Maria Concetta Olianas - Academia.edu (original) (raw)
Papers by Maria Concetta Olianas
Neuropsychopharmacology, 1999
We recently reported that clozapine behaves as a partial agonist at the cloned human m4 muscarini... more We recently reported that clozapine behaves as a partial agonist at the cloned human m4 muscarinic receptor subtype. In the present study, we investigated whether the drug could elicit similar effects at the cloned human m1, m2, and m3 muscarinic receptor subtypes expressed in the Chinese hamster ovary (CHO) cells. Clozapine elicited a concentration-dependent stimulation of [ 3 H]inositol phosphates accumulation in CHO cells expressing either the m1 or the m3 receptor subtype. Moreover, clozapine inhibited forskolin-stimulated cyclic AMP accumulation and enhanced [ 35 S] GTP ␥ S binding to membrane G proteins in CHO cells expressing the m2 receptor. These agonist effects of clozapine were antagonized by atropine. The intrinsic activity of clozapine was lower than that of the full cholinergic agonist carbachol, and, when the compounds were combined, clozapine potently reduced the receptor responses to carbachol. These data indicate that clozapine behaves as a partial agonist at different muscarinic receptor subtypes and may provide new hints for understanding the receptor mechanisms underlying the antipsychotic efficacy of the drug.
British Journal of Pharmacology, 2006
In the present study, we examined the pharmacological activity of the putative k 3-opioid recepto... more In the present study, we examined the pharmacological activity of the putative k 3-opioid receptor agonist naloxone benzoylhydrazone (NalBzoH) at recombinant human opioid receptors individually expressed in Chinese hamster ovary (CHO) cells and native opioid receptors present in rat striatum. 2 At the m-opioid receptor (MOR), NalBzoH stimulated guanosine-5 0-O-(3-[ 35 S]thio)triphosphate ([ 35 S]GTPgS) binding (pEC 50 ¼ 8.59) and inhibited cyclic AMP accumulation (pEC 50 ¼ 8.74) with maximal effects (E max) corresponding to 55 and 65% of those obtained with the MOR agonist DAMGO, respectively. The MOR antagonist CTAP blocked the stimulatory effects of NalBzoH and DAMGO with similar potencies. 3 At the k-opioid receptor (KOR), NalBzoH stimulated [ 35 S]GTPgS binding (pEC 50 ¼ 9.70) and inhibited cyclic AMP formation (pEC 50 ¼ 9.45) as effectively as the selective KOR agonist (À)-U-50,488. The NalBzoH effect was blocked by the KOR antagonist nor-binaltorphimine (nor-BNI) (pK i ¼ 10.30). 4 In CHO cells expressing the d-opioid receptor (DOR), NalBzoH increased [ 35 S]GTPgS binding (pEC 50 ¼ 8.49) and inhibited cyclic AMP formation (pEC 50 ¼ 8.61) almost as effectively as the DOR agonist DPDPE. Naltrindole (NTI), a selective DOR antagonist, completely blocked the response to NalBzoH (pK i of 10.40). 5 In CHO cells expressing the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), NalBzoH failed to exert agonist effects and antagonized the agonist-induced receptor activation. 6 When compared to other opioid receptor ligands, NalBzoH showed an efficacy that was lower than that of morphine at MOR, but higher at KOR and DOR. 7 In rat striatum, NalBzoH enhanced [ 35 S]GTPgS binding and inhibited adenylyl cyclase activity. These effects were antagonized by either CTAP, nor-BNI or NTI, each antagonist blocking a fraction of the NalBzoH response. 8 These data demonstrate that NalBzoH displays agonist activity at MOR, DOR and KOR expressed either in a heterologous cell system or in a native environment.
Biochemical pharmacology, Jan 15, 2015
Different lines of evidence indicate that the lysophosphatidic acid (LPA) receptor LPA1 is involv... more Different lines of evidence indicate that the lysophosphatidic acid (LPA) receptor LPA1 is involved in neurogenesis, synaptic plasticity and anxiety-related behaviour, but little is known on whether this receptor can be targeted by neuropsychopharmacological agents. The present study investigated the effects of different antidepressants on LPA1 signaling. We found that in Chinese hamster ovary (CHO)-K1 fibroblasts expressing endogenous LPA1 tricyclic and tetracyclic antidepressants and fluoxetine induced the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2) and CREB. This response was antagonized by either LPA1 blockade with Ki16425 and AM966 or knocking down LPA1 with siRNA. Antidepressants induced ERK1/2 phosphorylation in human embryonic kidney (HEK)-293 cells overexpressing LPA1, but not in wild type cells. In PathHunter™ assay measuring receptor-β-arrestin interaction, amitriptyline, mianserin and fluoxetine failed to induce activation of LPA2 and LPA3. stabl...
Neuroscience Letters, 1989
In homogenates of rat nucleus accumbens, quinpirole, a dopamine (DA) D2 receptor agonist, inhibit... more In homogenates of rat nucleus accumbens, quinpirole, a dopamine (DA) D2 receptor agonist, inhibited the activation of tyrosine hydroxylase (TH) elicited by either forskolin, an activator of adenylate cyclase, or rolipram, a cyclic nucleotide phosphodiesterase inhibitor. The inhibition produced by 1 microM quinpirole was completely antagonized by the D2 blocker L-sulpiride (2 microM). Quinpirole failed to inhibit the stimulation of TH elicited by dibutyryl cyclic AMP (2 mM), which acts independently of adenylate cyclase. Quinpirole (10 microM) significantly inhibited the stimulation of adenylate cyclase activity elicited by 1 microM forskolin. These results indicate that mesolimbic DA autoreceptors can regulate TH activity by inhibiting a presynaptic adenylate cyclase system.
Neuroscience Letters, 1992
Because some responses to forskolin are adenosine 3',5'-cyclic phosphate (cyclic AMP) independent... more Because some responses to forskolin are adenosine 3',5'-cyclic phosphate (cyclic AMP) independent, we investigated the involvement of adenylate cyclase in the stimulatory effect of forskolin on synaptosomal tyrosine hydroxylase (TH) activity of rat striatum. The t)rskolin analogue~ 1,9dideoxyt'orskolin, which mimics the cyclic AMP-independent effects of forskolin, was a very weak activator of both striatal adenylate cyclase and tyrosine hydroxylase activities, whereas forskolin stimulated the two enzymes effectively and with similar potencies. Moreover, exposure of synaptosomes to the specific cyclic AMP antagonist Rp-adenosine 3',5'-cyclic phosphorothioate reduced basal TH activity and counteracted the stimulatory effect on the enzyme activity by submaximal concentrations of forskolin. These results provide circumstantial evidence that in striatal dopaminergic nerve terminals a presynaptic adenylate cyclase mediates the stimulation of TH activity by forskolin.
