HFpEF) and its associated kidney disease and vascular endothelial dysfunction is still controversial. Whether females are protected from HFpEF and associated complications is not well established. Previous studies report conflicting prevalence between genders. We hypothesize that female mice are unprotected from HFpEF and its associated kidney disease and vascular endothelial dysfunction. Methods: Eight-week-old female mice were divided into four groups: control groups receiving a standard diet and water for either 5 or 16 weeks, and HFpEF groups fed a high-fat diet (HFD, Rodent Diet With 60 kcal% Fat) and N [w]-nitro-l-arginine methyl ester (L-NAME - 0.5 g/L) in the drinking water for 5 or 16 weeks. Various measurements and assessments were performed, including echocardiography, metabolic and hypertensive evaluations, markers of heart and kidney injury, and assessment of vascular endothelial function. Results: Female mice with HFD and L-NAME developed HFpEF at 5 weeks, evidenced by increased E/E' ratio, reduced cardiac index, left ventricular mass, and unchanged ejection fraction. After 16 weeks, HFpEF worsened. Metabolic disorders, hypertension, lung wet/kidney weight increase, exercise intolerance, and cardiac/renal injury markers were observed. Vascular endothelial dysfunction was associated with ER stress and fibrosis induction. Conclusions: We found that female mice are susceptible to the development of HFpEF and its associated kidney disease and vascular endothelial dysfunction. Our data support the concept that the female sex does not protect from HFpEF and its associated kidney disease and vascular endothelial dysfunction when disease risk factors are present.

Long-term effect of N-acetyl-seryl-aspartyl-lysyl-proline on left ventricular collagen deposition in rats with 2-kidney, 1-clip hypertension.

BACKGROUND: N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a natural inhibitor of pluripotent hematopoietic stem cell proliferation. Ac-SDKP plasma concentration is increased 5-fold after angiotensin-converting enzyme inhibition. Here we studied the effect of Ac-SDKP on monocyte/macrophage infiltration, fibroblast proliferation, and collagen deposition in the rat heart in renovascular hypertension. METHODS AND RESULTS: We investigated whether long-term Ac-SDKP administration would prevent left ventricular (LV) hypertrophy and interstitial collagen deposition in rats with 2-kidney, 1-clip (2K-1C) hypertension. Ac-SDKP (400 microgram. kg(-1). d(-1)) did not affect development of hypertension. Mean blood pressure was similar in rats with 2K-1C hypertension whether they were given vehicle or Ac-SDKP and was higher than in controls. Both LV weight and cardiomyocyte size were significantly increased in rats with 2K-1C hypertension compared with controls and were unaffected by Ac-SDKP. Proliferating cell nuclear antigen- and monocyte/macrophage-positive cells were increased in the LV of 2K-1C hypertensive rats; this increase was significantly blunted by Ac-SDKP (P<0.001). LV interstitial collagen fraction was also increased in 2K-1C hypertensive rats given vehicle (10.1+/-0.8%) compared with sham (5.3+/-0.1%, P<0.0001), and this increase was prevented by Ac-SDKP (5.4+/-0.4%, P<0.001). CONCLUSIONS: Ac-SDKP inhibited monocyte/macrophage infiltration, cell proliferation, and collagen deposition in the LV of hypertensive rats without affecting blood pressure or cardiac hypertrophy, suggesting that it may be partly responsible for the cardioprotective effect of angiotensin-converting enzyme inhibitors.

New selective bradykinin receptor antagonists and bradykinin B2 receptor characterization.

Substantial progress has been made recently in the field of kinin pharmacology with the identification of sensitive bioassay organs and the discovery of bradykinin B2 receptor antagonists. Data obtained with such compounds in various laboratories support the hypothesis that kinins act on multiple (at least two) receptor types. Domenico Regoli and colleagues review here the basic criteria of receptor characterization as they apply to kinins and present a critical analysis of the bioassay organs and B2 receptor antagonists currently used in kinin pharmacology.

Role of kinins and nitric oxide in the antihypertrophic effect of ramipril.

