Rat Cerebral Cortical Membranes Research Articles

Functional activation of Gαq/11 protein via α1 -adrenoceptor in rat cerebral cortical membranes.

Although it is recognized that α1 -adrenoceptors are coupled to diverse intracellular signalling pathways, its primary transduction mechanisms are evoked by activating phospholipase C in the cell membrane through Gαq/11 , resulting in production of inositol 1,4,5-trisphosphate and diacylglycerol. However, there have been few studies that indicate directly the involvement of Gαq/11 proteins in this signalling pathway in the central nervous system. In the current study, we tried to pharmacologically characterize (-)-adrenaline-stimulated [35 S]GTPγS binding to Gαq/11 in rat brain membranes. Functional activation of Gαq/11 coupled to α1 -adrenoceptor was investigated by using [35 S]GTPγS binding/immunoprecipitation assay in the membranes prepared from rat cerebral cortex, hippocampus, and striatum. The specific [35 S]GTPγS binding to Gαq/11 was stimulated by (-)-adrenaline in a concentration-dependent and saturable manner in rat cerebral cortical membranes. In hippocampal or striatal membranes, the stimulatory effects of (-)-adrenaline were scarce. The effect of (-)-adrenaline was potently inhibited by prazosin, a potent and selective α1 -adrenoceptor antagonist, but not by yohimbine, a selective α2 -adrenoceptor antagonist. The response was mimicked by cirazoline, but not by R(-)-phenylephrine. Although oxymetazoline also stimulated the specific [35 S]GTPγS binding to Gαq/11 as an apparent "super-agonist", detailed pharmacological characterization revealed that its agonistic properties in this experimental system were derived from off-target effects on 5-HT2A receptors, but not via α1 -adrenoceptors. In conclusion, functional coupling of α1 -adrenoceptors to Gαq/11 proteins are detectable in rat brain membranes by means of [35 S]GTPγS binding/immunoprecipitation assay. It is necessary to interpret the experimental data with caution when oxymetazoline is included as an agonist at α1 -adrenoceptors.

Dopamine-induced functional activation of Gαq mediated by dopamine D1-like receptor in rat cerebral cortical membranes

Although multiple roles of dopamine through D1-like (D1 and D5) and D2-like (D2, D3, and D4) receptors are initiated primarily through stimulation or inhibition of adenylyl cyclase via Gs/olf or Gi/o, respectively, there have been many reports indicating diverse signaling mechanisms that involve alternative G protein coupling. In this study, dopamine-induced Gαq activation in rat brain membranes was investigated. Agonist-induced Gαq activation was assessed by increase in guanosine-5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding to Gαq determined by [35S]GTPγS binding/immunoprecipitation assay in rat brain membranes. Dopamine-stimulated Gαq functionality was highest in cortex as compared to hippocampus or striatum. In cerebral cortical membranes, this effect was mimicked by benzazepine derivatives with agonist properties at dopamine D1-like receptors, that is, SKF83959, SKF83822, R(+)-SKF81297, R(+)-SKF38393, and SKF82958, but not by the compounds with dopamine D2-like receptor agonist properties except for aripiprazole. Against expectation, stimulatory effects were also induced by SKF83566, R(+)-SCH23390, and pergolide. The pharmacological profiling by using a series of antagonists indicated that dopamine-induced response was mediated through dopamine D1-like receptor, which was distinct from the receptor involved in 5-HT-induced response (5-HT2A receptor). Conversely, the responses induced by SKF83566, R(+)-SCH23390, and pergolide were most likely mediated by 5-HT2A receptor, but not by dopamine D1-like receptor. Caution should be paid when interpreting the experimental data, especially in behavioral pharmacological research, in which SKF83566 or R(+)-SCH23390 is used as a standard selective dopamine D1-like receptor antagonist. Also, possible clinical implications of the agonistic effects of pergolide on 5-HT2A receptor has been mentioned.

