Evidence for the participation of kinins in Freund's adjuvant-induced inflammatory and nociceptive responses in kinin B1 and B2 receptor knockout mice

Experiments were designed to investigate the role of kinin B₁ and B₂ receptors in Freund's adjuvant (CFA)-induced inflammation and nociception responses by the use of B₁ and B₂ null mutant mice. Intradermal (i.d.) injection of CFA produced time-dependent and marked hyperalgesic responses in both ipsilateral and contralateral paws of wild-type mice. Gene disruption of the kinin B₂ receptor did not interfere with CFA-induced hyperalgesia, but ablation of the gene of the B₁ receptor reduced the hyperalgesia in both ipsilateral (48±13%, at 12 h) and contralateral (91±22%, at 12 h) paws. Treatment of wild-type mice with the selective B₁ antagonist des-Arg⁹-[Leu⁸]-BK (150 nmol/kg, s.c.) reduced CFA-evoked thermal hyperalgesia, to an extent which was similar to that observed in mice lacking kinin B₁ receptor. I.d. injection of CFA produced a time-related and long-lasting (up to 72 h) increase in paw volume in wild-type mice. A similar effect was observed in B₁ knockout mice. In mice lacking B₂ receptor, the earlier stage of the CFA-induced paw oedema (6 h) was significantly greater compared with the wild-type animals, an effect which was almost completely reversed (76±5%) by des-Arg⁹-[Leu⁸]-BK. This data demonstrates that kinin B₁ receptor, but not B₂ receptor, exerts a critical role in controlling the persistent inflammatory hyperalgesia induced by CFA in mice, while B₂ receptor appears to have only a minor role in the amplification of the earlier stage of CFA-induced paw oedema formation. The results of the present study, taken together with those of previous studies, suggest that B₁ receptor antagonists represent a potential target for the development of new drugs to treat persistent inflammatory pain.     

Evidence for the participation of kinins in Freund's adjuvant-induced inflammatory and nociceptive responses in kinin B1 and B2 receptor knockout mice.

Experiments were designed to investigate the role of kinin B₁ and B₂ receptors in Freund's adjuvant (CFA)-induced inflammation and nociception responses by the use of B₁ and B₂ null mutant mice. Intradermal (i.d.) injection of CFA produced time-dependent and marked hyperalgesic responses in both ipsilateral and contralateral paws of wild-type mice. Gene disruption of the kinin B₂ receptor did not interfere with CFA-induced hyperalgesia, but ablation of the gene of the B₁ receptor reduced the hyperalgesia in both ipsilateral (48±13%, at 12 h) and contralateral (91±22%, at 12 h) paws. Treatment of wild-type mice with the selective B₁ antagonist des-Arg⁹-[Leu⁸]-BK (150 nmol/kg, s.c.) reduced CFA-evoked thermal hyperalgesia, to an extent which was similar to that observed in mice lacking kinin B₁ receptor. I.d. injection of CFA produced a time-related and long-lasting (up to 72 h) increase in paw volume in wild-type mice. A similar effect was observed in B₁ knockout mice. In mice lacking B₂ receptor, the earlier stage of the CFA-induced paw oedema (6 h) was significantly greater compared with the wild-type animals, an effect which was almost completely reversed (76±5%) by des-Arg⁹-[Leu⁸]-BK. This data demonstrates that kinin B₁ receptor, but not B₂ receptor, exerts a critical role in controlling the persistent inflammatory hyperalgesia induced by CFA in mice, while B₂ receptor appears to have only a minor role in the amplification of the earlier stage of CFA-induced paw oedema formation. The results of the present study, taken together with those of previous studies, suggest that B₁ receptor antagonists represent a potential target for the development of new drugs to treat persistent inflammatory pain.     

Effects of kinin B1 and B2 receptor antagonists on overactive urinary bladder syndrome induced by spinal cord injury in rats

Background and Purpose

Kinin B₁ and B₂ receptors have been implicated in physiological and pathological conditions of the urinary bladder. However, their role in overactive urinary bladder (OAB) syndrome following spinal cord injury (SCI) remains elusive.

Experimental Approach

We investigated the role of kinin B₁ and B₂ receptors in OAB after SCI in rats.

