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 (PAR(2)), a G protein-coupled receptor, we hypothesized that insulin would have an impact on the inflammatory actions of PAR(2). We found that low doses or concentrations of insulin in the subnanomolar range reduced PAR(2)-induced inflammation in a murine paw edema model, attenuated PAR(2)-induced leukocyte trafficking in mouse intestinal venules, and reduced PAR(2) calcium signaling in cultured dorsal root ganglion neurons and endothelial cells. This effect of insulin to attenuate PAR(2)-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 PAR(2) 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 PAR(2).

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 approximately= 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.

Neutrophils and the kallikrein-kinin system in proteinase-activated receptor 4-mediated inflammation in rodents.

1 We evaluated a potential role for proteinase-activated receptor 4 (PAR(4)) in a rodent paw inflammation model, with a focus on two main features of inflammation: (1) oedema and (2) granulocyte recruitment. 2 A PAR(4) antagonist (Pepducin P4pal-10; palmitoyl-SGRRYGHALR-NH(2)) reduced both the oedema and granulocyte recruitment induced by a localized administration of carrageenan in the rat hind paw, pointing to a key role for PAR(4) in this inflammation model. 3 Further, intraplantar injection in the mouse hind paw of a PAR(4) agonist (AYPGKF-NH(2)), but not its standard PAR(4)-inactive peptide control (YAPGKF-NH(2)), caused an inflammatory reaction characterized by oedema (increased paw thickness) and granulocyte recruitment (increased paw myeloperoxidase activity). The PAR(4) agonist-induced effects were inhibited in mice pretreated with pepducin P4pal10. 4 These PAR(4) agonist-mediated effects were not affected by pretreatment with inhibitors of either NO production or prostaglandin release (L-NAME and indomethacin, respectively). 5 However, selective immuno-depletion of neutrophils significantly reduced PAR(4) agonist-induced oedema formation. 6 Moreover, AYPGKF-NH(2)-induced oedema was also reduced by pretreatment with either a kinin B(2) receptor antagonist (icatibant) or a tissue or plasma kallikrein inhibitor (FE999024 and FE999026, respectively), but not with a kinin B(1) receptor antagonist (SSR240612). 7 We conclude: (1) that PAR(4) plays an important role in the inflammatory response as it mediates some of the hallmarks of inflammation and (2) that PAR(4)-mediated oedema is dependent on the recruitment of neutrophils and components of the kallikrein-kinin system.

Proteinases as hormone-like signal messengers.

Proteinases like thrombin and trypsin, long known for their ability to activate the coagulation cascade or to act as hormone-processing enzymes, are now recognised as hormone-like regulators of cell function. These serine proteinases activate cell signalling by triggering a novel four-member family of G-protein-coupled receptors, termed Proteinase-Activated Receptors (PARs). This review article summarises historically the discovery of PARs as well as their unique mechanism of activation and outlines a number of different pathophysiological settings in which PARs can act to regulate cell and tissue function. PARs can be seen to play a role in pathophysiological processes ranging from inflammation and pain to cardiovascular disease and cancer. Apart from activating PARs to cause their physiological effects in tissues, proteinases can also mediate cell signalling via a number of other mechanisms, including the activation of growth factor receptors, like the one for insulin. Therefore, this article also describes the non-PAR mechanisms whereby proteinases can have hormone-like actions in cells and tissues.

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.

Proteinase-activated receptor-4: evaluation of tethered ligand-derived peptides as probes for receptor function and as inflammatory agonists in vivo.

