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) [^125]I-Tyr8]bradykinin (BK) recognized bradykinin binding sites in guinea pig epithelium membranes with a K_d value of 1.6 nM and a B_max 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 K_i value of 32 nM. In the present study, we have radioiodinated the B₂ antagonists 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 K_d of 16.8 nM and a B_max 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,Leu^5.8,D-Phe⁷]BK, D-Arg-[Hyp³,Gly⁶,D-Phe⁷,Leu⁸]BK and D-Arg-[Hyp³,Thi^5.8,D-Phe⁷]BK inhibited ¹²⁵I-Tyr-D-Arg-[Hyp³,D-Phe⁷,Leu Leu⁸]BK binding with K_i values of 25.0, 20.9, 15.8, 64.6 and 6606.9 nM respectively. On the other hand, [Thi^5.8,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 (K_i = 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.     

Kinin-induced relaxations of the rat duodenum

Summary Both bradykinin (BK) and des-Arg⁹-BK induced relaxations of the isolated longitudinal smooth muscles of the rat duodenum. No contractile effects were observed with both peptides at concentrations up to 1 mol/l. Des-Arg⁹-BK was about 1000 times less potent than BK. The novel B₂ antagonist HOE 140 (d-Arg-[Hyp³, Thi⁵, D-Phe⁷, Oic⁸]-BK) potently inhibited the BK-induced relaxations, but did not affect the relaxations induced by des-Arg⁹-BK. Conversely, the B₁ receptor antagonist des-Arg⁹-[Leu⁸]-BK only inhibited des-Arg⁹-BK, but did not affect BK-induced relaxations.The relaxations induced by BK and by des-Arg⁹-BK were inhibited by apamin (1 mol/l) demonstrating that apamin-sensitive K⁺ channels are involved. In contrast, tetraethylammonium (1 mmol/l) did not inhibit the relaxations. BK-induced relaxations were reduced by about 25% in the presence of indomethacin (10 mol/l) although the concentration-response curve to BK was not shifted to the left. Prostaglandin E₁ caused relaxations with a pD₂ value of 9.2.It is concluded that both BK and des-Arg⁹-BK can elicit relaxations of the rat duodenum via pharmacologically distinct kinin receptor subtypes, but via similar effector mechanisms.     

Identification and characterization of B2 bradykinin receptors in sheep nasal turbinate membranes

Because bradykinin (BK) has been implicated as a mediator of upper respiratory tract symptomatology, specific ³H-BK binding was investigated in membrane homogenates prepared from sheep nasal turbinate tissue in order to identify and characterize the BK receptor subtype(s) present. ³H-BK saturation and Scatchard analyses revealed a single, high affinity, saturable site (K_D of 0.098 nM) with a density of 0.44 pmol/g wet weight tissue. Competition experiments using B₁ and B₂ receptor agents revealed a B₂-BK receptor pharmacology; the B₂ agents BK, Lys-BK, NPC-567, [D-Phe⁷]-BK and [Thi,^5,8 D-Phe⁷]-BK displayed nM affinity while the B₁ agents [des-Arg⁹]-BK and [Leu,⁸ des-Arg⁹]-BK competed in the uM range. The absolute and rank order of affinities in this tissue paralleled that found in the guinea pig ileum. No specific binding was found using the putative B₁ receptor radioligand ³H-[des-Arg⁹]-BK. Specific B₂-BK receptor binding was not effected by the addition of non-hydrolyzable guanine or adenine nucleotides. These data confirm the presence of B₂-BK receptors in this tissue and provide support for a role of BK in nasal function.     

Bradykinin-antagonists: Therapeutic perspectives

In vitro and in vivo studies of the actions of various antagonists and agonists of bradykinin (BK) B₁ and B₂ receptors have been reviewed and analyzed. It seems apparent that certain B₂-antagonists, such as [Thi^5,8,D-Phe⁷]-BK and D-Arg⁰-[Hyp³,Thi^5,8,D-Phe⁷]-BK will serve as valuable model substances for developing new anti-inflammatory/analgesic drugs. Further development of B₁-antagonists, based on modifications of the structure of des-Arg⁹,[Leu⁸]-BK, or combined B₁/B₂-antagonists might be useful in controlling disease states involving chronic tissue injury and/or prolonged vasopasm.     

Analysis of bradykinin receptor mediating relaxation of cat cerebral arteries in vivo and in vitro

Summary Bradykinin (BK), methionyl-lysyl-BK (M-L-BK) and des-Arg ⁹-BK produced, in decreasing potency, dose-related dilatations of the superficial pial arteries of the cat in vivo. The competitive, specific B₁-receptor antagonist, des-Arg ⁹-Leu⁸-BK was ineffective against BK-induced dilatations in this in vivo model.On the cat middle cerebral artery in vitro (but not the basilar artery), under resting tension and when contracted with 5-hydroxytryptamine (5-HT) or KCl, concentration related relaxations were produced by BK, M-L-BK, and des-Arg ⁹-BK, this being the order of relative potency of the three kinins.There was no increase in the sensitivity of either the middle cerebral or the basilar artery in vitro under resting tension or when contracted with 5-HT or KCl to BK or des-Arg ⁹-BK, concentration effect curves to which were produced at 2 h intervals over an 8 h period. The B₁-receptor antagonist des-Arg ⁹-Leu⁸-BK was ineffective against relaxations to BK or des-Arg ⁹-BK of the middle cerebral artery under resting tension or when contracted with 5-HT.The receptor mediating dilatation of the superficial pial arteries of the cat in vivo and relaxation of the middle cerebral artery in vitro to kinins is of the B₂-type. The cat basilar artery in vitro is relatively insensitive to the action of kinins and this is possibly due to an absence of receptors for kinins on this tissue.     

