Bradykinin receptors : agonists, antagonists, expression, signaling and adaptation to sustained stimulation

Bradykinin-related peptides, the kinins, are blood-derived peptides that stimulate 2 G protein–coupled receptors, the B1 and B2 receptors (B1R, B2R). The pharmacologic and molecular identities of these 2 receptor subtypes will be succinctly reviewed, with emphasis on drug development, receptor expression, signaling, and adaptation to persistent stimulation. Peptide and nonpeptide antagonists and fluorescent ligands have been produced for each receptor. The B2R is widely and constitutively expressed in mammalian tissues, whereas the B1R is mostly inducible under the effect of cytokines during infection and immunopathology. Both receptor subtypes mediate the vascular aspects of inflammation (vasodilation, edema formation). On this basis, icatibant, a peptide antagonist of the B2R, is approved in the management of hereditary angioedema attacks. Other clinical applications are still elusive despite the maturity of the medicinal chemistry efforts applied to kinin receptors. While both receptor subtypes are mainly coupled to the Gq protein and related second messengers, the B2R is temporarily desensitized by a cycle of phosphorylation/endocytosis followed by recycling, whereas the nonphosphorylable B1R is relatively resistant to desensitization and translocated to caveolae on activation

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 B2 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 B1 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

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

Mating tactics and mate choice in relation to age and social rank in male mountain goats

In polygynous mammals, mating success of males often depends on intense male-male competition and the use of alternative mating tactics. Because reproduction incurs substantial energetic costs and risks of fight injuries, mate selection by males should be expected, particularly when females vary in their ability to produce offspring but can only be defended 1 at a time. Here, we investigated during 3 ruts how age and social rank of male mountain goats (Oreamnos americanus) affected the formation of consort pairs with females (“tending” tactic) in a marked population at Caw Ridge, Alberta, Canada. Among consort pairs, we quantified the behaviors of males and females, and the use of an alternative mating tactic by competing males, “coursing,” which consists of disrupting the pair to gain temporarily access to the female, often by pursuing her. Mate choice was assessed by testing if old and dominant males observed in consort pairs tended experienced females more often than younger females, because reproductive success of females increases with age. Males in consort pairs were ≥4 years old and most (86%, n = 59) were in the top one-half of the dominance hierarchy. Age and social rank of males were positively related to age of females and the total number of young produced by the tended female. All observed matings (n = 32) occurred between 14 November and 2 December and 91% were between males and females in consort pairs. Subordinate males gained mating access to females through coursing, but this tactic was rare. Our study provides evidences of mate choice by males for experienced females in an ungulate and the 1st quantitative information on the rut of mountain goats

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 vectoralone-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 ≅ transcinnamoyl-LIGRLO-NH2 \u3e SLIGKV-NH2 \u3e 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. Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics

Insulin modulates protease-activated receptor 2 signaling: Implications for the innate immune response

Given the anti-inflammatory effects of insulin in human and animal studies done in vivo and given the signaling pathways in common between insulin and the protease-activated receptor 2 (PAR2), a G protein-coupled receptor, we hypothesized that insulin would have an impact on the inflammatory actions of PAR2. We found that low doses or concentrations of insulin in the subnanomolar range reduced PAR2-induced inflammation in a murine paw edema model, attenuated PAR2-induced leukocyte trafficking in mouse intestinal venules, and reduced PAR2 calcium signaling in cultured dorsal root ganglion neurons and endothelial cells. This effect of insulin to attenuate PAR2-mediated inflammation was reversed when cells were preincubated with LY294002 (a PI3K inhibitor) and GF 109203X (a pan-protein kinase C inhibitor). The enhanced inflammatory effect of PAR2 observed in vivo in an insulin-deficient murine type 1 diabetes model was attenuated by the local administration of insulin at the inflammatory site. Our data point to an anti-inflammatory action of insulin that targets the acute innate inflammatory response triggered by PAR2. Copyright © 2010 by The American Association of Immunologists, Inc

Proteinase-activated receptor-2 activating peptides: Distinct canine coronary artery receptor systems

In canine coronary artery preparations, the proteinase-activated receptor-2 (PAR2) activating peptides (PAR2-APs) SLIGRL-NH 2 and 2-furoyl-LIGRLO-NH2 caused both an endothelium-dependent relaxation and an endothelium-independent contraction. Relaxation was caused at peptide concentrations 10-fold lower than those causing a contractile response. Although trans-cinnamoyl-LIGRLO-NH2, like other PAR2-APs, caused relaxation, it was inactive as a contractile agonist and instead antagonized the contractile response to SLIGRL-NH 2. RT-PCR-based sequencing of canine PAR2 revealed a cleavage/activation (indicated by underlines) sequence (SKGR/SLIGKTDSSLQITGKG) that is very similar to the human PAR2 sequence (R/SLIGKV). As a synthetic peptide, the canine PAR-AP (SLIGKT-NH2) was a much less potent agonist than either SLIGRL-NH2 or 2-furoyl-LIGRLO-NH 2, either in the coronary contractile assay or in a Madin-Darby canine kidney (MDCK) cell PAR2 calcium signaling assay. In the MDCK signaling assay, the order of potencies was as follows: 2-furoyl-LIGRLO-NH 2 ≫ SLIGRL-NH2 = trans-cinnamoyl-LIGRLO-NH2 ≫ SLIGKT-NH2, as expected for PAR2 responses. In the coronary contractile assay, however, the order of potencies was very different: SLIGRL-NH2 ≫ 2-furoyl-LIGRLO-NH2 ≫ SLIGKT-NH 2, trans-cinnamoyl-LIGRLO-NH2 = antagonist. Because of 1) the distinct agonist (relaxant) and antagonist (contractile) activity of trans-cinnamoyl-LIGRLO-NH2 in the canine coronary contractile assays, 2) the different concentration ranges over which the peptides caused either relaxation or contraction in the same coronary preparation, and 3) the markedly distinct structure-activity profiles for the PAR-APs in the coronary contractile assay, compared with those for PAR2-mediated MDCK cell calcium signaling, we suggest that the canine coronary tissue possesses a receptor system for the PAR-APs that is distinct from PAR2 itself. Copyright © 2007 the American Physiological Society