Pharmacological characterisation of melatonin mt1 receptor-mediated stimulation of [35S]-GTPγs binding

The activation of G-proteins by melatonin mt₁ receptors was studied by measuring [³⁵S]-guanosine-5′-(3-thiotriphosphate) ([³⁵S]-GTPγS) binding to membranes prepared from Chinese hamster ovary (CHO) cells stably expressing human mt₁ receptors. Melatonin stimulated [³⁵S]-GTPγS binding in a concentration-dependent manner (pEC₅₀, 8.77 ± 0.02). The optimal (212 ± 4%) increase over basal levels of binding (basal = 100%) was observed following incubation of membranes (12.5 μg protein/well) for 120 min at 30° with [³⁵S]-GTPγS (0.1 nM), in the presence of GDP (10 μM), NaCl (100 mM), and MgCl₂ (10 mM). Melatonin analogues stimulated [³⁵S]-GTPγS binding with a rank order (2-iodomelatonin > melatonin = S20098 > GR196429 > 6-chloromelatonin = 6-hydroxymelatonin ≫ N-acetylserotonin ≥ GR135531 = mt₁ luzindole = 5-HT = 0), which was identical to their affinities for the high affinity state of the receptor (correlation coefficient 0.94). All agonists evoked similar maximum increases in [³⁵S]-GTPγS binding. EC₅₀ values were 14- to 63-fold lower than binding affinities. The melatonin receptor antagonist luzindole (0.1–10 μM) evoked a parallel rightward shift in the melatonin concentration-response curve, with a pK_B of 7.19 ± 0.13, which is similar to its affinity in radioligand binding studies for human mt₁ receptors. Stimulation of [³⁵S]-GTPγS binding was abolished by pretreatment of cells with pertussis toxin (18 hr, 100 ng/mL) prior to preparation of membranes. Melatonin was without effect in CHO cells which lacked the mt₁ receptor. Thus, melatonin and melatonin analogues stimulate [³⁵S]-GTPγS binding with a profile which is consistent with binding to mt₁ receptors causing activation of G_i/G_o G-proteins.     

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.

     

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.     

AM630 antagonism of cannabinoid-stimulated [35S]GTPγS binding in the mouse brain

This research was designed to determine the action of the novel aminoalkylindole AM630 (6-iodo-pravadoline) at the cannabinoid receptor by studying its interaction with the cannabinoid receptor agonist WIN 55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate) on guanosine-5′-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding in mouse brain. WIN 55,212-2 stimulated [³⁵S]GTPγS binding, while AM630 had no effect. AM630 antagonized WIN 55,212-2-induced [³⁵S]GTPγS binding and shifted the WIN 55,212-2 dose-response curve to the right. These results clearly demonstrate that AM630 exerts cannabinoid receptor antagonist properties in the brain.     

Subtype-selective mechanisms of negative allosteric modulators binding to group Ⅰ metabotropic glutamate receptors

Group Ⅰ metabotropic glutamate receptors(mGlu1 and mGlu5)are promising targets for multiple psychiatric and neurodegenerative disorders.Understanding the subtyp...     

Characterization of dopaminergic compounds at hD2short, hD4.2 and hD4.7 receptors in agonist-stimulated [35S]GTPγS binding assays

