Binding of GTPγ[35S] is regulated by GDP and receptor activation. Studies with the nociceptin/orphanin FQ receptor

Background and purpose:

We have examined the effects of ligand efficacy and receptor density on the binding of guanosine 5′-[γ-thio]triphosphate (GTPγS) and GDP to the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP)-coupled G-proteins.

Experimental approach:

In GTPγ[³⁵S] binding experiments, using stable (CHO_hNOP) and inducible (CHO_INDhNOP) recombinant human and rat NOP we have measured: (i) ligand-specific GDP requirements; (ii) the effects of receptor density on guanine nucleotide affinity/capacity; and (iii) the effect of ligand efficacy on GTPγS association kinetics.

Key results:

GTPγS competition curves were shallow and modelled by high- and low-affinity components that were relatively consistent between cell types and tissue preparations. In the presence of 1 µM N/OFQ a high-affinity GDP binding site was also present, but the fraction of total binding was reduced. In an efficacy-dependent manner, the partial agonists [F/G]N/OFQ(1-13)NH₂ ([Phe¹ψ(CH₂-NH)Gly²]-nociceptin(1-13)NH₂) and naloxone benzoylhydrazone both reduced the fraction of high-affinity sites for GDP (relative to basal). While the pIC₅₀ for high-affinity GDP binding site did not decrease in the presence of 1 µM N/OFQ, N/OFQ produced a significant reduction in pIC₅₀ for the low-affinity site. Agonist-mediated decrease in affinity for GDP binding was efficacy-dependent. GDP displayed three affinities: high, conserved in the presence and absence of ligand; intermediate, present as a low fraction under basal conditions; low (efficacy-dependent), present during receptor activation representing the majority of binding.

Conclusions and implications:

The affinity of GTPγ[³⁵S] was regulated by GDP and receptor activation caused increased binding of GTPγ[³⁵S] through a reduction in GDP affinity.     

Role of gonadal hormones on mu-opioid-stimulated [<Superscript>35</Superscript>S]GTPγS binding and morphine-mediated antinociception in male and female Sprague–Dawley rats

Rationale  

Male rats are more sensitive to morphine-mediated antinociception than female rats. A role for gonadal hormones in this sex difference has not been clearly defined.     

In vitro pharmacological characterisation of a novel cyclic nociceptin/orphanin FQ analogue c[Cys<Superscript>7,10</Superscript>]N/OFQ(1–13)NH<Subscript>2</Subscript>

Nociceptin/orphanin FQ (N/OFQ) is the endogenous 17 amino acid peptide ligand for the G_i-protein-coupled N/OFQ receptor (NOP). In an attempt to improve the metabolic stability of N/OFQ, we have produced a truncated cyclic analogue with cysteine residues at positions 7 and 10, c[Cys^7,10]N/OFQ(1–13)NH₂ (c[Cys^7,10]). c[Cys^7,10], the template N/OFQ(1–13)NH₂ and N/OFQ displaced the binding of [³H]N/OFQ to Chinese hamster ovary cells expressing recombinant human NOP (CHO_hNOP) with pK _i values of 9.98, 9.83 and 9.18, respectively. In addition, c[Cys^7,10], N/OFQ(1–13)NH₂ and N/OFQ stimulated the binding of guanosine triphosphate gamma [³⁵S] to CHO_hNOP cells with pEC₅₀/E _max (stimulation factor) of 9.16/5.5, 9.11/4.9 and 8.35/5.5, respectively. c[Cys^7,10], N/OFQ(1–13)NH₂ and N/OFQ inhibited forskolin-stimulated cyclic adenosine monophosphate (cAMP) formation with pEC₅₀ values of 10.08, 10.11 and 9.78, respectively. All ligands produced complete inhibition of cAMP formation. In both functional assays, c[Cys^7,10] was a full agonist. In a series of metabolism experiments, incubation of 1 nM c[Cys^7,10], N/OFQ(1–13)NH₂ and N/OFQ with a rat brain homogenate produced a time-dependent loss of peptide that was greatest for the native peptide N/OFQ. Amidation in N/OFQ(1–13)NH₂ produced some metabolic protection, but this was not significantly improved by further inclusion of c[Cys^7,10]. In summary, c[Cys^7,10] is a high-affinity, high-potency full agonist of the NOP receptor. However, we were unable to demonstrate clear metabolic protection.     