We examined the effect of non-antihypertensive doses of the angiotensin-converting enzyme inhibitor ramipril, kinins, and/or nitric oxide on left ventricular hypertrophy in rats with aortic coarctation. We investigated the effect of either HOE 140, a specific B2 receptor antagonist, or NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, on the antihypertrophic effect of ramipril at non-antihypertensive doses (10 micrograms/kg per day) failed to alter left ventricular hypertrophy significantly, although a small decrease was obtained. Given at a dose of 1 mg/kg per day for 6 weeks, ramipril prevented increased blood pressure and left ventricular hypertrophy after aortic coarctation. Neither of these effects was blocked by simultaneous administration of HOE 140 (500 micrograms/kg per day). In rats with aortic coarctation treated with L-NAME, blood pressure increased further but left ventricular weight did not. Ramipril (1 mg/kg per day) significantly reduced left ventricular hypertrophy, although blood pressure was still higher than in rats given water alone. The slope of the correlation between left ventricular weight and blood pressure in rats that received L-NAME was significantly lower than in rats that did not (0.52 versus 1.29; P = .008). This suggests that for each 1 mm Hg that the blood pressure increased, the increase in left ventricular weight was less in the L-NAME groups. Thus, only antihypertensive doses of ramipril possessed antihypertrophic activity. Kinins did not participate in the chronic antihypertensive and antihypertrophic effects of ramipril. In hypertension induced or aggravated by chronic nitric oxide synthase, L-NAME partially impaired development of left ventricular hypertrophy for reasons that are unclear.

Effect of ACE inhibitor on DOCA-salt- and aortic coarctation-induced hypertension in mice: do kinin B2 receptors play a role?

Kinins have been shown to play an important role in the cardioprotective effect of ACE inhibitors (ACEi) during heart failure and ischemia-reperfusion. However, it is controversial as to whether kinins oppose the hypertensinogenic effect of deoxycorticosterone acetate plus salt (DOCA-salt) or aortic coarctation and whether they mediate both chronic antihypertensive and cardiac antihypertrophic effects of ACEi in hypertension. Using normal 129/SvEvTac mice and mice lacking the bradykinin B2 receptor gene (B2-KO), we investigated whether (1) the hypertensinogenic effect of DOCA-salt or aortic coarctation is enhanced in B2-KO mice and (2) the chronic antihypertensive and antihypertrophic effects of an ACEi (ramipril, 4 mg. kg-1. d-1) are mediated by B2 receptors in aortic coarctation (6 weeks)- and DOCA-salt (4 weeks)-induced hypertension. Before surgery, there was no difference between 129/SvEvTac and B2-KO mice in terms of blood pressure and heart weight, suggesting that kinins are not essential to maintaining normal blood pressure. DOCA-salt (volume expansion) or aortic coarctation (renin-dependent) induced similar hypertension and left ventricular hypertrophy (LVH) in 129/SvEvTac and B2-KO mice, suggesting that kinins do not play an essential role in the development of DOCA-salt- or aortic coarctation-induced hypertension. We found that B2 receptors mediate only the early (1 week) but not the late phase (4 weeks) of the chronic hypotensive effect of ACEi in DOCA-salt hypertension. On the other hand, chronic ACE inhibition prevented the development of hypertension and LVH in both 129/SvEvTac and B2-KO mice given DOCA-salt or subjected to aortic coarctation, suggesting that kinins do not participate in the chronic antihypertensive and antihypertrophic effects of ACEi in these 2 models of hypertension. Thus, in mice, kinins acting via B2 receptors do not participate in (1) maintenance of normal basal blood pressure, (2) establishment and maintenance of hypertension induced by DOCA-salt or aortic coarctation, and (3) chronic antihypertensive and cardiac antihypertrophic effects of ACEi in DOCA-salt and aortic coarctation hypertension.

DuP 753 is a specific antagonist for the angiotensin receptor.