Characterization of a B2-bradykinin receptor in rat renal mesangial cells

The specific binding of bradykinin (BK) was investigated using membrane fractions from mesangial cells in primary culture, a cloned cell line, and in intact adherent cells with three different radiolabelled BK analogues: ¹²⁵I-[Tyr⁰]BK, ¹²⁵I-[Tyr⁵]BK and ¹²⁵I-[Tyr⁸]BK. The best radioligand was ¹²⁵I-[Tyr⁰]BK, and assay conditions were determined to ensure maximal stable binding. Binding appeared to be reversible and not to be inhibited by a wide variety of protease inhibitors including converting enzyme inhibitor and phosphoramidon. The maximum density of binding sites (B_max) was about 88 ± 18 fmol/mg protein, which is equivalent to about 6000 sites/cell, and the dissociation constant averaged 2 nM. No significant difference in B_max was observed between membranes from cells in primary culture and those from cloned cells. Of the BK analogues tested, unmodified BK exhibited the highest inhibition constant (close to 10⁻¹⁰ M). No displacement of ¹²⁵I-[Tyr⁰]BK was observed in the presence of the B₁ agonist des-Arg⁹-BK or several unrelated peptides, including atrial natriuretic factor and angiotensin I and II, whereas 50% inhibition of binding was achieved with the B₂ antagonist [D-Arg,Hyp³,D-Phe⁷]BK (10⁻⁹ M). Addition of BK for 3 min to the incubation medium of cloned mesangial cells induced a dose- and time-dependent increase in PGE₂ unlike des-Arg⁹-BK, which showed no such effect. The secretion was strongly inhibited by prior incubation with the B₂ antagonist [D-Arg,Hyp³,D-Phe⁷]BK. The pharmacological profile of the binding site determined with various BK agonists and antagonists, and the stimulating effect of binding site activation on prostaglandin release strongly suggest that B₂-kinin-like receptors are present in rat mesangial cells.     

Characterization of bradykinin receptors mediating catecholamine release in PC12 cells

The rat pheochromocytoma cell line PC12, which is a widely used model for analyzing stimulus-secretion coupling, was investigated for the effects of kinins on catecholamine release. Subtypes of kinin receptors were characterized using the B₁ agonist desArg⁹-bradykinin, the B₂ agonist bradykinin and the B₂ antagonists [Thi^5,8, D-Phe⁷]-bradykinin, D-Arg[Hyp³, D-Tic⁷, Oic⁸]-bradykinin (HOE 890307) and D-Arg-[Hyp³, Thi⁵, D-Tic⁷, Oic⁸]-bradykinin (HOE 140). The effectiveness of acute and chronic exposure to angiotensin I converting enzyme inhibitors as well as pretreatment of the cells with bacterial lipopolysaccharides in modulating B₁ or B₂ receptor systems was also tested.Bradykinin stimulated noradrenaline release from PC12 cells at low concentrations (EC₅₀ = 1 nM), maximally inducing a release of 43.7% of the cellular content within 15 min. In comparison with acetylcholine and K⁺-induced depolarization, bradykinin was the most effective stimulus. DesArg⁹-bradykinin was only effective at very high concentrations (> 30 M). Like in other neuronal cells, the B₂-specific partial antagonist [Thi^5,8, D-Phe⁷]-bradykinin acted as a low-affinity agonist without any antagonistic effects. The B₂ antagonists HOE 890307 and HOE 140 exerted no agonistic effects and concentration-dependently inhibited bradykinin-induced noradrenaline release, showing competitive antagonism with K_i values of 1.38 nM and 0.66 nM, respectively. Only at the highest concentration used (1 M), HOE 140 did depress the maximal response to bradykinin. HOE 890307 also abolished the effects of desArg⁹-bradykinin and [Thi^5,8, D-Phe⁷]-bradykinin. Acute or chronic inhibition of the angiotensin I converting enzyme or application of lipopolysaccharides, which all can lead to induction of the B₁ receptor subtype in vivo, did not alter the secretory response of PC12 cells to either bradykinin (0.1 and 30 nM) or desArg⁹-bradykinin (1 M).In conclusion, noradrenaline release from PC12 cells is stimulated via B₂, but not B₁, receptors. Despite the fact that the receptor system is highly susceptible to stimulation by low-affinity ligands, HOE 890307 and HOE 140 are pure antagonists, with only high concentrations of HOE 140 (> 1 M) showing a non-competitive type of inhibition. Induction of B₁ receptors which could stimulate noradrenaline release could not be demonstrated in this model. The possible role of bradykinin in modulating sympathetic neurotransmission during inhibition of angiotensin I converting enzyme is discussed.     