Dopamine receptor agonists and antagonists have been extensively characterized in radioligand binding assays; only a limited number of laboratories have characterized them using a functional assay at multiple receptor subtypes. Experiments were designed to assess four agonists and seven antagonists at three cloned human dopamine receptors using agonist-stimulated [35S]GTPgammaS binding assays in membranes to quantify the initial cellular event following ligand/receptor interaction. In this model there is constitutive G protein activity (agonist-independent [35S]GTPgammaS binding) and potentially constitutive dopamine receptor activity. Thus, discrimination between silent antagonists, partial agonists and inverse agonists is theoretically possible. It was anticipated that distinctions could be made regarding efficacy of the seven receptor antagonists to provide insight regarding the therapeutic use of antipsychotic drugs. In membranes prepared from CHO cells transfected to express high densities of human D2short, D4.2 or D4.7 receptors, the dopamine receptor agonists apomorphine, pergolide, quinelorane and quinpirole produced concentration-dependent increases in agonist-stimulated [35S]GTPgammaS binding. At the hD2short receptor, pergolide and apomorphine were essentially equipotent and more potent than quinelorane and quinpirole; all four agonists displayed similar efficacy at this receptor. At the hD4.2 and the hD4.7 receptors apomorphine was the most potent and pergolide the least efficacious of the four drugs. The ability (both potency and efficacy) of clozapine, haloperidol, olanzapine, quetiapine, risperidone, spiperone and ziprasidone to block apomorphine-stimulated [35S]GTPgammaS binding and alter basal [35S]GTPgammaS binding was also assessed. All of the antagonists inhibited apomorphine-stimulated [35S]GTPgammaS binding with potencies (Kb values) similar to and in rank order consistent with their affinities reported in the literature using radioligand binding assays. Additionally, none of the antagonists altered basal, agonist-independent [35S]GTPgammaS binding, thus they behaved as pure, silent antagonists at D2short, D4.2 and D4.7 receptors under our conditions. In summary, the data suggest that therapeutic distinctions between typical and atypical antipsychotic drugs cannot be made based on their function at D2short, D4.2 and D4.7 subtypes of dopamine receptors.     

(35)S]GTPγS binding and opioid tolerance and efficacy in mouse spinal cord

The present study examined efficacy of a series of opioid agonists and then using chronic in vivo treatment protocols, determined tolerance to opioid agonist stimulated [³⁵S]GTPγS (guanosine 5′-O-(3-[³⁵S] thio)triphosphate) binding in mouse spinal cord membranes and compared it directly to spinal analgesic tolerance. The [³⁵S]GTPγS binding assay was used to estimate efficacy (E_max and τ; Operational Model of Agonism) of a series of opioid agonists for G-protein activation in mouse spinal cord. The rank order of opioid agonist efficacy determined in the [³⁵S]GTPγS assay using the Operational Model and E_max was similar. These efficacy estimates correlated with historical analgesic efficacy estimates. For tolerance studies, mice were continuously treated s.c. for 7 days with morphine, oxycodone, hydromorphone, etorphine or fentanyl and [³⁵S]GTPγS studies were conducted in spinal cord membranes. Other mice were tested in i.t. analgesia dose response studies (tailflick). Tolerance to DAMGO ([D-Ala²,N-MePhe⁴,Gly-ol⁵]enkephalin) or morphine stimulated [³⁵S]GTPγS binding (decrease in E_max) was observed following etorphine and fentanyl treatment only. These treatment protocols downregulate μ-opioid receptor density whereas morphine, oxycodone and hydromorphone do not. Spinal analgesic tolerance was observed following all treatment protocols examined (morphine, oxycodone and etorphine). Opioid antagonist treatment that specifically upregulates (chronic naltrexone) or downregulates (clocinnamox) μ-opioid receptor density produced a corresponding change in opioid agonist stimulated [³⁵S]GTPγS binding. Although receptor downregulation and G-protein uncoupling are among potential mechanisms of opioid tolerance, the present results suggest that uncoupling in mouse spinal cord plays a minor role and that the [³⁵S]GTPγS assay is particularly responsive to changes in μ-opioid receptor density.     

