Alexandre Trifilieff

Structural requirements for neuropeptide Y in mast cell and G protein activation

Incubation of neuropeptide Y or its C-terminal fragments with rat peritoneal mast cells resulted in a dose-dependent histamine release. Fragment 18-36 of neuropeptide Y was the most biologically active peptide. EC25 value on rat mast cells was 7.2 +/- 2.2 nM. Neuropeptide Y was also able to induce a flare response after intradermal injection in humans. The histamine releasing effects of neuropeptide Y related peptides were greatly inhibited by pretreatment of rat mast cells with pertussis toxin or benzalkonium chloride. Neuropeptide Y and C-terminal related peptides also stimulated the GTPase activity of purified heterotrimeric G proteins in a dose-dependent manner from 1 to 50 microM. Binding studies with [125I]neuropeptide Y were unable to provide evidence for the presence of specific binding sites on the surface of mast cells. The alpha helical conformation of neuropeptide Y fragments was studied by measuring the circular dichroism spectra. Neuropeptide Y-(18-36) was the smallest fragment having a strong helical conformation. Our results demonstrate that neuropeptide Y activates mast cells through a non-specific process leading to G protein activation.

Comparative action of new highly potent bradykinin receptor antagonists in the guinea-pig trachea.

We investigated the effect of two new bradykinin receptor antagonists, D-Arg0[Hyp3,D-HypE(trans-propyl)7,Oic8]bradykinin (NPC 17731) and D-Arg0[Hyp3,D-HypE(trans-thiophenyl)7,Oic8]bradykinin (NPC 17761) on [3H]bradykinin binding and on bradykinin-induced contraction of the guinea-pig trachea. Both of these compounds inhibited [3H]bradykinin binding with an affinity similar to that of unlabelled bradykinin. NPC 17731 inhibited bradykinin-induced contraction in a non-competitive manner, whereas NPC 17761 showed competitive antagonism. Therefore, NPC 17761 could contribute to the investigation of the bradykinin receptors in guinea-pig airways.

Effects of WIN 64338, a nonpeptide bradykinin B2 receptor antagonist, on guinea‐pig trachea

We investigated the effect of the nonpeptide bradykinin receptor antagonist, [[4‐[[2‐[[bis(cyclohexylamino)methylene] amino]‐3‐(2‐naphthalenyl) 1‐oxopropyl]amino]‐phenyl]‐tributyl, chloride, monohydrochloride (WIN 64338), on [3H]‐bradykinin binding and on bradykinin‐induced contraction of the guinea‐pig trachea. This non peptide bradykinin receptor antagonist inhibited [3H]‐bradykinin binding with a nanomolar range of affinity, Ki = 50.9 ± 19 nM and inhibited bradykinin‐induced contraction in a noncompetitive manner with a KB value of 6.43 10−8±2.34 10−8 m.

Bradykinin binding sites in healthy and carcinomatous human lung.

1 Direct ligand binding techniques have been used to compare bradykinin receptors in squamous‐ or adeno‐carcinoma and healthy lung membranes removed from patients during operations. 2 The binding of [3H]‐bradykinin to healthy lung membrane is time‐dependent and saturable with a KD value of 1.08 ± 0.18 nm and a Bmax value of 46.1 ± 3.1 fmol mg−1protein (n = 10). In squamous‐carcinoma tissue (n = 8) the same amount of receptors are present, Bmax = 52.2 ± 3.3 fmol mg−1protein (P = 0.22) but the KD value is significantly higher 2.57 ± 0.40 nm (P = 0.004). Similar measurements were obtained for adeno‐carcinoma tissue (n = 3), KD = 2.80 ± 0.29 mm (P = 0.001) and Bmax = 49.8 ± 2.1 fmol mg−1protein (P = 0.56). 3 In both healthy and squamous‐carcinoma preparations, bradykinin analogues displace [3H]‐bradykinin binding with the following relative order of potency: Hoe 140 > bradykinin > kallidin > d‐Arg0[Hyp3,d‐Phe7]bradykinin >>>des‐Arg9‐bradykinin. Of the analogues used, bradykinin and d‐Arg0[Hyp3,d‐Phe7]bradykinin appear to be able to differentiate the bradykinin receptors present in both preparations. 4 It is concluded that bradykinin receptors present in healthy and carcinomatous human lung are of the B2 type.