2-n-Butyl-4-chloro-5-hydroxy-methyl-1-[(2'-(1H)-tetrazol-5-yl)biph enyl-4- yl)methyl]imidazol potassium salt (DuP 753) is a nonpeptide angiotensin II receptor antagonist that inhibits the contractile effects of angiotensin II competitively and shows pA2 values of 8.27 on the rabbit aorta and jugular vein, 8.66 on the rat portal vein and stomach, 8.19 on the rat urinary bladder, and 8.36 on human colon, ileum, and urinary bladder. This agent (more than 10(-5) M) exhibits no agonistic activity and does not affect the contractile effects of norepinephrine, acetylcholine, bradykinin, desArg9-bradykinin, substance P, neurokinin A, neurokinin B, or bombesin in the various tissues. The present results demonstrate that DuP 753 is a potent nonpeptide antagonist with high affinity, specificity, and selectivity for the angiotensin receptor.

Structure-activity studies of bradykinin and related peptides. B2-receptor antagonists.

Thirty-seven compounds were tested as antagonists of kinin B2- and B1-receptors to identify the chemical changes required to obtain antagonism, improve antagonist affinity, and eliminate residual agonistic activities. Apparent affinity of antagonists was evaluated in terms of pA2 on the rabbit jugular vein, the dog carotid and renal arteries, the hamster urinary bladder, the guinea pig ileum, the rat vas deferens, the guinea pig trachea, and the rabbit aorta, using bradykinin and desArg9-bradykinin as B2- and B1-receptor activators. Replacement of Pro7 of bradykinin with D-Phe leads to antagonism; substitution of Pro3 by Hyp and extension of the peptide chain at the N-terminal with a D-Arg residue improves the affinity of antagonists; acetylation of N-terminal amine function reduces residual agonistic activity; these changes, combined with the replacement of Phe8 by Leu as in Ac-D-Arg[Hyp3,D-Phe7,Leu8]-bradykinin, led to potent full B2-receptor antagonists. Affinity of antagonists differs markedly between highly sensitive (rabbit jugular vein, dog carotid and renal artery), moderately sensitive (hamster urinary bladder, guinea pig ileum, and rat vas deferens), and insensitive preparations (the guinea pig trachea) in which antagonists act as potent stimulants. High concentrations of antagonists block bradykinin completely in the rabbit jugular vein but not in the guinea pig ileum, suggesting that kinins stimulate the moderately sensitive tissues by two mechanisms, of which only one is blocked by antagonists. It thus appears that kinins act on various B2-receptor subtypes or by different action mechanisms.

Role of nNOS in blood pressure regulation in eNOS null mutant mice.

The role of neural nitric oxide synthase (nNOS) in regulating blood pressure (BP) remains uncertain. Recently it was reported that in mice lacking functional endothelial NOS (eNOS) genes (-/-), acute administration of a nonselective NOS inhibitor, Nw-nitro-L-arginine, decreased mean BP, suggesting that NO released by non-eNOS isoforms increases BP. Because the inducible NOS isoform is not constitutively expressed and when induced causes hypotension, we hypothesize that it is NO produced by nNOS that increases BP in the absence of eNOS activity. To test this hypothesis, we studied the acute effect of selective and nonselective nNOS inhibitors on BP and cerebellar NOS activity in eNOS (-/-), wild-type (+/+), and heterozygous (+/-) mice as well as in +/+ mice with renovascular hypertension. Because it is not known whether the decrease in BP caused by acute NOS inhibition in -/- mice can occur chronically, we also studied the effect of chronic NOS inhibition on both BP and cerebellar NOS activity. eNOS (-/-) mice had higher BP than +/+ or +/-mice, and acute administration of the selective nNOS inhibitor 7-nitroindazole (7-NI) decreased their mean BP from 137+/-13 to 124+/-12 mm Hg (P<0.01). In +/+, +/-, or renovascular hypertensive +/+ mice, 7-NI caused a small but insignificant rise from 105+/-5 to 110+/-6 mm Hg, from 115+/-9 to 119+/-13 mm Hg, and from 146+/-6 to 150+/-6 mm Hg, respectively. Fifteen minutes after administration of 7-NI, cerebellar NOS activity decreased by 70%; however, this inhibitory effect was brief, since 2 hours after 7-NI administration NOS returned toward control values. Chronic oral or intraperitoneal administration of 7-NI did not inhibit cerebellar NOS activity, whereas the nonselective NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) decreased this activity by 50%. Therefore, we studied the effect of chronic L-NAME administration (4 weeks) on BP. In -/- mice, chronic L-NAME administration decreased BP from 135+/-4 to 120+/-3 mm Hg (P<0.05), whereas in +/+ and +/-mice, as expected, it increased BP from 109+/-2 to 125+/-3 mm Hg (P<0.001) and from 107+/-6 to 119+/-5 mm Hg (P<0.02), respectively. After L-NAME administration was stopped, BP returned to baseline. These results suggest that in eNOS -/- mice, NO derived from nNOS increases BP both acutely and chronically.

