François Marceau

Oxidative Stress-Induced Actin Reorganization Mediated by the p38 Mitogen-Activated Protein Kinase/Heat Shock Protein 27 Pathway in Vascular Endothelial Cells

Vascular endothelial cells are constantly in contact with oxyradicals and must be especially well equipped to resist their toxic effects and generate appropriate physiological responses. Despite the importance of oxyradicals in the physiopathology of the vascular endothelium, the mechanisms regulating the oxidative response of endothelial cells are poorly understood. In the present study, we observed that H2O2 in concentrations that induced severe fragmentation of F-actin in fibroblasts rather induced a reorganization of F-actin in primary cultures of human umbilical vein endothelial cells (HUVECs) that was characterized by the accumulation of stress fibers, the recruitment of vinculin to focal adhesions, and the loss of membrane ruffles, H2O2 also induced in these cells a strong (10- to 14-fold) activation of the p38 mitogen-activated protein (MAP) kinase, which resulted in activation of MAP kinase-activated protein kinase-2/3 and phosphorylation of the F-actin polymerization modulator, heat shock protein 27 (HSP27). The MAP kinases extracellular-regulated kinase, and c-Jun N-terminal kinase/stress-activated protein kinase were only slightly increased by these treatments. Inhibiting p38 activity with the highly specific inhibitor SB203580 blocked the H2O2-induced endothelial microfilament responses. Moreover, fibroblasts acquired an endothelium-like SB203580-sensitive actin response when HSP27 concentration was increased by gene transfection to the same high level as found in HUVECs. The results indicate that activation of p38 MAP kinase in cells such as endothelial cells, which naturally express high level of HSP27, plays a central role in modulating microfilament responses to oxidative stress. Consequently, the p38 MAP kinase pathway may participate in the several oxyradical-activated functions of the endothelium that are associated with reorganization of microfilament network.

Kinin B1 receptors: a review

The kinin B1 receptor has been initially defined as the one mediating the contractile effect of bradykinin (BK)-related peptides in the isolated rabbit aorta. The B1 receptor is selectively sensitive to kinin metabolites without the C-terminal arginine residue, e.g. des-Arg9-BK and Lys-des-Arg9-BK; it is apparently rapidly up-regulated in immunopathology under the influence of cytokines and is further regulated by growth factors. Progress in the understanding of this pharmacologic entity is reviewed, including the development of B1 receptor agonists and antagonists, binding assays, physiopathological applications and the recent cloning and sequencing of the receptor cDNA.

Bradykinin receptor ligands: therapeutic perspectives

Kinins, which are produced by the action of kallikrein enzymes, are blood-derived local-acting peptides that have broad effects mediated by two related G-protein-coupled receptors termed the bradykinin receptors. The endogenous kallikrein–kinin system controls blood circulation and kidney function, and promotes inflammation and pain in pathological conditions, which has led to interest in developing modulators of bradykinin receptors as potential therapeutics. This review discusses recent progress in our understanding of the genetics, molecular biology and pathophysiology of kinins and their receptors, as well as developments in medicinal chemistry, which have brought us closer to therapeutic applications of kinin receptor ligands in various indications. The potential of kinin receptor antagonists as novel analgesic agents that do not result in tolerance or have a liability for abuse has attracted particular interest.

Studies on the induction of pharmacological responses to des-Arg9-bradykinin in vitro and in vivo.

1 The mechanisms by which agents modulate the induction of kinin B1‐receptors were investigated by studying the effects of kinins in vitro, by use of the rabbit isolated aorta, and in vivo by measuring the blood pressure of anaesthetized rabbits. 2 The contractile response of the rabbit isolated aorta to kinins increased in a time‐dependent manner in vitro. This effect was abolished by continuous exposure to the protein synthesis inhibitor cycloheximide (71 μm). 3 Several substances were found to increase specifically the rate of sensitization to des‐Arg9‐bradykinin (des‐Arg9‐Bk), when applied continuously in vitro to tissues isolated from normal animals: bacterial lipopolysaccharide (LPS; 1 μg ml−1), muramyl‐dipeptide (MDP; 2 μg ml−1), phorbol myristate acetate (PMA; 320 nm), epidermal growth factor (EGF; 100 ng ml−1) and endothelial cell growth factor (150 μg ml−1). 4 The protease inhibitors phenylmethylsulphonyl fluoride and aprotinin, a non‐adjuvant isomer of MDP, rabbit purified leukocyte interferon, fibroblast growth factor and the chemotactic peptide N‐formyl‐l‐methionyl‐l‐leucyl‐l‐phenylalanine (FMLP) did not have this effect. 5 It has been demonstrated that LPS induces B1‐receptors in rabbits enabling des‐Arg9‐Bk to act as a hypotensive agent. In these experiments neutropenia induced by nitrogen mustard, did not prevent the in vivo effect of LPS. MDP (300 μg) and PMA (100 μg) were also found to induce a state of responsiveness to des‐Arg9‐Bk in vivo. FMLP (1 mg i.v.) induced a temporary decrease in blood neutrophil counts but had no effect on the induction of responses to des‐Arg9‐Bk. 6 The development of responses mediated by the B1‐receptor in the two experimental systems seems to be unrelated to the activation of neutrophil leukocytes, but may be related to the activation of tissue macrophages. Approximately 3% of cultured adherent cells derived from rabbit aorta strips following protease digestion were stained for non‐specific esterase, supporting such a possibility.