The expression of functionally-coupled B2-bradykinin receptors in human corneal epithelial cells and their pharmacological characterization with agonists and antagonists

1. Bradykinin (BK) and Lys-BK are peptides which are released at high nanomolar concentrations into the tear-film of ocular allergic patients. We hypothesized that these peptides may activate specific receptors on the ocular surface, especially the corneal epithelium (CE) and thus the CE cells may represent a potential target tissue for these kinins.
2. The purpose of the present studies, therefore, was to determine the presence of and the pharmacological characteristics of bradykinin receptors on normal cultured primary and SV40 virus-transformed human corneal epithelial (CEPI) cells by use of the accumulation of [³H]-inositol phosphates ([³H]-IPs) as a bioassay.
3. Bradykinin (BK) induced a maximal 1.95±0.24 fold (n=17) and 2.51±0.29 fold (n=26) stimulation of [³H]-IPs accumulation in normal, primary (P-CEPI) and SV40-immortalized (CEPI-17-CL4) cells, respectively. This contrasted with a maximal 3.2–4.5 fold and 2.0–2.9 fold stimulation by histamine (100 μM) and platelet activating factor (100 nM) in both cell-types, respectively.
4. The molar potencies of BK and some of its analogues in the CEPI-17-CL4 cells were as follows: BK (EC₅₀=3.26±0.61 nM, n=18), Lys-BK (EC₅₀=0.95±0.16 nM, n=5), Met-Lys-BK (EC₅₀=2.3± 0.42 nM, n=5), Ile-Ser-BK (EC₅₀=5.19±1.23 nM, n=6), Ala³-Lys-BK (EC₅₀=12.7±2.08 nM, n=3), Tyr⁸-BK (EC₅₀=19.3±0.77 nM, n=3), Tyr⁵-BK (EC₅₀=467±53 nM, n=4) and des-Arg⁹-BK (EC₅₀=14.1±2.7 μM, n=4). The potencies of BK-related peptides in normal, P-CEPI cells were similar to those found in transformed cells, thus: BK, EC₅₀=2.02±0.69 nM (n=7), Tyr⁸-BK, EC₅₀=14.6±2.7 nM (n=3), Tyr⁵=BK, EC₅₀=310±70 nM (n=4) and des-Arg⁹-BK, EC₅₀=12.3± 3.8 μM (n=3).
5. The bradykinin-induced responses were competitively antagonized by the B₂-receptor selective BK antagonists, Hoe-140 (D-Arg-[Hyp³,Thi⁵,D-Tic⁷, Oic⁸]BK; Icatibant; molar antagonist potency=2.9 nM; pA₂=8.54±0.06, n=4; and slope=1.04±0.08) and D-Arg⁰[Hyp³,Thi^5,8, DPhe⁷]-BK (K_B=371 nM; pK_B=6.43±0.08, n=4) in CEPI-17-CL4 cells. The antagonist potency of Hoe-140 against BK in normal, P-CEPI cells was 8.4±1.8 nM (pK_i=8.11±0.12, n=4), this being similar to the potency observed in the immortalized cells.
6. This rank order of potency of agonist BK-related peptides, coupled with the antagonism of the BK-induced [³H]-IPs by the specific B₂-receptor antagonists, strongly suggests that a B₂-receptor subtype is involved in mediating functional phosphoinositide (PI) responses in the CEPI-17-CL4 and P-CEPI cells.
7. In conclusion, these data indicate that the P-CEPI and CEPI-17-CL4 cells express BK receptors of the B₂-subtype coupled to the PI turnover signal transduction pathway. The CEPI-17-CL4 cells represent a good in vitro model of the human corneal epithelium in which to study further the role of BK receptors in its physiology and pathology, such as in allergic/inflammatory conditions, potential wound healing and other functions of the cornea.

     

Effect of chronic estradiol and haloperidol treatment on striatal dopamine receptors

The intravenous injection of 10 μg of a lipopolysaccharide extracted from E. Coli to rabbits leads to the appearance of a hypotensive effect for des-Arg⁹-BK and increases significantly the vasodilator effect of this peptide in isolated hearts and its contractile effects in strips of large arteries and veins. LPS elicits these responses when administered 5 or 20 h before anaesthesia; the hypotensive response of animals receiving LPS just before anaesthesia is similar to that of untreated rabbits. All actions of des-Arg⁹-BK in vivo, in isolated hearts and in isolated tissues are blocked by des-Arg¹⁰,[Leu⁹]-kallidin (KD), a specific inhibitor of kinins B₁-receptor. These data are taken as evidence of the appearance of B₁-response to kinins in the few hours following LPS injection. The response of the animals, perfused organs and isolated tissues to other agonists, such as substance P or [Tyr(Me)⁸]-BK (an activator of B₂-receptors for kinins) are not affected by the treatment with LPS nor are they modified by the antagonist des-Arg¹⁰,[Leu⁹]-KD. The present data, together with previous studies on the sensitization mechanism of B₁-receptor containing preparations, suggest that LPS induces the formation of B₁-receptors in the rabbit, within a few hours. The activation of B₁-receptors by des-Arg⁹-BK produces hypotension, coronary vasodilation and stimulation of large arteries and veins isolated and suspended in vitro. Some large arteries and veins (e.g. the aorta and the anterior mesenteric vein) as well as some peripheral vascular beds (e.g. the coronary vessels) have the ability of generating B₁-receptors, while other organs (e.g. the external jugular vein) have not or very little. The reason for this phenomenon as well as the intimate mechanism by which LPS induces the formation of B₁-receptors remain to be elucidated.     