Key Results

SCI was associated with a marked inflammatory response and functional changes in the urinary bladder. SCI resulted in an up-regulation of B₁ receptor mRNA in the urinary bladder, dorsal root ganglion and spinal cord, as well as in B₁ protein in the urinary bladder and B₁ and B₂ receptor protein in spinal cord. Interestingly, both B₁ and B₂ protein expression were similarly distributed in detrusor muscle and urothelium of animals with SCI. In vitro stimulation of urinary bladder with the selective B₁ or B₂ agonist elicited a higher concentration-response curve in the SCI urinary bladder than in naive or sham urinary bladders. Cystometry revealed that treatment of SCI animals with the B₂ selective antagonist icatibant reduced the amplitude and number of non-voiding contractions (NVCs). The B₁ antagonist des-Arg⁹-[Leu⁸]-bradykinin reduced the number of NVCs while the non-peptide B₁ antagonist SSR240612 reduced the number of NVCs, the urinary bladder capacity and increased the voiding efficiency and voided volume.

Conclusions and Implications

Taken together, these data show the important roles of B₁ and B₂ receptors in OAB following SCI in rats and suggest that blockade of these receptors could be a potential therapeutic target for controlling OAB.     

Peripheral kinin B(1) and B(2) receptor-operated mechanisms are implicated in neuropathic nociception induced by spinal nerve ligation in rats

The kinin system can contribute distinctly to the sensory changes associated with different models of nerve injury-induced neuropathic pain. This study examines the roles of kinin B(1) and B(2) receptor-operated mechanisms in alterations in nociceptive responses of rats submitted to unilateral L5/L6 spinal nerve ligation (SNL) injury. Behavioural responses to ipsilateral hind paw stimulation with acetone (evaporation-evoked cooling), radiant heat (Hargreaves method) or von Frey hairs revealed that SNL rats developed long-lasting cold allodynia (from Days 3 to 40 post-surgery, peak on Day 6), heat hyperalgesia (stable peak from Days 9 to 36) and tactile allodynia (stable peak from Days 3 to 51). SNL rats manifested nocifensive responses to intraplantar injections on Day 12 of the selective B(1) receptor agonist des-Arg(9)-bradykinin (DABK) and augmented responses to the selective B(2) receptor agonist bradykinin (BK; each at 0.01-1nmol/paw). Systemic treatment of SNL rats with des-Arg(9)-Leu(8)-BK or HOE 140 (peptidic B(1) and B(2) receptor antagonists, respectively; 0.1-1mumol/kg, i.p.) selectively blocked responses triggered by DABK and BK (1nmol/paw) and alleviated partially and transiently established cold allodynia, heat hyperalgesia and (to a lesser extent) tactile allodynia. Western blot analysis revealed enhanced expression of kinin B(1) and B(2) receptor protein in ipsilateral L4-L6 spinal nerve and hind paw skin samples collected on Day 12 after SNL surgery. These results indicate that peripheral pronociceptive kinin B(1) and B(2) receptor-operated mechanisms contribute significantly to the maintenance of hind paw cold and mechanical allodynia and heat hyperalgesia induced by L5/L6 SNL in rats.     

Stable expression of human kinin B1 receptor in 293 cells: pharmacological and functional characterization

1. We compared the binding properties of [³H]-desArg¹⁰-[Leu⁹]-kallidin, a radiolabelled kinin B₁ receptor antagonist, to membranes from IMR-90 human embryonic fibroblasts and from 293 cells transiently or stably transfected with the human B₁ receptor.
2. The dissociation constant (K_D) of [³H]-desArg¹⁰-[Leu⁹]-kallidin and the affinity of several kinin receptor agonists and antagonists were similar between the native and cloned receptor, either transiently or stably expressed in 293 cells. In IMR-90 cells, the rank order of potency was that expected for a kinin B₁ receptor.
3. The receptors transiently or stably expressed in 293 cells were fully functional with respect to their signalling properties. Phosphoinositide hydrolysis was increased in a concentration-dependent manner by the B₁ receptor agonist, desArg¹⁰-kallidin. Functional coupling to the calcium pathway was also demonstrated for the native and stably expressed human B₁ receptor.
4. In conclusion, the established stable and functional 293 cell clone may provide an important tool for further analysis of the molecular mechanisms involved in binding, activation, and coupling of the kinin B₁ receptor.

