Pier Luigi Onali

Effects of clozapine on rat striatal muscarinic receptors coupled to inhibition of adenylyl cyclase activity and on the human cloned m4 receptor

1 Clozapine has recently been claimed to behave as a selective and full agonist at the cloned m4 muscarinic receptor artificially expressed in Chinese hamster ovary (CHO) cells. In the present study we have investigated whether clozapine could activate the rat striatal muscarinic receptors coupled to the inhibition of adenylyl cyclase activity, considered as pharmacologically equivalent to the m4 gene product. In addition, we have examined the effect of the drug on various functional responses following the activation of the cloned m4 receptor expressed in CHO cells. 2 In rat striatum, clozapine (1u2003nM–10u2003μM) caused a slight inhibition of forskolin‐stimulated adenylyl cyclase activity, which was not counteracted by 10u2003μM atropine. On the other hand, clozapine antagonized the inhibitory effect of acetylcholine with a pA2 value of 7.51. Moreover, clozapine (1u2003μM) failed to inhibit dopamine D1 receptor stimulation of adenylyl cyclase activity, but counteracted the inhibitory effect of carbachol (CCh). Clozapine displaced [3H]‐N‐methylscopolamine ([3H]‐NMS) bound to striatal M4 receptors with a monophasic inhibitory curve and a pKi value of 7.69. The clozapine inhibition was not affected by the addition of guanosine‐5′‐O‐(thio)triphosphate (GTPγS). 3 In intact CHO cells, clozapine inhibited forskolin‐stimulated cyclic AMP accumulation with an EC50 of 31u2003nM. This effect was antagonized by atropine. CCh produced a biphasic effect on cyclic AMP levels, inhibiting at concentrations up to 1u2003μM (EC50=50u2003nM) and stimulating at higher concentrations (EC50=7u2003μM). Clozapine (0.3–5u2003μM) antagonized the CCh stimulation of cyclic AMP with a pKi value of 7.47. Similar results were obtained when the adenylyl cyclase activity was assayed in CHO cell membranes. 4 In CHO cells pretreated with the receptor alkylating agent 1‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline (10u2003μM), the maximal inhibitory effect of clozapine on cyclic AMP formation was markedly reduced, whereas the CCh inhibitory curve was shifted to the right with no change in the maximum. 5 As in rat striatum, in CHO cell membranes the displacement of [3H]‐NMS binding by clozapine yielded a monophasic curve which was not affected by GTPγS. 6 Clozapine (10u2003nM–10u2003μM) had a small stimulant effect (∼20%) on the binding of [35S]‐GTPγS to CHO cell membranes, whereas CCh caused a 250% increase of radioligand binding. Moreover, clozapine (50u2003nM–5u2003μM) antagonized the CCh‐stimulated [35S]‐GTPγS binding with a pA2 value of 7.48. 7 These results show that at the striatal M4 receptors clozapine is a potent and competitive antagonist, whereas at the cloned m4 receptor it elicits both agonist and antagonist effects. Thus, clozapine behaves as a partial agonist, rather than as a full agonist, at the m4 receptor subtype, with intrinsic activity changing as a function of the coupling efficiency of the receptor to effector molecules.

Muscarinic M4 receptor inhibition of dopamine D1-like receptor signalling in rat nucleus accumbens.

Several studies have indicated the occurrence of an antagonistic interaction between muscarinic and dopamine D1-like receptors in the ventral striatum, but the subtype(s) of muscarinic receptor involved has not been characterized. We show that in membranes of rat nucleus accumbens, carbachol inhibited the stimulation of adenylyl cyclase activity by dopamine and the dopamine D1-like receptor agonist (+/-)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine without affecting the binding properties of dopamine to dopamine D1-like receptors. The carbachol inhibition was competitively counteracted by receptor antagonists with a rank order of potency typical of the involvement of the muscarinic M(4) receptor subtype. Moreover, muscarinic toxin 3, a selective muscarinic M(4) receptor antagonist, completely blocked the carbachol inhibition, whereas muscarinic toxin 7, a selective muscarinic M(1) receptor antagonist, had no effect. The muscarinic inhibition occurred to a similar extent in the core and shell regions. These data demonstrate that in nucleus accumbens, muscarinic M(4) receptors exert a direct inhibitory control on dopamine D1-like receptor signalling.

