Steeve Houle

Kinin receptors: functional aspects

Two types of receptors (B1R, B2R) for kinins are defined in mammalian species. Comparative experiments involving recombinant fusion proteins consisting of rabbit B1R or B2R fused to GFP-related proteins are exploited to study the regulation of the response to kinins at the receptor level. The following points will be briefly reviewed and supported by some novel data. (1) The constitutive B2Rs are internalized upon agonist stimulation, but completely recycled to the cell surface; however, B2R destruction can be achieved following limited proteolysis (extracellular trypsin, neutrophil proteases), a plausible down-regulation mechanism in pathology. (2) The inducible B1Rs, stimulated by des-Arg9-kinins, are not phosphorylated nor internalized upon agonist stimulation, but rather undergo a reversible redistribution to caveolae-related rafts. B2Rs are also subjected to this translocation, but only transiently (before endocytosis). (3) Based on the analysis of rabbit aortic smooth muscle cells, B1R induction by cytokines is dependent on nuclear factor KB in rabbit vascular tissue, but exogenous kinins acting on either receptor type do not induce B1R expression.

Antagonist-Induced Intracellular Sequestration of Rabbit Bradykinin B2 Receptor

In a contractility assay based on the rabbit jugular vein, the structurally related drugs NPC 17731 or icatibant (1 to 3 nmol/L) were insurmountable antagonists of bradykinin (BK) B(2) receptors (B(2)Rs). After ample washing (3 hours), the antagonism exerted by these peptides was not reversible. By contrast, the antagonist LF 16. 0687 (30 to 100 nmol/L) was competitive and reversible. A rabbit B(2)R-green fluorescent protein (B(2)R-GFP) conjugate was expressed in mammalian cells. In COS-1 cells, it exhibited an affinity for [3H]BK (K(D)=1.61 nmol/L) similar to that of the wild-type rabbit B(2)R. The stably expressed construction in HEK-293 cells was functionally active (phospholipase A(2) assay), and the antagonists mentioned above retained their respective surmountable or insurmountable behavior. Competition of [(3)H]BK binding to B(2)R-GFP by the antagonists or BK was largely reversible after a 3-hour washout period at 0 degrees C; at 37 degrees C, icatibant or NPC 17731 effects were not reversible. B(2)R-GFP was visualized in the plasma membranes of HEK-293 cells and rapidly internalized in response to BK. NPC 17731 or icatibant slowly translocated B(2)R-GFP into cells over 24 hours, whereas LF 16.0687 had no effect on the subcellular distribution of B(2)R-GFP. Cell extract immunoblotting with anti-GFP antibodies revealed a 101- to 105-kDa protein that was not significantly degraded on 24 hours of cell treatment with any of the ligands but was translocated in part to the 15 000-g pellet of the extract on treatment with BK or the noncompetitive antagonists. NPC 17731 and icatibant are noncompetitive, nonequilibrium antagonists that promote the cellular sequestration of rabbit B(2)R expressed in an heterologous system.

Altered frequency of a promoter polymorphic allele of the kinin B1 receptor gene in inflammatory bowel disease

BACKGROUND & AIMS Evidence of kinin-mediated inflammation in the gastrointestinal tract is accumulating. The genes and some polymorphic sites have been characterized for both kinin B1 and B2 receptors. These candidate genes were studied for their possible association with inflammatory bowel disease (IBD). METHODS In a retrospective study, the prevalence of allele pairs for four polymorphic sites of the two kinin receptor genes was determined in 53 patients with IBD and in 110 healthy volunteers similar in age, body weight, and gender proportions, using polymerase chain reaction and other techniques. RESULTS Only the B1 receptor promoter polymorphism (G-699-->C) exhibited a significantly different allele frequency between the two groups (prevalence of the C allele of 5.7% in patients with IBD compared with 33.6% in controls; P = 0.0002) or between the controls and either etiologic subgroup (ulcerative colitis and Crohns disease). Allelic polymorphisms affecting exon 3 of the B1 receptor gene (A1098-->G) or exon 2 (C181-->T) or 1 (a 9-base pair deletion) of the B2 receptor gene were found to be neutral. CONCLUSIONS The gene corresponding to the B1 receptor for kinins may be a nonetiologic marker of symptomatic IBD, as suggested by the altered prevalence of a polymorphism presumably affecting its regulation.