Pharmacological profile of the cyclic nociceptin/orphanin FQ analogues c[Cys<Superscript>10,14</Superscript>]N/OFQ(1–14)NH<Subscript>2</Subscript> and c[Nphe<Superscript>1</Superscript>,Cys<Superscript>10,14</Superscript>]N/OFQ(1–14)NH<Subscript>2</Subscript>

In this study we describe the activity of two cyclic nociceptin/orphanin FQ (N/OFQ) peptides; c[Cys^10,14]N/OFQ(1–14)NH₂ (c[Cys^10,14]) and its [Nphe¹] derivative c[Nphe¹,Cys^10,14]N/OFQ(1–14)NH₂ (c[Nphe¹,Cys^10,14]) in native rat and mouse and recombinant human N/OFQ receptors (NOP). Cyclisation may protect the peptide from metabolic degradation.In competition binding studies of rat, mouse and human NOP the following rank order pK_i was obtained: N/OFQ(1–13)NH₂(reference agonist)>N/OFQ=c[Cys^10,14]>>c[Nphe¹Cys^10,14]. In GTP³⁵S studies of Chinese hamster ovary cells expressing human NOP (CHO_hNOP) c[Cys^10,14] (pEC₅₀ 8.29) and N/OFQ(1–13)NH₂ (pEC₅₀ 8.57) were full agonists whilst c[Nphe¹Cys^10,14] alone was inactive. Following 30 min pre-incubation c[Nphe¹Cys^10,14] competitively antagonised the effects of N/OFQ(1–13)NH₂ with a pA₂ and slope factor of 6.92 and 1.01 respectively. In cAMP assays c[Cys^10,14] (pEC₅₀ 9.29, E_max 102% inhibition of the forskolin stimulated response), N/OFQ(1–13)NH₂ (pEC₅₀ 10.16, E_max 103% inhibition) and c[Nphe¹Cys^10,14] (~80% inhibition at 10 M) displayed agonist activity. In the mouse vas deferens c[Cys^10,14] (pEC₅₀ 6.82, E_max 89% inhibition of electrically evoked contractions) and N/OFQ(1–13)NH₂ (pEC₅₀ 7.47, E_max 93% inhibition) were full agonists whilst c[Nphe¹Cys^10,14] alone was inactive. c[Nphe¹Cys^10,14] (10 M) competitively antagonised the effects of N/OFQ(1–13)NH₂ with a pK_B of 5.66. In a crude attempt to assess metabolic stability, c[Cys^10,14] was incubated with rat brain membranes and then the supernatant assayed for remaining peptide. Following 60 min incubation 64% of the 1 nM added peptide was metabolised (compared with 54% for N/OFQ-NH₂).In summary, we report that c[Cys^10,14] is a full agonist with a small reduction in potency but no improvement in stability whilst c[Nphe¹Cys^10,14] displays tissue (antagonist in the vas deferens) and assay (antagonist in the GTP³⁵S assay and agonist in cAMP assay) dependent activity.Presented in part to The British Pharmacological Society at the Brighton, UK Meeting January 2003     

Autoradiographic analysis of cannabinoid receptor binding and cannabinoid agonist-stimulated [35S]GTPγS binding in morphine-dependent mice

