The actions of kinin antagonists on B1 and B2 receptor systems

The newly discovered bradykinin antagonist [Thi5,8,D-Phe7]Bradykinin, supplied by J.-M. Stewart and three other compounds, [D-Phe7]BK, [Thi5,8,D-Phe7]Bradykinin and [Thi6,9,D-Phe8]Kallidin synthesized in our laboratory, were tested for their ability to antagonize bradykinin in four B2 receptor systems, the guinea-pig ileum, the rabbit jugular vein, the dog carotid artery and the dog urinary bladder as well as against desArg9-bradykinin in the rabbit aorta (a B1 receptor system). [D-Phe7]Bradykinin is a partial agonist, while [Thi5,8,D-Phe7]Bradykinin and [Thi6,9,D-Phe8]Kallidin are pure antagonists, the second one showing little BK-like activity on three of the four preparations. The kallidin analogue is more potent in all preparations than the bradykinin one. The two [Thi5,8,D-Phe7]BK (that supplied by J.-M. Stewart and that prepared in our laboratory) show very similar affinities in all preparations. The bradykinin analogue as well as the kallidin one are also active against desArg9-bradykinin in the rabbit aorta, at concentrations similar to those active on B2 receptor systems. The kinin antagonists are however specific for the kinins, since they do not interfere with the myotropic effects of angiotensin or substance P (SP) in the various preparations.     

Non-peptide antagonists and agonists of the bradykinin B(2) receptor

Stimulation of the bradykinin (BK) B(2) receptor by kinins is associated with pathophysiological as well as pronounced beneficial effects. Consequently, interference with BK B(2) receptors by either antagonism or agonism offers promising therapeutic approaches for the development of drugs for the treatment of various human diseases. BK B(2) receptor antagonists may prove useful for the treatment of pathological situations caused by excessively increased local kinin concentrations, such as inflammation, tissue injury and pain. Beneficial effects of peptide BK B(2) receptor antagonists in perennial rhinitis, asthma and brain edema have already been demonstrated in clinical trials. On the other hand, kinins have also been identified as potent vasodilatory and organ-protective peptides. Therefore, BK B(2) receptor agonists may have the potential to become valuable therapeutics in the treatment of cardiovascular diseases such as hypertension, myocardial hypertrophy, myocardial infarction and arrhythmias as well as diabetic disorders. For both approaches, potent, selective and even orally active non-peptide compounds have been discovered recently. Prototypes of these novel third generation classes of compounds are the alkylphosphonium salt WIN-64338, the pseudopeptide NPC-18884, the thiosemicarbazide bradyzide and especially the imidazo[1,2-a]pyridine FR-167344 and the quinolines FR-173657 and LF-16.0687 as non-peptide BK B(2) receptor antagonists, whereas the 4-(2-pyridylmethoxy)-substituted quinoline FR-190997 and the 3-(2-pyridylmethyl)-substituted benzimidazole FR-191413 emerged as non-peptide BK B(2) receptor agonists. These antagonists and agonists of the BK B(2) receptor have already demonstrated efficacy a various animal models of human diseases, which offers promising therapeutic approaches for the development of drugs for the treatment or even prevention of a variety of severe human diseases either via stimulation or via blockade of BK B(2) receptors.     

In vivo and in vitro activity of selective 5-hydroxytryptamine2 receptor antagonists

The abilities of ketanserin, ritanserin, R56413 and LY53857 to inhibit 5-hydroxytryptamine (5-HT) and noradrenaline-induced vasoconstrictor responses both in vitro and in vivo and to lower blood pressure in the rat, were compared. In the isolated perfused mesenteric artery preparation of the rat all of the compounds tested were found to be potent inhibitors of 5-HT-induced vasoconstrictor responses. Ritanserin was the most potent compound, producing more than 50% inhibition of a near maximal response to 5-HT at a concentration of 10(-11) M. All four compounds were found to be competitive antagonists of noradrenaline; ketanserin being the most potent with a pA2 value of 7.64 +/- 0.06. 5-HT-induced pressor responses in the pithed rat were inhibited by low doses (0.3-10 micrograms kg-1) of the four compounds. Ketanserin, at doses of 0.1-3.0 mg kg-1, resulted in rightward shifts of the control dose-response curve to noradrenaline in the pithed rat. None of the other compounds had any significant effect on the noradrenaline-induced pressor responses. Ketanserin (0.1-1 mg kg-1) produced a dose-dependent decrease in the mean arterial blood pressure of anaesthetized rats. The maximum decrease in blood pressure observed following a dose of 1 mg kg-1 ketanserin was 73.7 +/- 4.7 mmHg. The other compounds at doses of 1.0-3.0 mg kg-1 produced a decrease in blood pressure of a lesser magnitude than that following ketanserin. In addition, this effect did not appear to be dose-dependent. It is suggested that the acute hypotensive effect of ketanserin results predominantly from alpha 1-adrenoceptor blockade.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological characterization of the cardiovascular responses elicited by kinin B(1) and B(2) receptor agonists in the spinal cord of streptozotocin-diabetic rats