     

The role of kinin B1 and B2 receptors in the scratching behaviour induced by proteinase-activated receptor-2 agonists in mice

Background and purpose:

Activation of the proteinase-activated receptor-2 (PAR-2) induces scratching behaviour in mice. Here, we have investigated the role of kinin B₁ and B₂ receptors in the pruritogenic response elicited by activators of PAR-2.

Experimental approach:

Scratching was induced by an intradermal (i.d.) injection of trypsin or the selective PAR-2 activating peptide SLIGRL-NH₂ at the back of the mouse neck. The animals were observed for 40 min and their scratching response was quantified.

Key results:

I.d. injection of trypsin or SLIGRL-NH₂ evoked a scratching behaviour, dependent on PAR-2 activation. Mice genetically deficient in kinin B₁ or B₂ receptors exhibited reduced scratching behaviour after i.d. injection of trypsin or SLIGRL-NH₂. Treatment (i.p.) with the non-peptide B₁ or B₂receptor antagonists SSR240612 and {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657, respectively, prevented the scratching behaviour caused by trypsin or SLIGRL-NH₂. Nonetheless, only treatment i.p. with the peptide B₂receptor antagonist, Hoe 140, but not the B₁receptor antagonist (DALBK), inhibited the pruritogenic response to trypsin. Hoe 140 was also effective against SLIGRL-NH₂-induced scratching behaviour when injected by i.d. or intrathecal (i.t.) routes. Also, the response to SLIGRL-NH₂ was inhibited by i.t. (but not by i.d.) treatment with DALBK. Conversely, neither Hoe 140 nor DALBK were able to inhibit SLIGRL-NH₂-induced scratching behaviour when given intracerebroventricularly (i.c.v.).

Conclusions and implications:

The present results demonstrated that kinins acting on both B₁ and B₂ receptors played a crucial role in controlling the pruriceptive signalling triggered by PAR-2 activation in mice.     

Effects of kinins on isolated stomachs of control and transgenic knockout B2 receptor mice

The aim of this study was to investigate the pharmacological profile of the kinin B₁ and B₂ receptors in isolated stomachs from wild-type control and B₂ receptor knockout mice. Isometric contractions evoked by bradykinin (BK) (9 nM) and desArg⁹BK (28 nM) were shown to be different. The contraction induced by desArg⁹BK had a longer duration than that evoked by BK and increased during incubation in vitro in stomachs of wild-type controls, while in the transgenic B₂ receptor knockout mice, the contractions evoked by desArg⁹BK and BK were similar and followed the B₁ receptor agonist pattern. BK but not the carboxypeptidase-resistant analog, [Phe⁸ψ(CH₂-NH)Arg⁹]BK, was found to be active in the stomach of B₂ receptor knockout mice. BK-induced contractions were prevented by mergetpa (a carboxypeptidase M inhibitor) (10 μM) and by a the B₁ receptor antagonist, AcLys[DβNal⁷,Ile⁸]desArg⁹BK (R 715) (0.88 μM), while not being influenced by the B₂ receptor antagonist HOE 140 (0.38 μM). BK and [Phe⁸ψ(CH₂-NH)Arg⁹]BK were potent contractants of the wild-type mice stomach and their effects were not influenced by mergetpa or by the B₁ receptor antagonist: they were reduced by HOE 140. After incubation in vitro for 3–4 hours, the tissues were treated with HOE 140 (4 μM) and FR-173657 (17 μM) to eliminate B₂ receptor function. In these tissues, BK evoked a B₁-like contraction which was inhibited by mergetpa (10 μM) and antagonized by R 715 (8 μM). The results indicate that BK acts primarily on B₂ receptors. However, after intramural conversion to desArg⁹BK, activation of B₁ receptors of the mice stomach occurs. In the tissues of B₂ receptor knockout mice, BK behaves as a pure B₁ receptor agonist while in stomachs of control animals, the B₂ receptor contribution is overwhelming. After complete blockade of the B₂ receptor, BK is able to evoke B₁-mediated responses similar to those observed in tissues of B₂ receptor knockout mice. It is concluded that the disruption of the B₂ receptor gene eliminates the B₂ receptor without influencing the B₁ receptor system. Received: 18 September 1997 / Accepted: 21 November 1997     