Adenosine A1 receptors are selectively coupled to Gαi-3 in postmortem human brain cortex: Guanosine-5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding/immunoprecipitation study

By means of guanosine-5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding assay combined with immunoprecipitation using anti-Gα subunit antibody, we recently reported 5-HT2A receptor- and M1 muscarinic acetylcholine receptor-mediated Gαq activation in rat cerebral cortical membranes (Odagaki et al., 2014). In the present study, this method has been applied to postmortem human brains, with focusing on adenosine receptor-mediated G-protein activation. In the exploratory experiments using a series of agonists and the antibodies specific to each Gα subtypes in the presence of low (10nM) or high (50μM) concentration of GDP, the most prominent increases in specific [35S]GTPγS binding in the membranes prepared from human prefrontal cortex were obtained for the combinations of adenosine (1mM)/anti-Gαi-3 in the presence of 50μM GDP as well as 5-HT (100μM)/anti-Gαq and carbachol (1mM)/anti-Gαq in the presence of 10nM GDP. Adenosine-induced activation of Gαi-3 emerged only when GDP concentrations were increased higher than 10μM, and the following experiments were performed in the presence of 300μM GDP. Adenosine increased specific [35S]GTPγS binding to Gαi-3 in a concentration-dependent manner to 251.4% of the basal unstimulated binding, with an EC50 of 1.77μM. The involvement of adenosine A1 receptor was verified by the experiments using selective agonists and antagonists at adenosine A1 or A3 receptor. Among the α subunits of Gi/o class (Gαi-1, Gαi-2, Gαi-3, and Gαo.), only Gαi-3 was activated by 1mM adenosine, indicating that human brain adenosine A1 receptor is coupled preferentially, if not exclusively, to Gαi-3.

Functional activation of Gαq via serotonin2A (5-HT2A) and muscarinic acetylcholine M1 receptors assessed by guanosine-5׳-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding/immunoprecipitation in rat brain membranes

Functional coupling between serotonin2A (5-HT2A) receptors and Gαq proteins in native brain membranes has been sparsely reported thus far. In the present study, the guanosine-5׳-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding assay combined with immunoprecipitation using magnetic beads (Dynabeads Protein A) coated with anti-Gαq antibody was developed. Under experimental conditions optimised for assay constituents (GDP, MgCl2, and NaCl), for contents of membrane protein, anti-Gαq antibody, and Dynabeads Protein A, and for the incubation period, 5-HT stimulated specific [35S]GTPγS binding to Gαq in rat cerebral cortical membranes in a concentration-dependent and saturable manner, with a signal/noise ratio that was sufficiently high for further detailed pharmacological characterisation. This characterisation revealed an involvement of 5-HT2A receptors. Activation of Gαq proteins was also detectable by the addition of carbachol via muscarinic acetylcholine M1 receptors, (-)-epinephrine, and dopamine, but not by L-glutamate or (±)-baclofen. When 5-HT2A receptors and M1 receptors were stimulated simultaneously, there were non-additive effects, indicating that the two receptors were coupled to the same components of Gαq proteins in the rat cerebral cortex. This method will serve as an efficacious strategy for neurobiological investigations aimed at elucidating the physiological and pathological implications of signal transduction systems mediated via Gαq proteins coupled with 5-HT2A receptors and muscarinic acetylcholine M1 receptors.

Activation of Gq Proteins Coupled with 5-HT2 Receptors in Rat Cerebral Cortical Membranes Assessed by Antibody-Capture Scintillation Proximity Assay/[35S]GTPγS Binding

Background/Aims: Functional activation of Gq coupled with 5-HT₂ receptors was investigated in rat cerebral cortical membranes. Methods: Antibody-capture scintillation proximity assay (SPA)/[³⁵S]GTPγS binding with anti-Gα_q antibody was performed. Results: The specific [³⁵S]GTPγS binding to Gα_q was increased by 5-hydroxytryptamine (5-HT) in a concentration-dependent but unsaturable manner. The increase elicited by micromolar concentrations of 5-HT was inhibited completely by ketanserin, whereas it inhibited the response by submillimolar to millimolar concentrations of 5-HT only partially. Analysis of the concentration-dependent increases by 5-HT in the absence and presence of ketanserin, methiothepin, WAY100635, and pirenzepine clearly indicates that there are two distinct components of 5-HT-stimulated [³⁵S]GTPγS binding, one of which is a pharmacologically relevant increase elicited by lower concentrations (-30 μmol/l) of 5-HT mediated through 5-HT₂ receptors and the other is pharmacologically undefined stimulation by higher concentrations of 5-HT. When 5-HT and carbachol were added simultaneously, there was apparently lack of additivity. Conclusion: It is concluded that by means of antibody-capture SPA/[³⁵S]GTPγS binding it is possible to detect two distinct components of 5-HT-elicited activation of Gq shared by M₁ muscarinic receptors, one of which is mediated through 5-HT₂ receptors and the other is derived from unknown origin in rat cerebral cortical membranes.