Direct Agonist Activity of Tricyclic Antidepressants at Distinct Opioid Receptor Subtypes

Tricyclic antidepressants (TCAs) have been reported to interact with the opioid system, but their pharmacological activity at opioid receptors has not yet been elucidated. In the present study, we investigated the actions of amoxapine, amitriptyline, nortriptyline, desipramine, and imipramine at distinct cloned and native opioid receptors. In Chinese hamster ovary (CHO) cells expressing δ-opioid receptors (CHO/DOR), TCAs displaced [3H]naltrindole binding and stimulated guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding at micromolar concentrations with amoxapine displaying the highest potency and efficacy. Amoxapine and amitriptyline inhibited cyclic AMP formation and induced the phosphorylation of signaling molecules along the extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol-3 kinase pathways. Amoxapine also activated δ-opioid receptors in rat dorsal striatum and nucleus accumbens and human frontal cortex. In CHO cells expressing κ-opioid receptors (CHO/KOR), TCAs, but not amoxapine, exhibited higher receptor affinity and more potent stimulation of [35S]GTPγS binding than in CHO/DOR and effectively inhibited cyclic AMP accumulation. Amitriptyline regulated ERK1/2 phosphorylation and activity in CHO/KOR and C6 glioma cells endogenously expressing κ-opioid receptors, and this effect was attenuated by the κ-opioid antagonist nor-binaltorphimine. In rat nucleus accumbens, amitriptyline slightly inhibited adenylyl cyclase activity and counteracted the inhibitory effect of the full κ agonist trans-(−)-3,4dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U50,488). At the cloned μ-opioid receptor, TCAs showed low affinity and no significant agonist activity. These results show that TCAs differentially regulate opioid receptors with a preferential agonist activity on either δ or κ subtypes and suggest that this property may contribute to their therapeutic and/or side effects.

Bimodal regulation of cyclic AMP by muscarinic receptors. Involvement of multiple G proteins and different forms of adenylyl cyclase.

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.

N-Desmethylclozapine, a Major Clozapine Metabolite, Acts as a Selective and Efficacious |[delta]|-Opioid Agonist at Recombinant and Native Receptors

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 δ-opioid receptor (CHO/DOR), NDMC behaved as a full agonist both in stimulating [35S]GTPγS binding (pEC50=7.24) and in inhibiting cyclic AMP formation (pEC50=6.40). NDMC inhibited [3H]naltrindole binding to CHO/DOR membranes with competition curves that were modulated by guanine nucleotides in an agonist-like manner. Determination of intrinsic efficacies by taking into consideration both the maximal [35S]GTPγS 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 δ-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 κ-opioid receptor and was inactive at μ-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 δ-opioid receptor. Moreover, in membranes of different brain regions, NDMC stimulated [35S]GTPγS 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 δ-opioid receptor agonist and suggest that this unique property may contribute, at least in part, to the clinical actions of the atypical antipsychotic clozapine.

Allosteric modulation of GABAB receptor function in human frontal cortex

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.

Type I interferons impair BDNF-induced cell signaling and neurotrophic activity in differentiated human SH-SY5Y neuroblastoma cells and mouse primary cortical neurons

J. Neurochem. (2012) 122, 58–71.

δ-Opioid receptors stimulate GLUT1-mediated glucose uptake through Src- and IGF-1 receptor-dependent activation of PI3-kinase signalling in CHO cells

BACKGROUND AND PURPOSE Although opioids have been reported to affect glucose homeostasis, relatively little is known on the role of δ‐opioid receptors. We have investigated the regulation of glucose transport by human δ‐opioid receptors expressed in Chinese hamster ovary cells.

Agonist activity of N-desmethylclozapine at δ-opioid receptors of human frontal cortex

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.

Agonist activity of naloxone benzoylhydrazone at recombinant and native opioid receptors.

In the present study, we examined the pharmacological activity of the putative κ3‐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. At the μ‐opioid receptor (MOR), NalBzoH stimulated guanosine‐5′‐O‐(3‐[35S]thio)triphosphate ([35S]GTPγS) binding (pEC50=8.59) and inhibited cyclic AMP accumulation (pEC50=8.74) with maximal effects (Emax) 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. At the κ‐opioid receptor (KOR), NalBzoH stimulated [35S]GTPγS binding (pEC50=9.70) and inhibited cyclic AMP formation (pEC50=9.45) as effectively as the selective KOR agonist (−)‐U‐50,488. The NalBzoH effect was blocked by the KOR antagonist nor‐binaltorphimine (nor‐BNI) (pKi=10.30). In CHO cells expressing the δ‐opioid receptor (DOR), NalBzoH increased [35S]GTPγS binding (pEC50=8.49) and inhibited cyclic AMP formation (pEC50=8.61) almost as effectively as the DOR agonist DPDPE. Naltrindole (NTI), a selective DOR antagonist, completely blocked the response to NalBzoH (pKi of 10.40). 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. 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. In rat striatum, NalBzoH enhanced [35S]GTPγS 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. 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.