![Research paper thumbnail of [Phe1 Φ(CH2-H)Gly2]nociceptin-(1-13)-NH2 acts as a partial agonist at ORL1 receptor endogenously expressed in mouse N1E-115 neuroblastoma cells](https://attachments.academia-assets.com/117136152/thumbnails/1.jpg)
](NeuroReport, 1999
THE nociceptin derivative [Phe 1 Ö(CH 2-NH)Gly 2 ]nociceptin-(1-13)-NH 2 (PheÖnoc) has been repor... more THE nociceptin derivative [Phe 1 Ö(CH 2-NH)Gly 2 ]nociceptin-(1-13)-NH 2 (PheÖnoc) has been reported to act either as a simple antagonist or as a full agonist at the opioid receptor-like (ORL1) receptor. In the present study, we identi®ed the expression of the ORL1 receptor in murine N1E-115 neuroblastoma cells and used this neuronal system to investigate the pharmacological activity of PheÖnoc. Like nociceptin, PheÖnoc stimulated the binding of [ 35 S]GTPãS (EC 50 120 nM) and inhibited forskolin-stimulated [ 3 H]cAMP formation (EC 50 3.3 nM). However, PheÖnoc elicited maximal effects lower than those induced by nociceptin, and when combined with nociceptin potently antagonized the responses to the natural agonist (K i 0.9 nM). These data indicate that PheÖnoc acts as a partial agonist at the ORL1 receptor endogenously expressed in N1E-115 cells. NeuroReport 10:1127±1131 # 1999 Lippincott Williams & Wilkins.
Neuropsychopharmacology, 2006
The present study examined the effects of N-desmethylclozapine (NDMC), a biologically active meta... more The present study examined the effects of N-desmethylclozapine (NDMC), a biologically active metabolite of the atypical antipsychotic clozapine, at cloned human opioid receptors stably expressed in Chinese hamster ovary (CHO) cells and at native opioid receptors present in NG108-15 cells and rat brain. In CHO cells expressing the d-opioid receptor (CHO/DOR), NDMC behaved as a full agonist both in stimulating [ 35 S]GTPgS binding (pEC 50 ¼ 7.24) and in inhibiting cyclic AMP formation (pEC 50 ¼ 6.40). NDMC inhibited [ 3 H]naltrindole binding to CHO/DOR membranes with competition curves that were modulated by guanine nucleotides in an agonistlike manner. Determination of intrinsic efficacies by taking into consideration both the maximal [ 35 S]GTPgS binding stimulation and the extent of receptor occupancy at which half-maximal effect occurred indicated that NDMC had an efficacy value equal to 82% of that of the full d-opioid receptor agonist DPDPE, whereas clozapine and the other clozapine metabolite clozapine N-oxide displayed much lower levels of agonist efficacy. NDMC exhibited poor agonist activity and lower affinity at the k-opioid receptor and was inactive at m-opioid and NOP receptors. In NG108-15 cells, NDMC inhibited cyclic AMP formation and stimulated the phosphorylation of extracellular signal-regulated kinase 1/2 by activating the endogenously expressed d-opioid receptor. Moreover, in membranes of different brain regions, NDMC stimulated [ 35 S]GTPgS binding and regulated adenylyl cyclase activity and the effects were potently antagonized by naltrindole. These data demonstrate for the first time that NDMC acts as a selective and efficacious d-opioid receptor agonist and suggest that this unique property may contribute, at least in part, to the clinical actions of the atypical antipsychotic clozapine.
Neuropharmacology, 2014
Both type I interferons (IFNs) and neurotrophins regulate neuroadaptive responses, but relatively... more Both type I interferons (IFNs) and neurotrophins regulate neuroadaptive responses, but relatively little is known on the interaction between these two classes of regulatory proteins. Here we investigated the effect of IFN-β on the expression and functional activity of the common neurotrophin receptor p75NTR and the nerve growth factor (NGF) receptor TrkA. In differentiated human SH-SY5Y neuroblastoma cells prolonged exposure to IFN-β up-regulated p75NTR and TrkA levels, failed to affect the content of sortilin, a p75NTR co-receptor, and, consistent with our previous finding, down-regulated the brain-derived neurotrophic factor receptor TrkB. Quantitative real time RT-PCR indicated that IFN-β increased p75NTR and TrkA mRNA levels. In control and IFN-β treated cells proNGF failed to induce c-Jun N-terminal kinase and nuclear factor/kB activation, two p75NTR/sortilin signalling pathways mediating neuronal death. On the other hand, IFN-β treatment enhanced TrkA autophosphorylation and signalling induced by NGF and proNGF. Knockdown of p75NTR by siRNA reduced TrkA activation by proNGF and a subnanomolar concentration of NGF, whereas co-immunoprecipitation indicated close association of p75NTR and TrkA. Co-treatment with either NGF or proNGF reduced IFN-β pro-apoptotic and anti-neurotrophic effects. Similarly, in primary mouse hippocampal neurons IFN-β increased p75NTR and TrkA expression, down-regulated TrkB and enhanced NGF-induced phosphorylation of the pro-survival protein kinase Akt. The data demonstrate that in neuronal cells IFN-β differentially affects the expression and signalling of neurotrophin receptors and suggest that the up-regulation of the p75NTR/TrkA signalling complex may constitute a novel mechanism by which this cytokine selectively attenuates its pro-apoptotic effect in NGF-responsive cells.
Neurochemistry International, 2005
In the present study, the effects of different allosteric modulators on the functional activity o... more In the present study, the effects of different allosteric modulators on the functional activity of gamma-aminobutyric acid (GABA)B receptors in membranes of post-mortem human frontal cortex were examined. Western blot analysis indicated that the tissue preparations expressed both GABA(B1) and GABA(B2) subunits of the GABA(B) receptor heterodimer. In [35S]-GTPgammaS binding assays, Ca2+ ion (1 mM) enhanced the potency of the agonists GABA and 3-aminopropylphosphinic acid (3-APA) and that of the antagonist CGP55845, but not that of the GABA(B) receptor agonist (-)-baclofen. CGP7930 (2,6-di-t-Bu-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol), a positive allosteric modulator of GABA(B) receptors, potentiated both GABA(B) receptor-mediated stimulation of [35S]-GTPgammaS binding and inhibition of forskolin (FSK)-stimulated adenylyl cyclase activity. Chelation of Ca2+ ion by EGTA reduced the CGP7930 enhancement of GABA potency in stimulating [35S]-GTPgammaS binding by two-fold. Fendiline, also reported to act as a positive allosteric modulator of GABA(B) receptors, failed to enhance GABA stimulation of [35S]-GTPgammaS binding but inhibited the potentiating effect of CGP7930. The inhibitory effect was mimicked by the phenothiazine antipsychotic trifluoperazine (TFP), but not by other compounds, such as verapamil or diphenydramine (DPN). These data demonstrate that the function of GABA(B) receptors of human frontal cortex is positively modulated by Ca2+ ion and CGP7930, which interact synergistically. Conversely, fendiline and trifluoperazine negatively affect the allosteric regulation by CGP7930.