Pharmacological characterization of M1 muscarinic acetylcholine receptor-mediated Gq activation in rat cerebral cortical and hippocampal membranes

This study aimed to pharmacologically characterize the response derived from functional activation of Gq proteins coupled with native muscarinic acetylcholine receptors in rat cerebral cortex and hippocampus. Rat cerebral cortical and hippocampal membranes were prepared, and the effects of a range of mAChR agonists and antagonists, allosteric modulators, and muscarinic toxins were determined by an antibody-capture scintillation proximity assay combined with [(35)S]GTPγS binding, using the anti-Gαq antibody sc-393. Increased specific [(35)S]GTPγS binding, elicited by carbachol (CCh), was selectively inhibited by the muscarinic toxin MT7, and was resistant to membrane pretreatment with N-ethylmaleimide, indicating that the response derived exclusively from Gαq, selectively coupled with the M1 mAChR. In addition to CCh, many mAChR agonists, including oxotremorine, arecholine, and methacholine, stimulated binding in a concentration-dependent manner with varied potencies and efficacies. The intrinsic activities of partial M1 mAChR agonists in the present study were generally lower than previously reported in M1-expressing cells. Xanomeline and N-desmethylclozapine had negligible or minimal agonist properties. CCh-stimulated [(35)S]GTPγS binding to Gαq was inhibited by mAChR antagonists, including scopolamine, ipratropium, atropine, 4-DAMP, pirenzepine, and AF-DX 116, with a rank order of potency consistent with previous studies of M1-expressing cells. There was a highly significant correlation between the potencies of 13 agonists and 19 antagonists in the cerebral cortex and hippocampus. The effects of several allosteric mAChR modulators were also investigated. These data provide a comprehensive pharmacological profile of the Gq-coupled M1 mAChR subtype natively expressed at physiological levels in rat cerebral cortex and hippocampus.

Functional Coupling between Metabotropic Glutamate Receptors and G‐Proteins in Rat Cerebral Cortex Assessed by Guanosine‐5′‐O‐(3‐[35S]thio)triphosphate Binding Assay

Stimulation of specific guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding by l-glutamate was pharmacologically characterized in rat cerebral cortical membranes. Optimization of the experimental conditions with respect to the concentrations of GDP, MgCl(2) and NaCl in assay buffer prompted us to adopt the incubation of rat cerebral cortical membranes with 0.2 nM [(35)S]GTPγS at 30°C for 60 min. in the presence of 20 μM GDP, 5 mM MgCl(2) and 100 mM NaCl as a standard condition. Specific [(35)S]GTPγS binding was stimulated by l-glutamate in a concentration-dependent manner but not by ionotropic glutamate receptor agonists. The stimulatory responses were also elicited by many agonists for metabotropic glutamate (mGlu) receptor, with (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268) being the most potent. l-glutamate-stimulated [(35)S]GTPγS binding was inhibited by several mGlu antagonists, with (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495) being the most potent. The pharmacological properties of a series of agonists and antagonists indicated the involvement of group II mGlu receptors, especially mGlu2. Supportive of this notion was the finding that l-glutamate-stimulated specific [(35)S]GTPγS binding was augmented by 2,2,2-trifluoro-N-[4-(2-methoxyphenoxy)phenyl]-N-(3-pyridinylmethyl)ethanesulphonamide hydrochloride (LY487379), a reportedly selective allosteric positive modulator for mGlu2, by means of upward and leftward shift of the concentration-response curve. In addition, LY487379 per se stimulated [(35)S]GTPγS binding, though, through a mechanism different from the stimulation by l-glutamate. Pre-treatment of the membranes with N-ethylmaleimide (NEM) cancelled l-glutamate-stimulated [(35)S]GTPγS binding in a concentration- and incubation time-dependent manner. Taken altogether, l-glutamate-stimulated [(35)S]GTPγS binding serves as a useful functional assay for the activation of NEM-sensitive G(i/o) -mediated group II mGlu receptors in rat cerebral cortical membranes.