Corticosterone responses in 5-HT1B receptor knockout mice to stress or 5-HT1A receptor activation are normal

RATIONALE: Previous research found no adaptations in presynaptic 5-HT1A receptors in mice lacking 5-HT1B receptors (5-HT1B KO). Stress and 5-HT1A receptor agonists induce corticosterone release in mice via hypothalamus-pituitary-adrenal (HPA) axis activation. 5-HT1B KO mice are hyperreactive to mild stressors and this might be reflected in altered postsynaptic 5-HT1A receptor sensitivity. OBJECTIVES: Our aim was to determine whether the activity of the HPA axis was increased in 5-HT1B KO mice in response to mild stress and pharmacological activation of 5-HT1A receptors as an indication of putative adaptive changes in postsynaptic 5-HT1A receptor function. METHODS: The effect of mild stress [i.e., the stress-induced hyperthermia (SIH) paradigm], induced by rectal temperature measurement, was determined on temperature and corticosterone over time (0, 5, 10, 20, 30, 60, and 90 min) in 5-HT1B KO and wildtype mice. In addition, corticosterone was measured 60 min after 5-HT1A receptor activation by flesinoxan (0, 0.03, 0.1, 0.3, 1, and 3 mg/kg s.c.). Blood was collected and plasma corticosterone levels were determined by radioimmunoassay. RESULTS: Both genotypes showed comparable time-dependent SIH responses, whereas basal temperature was higher in 5-HT1B KO mice. The effect of SIH on temperature was mirrored by mild increases in plasma corticosterone. Activation of 5-HT1A receptors caused a strong dose-dependent release of corticosterone in both genotypes. Neither response observed showed differences between both genotypes. CONCLUSIONS: Although 5-HT1B KO mice are hyperreactive to mild stress, this reactivity is not reflected by stronger corticosterone responses in the SIH paradigm. The lack of shift in dose-response curves for flesinoxan suggests that postsynaptic 5-HT1A receptor function is unaffected in 5-HT1B KO mice.     

The endocannabinoid anandamide inhibits kinin B1 receptor sensitization through cannabinoid CB1 receptor stimulation in human umbilical vein

The possible inhibition of kinin B₁ receptor up-regulation by arachidonoylethanolamide (anandamide) was evaluated in isolated human umbilical vein. Anandamide and its metabolically stable analogue, R-N-(2-Hydroxy-1-methylethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (R-(+)-methanandamide), produced a selective and dose-dependent inhibition of kinin B₁ receptor-sensitized contractile responses. The inhibitory effect of anandamide on B₁ receptor-sensitized responses failed to be modified either by 5-biphenyl-4-ylmethyl-tetrazole-1-carboxylic acid dimethylamide (LY2183240), a selective anandamide uptake inhibitor, or 6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-y l](4-methoxyphenyl) methanone (AM630), selective cannabinoid CB₂ receptor antagonist. However, the cannabinoid CB₁ receptor antagonist, N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophen yl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), abolished anandamide effects on kinin B₁ receptor sensitization. The present results provide strong pharmacological evidence indicating that endocannabinoid anandamide inhibits kinin B₁ receptor up-regulation through cannabinoid CB₁ receptor stimulation in human umbilical vein.     

Role of spinal serotonin1 receptor subtypes in thermally and mechanically elicited nociceptive reflexes