The effect of kinin agonists and antagonists on the pain response of the human blister base

Summary 1. The effect of bradykinin (BK) and some analogues of BK on the human blister base was studied. 2. BK produced reproducible dose-related increases in pain responses. A characteristic delay, which was not dose-related occurred between application of BK and the resultant response. 3. The rank order of potency of several kinin analogues on the pain response was BK > > > -cyclo-(Lys¹-Gly⁶)-BK = -cyclo-kallidin > des-Arg⁹-BK. 4. No increase in pain response was seen with repeated application of the selective B₁ receptor agonist des-Arg⁹-BK to the same blister base at 4 h intervals. The B₁ receptor antagonist des-Arg⁹-Leu⁸-BK was without effect against BK-induced responses. 5. The B₂ receptor antagonists, d-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-d-Phe-Thi-Arg-TFA and d-Pro-Phe-Arg-heptylamide produced significant antagonism of the bradykinin-induced pain responses at doses which had no effect against 5-hydroxytryptamine or potassium chloride. 6. It is concluded that the kinin receptor mediating pain on the human blister base is of the B₂ type. Send offprint requests to E. T. Whalle     

Analysis of the mechanism of action of bradykinin on human basilar artery in vitro

Summary Bradykinin (BK) initially produced concentration-related relaxations of human basilar artery in vitro. Concentration-effect curves constructed at 2 h intervals to BK over an 8 h period were reproducible. The rank order of potency of three kinins on the human basilar artery was found to be BK > methionyl-lysyl-BK > des-Arg⁹-BK. The B₂-receptor antagonist Thi^5,8 d-Phe⁷-BK but not the B₁-receptor antagonist des-Arg⁹-Leu⁸-BK selectively blocked BK-induced relaxations of the human basilar artery.The relaxant effects of bradykinin and acetylcholine but not papaverine were attenuated after removal of the endothelium or treating the tissues with BW755C. Indomethacin was without effect. Concentration-effect curves to angiotensin I were markedly attenuated by captopril at a concentration which had no effect on BK, angiotensin II or 5-hydroxytryptamine responses. It is concluded that BK induced relaxations of the human basilar artery are mediated via activation of a B₂ receptor and the response is dependent upon the release of a factor present in the endothelium. Angiotensin converting enzyme is present in the human basilar artery and is important for the conversion of angiotensin I to angiotensin II but apparently not for the degradation of BK. It is likely that other kininases are present and active in the tissue. Send offprint requests to E. T. Whalley at the above address     

Met-Lys-bradykinin-Ser-Ser, a peptide produced by the neutrophil from kininogen, is metabolically activated by angiotensin converting enzyme in vascular tissue

Bradykinin (BK) is a vasoactive nonapeptide cleaved from circulating kininogens and that is degraded by angiotensin converting enzyme (ACE). It has been reported that the PR3 protease from human neutrophil releases an alternate peptide of 13 amino acids, Met-Lys-BK-Ser-Ser, from high molecular weight kininogen. We have studied vascular actions of this kinin. Its affinity for recombinant B₁ and B₂ receptors is very low, as assessed by the binding competition of [³H]Lys-des-Arg⁹-BK and [³H]BK, respectively, but Met-Lys-BK-Ser-Ser effectively displaced a fraction of [³H]enalaprilat binding to recombinant ACE. Mutant recombinant ACE constructions revealed that affinity gap between BK and Met-Lys-BK-Ser-Ser is larger for the N-terminal catalytic site than for the C-terminal one, based on competition for the substrate Abz-Phe-Arg-Lys(Dnp)-Pro-OH in an enzymatic assay. Met-Lys-BK-Ser-Ser is a low potency stimulant of the rabbit aorta (bioassay for B₁ receptors), but the human isolated umbilical vein, a contractile bioassay for the B₂ receptors, responded to Met-Lys-BK-Ser-Ser more than expected from the radioligand binding assay, this agonist being ∼30-fold less potent than BK in the vein. Venous tissue treatment with the ACE inhibitor enalaprilat reduced the apparent potency of Met-Lys-BK-Ser-Ser by 15-fold, while not affecting that of BK. In the rabbit isolated jugular vein, Met-Lys-BK-Ser-Ser is nearly as potent as BK as a contractile stimulant of endogenous B₂ receptors (EC₅₀ values of 16.3 and 10.5 nM, respectively), but enalaprilat reduced the potency of Met-Lys-BK-Ser-Ser 13-fold while increasing that of BK 5.3-fold. In vascular tissue, ACE assumes a paradoxical activating role for Met-Lys-BK-Ser-Ser.     