Alpha-adrenoceptor interaction of tetrandrine and isotetrandrine in the rat: functional and binding assays.

The action of 1S,1'S-tetrandrine, a bisbenzyltetrahydroisoquinoline alkaloid, on alpha1-adrenoceptors has been compared with that of its isomer 1R,1'S-isotetrandrine. The work includes binding assays to analyse the affinity of these products for the [3H]prazosin binding site of rat cerebral cortical membranes and functional studies on rat isolated aorta to examine the effects of both alkaloids on intracellular calcium processes related or not to alpha-adrenoceptor activation. A radioligand receptor-binding study showed that both compounds interacted with the alpha1-adrenoceptors displacing [3H]prazosin from the specific binding site. The Ki values (inhibition constants) were 0.69+/-0.12 and 1.6+/-0.4 microM for tetrandrine and isotetrandrine, respectively. The functional studies showed that both alkaloids concentration-dependently inhibited noradrenaline-induced contraction in Ca2+-free solution (IC50 values, i.e. the concentrations needed to induce 50% inhibition, were 252.8 and 174.9 microM for tetrandrine and isotetrandrine, respectively), the spontaneous contractile response elicited by extracellular calcium after depletion of noradrenaline-sensitive intracellular stores (increase in resting tone; IC50 values 11.6 and 19.6 microM for tetrandrine and isotetrandrine, respectively) and the refilling of intracellular Ca2+ stores sensitive to noradrenaline (IC50 values 7.4 and 14.9 microM for tetrandrine and isotetrandrine, respectively). The results show that tetrandrine and isotetrandrine interact with alpha1-adrenoceptors by displacing the [3H]prazosin binding site and that both compounds inhibit mainly the Ca2+-dependent process and have less action on alpha1-adrenoceptors. Tetrandrine is more potent than isotetrandrine.

Pharmacological characterization of α 2D-adrenergic receptor-mediated [ 35S]GTPγS binding in rat cerebral cortical membranes

Using the rat cerebral cortical membranes that had once been frozen and stored at −80 °C, the stimulation of specific guanosine-5′- O-(3-[ 35S]thio)triphosphate ([ 35S]GTPγS) binding mediated through α 2-adrenergic receptors (α 2-ARs) was pharmacologically characterized. The stimulatory effects of (−)-noepinephrine ((−)-NE) and 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK-14,304) at maximally effective concentrations were prominent only in the presence of micromolar to submillimolar levels of GDP, with the greatest signal/noise ratio achieved at 100 and 30–100 μM GDP for (−)-NE and UK-14,304, respectively. The α 2-AR-mediated [ 35S]GTPγS binding was also critically dependent on the presence of millimolar concentrations of MgCl 2 in assay medium. The maximum stimulation induced by UK-14,304 was equivalent to, or even greater than, that of an endogenous ligand (−)-NE at lower concentrations of NaCl, while it became a partial agonist with higher concentrations of NaCl. Concentration–response curves for several agonists revealed that the omission of NaCl from incubation buffer generally shifted the curves leftward and increased the relative efficacies as compared to the endogenous ligand. The pharmacological profile characterized with a series of adrenergic agonists and antagonists indicated that this response was derived from activation of G proteins coupled with α 2-ARs, in particular α 2D-AR subtype, though the possible involvement of α 2C-ARs was not completely excluded. However, the stimulatory effects of several adrenergic compounds, such as rauwolscine and yohimbine, were ascribed to their agonist properties at 5-HT 1A receptors. This method serves as a simple but useful strategy for investigating the functional interaction between α 2D-ARs and their coupled G proteins in rat native cerebral cortical membranes, especially in the field of psychopharmacology and biological psychiatry.

Thiocolchicoside inhibits the activity of various subtypes of recombinant GABA A receptors expressed in Xenopus laevis oocytes