Life Sciences, 2005
The mamba toxin MT-7 is the most selective ligand currently available for the muscarinic M 1 rece... more The mamba toxin MT-7 is the most selective ligand currently available for the muscarinic M 1 receptor subtype. The toxin binds stably to the receptor and blocks the agonist-induced activation non-competitively. Although its mode of action on M 1 receptors is not yet fully understood, some of the toxin properties support an allosteric mechanism. Thus, the toxin fails to elicit a complete inhibition of the binding of either the muscarinic antagonist [ 3 H]N-methyl-scopolamine ([ 3 H]NMS) or the agonist [ 3 H]acetylcholine ([ 3 H]ACh). When added to ligandoccupied M 1 receptors, the toxin slows the dissociation rate of [ 3 H]NMS and increases that of [ 3 H]ACh. Sitedirected mutagenesis studies have provided important information about the toxin amino acid residues which are critical for the stable binding to the receptor and for the allosteric modulation of antagonist dissociation. In vivo studies have shown that the intracerebral injection of MT-7 causes a long-lasting blockade of M 1 receptor, thus providing a tool for the characterization of the functional role of this receptor subtype in discrete brain areas.
Life Sciences, 1995
In membranes of rat olfactory bulb, muscarinic receptor agonists stimulate basal adenylyl cyclase... more In membranes of rat olfactory bulb, muscarinic receptor agonists stimulate basal adenylyl cyclase activity . This response is inhibited by a number of muscarinic receptor antagonists with a rank order of potency suggesting the involvement of the M4 muscarinic receptor subtype. The stimulatory effect does not require Ca2+ and occurs independently of activation of phosphoinositide hydrolysis. Pretreatment with pertussis toxin completely prevents the muscarinic stimulation of adenylyl cyclase, indicating the participation of G proteins of the Gi/Go family. Immunological impairment of the G protein, Gs, also reduces the muscarinic response, whereas concomitant activation of Gs-coupled receptors by CRH or VIP results in a synergistic stimulation of adenylyl cyclase activity. Although these data suggest a role for Gs, a body of evidence indicates that the muscarinic receptors do not interact directly with this G protein. Moreover, the Ca2+/calmodulin (Ca2+/CaM)- and forskolin-stimulated enzyme activities are inhibited by muscarinic receptor activation in a pertussis toxin-sensitive manner and with a pharmacological profile similar to that observed for the stimulatory response. These data indicate that in rat olfactory bulb M4 muscarinic receptors exert a bimodal control on cyclic AMP formation through a sequence of events that may involve activation of Gi/Go proteins, synergistic interaction with Gs and differential modulation of Ca2+/CaM-independent and -dependent forms of adenylyl cyclase.
Journal of Neurochemistry, 2008
Nociceptin/orphanin FQ (N/OFQ) has been reported to inhibit dopamine (DA) release in basal gangli... more Nociceptin/orphanin FQ (N/OFQ) has been reported to inhibit dopamine (DA) release in basal ganglia mainly by acting on NOP receptors in substantia nigra and ventral tegmental area. We investigated whether N/OFQ could affect DA transmission by acting at either DA nerve endings or DA‐targeted post‐synaptic neurons. In synaptosomes of rat nucleus accumbens and striatum N/OFQ inhibited DA synthesis and tyrosine hydroxylase (TH) phosphorylation at Ser40 via NOP receptors coupled to inhibition of the cAMP/protein kinase A pathway. Immunofluorescence studies showed that N/OFQ preferentially inhibited phospho‐Ser40‐TH in nucleus accumbens shell and that in this subregion NOP receptors partly colocalized with either TH or DA D1 receptor positive structures. In accumbens and striatum N/OFQ inhibited DA D1 receptor‐stimulated cAMP formation, but failed to affect either adenosine A2A or DA D2 receptor regulation of cAMP. In accumbens slices, N/OFQ inhibited DA D1‐induced phosphorylation of NMDA...
![Research paper thumbnail of Stimulation of Guanosine 5′‐O‐(3‐[35S]Thiotriphosphate) Binding by Cholinergic Muscarinic Receptors in Membranes of Rat Olfactory Bulb](https://attachments.academia-assets.com/117136154/thumbnails/1.jpg)
](Journal of Neurochemistry, 2002
Journal of Neurochemistry, 1993
We reported previously that in homogenates of rat olfactory bulb muscarinic and opioid receptor a... more We reported previously that in homogenates of rat olfactory bulb muscarinic and opioid receptor agonists stimulate adenylyl cyclase activity. In the present study we show that carbachol (CCh) and Leu-Enkephalin act synergistically with vasoactive intestinal peptide (VIP) and corticotropin-releasing hormone (CRH), but not with I-isoproterenol, in increasing cyclic AMP formation. The synergistic interaction consists of an increase in the maximal adenylyl cyclase activation without a significant change in the potency of each agonist. CCh also fails to affect CRH binding to olfactory bulb membranes. The synergism requires micromolar concentrations of GTP. Substitution of the stable GTP analog guanosine 5'-0-(3'-thiotriphosphate) for GTP allows the CRH stimulation, but abolishes the CCh enhancement of both basal and CRH-stimulated enzyme activities. Moreover, in vivo treatment of olfactory bulbs with pertussis toxin completely prevents the muscarink and opioid effects. Thus, the synergistic interaction appears to result from opioid-and muscarinic-induced activation of a pertussis toxin-sensitive GTP-binding protein which may potentiate the adenylyl cyclase stimulation by the stimulatory GTP-binding protein activated by either VIP or CRH receptors.
Journal of Neurochemistry, 2002
: In rat olfactory bulb, muscarinic and opioid receptor agonists stimulate basal adenylyl cyclase... more : In rat olfactory bulb, muscarinic and opioid receptor agonists stimulate basal adenylyl cyclase activity in a GTP‐dependent and pertussis toxin‐sensitive manner. However, in the present study, we show that in the same brain area activation of these receptors causes inhibition of adenylyl cyclase activity stimulated by Ca2+ and calmodulin (CaM) and by forskolin (FSK), two direct activators of the catalytic unit of the enzyme. The opioid and muscarinic inhibitions consist of a decrease of the maximal stimulation elicited by either CaM or FSK, without a change in the potency of these agents. [Leu5]Enkephalin and selective δ‐ and μ‐, but not κ‐, opioid receptors agonists inhibit the FSK stimulation of adenylyl cyclase activity with the same potencies displayed in stimulating basal enzyme activity. Similarly, the muscarinic inhibition of FSK‐stimulated adenylyl cyclase activity shows agonist and antagonist sensitivities similar to those characterizing the muscarinic stimulation of basal enzyme activity. Fluoride stimulation of adenylyl cyclase is not affected by either carbachol or [Leu5]enkephalin. In vivo treatment of olfactory bulb with pertussis toxin prevents both opioid and muscarinic inhibition of Ca2+/CaM‐ and FSK‐stimulated enzyme activities. These results indicate that in rat olfactory bulb δ‐ and μ‐opioid receptors and muscarinic receptors, likely of the M4 subtype, can exert a dual effect on cyclic AMP formation by interacting with pertussis toxin‐sensitive GTP‐binding protein(s) and possibly by affecting different molecular forms of adenylyl cyclase.