Kinin receptor agonists and antagonists at the B(1) and B(2) receptors were injected intrathecally (i.t., at T-9 spinal cord level) to conscious unrestrained rats and their effects on mean arterial pressure (MAP) and heart rate (HR) were compared in streptozotocin (STZ)-diabetic rats (65 mg kg(-1) STZ, i.p. 3 weeks earlier) and aged-matched control rats. The B(1) receptor agonist, des-Arg(9)-Bradykinin (BK) (3.2 - 32.5 nmol), evoked dose-dependent increases in MAP and tachycardia during the first 10 min post-injection in STZ-diabetic rats only. The cardiovascular response to 6.5 nmol des-Arg(9)-BK was reversibly blocked by the prior i.t. injection of antagonists for the B(1) receptor ([des-Arg(10)]-Hoe 140, 650 pmol or [Leu(8)]-des-Arg(9)-BK, 65 nmol) and B(2) receptor (Hoe 140, 81 pmol or FR173657, 81 pmol) or by indomethacin (5 mg kg(-1), i.a.). The i.t. injection of BK (8.1 - 810 pmol) induced dose-dependent increases in MAP which were accompanied either by tachycardiac (STZ-diabetic rats) or bradycardiac (control rats) responses. The pressor response to BK was significantly greater in STZ-diabetic rats. The cardiovascular response to 81 pmol BK was reversibly blocked by 81 pmol Hoe 140 or 81 pmol FR173657 but not by B(1) receptor antagonists nor by indomethacin in STZ-diabetic rats. The data suggest that the activation of kinin B(1) receptor in the spinal cord of STZ-diabetic rats leads to cardiovascular changes through a prostaglandin mediated mechanism. Thus, this study affords an accessible model for studying the expression, the pharmacology and physiopathology of the B(1) receptor in the central nervous system.     

In vivo effects of noradrenaline and noradrenergic receptor agonists and antagonists on rat cerebellar cyclic GMP levels

Cerebellar cyclic GMP levels can be altered by neurotransmitters and their receptor agonists and antagonists. In this study, we investigated the action of noradrenaline and certain drugs affecting alpha- and beta-adrenoceptors on rat cerebellaceptor agonists such as methoxamine and phenylephrine increased cGMP levels. alpha-Adrenoceptor antagonists such as phentolamine, phenoxybenzamine and ARC 239 decreased cGMP levels, whereas yohimbine and piperoxane which are known to act as presynaptic alpha-adrenoceptor antagonists had no effect. The action of clonidine which decreased cGMP levels at low doses was probably due to the fact that this adrenoceptor agonist inhibited the release of noradrenaline from adrenergic nerve terminals, since piperoxane injected prior to clonidine antagonized the effect of clonidine on cerebellar cGMP, and since pretreatment of the animals with 6-hydroxydopamine partially antagonized the effect of clonidine. Isoproterenol, a beta-noradrenergic agonist had no effect on cerebellar cGMP levels. Propranolol, a beta-noradrenergic antagonist decreased cGMP levels. Phenoxybenzamine or propranolol injected prior to noradrenaline decreased cerebellar cGMP.     

Comparison of kinin B(1) and B(2) receptor expression in neutrophils of asthmatic and non-asthmatic subjects