Thiocolchicoside is a myorelaxant drug with anti-inflammatory and analgesic properties as well as pronounced convulsant activity. To characterize the mechanisms of action of this drug at the molecular level, we examined its effects on the function of various recombinant neurotransmitter receptors expressed in Xenopus oocytes. Electrophysiological recordings from recombinant human γ-aminobutyric acid type A (GABA A) receptors consisting of α1β1γ2L, α1β2γ2L, or α2β2γ2L subunit combinations revealed that thiocolchicoside inhibited GABA-evoked Cl − currents with similar potencies (median inhibitory concentrations of 0.13 to 0.2 μM) and in a competitive manner. Consistent with previous observations, thiocolchicoside also inhibited the binding of GABA to rat cerebral cortical membranes. Thiocolchicoside inhibited the function of recombinant human strychnine-sensitive glycine receptors composed of the α1 subunit with a potency (median inhibitory concentration of 47 μM) lower than that apparent with recombinant GABA A receptors. It also inhibited the function of human nicotinic acetylcholine receptors composed of the α4 and β2 subunits, but this effect was only partial and apparent at high concentrations. In contrast, thiocolchicoside had no effect on the function of 5-HT 3A serotonin receptors. Our results thus provide molecular evidence that the epileptogenic activity of thiocolchicoside might be due to inhibition of the function of inhibitory receptors in the central nervous system, especially that of GABA A receptors.

Increased levels of pregnenolone and its neuroactive metabolite allopregnanolone in autopsied brain tissue from cirrhotic patients who died in hepatic coma

It has been suggested that neurosteroids with agonist properties at the central GABA-A receptor are implicated in the pathogenesis of hepatic encephalopathy (HE) in chronic liver disease. In order to address this issue, gas chromatography/mass spectrometry was used to measure the neurosteroids pregnenolone, allopregnanolone, and tetrahydrodeoxycorticosterone (THDOC) in postmortem brain tissue from controls, cirrhotic patients who died without HE, a patient who died in uremic coma, and cirrhotic patients who died in hepatic coma. Exposure of rat cerebral cortical membranes to brain extracts from hepatic coma patients resulted in a 53% ( p < 0.001) increase in binding of [ 3H]muscimol, a GABA-A receptor ligand. Subsequent GC/MS analysis showed that concentrations of the GABA-A receptor agonist neurosteroid allopregnanolone were significantly increased in brain tissue from hepatic coma patients compared to patients without HE or controls ( p < 0.001). Brain allopregnanolone concentrations were significantly correlated with the magnitude of induction of [ 3H]muscimol binding ( r 2 = 0.82, p < 0.0001). Concentrations of allopregnanolone comparable to those observed in hepatic coma brains are pathophysiologically relevant. Concentrations of the neurosteroid precursor pregnenolone were also increased in brain tissue from hepatic coma patients, while those of a second neurosteroid THDOC were below the levels of detection in all groups. Brain concentrations of benzodiazepine receptor ligands estimated by radioreceptor assay were not significantly increased in cirrhotic patients with or without hepatic coma. These findings suggest that increased levels of allopregnanolone rather than “endogenous benzodiazepines” offer a cogent explanation for the phenomenon of “increased GABAergic tone” previously proposed in HE.

5-HT 1A receptor-mediated G protein activation assessed by [ 35S]GTPγS binding in rat cerebral cortex

To date, 5-hydroxytryptamine 1A (5-HT 1A) receptor-mediated functional assays (adenylyl cyclase inhibition, high-affinity GTPase activity and [ 35S]guanosine-5′- O-(γ-thio)-triphosphate ([ 35S]GTPγS) binding) have been performed mainly in hippocampal membranes. In the current study, 5-HT-stimulated G protein activation assays were carried out in rat cerebral cortical membranes. High-affinity GTPase activity was stimulated by 5-HT, but not by 8-hydroxy-2-(di- n-propylamino)tetralin (8-OH-DPAT). By contrast, 5-HT- and 8-OH-DPAT-stimulated [ 35S]GTPγS binding displayed sufficient dynamic range enough to warrant further pharmacological analysis. Under standard conditions, which were determined precisely in terms of the concentrations of GDP, MgCl 2 and NaCl, the profile of 5-HT-stimulated [ 35S]GTPγS binding investigated using a series of 5-HT receptor agonists and antagonists clearly indicated the involvement of the 5-HT 1A receptor subtype. There appeared to be no evidence supporting the presence of regional heterogeneity in coupling efficiency between 5-HT 1A and G proteins in the hippocampus or cortex. This method is a useful tool for investigating functional coupling between postsynaptic 5-HT 1A receptors and G proteins in cerebral cortical membranes.