The present study was designed to test the possible existence of changes in brain cannabinoid receptors in morphine-dependent mice. To this end, we compared cannabinoid receptor binding and WIN 55,212-2-stimulated [³⁵S]guanylyl-5′-O-(γ-thio)-triphosphate ([³⁵S]GTPγS) binding in several brain regions of mice chronically exposed to morphine or saline. The existence of opiate dependence in morphine-injected mice was assessed by analyzing the well-known jumping behavior induced by the blockade of opioid receptors with naloxone, whereas these animals were unresponsive to the blockade of cannabinoid receptors with SR141716. The different structures analyzed exhibited similar cannabinoid receptor binding levels in morphine-dependent and control mice, with the only exception of the globus pallidus, which exhibited a very small, but statistically significant, increase. In addition, the activation of cannabinoid receptors with WIN 55,212-2 increased [³⁵S]GTPγS binding in most of the structures examined. The increase was of similar magnitude in morphine-dependent and control mice, except in the substantia nigra, where morphine-dependent mice exhibited lesser [³⁵S]GTPγS binding levels in basal conditions, although a significantly higher WIN 55,212-2-stimulated binding. Other structures, such as the central gray substance, where there was a poor agonist-induced stimulation in control mice, exhibited, however, higher levels of WIN 55,212-2-stimulated [³⁵S]GTPγS binding in morphine-dependent mice, whereas these animals tended to exhibit a higher [³⁵S]GTPγS binding levels in basal conditions, although a lesser and not statistically significant WIN 55,212-2-stimulated binding, in the deep layers of the cerebral cortex. Thus, the data support the potential existence of a specific effect of morphine in the coupling of cannabinoid receptors to GTP-binding proteins, rather than on receptor binding, although this was observed only in the substantia nigra and central gray substance.     

Agonist and antagonist modulation of [35S]-GTPγS binding in transfected CHO cells expressing the neurotensin receptor

1. The functional interaction of the cloned rat neurotensin receptor with intracellular G-proteins was investigated by studying the binding of the radiolabelled guanylyl nucleotide analogue [³⁵S]-GTPγS induced by neurotensin to membranes prepared from transfected Chinese hamster ovary (CHO) cells.
2. The agonist-induced binding of [³⁵S]-GTPγS was only detected in the presence of NaCl in the incubation buffer. However, it was also demonstrated that the binding of [³H]-neurotensin to its receptor was inhibited by NaCl. In the presence of 50 mM NaCl, the binding of the labelled nucleotide was about 2 fold increased by stimulation with saturating concentrations of neurotensin (EC₅₀ value of 2.3±0.9 nM).
3. The stimulation of [³⁵S]-GTPγS binding by neurotensin was mimicked by the stable analogue of neurotensin, JMV-449 (EC₅₀ value of 1.7±0.4 nM) and the neurotensin related peptide neuromedin N (EC₅₀ value of 21±6 nM).
4. The NT-induced [³⁵S]-GTPγS binding was competitively inhibited by SR48692 (pA₂ value of 9.55±0.28), a non-peptide neurotensin receptor antagonist. SR48692 alone had no effect on the specific binding of [³⁵S]-GTPγS.
5. The response to neurotensin was found to be inhibited by the aminosteroid U-73122, a putative inhibitor of phospholipase C-dependent processes, indicating that this drug may act at the G-protein level.
6. Taken together, these results constitute the first characterization of the exchange of guanylyl nucleotides at the G-protein level that is induced by the neuropeptide neurotensin after binding to its receptor.

     

A comparative autoradiographic study of the density of [<Superscript>3</Superscript>H]SR95531, [<Superscript>3</Superscript>H]MK-801 and [<Superscript>3</Superscript>H]cGMP binding in the locus coeruleus and central pontine grey of spontaneously hypertensive and Wistar–Kyoto rats

The Spontaneously Hypertensive rat (SHR) has been previously shown to have a host of neurochemical differences compared with their normotensive counterpart, the Wistar–Kyoto (WKY) rat. Using quantitative receptor autoradiography, the density of GABA_A and NMDA receptors and [³H]cGMP binding within the locus coeruleus (LC) and central pontine grey (CGPn) were compared in the SHR and WKY rat using the radioligands [³H]SR95531, [³H]MK-801 and [³H]cGMP respectively. It was found that [³H]SR95531 binding was significantly greater in both the LC and CGPn of the SHR compared with the WKY rat (unpaired t test; P<0.05). Greater binding densities of [³H]MK-801 and [³H]cGMP were also observed in the LC of the SHR compared with the WKY rat; however, no differences in the binding density of these two ligands were observed in the CGPn. It is suggested that these neurochemical differences within the LC of the SHR may relate to phenotypic differences between SHR and WKY rats that have previously been reported.     