Kinins have been implicated in the pathophysiology of asthma and activation of kinin receptors stimulates neutrophil chemotaxis. However, the expression of kinin receptors on neutrophils of asthmatic subjects has not been assessed. The aim of this study was to compare the expression of kinin B(1) and B(2) receptor mRNA and proteins in neutrophils of asthmatic and non-asthmatic subjects, and to assess whether inhaled corticosteroid treatment may influence expression of the kinin receptors. Neutrophils were isolated from peripheral blood of asthmatic (n=27) and non-asthmatic subjects (n=14). The presence of kinin B(1) and B(2) receptor protein on neutrophils was confirmed by immunolabeling with specific antibodies followed by immunoperoxidase, immunofluorescence and FACS detection. Kinin B(1) and B(2) receptor mRNA expression was assessed by RT-PCR. Quantitative image analysis of fluorescence immunolabeled neutrophils showed no differences in kinin B(1) or B(2) receptor protein expression between asthmatic and non-asthmatic subjects. Similarly, quantitative real time RT-PCR analysis demonstrated no differences in expression of mRNA for the kinin B(1) or B(2) receptors between asthmatic and non-asthmatic subjects. However, B(1) receptor mRNA expression was significantly lower in asthmatic subjects using > or =2000 microg of inhaled corticosteroid per day (p<0.05) and B(1) receptor protein levels also tended to be lower in these subjects. Corticosteroids may have a beneficial anti-inflammatory effect in asthma by down-regulating B(1) receptor expression on neutrophils, thereby decreasing the migration of these inflammatory cells into the airways.     

Haemodynamic and cardiac effects of kinin B1 and B2 receptor stimulation in conscious instrumented dogs.

1. Mongrel dogs were chronically instrumented with an intra-aortic catheter, a Königsberg intraventricular pressure transducer and a Döppler flow probe around the left coronary artery. After ganglionic blockade with hexamethonium, the cardiovascular effects of bradykinin B1 and B2 receptor agonists, des-Arg9-bradykinin and bradykinin (BK), were investigated in the presence and absence of specific antagonists. The contribution of nitric oxide (NO) and prostanoids to the cardiovascular effects of kinins was also examined. 2. BK (1 microgram kg-1 min-1) and des-Arg9-BK (1 microgram kg-1 min-1) both given as a 2 min i.v. infusion, produced a significant decrease in mean arterial pressure (MAP, -34 +/- 4% for BK and -45 +/- 2% for des-Arg9-BK) and coronary vascular resistance (CVR, -37 +/- 5% for BK and -50 +/- 2% for des-Arg9-BK), without affecting cardiac contractility, left ventricular end diastolic pressure, and coronary velocity. BK caused a significantly greater decrease in MAP and CVR than des-Arg9-BK (P < 0.05). 3. Pretreatment with the B1 receptor antagonist, des-Arg9-[Leu8]-BK (25 micrograms kg-1) significantly inhibited the decrease in MAP and CVR produced by des-Arg9-BK but not by BK. Infusion of des-Arg9-[Leu8]-BK alone also induced a significant decrease in MAP and CVR (P < 0.05). In the presence of the B2 receptor antagonist, Hoe 140 (25 micrograms kg-1), only the decreases in MAP and CVR caused by BK were significantly reduced (P < 0.05). 4. Inhibition of NO synthase with N omega-nitro-L-arginine (L-NOARG, 45 mg kg-1) significantly (P < 0.05) prevented the decrease in CVR but not MAP induced by des-Arg9-BK, whilst responses to BK were not affected by L-NOARG pretreatment. Inhibition of prostanoid synthesis with indomethacin (25 mg kg-1) did not affect the reductions in MAP and CVR induced by des-Arg9-BK or BK. 5. In conclusion, i.v. des-Arg9-BK and BK administration induced reductions in MAP and CVR suggesting that in conscious instrumented dogs both B1 and B2 receptors are present and can affect systemic blood pressure and coronary resistance regulation. Our results also suggest that prostanoids are not involved in the vascular response to kinins and that coronary vascular B1 receptors are at least in part coupled to the release of NO.     

Recent developments in ghrelin receptor (GHS-R1a) agonists and antagonists

The 28 amino acid polypeptide, ghrelin, is a novel neuroendocrine hormone synthesised primarily in the stomach. It stimulates growth hormone (GH) secretion, increases food intake and appetite and promotes body weight gain by decreasing fat mass metabolism, particularly in rodent models. The pharmacological properties of this peptide are mediated by the type 1a growth hormone secretagogue receptor (GHS-R1a), a G-protein-coupled receptor localised predominantly in the pituitary gland and hypothalamus. The discovery of new agents that either mimic or modulate the actions of ghrelin via the GHS-R1a has attracted considerable interest in recent years. Many peptidyl and peptidomimetic GHS-R1a agonists such as growth hormone-releasing peptides (GHRPs) and growth hormone secretagogues (GHSs) that stimulate GH secretion and increase plasma levels of insulin-like growth factor-1 (IGF-1) have been identified. First generation GHSs have undergone extensive clinical evaluation in humans, but have shown disappointing results in the treatment of diseases such as frailty, the age-related decline in body composition. Several of these compounds were found to increase adiposity in rodent models. Second generation dipeptidyl and third generation small molecule GHSs have recently been profiled in various animal models. A third generation of GHS with partial agonist activity at the GHS-R1a showed anabolic activity in rodent models, increasing fat-free (muscle) mass rather than fat mass. A few GHS-R1a receptor antagonists have been disclosed, although these compounds have exhibited poor selectivities or weak affinities for the receptor. Since endogenous ghrelin appears to play an important role in the long-term regulation of energy balance, GHS-R1a antagonists may be useful in the prevention of weight gain, following weight loss, or in the treatment of obesity, a pathological condition characterised by excess adiposity. This review describes the pharmacology of select ghrelin receptor modulators and highlights new ghrelin receptor agonists and antagonists that have appeared in the literature from 1999 – 2002.     