8-NH2-Boldine, an Antagonist of α1Aand α1BAdrenoceptors without Affinity for the α1DSubtype: Structural Requirements for Aporphines at α1-Adrenoceptor Subtypes

Structure-activity analysis of 21 aporphine derivatives was performed by examining their affinities for cloned human alpha (1A), alpha (1B) and alpha (1D) adrenoceptors (AR) using membranes prepared from rat-1 fibroblasts stably expressing each alpha (1)-AR subtype. All the compounds tested competed for [ (125)I]-HEAT binding with steep and monophasic curves. The most interesting compound was 8-NH (2)-boldine, which retains the selective affinity for alpha(1A)-AR (pKi = 6.37 +/- 0.21) vs. alpha(1B)-AR (pKi = 5.53 +/- 0.11) exhibited by 1,2,9,10-tetraoxygenated aporphines, but shows low affinity for alpha(1D)-AR (pKi < 2.5). Binding studies on native adrenoceptors present in rat cerebral cortex confirms the results obtained for human cloned alpha (1)-AR subtypes. The compounds selective for the alpha (1A) subtype discriminate two binding sites in rat cerebral cortex confirming a mixed population of alpha (1A)- and alpha (1B)-AR in this tissue. All compounds are more selective as inhibitors of [ (3)H]-prazosin binding than of [ (3)H]-diltiazem binding to rat cerebral cortical membranes. A close relationship was found between affinities obtained for cloned alpha (1A)-AR and inhibitory potencies on noradrenaline-induced contraction or inositol phosphate accumulation in tail artery, confirming that there is a homogeneous functional population of alpha(1A)-AR in this vessel. On the contrary, a poor correlation seems to exist between the affinity of 8-NH (2)-boldine for cloned alpha (1D)-AR and its potency as an inhibitor of noradrenaline-induced contraction or inositol phosphate accumulation in rat aorta, which confirms that a heterogeneous population of alpha (1)-AR mediates the adrenergic response in this vessel.

Ethyl 2-(4-bromophenyl)-1-(2,4-dichlorophenyl)-1H-4-imidazolecarboxylate is a novel positive modulator of GABAA receptors

Ethyl 2-(4-bromophenyl)-1-(2,4-dichlorophenyl)-1H-4-imidazolecarboxylate (TG41) enhanced the binding both of γ-aminobutyric acid (GABA) and of flunitrazepam to rat cerebral cortical membranes. Electrophysiological recordings from Xenopus oocytes expressing various recombinant GABAA receptor subtypes revealed that TG41 enhanced the function of all receptor subunit combinations tested. The potency of TG41 at receptors containing α1, β2, and γ2L subunits was greater than that of alphaxalone, etomidate, propofol, or pentobarbital. The potency of TG41 was also greater at receptors containing α1 or α2 subunits than at those containing α4 and it was markedly higher at receptors containing β2 or β3 subunits than at those containing β1. This drug induced a reversible loss of the righting reflex in Xenopus tadpoles and it elicited hypnosis (5 mg/kg) after intravenous administration in rats. These results indicate that the pharmacological profile of TG41 is similar to that of general anesthetics which potentiate the activity of GABAA receptors containing the β2 or β3 subunit.

Imidazole H3-antagonists: relationship between structure and ex vivo binding to rat brain H3-receptors.

H3-antagonists possess promising pharmacological effects on awakening, learning and memory, but few data on their access to the central nervous system (CNS) have been reported so far. The purpose of this work was to investigate the relationships between structure and brain penetration of a series of H3-antagonists, using ex vivo binding experiments in rats. H3-antagonists belonging to different chemical classes but all having an imidazole ring, an alkyl spacer, a polar fragment and a lipophilic ending group, were selected among the numerous H3-antagonists recently described by us. Ex vivo binding studies were performed by inhibiting specific [3H]-(R)-alpha-methylhistamine ([3H]-RAMHA) binding to rat cerebral cortical membranes following H3-antagonist peripheral administration. Ionization constants and partition coefficients in n-octanol/water and 1,2-dichloroethane/water were determined by the potentiometric pH-metric method and were compared to the ex vivo binding potencies to analyse structure-property relationships (SPR). In the ex vivo assay, the H3-antagonists showed different potencies (pED50) not correlated to their in vitro H3-receptor binding affinities (pKi). Compound 4a, having a benzothiazol-2-yl-thioethyl chain, showed high ex vivo potency (ED50=1.35 mg kg(-1) i.p.) and a fast brain penetration, eliciting maximal displacement of [3H]-RAMHA already 5 min after i.v. or i.p. administration. Ex vivo binding assays of three compounds, following i.v. and i.p. administration, showed that the observed i.p. ex vivo potencies were not significantly affected by biotransformation. Within the set of compounds, those having a better ability to reach the CNS had a logDoct(7.4) in the range 2-3.5, and a DeltalogPoct-dce < 2. The combined use of two easily measurable physicochemical descriptors, namely logDoct(7.4) (apparent lipophilicity at pH 7.4) and DeltalogPoct-dce (a descriptor of H-bond donor capacity) allowed to model brain permeation of the majority of the compounds examined.