Journal of Neurochemistry, 2006
4P-Phorbol 12-myristate 13-acetate (FMA), added to a lysed mitochondria1 fraction of rat striatum... more 4P-Phorbol 12-myristate 13-acetate (FMA), added to a lysed mitochondria1 fraction of rat striatum, stimulates adenylate cyclase activity with an apparent time lag of-30 s. Half-maximal and maximal enzyme stimulations are obtained with 8 and 200 nM PM,4, respectively. The PMA stimulation is GTP dependent, reaching a maximum of-60% at 50 p L M GTP, and is associated with disappearance of the enzyme inhibition induced by micromolar concentrations of GTP. Enhancement of enzyme activity by cholera toxin and 3,4-dihydroxyphenylethylamine is amplified by PMA only at micromolar concentrations of GTP. PMA does not affect the enzyme stimulation by forskolin but reverses the inhibition of forskolin-stimulated enzyme by GTP. When guanyl-5'-yl-imidodiphosphate is substituted for GTP, PMA does not modify adenylate cyclase activity. Enzyme inhibition by acetylcholine, Leu-enkephalini, and R(-)N6-(2-phenylisopropyl)adenosine is magnified by PMA. Stimulation of adenylate cyclase by PMA is markedly reduced following EGTA treatment, is not observed when adenyl-5'-yl-imidodiphosphate is substituted for ATP as substrate for adenylate cyclase, and is enhanced by L-a-phosphatidyl-L-serine. Like PMA, 4P-phorbol 12,13-dibutyrate and l-oleoyl-2-acetylglycerol stimulate striatal adenylate cyclase, whereas 4P-phorbol and 4pphorbol 13-acetate are ineffective. The results indicate that phorbol esters increase striatal adenylate cyclase activity by reducing the GTP-induced inhibition of the enzyme, presumably as a result of protein kinase C activation.
Journal of Neural Transmission, 1992
In homogenates of female rat anterior pituitary, the azepine derivative B-HT 920 inhibited the fo... more In homogenates of female rat anterior pituitary, the azepine derivative B-HT 920 inhibited the forskolin-stimulated adenylate cyclase activity with an EC50 value of 0.35 microM. In male rat anterior pituitary, B-HT 920 curtailed the stimulation of adenylate cyclase activity by vasoactive intestinal peptide with an EC50 of 0.20 microM. In synaptic plasma membranes of rat striatum, B-HT 920 significantly reduced basal adenylate cyclase activity with an EC50 of 0.68 microM. Both in pituitary and striatum, the B-HT 920 inhibition was counteracted by the dopamine (DA) D2 receptor antagonist 1-sulpiride, but not by the alpha 2-adrenergic antagonist yohimbine. These results indicate that B-HT 920 is capable of activating DA D2 receptors negatively coupled to adenylate cyclase activity.
European Journal of Pharmacology, 2009
The clozapine metabolite N-desmethylclozapine (NDMC) has been recently shown to act at different ... more The clozapine metabolite N-desmethylclozapine (NDMC) has been recently shown to act at different neurotransmitter receptors and to display both antagonist and agonist activities. We have previously reported that in cells over-expressing the recombinant delta-opioid receptor NDMC behaved as partial agonist with high intrinsic activity, but its action at the receptors naturally expressed in human brain remained to be investigated. In the present study, we examined whether NDMC was able to bind to and activate delta-opioid receptors in membranes of post-mortem human frontal cortex. In radioligand binding assays, NDMC competition curves displayed high- (K(i)=26 nM) and low-affinity (K(i)=3 microM) components, whose proportion was regulated by guanine nucleotides in an agonist-like fashion. In functional assays, NDMC stimulated [(35)S]GTPgammaS binding (EC(50)=905 nM) and inhibited cyclic AMP formation (EC(50)=590 nM) as effectively as delta-opioid agonists, whereas clozapine was much less potent and efficacious and clozapine N-oxide was completely inactive. The NDMC agonist activity was potently antagonized by the delta-opioid antagonist naltrindole, but not by the micro-opioid receptor antagonist CTAP (D-phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2)) or the kappa-opioid antagonist nor-binaltorphimine. Moreover, blockade of either acetylcholine muscarinic, dopamine D(2) or serotonin 5HT(1A) receptors failed to affect NDMC agonist activity. These data demonstrate that at clinically relevant concentrations NDMC behaves as an efficacious agonist at delta-opioid receptors of human frontal cortex.
European Journal of Pharmacology, 1998
In membranes of olfactory tubercle and striatum, the selective muscarinic M4 receptor antagonist ... more In membranes of olfactory tubercle and striatum, the selective muscarinic M4 receptor antagonist muscarinic toxin 3 completely antagonized the acetylcholine-induced inhibition of forskolin- and dopamine D1 receptor-stimulated cyclic AMP formation with Ki values of 7 and 4 nM, respectively. In olfactory bulb, where acetylcholine stimulated basal adenylyl cyclase activity and inhibited forskolin-stimulated enzyme activity, muscarinic toxin 3 caused a partial antagonism of both acetylcholine effects with high potencies (Ki values = 4-6 nM). In frontal cortex, muscarinic toxin 3 counteracted the acetylcholine-induced potentiation of corticotropin-releasing hormone-stimulated cyclic AMP with a Ki of 58 nM, which is close to the toxin affinity for the muscarinic M1 receptor. In the same brain region, the acetylcholine inhibition of forskolin-stimulated enzyme activity was not affected by muscarinic toxin 3. In microdissected regions of the hippocampus, a significant portion (33-48%) of the acetylcholine inhibition of forskolin-stimulated adenylyl cyclase activity was blocked by muscarinic toxin 3 with Ki values (6-8 nM) consistent with the involvement of muscarinic M4 receptors. These data show that muscarinic toxin 3 discriminates between adenylyl cyclase-coupled muscarinic receptors and demonstrate the utility of the toxin in identifying the relative contribution by the muscarinic M4 receptor subtype.
European Journal of Pharmacology, 2003
The ability of 2,6 Di-tert-butyl-4-(-hydroxy-2,2-dimethyl-propyl)-phenol (CGP7930), a positive al... more The ability of 2,6 Di-tert-butyl-4-(-hydroxy-2,2-dimethyl-propyl)-phenol (CGP7930), a positive allosteric modulator of GABA(B) receptors, to regulate GABA(B) receptor-induced stimulation and inhibition of adenylyl cyclase activity in rat brain was investigated. In olfactory bulb granule cell layer and in frontal cortex, CGP7930 potentiated the stimulatory effects of (-)-baclofen and gamma-aminobutyric acid (GABA) on basal and corticotropin-releasing hormone-stimulated adenylyl cyclase activities, respectively. In these stimulatory responses, CGP7930 enhanced both agonist potencies and maximal effects. When GABA(B) receptor-mediated inhibition of forskolin-stimulated adenylyl cyclase activity of frontal cortex was examined, CGP7930 increased the agonist potencies but failed to affect the maximal effect of (-)-baclofen and modestly increased that of GABA. Similar results were obtained for the inhibition of Ca(2+)/calmodulin-stimulated adenylyl cyclase in striatum and cerebellum. Western blot analysis of each membrane preparation showed the presence of GABA(B2) receptor subunit, a putative site of action of CGP7930. These data indicate that CGP7930 positively modulates brain GABA(B) receptors coupled to either stimulation or inhibition of cyclic AMP signalling.