Synthesis and biological evaluation of bradykinin B(1)/B(2) and selective B(1) receptor antagonists

We recently described a potent bradykinin B(2) receptor agonist (JMV1116) obtained by replacing the D-Tic-Oic dipeptide moiety of HOE140 by a (3S)-amino-5-(carbonylmethyl)-2,3-dihydro-1, 5-benzothiazepin-4(5H)-one (D-BT) moiety. This compound inhibited the specific binding of [(3)H]BK on membranes of CHO cells expressing the human cloned B(2) receptor with nanomolar affinity and contracted both isolated rat uterus and human umbilical vein. These data demonstrated that D-BT could be a good mimic of the Pro-Phe dipeptide. In the present study we characterized B(1) receptor antagonists containing the D-BT moiety. We prepared an analogue of compound JMV1116 deleting the C-terminal arginine residue. The resulting compound (1) had an affinity of 83 nM for the human cloned B(1) receptor. The most remarkable property of 1 is its ability to bind also the B(2) receptor with an affinity of 4.4 nM despite the absence of the C-terminal arginine residue. Modifications at the N-terminal part of 1 associated with the substitution of the thienylalanine residue by alpha-(2-indanyl)glycine resulted in analogues selectively binding to the B(1) receptor with an affinity in the picomolar range.     

In vitro and in vivo pharmacological characterization of two novel selective cannabinoid CB2 receptor inverse agonists

We have previously developed quinolone-3-carboxamides with the aim of obtaining new ligands for both cannabinoid receptors, CB₁ and CB₂. Our preliminary screening led to the identification of cannabinoid receptor ligands characterized by high affinity and, in some cases, also selectivity for CB₂ receptors. Specifically, three compounds, 1, 2 and 3 showed high affinity for CB₂ as well as high selectivity over CB₁ receptors. In addition, the activity shown by 1 against the formalin-induced nocifensive response in mice, reported in our previous paper, suggests that quinolone-3-carboxamides possess anti-nociceptive properties. In the present work, we have performed functional in vitro bioassays with the aim of investigating the functional activity in the [³⁵S]GTPγS binding assay of the other two compounds that, like 1, behave as CB₂ selective ligands, and their potential analgesic actions in vivo. We found that both 2 and 3 behave in vitro as CB₂ inverse agonists and are able to decrease nociceptive behaviour in the late phase of the formalin test only at the highest dose tested, although, at lower doses, they prevent the anti-nociceptive effects of a selective CB₂ partial agonist in the formalin test. These results identify in 2 and 3 two novel, potent and selective CB₂ antagonists/inverse agonists and confirm previous reports in the literature that, in addition to agonists at cannabinoid CB₂ receptors, also inverse agonists/antagonists at these receptors show promise as anti-inflammatory agents.     

Cannabinoid receptor 2 (CB2) agonists and antagonists: a patent update

Introduction: Modulation of the CB₂ receptor is an interesting approach for pain and inflammation, arthritis, addictions, neuroprotection, and cancer, among other possible therapeutic applications, and is devoid of central side effects.

Areas covered: This review highlights the novel scaffolds for CB₂ ligands and the diverse therapeutic applications for CB₂ modulators disclosed in patents published since 2012.

Expert opinion: Structural diversity of CB₂ modulator scaffolds characterized the patent literature. Several CB₂ agonists reached clinical Phase II for pain management and inflammation. Other therapeutic applications need to be explored such as neuroprotection and/or neurodegeneration.     

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.     

Development of hyperthermia following intracerebroventricular administration of endotoxin in the rat: effect of kinin B1 and B2 receptor antagonists.