“In vivo” administration of valproate decreases t-[35S]butylbicyclophosphorothionate binding in the rat brain

Summary The effect of the in vivo administration of sodium valproate on t-[³⁵S]butylbicyclophosphorothionate (³⁵S-TBPS) binding measured ex vivo in the rat cerebral cortex was investigated. Sodium valproate produced a decrease of ³⁵S-TBPS binding. The maximal effect (–32%) was reached with the dose of 400 mg/kg i. p., 60 min after the administration of the drug. Saturation experiments revealed that the effect of sodium valproate was due to a decrease in the total number of binding sites with no changes in the affinity constant. A small dose of diazepam (0.5 mg/kg, i. p.), which per se does not modify ³⁵S-TBPS binding, markedly potentiated the inhibitory effect of sodium valproate on ³⁵S-TBPS binding. Moreover, the in vitro addition of sodium valproate to cortical membranes failed to modify ³⁵S-TBPS binding, indicating that the effect of the in vivo administration of this drug is not due to its direct interaction with the chloride associated binding sites. These results strongly suggest that this drug enhances the function of GABAergic synapses at the level of the GABA-coupled chloride channel. This conclusion supports the hypothesis that an enhancement of GABAergic transmission plays a role in the molecular mechanism involved in the antiepileptic action of sodium valporate.     

Estrogen receptors and type 1 metabotropic glutamate receptors are interdependent in protecting cortical neurons against β-amyloid toxicity

We examined the interaction between estrogen receptors (ERs) and type 1 metabotropic glutamate receptors (mGlu1 receptors) in mechanisms of neurodegeneration/neuroprotection using mixed cultures of cortical cells challenged with β-amyloid peptide. Both receptors were present in neurons, whereas only ERα but not mGlu1 receptors were found in astrocytes. Addition of 17β-estradiol (17βE2) protected cultured neurons against amyloid toxicity, and its action was mimicked by the selective ERα agonist, 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) as well as by a cell-impermeable bovine serum albumin conjugate of 17βE2. The selective ERβ agonist, diarylpropionitrile (DPN), was only slightly neuroprotective. The mGlu1/5 receptor agonist, 3,5-dihydroxyphenylglycine (DHPG), was also neuroprotective against amyloid toxicity, and its action was abolished by the mGlu1 receptor antagonist, (3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4-methoxycyclohexyl)-methanone (JNJ 16259685). Neuroprotection by 17βΕ2 or PPT (but not DPN) and DHPG was less than additive, suggesting that ERα and mGlu1 receptors activate the same pathway of cell survival. More important, neuroprotection by 17βΕ2 was abolished not only by the ER antagonist fulvestrant (ICI 182,780) but also by JNJ 16259685, and neuroprotection by DHPG was abolished by ICI 182,780. ERα and mGlu1 receptors were also interdependent in activating the phosphatidylinositol-3-kinase pathway, and pharmacological blockade of this pathway abolished neuroprotection by 17βE2, DHPG, or their combination. These data provide the first evidence that ERα and mGlu1 receptors critically interact in promoting neuroprotection, information that should be taken into account when the impact of estrogen on neurodegeneration associated with central nervous system disorders is examined.     

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.     

Pharmacological characterization of (10bS)-1,2,3,5,6,10b-hexahydropyrrolo[2,1-a]isoquinoline oxalate (YSL-3S) as a new α2-adrenoceptor antagonist