Neuropsychopharmacology, 1999
We recently reported that clozapine behaves as a partial agonist at the cloned human m4 muscarini... more We recently reported that clozapine behaves as a partial agonist at the cloned human m4 muscarinic receptor subtype. In the present study, we investigated whether the drug could elicit similar effects at the cloned human m1, m2, and m3 muscarinic receptor subtypes expressed in the Chinese hamster ovary (CHO) cells. Clozapine elicited a concentration-dependent stimulation of [ 3 H]inositol phosphates accumulation in CHO cells expressing either the m1 or the m3 receptor subtype. Moreover, clozapine inhibited forskolin-stimulated cyclic AMP accumulation and enhanced [ 35 S] GTP ␥ S binding to membrane G proteins in CHO cells expressing the m2 receptor. These agonist effects of clozapine were antagonized by atropine. The intrinsic activity of clozapine was lower than that of the full cholinergic agonist carbachol, and, when the compounds were combined, clozapine potently reduced the receptor responses to carbachol. These data indicate that clozapine behaves as a partial agonist at different muscarinic receptor subtypes and may provide new hints for understanding the receptor mechanisms underlying the antipsychotic efficacy of the drug.
British Journal of Pharmacology, 2006
In the present study, we examined the pharmacological activity of the putative k 3-opioid recepto... more In the present study, we examined the pharmacological activity of the putative k 3-opioid receptor agonist naloxone benzoylhydrazone (NalBzoH) at recombinant human opioid receptors individually expressed in Chinese hamster ovary (CHO) cells and native opioid receptors present in rat striatum. 2 At the m-opioid receptor (MOR), NalBzoH stimulated guanosine-5 0-O-(3-[ 35 S]thio)triphosphate ([ 35 S]GTPgS) binding (pEC 50 ¼ 8.59) and inhibited cyclic AMP accumulation (pEC 50 ¼ 8.74) with maximal effects (E max) corresponding to 55 and 65% of those obtained with the MOR agonist DAMGO, respectively. The MOR antagonist CTAP blocked the stimulatory effects of NalBzoH and DAMGO with similar potencies. 3 At the k-opioid receptor (KOR), NalBzoH stimulated [ 35 S]GTPgS binding (pEC 50 ¼ 9.70) and inhibited cyclic AMP formation (pEC 50 ¼ 9.45) as effectively as the selective KOR agonist (À)-U-50,488. The NalBzoH effect was blocked by the KOR antagonist nor-binaltorphimine (nor-BNI) (pK i ¼ 10.30). 4 In CHO cells expressing the d-opioid receptor (DOR), NalBzoH increased [ 35 S]GTPgS binding (pEC 50 ¼ 8.49) and inhibited cyclic AMP formation (pEC 50 ¼ 8.61) almost as effectively as the DOR agonist DPDPE. Naltrindole (NTI), a selective DOR antagonist, completely blocked the response to NalBzoH (pK i of 10.40). 5 In CHO cells expressing the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), NalBzoH failed to exert agonist effects and antagonized the agonist-induced receptor activation. 6 When compared to other opioid receptor ligands, NalBzoH showed an efficacy that was lower than that of morphine at MOR, but higher at KOR and DOR. 7 In rat striatum, NalBzoH enhanced [ 35 S]GTPgS binding and inhibited adenylyl cyclase activity. These effects were antagonized by either CTAP, nor-BNI or NTI, each antagonist blocking a fraction of the NalBzoH response. 8 These data demonstrate that NalBzoH displays agonist activity at MOR, DOR and KOR expressed either in a heterologous cell system or in a native environment.
Biochemical pharmacology, Jan 15, 2015
Different lines of evidence indicate that the lysophosphatidic acid (LPA) receptor LPA1 is involv... more Different lines of evidence indicate that the lysophosphatidic acid (LPA) receptor LPA1 is involved in neurogenesis, synaptic plasticity and anxiety-related behaviour, but little is known on whether this receptor can be targeted by neuropsychopharmacological agents. The present study investigated the effects of different antidepressants on LPA1 signaling. We found that in Chinese hamster ovary (CHO)-K1 fibroblasts expressing endogenous LPA1 tricyclic and tetracyclic antidepressants and fluoxetine induced the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2) and CREB. This response was antagonized by either LPA1 blockade with Ki16425 and AM966 or knocking down LPA1 with siRNA. Antidepressants induced ERK1/2 phosphorylation in human embryonic kidney (HEK)-293 cells overexpressing LPA1, but not in wild type cells. In PathHunter™ assay measuring receptor-β-arrestin interaction, amitriptyline, mianserin and fluoxetine failed to induce activation of LPA2 and LPA3. stabl...
Neuroscience Letters, 1989
In homogenates of rat nucleus accumbens, quinpirole, a dopamine (DA) D2 receptor agonist, inhibit... more In homogenates of rat nucleus accumbens, quinpirole, a dopamine (DA) D2 receptor agonist, inhibited the activation of tyrosine hydroxylase (TH) elicited by either forskolin, an activator of adenylate cyclase, or rolipram, a cyclic nucleotide phosphodiesterase inhibitor. The inhibition produced by 1 microM quinpirole was completely antagonized by the D2 blocker L-sulpiride (2 microM). Quinpirole failed to inhibit the stimulation of TH elicited by dibutyryl cyclic AMP (2 mM), which acts independently of adenylate cyclase. Quinpirole (10 microM) significantly inhibited the stimulation of adenylate cyclase activity elicited by 1 microM forskolin. These results indicate that mesolimbic DA autoreceptors can regulate TH activity by inhibiting a presynaptic adenylate cyclase system.
Neuroscience Letters, 1992
Because some responses to forskolin are adenosine 3',5'-cyclic phosphate (cyclic AMP) independent... more Because some responses to forskolin are adenosine 3',5'-cyclic phosphate (cyclic AMP) independent, we investigated the involvement of adenylate cyclase in the stimulatory effect of forskolin on synaptosomal tyrosine hydroxylase (TH) activity of rat striatum. The t)rskolin analogue~ 1,9dideoxyt'orskolin, which mimics the cyclic AMP-independent effects of forskolin, was a very weak activator of both striatal adenylate cyclase and tyrosine hydroxylase activities, whereas forskolin stimulated the two enzymes effectively and with similar potencies. Moreover, exposure of synaptosomes to the specific cyclic AMP antagonist Rp-adenosine 3',5'-cyclic phosphorothioate reduced basal TH activity and counteracted the stimulatory effect on the enzyme activity by submaximal concentrations of forskolin. These results provide circumstantial evidence that in striatal dopaminergic nerve terminals a presynaptic adenylate cyclase mediates the stimulation of TH activity by forskolin.
