Discriminative stimulus effects of ethyl-β-carboline-3-carboxylate at two training doses in rats

Rationale and objectives: The role of benzodiazepine (BZ) receptor mechanisms in modulating the stimulus effects of the BZ partial inverse agonist ethyl-β-carboline-3-carboxylate (β-CCE) are not well understood. The purpose of the present experiments was to assess the role of BZ and non-BZ receptor stimulation in the discriminative stimulus effects of β-CCE in rats. Methods: Adult male rats were trained to discriminate either a relatively high dose (10 mg/kg, n=8) or a relatively low dose (5.0 mg/kg, n=7) of β-CCE from saline under a fixed-ratio 10 schedule of food presentation. Results: Under the high-dose training condition, β-CCE engendered an increase in responding on the drug-paired lever up to 100% drug-lever responding, with no decrease in response rate. Diazepam, pentobarbital, flumazenil, (+)-amphetamine, and morphine did not share stimulus effects with 10 mg/kg β-CCE up to doses that suppressed rate of responding. The BZ full inverse agonist dimethoxy-4-ethyl-β-carboline-3-carboxylate also did not engender ≥80% β-CCE-lever responding up to doses that suppressed response rate and produced seizures in some animals. The BZ partial inverse agonists Ro 15-4513 and sarmazenil fully reproduced the stimulus effects of β-CCE. Flumazenil antagonized the effects of β-CCE with an in vivo apparent pA₂ value of 6.1 (slope=–0.86). Under the low-dose condition, β-CCE engendered an increase in drug-lever responding, with no changes in response rate. In contrast to the high-dose condition, diazepam, pentobarbital, and (+)-amphetamine engendered high levels of β-CCE-lever responding (up to 77, 96, and 75%, respectively), whereas flumazenil and morphine did not engender full β-CCE- lever responding. Conclusions: These results indicated that the stimulus effects of the high dose of β-CCE appeared consistent with mediation by the drug’s partial inverse agonist effects at BZ receptors. The discriminative stimulus effects of β-CCE at the lower training dose, however, appeared to be relatively non-specific. Received: 12 November 1998 / Final version: 15 March 1999     

Discriminative stimulus properties of β-carbolines characterized as agonists and inverse agonists at central benzodiazepine receptors

The discriminative stimulus properties of three -carboline derivatives were studied in three groups of rats trained, respectively, to discriminate diazepam (2.5 mg/kg IP), chlordiazepoxide (CDP, 5 mg/kg IP) or pentylenetetrazol (PTZ, 15 mg/kg IP) from saline in standard procedures employing two-lever operant chambers. Two -carbolines, ZK 91296 and ZK 93423, substituted for the benzodiazepines in both CDP- and diazepam-trained rats. The neutral benzodiazepine antagonist Ro 15-1788 blocked the diazepam discriminative stimulus and the ability of ZK 91296 to substitute for diazepam. A third -carboline, FG 7142, was not identified as benzodiazepine-like in generalization tests in either diazepam- or CDP-trained rats, but when administered together with CDP antagonized the benzodiazepine discriminative stimulus. In rats trained to discriminate PTZ from saline (a discrimination which is thought to depend on the anxiogenic properties of PTZ) the PTZ cue was antagonized by diazepam and ZK 93423, and partially antagonized by ZK 91296. The PTZ cue generalized to FG 7142 and this generalization was partially antagonized by Ro 15-1788. These results suggest that the three -carbolines provide more than one kind of discriminative stimulus, consistent with the classification of ZK 93423 as an agonist at central benzodiazepine receptors, with ZK 91 296 as a partial agonist, and with FG 7142 as an inverse agonist. Pharmacologically, ZK 93 423 and ZK 91 296 may exhibit anxiolytic qualities, whereas FG 7142 produces anxiogenic effects.     

