Pierluigi Onali

Stress-induced increase in 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the cerebral cortex and in n. accumbens: reversal by diazepam.

Abstract The effect of electrical foot shock stress on dopamine and DOPAC levels was examined in the frontal cortex, nucleus accumbens, striatum, substantia nigra and medial basal hypothalamus of rats. DA content did not change after stress in any of the structures analyzed except in the substantia nigra in which DA level decreased by about 35% following 20, 60 or 180 min of stress. DOPAC level did not change in the striatum, medial basal hypothalamus and substantia nigra, but increased in the frontal cortex and in n. accumbens by about 75% and 40%, respectively. Pretreatment with diazepam, but not with pentobarbital, prevented stress-induced increased in DOPAC levels.

Snake toxins with high selectivity for subtypes of muscarinic acetylcholine receptors.

There are five subtypes of muscarinic acetylcholine receptors (M(1) to M(5)) which control a large number of physiological processes, such as the function of heart and smooth muscles, glandular secretion, release of neurotransmitters, gene expression and cognitive functions as learning and memory. A selective ligand is very useful for studying the function of a subtype in presence of other subtypes, which is the most common situation, since a cell or an organ usually has several subtypes. There are many non-selective muscarinic ligands, but only few selective ones. Mambas, African snakes of genus Dendroaspis have toxins, muscarinic toxins, that are selective for M(1), M(2) and M(4) receptors. They consist of 63-66 amino acids and four disulfides which form four loops. They are members of a large group of snake toxins, three-finger toxins; three loops are extended like the middle fingers of a hand and the disulfides and the shortest loop are in the palm of the hand. Some of the toxins target the allosteric site which is located in a cleft of the receptor molecule close to its extracellular part. A possible explanation to the good selectivity is that the toxins bind to the allosteric site, but because of their size they probably also bind to extracellular parts of the receptors which are rather different in the various subtypes. Some other allosteric ligands also have good selectivity, the alkaloid brucine and derivatives are selective for M(1), M(3) and M(4) receptors. Muscarinic toxins have been used in several types of experiments. For instance radioactively labeled M(1) and M(4) selective toxins were used in autoradiography of hippocampus from Alzheimer patients. One significant change in the receptor content was detected in one region of the hippocampus, dentate gyrus, where M(4) receptors were reduced by 50% in patients as compared to age-matched controls. Hippocampus is essential for memory consolidation. M(4) receptors in dentate gyrus may play a role, since they decreased in Alzheimers disease which destroys the memory. Another indication of the role of M(4) receptors for memory is that injection of the M(4) selective antagonist muscarinic toxin 3 (M(4)-toxin 1) into rat hippocampus produced amnesia.

Rat striatal muscarinic receptors coupled to the inhibition of adenylyl cyclase activity: potent block by the selective m4 ligand muscarinic toxin 3 (MT3)

1 In rat striatal membranes, muscarinic toxin 3 (MT3), a selective ligand of the cloned m4 receptor subtype, antagonized the acetylcholine (ACh) inhibition of forskolin‐and dopamine D1 receptor‐stimulated adenylyl cyclase activities with pA2 values of 8.09 and 8.15, respectively. 2 In radioligand binding experiments, MT3 increased the Ki but did not change the Bmax value of [3H]‐ N‐methylscopolamine (3H]‐NMS) binding to rat striatal muscarinic receptors. The toxin displaced the major portion of the [3H]‐NMS binding sites with a Ki of 8.0 nM. 3 In rat myocardium, MT3 antagonized the ACh inhibition of adenylyl cyclase with a Ki value of 860 nM. 4 In rat cerebral cortical membranes prelabelled with [3H]‐myo‐inositol, MT3 counteracted the methacholine stimulation of [3H]‐inositol phosphates formation with a Ki value of 113 nM. 5 The present study shows that MT3 is a potent antagonist of the striatal muscarinic receptors coupled to inhibition of adenylyl cyclase activity. This finding provides strong evidence for the classification of these receptors as pharmacologically equivalent to the m4 gene product (M4). On the other hand, the weaker potencies of MT3 in antagonizing the muscarinic responses in cerebral cortex and in the heart are consistent with the reported lower affinities of the toxin for the cloned ml and m2 receptor subtypes, respectively.

Evidence that adenosine A2 and dopamine autoreceptors antagonistically regulate tyrosine hydroxylase activity in rat striatal synaptosomes

Incubation of rat striatal synaptosomes with the adenosine receptor agonist 2-chloroadenosine (2-CADO) produced a concentration-dependent increase of dopamine (DA) synthesis (about 50% of control value). The effect was not additive with the stimulation produced by either 10 microM forskolin or 2 mM dibutyryl cyclic AMP. Pretreatment of striatal synaptosomes with 2-CADO produced an activation of tyrosine hydroxylase (TH) which withstood washing and lysing of the tissue. This activation was largely independent of the presence of Ca2+ ion in the preincubation medium and, when analyzed as a function of different concentrations of the pterin cofactor 6-methyl-5,6,7,8-tetrahydropterin (0.08-0.4 mM), it was associated with an apparent increase in the Vmax of the enzyme. Quinpirole, a selective D2 DA receptor agonist, reduced control synaptosomal DA synthesis and caused a persistent inhibition of TH activity. When added together with 2-CADO, quinpirole depressed the stimulation of DA synthesis and TH activity produced by the adenosine analog. The effect of quinpirole was stereospecifically antagonized by the D2 DA antagonist sulpiride. Quinpirole also inhibited the activation of TH elicited by a submaximal concentration of forskolin, but not that produced by dibutyryl cyclic AMP. The inhibitory effect of quinpirole on basal and 2-CADO-stimulated TH activities was mimicked by DA. These results indicate that presynaptic DA autoreceptors and adenosine A2 receptors interact antagonistically in controlling DA synthesis in rat striatal synaptosomes presumably by exerting opposite inputs on a presynaptic adenylate cyclase system.