NeuroReport, 1999
THE nociceptin derivative [Phe 1 Ö(CH 2-NH)Gly 2 ]nociceptin-(1-13)-NH 2 (PheÖnoc) has been repor... more THE nociceptin derivative [Phe 1 Ö(CH 2-NH)Gly 2 ]nociceptin-(1-13)-NH 2 (PheÖnoc) has been reported to act either as a simple antagonist or as a full agonist at the opioid receptor-like (ORL1) receptor. In the present study, we identi®ed the expression of the ORL1 receptor in murine N1E-115 neuroblastoma cells and used this neuronal system to investigate the pharmacological activity of PheÖnoc. Like nociceptin, PheÖnoc stimulated the binding of [ 35 S]GTPãS (EC 50 120 nM) and inhibited forskolin-stimulated [ 3 H]cAMP formation (EC 50 3.3 nM). However, PheÖnoc elicited maximal effects lower than those induced by nociceptin, and when combined with nociceptin potently antagonized the responses to the natural agonist (K i 0.9 nM). These data indicate that PheÖnoc acts as a partial agonist at the ORL1 receptor endogenously expressed in N1E-115 cells. NeuroReport 10:1127±1131 # 1999 Lippincott Williams & Wilkins.
Neuropsychopharmacology, 2006
The present study examined the effects of N-desmethylclozapine (NDMC), a biologically active meta... more The present study examined the effects of N-desmethylclozapine (NDMC), a biologically active metabolite of the atypical antipsychotic clozapine, at cloned human opioid receptors stably expressed in Chinese hamster ovary (CHO) cells and at native opioid receptors present in NG108-15 cells and rat brain. In CHO cells expressing the d-opioid receptor (CHO/DOR), NDMC behaved as a full agonist both in stimulating [ 35 S]GTPgS binding (pEC 50 ¼ 7.24) and in inhibiting cyclic AMP formation (pEC 50 ¼ 6.40). NDMC inhibited [ 3 H]naltrindole binding to CHO/DOR membranes with competition curves that were modulated by guanine nucleotides in an agonistlike manner. Determination of intrinsic efficacies by taking into consideration both the maximal [ 35 S]GTPgS binding stimulation and the extent of receptor occupancy at which half-maximal effect occurred indicated that NDMC had an efficacy value equal to 82% of that of the full d-opioid receptor agonist DPDPE, whereas clozapine and the other clozapine metabolite clozapine N-oxide displayed much lower levels of agonist efficacy. NDMC exhibited poor agonist activity and lower affinity at the k-opioid receptor and was inactive at m-opioid and NOP receptors. In NG108-15 cells, NDMC inhibited cyclic AMP formation and stimulated the phosphorylation of extracellular signal-regulated kinase 1/2 by activating the endogenously expressed d-opioid receptor. Moreover, in membranes of different brain regions, NDMC stimulated [ 35 S]GTPgS binding and regulated adenylyl cyclase activity and the effects were potently antagonized by naltrindole. These data demonstrate for the first time that NDMC acts as a selective and efficacious d-opioid receptor agonist and suggest that this unique property may contribute, at least in part, to the clinical actions of the atypical antipsychotic clozapine.
Neuropharmacology, 2014
Both type I interferons (IFNs) and neurotrophins regulate neuroadaptive responses, but relatively... more Both type I interferons (IFNs) and neurotrophins regulate neuroadaptive responses, but relatively little is known on the interaction between these two classes of regulatory proteins. Here we investigated the effect of IFN-β on the expression and functional activity of the common neurotrophin receptor p75NTR and the nerve growth factor (NGF) receptor TrkA. In differentiated human SH-SY5Y neuroblastoma cells prolonged exposure to IFN-β up-regulated p75NTR and TrkA levels, failed to affect the content of sortilin, a p75NTR co-receptor, and, consistent with our previous finding, down-regulated the brain-derived neurotrophic factor receptor TrkB. Quantitative real time RT-PCR indicated that IFN-β increased p75NTR and TrkA mRNA levels. In control and IFN-β treated cells proNGF failed to induce c-Jun N-terminal kinase and nuclear factor/kB activation, two p75NTR/sortilin signalling pathways mediating neuronal death. On the other hand, IFN-β treatment enhanced TrkA autophosphorylation and signalling induced by NGF and proNGF. Knockdown of p75NTR by siRNA reduced TrkA activation by proNGF and a subnanomolar concentration of NGF, whereas co-immunoprecipitation indicated close association of p75NTR and TrkA. Co-treatment with either NGF or proNGF reduced IFN-β pro-apoptotic and anti-neurotrophic effects. Similarly, in primary mouse hippocampal neurons IFN-β increased p75NTR and TrkA expression, down-regulated TrkB and enhanced NGF-induced phosphorylation of the pro-survival protein kinase Akt. The data demonstrate that in neuronal cells IFN-β differentially affects the expression and signalling of neurotrophin receptors and suggest that the up-regulation of the p75NTR/TrkA signalling complex may constitute a novel mechanism by which this cytokine selectively attenuates its pro-apoptotic effect in NGF-responsive cells.
Neurochemistry International, 2005
In the present study, the effects of different allosteric modulators on the functional activity o... more In the present study, the effects of different allosteric modulators on the functional activity of gamma-aminobutyric acid (GABA)B receptors in membranes of post-mortem human frontal cortex were examined. Western blot analysis indicated that the tissue preparations expressed both GABA(B1) and GABA(B2) subunits of the GABA(B) receptor heterodimer. In [35S]-GTPgammaS binding assays, Ca2+ ion (1 mM) enhanced the potency of the agonists GABA and 3-aminopropylphosphinic acid (3-APA) and that of the antagonist CGP55845, but not that of the GABA(B) receptor agonist (-)-baclofen. CGP7930 (2,6-di-t-Bu-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol), a positive allosteric modulator of GABA(B) receptors, potentiated both GABA(B) receptor-mediated stimulation of [35S]-GTPgammaS binding and inhibition of forskolin (FSK)-stimulated adenylyl cyclase activity. Chelation of Ca2+ ion by EGTA reduced the CGP7930 enhancement of GABA potency in stimulating [35S]-GTPgammaS binding by two-fold. Fendiline, also reported to act as a positive allosteric modulator of GABA(B) receptors, failed to enhance GABA stimulation of [35S]-GTPgammaS binding but inhibited the potentiating effect of CGP7930. The inhibitory effect was mimicked by the phenothiazine antipsychotic trifluoperazine (TFP), but not by other compounds, such as verapamil or diphenydramine (DPN). These data demonstrate that the function of GABA(B) receptors of human frontal cortex is positively modulated by Ca2+ ion and CGP7930, which interact synergistically. Conversely, fendiline and trifluoperazine negatively affect the allosteric regulation by CGP7930.