The ability of 5-HT_1A and 5-HT_1B agonists to alter a spinal animal's nociceptive threshold was examined using two analgesiometric tests. In the spinal withdrawal reflex test, administration of the selective 5-HT_1A agonists ipsapirone, gepirone and PAPP resulted in significant dose-dependent increases in receptive field (RF) area for withdrawal reflexes when compared to predrug baseline values, indicating an increase in nociceptive sensitivity. The average overall percent maximal increase in RF area following administration of 5-HT_1A selective compounds was: 80±16% for the ventroflexion reflex, 90±6% for the dorsiflexion reflex and 87±8% for the lateral flexion reflex. Similar to the effects noted with 5-HT_1A agonists, administration of 5-HT_1B agonists RU24969, mCPP and TFMPP resulted in a hyperalgesic response with an overall percent maximal increase of 43±6% for the ventroflexion reflex, 51±6% for the dorsiflexion reflex and 38±9% for the lateral flexion reflex. In the tail-flick analgesiometric test, administration of the 5-HT_1A agonists 8-OH-DPAT and ipsapirone and the 5-HT_1B agonists RU24969 and mCPP resulted in a significant dose-dependent increase in tail-flick latencies when compared to predrug baseline values, indicating a decrease in nociceptive sensitivity to noxious thermal stimuli. No differences in magnitude of the effect of the two receptor subtypes were found, indicating that stimulation of either 5-HT_1A or 5-HT_1B receptors was equipotent in producing the antinociceptive tail-flick response. Administration of the 5-HT_1A antagonist meter-goline completely reversed the increase in TFL produced by RU24969, providing further evidence that the effects seen here are mediated by spinal 5-HT receptors. The results of this study indicate that spinal 5-HT₁ receptor subtypes may either facilitate or inhibit nociceptive input depending upon the type of nociceptor that is activated, as opposed to the type of receptor subtype that is stimulated.     

Hyperalgesia in non-obese diabetic (NOD) mice: a role for the inducible bradykinin B1 receptor

Most studies performed to investigate the role of the inducible bradykinin B(1) receptor in the pathology and complications of type 1 diabetes have been carried out using the model of streptozotocin (STZ)-induced diabetes. The model of spontaneous autoimmune diabetes in non-obese diabetic (NOD) mice involves a long-term inflammatory process that closely resembles the human type 1 diabetes. In the present study, we aimed at establishing the correlation between the progress of diabetic hyperalgesia and the incidence of diabetes, as a function of age, in NOD mice. We also evaluated the implication of the bradykinin B(1) receptor, a receptor up-regulated during the inflammatory progress of diabetes, in the development of diabetic hyperalgesia in NOD mice. Female NOD mice were followed up from the 4th to the 32nd week of age for the incidence of diabetes. Only NOD mice with plasma glucose concentration >20 mmol/l were considered diabetic. The nociception was assessed using the hot plate and the tail immersion pain tests and the effect of acute and chronic administration of the selective bradykinin B(1) receptor agonist, desArg(9)bradykinin and its selective antagonists, R-715 (Ac-Lys-[D-beta Nal(7), Ile(8)]desArg(9)bradykinin) and R-954 (Ac-Orn-[Oic(2), alpha-MePhe(5), D-beta Nal(7), Ile(8)]desArg(9)bradykinin), on the development of diabetic hyperalgesia was studied. Diabetic NOD mice developed a significant time-dependent hyperalgesia, as measured in both tests, starting from the 8th week of age with the maximum effect observed over 16 to 20 weeks, whereas the incidence of diabetes in the tested NOD mice was only 40.16% at the age of 16 weeks and reached a maximum of 73.23% at the age 24 weeks. Both acute and chronic administration of desArg(9)bradykinin (400 microg/kg) markedly increased the hyperalgesic activity in diabetic NOD mice in the hot plate and tail immersion nociceptive tests. The selective bradykinin B(1) receptor antagonist R-715 (400 microg/kg) and its more potent and long acting analogue R-954 (200 microg/kg), administered in acute or chronic manner, significantly attenuated diabetic hyperalgesia in NOD mice in both thermal pain tests and restored nociceptive responses to values observed in control non-diabetic siblings. Our results bring the first evidence that the development of hyperalgesia in NOD mice, a model of spontaneous type 1 diabetes, precedes the occurrence of hyperglycemia and is mediated by the bradykinin B(1) receptor. It is suggested that bradykinin B(1) receptor antagonism could become a novel therapeutic approach to the treatment of diabetic neuropathic complications.     

Discovery and therapeutic potential of kinin receptor antagonists

Introduction: Kinins are bioactive peptide hormones that exert biological effects by activating two types of G protein-coupled receptors namely, B₁ (B₁R) and B₂ (B₂R). These modulate normal physiological cellular functions, inflammatory disorders and carcinogenesis. New and novel kinin receptor antagonists have been synthesized and their efficacy evaluated.