Alpha2-adrenoceptor antagonists, which can enhance synaptic norepinephrine levels by blocking feedback inhibition processes, are potentially useful in the treatment of disease states such as depression, memory impairment, impotence and sexual dysfunction. (10bS)-1,2,3,5,6,10b-Hexahydropyrrolo[2,1-a]isoquinoline oxalate (YSL-3S) was evaluated in several in vitro biological tests to establish its pharmacological profile of activities as an alpha2-adrenoceptor antagonist. Saturation binding assay revealed that [3H]rauwolscine bound to the alpha2-adrenoceptors with a Kd value of 6.3+/-0.5 nM and a Bmax value of 251+/-39 fmol/mg protein in rat cortical synaptic membranes. Competitive binding assay showed that YSL-3S inhibited the binding of [3H]rauwolscine (1 nM) in a concentration-dependent manner with a Ki value of 98.2+/-12.1 nM while it did not inhibit the binding of [3H]cytisine (1.25 nM) to neuronal nicotinic cholinergic receptors. The Ki values of yohimbine, clonidine and norepinephrine for [3H]rauwolscine binding were 15.8+/-1.0, 40.1+/-5.9 and 40.0+/-11.5 nM, respectively. In addition, the binding affinity of YSL-3S for alpha2-adrenoceptors was higher than that of its antipode and the racemic mixture. The functional activity of YSL-3S at the presynaptic alpha2-adrenoceptors was assessed using the prostatic portion of the rat vas deferens. Clonidine inhibited field-stimulated contractions of the vas deference in a dose-dependent manner. The presence of YSL-3S or yohimbine caused a parallel, rightward the dose-response curve of clonidine in a dose-dependent manner, indicating an antagonistic action at the presynaptic alpha2-adrenoceptors. The pA2 values of yohimbine and YSL-3S were 7.66+/-0.13 and 6.64+/-0.18, respectively. The results indicate that YSL-3S acts as a competitive antagonist at presynaptic alpha2-adrenoceptors with a potency approximately ten times lower than yohimbine, but is devoid of binding affinity for neuronal nicotinic cholinergic receptors.     

Pindolol antagonises G-protein activation at both pre- and postsynaptic serotonin 5-HT1A receptors: a [35S]GTPγS autoradiography study

The arylalkylamine, pindolol, may potentiate the clinical actions of antidepressant agents. Although it is thought to act via blockade of 5-HT1A autoreceptors, its efficacy at these sites remains controversial. Herein, we evaluated the actions of pindolol at 5-HT1A autoreceptors and specific populations of postsynaptic 5-HT1A receptors employing [35S]GTPgammaS autoradiography, a measure of receptor-mediated G-protein activation. Both 8-OH-DPAT (1 microM) and 5-HT (10 microM) elicited a pronounced increase in [35S]GTPyS binding in the dorsal raphe nucleus, which contains serotonergic cell bodies bearing 5-HT1A autoreceptors. Pindolol abolished their actions. In the dentate gyrus, lateral septum and entorhinal cortex, structures enriched in postsynaptic 5-HT1A receptors, 8-OH-DPAT (1 microM) and 5-HT (10 microM) also elicited a marked increase in [35S]GTPgammaS binding which was likewise blocked by pindolol. The antagonism of 5-HT-induced [35S]GTPgammaS labelling in the dentate gyrus was shown to be concentration-dependent, yielding a pIC50 of 5.82. Pindolol did not, itself, affect [35S]GTPgammaS binding in any brain region examined. In conclusion, these data suggest that, as characterised by [35S]GTPgammaS autoradiography, and compared with 5-HT and 8-OH-DPAT, pindolol possesses low efficacy at both pre- and postsynaptic 5-HT1A receptors.     

Stereoselective binding of niguldipine enantiomers to α1A-adrenoceptors labeled with [3H]5-methyl-urapidil

[³H]5-Methyl-urapidil, a potent antihypertensive derivative of urapidil, binds to α_1A-adrenoceptors in rat brain cortex membranes with a dissociation constant (K_D) of 0.89 nM and a B_max of 116 fmol/mg protein. The ligand does not bind to purified liver cell membranes (α_1B-adrenoceptors. [³H]5-Methyl-urapidil also labels 5-HT_1A receptors in brain membranes (K_D: 0.84 nM and B_max: 235 fmol/mg protein). (±)-Niguldipine, a novel 1,4-dihydropyridine with Ca²⁺-antagonistic as well as α_1A-adrenoceptor blocking properties, is a competitive inhibitor of [³H]5-methyl-urapidil binding to α_1A-adrenoceptors. In contrast to those for prazosin, the K_i values for niguldipine were highly dependent on the membrane protein concentration, indicating partitioning of niguldipine into hydrophobic compartments unavailable for α-adrenoceptor interaction. The extrapolated, ‘true’ K_i values were as follows: (±)-niguldipine: 0.298 nM, (−)-niguldipine: 3.12 nM, (+)-niguldipine: 0.145 nM.     