Life Sciences, 2005
The mamba toxin MT-7 is the most selective ligand currently available for the muscarinic M 1 rece... more The mamba toxin MT-7 is the most selective ligand currently available for the muscarinic M 1 receptor subtype. The toxin binds stably to the receptor and blocks the agonist-induced activation non-competitively. Although its mode of action on M 1 receptors is not yet fully understood, some of the toxin properties support an allosteric mechanism. Thus, the toxin fails to elicit a complete inhibition of the binding of either the muscarinic antagonist [ 3 H]N-methyl-scopolamine ([ 3 H]NMS) or the agonist [ 3 H]acetylcholine ([ 3 H]ACh). When added to ligandoccupied M 1 receptors, the toxin slows the dissociation rate of [ 3 H]NMS and increases that of [ 3 H]ACh. Sitedirected mutagenesis studies have provided important information about the toxin amino acid residues which are critical for the stable binding to the receptor and for the allosteric modulation of antagonist dissociation. In vivo studies have shown that the intracerebral injection of MT-7 causes a long-lasting blockade of M 1 receptor, thus providing a tool for the characterization of the functional role of this receptor subtype in discrete brain areas.
Life Sciences, 1995
In membranes of rat olfactory bulb, muscarinic receptor agonists stimulate basal adenylyl cyclase... more In membranes of rat olfactory bulb, muscarinic receptor agonists stimulate basal adenylyl cyclase activity . This response is inhibited by a number of muscarinic receptor antagonists with a rank order of potency suggesting the involvement of the M4 muscarinic receptor subtype. The stimulatory effect does not require Ca2+ and occurs independently of activation of phosphoinositide hydrolysis. Pretreatment with pertussis toxin completely prevents the muscarinic stimulation of adenylyl cyclase, indicating the participation of G proteins of the Gi/Go family. Immunological impairment of the G protein, Gs, also reduces the muscarinic response, whereas concomitant activation of Gs-coupled receptors by CRH or VIP results in a synergistic stimulation of adenylyl cyclase activity. Although these data suggest a role for Gs, a body of evidence indicates that the muscarinic receptors do not interact directly with this G protein. Moreover, the Ca2+/calmodulin (Ca2+/CaM)- and forskolin-stimulated enzyme activities are inhibited by muscarinic receptor activation in a pertussis toxin-sensitive manner and with a pharmacological profile similar to that observed for the stimulatory response. These data indicate that in rat olfactory bulb M4 muscarinic receptors exert a bimodal control on cyclic AMP formation through a sequence of events that may involve activation of Gi/Go proteins, synergistic interaction with Gs and differential modulation of Ca2+/CaM-independent and -dependent forms of adenylyl cyclase.
Journal of Neurochemistry, 2008
Nociceptin/orphanin FQ (N/OFQ) has been reported to inhibit dopamine (DA) release in basal gangli... more Nociceptin/orphanin FQ (N/OFQ) has been reported to inhibit dopamine (DA) release in basal ganglia mainly by acting on NOP receptors in substantia nigra and ventral tegmental area. We investigated whether N/OFQ could affect DA transmission by acting at either DA nerve endings or DA‐targeted post‐synaptic neurons. In synaptosomes of rat nucleus accumbens and striatum N/OFQ inhibited DA synthesis and tyrosine hydroxylase (TH) phosphorylation at Ser40 via NOP receptors coupled to inhibition of the cAMP/protein kinase A pathway. Immunofluorescence studies showed that N/OFQ preferentially inhibited phospho‐Ser40‐TH in nucleus accumbens shell and that in this subregion NOP receptors partly colocalized with either TH or DA D1 receptor positive structures. In accumbens and striatum N/OFQ inhibited DA D1 receptor‐stimulated cAMP formation, but failed to affect either adenosine A2A or DA D2 receptor regulation of cAMP. In accumbens slices, N/OFQ inhibited DA D1‐induced phosphorylation of NMDA...
Journal of Neurochemistry, 2002
Journal of Neurochemistry, 1993
We reported previously that in homogenates of rat olfactory bulb muscarinic and opioid receptor a... more We reported previously that in homogenates of rat olfactory bulb muscarinic and opioid receptor agonists stimulate adenylyl cyclase activity. In the present study we show that carbachol (CCh) and Leu-Enkephalin act synergistically with vasoactive intestinal peptide (VIP) and corticotropin-releasing hormone (CRH), but not with I-isoproterenol, in increasing cyclic AMP formation. The synergistic interaction consists of an increase in the maximal adenylyl cyclase activation without a significant change in the potency of each agonist. CCh also fails to affect CRH binding to olfactory bulb membranes. The synergism requires micromolar concentrations of GTP. Substitution of the stable GTP analog guanosine 5'-0-(3'-thiotriphosphate) for GTP allows the CRH stimulation, but abolishes the CCh enhancement of both basal and CRH-stimulated enzyme activities. Moreover, in vivo treatment of olfactory bulbs with pertussis toxin completely prevents the muscarink and opioid effects. Thus, the synergistic interaction appears to result from opioid-and muscarinic-induced activation of a pertussis toxin-sensitive GTP-binding protein which may potentiate the adenylyl cyclase stimulation by the stimulatory GTP-binding protein activated by either VIP or CRH receptors.
Journal of Neurochemistry, 2002
: In rat olfactory bulb, muscarinic and opioid receptor agonists stimulate basal adenylyl cyclase... more : In rat olfactory bulb, muscarinic and opioid receptor agonists stimulate basal adenylyl cyclase activity in a GTP‐dependent and pertussis toxin‐sensitive manner. However, in the present study, we show that in the same brain area activation of these receptors causes inhibition of adenylyl cyclase activity stimulated by Ca2+ and calmodulin (CaM) and by forskolin (FSK), two direct activators of the catalytic unit of the enzyme. The opioid and muscarinic inhibitions consist of a decrease of the maximal stimulation elicited by either CaM or FSK, without a change in the potency of these agents. [Leu5]Enkephalin and selective δ‐ and μ‐, but not κ‐, opioid receptors agonists inhibit the FSK stimulation of adenylyl cyclase activity with the same potencies displayed in stimulating basal enzyme activity. Similarly, the muscarinic inhibition of FSK‐stimulated adenylyl cyclase activity shows agonist and antagonist sensitivities similar to those characterizing the muscarinic stimulation of basal enzyme activity. Fluoride stimulation of adenylyl cyclase is not affected by either carbachol or [Leu5]enkephalin. In vivo treatment of olfactory bulb with pertussis toxin prevents both opioid and muscarinic inhibition of Ca2+/CaM‐ and FSK‐stimulated enzyme activities. These results indicate that in rat olfactory bulb δ‐ and μ‐opioid receptors and muscarinic receptors, likely of the M4 subtype, can exert a dual effect on cyclic AMP formation by interacting with pertussis toxin‐sensitive GTP‐binding protein(s) and possibly by affecting different molecular forms of adenylyl cyclase.