Areas covered: The authors provide a comprehensive review on the cellular and molecular biology of kinins and their receptors is delineated along with evolution and discovery of selective peptide and non-peptide antagonists. The authors describe the in vitro and in vivo methods used to understand the relative functional roles of B₁R and B₂R in physiology and pathohysiology. Furthermore, the authors translate the evaluation of kinin antagonists in selected preclinical models and associated clinical indications. Literature was surveyed from original publications, standard sources, SciFinder, patent applications and clinical trials.

Expert opinion: The authors suggest that several key areas of functional biology need consideration, namely: re-evaluation, particularly in vivo, of the mechanism of action and relative functional roles of the B₁R and B₂R in physiology and acute and chronic disease in animals and man; need for improved animal models with increased use of humanized and human systems; development of fluorescent probes for use in vivo in animals and man using advanced imaging techniques; combination of kinin receptor antagonists and traditional chemotherapy for various cancers.     

Site-directed mutagenesis at the human B2 receptor and molecular modelling to define the pharmacophore of non-peptide bradykinin receptor antagonists

Combining site-directed mutagenesis with information obtained from molecular modelling of the bradykinin (BK) human B2 receptor (hB2R) as derived from the bovine rhodopsin crystal structure [Science 289 (2000) 739], we previously defined a putative binding mode for the non-peptide B2 receptor antagonists, FR173657 and LF16-0687 [Can J Physiol Pharmacol 80 (2002) 303]. The present work is aimed to define the specific role of the quinoline moiety in the pharmacophore of these non-peptide antagonists. The effect of the mutations I110A, L114A (TM, transmembrane 3), W256A (TM6), F292A, Y295A and Y295F (TM7) was evaluated. None of the mutations affected the binding interaction of peptide ligands: the agonist BK and the peptide antagonist MEN 11270. The affinities in competing for [3H]-BK binding and in blocking the BK-induced IP production by the non-peptide antagonists LF16-0687 and FR173657 at the wild type and mutant receptors were analysed. While the affinities of LF16-0687 and FR173657 were crucially decreased at the I110A, Y295A, and Y295F mutants, the W256A mutation affected the affinity of the LF16-0687 only. The important contribution of the quinoline moiety was shown by the inability of an analogue of LF16-0687, lacking this moiety, to affect BK binding at the wild type receptor. On the other hand, the benzamidine group did not interact with mutated residues, since LF16-0687 analogues without this group or with an oxidated benzamidine displayed pairwise loss of affinity on wild type and mutated receptors. Further differences between FR173657 and LF16-0687 were highlighted at the I110 and Y295 mutants when comparing binding (pK(i)) and functional antagonist (pKB) affinity. First, the I110A mutation similarly impaired their binding affinity (250-fold), but at a less extent the antagonist potency of FR173657 only. Second, both the hydroxyl and the phenyl moieties of the Y295 residue had a specific role in the LF16-0687 interaction with the receptor, as demonstrated at the Y295F and Y295A mutants, respectively, but not in that of FR173657. Present data identify a receptor binding pocket comprised among TM3, 6, and 7, which concerns the interaction of the non-peptide antagonists FR173657 and LF16-0687, but not that of the peptide agonist or antagonist. Results indicate the quinoline group as the involved pharmacophoric element, and that the studied residues are differently involved in the interaction. The analysis performed by means of the GRID software led us to propose different spatial orientations of the quinoline moieties and partially overlapping binding pockets for the two ligands: that of LF16-0687 is located in the lipophilic environment amongst I110 (TM3), W256 (TM6), and Y295 (TM7) residues, whereas that of FR173657 lies essentially between I110 and Y295.     