Effect of glucocorticoids, monokines and growth factors on the spontaneously developing responses of the rabbit isolated aorta to des‐Arg9‐bradykinin

1 The mechanisms modulating the spontaneous induction of contractile responses to agonists of the B1‐receptors for kinins have been studied by submitting the rabbit isolated aorta preparation to various in vitro treatments. Des‐Arg9‐bradykinin (Des‐Arg9‐BK), applied after 6 h of in vitro incubation was the standard stimulus to monitor this up‐regulation process. 2 Specific inhibition of the development of the contractile response to des‐Arg9‐BK was obtained by exposing tissues continuously to dexamethasone, dexamethasone sodium phosphate (DSP) or cortisol, but not to oestradiol. The maximal extent of the inhibition obtained at high concentrations of glucocorticoids was 86%. 3 No gross inhibition of protein synthesis was observed in the presence of DSP as monitored by [35S]‐methionine incorporation into incubated pieces of rabbit aorta. 4 In vivo pretreatment of rabbits with DSP did not reduce further the development of the responses in vitro. DSP applied 15 min before the 6 h recording did not antagonize the contractile effect of the BK fragment. 5 Interleukin 1 (IL‐1) and interleukin 2 (IL‐2) applied in vitro for the first 3 h of incubation increased the development of the contractile response to des‐Arg9‐BK. 6 Arachidonic acid (AA), nordihydroguaiaretic acid, tumour necrosis factor‐α (TNF) and transforming growth factor‐β (TGF‐β) failed to influence the spontaneous development of the response to kinins. 7 Continuous exposure to DSP (100 μm) markedly inhibited the action of stimulants in this system: IL‐1, IL‐2, epidermal growth factor and muramyl dipeptide. Moreover, the presence of AA (30 μm) did not prevent the inhibitory effect of DSP (10 μm). 8 None of the treatments applied singly or in combination modified the contractile response of the rabbit aorta to noradrenaline. 9 The results are discussed in terms of the possible involvement of immunocompetent cells in the up‐regulation of vascular responsiveness to B1 receptor agonists.

The kallikrein-kininogen-kinin system: lessons from the quantification of endogenous kinins.

The purpose of the present review is to describe the place of endogenous kinins, mainly bradykinin (BK) and des-Arg(9)-BK in the kallikrein-kininogen-kinin system, to review and compare the different analytical methods reported for the assessment of endogenous kinins, to explain the difficulties and the pitfalls for their quantifications in biologic samples and finally to see how the results obtained by these methods could complement and extend the pharmacological evidence of their pathophysiological role.

Pulse exposure to protein synthesis inhibitors enhances vascular responses to des-Arg9-bradykinin: possible role of interleukin-1.

1 The modulation of the spontaneous increase in contractile responses to des‐Arg9‐bradykinin (des‐Arg9‐BK) of rabbit aortic strips incubated in vitro was studied. Rapid hypotensive responses to exogenous kinins were also measured in rabbits anaesthetized 5 h following pretreatment. 2 Continuous exposure to the protein synthesis inhibitors cycloheximide (71 μm) or anisomycin (3.8 μm) profoundly inhibited the sensitization to des‐Arg9‐BK in incubated aortic strips. However, temporary (3 h) inhibition of protein synthesis in vitro followed by further incubation (3 h) of tissues without inhibitor, paradoxically enhanced both the maximal contractile responses to des‐Arg9‐BK (1.7 μm) and the apparent affinity of the kinin without affecting contractions to noradrenaline (NA, 100 nm) at 6.5 h. 3 The stimulatory activity of the short treatment (pulse) with cycloheximide was abolished in the presence of dexamethasone sodium phosphate (100 μm throughout the incubation). The function of receptors for kinins did not appear to be altered directly by the steroid treatment. 4 Interleukin‐1β (IL‐1β), applied at low concentrations (100–250 pg ml−1) on aortic strips between 3 h and 6.5 h of incubation time, mimicked the selective stimulatory effect of the cycloheximide pulse on responses to des‐Arg9‐BK. Higher concentrations of IL‐1β (0.5–5 ng ml−1) did not further amplify the responses to des‐Arg9‐BK but decreased the contractile responses to NA. 5 The modulation by IL‐1β of vascular sensitivity to des‐Arg9‐BK and to NA was prevented by blockade of protein synthesis. 6 The induction in vivo by IL‐1β (5 μg kg−1) or by cycloheximide (10 mg kg−1) of cardiovascular responsiveness to des‐Arg9‐BK was demonstrated with a blood pressure assay of exogenous kinins or with tissues isolated ex vivo 5 h after pretreatment of animals. Evidence of active disposition of cycloheximide in vivo was also obtained. 7 We propose the production of endogenous IL‐1 as a possible mechanism for the enhancement of responsiveness to des‐Arg9‐BK observed in tissues pulsed with a protein synthesis inhibitor and for the inducing effect of cycloheximide or E. coli lipopolysaccharide in vivo. These results suggest that effects mediated by the BK1 receptor for kinins are potentially present in pathological conditions associated with IL‐1 production.