The effect of kinins on paw oedema and uterus in rats

Summary Bradykinin (BK) produced a dose-related increase in the paw volume of the rat. Responses to BK at all doses used were not affected by pretreating the rats with diphenhydramine, 1 mg kg^–1, or indomethacin, 2.5 and 5 mg kg^–1. Indomethacin, 10 mg kg^–1 produced a small but significant reduction in the responses to BK. Captopril 1 mg kg^–1 enhanced responses to low but not to high doses of BK.The rank order of potency of various kinin analogues to increase paw volume was found to be methionyl-lysyl-BK (met-lys-BK) > BK > kysyl-BK (Kallidin) des-Arg⁹-BK. The B₁-receptor antagonist des-Arg⁹-Leu⁸-BK did not affect responses to BK on paw volume.Two modified kinin fragments S2302 (H-D-Pro-Phe-Arg-p-Nitroaniline) and S2441 (H-D-Pro-Phe-Arg-NH-heptyl) produced dose-related increases in paw volume both having approximately half the potency of BK. These responses were not anagonised by diphenhydramine, 1 mg kg^–1 which reduced significantly the response to histamine.On the isolated rat uterus the rank order of potency of various kinins was BK > Kallidin > met-lys-BK > des-Arg⁹-BK. The two modified kinin fragments S2302 and S2441 (but not des-Arg⁹-Leu⁸-BK) antagonised BK induced contractions of the rat uterus.From the rank order of potency studies the receptor mediating contraction of the rat uterus in vitro and increase in rat paw volume to BK appear to be of the same type. Whether the differences in the action of S2302 and S2441 in the rat paw and rat uterus reflect a real difference in receptor type requires further study.     

Receptors mediating the increase in vascular permeability to kinins: comparative studies in rat,guinea-pig and rabbit

Summary The effect of bradykinin (BK) and a variety of kinin analogues and modified kinin fragments were assessed in several models representing the vascular permeability aspect of the inflammatory response. The rank order of potency of various kinin analogues to increase paw volume and skin vascular permeability in rats was \gZ-cyclo-(Lys¹-Gly⁶)-BK = \gZ-cyclo-kallidin > BK > kallidin = methionyllysyl-BKdes-Arg⁹-BK. The same order was seen for skin responses in day 21, rheumatoid-arthritic rats. Mepyramine, 10 mg · kg^–1 almost completely inhibited rat skin vascular responses to histamine, had no effect on BK and produced a small but significant inhibition of the responses to both cyclic-kinins. A different rank order of potency for the kinins was produced in both the guinea-pig and rabbit skin; this being kallidin > methionyl-lysyl-BK > BK >> Z-cyclo-(Lys¹-Gly⁶)-BK = Z-cyclo-kallidin>des-Arg⁹-BK. Neither of the cyclic kinins antagonised BK-induced increases in skin vascular permeability in guinea-pig or rabbit. Two modified kinin fragments, D-Pro-Phe-Arg-paranitroaniline and D-Pro-Phe-Arg-heptylamide, which have previously been demonstrated to be putative B2 receptor antagonists on in vitro tissues, enhanced the effect of BK in rat skin and when injected alone produced dose-related increases in skin vascular permeability in normal and rheumatoid-arthritic rats, both having approximately half the potency of BK. Neither of these fragments possessed agonist or antagonist action against BK in guinea-pig or rabbit skin. The B₁. receptor antagonist, des-Arg⁹-Leu⁸-BK, had no effect against BK-induced responses in any of the models used. It is concluded that the kinin receptor mediating the increase in paw volume of rat and the increase in vascular permeability in rat, rabbit and guinea-pig is not of the B₁-type. The difference in effects of the cyclic kinins and the modified kinin fragments may reflect a difference in the receptor mediating paw oedema and skin permeability in the rat compared to that mediating the same response in rabbit and guinea-pig skin. This requires further study. Send offprint requests to E. T. Whalley at the above address     

Characterization of the receptor and the mechanisms underlying the inflammatory response induced by des-Arg9-BK in mouse pleurisy