Discriminative stimulus properties of 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane [(±)DOI] in C57BL/6J mice

Abstract Rationale. The drug discrimination procedure has proven to be a valuable tool for studying the mechanism of action of psychoactive drugs. Recently, mice with targeted gene mutations have been developed that may also prove useful in evaluating the role of specific receptors in mediating the actions of drugs. We were interested in studying the effects of hallucinogens in genetically modified mice using the drug discrimination procedure. Objective. To establish the training procedures and characterize the stimulus properties of 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane [(±)DOI] versus saline in wild-type mice. Methods. Using a two-lever drug discrimination procedure, C57BL/6J mice were trained to discriminate (±)DOI (2.5 mg/kg) from saline on a VI 30-s schedule of reinforcement. Results. The acquisition function was orderly and similar to that found previously with rats, although the training dose required for the mice was four times higher (2.5 versus 0.75 mg/kg). The dose-response relationship indicated that percent drug lever responding was dose-dependent. Two other hallucinogens, LSD and (–)DOB, substituted fully for (±)DOI. Mice were tested for their ability to discriminate (±)DOI following pretreatment with the 5-HT_2A receptor antagonist MDL 100,907, or with 5-HT_2C selective antagonists, SB 206,553 or SB 242,084. A dose of 0.25 mg/kg MDL 100,907 essentially completely blocked the discriminative stimulus effects of 2.5 mg/kg (±)DOI. Surprisingly, both SB 206,553 and SB 242,084 also attenuated the effect of 2.5 mg/kg (±)DOI. The effect of SB 206,553 was surmountable at 5.0 mg/kg (±)DOI. Conclusions. These data agree with the results from studies with rats indicating a prominent role for the 5-HT_2A receptors in mediating the discriminative stimulus effects of (±)DOI but in addition, suggest a small but significant role for the 5-HT_2C receptor in mice. Electronic Publication     

Antagonism of stimulus properties of nicotine by dihydro-β-erythroidine (DHβE) in rats

Rationale: Previous work has shown that a dose of DHβE, a competitive nicotinic receptor antagonist that blocked the discriminative stimulus properties of nicotine, was insufficient to block locomotor depression or operant rate-reducing effects of nicotine in rats. Examination of DHβE against other behavioural effects of nicotine may help in understanding its diverse actions. Objective: The present experiments examine the aversive stimulus properties of nicotine, a function implicated in the regulation of nicotine intake. Furthermore, to characterise the duration of pharmacological blockade produced by DHβE, the antagonist was examined in the drug discrimination (DD) procedure. Methods: Using the conditioned taste aversion (CTA) paradigm, male hooded rats were trained to avoid one of two distinctively flavoured solutions paired with nicotine (0.2 or 0.4 mg/kg) administration. In rats trained to discriminate 0.2 mg/kg SC nicotine in a two-lever procedure maintained under a tandem VI60”-FR10 schedule of food reinforcement, the offset of antagonism by DHβE was examined 5, 15 and 30 min following injection of nicotine (0.2 or 0.4 mg/kg SC) or vehicle. Results: Administration of DHβE (0.5, 1.6 and 5.0 mg/kg SC) 30 min before nicotine failed to block nicotine (0.4 mg/kg) CTA, while co-administration of DHβE (5.0 mg/kg SC) with nicotine (0.2 and 0.4 mg/kg SC) prevented the development of CTAs. This blockade complemented nicotine discrimination data in which DHβE blocked the discriminative stimulus effect of nicotine (0.2 or 0.4 mg/kg SC) for 45 min after its administration. Conclusions: These observations of DHβE’s short-lasting antagonism against the aversive and discriminative stimulus effects of nicotine support the involvement of the similar subtypes of nicotinic receptor in the mediation of these diverse behavioural effects. Received: 19 June 1999 / Final version: 11 November 1999     

Discriminative stimulus effects of ω (BZ) receptor ligands: correlation with in vivo inhibition of [3H]-flumazenil binding in different regions of the rat central nervous system