Mixed Agonist–Antagonist Properties of Clozapine at Different Human Cloned Muscarinic Receptor Subtypes Expressed in Chinese Hamster Ovary Cells

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 [3H]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 [35S] GTPγS 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.

Inhibition of acetylcholine muscarinic M1 receptor function by the M1‐selective ligand muscarinic toxin 7 (MT‐7)

MT‐7 (1–30 nM), a peptide toxin isolated from the venom of the green mamba Dendroaspis angusticeps and previously found to bind selectively to the muscarinic M1 receptor, inhibited the acetylcholine (ACh)‐stimulated [35S]‐guanosine‐5′‐O‐(3‐thio)triphosphate ([35S]‐GTPγS) binding to membranes of Chinese hamster ovary (CHO) cells stably expressing the cloned human muscarinic M1 receptor subtype. MT‐7 failed to affect the ACh‐stimulated [35S]‐GTPγS binding in membranes of CHO cells expressing either the M2, M3 or M4 receptor subtype. In N1E‐115 neuroblastoma cells endogenously expressing the M1 and M4 receptor subtypes, MT‐7 (0.3–3.0 nM) inhibited the carbachol (CCh)‐stimulated inositol phosphates accumulation, but failed to affect the CCh‐induced inhibition of pituitary adenylate cyclase activating polypeptide (PACAP) 38‐stimulated cyclic AMP accumulation. In both CHO/M1 and N1E‐115 cells the MT‐7 inhibition consisted in a decrease of the maximal agonist effect with minimal changes in the agonist EC50 value. In CHO/M1 cell membranes, MT‐7 (0.05–25 nM) reduced the specific binding of 0.05, 1.0 and 15 nM [3H]‐N‐methylscopolamine ([3H]‐NMS) in a concentration‐dependent manner, but failed to cause a complete displacement of the radioligand. Moreover, MT‐7 (3 nM) decreased the dissociation rate of [3H]‐NMS by about 5 fold. CHO/M1 cell membranes preincubated with MT‐7 (10 nM) and washed by centrifugation and resuspension did not recover control [3H]‐NMS binding for at least 8 h at 30°C. It is concluded that MT‐7 acts as a selective noncompetitive antagonist of the muscarinic M1 receptors by binding stably to an allosteric site.

Interferon‐β induces apoptosis in human SH‐SY5Y neuroblastoma cells through activation of JAK–STAT signaling and down‐regulation of PI3K/Akt pathway

J. Neurochem. (2010) 115, 1421–1433.

Positive regulation of GABAB receptors dually coupled to cyclic AMP by the allosteric agent CGP7930

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.

Neuropeptide Y inhibits forskolin-stimulated adenylate cyclase activity in rat hippocampus

Neuropeptide Y inhibited the forskolin-stimulated adenylate cyclase activity in rat hippocampus with the half-maximal effect occurring at 73 nM. The maximal inhibition corresponded to a 17-22% decrease of the control level of enzyme activity. The effect of neuropeptide Y was mimicked by the peptide YY but not by the avian pancreatic polypeptide and required micromolar concentrations of GTP. These results indicate that, in the brain, the inhibition of adenylate cyclase activity constitutes a mechanism by which the receptor for neuropeptide Y transduces its signal.

GABAB receptor‐mediated stimulation of adenylyl cyclase activity in membranes of rat olfactory bulb

Previous studies have shown that GABAB receptors facilitate cyclic AMP formation in brain slices likely through an indirect mechanism involving intracellular second messengers. In the present study, we have investigated whether a positive coupling of GABAB receptors to adenylyl cyclase could be detected in a cell‐free preparation of rat olfactory bulb, a brain region where other Gi/Go‐coupled neurotransmitter receptors have been found to stimulate the cyclase activity. The GABAB receptor agonist (−)‐baclofen significantly increased basal adenylyl cyclase activity in membranes of the granule cell and external plexiform layers, but not in the olfactory nerve‐glomerular layer. The adenylyl cyclase stimulation was therefore examined in granule cell layer membranes. The (−)‐baclofen stimulation (pD2=4.53) was mimicked by 3‐aminopropylphosphinic acid (pD2=4.60) and GABA (pD2=3.56), but not by (+)‐baclofen, 3‐aminopropylphosphonic acid, muscimol and isoguvacine. The stimulatory effect was counteracted by the GABAB receptor antagonists CGP 35348 (pA2=4.31), CGP 55845 A (pA2=7.0) and 2‐hydroxysaclofen (pKi=4.22). Phaclofen (1 mM) was inactive. The (−)‐baclofen stimulation was not affected by quinacrine, indomethacin, nordihydroguaiaretic acid and staurosporine, but was completely prevented by pertussis toxin and significantly reduced by the α subunit of transducin, a βγ scavenger. The βγ subunits of transducin stimulated the cyclase activity and this effect was not additive with that produced by (−)‐baclofen. In the external plexiform and granule cell layers, but not in the olfactory nerve‐glomerular layer, (−)‐baclofen enhanced the adenylyl cyclase stimulation elicited by the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) 38. Conversely, the adenylyl cyclase activity stimulated by either forskolin or Ca2+/calmodulin‐(Ca2+/CaM) was inhibited by (−)‐baclofen in all the olfactory bulb layers examined. These data demonstrate that in specific layers of rat olfactory bulb activation of GABAB receptors enhances basal and neurotransmitter‐stimulated adenylyl cyclase activities by a mechanism involving βγ subunits of Gi/Go. This positive coupling is associated with a widespread inhibitory effect on forskolin‐ and Ca2+/CaM‐stimulated cyclic AMP formation.