Characterization of bombesin receptors in peripheral contractile organs.

1 Guinea-pig and rat urinary bladders, rat stomach and the guinea-pig gall bladder, four isolated organs that show high sensitivity to bombesin, were used to characterize bombesin receptors in peripheral organs. 2 The order of potency of agonists was determined with several naturally occurring peptides of the bombesin series, namely bombesin (BBS), litorin (Lit), neuromedin B (NMB), the gastrin-releasing peptide (GRP 18-27), neuromedin C (NMC) and with some bombesin fragments. It was found that bombesin, neuromedin C, litorin and two bombesin fragments, BBS (6-14) and AcBBS (6-14) had similar activities in the four preparations, while neuromedin B and [Phe6]-neuromedin C were more active on the rat urinary bladder than on the other tissues. 3 The order of potency of agonists determined in the rat urinary bladder was as follows: BBS = NMB greater than Lit greater than NMC greater than [Phe6]NMC = GRP and it was found to be different from that observed in the other preparations: BBS greater than GRP = Lit greater than or equal to NMC much greater than NMB greater than [Phe6]NMC, suggesting the existence of two different bombesin receptors, BBS1 and BBS2. 4 This interpretation was convalidated by the finding that bombesin antagonists, namely Ac.GRP(20-26)OCH3 and Ac.GRP(20-26)OC2H5 reduced or blocked the effects of bombesin-related peptides on BBS2 receptor systems while being completely inactive on the rat urinary bladder (BBS1 system).

Structure-activity studies on bradykinin and related peptides: agonists.

1. Bradykinin, kallidin, T-kinin, [Hyp3]-bradykinin and several analogues were prepared by solid-phase synthesis and purified by high performance liquid chromatography. 2. The various peptides were tested for their abilities to relax the dog carotid and renal arteries, or to contract the rabbit jugular vein and aorta, in order to measure their activities on BK2 (the first three preparations) or BK1 (the rabbit aorta) receptors. The dog renal artery without endothelium was also used as a BK1 receptor system. 3. T-kinin was found to be less active than bradykinin, while the replacement of Pro3 with Hyp favoured BK2 receptor occupation. [Hyp3,Tyr(Me)8]-BK was found to be a selective BK2 receptor agonist. 4. Amidation or methylation of the C-terminal carboxyl decreased activity, while extension of the N-terminal with Sar or D-Arg increased affinity and selectivity for BK1 (Sar) and affinity for BK2 (D-Arg) receptors. Acetylation of N-terminal amide brought affinity down to 10% or less. 5. Replacement of the peptide bonds Phe8-Arg9 to protect from kininase I and II, decreased affinities slightly, but was incompatible with additional changes at the N-terminal or in the peptide bond Gly4-Phe5. 6. Substitution of C-terminal Phe in desArg9-BK (the BK1 receptor stimulant) with D-Phe increased potency and selectivity for BK1 receptors while protecting from carboxypeptidases. Sar[D-Phe8]desArg9-BK was found to be a potent and selective BK1 receptor agonist.

Antagonists for the neurokinin NK-3 receptor evaluated in selective receptor systems.