Journal of Neurochemistry, 2006
4P-Phorbol 12-myristate 13-acetate (FMA), added to a lysed mitochondria1 fraction of rat striatum... more 4P-Phorbol 12-myristate 13-acetate (FMA), added to a lysed mitochondria1 fraction of rat striatum, stimulates adenylate cyclase activity with an apparent time lag of-30 s. Half-maximal and maximal enzyme stimulations are obtained with 8 and 200 nM PM,4, respectively. The PMA stimulation is GTP dependent, reaching a maximum of-60% at 50 p L M GTP, and is associated with disappearance of the enzyme inhibition induced by micromolar concentrations of GTP. Enhancement of enzyme activity by cholera toxin and 3,4-dihydroxyphenylethylamine is amplified by PMA only at micromolar concentrations of GTP. PMA does not affect the enzyme stimulation by forskolin but reverses the inhibition of forskolin-stimulated enzyme by GTP. When guanyl-5'-yl-imidodiphosphate is substituted for GTP, PMA does not modify adenylate cyclase activity. Enzyme inhibition by acetylcholine, Leu-enkephalini, and R(-)N6-(2-phenylisopropyl)adenosine is magnified by PMA. Stimulation of adenylate cyclase by PMA is markedly reduced following EGTA treatment, is not observed when adenyl-5'-yl-imidodiphosphate is substituted for ATP as substrate for adenylate cyclase, and is enhanced by L-a-phosphatidyl-L-serine. Like PMA, 4P-phorbol 12,13-dibutyrate and l-oleoyl-2-acetylglycerol stimulate striatal adenylate cyclase, whereas 4P-phorbol and 4pphorbol 13-acetate are ineffective. The results indicate that phorbol esters increase striatal adenylate cyclase activity by reducing the GTP-induced inhibition of the enzyme, presumably as a result of protein kinase C activation.
Journal of Neural Transmission, 1992
In homogenates of female rat anterior pituitary, the azepine derivative B-HT 920 inhibited the fo... more In homogenates of female rat anterior pituitary, the azepine derivative B-HT 920 inhibited the forskolin-stimulated adenylate cyclase activity with an EC50 value of 0.35 microM. In male rat anterior pituitary, B-HT 920 curtailed the stimulation of adenylate cyclase activity by vasoactive intestinal peptide with an EC50 of 0.20 microM. In synaptic plasma membranes of rat striatum, B-HT 920 significantly reduced basal adenylate cyclase activity with an EC50 of 0.68 microM. Both in pituitary and striatum, the B-HT 920 inhibition was counteracted by the dopamine (DA) D2 receptor antagonist 1-sulpiride, but not by the alpha 2-adrenergic antagonist yohimbine. These results indicate that B-HT 920 is capable of activating DA D2 receptors negatively coupled to adenylate cyclase activity.
European Journal of Pharmacology, 2009
The clozapine metabolite N-desmethylclozapine (NDMC) has been recently shown to act at different ... more The clozapine metabolite N-desmethylclozapine (NDMC) has been recently shown to act at different neurotransmitter receptors and to display both antagonist and agonist activities. We have previously reported that in cells over-expressing the recombinant delta-opioid receptor NDMC behaved as partial agonist with high intrinsic activity, but its action at the receptors naturally expressed in human brain remained to be investigated. In the present study, we examined whether NDMC was able to bind to and activate delta-opioid receptors in membranes of post-mortem human frontal cortex. In radioligand binding assays, NDMC competition curves displayed high- (K(i)=26 nM) and low-affinity (K(i)=3 microM) components, whose proportion was regulated by guanine nucleotides in an agonist-like fashion. In functional assays, NDMC stimulated [(35)S]GTPgammaS binding (EC(50)=905 nM) and inhibited cyclic AMP formation (EC(50)=590 nM) as effectively as delta-opioid agonists, whereas clozapine was much less potent and efficacious and clozapine N-oxide was completely inactive. The NDMC agonist activity was potently antagonized by the delta-opioid antagonist naltrindole, but not by the micro-opioid receptor antagonist CTAP (D-phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2)) or the kappa-opioid antagonist nor-binaltorphimine. Moreover, blockade of either acetylcholine muscarinic, dopamine D(2) or serotonin 5HT(1A) receptors failed to affect NDMC agonist activity. These data demonstrate that at clinically relevant concentrations NDMC behaves as an efficacious agonist at delta-opioid receptors of human frontal cortex.
European Journal of Pharmacology, 1998
In membranes of olfactory tubercle and striatum, the selective muscarinic M4 receptor antagonist ... more In membranes of olfactory tubercle and striatum, the selective muscarinic M4 receptor antagonist muscarinic toxin 3 completely antagonized the acetylcholine-induced inhibition of forskolin- and dopamine D1 receptor-stimulated cyclic AMP formation with Ki values of 7 and 4 nM, respectively. In olfactory bulb, where acetylcholine stimulated basal adenylyl cyclase activity and inhibited forskolin-stimulated enzyme activity, muscarinic toxin 3 caused a partial antagonism of both acetylcholine effects with high potencies (Ki values = 4-6 nM). In frontal cortex, muscarinic toxin 3 counteracted the acetylcholine-induced potentiation of corticotropin-releasing hormone-stimulated cyclic AMP with a Ki of 58 nM, which is close to the toxin affinity for the muscarinic M1 receptor. In the same brain region, the acetylcholine inhibition of forskolin-stimulated enzyme activity was not affected by muscarinic toxin 3. In microdissected regions of the hippocampus, a significant portion (33-48%) of the acetylcholine inhibition of forskolin-stimulated adenylyl cyclase activity was blocked by muscarinic toxin 3 with Ki values (6-8 nM) consistent with the involvement of muscarinic M4 receptors. These data show that muscarinic toxin 3 discriminates between adenylyl cyclase-coupled muscarinic receptors and demonstrate the utility of the toxin in identifying the relative contribution by the muscarinic M4 receptor subtype.
European Journal of Pharmacology, 2003
The ability of 2,6 Di-tert-butyl-4-(-hydroxy-2,2-dimethyl-propyl)-phenol (CGP7930), a positive al... more The ability of 2,6 Di-tert-butyl-4-(-hydroxy-2,2-dimethyl-propyl)-phenol (CGP7930), a positive allosteric modulator of GABA(B) receptors, to regulate GABA(B) receptor-induced stimulation and inhibition of adenylyl cyclase activity in rat brain was investigated. In olfactory bulb granule cell layer and in frontal cortex, CGP7930 potentiated the stimulatory effects of (-)-baclofen and gamma-aminobutyric acid (GABA) on basal and corticotropin-releasing hormone-stimulated adenylyl cyclase activities, respectively. In these stimulatory responses, CGP7930 enhanced both agonist potencies and maximal effects. When GABA(B) receptor-mediated inhibition of forskolin-stimulated adenylyl cyclase activity of frontal cortex was examined, CGP7930 increased the agonist potencies but failed to affect the maximal effect of (-)-baclofen and modestly increased that of GABA. Similar results were obtained for the inhibition of Ca(2+)/calmodulin-stimulated adenylyl cyclase in striatum and cerebellum. Western blot analysis of each membrane preparation showed the presence of GABA(B2) receptor subunit, a putative site of action of CGP7930. These data indicate that CGP7930 positively modulates brain GABA(B) receptors coupled to either stimulation or inhibition of cyclic AMP signalling.