Rats can be trained to discriminate benzodiazepines (BZ) from vehicle and there is considerable evidence that the stimulus effects of these drugs are mediated by activity at (BZ) modulatory sites of the GABA_A receptor complex. A number of recent studies, however, have indicated that differences may exist between the discriminative stimulus effects of benzodiazepines and those of certain non-benzodiazepine ligands for the (BZ) receptors (e.g. zolpidem, abecarnil). As it is known that several subtypes of (BZ) sites are found in the central nervous system, and that drugs such as zolpidem have selectivity for certain subtypes, it is possible that differential stimulus effects may be associated with receptor selectivity. In the present study, correlations were calculated between the potencies of nine compounds with affinity for receptors (diazepam, lorazepam, triazolam, clonazepam, alprazolam, zopiclone, suriclone, CL 218, 872 and zolpidem) to substitute for chlordiazepoxide in rats trained to discriminate a dose (5 mg/kg) of this benzodiazepine and the ability of the same compounds to inhibit the binding of [³H]-flumazenil from different structures in the rat central nervous system in vivo. The correlations obtained were: cerebellum 0.46, cortex 0.39, striatum 0.78 (P<0.05), hippocampus 0.79 (P<0.05) and spinal cord 0.95 (P<0.001). These different structures are known to contain different relative concentrations of ₁ (BZ₁) and ₂ (BZ₂) sites with the spinal cord containing the greatest (80%) and cerebellum the lowest (5%) concentration of ₂ (BZ₂) sites. Good correlations were also observed between the ability of these compounds to substitute for chlordiazepoxide and their potency to inhibit (³H)-flumazenil binding in both cerebellar (r=0.88) and spinal cord (r=0.91) membranes in vitro indicating that the physiological relevance of receptor subtypes cannot be deduced from in vitro studies. The present results are consistent with the possibility that the discriminative stimulus produced by chlordiazepoxide is mediated by activity at ₂ (BZ₂) sites. No correlations were observed between inhibition of [³H]-flumazenil binding and response rate decreases, suggesting that the mechanism underlying this behavioural effect is different from that mediating the discriminative stimulus.     

The Biotransformation of 3-(γ-Morpholino-β-3′,4′,5′-trimethoxybenzoxypropyl)-4-methyl-7,8-dimethoxy- coumarin hydrochloride in the Rabbit

1. Following single oral doses (50?mg/kg) of 3-(γ-morpholino-β-3′,4′,5′-trimethoxybenzoxypropyl)-4-methyl-7,8-dimethoxy[4-¹⁴C]courmarin hydrochloride (ABBOTT-37624) to female New Zealand white rabbits, the major metabolite found in urine and faeces was the 7-O-demethylated, de-esterified compound (54.9% of dose). Another major fraction consisted of polar compounds, evenly distributed between urine and faeces (14.8% and 14.3% of dose respectively). Trace amounts of parent compound and the product of its de-esterification were found in rabbit faeces.

2. Glucuronide formation appears to be the principal form of conjugation of the metabolites in this species.     

Adaptation and validation of the revised Patients’ Attitudes towards Deprescribing (rPATD) questionnaire for benzodiazepine receptor agonists

BackgroundThe revised Patients' Attitudes Towards Deprescribing (rPATD) questionnaire explores older adults’ views on deprescribing in general. Those views may differ, however, when the target is a specific drug such as benzodiazepine receptor agonists (BZRA).ObjectiveThis study aimed to adapt the 22-item French rPATD questionnaire to create a BZRA-specific instrument and to assess the psychometric properties of this new tool.MethodsThe adaptation of the questionnaire comprised 3 steps: 1) item transformation during group discussions with 8 healthcare providers and 8 BZRA users (aged ≥65 years), 2) pre-test of the questionnaire with 12 other older adults to ensure items understanding, 3) evaluation of the psychometric properties of the new questionnaire with 221 older BZRA users recruited in Belgium, France, and Switzerland. Construct validity was assessed using exploratory factor analysis (EFA), internal consistency with Cronbach's alpha, and test-retest reliability with intraclass correlation coefficient (ICC).ResultsAfter the pre-test, the questionnaire had 24 items (19 adapted from the French rPATD, 3 removed, and 5 added). The EFA, however, found that several items performed poorly. Eleven items were consequently removed, based on statistical performance and clinical relevance. Three factors were extracted from the EFA performed on the 11 retained items and were named “Concerns about stopping BZRA”, “BZRA inappropriateness”, and “Dependence on BZRA”. The questionnaire also includes two global questions about willingness to reduce BZRA dosage and willingness to discontinue BZRA. All factors showed acceptable internal consistency (0.68 ≤ Cronbach's alpha ≤0.74). Two factors showed acceptable test-retest reliability. The “Concerns about stopping BZRA” factor was found to vary over time (ICC [95%CI]: 0.35[-0.02; 0.64]).ConclusionsWe developed and validated a 13-item questionnaire to evaluate the attitudes of older people towards BZRA deprescribing. Despite some limitations, this questionnaire appears to be a useful tool for facilitating shared decision-making on BZRA deprescribing.     