Four isolated vessels that are monoreceptor systems for neurokinins, the dog carotid artery and rabbit jugular vein (NK-1), the rabbit pulmonary artery (NK-2) and the rat portal vein (NK-3), were used to compare the activities of selective neurokinin agonists and evaluate the affinities of new NK-3 antagonists. Chemical modifications in the partial sequences NKA (4-10) and NKB (4-10), particularly the replacement of Val7 with an aromatic residue (Tyr, MePhe or Trp) and the extension of the peptide backbone in position 8, obtained with beta-Ala, led to compounds that maintain weak agonistic activities on the NK-1 and NK-2, and some of them also on NK-3 receptors but exert potent antagonism against NKB on the NK-3 receptor of the rat portal vein. Antagonistic affinity is the highest when Trp is used in position 7 of [beta-Ala8]-NKA (4-10) and MePhe in position 7 of [beta-Ala8]-NKB (4-10). Antagonism is selective for NKB or [MePhe7]-NKB, and appears to be specific, since the most active compound [Trp7, beta-Ala8]-NKA (4-10) is inactive against bradykinin on the rabbit jugular vein (B2 receptor), against SP on the rabbit jugular vein (NK-1 receptor), against desArg9-bradykinin on the rabbit aorta (B1 receptor), and against angiotensin II and histamine (AT and H receptors, respectively) in the rabbit aorta. The new NK-3 receptor antagonists described in the present study provide useful tools for neurokinin receptor characterization and for determining the roles of neurokinins in physiopathology.

Selective agonists for substance P and neurokinin receptors.

A series of neurokinin analogues and fragments have been prepared in an attempt to identify selective agonists for NK-P, NK-A and NK-B receptors. The compounds have been tested on the dog carotid artery (NK-P receptor system), the rabbit pulmonary artery (NK-A) and the rat portal vein (NK-B). C-terminal substituted analogues of the three neurokinins have provided indication that NK-P receptor selectivity is improved by the oxidation of methionine to Met(O2), while selectivity for NK-A is favoured by replacing Met with NIe. Selectivity for NK-P receptors is further improved by the replacement of Gly9 with Sar. Selectivity and affinity for NK-B receptors is markedly increased when Val7 is replaced with MePhe in both the fragment NKB (4-10) and NKB. The results of the present study indicate that a) [Sar9,Met(O2)11]SP is a potent and selective agonist for the NK-P receptors of the dog carotid artery; b) [MePhe7]NKB is a very potent and selective stimulant of receptors for neurokinin B and c) [Nle10]NKA (4-10) is a promising compound, showing some selectivity for NK-A receptor; further modifications are however needed to improve its affinity.

Structure-activity studies of neurokinin A.

A structure-activity study on neurokinin A and its C-terminal fragment NKA (4-10) has been performed in order to find selective agonists for the NK-2 receptor and identify chemical modifications suitable for protecting the peptides from degradation, while maintaining activity. Five series of compounds have been prepared and tested: 1. the complete series of the L-Ala monosubstituted analogues of NKA; 2. a series of NKA fragments from the C- or N-terminal; 3. the complete series of NKA (4-10) analogues monosubstituted with beta-Ala; 4. a series of NKA (4-10) analogues with monosubstitutions in pos. 4, 8, 10 or multisubstitutions in two or more of the same positions; and 5. a series of 6 NKA (4-10) analogues monosubstituted with 1-amino,1-cyclohexane carboxylic acid residue. It has been found that the most selective agonists for the NK-2 receptor system are [beta Ala8]NKA (4-10) and [Nle10]NKA (4-10). Protection from aminopeptidase may be obtained by acetylation of the N-terminal amide of NKA (4-10), while partial protection from endopeptidases should be expected from the presence of beta-Ala in position 8. Conformational constraints induced with 1,amino,1-cyclohexane carboxylic acid residue gave weakly active compounds. Multiple substitutions reduce rather than potentiating the favorable effects of the corresponding monosubstituted compounds.

Characterization of a novel binding site for 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]bradykinin on epithelial membranes of guinea pig ileum.