1. We evaluated the ability of a number of peptides based on the tethered ligand sequences of human, rat and murine proteinase-activated receptor-4 (PAR(4)), to serve as receptor-activating probes or antagonists for bioassays carried out in vitro and for in vivo models of inflammation. 2. In a rat PAR(4)-dependent platelet aggregation assay, the relative potencies of the active sequences (AYPGKF-NH(2)>GYPGKF-NH(2)>GYPGFK-NH(2)>GFPGKP-NH(2)) were consistent with an activation of PAR(4). 3. In the aggregation assay, the reverse or partial reverse-sequence peptides (VQGPYG-NH(2), YAPGKF-NH(2) and FKGPYA-NH(2)) were inactive, while trans-cinnamoyl (Tc)-YPGKF-NH(2), Tc-APGKF-NH(2) and N-palmitoyl-SGRRYGHALR-NH(2) (pepducin P4pal-10) were antagonists. 4. However, in an endothelium-dependent NO-mediated rat aorta (RA) relaxation assay and in a gastric longitudinal muscle (LM) contraction assay, these antagonist peptides were agonists as were most other peptides, with distinct orders of potencies that differed for both the RA and LM assays and from the platelet assay. 5. We conclude that PAR(4)-derived tethered ligand peptide agonists can act at receptors other than PAR(4) and that a judicious choice of ligands is required to probe for PAR(4) function in bioassay systems and in particular for in vivo models. 6. By selecting from these peptides the ones most reliably reflecting PAR(4) activation (AYPGKF-NH(2) as a standard agonist; YAPGKF-NH(2) as a PAR(4)-inactive standard), we were able to establish an inflammatory role for the PAR(4)-activating peptides acting via a non-neurogenic mechanism in a rat paw oedema model.

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.

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.

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

In canine coronary artery preparations, the proteinase-activated receptor-2 (PAR(2)) activating peptides (PAR(2)-APs) SLIGRL-NH(2) and 2-furoyl-LIGRLO-NH(2) 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-NH(2), like other PAR(2)-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 PAR(2) revealed a cleavage/activation (indicated by underlines) sequence (SKGR/SLIGKTDSSLQITGKG) that is very similar to the human PAR(2) sequence (R/SLIGKV). As a synthetic peptide, the canine PAR-AP (SLIGKT-NH(2)) was a much less potent agonist than either SLIGRL-NH(2) or 2-furoyl-LIGRLO-NH(2), either in the coronary contractile assay or in a Madin-Darby canine kidney (MDCK) cell PAR(2) calcium signaling assay. In the MDCK signaling assay, the order of potencies was as follows: 2-furoyl-LIGRLO-NH(2) >> SLIGRL-NH(2) = trans-cinnamoyl-LIGRLO-NH(2) >> SLIGKT-NH(2), as expected for PAR(2) responses. In the coronary contractile assay, however, the order of potencies was very different: SLIGRL-NH(2) >> 2-furoyl-LIGRLO-NH(2) >> SLIGKT-NH(2), trans-cinnamoyl-LIGRLO-NH(2) = antagonist. Because of 1) the distinct agonist (relaxant) and antagonist (contractile) activity of trans-cinnamoyl-LIGRLO-NH(2) 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 PAR(2)-mediated MDCK cell calcium signaling, we suggest that the canine coronary tissue possesses a receptor system for the PAR-APs that is distinct from PAR(2) itself.

Tissue kallikrein actions at the rabbit natural or recombinant kinin B2 receptors.

We have examined whether exogenous human tissue kallikrein exerts pharmacological actions via the bradykinin B2 receptor; specifically, whether the protease can bind to, cleave, internalize, and/or activate a fusion protein composed of the rabbit B2 receptor conjugated to the green fluorescent protein (B2R-GFP). The enzyme partially digested the fusion protein at 1 micromol/L, but not 100 nmol/L, and promoted B2R-GFP endocytosis in HEK 293 cells (> or =50 nmol/L). Trypsin and endoproteinase Lys-C, but not plasma kallikrein, also cleaved B2R-GFP. Phospholipase A2 was activated by 50 nmol/L tissue kallikrein in HEK 293 cells expressing B2R-GFP, and this was mediated by the receptor, as shown by the effect of a B2 receptor antagonist and by the lack of response in untransfected cells. However, 500 nmol/L kallikrein elicited a strong receptor-independent activation of phospholipase A2. Tissue kallikrein competed for [3H]bradykinin binding to B2R-GFP only at 1 micromol/L. A simulation involving kallikrein treatment of HEK 293 cells, pretreated or not with human plasma, evidenced the formation of immunoreactive bradykinin. The enzyme (50 nmol/L) contracted the rabbit isolated jugular vein via its endogenous B2 receptors, but the effect was tachyphylactic, and there was no cross-desensitization with bradykinin effects. Aprotinin prevented all pharmacological responses to tissue kallikrein, indicating that the enzyme activity is required for its effect. The local generation of kinins is a plausible mechanism for the pharmacological effects of lower concentrations of tissue kallikrein (50 to 100 nmol/L); higher levels (0.5 to 1 micromol/L) can not only initiate the degradation of rabbit B2 receptors but also exert nonreceptor-mediated effects.