Modulation of neuroplasticity pathways and antidepressant-like behavioural responses following the short-term (3 and 7 days) administration of the 5-HT₄ receptor agonist RS67333

It has been recently suggested that activation of 5-HT? receptors might exert antidepressant-like effects in rats after 3 d treatment, suggesting a new strategy for developing faster-acting antidepressants. We studied the effects of 3 d and 7 d treatment with the 5-HT? receptor partial agonist RS67333 (1.5 mg/kg.d) in behavioural tests of chronic efficacy and on neuroplastic-associated changes, such as adult hippocampal neurogenesis, expression of CREB, BDNF, β-catenin, AKT and 5-HT? receptor functionality. RS67333 treatment up-regulated hippocampal cell proliferation, β-catenin expression and pCREB/CREB ratio after 3 d treatment. This short-term treatment also reduced immobility time in the forced swim test (FST), together with a partial reversion of the anhedonic-like state (sucrose consumption after chronic corticosterone). Administration of RS67333 for 7 d resulted in a higher increase in the rate of hippocampal cell proliferation, a significant desensitization of 5-HT? receptor-coupled adenylate cyclase activity and a more marked increase in the expression of neuroplasticity-related proteins (BDNF, CREB, AKT): these changes reached the same magnitude as those observed after 3 wk administration of classical antidepressants. Consistently, a positive behavioural response in the novelty suppressed feeding (NSF) test and a complete reversion of the anhedonic-like state (sucrose consumption) were also observed after 7 d treatment. These results support the antidepressant-like profile of RS67333 with a shorter onset of action and suggest that this time period of administration (3-7 d) could be a good approximation to experimentally predict the onset of action of this promising strategy.     

β(2)-Adrenoceptors increase translocation of GLUT4 via GPCR kinase sites in the receptor C-terminal tail

BACKGROUND AND PURPOSE

β-Adrenoceptor stimulation induces glucose uptake in several insulin-sensitive tissues by poorly understood mechanisms.

EXPERIMENTAL APPROACH

We used a model system in CHO-K1 cells expressing the human β₂-adrenoceptor and glucose transporter 4 (GLUT4) to investigate the signalling mechanisms involved.

KEY RESULTS

In CHO-K1 cells, there was no response to β-adrenoceptor agonists. The introduction of β₂-adrenoceptors and GLUT4 into these cells caused increased glucose uptake in response to β-adrenoceptor agonists. GLUT4 translocation occurred in response to insulin and β₂-adrenoceptor stimulation, although the key insulin signalling intermediate PKB was not phosphorylated in response to β₂-adrenoceptor stimulation. Truncation of the C-terminus of the β₂-adrenoceptor at position 349 to remove known phosphorylation sites for GPCR kinases (GRKs) or at position 344 to remove an additional PKA site together with the GRK phosphorylation sites did not significantly affect cAMP accumulation but decreased β₂-adrenoceptor-stimulated glucose uptake. Furthermore, inhibition of GRK by transfection of the βARKct construct inhibited β₂-adrenoceptor-mediated glucose uptake and GLUT4 translocation, and overexpression of a kinase-dead GRK2 mutant (GRK2 K220R) also inhibited GLUT4 translocation. Introducing β₂-adrenoceptors lacking phosphorylation sites for GRK or PKA demonstrated that the GRK sites, but not the PKA sites, were necessary for GLUT4 translocation.

CONCLUSIONS AND IMPLICATIONS

Glucose uptake in response to activation of β₂-adrenoceptors involves translocation of GLUT4 in this model system. The mechanism is dependent on the C-terminus of the β₂-adrenoceptor, requires GRK phosphorylation sites, and involves a signalling pathway distinct from that stimulated by insulin.     

In vivo and in vitro evaluation of the estrogenic properties of the 17β-(butylamino)-1,3,5(10)-estratrien-3-ol (buame) related to 17β-estradiol