HTLV-1 Infection and Neuropathogenesis in the Context of Rag1<Superscript>-/-</Superscript>γc<Superscript>-/-</Superscript> (RAG1-Hu) and BLT Mice

To date, the lack of a suitable small animal model has hindered our understanding of Human T-cell lymphotropic virus (HTLV)-1 chronic infection and associated neuropathogenesis defined as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The host immune response plays a critical role in the outcome of HTLV-1 infection, which could be better tested in the context of humanized (hu) mice. Thus, we employ here the Balb/c-Rag1^?/?γc^?/? or Rag1 as well as Bone marrow-Liver-Thymic (BLT) mouse models for engraftment of human CD34⁺ hematopoietic stem cells. Flow cytometry and histological analyses confirmed reconstitution of Rag1 and BLT mice with human immune cells. Following HTLV-1 infection, proviral load (PVL) was detected in the blood of Rag-1 and BLT hu-mice as early as 2 weeks post-infection (wpi) with sustained elevation in the subsequent weeks followed by Tax expression. Additionally, infection was compared between adult and neonatal Rag1 mice with both PVL and Tax expression considerably higher in the adult Rag1 mice as compared to the neonates. Establishment of peripheral infection led to lymphocytic infiltration with concomitant Tax expression and resulting myelin disruption within the central nervous system of infected mice. In addition, up-regulation in the expression of several immune checkpoint mediators such as programmed cell death-1 (PD-1), T-cell Ig and ITIM domain (TIGIT), and T cell Ig and mucin domain-3 protein (Tim-3) were observed on CD8⁺ T cells in various organs including the CNS of infected hu-mice. Collectively, these studies represent the first attempt to establish HTLV-1 neuropathogenesis in the context of Rag-1 and BLT hu-mice as potential novel tools for understanding HTLV-1 neuropathogenesis and testing of novel therapies such as immune checkpoint blockade in the amelioration of chronic HTLV-1 infection.     

Identification of a novel snake peptide toxin displaying high affinity and antagonist behaviour for the ��2-adrenoceptors

BACKGROUND AND PURPOSE

Muscarinic and adrenergic G protein-coupled receptors (GPCRs) are the targets of rare peptide toxins isolated from snake or cone snail venoms. We used a screen to identify novel toxins from Dendroaspis angusticeps targeting aminergic GPCRs. These toxins may offer new candidates for the development of new tools and drugs.

EXPERIMENTAL APPROACH

In binding experiments with ³H-rauwolscine, we studied the interactions of green mamba venom fractions with α₂-adrenoceptors from rat brain synaptosomes. We isolated, sequenced and chemically synthesized a novel peptide, ρ-Da1b. This peptide was pharmacologically characterized using binding experiments and functional tests on human α₂-adrenoceptors expressed in mammalian cells.

KEY RESULTS

ρ-Da1b, a 66-amino acid peptide stabilized by four disulphide bridges, belongs to the three-finger-fold peptide family. Its synthetic homologue inhibited 80% of ³H-rauwolscine binding to the three α₂-adrenoceptor subtypes, with an affinity between 14 and 73 nM and Hill slopes close to unity. Functional experiments on α_2A-adrenoceptor demonstrated that ρ-Da1b is an antagonist, shifting adrenaline activation curves to the right. Schild regression revealed slopes of 0.97 and 0.67 and pA₂ values of 5.93 and 5.32 for yohimbine and ρ-Da1b, respectively.

CONCLUSIONS AND IMPLICATIONS

ρ-Da1b is the first toxin identified to specifically interact with α₂-adrenoceptors, extending the list of class A GPCRs sensitive to toxins. Additionally, its affinity and atypical mode of interaction open up the possibility of its use as a new pharmacological tool, in the study of the physiological roles of α₂-adrenoceptor subtypes.     

Binding of [<Superscript>3</Superscript>H]prazosin to α<Subscript>1A</Subscript>- and α<Subscript>1B</Subscript>-adrenoceptors,and to a cimetidine-sensitive non-α<Subscript>1</Subscript> binding site in rat kidney membranes

[(3)H]Prazosin bound to alpha(1A)- and alpha(1B)-adrenoceptors, as well as to a cimetidine-sensitive non-alpha(1)-adrenoceptor binding site in rat kidney membranes. An experimental design is presented where the alpha(1)-adrenoceptors are selectively exposed by blocking the non-alpha(1) binding site with 60 microM cimetidine. Conversely, the non-alpha(1) binding site can be selectively exposed by blocking the alpha(1)-adrenoceptors with 600 nM metitepine. The identity of the non-alpha(1) binding site for [(3)H]prazosin in the rat kidney, herein pharmacologically characterized by 33 competing substances, is still unknown.     