Involvement of B2 receptors in the bradykinin‐induced relaxation of guinea‐pig isolated trachea

1 The aim of this study was to determine the receptor type and involvement of arachidonic acid metabolites in bradykinin‐induced relaxation of the guinea‐pig isolated trachea. 2 In the resting tracheal preparation, bradykinin (0.1 nm − 30 μm induced a concentration‐related contractile response (pD2 = 8.8 ± 0.3). The maximal tension (1056 ± 321 mg) was observed at 0.3 μm bradykinin. In contrast, when tracheal preparations were pre‐contracted with histamine (30 μm leading to a half‐maximum response), a concentration‐related relaxation was observed with bradykinin. At the highest concentration of bradykinin used (3 μm), a reversal of 63 ± 13% of the contractile response to histamine was observed. Both effects of bradykinin were inhibited by the cyclo‐oxygenase inhibitor, indomethacin (1 μm). In concentration‐response curves, melittin (10 nm − 1 μm), a direct activator of phospholipase A2, mimicked both effects of bradykinin. The highest concentration of melittin used (1 μm), induced a tension of 813 ± 120 mg and led to the reversal of 41 ± 8% of the contractile response to histamine. The contractile effect of melittin was inhibited in the presence of both indomethacin (1 μm) and AA861 (1 μm), a 5‐lipoxygenase inhibitor. 3 [Des Arg9]‐bradykinin (1 nm‐3 μm), a B1‐receptor agonist, was unable to relax precontracted guinea‐pig tracheal preparations. The relaxation induced by bradykinin was antagonized by the B2 receptor antagonists, Hoe 140 (d‐Arg0[Hyp3, Thi5, d‐Tic7, Oic8]bradykinin) and NPC 17761 (d‐Arg0[Hyp3, d‐HypE(trans‐thiophenyl)7, Oic8]bradykinin). Hoe 140 (0.1 μm to 0.6 μm) behaved as a non‐competitive antagonist with an apparent pA2 = 7.2 ± 0.4, whereas NPC 17761 (0.3 to 1 μm) competitively antagonized bradykinin‐induced relaxation with a pKB = 7.3 ± 0.2. The Schild regression slope did not differ from unity, 0.96 ± 0.20, P < 0.05. 4 These data demonstrate that bradykinin‐induced relaxation of guinea‐pig trachea occurs via the activation of bradykinin B2‐receptors. The stimulation of B2‐bradykinin receptors induces the activation of the cyclo‐oxygenase pathway, leading either to contraction or relaxation depending on the tone of the trachea.

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 Nω-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.

Co-solubilization of bradykinin B2 receptors and angiotensin-converting enzyme from guinea pig lung membranes

Bradykinin B2 receptor-like binding activity was solubilized from guinea pig lung using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (Chaps). The binding of [3H]bradykinin to the soluble fraction was time-dependent and saturable. Scatchard analysis of equilibrium binding data indicated that the soluble extract contained a single class of binding sites with a Kd of 696 pM and a Bmax of 57 fmol/mg protein. Unlabelled bradykinin and B2 antagonists inhibited the binding of [3H]bradykinin to Chaps-solubilized extracts with relative potencies similar to those observed with the low-affinity membrane-bound binding sites. Following partial purification of the soluble preparation, using anion exchange (DEAE-Sephacel) and gel filtration (Aca 34) column chromatography steps, two peaks eluted off the column were able to bind [3H]bradykinin and have molecular masses of 168 and 98.5 kDa. The former seems to represent binding of bradykinin to angiotensin converting enzyme (ACE, EC 3.4.15.1) and the latter binding to bradykinin receptor. Using purified commercial ACE, we show that the binding of [3H]bradykinin to ACE can easily be distinguished from that of the bradykinin receptor, since both B1 and B2 ligands were able to inhibit bradykinin binding with affinities clearly different from that expected for a bradykinin receptor.

Bombesin Receptor Subtypes: Characterization and Solubilization

Publisher Summary Bombesin (BBS) is a 14-amino acid peptide that was originally isolated from the skin of the European frog, Bombina bombina. Various BBS-related peptides have been purified from amphibians. These amphibian skin peptides have been classified into BBS, litorin/ranatensin, and phyllolitorin subfamilies. This classification is based on their receptor affinities and on the primary amino acid sequences of their amidated carboxy-terminal octapeptide domains, which are the critical regions for receptor binding and biological activity. The BBS subfamily is characterized by the C-terminal tetrapeptide -Gly-His-Leu-Met-NH2; the litorin/ranatensin subfamily by the tetrapeptide -Gly-His-Phe-Met-NH2; and the phyllolitorin subfamily by the tetrapeptide -Gly-Ser-Phe(Leu)-Met-NH2. At present, only the BBS and litorin/ranatensin subfamilies have counterparts in mammalian tissues. Two mammalian BBS-like peptides, gastrin-releasing peptide (GRP), and neuromedin B (NMB), have been identified and characterized. GRP can be present in various forms having different molecular sizes. This chapter discusses the methodology used to identify and characterize the BBS receptor population in guinea pig lung, where high levels of BBS-like immunoreactivity have been reported.