BackgroundBuame [17β-(butylamino)-1,3,5(10)-estratrien-3-ol] possesses anticoagulant and antiplatelet activities that are potentially antithrombotic. Since its estrogenicity is unknown, it was evaluated by established methods.MethodsBuame (10, 100, 500, and 1,000 μg/kg), 17β-estradiol (E₂) (100 μg/kg), or propylene glycol (10 ml/kg) were subcutaneously (sc) administered for three days to immature Wistar female rats (21 days old). The relative uterotrophic effect to E₂ was 78 (E₂ = 100) with a relative uterotrophic potency of 1.48 (E₂ = 100). Adult ovariectomized Wistar rats received an sc injection at 8:00 h (reversed cycle) of: 7.5 μg of E₂ (≈ 30 μg/kg), buame (≈ 750, 1,500, 3,000 μg/kg), or corn oil (≈ 1.2 ml/kg). After 24 h, progesterone (4–5 mg/kg) was administered. Sexual receptivity was assessed 5 to 7 h later, and the lordosis quotient (LQ; number lordosis/number mounts × 100) was evaluated.ResultsBuame induced lordosis (LQmax 85 ± 9; ED50 952 ± 19 μg/kg) and E₂ LQmax 56 ± 8; ED50 10 ± 2 μg/kg; the relative LQpotency was 0.51 (E₂ = 100). Buame competed with [³H]E₂ for the estrogen receptor (Buame RBA = 0.15 and Ki = 5.9 × 10^?7 M; E₂ RBA = 100; Ki = 6.6 × 10^?9 M). Buame increased MCF-7 cells proliferation, from 10^?11 to 10^?9 M, its proliferative effect was 1.73–1.79 (E₂ = 3.0–3.9); its relative proliferative effect to E₂ was 33–40% (E₂ = 100%) and relative potency 10.4–10.7 (E₂ = 100). Tamoxifen and fulvestrant (ICI 182,780) inhibited buame's proliferation indicating mediation through estrogen receptors in this response.ConclusionBuame is therefore an estrogen partial agonist with a weak estrogenic activity.     

Functional characterization of the α5(Asn398) variant associated with risk for nicotine dependence in the α3β4α5 nicotinic receptor

Smoking is a major cause for premature death. Work aimed at identifying genetic factors that contribute to nicotine addiction has revealed several single nucleotide polymorphisms (SNPs) that are linked to smoking-related behaviors such as nicotine dependence and level of smoking. One of these SNPs leads to an aspartic acid-to-asparagine substitution in the nicotinic receptor α5 subunit at amino acid position 398 [rs16969968; α5(Asn398)]. The α5 subunit is expressed both in the brain and in the periphery. In the brain, it associates with the α4 and β2 subunits to form α4β2α5 receptors. In the periphery, the α5 subunit combines with the α3 and β4 subunits to form the major ganglionic postsynaptic nicotinic receptor subtype. The α3β4α5 receptor regulates a variety of autonomic responses such as control of cardiac rate, blood pressure, and perfusion. In this paradigm, the α5(Asn398) variant may act by regulating autonomic responses that may affect nicotine intake by humans. Here, we have investigated the effect of the α5(Asn398) variant on the function of the α3β4α5 receptor. The wild-type or variant α5 subunits were coexpressed with the α3 and β4 subunits in human embryonic kidney 293 cells. The properties of the receptors were studied using whole-cell and single-channel electrophysiology. The data indicate that the introduction of the α5(Asn398) mutation has little effect on the pharmacology of receptor activation, receptor desensitization, or single-channel properties. We propose that the effect of the α5(Asn398) variant on nicotine use is not mediated by an action on the physiological or pharmacological properties of the α3β4α5 subtype.     

Use of an α3β4 nicotinic acetylcholine receptor subunit concatamer to characterize ganglionic receptor subtypes with specific subunit composition reveals species-specific pharmacologic properties

Drug development for nicotinic acetylcholine receptors (nAChR) is challenged by subtype diversity arising from variations in subunit composition. On-target activity for neuronal heteromeric receptors is typically associated with CNS receptors that contain α4 and other subunits, while off-target activity could be associated with ganglionic-type receptors containing α3β4 binding sites and other subunits, including β4, β2, α5, or α3 as a structural subunit in the pentamer. Additional interest in α3 β4 α5-containing receptors arises from genome-wide association studies linking these genes, and a single nucleotide polymorphism (SNP) in α5 in particular, to lung cancer and heavy smoking. While α3 and β4 readily form receptors in expression system such as the Xenopus oocyte, since α5 is not required for function, simple co-expression approaches may under-represent α5-containing receptors. We used a concatamer of human α3 and β4 subunits to form ligand-binding domains, and show that we can force the insertions of alternative structural subunits into the functional pentamers. These α3β4 variants differ in sensitivity to ACh, nicotine, varenicline, and cytisine. Our data indicated lower efficacy for varenicline and cytisine than expected for β4-containing receptors, based on previous studies of rodent receptors. We confirm that these therapeutically important α4 receptor partial agonists may present different autonomic-based side-effect profiles in humans than will be seen in rodent models, with varenicline being more potent for human than rat receptors and cytisine less potent. Our initial characterizations failed to find functional effects of the α5 SNP. However, our data validate this approach for further investigations.     