The antipsychotic potential of l-stepholidine—a naturally occurring dopamine receptor D<Subscript>1</Subscript> agonist and D<Subscript>2</Subscript> antagonist

RATIONALE: l-Stepholidine, a dopamine D(2) antagonist with D(1) agonist activity, should in theory control psychosis and treat cognitive symptoms by enhancing cortical dopamine transmission. Though several articles describe its impact on the dopamine system, it has not been systematically evaluated and compared to available antipsychotics. MATERIALS AND METHODS: We examined its in vitro interaction with dopamine D(2) and D(1) receptors and compared its in vivo pharmacokinetic profile to haloperidol (typical) and clozapine (atypical) in animal models predictive of antipsychotic activity. RESULTS: In vitro, l-stepholidine showed significant activity on dopamine receptors, and in vivo, l-stepholidine demonstrated a dose-dependent striatal receptor occupancy (RO) at D(1) and D(2) receptors (D(1) 9-77%, 0.3-30 mg/kg; D(2) 44-94%, 1-30 mg/kg), though it showed a rather rapid decline of D(2) occupancy related to its quick elimination. In tests of antipsychotic efficacy, it was effective in reducing amphetamine- and phencyclidine-induced locomotion as well as conditioned avoidance response, whereas catalepsy and prolactin elevation, the main side effects, appeared only at high D(2)RO (>80%). This preferential therapeutic profile was supported by a preferential immediate early gene (Fos) induction in the nucleus accumbens over dorsolateral striatum. We confirmed its D(1) agonism in vitro, and then using D(2) receptor, knockout mice showed that l-stepholidine shows D(1) agonism in the therapeutic dose range. CONCLUSIONS: Thus, l-stepholidine shows efficacy like an "atypical" antipsychotic in traditional animal models predictive of antipsychotic activity and shows in vitro and in vivo D(1) agonism, and, if its rapid elimination does not limit its actions, it could provide a unique therapeutic approach to schizophrenia.     

Generation and Disease Model Relevance of a Manganese Enhanced Magnetic Resonance Imaging-Based NOD/scid-IL-2R<Emphasis Type="Italic">γ</Emphasis><Subscript>c</Subscript><Superscript><Emphasis Type="Italic">null</Emphasis></Superscript> Mouse Brain Atlas

Strain specific mouse brain magnetic resonance imaging (MRI) atlases provide coordinate space linked anatomical registration. This allows longitudinal quantitative analyses of neuroanatomical volumes and imaging metrics for assessing the role played by aging and disease to the central nervous system. As NOD/scid-IL-2Rγ _c ^null (NSG) mice allow human cell transplantation to study human disease, these animals are used to assess brain morphology. Manganese enhanced MRI (MEMRI) improves contrasts amongst brain components and as such can greatly help identifying a broad number of structures on MRI. To this end, NSG adult mouse brains were imaged in vivo on a 7.0 Tesla MR scanner at an isotropic resolution of 100 μm. A population averaged brain of 19 mice was generated using an iterative alignment algorithm. MEMRI provided sufficient contrast permitting 41 brain structures to be manually labeled. Volumes of 7 humanized mice brain structures were measured by atlas-based segmentation and compared against non-humanized controls. The humanized NSG mice brain volumes were smaller than controls (p?<?0.001). Many brain structures of humanized mice were significantly smaller than controls. We posit that the irradiation and cell grafting involved in the creation of humanized mice were responsible for the morphological differences. Six NSG mice without MnCl₂ administration were scanned with high resolution T₂-weighted MRI and segmented to test broad utility of the atlas.     