1. E. coli lipopolysaccharide (LPS) produced a dose-dependent (dose range: 0.02-150 micrograms) increase in rat core temperature that was maximal 6 h after intracerebroventricular (i.c.v.) administration. LPS (200 ng) increased core temperature by 1.0 +/- 0.2 degrees C, 6 h following administration, as compared to vehicle-treated controls (-0.2 +/- 0.2 degrees C). 2. LPS-induced (200 ng) hyperthermia was prevented by co-administration of the bradykinin (BK) B2 receptor antagonist, Hoe 140 (10 and 30 pmol, i.c.v.) or by indomethacin (10 nmol, i.c.v.). 3. Systemic administration of Hoe 140 at doses up to 1 mumol kg-1, s.c., did not attenuate LPS-induced (200 ng, i.c.v.) hyperthermia. However, LPS hyperthermia was significantly reduced by systemic administration of indomethacin (1 mumol kg-1, i.v.). 4. Co-administration of the selective B1 receptor antagonists, [des-Arg9, Leu8]BK (0.1 - 1 nmol, i.c.v.) or [des-Arg10] Hoe 140 (0.1 - 1 nmol, i.c.v.), did not prevent LPS-induced hyperthermia. 5. It is concluded that the development of hyperthermia following central administration of endotoxin requires activation of central, but not peripheral bradykinin B2 receptors. The formation of kinins within the CNS may be an important initial component of CNS inflammation following infection.     

Vascular kinin B(1) and B(2) receptor-mediated effects in the rat isolated perfused kidney - differential regulations

1. Bradydykinin (BK) and analogs acting preferentially at kinin B(1) or B(2) receptors were tested on the rat isolated perfused kidney. Kidneys were perfused in an open circuit with Tyrode's solution. Kidneys preconstricted with prostaglandin F(2alpha) were used for the analysis of vasodilator responses. 2. BK induced a concentration-dependent renal relaxation (pD(2)=8.9+/-0.4); this vasodilator response was reproduced by a selective B(2) receptor agonist, Tyr(Me)(8)-BK (pD(2)=9.0+/-0.1) with a higher maximum effect (E(max)=78.9+/-6.6 and 55.8+/-4.3% of ACh-induced relaxation respectively, n=6 and 19, P<0.02). Icatibant (10 nM), a selective B(2) receptor antagonist, abolished BK-elicited relaxation. Tachyphylaxis of kinin B(2) receptors appeared when repeatedly stimulated at 10 min intervals. 3. Des-Arg(9)-BK, a selective B(1) receptor agonist, induced concentration-dependent vasoconstriction at micromolar concentration. Maximum response was enhanced in the presence of lisinopril (1 microM) and inhibited by R 715 (8 microM), a selective B(1) receptor antagonist. Des-Arg(9)-[Leu(8)]-BK behaved as an agonist. 4. A contractile response to des-Arg(9)-BK occurred after 1 of perfusion and increased with time by a factor of about three over a 3 h perfusion. This post-isolation sensitization to des-Arg(9)-BK was abolished by dexamethasone (DEX, 30 mg kg(-1) i.p., 3 h before the start of the experiment and 10 microM in perfusate) and actinomycin D (2 microM). Acute exposure to DEX (10 microM) had no effect on sensitized des-Arg(9)-BK response, in contrast to indomethacin (30 microM) that abolished it. DEX pretreatment however had no effect on BK-induced renal vasodilation. 5. Present results indicate that the main renal vascular response to BK consists of relaxation linked to the activation of kinin B(2) receptors which rapidly desensitize. Renal B(1) receptors are also present and are time-dependently sensitized during the in vitro perfusion of the rat kidneys.     

Lysophosphatidic acid receptor agonists and antagonists (WO2010051053)

Lysophosphatidic acid (LPA) is a bioactive lipid involved in signaling pathways that result in cell survival, proliferation, migration and invasion. These cellular responses are a critical element of both normal development as well as pathophysiology. In particular, dysregulated LPA production and function have been linked with cancer and cardiovascular disease. RxBio, Inc. has generated several series of LPA analogs with varied agonist/antagonist function at the LPA_1–3 GPCR targets of LPA signaling. These analogs are simplified relative to LPA through deletion of the glycerol moiety linking the LPA phosphate and fatty acid groups. One of the example compounds was shown to protect intestinal crypt cells from radiation-induced apoptosis in mice when whole body irradiation occurred 2 h after oral dosing.     