We have recently shown that (a) [125I-Tyr8]bradykinin (BK) recognized bradykinin binding sites in guinea pig epithelium membranes with a Kd value of 1.6 nM and a Bmax of 156 fmol/mg protein, and (b) B2 agonists and some B2 antagonists, such as D-Arg-[Hyp3,D-Phe7,Leu8]BK, inhibited this specific binding with a Ki value of 32 nM. In the present study, we have radioiodinated the B2 antagonist Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK and have performed a full characterization of the binding properties of this tracer in the same membrane preparation. Equilibrium experiments performed in the absence or presence of an excess of BK (10(-5) M) showed that 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK specifically labelled two different sites. One of these is the same as the site labelled by [125I-Tyr8]BK, and this indicates that 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK interacts specifically with kinin B2 receptors. Equilibrium experiment performed in the presence of an excess of BK (10(-5) M) indicated that specific binding of 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK to the second site is also saturable and Scatchard analysis showed that the site is of high affinity with a Kd of 16.8 nM and a Bmax of 2.08 pmol/mg protein. Surprisingly, unlabelled B2 agonists such as bradykinin, [Tyr8]BK, [Leu8]BK, [Hyp3,Tyr8(OMe)]BK, D-Arg-[Hyp3]BK and kallidin were found to be inactive on this second site. A series of B2 receptor antagonists, Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK, D-Arg-[Hyp3,D-Phe7,Leu8]BK, D-Arg-[Hyp3,Leu5,8,D-Phe7]BK, D-Arg-[Hyp3,Gly6,D-Phe7,Leu8]BK and D-Arg-[Hyp3,Thi5,8,D-Phe7]BK inhibited 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK binding with Ki values of 25.0, 20.9, 15.8, 64.6 and 6606.9 nM respectively. On the other hand, [Thi5,8,D-Phe7]BK did not interfere with 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK but was found to be a potent inhibitor of [125I-Tyr8]BK binding (Ki = 53.7 nM). As expected, B1 receptor agonists, antagonists and peptides non-related to BK such as substance P, neurokinin A, neurokinin B, angiotensin II, bombesin, vasopressin and the calcitonin gene related peptide were unable to compete with 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK. The results show that 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK is interacting with two distinct binding sites in the guinea pig epithelium: one is the well known bradykinin B2 receptor and the other is a new, non-characterized binding site that interacts exclusively with some bradykinin receptor antagonists.

Bradykinin antagonism: differentiation between peptide antagonists and antiinflammatory agents.

Three non-steroidal antiinflammatory agents were tested for their ability to antagonize bradykinin in the rabbit jugular vein, the dog carotid artery and the guinea pig trachea. The new agents were compared with indomethacin, as well as with [Thi5,8,D-Phe7] bradykinin and [Thi6,9,D-Phe8] kallidin, two B2 receptor antagonists. The antiinflammatory agents did not alter the effects of bradykinin in the two isolated vessels while they completely blocked (like indomethacin) the relaxation induced by bradykinin in guinea pig tracheae contracted with histamine or neurokinin A. The two bradykinin antagonists significantly reduced the contractions of the rabbit jugular vein and the relaxation of the dog carotid artery (with endothelium) in response to bradykinin: these antagonists were however inactive against bradykinin in the guinea pig trachea. The data now reported suggest that B2 receptors are involved in the opposite (stimulant or inhibitor) effects of the kinins in the two isolated vessels while the relaxation of the guinea pig trachea is a prostaglandin-dependent phenomenon which does not involve the activation of B1 or B2 receptors.

Receptors for kinins in isolated arterial vessels of dogs.