The muscarinic M(4) receptor is the functionally predominant subtype in rat and mouse striatum as demonstrated using [(35)S] GTPγS binding

The peroxisome proliferator-activated receptor γ (PPARγ) has important effects on insulin sensitivity, obesity and diabetes. Pioglitazone improves insulin sensitivity by activating PPARγ. In view of inter-individual variability in therapeutic response to pioglitazone, this study was designed to search for an association between type 2 diabetes mellitus and Pro12Ala single-nucleotide polymorphism (SNP) in PPARγ (SNP rs1801282) and to investigate whether these genetic variants affect pioglitazone response in an Iranian population. A total of 101 patients with type 2 diabetes were treated for 12 weeks with pioglitazone (15 mg/day). Paraclinical parameters were measured before and after therapy. We genotyped 128 control participants without diabetes and all patients with type 2 diabetes. The Pro12Ala polymorphism in PPARγ was detected with real-time PCR. The Ala allele was found in 7% of the control participants vs. 3% of those with type 2 diabetes (P=0.04). The genotypic frequencies of Pro/Ala were 14.06% in the former group vs. 5.94% in the latter (P=0.036). There were significant changes in some laboratory values and biochemical markers of insulin sensitivity after pioglitazone therapy. The Pro12Ala polymorphism was associated with significant changes in insulin-to-glucose ratio after treatment (P=0.015 and P=0.005). Our findings suggest that in carriers of the 12Ala variant, pioglitazone significantly reduced the risk of type 2 diabetes, and in diabetic patients with the Pro12Ala genotype, the therapeutic response to treatment was better than in patients with the Pro12Pro genotype, although the difference between groups did not reach statistical significance.     

Pharmacological evidence that tetraethylammonium-sensitive,iberiotoxin-insensitive K<Superscript>+</Superscript> channels function as a negative feedback element for sympathetic neurotransmission by suppressing ω-conotoxin-GVIA-insensitive Ca<Superscript>2+</Superscript> channels in the relaxation of rabbit facial vein

The facial vein isolated from various species relaxes in response to electrical field stimulation (EFS). EFS-elicited relaxation of the facial vein is mediated through the release of noradrenaline (NA) from sympathetic nerve endings and the subsequent activation of smooth muscle beta-adrenoceptors. The release of NA from sympathetic nerve endings in arterial tissues requires transmembrane Ca2+ influx, mediated predominantly by voltage-gated N-type Ca2+ channels. The present pharmaco-mechanical study was undertaken to determine whether the N-type channel is the exclusive pre-junctional Ca2+ channel mediating NA release from sympathetic nerve endings in the rabbit facial vein. Possible roles of K+ channels in the sympathetic neurotransmission were also examined, especially focusing on the contribution of voltage-dependent, Ca2+-activated K+ (BKCa) channels. An isolated ring preparation of the rabbit facial vein exhibited intrinsic myogenic tone which lasted for several hours when stretched. EFS produced frequency-dependent (0.25-2 Hz) relaxation in this preparation. EFS-elicited relaxation was abolished by tetrodotoxin (TTX, 1 microM), guanethidine (5 microM) or propranolol (1 microM), indicating that NA released from sympathetic nerve endings was mediating the relaxant response. NA-mediated neurogenic relaxation was almost eliminated by omega-conotoxin-GVIA (1 microM), an N-type Ca2+ channel blocker. On the other hand, tetraethylammonium (TEA, 2 mM) strongly potentiated EFS-elicited relaxation without affecting the relaxation induced by exogenously applied NA. This potentiation by TEA was not profoundly diminished by omega-conotoxin-GVIA (1 microM) alone or omega-conotoxin-GVIA (1 microM) plus omega-agatoxin IVA (10 nM, P-type channel blocker), but was almost abolished by omega-conotoxin-GVIA (1 microM) plus omega-agatoxin IVA (10 nM) plus omega-conotoxin-MVIIC (3 microM, N-, P- and Q-type channel blocker). The potentiating effect of TEA was not mimicked by iberiotoxin (100 nM) or charybdotoxin (3 microM), both of which block BKCa channels. These findings suggest that pre-junctional N-type Ca2+ channels play the predominant role in the sympathetic nerve transmission in the rabbit facial vein, as in peripheral arterial vascular beds. In addition, Ca2+ channels resistant to 1 microM omega-conotoxin-GVIA, most probably Q-type channels, appear to be present at the sympathetic nerve endings in the rabbit facial vein and contribute substantially to the regulation of NA release from the nerve endings. Prejunctional K+ channels, sensitive to TEA but pharmacologically distinct from iberiotoxin-sensitive BKCa channels, seem to be functionally coupled intimately with the omega-conotoxin-GVIA-resistant Ca2+ channels, and thus function as a negative feedback element in sympathetic neurotransmission in the rabbit facial vein.     