Vascular activity of (-)-anonaine, (-)-roemerine and (-)-pukateine, three natural 6a(R)-1,2-methylenedioxyaporphines with different affinities for alpha1-adrenoceptor subtypes.

We have studied the mechanism of action of three 6a( R)-1,2-methylenedioxyaporphines as vasorelaxant compounds. The alkaloids assayed showed different affinities for the three human cloned alpha (1)-adrenoceptor (AR) subtypes stably expressed in rat-1 fibroblasts, showing lower affinity for alpha(1B)-AR with regard to the alpha(1A)- or alpha(1D)-subtypes. These three natural compounds are more potent inhibitors of [ (3)H]-prazosin binding than of [ (3)H]-diltiazem binding to rat cerebral cortical membranes. As all these alkaloids inhibited noradrenaline (NA)-induced [ (3)H]-inositol phosphate formation in cerebral cortex and rat tail artery, they may be safely viewed as alpha (1)-AR antagonists, as is demonstrated by the vasorelaxant responses observed in isolated rat tail artery and/or aorta precontracted with NA. The alkaloids also inhibited the contractile response evoked by KCl (80 mM) but with a lower potency than that shown against NA-induced contraction. We have also examined their ability to inhibit the different forms of cyclic nucleotide phosphodiesterases (PDE) isolated from bovine aortic smooth muscle and endothelial cells, with negative results. We conclude that N-methylation favours the interaction of (R)-aporphines with all alpha (1)-AR subtypes, and that the topography of the binding site recognizing the basic or protonated nitrogen atom is similar in all three alpha (1)-AR subtypes. The presence of a hydroxy group at C-11 has different effects on the affinity for each alpha (1)-AR subtype but decreases the affinity for Ca (2+) channels. These results confirm and extend the view that subtle changes in the hydroxylation patterns on the aromatic ring of the aporphine structure affect the interactions of these compounds with the three alpha (1)-AR subtypes in different ways, suggesting that the binding site recognizing the aporphine skeleton is different in each of the three subtypes.

ALpha1-adrenoceptor antagonist properties of CGP 12177A and other beta-adrenoceptor ligands: evidence against beta(3)- or atypical beta-adrenoceptors in rat aorta.

1. The alpha(1)-adrenoceptor antagonist properties of the beta-adrenoceptor nonconventional partial agonist, CGP 12177A, was investigated in functional assays in rat aorta and in radioligand binding assays in rat cerebral cortical membranes. In addition, binding affinities of other beta-adrenoceptor ligands were measured to investigate any correlation between alpha(1)-adrenoceptor affinity and relaxant potency in phenylephrine-constricted rings. 2. In functional studies, CGP 12177A produced parallel rightward shifts of the phenylephrine CRC with no reduction in the maximum responses. Schild regression analysis gave a straight line with a slope of 0.95 (95% CL: 0.87-1.04), suggesting reversible competitive antagonism, and gave a pK(B) value of 5.26. In contrast, CGP 12177A (<or=300 microm) had no effect on contraction induced by the thromboxane-mimetic, U46619. 3. In binding studies, CGP 12177A competed monophasically with [(3)H]prazosin binding (Hill slope, 0.95, 95% CL: 0.76-1.13), giving a pK(i) value of 5.48, in good agreement with the pK(B) from functional studies. 4. Competition experiments with various other beta-adrenoceptor ligands showed that they all displaced [(3)H]prazosin in a manner consistent with one-site competition. pK(i) values were as follows: SR 59230A, 6.25; cyanopindolol, 6.33; bupranolol, 6.35; alprenolol, 5.90; propranolol, 5.80; BRL 37344, 5.50; ICI 118551, 5.55; CGP 20712A, 5.26. The pK(i) values correlated well with the pEC(50) values for relaxation of phenylephrine-constricted rat aorta obtained previously (r(2)=0.984, P<0.0001). 5. In conclusion, relaxant effects of CGP 12177A and other beta-adrenoceptor ligands in phenylephrine-constricted rat aorta can be attributed to alpha(1)-adrenoceptor blockade and are unrelated to effects at beta(3)-adrenoceptors or atypical beta-adrenoceptors.