1. The characterization of the B₁ kinin receptor, and some mediators involved in the inflammatory response elicited by intrathoracic (i.t.) administration of des-Arg⁹-bradykinin (BK) in the mouse model of pleurisy, was investigated.
2. An i.t. injection of des-Arg⁹-BK (10–100 nmol per site), a selective B₁ agonist, caused a significant and dose-related increase in the vascular permeability observed after 5 min, which peaked at 1 h, associated with an increase in cell influx, mainly neutrophils, and, to a lesser extent, mononuclear cell influx, peaking at 4 h and lasting for up to 48 h. The increase in fluid leakage caused by des-Arg⁹-BK was completely resolved 4 h after peptide injection. I.t. injection of Lys-des-Arg⁹-BK (30 nmol per site) caused a similar inflammatory response.
3. Both the exudation and the neutrophil influx elicited by i.t. injection of des-Arg⁹-BK were significantly antagonized (P<0.01) by an i.t. injection of the selective B₁ antagonists des-Arg⁹-[Leu⁸]-BK (60 and 100 nmol per site) or des-Arg⁹-NPC 17731 (5 nmol per site), administered in association with des-Arg⁹-BK (P<0.01), or 30 and 60 min before the cellular peak, respectively. In contrast, an i.t. injection of the B₂ bradykinin selective receptor antagonist Hoe 140 (30 nmol per site), at a dose which consistently antagonized bradykinin (10 nmol per site)-induced pleurisy, had no significant effect on des-Arg⁹-BK-induced pleurisy.
4. An i.t. injection of the selective tachykinin receptor antagonists (NK₁) FK 888 (1 nmol per site), (NK₂) SR 48968 (20 nmol per site) or (NK₃) SR 142801 (10 nmol per site), administered 5 min before pleurisy induction, significantly antagonized neutrophil migration caused by i.t. injection of des-Arg⁹-BK. In addition, FK 888 and SR 142801, but not SR 48968, also prevented the influx of mononuclear cells in response to i.t. injection of des-Arg⁹-BK (P<0.01). However, the NK₃ receptor antagonist SR 142801 (10 nmol per site) also significantly inhibited des-Arg⁹-BK-induced plasma extravasation. An i.t. injection of the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP_8–37 (1 nmol per site), administered 5 min before pleurisy induction, inhibited des-Arg⁹-BK-induced plasma extravasation (P<0.01), without significantly affecting the total and differential cell migration.
5. The nitric oxide synthase inhibitors L-NOARG and L-NAME (1 pmol per site), administered 30 min beforehand, almost completely prevented des-Arg⁹-BK (i.t.)-induced neutrophil cell migration (P<0.01), and, to a lesser extent, mononuclear cell migration (P<0.01). The D-enantiomer D-NAME had no effect on des-Arg⁹-BK-induced pleurisy. At the same dose range, L-NOARG and L-NAME inhibited the total cell migration (P<0.01). L-NAME, but not L-NOARG caused significant inhibition of des-Arg⁹-BK-induced fluid leakage. Indomethacin (1 mg kg⁻¹, i.p.), administered 1 h before des-Arg⁹-BK (30 nmol per site), inhibited the mononuclear cell migration (P<0.05), but, surprisingly, increased the neutrophil migration at 4 h without interfering with plasma extravasation. The administration of terfenadine (50 mg kg⁻¹, i.p.), 30 min before des-Arg⁹-BK (30 nmol per site), did not interfere significantly with the total cell migration or with the plasma extravasation in the mouse pleurisy caused by i.t. injection of des-Arg⁹-BK.
6. Pretreatment of animals with the lipopolysaccharide of E. coli (LPS; 10 μg per animal, i.v.) for 24 h did not result in any significant change of the inflammatory response induced by i.t. injection of des-Arg⁹-BK compared with the saline treated group. However, the identical treatment of mice with LPS resulted in a marked enhancement of des-Arg⁹-BK induced paw oedema (P<0.01).
7. In conclusion, we have demonstrated that the inflammatory response induced by i.t. injection of des-Arg⁹-BK, in a murine model of pleurisy, is mediated by stimulation of constitutive B₁ receptors. These responses are largely mediated by release of neuropeptides such as substance P or CGRP and also by NO, but products derived from cyclo-oxygenase pathway and histamine seem not to be involved. Therefore, these results further support the notion that the B₁ kinin receptor has an important role in modulating inflammatory responses, and it is suggested that selective B₁ antagonists may provide therapeutic benefit in the treatment of inflammatory and allergic conditions.

     

Kinin B1 and B2 receptors in pig vessels: characterization of two monoreceptor systems

The coronary artery and renal vein of the adult pig are sensitive and reliable monoreceptor systems for studying kinin receptors. The pig coronary artery with intact endothelium is highly sensitive to bradykinin (BK, pEC₅₀ 8.6), while being insensitive to the B₁ receptor agonist, LysdesArg⁹BK. The tissue responds to BK with concentration-dependent relaxation, which is prevented by B₂ receptor antagonists, particularly dArg[Hyp³, Thi⁵, dTic⁷, Oic⁸]BK (HOE 140, pK_B 9.3), (E)-3-(6-acetoamido-3-pyridyl)-N-(N-{2, 4-dichloro-3-[(2-methyl-8-quinolinyl)oxy-methyl]phenyl}-N-methylaminocarbonylmethyl)acrylamide (FR 173657), a new non peptide compound (pK _B 9.3), while B₁ receptor antagonists (e.g. Lys[Leu⁸]desArg⁹BK) are inactive. The order of potency of kinin-related peptides in this vessel is: LysBK ≥ BK > [Hyp³]BK>[Aib⁷]BK, a sequence typical of a B₂ receptor system. Antagonists such as HOE 140 and FR 173657, at high concentrations reduce the maximum effect of BK and thus behave as noncompetitive antagonists. The kinin B₁ receptor was studied in the pig renal vein without endothelium and incubated for several hours in order to allow for the de novo formation of this functional site. After 7–8 h in vitro incubation, the vessel shows high sensitivity to LysdesArg⁹BK (pEC₅₀ 8.3) and is insensitive to BK. The pig renal vein responds to B₁ receptor agonists with concentration-dependent contraction which maintains a stable plateau and is prevented by selective B₁ receptor antagonists such as Lys[Leu⁸]desArg⁹BK (pK_B 6.7). The most active antagonist has been found to be desArg⁹HOE 140 (pA₂ 7.6) which acts as competitive antagonist in this preparation. Some B₂ antagonists (e.g. HOE 140) show weak (pK _B 6.1) anti-B₁ receptor activity while the non-peptide compound FR 173657 is inactive on the B₁ receptor and therefore acts as a potent and selective kinin B₂ receptor antagonist in the pig. The data obtained in this study allow us to compare the porcine B₂ and B₁ receptors with those of other species including man, and underline some interesting features that are unique to the porcine functional sites. Received: 9 April / Accepted: 20 June 1997     