Cannabinoid agonists differentially substitute for the discriminative stimulus effects of Δ<Superscript>9</Superscript>-tetrahydrocannabinol in C57BL/6J mice

RATIONALE: A variety of behavioral procedures have been developed to assess cannabinoid activity in mice; however, the feasibility of establishing Delta(9)-THC as a discriminative stimulus in mice has not been documented. OBJECTIVE: One goal was to establish Delta(9)-THC as a discriminative stimulus in mice; after having done so, another goal was to examine the in vivo mechanism of action of Delta(9)-THC with other cannabinoids and noncannabinoids. MATERIALS AND METHODS: C57BL/6J mice (n = 8) were trained to discriminate Delta(9)-THC (10 mg/kg i.p.) from vehicle while responding under a fixed ratio 30 schedule of food presentation. RESULTS: Mice satisfied the discrimination criteria in 18-98 (median = 67) sessions and the discriminative stimulus effects of Delta(9)-THC were dose-dependent (ED(50) = 2.6 mg/kg). CP 55940 and WIN 55212-2 dose-dependently increased Delta(9)-THC-appropriate responding to 100% (ED(50) = 0.032 and 0.45 mg/kg, respectively), whereas methanandamide and a variety of noncannabinoids (cocaine, ethanol, and ketamine) produced a maximum of 34% Delta(9)-THC-appropriate responding. The cannabinoid CB(1) antagonist SR 141716A (rimonabant) surmountably antagonized the discriminative effects of Delta(9)-THC, CP 55940, and WIN 55212-2; methanandamide did not significantly modify the Delta(9)-THC discriminative stimulus. CONCLUSIONS: The discriminative stimulus effects of Delta(9)-THC, CP 55940, and WIN 55212-2 are mediated by the same (i.e., CB(1)) receptors, whereas the effects of methanandamide or a metabolite of methanandamide are mediated at least in part by non-CB(1) receptors. The discriminative stimulus effects of Delta(9)-THC in mice could be used to evaluate mechanisms of cannabinoid activity with approaches (e.g., inducible knockouts) currently unavailable in nonmurine species.     

Cardioprotective effects of the novel Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger-1 inhibitor KR-32560 in a perfused rat heart model of global ischemia and reperfusion: Involvement of the Akt-GSK-3β cell survival pathway and antioxidant enzyme

To investigate the cardioprotective effects and mechanism of action of KR-32560 {[5-(2-methoxy-5-fluorophenyl)furan-2-ylcarbonyl]guanidine}, a newly synthesized NHE-1 inhibitor, we evaluated the effects of KR-32560 on cardiac function in a rat model of ischemia/reperfusion (I/R)-induced heart injury as well as the role antioxidant enzymes and pro-survival proteins play these observed effects. In isolated rat hearts subjected to 25 min of global ischemia followed by 30 min of reperfusion, KR-32560 (3 and 10 μM) significantly reversed the I/Rinduced decrease in left ventricular developed pressure and increase in left ventricular enddiastolic pressure. In rat hearts reperfused for 30 min, KR-32560 (10 μM) significantly decreased the malondialdehyde content while increasing the activities of both glutathione peroxidase and catalase, two important antioxidant enzymes. Western blotting analysis of left ventricles subjected to I/R showed that KR-32560 significantly increased phosphorylation of both Akt and GSK-3β in a dose-dependent manner, with no effect on the phosphorylation of eNOS. These results suggest that KR-32560 exerts potent cardioprotective effects against I/Rinduced rat heart injury and that its mechanism involves antioxidant enzymes and the Akt-GSK-3β cell survival pathway.     