N-(3,4-dimethoxyphenethyl)-4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2[1H]-yl)-6,7-dimethoxyquinazolin-2-amine (CP-100,356) as a “chemical knock-out equivalent” to assess the impact of efflux transporters on oral drug absorption in the rat

The utility of the diaminoquinazoline derivative CP-100,356 as an in vivo probe to selectively assess MDR1/BCRP-mediated drug efflux was examined in the rat. CP-100,356 was devoid of inhibition (IC₅₀ >50 µM) against major human P450 enzymes including P4503A4. In human MDR1-transfected MDCKII cells, CP-100,356 inhibited acetoxymethyl calcein (calcein-AM) uptake (IC₅₀ ～0.5 ± 0.07 µM) and digoxin transport (IC₅₀ ～1.2 ± 0.1 µM). Inhibition of prazosin transport (IC₅₀ ～1.5 ± 0.3 µM) in human BCRP-transfected MDCKII cells by CP-100,356 confirmed the dual MDR1/BCRP inhibitory properties. CP-100,356 was a weak inhibitor of OATP1B1 (IC₅₀ ～66 ± 1.1 µM) and was devoid of MRP2 inhibition (IC₅₀ >15 µM). In vivo inhibitory effects of CP-100,356 in rats were examined after coadministration with MDR1 substrate fexofenadine and dual MDR1/BCRP substrate prazosin. Coadministration with increasing doses of CP-100,356 resulted in dramatic increases in systemic exposure of fexofenadine (36- and 80-fold increase in C_max and AUC at a CP-100,356 dose of 24 mg/kg). Significant differences in prazosin pharmacokinetics were also discernible in CP-100,356-pretreated rats as reflected from a 2.6-fold increase in AUC. Coadministration of CP-100,356 and P4503A substrate midazolam did not result in elevations in systemic exposure of midazolam in the rat. The in vivo methodology should have utility in drug discovery in selective and facile assessment of the role of MDR1 and BCRP efflux transporters in oral absorption of new drug candidates. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4914–4927, 2009     

The additional ACh binding site at the α4(+)/α4(?) interface of the (α4β2)2α4 nicotinic ACh receptor contributes to desensitization

BACKGROUND AND PURPOSE

Nicotinic ACh (α4β2)₂α4 receptors are highly prone to desensitization by prolonged exposure to low concentrations of agonist. Here, we report on the sensitivity of the three agonist sites of the (α4β2)₂α4 to desensitization induced by prolonged exposure to ACh. We present electrophysiological data that show that the agonist sites of the (α4β2)₂α4 receptor have different sensitivity to desensitization and that full receptor occupation decreases sensitivity to desensitization.

EXPERIMENTAL APPROACH

Two-electrode voltage-clamp electrophysiology was used to study the desensitization of concatenated (α4β2)₂α4 receptors expressed heterologously in Xenopus oocytes. Desensitization was assessed by measuring the degree of functional inhibition caused by prolonged exposure to ACh, as measured under equilibrium conditions. We used the single-point mutation α4W182A to measure the contribution of individual agonist sites to desensitization.

KEY RESULTS

(α4β2)₂α4 receptors are less sensitive to activation and desensitization by ACh than (α4β2)₂β2 receptors. Incorporation of α4W182A into any of the agonist sites of concatenated (α4β2)₂α4 receptors decreased sensitivity to activation and desensitization but the effects were more pronounced when the mutation was introduced into the α4(+)/α4(−) interface.

CONCLUSIONS AND IMPLICATIONS

The findings suggest that the agonist sites in (α4β2)₂α4 receptors are not functionally equivalent. The agonist site at the α4(+)/α4(−) interface defines the sensitivity of (α4β2)₂α4 receptors to agonist-induced activation and desensitization. Functional differences between (α4β2)₂α4 and (α4β2)₂β2 receptors might shape the physiological and behavioural responses to nicotinic ligands when the receptors are exposed to nicotinic ligands for prolonged periods of times.     