Effects of bradykinin on signal transduction,cell proliferation,and cytokine,prostaglandin E2 and collagenase-1 release from human corneal epithelial cells

1. We recently demonstrated the presence of phospholipase C-coupled bradykinin (BK) B₂-receptors in human primary and SV40 virus-immortalized corneal epithelial (CEPI) cells.
2. The aims of the present studies were to demonstrate the specific binding of [³H]-BK to CEPI cell membranes and to study its pharmacological characteristics. In addition, we wished to study the functional coupling of the BK receptors to various physiological and pathological mechanisms in the CEPI cells, including phosphoinositide (PI) turnover, intracellular Ca²⁺-mobilization ([Ca²⁺]_i), cell proliferation (via [³H]-thymidine incorporation), and the release of various cytokines, collagenase-1 (matrix metalloproteinase-1) and prostaglandin E₂ (PGE₂).
3. Specific [³H]-BK binding comprised 83±2% of the total binding, and was of high affinity (K_d=1.66±0.52 nM, n=5), saturable (B_max=640±154 fmol g⁻¹ wet weight) and reversible. Competition studies yielded the following affinity values for BK and a number of BK-related peptides: Hoe-140 (D-Arg-[Hyp³,Thi⁵,D-Tic⁷,Oic⁸]BK; icatibant): K_i=0.17±0.07 nM; BK: K_i=1.0±0.11 nM; [Tyr⁸]-BK: K_i=12.9±2.3 nM; [des-Arg⁹]-BK: K_i>9,200 nM (all n=3–5)).
4. BK potently stimulated PI turnover (EC₅₀=2.3±0.3 nM; n=7) and [Ca²⁺]_i mobilization (EC₅₀=8–20 nM) in CEPI cells and both responses were inhibited in a concentration-dependent manner by 100 nM–10 μM Hoe-140, a selective B₂-receptor antagonist, and also inhibited by the selective phospholipase C (PLC) inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) (IC₅₀=3.0±1.6 μM). BK-induced [Ca²⁺]_i mobilization was reduced by about 30% in the presence of 4 mM EGTA, but was not significantly affected by 100 nM nifedipine.
5. BK (0.1 nM–10 μM) significantly (P<0.05–0.001) stimulated [³H]-thymidine incorporation into CEPI cellular DNA. However, while interleukin-1α (IL-1α; 10 ng ml⁻¹) potently stimulated the release of IL-6, IL-8 and granulocyte macrophage colony-stimulating factor from CEPI cells, BK (0.1 nM–10 μM) was without effect.
6. Whilst phorbol-12-myristate-13-acetate (PMA; 3 μg ml⁻¹) and 10% foetal bovine serum (positive control agents) significantly stimulated the release of both MMP-1 and PGE₂ from CEPI cells, BK (0.1 nM–10 μM) was without any significant effect under these conditions.
7. In conclusion, these data indicate that the CEPI cells express high-affinity [³H]-BK binding sites representing B₂-subtype BK receptors coupled to PI turnover and [Ca²⁺]_i mobilization which appear to stimulate [³H]-thymidine incorporation into cellular DNA. In contrast, BK failed to elicit the release of PGE₂, various cytokines and MMP-1 from CEPI cells. These results suggest that BK may have a potential role in corneal epithelium wound healing by stimulating cell proliferation.

     

Effects of acute and chronic desipramine treatment on somatostatin receptors in brain

The effects of acute (5 mg/kg, IP twice daily for 2 days) and chronic (5 mg/kg IP twice daily for 21 days) administration of desipramine (DMI) on [¹²⁵I]-Tyr¹¹-somatostatin binding sites in brain were examined. There was no change in [¹²⁵I]Tyr¹¹-somatostatin binding in membranes prepared from the frontal cortex, striatum, and hippocampus of rats acutely or chronically treated with DMI as compared to non treated animals. [¹²⁵I]Tyr¹¹-Somatostatin binding was increased in membranes prepared from the rat nucleus accumbens only after chronic DMI administration. Scatchard analysis of the binding data from the nucleus accumbens showed that [¹²⁵I]Tyr¹¹-somatostatin labels a single population of somatostatin binding sites with an affinity constant, K_d, of 1.8±0.60 nM and a B_max of 330±90 fmol/mg protein. Chronic treatment with DMI increased the B_max (500±140 fmol/mg protein) but had no effect on the K_d. This finding shows a regional effect of DMI on [¹²⁵I]Tyr¹¹-somatostatin binding sites in rat brain and suggests that somatostatin may play a role in the pathophysiology of depression.     