Biochemical and behavioural effects of isamoltane,a β-adrenoceptor antagonist with affinity for the 5-HT1B receptor of rat brain

Summary The biochemical and behavioural effects of isamoltane, a \-adrenoceptor and 5-HT_1B receptor antagonist that has higher affinity for 5-HT_1B receptors than for 5-HTIA receptors, on 5-HT neurotransmission in the rat brain were examined. In binding experiments isamoltane was found to be about five times more potent as a ligand for the 5-HT_1B receptor than for the 5-HT_1A receptor (K_i values 21 and 112 nmol/l, respectively). Isamoltane increased the K⁺-evoked overflow of ³H from ³H-5-HT loaded slices of rat occipital cortex at 0.1 mol/l, consistent with inhibition of the terminal 5HT autoreceptor. In vivo, isamoltane significantly increased the concentration of 5-hydroxyindoleacetic acid in hypothalamus and hippocampus indicating an increased 5-HT turnover with a maximal effect at 3 mg/kg s.c. A higher dose produced a less pronounced effect. This effect did not seem to be due to the -adrenoceptor blocking action of isamoltane since the -adrenoceptor antagonists, (–)-alprenolol, betaxolol or ICI 118,551 had no significant effects on 5-HT turnover at 5 mg/kg s.c. Isamoltane at 3 mg/kg s.c. induced the wet-dog shake response which was blocked by the tryptophan hydroxylase inhibitor p-chlorophenylalanine. In contrast, the same response induced by the 5-HT₂ receptor agonist quipazine was not blocked by pretreatment with p-chlorophenylalanine. The wet-dog shakes evoked by isamoltane and quipazine were blocked by ritanserin, which indicates that 5-HT₂ receptors are involved in their expression. These observations indicate that isamoltane, by inhibiting the terminal 5-HT autoreceptors, increased the synaptic concentration of 5-HT to a level that induced a behavioural response. Send of offprint requests to S. B. Ross at the above addressThe present results have been presented in part at the Second IUPHAR Satellite Meeting on Serotonin, Basel, Switzerland, July 11–13, 1990     

Efficacy and safety of the novel α<Subscript>4</Subscript>β<Subscript>2</Subscript> neuronal nicotinic receptor partial agonist ABT-089 in adults with attention-deficit/hyperactivity disorder: a randomized,double-blind,placebo-controlled crossover study

Rationale  

α₄β₂ Neuronal nicotinic receptors (NNRs) are implicated in the pathophysiology of attention-deficit/hyperactivity disorder (ADHD).     

Reversal of a cholinergic-induced deficit in a rodent model of recognition memory by the selective 5-HT<Subscript>6</Subscript> receptor antagonist,Ro 04-6790

Rationale Accumulating evidence suggests a potential role for the 5-HT₆ receptor in cognitive function and the potential use of 5-HT₆ receptor antagonists in the treatment of learning and memory disorders.Objectives The aim of the current study was to investigate the effect of the selective 5-HT₆ receptor antagonist, Ro 04-6790, on both the performance of normal adult rats and restoration of a pharmacological disruption of memory function produced by the non-selective muscarinic receptor antagonist, scopolamine, or the dopamine D₂ receptor antagonist, raclopride, in a rodent model of recognition memory.Methods Passive, perceptually based, recognition memory was assessed using a novel object discrimination task. Following habituation to an arena, rats were presented with two identical objects during trial 1 (T₁) and a novel and familiar object during trial 2 (T₂). The time spent exploring the two objects in each trial was measured and novel object discrimination assessed in T₂.Results In the absence of drug all rats spent an equal time exploring the two identical objects in T₁ but more time exploring the novel object in T₂. Scopolamine (but not N-methylscopolamine) and raclopride both produced a dose-dependent reduction in novel object discrimination whilst the 5-HT₆ receptor antagonist, Ro 04-6790, had no effect on discrimination when given alone but completely reversed the scopolamine- but not the raclopride-induced deficit.Conclusion This study demonstrates that acute administration of Ro 04-6790 reverses a cholinergic but not a dopaminergic deficit in a rodent model of recognition memory and provides further support for a role of the 5-HT₆ receptor in the regulation of cognitive function.