Effect of endogenous kinins, prostanoids, and NO on kinin B1 and B2 receptor expression in the rabbit

To determine whether kinin receptor expression is regulated by kinins, prostaglandins, and/or nitric oxide (NO), rabbits were treated with a B1 receptor (B1R) antagonist, a B2 receptor (B2R) antagonist, a prostacyclin mimetic, or inhibitors of NO synthase, cyclooxygenase, or angiotensin-converting enzyme. The mRNA concentrations for B1R and B2R (multiplex RT-PCR) were measured in several organs. The B2R mRNA expression was not significantly upregulated by any of the treatments; it was notably downregulated by angiotensin-converting enzyme or cyclooxygenase blockade or B2R antagonism in the heart and duodenum. A treatment with bacterial lipopolysaccharide (LPS), known to induce B1R expression, has also been applied and was the most consistent in upregulating the expression of B1R mRNA (kidney, duodenum, and striated muscle). The contractile responses mediated by kinin receptors in blood vessels isolated from the treated rabbits also indicated that LPS was the only B1R inducer (aorta). Icatibant, a nonequilibrium antagonist of the rabbit B2R, was the sole tested drug to alter the contractions mediated by the B2R in the jugular vein or the intensity of the immunohistochemical B2R staining in several organs (inhibition in both cases). B2R mRNA expression was downregulated in some organs by several of the applied treatments, but the data did not support generally applicable feedback for the regulation of B2R expression involving endogenous kinins, prostanoids, or NO. There was no indication of compensatory or reciprocal regulation of B1Rs, relative to B2Rs, inasmuch as B1R expression was restricted to LPS-treated animals.

Wortmannin alters the intracellular trafficking of the bradykinin B2 receptor: role of phosphoinositide 3-kinase and Rab5

Wortmannin reportedly induces the formation of enlarged cytoplasmic endosomes. Such vesicles were observed in a definite time window after wortmannin treatment (250 nM) in HEK-293 cells stably expressing a B2R (B2 receptor)--green fluorescent protein conjugate and other cell types. The alternative PI3K (phosphoinositide 3-kinase) inhibitor LY 294002 (100 microM) and a dominant-negative form of the enzyme (p85alpha DeltaiSH2) induce a more modest vesicle enlargement. PI3K inhibition by drugs did not affect agonist-induced [3H]arachidonate release. The wortmannin-induced formation of giant endosomes also involves Rab5 activity, since a dominant-negative form of this GTPase (Rab5 S34N) partially inhibits the wortmannin effect and a constitutively active form of Rab5 (Rab5 Q79L) induces the formation of enlarged endosomes. Moreover, agonist stimulation targeted B2R-green fluorescent protein towards the periphery of the giant vesicles and led to partial receptor degradation only in wortmannin-treated cells. Receptor degradation was decreased by protease inhibitors and by bafilomycin A1, a drug that inhibits lysosome function. Accumulation of fluorescent material inside the enlarged endosomes was observed in cells treated with bafilomycin A1, wortmannin and an agonist. [3H]Bradykinin binding was decreased in HEK-293 cells treated with both wortmannin and the agonist, but not with either separately. Furthermore, a wortmannin-induced functional down-regulation of B2R was observed in rabbit jugular veins after repeated agonist stimulation (contractility assay). This is the first report of a G-protein-coupled receptor down-regulation induced by an alteration of its usual routing in the cell. These results suggest that both PI3K and Rab5 influence B2R intracellular trafficking.