Characterisation of human recombinant somatostatin receptors. 2. Modulation of GTPγS binding

G protein activation by somatostatin (somatotropin release inhibiting factor, SRIF), cortistatin (CST) and analogues of these neuropeptides was investigated at human somatostatin receptor subtypes 1-5 (sst1-5) stably expressed in CCL39 Chinese hamster lung fibroblast cells by measuring agonist-stimulated [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) binding. [35S]GTPgammaS binding was assessed in the presence of 100 mM NaCl and 1 microM GDP, although higher Emax and/or pEC50 values may have been obtained under other conditions, but at the expense of lower absolute stimulation or signal/noise ratio. SRIF14 stimulated [35S]GTPgammaS binding to 162, 220, 148 and 266% of control levels via sst2, sst3, sst4 and sst5 receptors, respectively. At sst1 receptors, SRIF14 produced only a limited stimulation (Emax 115%). Hence sst1 receptors were not subjected to further [35S]GTPgammaS binding experiments. [35S]GTPgammaS binding assays were then performed with sst2-5 receptors. Most of the peptide analogues stimulated [35S]GTPgammaS binding in sst2-5 receptor-expressing cells. BIM 23056 behaved as an antagonist on SRIF14-induced [35S]GTPgammaS binding with an apparent pKBs of 6.33 and 5.84 at hsst3 and hsst5 receptors respectively, whereas neither agonism nor antagonism could be shown (at 1 microM) at sst2 or sst4 receptors. The effect at sst5 receptors was not surmountable and needs further investigations. The so-called "antagonist" SA, was devoid of antagonist activity at sst2 or sst3 receptors, whereas it was almost a full agonist at sst4 and sst5 receptor-mediated [35S]GTPgammaS binding. The [35S]GTPgammaS-binding profiles of hsst2-5 receptors were compared with their respective radioligand binding profiles. For sst4 and sst5 receptors, the rank order of affinity of all tested radioligands correlated highly significantly with [35S]GTPgammaS binding (r = 0.814-0.897). At sst3 receptors, [35S]GTPgammaS binding correlated somewhat less with binding profiles obtained with [125I][Tyr10]CST14 and [125I]CGP 23996 than with [125I]LTT-SRIF28 (r = 0.743, 0.757 and 0.882, respectively). At sst2 receptors, [35S]GTPgammaS binding correlated with [125I]LTT-SRIF28, [125I]CGP 23996 and [125I][Tyr3]octreotide binding profiles (r = 0.596-0.699), but not with [125I][Tyr10]CST14 binding. The present [35S]GTPgammaS binding data combined to previous radioligand binding results obtained in cells expressing human SRIF receptors, suggest that at any given receptor, agonists' rank orders of potency (not to mention absolute affinity values which vary profoundly) are not as strictly ordered as may be anticipated. We are investigating these aspects further by analysing additional signalling pathways.     

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.     

Discovery of a series of imidazo[4,5-b]pyridines with dual activity at angiotensin II type 1 receptor and peroxisome proliferator-activated receptor-γ

Mining of an in-house collection of angiotensin II type 1 receptor antagonists to identify compounds with activity at the peroxisome proliferator-activated receptor-γ (PPARγ) revealed a new series of imidazo[4,5-b]pyridines 2 possessing activity at these two receptors. Early availability of the crystal structure of the lead compound 2a bound to the ligand binding domain of human PPARγ confirmed the mode of interaction of this scaffold to the nuclear receptor and assisted in the optimization of PPARγ activity. Among the new compounds, (S)-3-(5-(2-(1H-tetrazol-5-yl)phenyl)-2,3-dihydro-1H-inden-1-yl)-2-ethyl-5-isobutyl-7-methyl-3H-imidazo[4,5-b]pyridine (2l) was identified as a potent angiotensin II type I receptor blocker (IC(50) = 1.6 nM) with partial PPARγ agonism (EC(50) = 212 nM, 31% max) and oral bioavailability in rat. The dual pharmacology of 2l was demonstrated in animal models of hypertension (SHR) and insulin resistance (ZDF rat). In the SHR, 2l was highly efficacious in lowering blood pressure, while robust lowering of glucose and triglycerides was observed in the male ZDF rat.     