Effect of thuringiensin on adenylate cyclase in rat cerebral cortex

The purpose of this work is to evaluate the effect of thuringiensin on the adenylate cyclase activity in rat cerebral cortex. The cyclic adenosine 3′5′-monophosphate (cAMP) levels were shown to be dose-dependently elevated 17–450% or 54–377% by thuringiensin at concentrations of 10 μM–100 mM or 0.5–4 mM, due to the activation of basal adenylate cyclase activity of rat cerebral cortical membrane preparation. Thuringiensin also activated basal activity of a commercial adenylate cyclase from Escherichia coli. However, the forskolin-stimulated adenylate cyclase activity in rat cerebral cortex was inhibited by thuringiensin at concentrations of 1–100 μM, thus cAMP production decreased. Furthermore, thuringiensin or adenylate cyclase inhibitor (MDL-12330A) reduced the forskolin (10 μM)-stimulated adenylate cyclase activity at concentrations of 10 μM, 49% or 43% inhibition, respectively. In conclusion, this study demonstrated that thuringiensin could activate basal adenylate cyclase activity and increase cAMP concentrations in rat cerebral cortex or in a commercial adenylate cyclase. Comparing the dose-dependent effects of thuringiensin on the basal and forskolin-stimulated adenylate cyclase activity, thuringiensin can be regarded as a weak activator of adenylate cyclase or an inhibitor of forskolin-stimulated adenylate cyclase.

5,7-Dihydroxy-6-methoxyflavone, a benzodiazepine site ligand isolated from Scutellaria baicalensis Georgi, with selective antagonistic properties

As part of an effort to identify naturally occurring GABA A receptor benzodiazepine binding site (BDS) ligands from traditional medicinal herbs, we previously reported that flavonoid derivatives isolated from Scutellaria baicalensis ( S. baicalensis) Georgi exhibited significant affinities for the BDS. The present study describes the characterization of 5,7-dihydroxy-6-methoxyflavone (oroxylin A), one of the major components of the herbal extract. Oroxylin A inhibited [ 3 H ]flunitrazepam binding to rat cerebral cortical membrane with a ic 50 value of 1.09±0.07 μM. A GABA ratio of 1.09±0.04 suggests that oroxylin A interacts as an antagonist at the recognition site. In neuropharmacological studies, oral administration of oroxylin A (3.75–60 mg kg −1) did not result in significant changes in animal models routinely employed for benzodiazepine (BD) evaluation. However, oroxylin A selectively abolished the anxiolytic, myorelaxant and motor incoordination, but not the sedative and anticonvulsant effects elicited by diazepam, a BDS agonist. These results add oroxylin A to the list of CNS active flavonoids, and as the first naturally occurring member endowed with selective antagonistic actions via the BDS.

Caffeine analogs: effects on ryanodine-sensitive calcium-release channels and GABAA receptors.

1. Caffeine at 0.3-10 mM enhanced the binding of [3H]ryanodine to calcium-release channels of rabbit muscle sarcoplasmic reticulum. A variety of other xanthines were as efficacious as caffeine or nearly so, but none appeared markedly more potent. 2. Caffeine at 1 mM markedly inhibited binding of [3H]diazepam to GABAA receptors in rat cerebral cortical membranes. 3. Other xanthines also inhibited binding with certain dimethylpropargylxanthines being nearly fivefold more potent than caffeine. 4. Caffeine at 1 mM stimulated binding of [35S]TBPS to GABAA receptors as did certain other xanthines. 5. The dimethylpropargylxanthines had little effect. 1,3-Dipropyl-8-cyclopentylxanthine at 100 microM had no effect on [3H]diazepam binding, but markedly inhibited [35S]TBPS binding. 6. Structure-activity relationships for xanthines do differ for calcium-release channels and and for different sites on GABAA receptors, but no highly selective lead compounds were identified.