Aspirin inhibits human bradykinin B2 receptor ligand binding function

The bradykinin B2 receptor, a member of the G protein-coupled receptors superfamily, is involved in a variety of physiological functions, including vasodilation, electrolyte transfer in epithelia, mediation of pain, and inflammation. The effect of aspirin on bradykinin binding to cell-surface receptor and on signal transduction were studied in CHO-K1 cells, stably expressing the human B2 receptor. Cell-surface organization of the receptor was assessed by immunoprecipitation and Western blot analysis in CHO-K1 cells expressing N-terminally V5-tagged B2 receptor. We found that the widely used analgesic, anti-thrombotic, and anti-inflammatory drug aspirin alters the B2 receptor ligand binding properties. Aspirin reduces the apparent affinity of the receptor for [3H]-bradykinin by accelerating the dissociation rate of [3H]-bradykinin-receptor complexes. In addition, aspirin reduces the capacity of unlabeled bradykinin or the B2 receptor antagonist icatibant to destabilize pre-formed [3H]-bradykinin-receptor complexes. Kinetic and reversibility studies are consistent with an allosteric type of mechanism. Aspirin effect on B2 receptor binding properties is not accompanied by alteration of the cell-surface organization of the receptor in dimers and monomers. Aspirin does not influence the receptor ability to transduce bradykinin binding into activation of G-proteins and phospholipase C. These results suggest that aspirin is an allosteric inhibitor of the B2 receptor, a property that may be involved in its therapeutic actions.     

Pharmacological characterization of the first potent and selective antagonist at the cysteinyl leukotriene 2 (CysLT2) receptor

Background and purpose:

Cysteinyl leukotrienes (CysLTs) have been implicated in the pathophysiology of inflammatory and cardiovascular disorders. Their actions are mediated by CysLT₁ and CysLT₂ receptors. Here we report the discovery of 3-({[(1S,3S)-3-carboxycyclohexyl]amino}carbonyl)-4-(3-{4-[4-(cyclo-hexyloxy)butoxy]phenyl}propoxy) benzoic acid (HAMI3379), the first potent and selective CysLT₂ receptor antagonist.

Experimental approach:

Pharmacological characterization of HAMI3379 was performed using stably transfected CysLT₁ and CysLT₂ receptor cell lines, and isolated, Langendorff-perfused, guinea pig hearts.

Key results:

In a CysLT₂ receptor reporter cell line, HAMI3379 antagonized leukotriene D₄- (LTD₄-) and leukotriene C₄- (LTC₄-) induced intracellular calcium mobilization with IC₅₀ values of 3.8 nM and 4.4 nM respectively. In contrast, HAMI3379 exhibited very low potency on a recombinant CysLT₁ receptor cell line (IC₅₀ > 10 000 nM). In addition, HAMI3379 did not exhibit any agonistic activity on both CysLT receptor cell lines. In binding studies using membranes from the CysLT₂ and CysLT₁ receptor cell lines, HAMI3379 inhibited [³H]-LTD₄ binding with IC₅₀ values of 38 nM and >10 000 nM respectively. In isolated Langendorff-perfused guinea pig hearts HAMI3379 concentration-dependently inhibited and reversed the LTC₄-induced perfusion pressure increase and contractility decrease. The selective CysLT₁ receptor antagonist zafirlukast was found to be inactive in this experimental setting.

Conclusions and implications:

HAMI3379 was identified as a potent and selective CysLT₂ receptor antagonist, which was devoid of CysLT receptor agonism. Using this compound, we showed that the cardiac effects of CysLTs are predominantly mediated by the CysLT₂ receptor.     

UTP reduces infarct size and improves mice heart function after myocardial infarct via P2Y2 receptor

Pyrimidine nucleotides are signaling molecules, which activate G protein-coupled membrane receptors of the P2Y family. P2Y₂ and P2Y₄ receptors are part of the P2Y family, which is composed of 8 subtypes that have been cloned and functionally defined. We have previously found that uridine-5′-triphosphate (UTP) reduces infarct size and improves cardiac function following myocardial infarct (MI). The aim of the present study was to determine the role of P2Y₂ receptor in cardiac protection following MI using knockout (KO) mice, in vivo and wild type (WT) for controls. In both experimental groups used (WT and P2Y₂^−/− receptor KO mice) there were 3 subgroups: sham, MI, and MI + UTP. 24 h post MI we performed echocardiography and measured infarct size using triphenyl tetrazolium chloride (TTC) staining on all mice. Fractional shortening (FS) was higher in WT UTP-treated mice than the MI group (44.7 ± 4.08% vs. 33.5 ± 2.7% respectively, p < 0.001). However, the FS of P2Y₂^−/− receptor KO mice were not affected by UTP treatment (34.7 ± 5.3% vs. 35.9 ± 2.9%). Similar results were obtained with TTC and hematoxylin and eosin stainings. Moreover, troponin T measurements demonstrated reduced myocardial damage in WT mice pretreated with UTP vs. untreated mice (8.8 ± 4.6 vs. 12 ± 3.1 p < 0.05). In contrast, P2Y₂^−/− receptor KO mice pretreated with UTP did not demonstrate reduced myocardial damage. These results indicate that the P2Y₂ receptor mediates UTP cardioprotection, in vivo.     