Pharmacological and functional characterization of bradykinin receptors in canine cultured tracheal epithelial cells.

1. A direct [3H]-bradykinin ([3H]-BK) binding assay has been used to characterize the BK receptors in canine cultured tracheal epithelial cells (TECs). Based on receptor binding assay, TECs have specific, saturable, high-affinity binding sites for [3H]-BK. 2. The specific [3H]-BK binding was time- and temperature-dependent. Equilibrium of association of [3H]-BK with the BK receptors was attained within 30 min at room temperature and 1 h at 4 degrees C, respectively. 3. Analysis of binding isotherms yielded an apparent equilibrium dissociation constant (KD) of 1.5 +/- 0.2 nM and a maximum receptor density (Bmax) of 53.2 +/- 5.2 fmol mg-1 protein. The Hill coefficient for [3H]-BK binding was 1.00 +/- 0.02. The association (K1) and dissociation (K-1) rate constants were (7.6 +/- 1.1) x 10(6) M-1 min-1 and (9.2 +/- 1.5) x 10 M-3 min-1, respectively. KD, calculated from the ratio of K-1 and K1, was 1.2 +/- 0.3 nM, a value close to that calculated from Scatchard plots of binding isotherms. 4. Neither a B1 receptor selective agonist (des-Arg9-BK, 0.1 nM - 10 microM) nor antagonist ([Leu8, des-Arg9]-BK, 0.1 nM - 10 microM) significantly inhibited [3H]-BK binding to TECs, which excludes the presence of B1 receptors in canine TECs. 5. The specific binding of [3H]-BK to canine TECs was inhibited by the B2 receptor selective antagonists ([D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]-BK (Hoe 140, 0.1 nM-10 microM) and [D-Arg0, Hyp3, Thi5.8, D-Phe7]-BK, 0.1 nM - 10 microM) and agonists (BK and kallidin, 0.1 nM-10 microM) with a best fit by a one-binding site model. The order of potency for the inhibition of [3H]-BK binding was kallidin = BK = Hoe 140 > [D-Arg0, Hyp3, Thi5,8, D-Phe7]-BK. 6. BK and kallidin significantly induced concentration-dependent accumulation of IPs with a half-maximal response (EC50) at 17.6 +/- 3.5 and 26.6 +/- 5.3 nM, respectively, while the B1-selective agonist, des-Arg9-BK did not stimulate IPs accumulation and the B1-selective antagonist [Leu8, des-Arg9]-BK did not inhibit BK-induced IPs accumulation. Two B2-selective antagonists, Hoe 140 and [D-Arg0, Hyp3, Thi5,8, D-Phe7]-BK, inhibited BK-stimulated IPs accumulation with apparent pKB values of 8.8 +/- 0.3 and 7.0 +/- 0.3, respectively. 7. It is concluded that the pharmacological characteristics of the BK receptors in canine cultured TECs are primarily of the B2 receptor subtype which might regulate the function of tracheal epithelium through the activation of this receptor subtype coupling to PI hydrolysis.     

Bradykinin-induced contraction of guinea pig lung in vitro

We have investigated the contractile effect of bradykinin (BK) in guinea pig lung in vitro. BK induces a dose-related contraction of lung parenchymal strips which is increased significantly in the presence of 10^–5 M captopril (an angiotensin converting enzyme inhibitor) or 10^–5 M dl-thiorphan (a neutral endopeptidase inhibitor). The kininase I inhibitor, dl-2-mercaptomethyl-3-guanidino-ethylthiopropionic acid (MGTPA), has no effect on the BK-induced contraction. BK is more potent in contracting parenchymal lung strips than other contractile agents (histamine, carbachol and substance P), however the BK-induced maximal contraction is lower than those obtained with histamine and carbachol. The B₁ agonist, des-Arg⁹-BK, does not contract lung parenchymal strips. The new BK B₂ receptor antagonists (Hoe 140, NPC 17731 and NPC 17761), which possess binding affinities in the nanomolar range, inhibit the BK-induced contractile response in a dose-dependent manner. The BK-induced contraction was unaffected by propranolol, atropine, tetrodotoxin, capsaicin pre-treatment, triprolidine, methysergide, Ro 19-3704 and N-nitro-l-arginine-methyl-ester (l-NAME), excluding the involvement of nervous pathways, preformed mast cell mediators, platelet-activating factor and nitric oxide. However, indomethacin, a cyclooxygenase inhibitor, AA-861, a 5-lipoxygenase inhibitor, and furegrelate, a thromboxane A₂ synthase inhibitor, decreased the contractile response to BK, suggesting that both cyclooxygenase and 5-lipoxygenase products are involved in this contraction. Thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺ ATPase, abolished the BK-induced contraction demonstrating an intracellular calcium-dependent mechanism. Moreover, on a mixed lung cell suspension, obtained by enzymatic digestion, BK is able to induce phosphoinositide production. We conclude that BK, acting on B2 receptors, is a powerful contractile agent of the guinea pig lung in vitro. The BK-induced contraction, modulated by kininases II, is not dependent on neural mechanisms whereas both eicosanoids and intracellular calcium are involved. Correspondence to: J. P. Gies at the above address