NTPDase1 (CD39) controls nucleotide-dependent vasoconstriction in mouse

AIMS Extracellular nucleotides are vasoactive molecules. The concentrations of these molecules are regulated by ectonucleotidases. In this study, we investigated the role of the blood vessel ectonucleotidase NTPDase1, in the vasoconstrictor effect of nucleotides using Entpd1(-/-) mice. METHODS AND RESULTS Immunofluorescence, enzyme histochemistry, and HPLC analysis were used to evaluate both NTPDase expression and activity in arteries and isolated vascular smooth muscle cells (VSMCs). Vascular reactivity was evaluated in vitro and mean arterial blood pressure was recorded in anesthetized mice after nucleotide i.v. infusion. Expression of nucleotide receptors in VSMCs was determined by RT-PCR. Entpd1(-/-) mice displayed a dramatic deficit of nucleotidase activity in blood vessel wall in situ and in VSMCs in comparison to control mice. In aortic rings from Entpd1(-/-) mice, UDP and UTP induced a potent and long-lasting constriction contrasting with the weak response obtained in wild-type rings. This constriction occurred through activation of P2Y(6) receptor and was independent of other uracil nucleotide-responding receptors (P2Y(2) and P2Y(4)). UDP infusion in vivo increased blood pressure and this effect was potentiated in Entpd1(-/-) mice. In addition, pressurized mesenteric arteries from Entpd1(-/-) mice displayed an enhanced myogenic response, consistent with higher local concentrations of endogenously released nucleotides. This effect was inhibited by the P2 receptor antagonist RB-2. CONCLUSION NTPDase1 is the major enzyme regulating nucleotide metabolism at the surface of VSMCs and thus contributes to the local regulation of vascular tone by nucleotides.

Quantification of des-Arg9-bradykinin using a chemiluminescence enzyme immunoassay: application to its kinetic profile during plasma activation.

There is a renewed interest in the kininase I pathway of kinin metabolism, because des-Arg9-bradykinin (des-Arg9-BK) and des-Arg10-Lys-BK are selective and potent agonists of the B1 receptors, that are apparently upregulated by tissue injury. We have developed a polyclonal rabbit antiserum against des-Arg10-Lys-BK. In a radioimmunoassay for des-Arg10-Lys-BK, this antiserum exhibited high specificity. Notably, native kinins with the C-terminal Arg residue, bradykinin (BK) and Lys-BK, did not cross-react to a significant extent, whereas des-Arg9-BK and digoxigenin (DIG)-des-Arg9-BK exhibited a complete cross-reactivity. The antibodies were used to set up a sensitive chemiluminescence enzyme immunoassay (CLEIA) using the DIG-anti-DIG system as intermediate for the revelation of the immune complexes. The detection limit and the half-maximal saturation concentration for des-Arg9-BK were 27 and 1530 fmol/ml respectively. This assay, as well as another for BK quantification, have been applied in vitro to rabbit plasma activated by kaolin. The conversion of BK into des-Arg9-BK was generally efficient, and the persistence and concentration of both peptides were increased in the presence of enalaprilat an inhibitor of the angiotensin converting enzyme (ACEI). Rabbits treated with bacterial lipopolysaccharide exhibited an increase of plasma immunoreactive des-Arg9-BK that was potentiated in animals also treated with ACEI. This CLEIA for des-Arg9-BK is a new analytical tool applicable to analyze of the kininase I metabolites of kinins in vitro and in vivo. Measurements of des-Arg9-BK may be useful indicators of the kallikrein-kinin system activation.

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.