Non‐competitive pharmacological antagonism at the rabbit B1 receptor

The B1 receptor for kinins, stimulated by kinin metabolites without the C‐terminal Arg residue (e.g., des‐Arg9‐bradykinin (BK) and Lys‐des‐Arg9‐BK), is an increasingly recognized molecular target for the development of analgesic and anti‐inflammatory drugs. Recently developed antagonists of this receptor were compared to a conventional antagonist, Ac‐Lys‐[Leu8]‐des‐Arg9‐BK, in pharmacological assays based on the rabbit B1 receptor. B‐9858 (Lys‐Lys‐[Hyp3, Igl5, D‐Igl7, Oic8]des‐Arg9‐BK) and three other analogues possessing the α‐2‐indanylglycine5 (Igl5) residue (order of potency B‐9858 ∼ B‐10146>B‐10148>B‐10050) were partially insurmountable antagonists of des‐Arg9‐BK in the contractility assay based on rabbit aortic rings. B‐9858‐induced depression of the maximal effect was more pronounced in tissues treated with the protein synthesis inhibitor cycloheximide to block the spontaneous increase of response attributed to the post‐isolation formation of B1 receptors, and only partly reversible on washing. By comparison, Ac‐Lys‐[Leu8]des‐Arg9‐BK was a surmountable antagonist (pA2 7.5), even in cycloheximide‐treated tissues. B‐9958 (Lys‐[Hyp3, CpG5, D‐Tic7, CpG8]des‐Arg9‐BK) was also surmountable (pA2 8.5). The binding of [3H]‐Lys‐des‐Arg9‐BK to recombinant rabbit B1 receptors expressed in COS‐1 cells was influenced by two of the antagonists: while Ac‐Lys‐[Leu8]des‐Arg9‐BK competed for the radioligand binding without affecting the Bmax, B‐9858 decreased the Bmax in a time‐dependent and washout‐resistant manner. B‐9858 and analogues possessing Igl5 are the first reported non‐competitive, non‐equilibrium antagonists of the kinin B1 receptor.

Derivatized 2-Furoyl-LIGRLO-amide, a Versatile and Selective Probe for Proteinase-Activated Receptor 2. Binding and Visualization

The proteinase-activated receptor-2 (PAR2)-activating peptide with an N-terminal furoyl group modification, 2-furoyl-LIGRLO-NH2 (2fLI), was derivatized via its free ornithine amino group to yield [3H]propionyl-2fLI and Alexa Fluor 594-2fLI that were used as receptor probes for ligand binding assays and receptor visualization both for cultured cells in vitro and for colonic epithelial cells in vivo. The binding of the radiolabeled and fluorescent PAR2 probes was shown to be present in PAR2-transfected Kirsten normal rat kidney cells, but not in vector-alone-transfected cells, and was abolished by pretreatment of cells with saturating concentrations of receptor-selective PAR2 peptide agonists such as SLIGRL-NH2 and the parent agonist 2fLI but not by reverse-sequence peptides such as 2-furoyl-OLRGIL-NH2 that cannot activate PAR2. The relative orders of potencies for a series of PAR2 peptide agonists to compete for the binding of [3H]propionyl-2fLI (2fLI >> SLIGRL-NH2 ≅ trans-cinnamoyl-LIGRLO-NH2 > SLIGKV-NH2 > SLIGKT-NH2) mirrored qualitatively their relative potencies for PAR2-mediated calcium signaling in the same cells or for vasorelaxation in a rat aorta vascular assay. In the vascular assay, the potency of Alexa Fluor 594-2fLI was the same as 2fLI. We conclude that ornithine-derivatized 2fLI peptides are conveniently synthesized PAR2 probes that will be of value for future studies of receptor binding and visualization.