Lysophosphatidic acid receptor agonists and antagonists (WO2010051053)

Lysophosphatidic acid (LPA) is a bioactive lipid involved in signaling pathways that result in cell survival, proliferation, migration and invasion. These cellular responses are a critical element of both normal development as well as pathophysiology. In particular, dysregulated LPA production and function have been linked with cancer and cardiovascular disease. RxBio, Inc. has generated several series of LPA analogs with varied agonist/antagonist function at the LPA(1-3) GPCR targets of LPA signaling. These analogs are simplified relative to LPA through deletion of the glycerol moiety linking the LPA phosphate and fatty acid groups. One of the example compounds was shown to protect intestinal crypt cells from radiation-induced apoptosis in mice when whole body irradiation occurred 2 h after oral dosing.     

In vitro interactions of agonists and antagonists with beta-adrenergic receptors

Neurotransmitters and hormones mediate their effects through interaction with specific receptors. A complete understanding of the effects of these chemical signals requires detailed knowledge, at the molecular level, of agonist/receptor interactions. It is likely that agonists and antagonists interact with the same site on a receptor. Agonists, however, are by definition different from antagonists in that agonists are responsible for transducing information across the cell membrane, ultimately resulting in a biological response, while antagonists appear to act through passive occupancy of receptors. Implicit in this concept is the idea that these fundamental differences between agonists and antagonists arise from the sequelae induced by agonist-specific interactions with receptors.     

Agonist-induced translocation of the kinin B(1) receptor to caveolae-related rafts

The kallikrein-kinin system, activated during inflammatory conditions and the regulation of specific cardiovascular and renal functions, includes two G protein-coupled receptors for bradykinin (BK)-related peptides. The B(1) receptor (B(1)R) subtype is not believed to undergo agonist-induced phosphorylation and endocytosis. A conjugate made of the rabbit B(1)R fused with the yellow variant of green fluorescent protein (YFP) was expressed in mammalian cells. In COS-1 or human embryonic kidney (HEK) 293 cells, the construction exhibited a nanomolar affinity for the agonist radioligand [(3)H]Lys-des-Arg(9)-BK or the antagonist ligand [(3)H]Lys-[Leu(8)]des-Arg(9)-BK and a pharmacological profile virtually identical to that of wild-type B(1)R. Lys-des-Arg(9)-BK stimulation of HEK 293 cells stably expressing B(1)R-YFP but not stimulation of untransfected cells released [(3)H]arachidonate in a phospholipase A(2) assay. B(1)R-YFP was visualized as a continuous labeling of the plasma membranes in stably transfected HEK 293 cells (confocal microscopy). Addition of Lys-des-Arg(9)-BK (1-100 nM) rapidly concentrated the receptor-associated fluorescence into multiple aggregates that remained associated with the plasma membrane (no significant internalization) and colocalized with caveolin-1. This reaction was slowly reversible upon agonist washing at 37 degrees C and prevented pretreatment with a B(1)R antagonist. beta-Cyclodextrin treatment, which extracts cholesterol from membranes and disrupts caveolae-related rafts, prevented agonist-induced redistribution of B(1)R-YFP but not the PLA(2) activation mediated by this receptor. The agonist radioligand copurified with caveolin-1 to a greater extent than the tritiated antagonist in buoyant fractions of HEK 293 cells treated with the ligands. Agonist-induced cellular translocation of the kinin B(1)R to caveolae-related rafts without endocytosis is a novel variation on the theme of G protein-coupled receptor adaptation.     

In vitro selectivity of agonists and antagonists for beta 1- and beta 2-adrenoceptor subtypes in rat brain

A radioreceptor binding assay was developed to determine the selectivity of beta-adrenoceptor agents in rat brain. This was achieved by using the highly selective unlabelled antagonists CGP 20712A and ICI 118-551 to block beta 1- or beta 2-sub-populations respectively in rat cerebral cortex membranes. This permitted the selective labelling of beta-adrenoceptors with the antagonist (-)-[125I]pindolol. Using this method, compounds could be routinely screened and selectivity profiles for binding in the CNS determined with a high degree of sensitivity and resolution.     

From bradykinin B2 receptor antagonists to orally active and selective bradykinin B1 receptor antagonists

The bradykinin (BK) B1 receptor is an attractive target for the treatment of chronic pain and inflammation. Starting from a dual B1 and B2 antagonist, novel antagonists were designed that display low-nanomolar affinity for human B1 receptor and selectivity over B2. Initially, potent imidazoline derivatives were studied, but these compounds suffered from low bioavailability. This issue could be overcome by the use of less basic amino derivatives leading to orally active compounds.