A variety of kinin peptides, agonists and antagonists were tested with dog carotid and renal arteries in order to characterize kinin receptor types and functions. The dog carotid artery responds to bradykinin with concentration-dependent relaxation only when the endothelium is intact but des-Arg9-bradykinin is practically inactive. The effect of bradykinin is blocked by B2 receptor antagonists, suggesting that the dog carotid artery has B2 receptors in the endothelium. These receptors mediate relaxation of the arterial smooth muscles by promoting the release of an endothelium-derived relaxing factor whose action is prevented by methylene blue. Kinins relax the dog renal artery with or without endothelium. Methylene blue prevents the effect of bradykinin only, suggesting that B2 receptors, promoting the release of endothelium-derived relaxing factor, are present in the endothelium of the dog renal artery. Moreover, the dog renal artery appears to have both B2 and B1 receptors mediating relaxation of the arterial smooth muscle. The presence of the two receptor types has been demonstrated by means of specific agonists and antagonists. Indomethacin blocks the effects of both bradykinin and des-Arg9-bradykinin on the dog renal artery without endothelium, suggesting that muscular B1 and B2 receptors act by promoting the release of prostaglandins. Captopril, a kininase II inhibitor, potentiates the effect of bradykinin on the dog carotid artery more than on the dog renal artery.

Pharmacological characterization of a new highly potent B2 receptor antagonist (HOE 140: D-Arg-[Hyp3,Thi5,D-Tic7,Qic8]bradykinin).

HOE 140 (D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin), a new B2 antagonist, was compared to R-493 (D-Arg[Hyp3-D-Phe7,Leu8]bradykinin) with respect to inhibition of the responses of seven isolated smooth muscle preparations to bradykinin. R-493 was found to exert: (a) high antagonistic activity on the rabbit jugular vein (pA2 of 8.86), (b) moderate activity on the rabbit aorta, guinea-pig ileum, hamster urinary bladder and human urinary bladder (pA2 of 5.76, 6.77, 7.16 and 7.15, respectively) and (c) a stimulatory effect on the guinea-pig trachea. On the other hand, HOE 140 showed identical apparent affinities (8.36-9.12) on all preparations except the rabbit aorta where it was inactive and the guinea-pig trachea where the compound was an antagonist (pA2: 7.42) without agonistic effect. HOE 140 is specific and selective for B2 receptors since it was inactive against angiotensin II, substance P, neurokinin A, desArg9-bradykinin, noradrenaline or acetylcholine in the various preparations. R-493 inhibited the contractile effects of bradykinin competitively, while HOE 140 was not competitive even at low concentrations (7.7 x 10(-9) M). These results demonstrate that HOE 140 is a potent B2 antagonist with high affinity, specific for kinin receptors and selective for the B2 receptor type, but is non-competitive. HOE 140 is the first bradykinin receptor antagonist that acts as such on the guinea-pig trachea without showing any agonistic activity.

Receptors for substance P and neurokinins. Correlation between binding and biological activities.

The biological activities of neurokinin-related peptides were compared with their binding affinities measured in various laboratories. A positive significant correlation was demonstrated between the relaxation of the dog carotid artery and the binding of Bolton-Hunter [125I]substance P to rat brain synaptosomes, the contractions of the rat duodenum and the rabbit pulmonary artery and the binding of Bolton-Hunter [125I]neurokinin A to duodenum smooth muscle plasma membranes, and the contraction of the rat portal vein and the binding of [125I]Bolton-Hunter NH-senktide to rat cerebral cortex membranes.

Specific agonists for neurokinin B receptors.

A series of analogues of the partial sequence NKB-(4-10) (H-Asp-Phe-Phe-Val-Gly-Leu-Met.NH2) was prepared in an attempt to identify selective agonists for the neurokinin B receptor type. The compounds were tested in the dog carotid artery, the rabbit pulmonary artery and the rat portal vein to evaluate their affinity for the receptors of substance P, neurokinin A and neurokinin B respectively. It has been shown that the replacement of Val7 with MePhe increased significantly the affinity of NKB-(4-10) for the neurokinin B receptor and confered marked selectivity. [MePhe7]NKB-(4-10) was practically inactive as stimulant of the receptor for NKA and was a weak agonist on the receptor for SP. Such significant changes in the pharmacological spectrum of [MePhe7]NKB-(4-10) cannot be attributed to protection from metabolism and appear to be due to changes in the peptide conformation.