Antagonist, partial agonist and antiproliferative actions of B‐9870 (CU201) as a function of the expression and density of the bradykinin B1 and B2 receptors

A bradykinin (BK) B2 receptor (B2R) antagonist, B‐9870 (CU201), has been proposed to behave as a ‘biased agonist’ at B2Rs and to exert anti‐neoplasic effects. It was unclear whether these effects were determined by the activation of B2Rs by the drug. B‐9870 was evaluated for antagonism or stimulation of several responses mediated by the rabbit B2R or B1 receptor (B1R); its anti‐proliferative activity was also characterized.

Absence of ligand-induced regulation of kinin receptor expression in the rabbit

The induction of B1 receptors (B1Rs) and desensitization or down‐regulation of B2 receptors (B2Rs) as a consequence of the production of endogenous kinins has been termed the autoregulation hypothesis. The latter was investigated using two models based on the rabbit: kinin stimulation of cultured vascular smooth muscle cells (SMCs) and in vivo contact system activation (dextran sulphate intravenous injection, 2 mg kg−1, 5 h). Rabbit aortic SMCs express a baseline population of B1Rs that was up‐regulated upon interleukin‐1β treatment ([3H]‐Lys‐des‐Arg9‐BK binding or mRNA concentration evaluated by RT–PCR; 4 or 3 h, respectively). Treatment with B1R or B2R agonists failed to alter B1R expression under the same conditions. Despite consuming endogenous kininogen (assessed using the kinetics of immunoreactive kinin formation in the plasma exposed to glass beads ex vivo) and producing hypotension mediated by B2Rs in anaesthetized rabbits, dextran sulphate treatment failed to induce B1Rs in conscious animals (RT–PCR in several organs, aortic contractility). By contrast, lipopolysaccharide (LPS, 50 μg kg−1, 5 h) was an effective B1R inducer (kidney, duodenum, aorta) but did not reduce kininogen reserve. We tested the alternate hypothesis that endogenous kinin participate in LPS induction of B1Rs. Kinin receptor antagonists (icatibant combined to B‐9858, 50 μg kg−1 of each) failed to prevent or reduce the effect of LPS on B1R expression. Dextran sulphate or LPS treatments did not persistently down‐regulate vascular B2Rs (jugular vein contractility assessed ex vivo). The kinin receptor autoregulation hypothesis is not applicable to primary cell cultures derived from a tissue known to express B1Rs in a regulated manner (aorta). The activation of the endogenous kallikrein‐kinin system is ineffective to induce B1Rs in vivo in an experimental time frame sufficient for B1R induction by LPS.

Vascular smooth muscle contractility assays for inflammatory and immunological mediators.

The blood vessels are one of the important target tissues for the mediators of inflammation and allergy; further cytokines affect them in a number of ways. We review the use of the isolated blood vessel mounted in organ baths as an important source of pharmacological information. While its use in the bioassay of vasoactive substances tends to be replaced with modern analytical techniques, contractility assays are effective to evaluate novel synthetic drugs, generating robust potency and selectivity data about agonists, partial agonists and competitive or insurmountable antagonists. For instance, the human umbilical vein has been used extensively to characterize ligands of the bradykinin B(2) receptors. Isolated vascular segments are live tissues that are intensely reactive, notably with the regulated expression of gene products relevant for inflammation (e.g., the kinin B(1) receptor and inducible nitric oxide synthase). Further, isolated vessels can be adapted as assays of unconventional proteins (cytokines such as interleukin-1, proteases of physiopathological importance, complement-derived anaphylatoxins and recombinant hemoglobin) and to the gene knockout technology. The well known cross-talks between different cell types, e.g., endothelium-muscle and nerve terminal-muscle, can be extended (smooth muscle cell interaction with resident or infiltrating leukocytes and tumor cells). Drug metabolism and distribution problems can be modeled in a useful manner using the organ bath technology, which, for all these reasons, opens a window on an intermediate level of complexity relative to cellular and molecular pharmacology on one hand, and in vivo studies on the other.