Locomotor response to MDMA is attenuated in knockout mice lacking the 5-HT1B receptor

3,4-Methylenedioxymethamphetamine (MDMA) is a psychoactive drug of abuse which is increasingly popular in human recreational drug use. In rats, the drug has been shown to stimulate locomotion while decreasing exploratory behavior. MDMA acts as an indirect agonist of serotonin (5-HT) receptors by inducing 5-HT release by a 5-HT reuptake transporter-dependent mechanism, although it is not known which 5-HT receptors are important for the behavioral effects of the drug. In order to examine the role of specific 5-HT receptors, we assessed the behavioral effects of MDMA on knockout mice lacking the 5-HT_1B receptor. Knockout animals show a reduced locomotor response to MDMA, although delayed locomotor stimulation is present in these animals. This finding indicates that the locomotor effects of MDMA are dependent upon the 5-HT_1B receptor, at least in part. In contrast, MDMA eliminates exploratory behavior in both normal and knockout mice, suggesting that the exploratory suppression induced by MDMA occurs through mechanisms other than activation of the 5-HT_1B receptor. To confirm these findings, we tested the effects of MDMA on the locomotor and exploratory behavior of wild-type mice pretreated with GR 127935, a 5-HT_1B/1D receptor antagonist. These mice had an attenuated locomotor response to MDMA, but still exhibited the drug-induced suppression of exploration. Received: 5 February 1998/Final version: 7 May 1998     

Increased susceptibility of annexin-A1 null mice to nociceptive pain is indicative of a spinal antinociceptive action of annexin-A1

BACKGROUND AND PURPOSE: Annexin-A1 (ANXA1), a glucocorticoid-regulated protein, mediates several of the anti-inflammatory actions of the glucocorticoids. Previous studies demonstrated that ANXA1 is involved in pain modulation. The current study, using ANXA1 knockout mice (ANXA1-/-), is aimed at addressing the site and mechanism of the modulatory action of ANXA1 as well as possible involvement of ANXA1 in mediating the analgesic action of glucocorticoids. EXPERIMENTAL APPROACH: The acetic acid-induced writhing response was performed in ANXA1-/- and wild-type (ANXA1+/+) mice with spinal and brain levels of prostaglandin E2 (PGE2) examined in both genotypes. The effect of the ANXA1 peptomimetic Ac2-26 as well as methylprednisolone on the writhing response and on spinal cord PGE2 of ANXA1+/+ and ANXA1-/- was compared. The expression of proteins involved in PGE2 synthesis, cytosolic phospholipase A2 (cPLA2) and cyclooxygenases (COXs), in the spinal cord of ANXA1+/+ and ANXA1-/- was also compared.Key results:ANXA1-/- mice exhibited a significantly greater writhing response and increased spinal cord levels of PGE2 compared with ANXA1+/+ mice. Ac2-26 produced analgesia and reduced spinal PGE2 levels in ANXA1+/+ and ANXA1-/- mice, whereas methylprednisolone reduced the writhing response and spinal PGE2 levels in ANXA1+/+, but not in ANXA1-/- mice. The expression of cPLA2, COX-1, COX-2 and COX-3 in spinal cord tissues was upregulated in ANXA1-/-compared with ANXA1+/+. CONCLUSIONS AND IMPLICATIONS: We conclude that ANXA1 protein modulates nociceptive processing at the spinal level, by reducing synthesis of PGE2 by modulating cPLA2 and/or COX activity. The analgesic activity of methylprednisolone is mediated by spinal ANXA1.