Characteristics of the [3H]-yohimbine binding on rat brain α2

Summary The labelling of rat cerebral cortex ₂-adrenoceptors with [³H]-yohimbine ([³H]-YOH) was investigated. At 25° C, binding equilibrium was reached in about 10 min and dissociation occurred with a half time of about 1 min. Saturation experiments gave an equilibrium K _D value of 10.13±1.95 nM and a maximum number of sites of 254±22 fmol/mg protein. The [³H]-YOH binding sites exhibited ₂-adrenergic receptor specificity; the order of potency for the antagonists was rauwolscine > yohimbine prazosin > corynanthine. For the agonists, the order was: oxymetazoline > clonidine > (–)-adrenaline > (–)-noradrenaline (–)-phenylephrine. Agonists exhibited shallow curves in inhibiting [³H]-YOH binding, with pseudo-Hill coefficients (n_H) of less than 1.0. These curves were shifted to lower overall affinity and steepened in the presence of 100 M GTP. Antagonist competition curves were also shallow but GTP had no significant effect.Divalent cations at millimolar concentrations decreased the [³H]-YOH binding: IC₅₀ values were about 6.0, 6.8 and 0.3 mM for Ca²⁺, Mg²⁺ and Mn²⁺ respectively.The maximal number of [³H]-YOH binding sites in the cortex was close to that labelled by the agonist [³H]-paraaminoclonidine ([³H]-PAC). The regional distribution of these sites in the brain, examined at a single concentration of [³H]-YOH and [³H]-PAC, showed a similar pattern except in the striatum. Taken together, the results indicate that like [³H]-PAC, [³H]-YOH labels ₂-adrenoceptors in rat brain cortex. They also show that [³H]-YOH is a useful tool for the study of the high and low affinity sites.     

Intracellular cAMP assay and Eu-GTP-γS binding studies of chimeric opioid peptide YFa

In our previous studies chimeric peptide of Met-enkephalin and FMRFa, YGGFMKKKFMRFamide (YFa), demonstrated concentration dependent κ– and μ–opioid receptor mediated antinociception without tolerance development. To gain further insight of the observed behavior of YFa, the present study was undertaken. The effect of chimeric peptide on forskolin-stimulated cAMP formation under acute and chronic treatment and stimulation of Eu-GTP-γS binding in CHO cells stably expressing κ– and μ–opioid receptors was assessed. YFa showed concentration dependent inhibition of forskolin-stimulated cAMP in both hKOR and hMOR-CHO cells; however, the inhibition at 1 nM was significantly higher in hKOR cells and comparable to DynA (1–13) than that shown at 20 nM in hMOR cells. Chronic treatment of YFa, similar to DynA (1–13), did not show significant change in forskolin-stimulated cAMP level in both hKOR and hMOR cells. However, chronic treatment of morphine and DAMGO showed an increase in forskolin-stimulated cAMP level in hMOR-CHO cells indicating superactivation of adenylyl cyclase. Eu-GTP-γS binding studies of YFa showed a concentration dependent adherent binding with κ– and μ–opioid receptors; however, the latter demonstrated significant binding at higher concentration. Thus the study indicates the chimeric opioid peptide YFa as a potent κ- receptor specific antinociceptive moiety, showing no tolerance and hence may serve as a lead in understanding the mechanism of tolerance development, antinociception and its modulation.