Johanne Bouthillier

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.

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.

Pharmacological modulation of the up-regulated responses to des-Arg9-bradykinin in vivo and in vitro

Des-Arg9-bradykinin (BK) is a selective agonist of the B1 type receptors for kinins. The biological responses to des-Arg9-BK are known to be selectively up-regulated following some types of tissue injury. Two models using rabbits were previously characterized. Firstly, in vivo, lipopolysaccharide (LPS) induces a state of sensitivity to des-Arg9-BK, which becomes a hypotensive peptide. Pretreatment of rabbits with dexamethasone sodium phosphate (DEX) did not modify the induction of cardiovascular sensitivity by LPS. Secondly, isolated rabbit aortic strips incubated in vitro exhibit a spontaneous, time-dependent increase in their responsiveness to des-Arg9-BK. This up-regulation process is selectively inhibited by DEX and stimulated by LPS applied in vitro. DEX application prevented the stimulant effect of LPS in vitro. A variety of growth factors and interleukins as well as interferon-gamma, serotonin, bradykinin and the metalloprotease inhibitor 2-mercaptoethanol were ineffective in modulating the spontaneous up-regulation of contractile responses to des-Arg9-BK. Of the known substances which do modulate this system, only epidermal growth factor (EGF) enhanced aortic contractions to des-Arg9-BK following an acute (15 min) exposure near the end of the 6-hour incubation period used in these studies. The possible involvement of des-Arg9-BK and related peptides in immunopathology is discussed.

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.

Inflammatory hyperalgesia induced by zymosan in the plantar tissue of the rat: effect of kinin receptor antagonists.

The Randall-Selitto paradigm (maximal tolerated pressure externally applied by a mechanical device) was used to develop a rat model of localized inflammatory hyperalgesia in order to compare the analgesic effects of bradykinin (BK) B1 and B2 receptor antagonists and of a non-steroidal anti-inflammatory drug (NSAID). Intra-plantar injection of zymosan (12.5 mg per paw) induced a considerable inflammation as evidenced from gross and histological evaluation and a mechanical hyperalgesia at 6 h. The contra-lateral paw of zymosan-treated animals or saline vehicle-injected paws did not exhibit a decreased pressure tolerance, relative to pre-injection measurements. Since the B1 receptor may be induced under inflammatory situations, we examined the amount of corresponding mRNA using quantitative RT-PCR. We found a significant increase of B1 receptor mRNA in the zymosan--but not the saline-injected paw at 6 h. Drugs were given subcutaneously 2 h before the 6 h readings to test their analgesic potential. The kinin B1 receptor antagonists [Leu8]des-Arg9-BK (3-30 nmol/kg) and R-715 (100 nmol/kg), the B2 receptor antagonists Hoe 140 (15 nmol/kg) and LF 16.0687 (3 and 10 mg/kg), as well as the NSAID diclofenac sodium (1 and 3 mg/kg) significantly reversed zymosan-induced hyperalgesia. We conclude that zymosan-induced hyperalgesia is a model suitable for the rapid evaluation of analgesic drugs with a peripheral site of action interfering either with kinin receptors or with prostanoid formation. In this regard, results of the present study confirm that blocking kinin B1 receptors is a novel approach for treatment of inflammatory pain.

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.

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.

Vacuolar ATPase-Mediated Cellular Concentration and Retention of Quinacrine: A Model for the Distribution of Lipophilic Cationic Drugs to Autophagic Vacuoles

The antiprotozoal agent quinacrine is a lipophilic cationic drug highly distributed to tissues. It has been used in the present experiments to examine whether the vacuolar and autophagic cytopathology induced by organic amines is independent from the therapeutic class. Furthermore, we tested the presence of the concentrated cationic drug itself in the enlarged vacuoles by exploiting the intense green fluorescence of quinacrine. Finally, the influence of lipophilicity on the apparent affinity of amine pseudotransport has been addressed by comparing quinacrine to another substituted triethylamine, procainamide. Quinacrine was concentration-dependently taken up by human smooth muscle cells (cytosolic granular-vacuolar morphology at and above 25 nM; in cell extracts, uptake nearly maximal in 2 h, apparent Km of 8.7 μM). The vacuolar (V)-ATPase inhibitor bafilomycin A1 prevented quinacrine uptake by cells or released the cell-associated drug in preloaded cells. The lipidated (II) form of microtubule-associated protein light chain 3 accumulated at and above a quinacrine concentration of 2.5 μM (4 h), indicating the conserved macroautophagic nature of the vacuolar cytopathology, although vacuole size was modest. The enlarged vacuoles containing quinacrine excluded cherry fluorescent protein; many vacuoles were lined with cherry fluorescent protein-conjugated Rab7, a GTPase associated with late endosomes/lysosomes. Taken together, these results are compatible with the transition of quinacrine-concentrating vacuoles toward an autophagolysosome identity. Quinacrine is concentrated in cells via V-ATPase-mediated ion trapping with an apparent affinity ∼500-fold higher than that of the less lipophilic drug procainamide, and, despite the small size of ensuing vacuoles, the macroautophagic signature of this cytopathology was observed.

Effects of inactivation-resistant agonists on the signalling, desensitization and down-regulation of bradykinin B2 receptors

Background and purpose:  A peptide bradykinin (BK) B2 receptor agonist partially resistant to degradation, B‐9972, down‐regulates this receptor subtype. We have used another recently described non‐peptide agonist, compound 47a, as a tool to study further the effects of metabolically more stable and thus persistent, agonists of the BK B2 receptor on signalling, desensitization and down‐regulation of this receptor.