Pharmacokinetics and Biologic Activity of the Novel Mast Cell Inhibitor, 4-(3′-Hydroxyphenyl)-amino-6,7-dimethoxyquinazoline in Mice

Purpose. The purpose of the present study was to examine the pharma-codynamic and pharmacokinetic features of the novel mast cell inhibitor 4-(3-Hydroxyphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P180) in mice. Methods. A high performance liquid chromatography (HPLC)-based quantitative detection method was used to measure plasma WHI-P180 levels in mice. The plasma concentration-time data was fit to a single compartment pharmacokinetic model by using the WinNonlin program to calculate the pharmacokinetic parameters. A cutaneous anaphylaxis model was used to examine the pharmacodynamic effects of WHI-P180 on anaphylaxis-associated vascular hyperpermeability. Results. The elimination half-life of WHI-P180 in CD-1 mice (BALB/ c mice) following i.v., i.p., or p.o. administration was less than 10 min. Systemic clearance of WHI-P180 was 6742 mL/h/kg in CD-1 mice and 8188 mL/h/kg in BALB/c mice. Notably, WHI-P180, when administered in two consecutive nontoxic i.p. bolus doses of 25 mg/kg, inhibited IgE/antigen-induced vascular hyperpermeability in a well-characterized murine model of passive cutaneous anaphylaxis. Conclusions. WHI-P180 is an active inhibitor of IgE-mediated mast cell responses in vitro and in vivo. Further preclinical characterization of WHI-P180 may improve the efficacy of WHI-P180 in vivo and provide the basis for design of effective treatment and prevention programs for mast cell mediated allergic reactions.     

The 5-HT3 receptor agonist 1-(m-chlorophenyl)-biguanide facilitates noradrenaline release by blockade of α2-adrenoceptors in the mouse brain cortex

We analyzed the facilitatory effect of the 5-HT₃ receptor agonist 1-(m-chlorophenyl)-biguanide (mCPBG) on the electrically evoked noradrenaline release in superfused mouse brain tissue. In addition, we determined the affinities of mCPBG and two other 5-HT receptor ligands, namely, 2-methyl-5-hydroxytryptamine (2-methyl-5-HT; also a 5-HT₃ receptor agonist) and 5-carboxamidotryptamine (5-CT; a 5-HT₁ receptor agonist) for ₂ binding sites. The latter two 5-HT receptor agonists were included because of the claimed involvement of ₂-adrenoceptors in their effects on noradrenaline release.In superfusion experiments on mouse brain cortex slices preincubated with ³H-noradrenaline, tritium overflow evoked by 2-min periods of electrical field stimula tion (3 Hz) was facilitated by mCPBG and, in addition, by rauwolscine (₂-adrenoceptor antagonist) and tetraethylammonium (K⁺ channel blocker) (which were examined for comparison). The effect of mCPBG was not affected by the 5-HT3 receptor antagonist tropisetron or by desipramine but was abolished by rauwolscine. In slices superfused with medium containing desipramine, the concentration-response curve of unlabelled noradrenaline for its inhibitory effect on the electrically (0.3 Hz) evoked overflow was shifted to the right by mCPBG and rauwolscine (apparent pA₂ 5.35 and 7.88, respectively). In another series of superfusion experiments, 4 electrical pulses, administered at 100 Hz, were used to evoke tritium overflow. Tritium overflow evoked by this stimulation procedure (under which an endogenous tone of noradrenaline does not develop) was not affected by mCPBG and rauwolscine but still increased by tetraethylammonium. The specific binding of 3H-rauwolscine to rat brain cortex homogenates was displaced monophasically by unlabelled rauwolscine, mCPBG, 2-methyl-5-HT and 5-CT (pK_i 8.59, 5.84, 5.05 and 5.86, respectively).In conclusion the present results indicate that mCPBG acts as a low-affinity antagonist at ₂-adrenoceptors. This property has to be considered in functional studies of 5-HT₃ receptor-mediated effects in tissues containing ₂-adrenoceptors as well.Abbreviations mCPBG 1-(m-chlorophenyl)-biguanide - 5-CT 5-carboxamidotryptamine - 2-methyl-5-HT 2-methyl-5-hydroxytryptamine - POP pseudo-one-pulse - TEA tetraethylammonium Correspondence to: E. Schlicker at the above address