AMP-activated protein kinase (AMPK) and δ-opioid receptors (DORs) are both involved in controllin... more AMP-activated protein kinase (AMPK) and δ-opioid receptors (DORs) are both involved in controlling cell survival, energy metabolism, and food intake, but little is known on the interaction between these two signaling molecules. Here we show that activation of human DORs stably expressed in Chinese hamster ovary (CHO) cells increased AMPK activity and AMPK phosphorylation on Thr172. DOR-induced AMPK phosphorylation was prevented by pertussis toxin, reduced by protein kinase A (PKA) activators, and unaffected by PKA, transforming growth factor-β-activated kinase 1, mitogen-activated protein kinase, and protein kinase C inhibitors. Conversely, the DOR effect was reduced by Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) inhibition, apyrase treatment, G(q/11) antagonism, and blockade of P2 purinergic receptors. Apyrase treatment also depressed DOR stimulation of intracellular Ca(2+) concentration, whereas P2 receptor antagonism blocked DOR stimulation of inositol phosphate acc...
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, 2010
Tricyclic antidepressants (TCAs) have been reported to interact with the opioid system, but their... more 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 -opioid receptors (CHO/DOR), TCAs displaced [3H]naltrindole binding and stimulated guanosine 5-O- (3-[35S]thio)triphosphate ([35S]GTPS) 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 -opioid receptors in rat dorsal striatum and nucleus accumbens and human frontal cortex. In CHOcells expressing -opioid recep...
Background and purpose
Although clinically useful for their immunomodulatory, antiproliferative ... more Background and purpose
Although clinically useful for their immunomodulatory, antiproliferative and antiviral properties, type I interferons (IFNs) are involved in the pathogenesis of several neurodegenerative/neuroinflammatory diseases. In the present study, we investigated the ability of cholinergic stimulation to protect from IFN-β-induced neuronal apoptosis. Experimental approach
The effects of the cholinergic agonist carbachol (CCh) on IFN-β-induced apoptosis of human SH-SY5Y neuroblastoma cells were examined by using Western blot, immunofluorescence and cytofluorimetry. The involvement of muscarinic acetylcholine receptors (mAChRs) was assessed by using selective antagonists and siRNA transfection. Pharmacological inhibitors and over-expression of ERK2 and an ERK2 constitutively active form (ERK2-CA) were employed to study ERK1/2 signalling. The effects of oxotremorine-M (Oxo-M) on IFN-β-induced apoptosis of mouse hippocampal neurons were examined by measuring cleaved caspase 3 expression. Key results
In SH-SY5Y cells, CCh inhibited IFN-β-induced mitochondrial cytochrome c release, activation of caspases 9, 7 and 3, PARP cleavage and DNA fragmentation. The anti-apoptotic effect of CCh was mediated by M3 mAChRs, blocked by the Gq/11 antagonist YM254890 and the protein kinase C inhibitor Go 6983, impaired by inhibition of ERK1/2 pathway, potentiated by over-expression of ERK2, and mimicked by ERK2-CA. Blockade of JNK activation enhanced the CCh anti-apoptotic response. IFN-β inhibited JNK activation and up-regulated CCh-induced ERK1/2 signalling. In hippocampal neurons, IFN-β-induced apoptosis was reduced by Oxo-M and the effect was antagonized by blockade of M1/M3 mAChRs and ERK1/2. Conclusions and implications
Stimulation of mAChRs counteracted IFN-β-induced neuronal apoptosis through the activation of ERK1/2 signalling. The data indicate that activation of ERK1/2-coupled mAChRs may be an effective strategy for preventing IFNs neurotoxicity.
Although clinically useful for the treatment of various diseases, type I interferons (IFNs) have ... more Although clinically useful for the treatment of various diseases, type I interferons (IFNs) have been implicated as causative factors of a number of neuroinflammatory disorders characterized by neuronal damage and altered CNS functions. As neurotrophin 3 (NT3) plays a critical role in neuroprotection, we examined the effects of IFN-β on the signalling and functional activity of the NT3/TrkC system. We found that prolonged exposure of differentiated human SH-SY5Y neuroblastoma cells to IFN-β impaired the ability of NT3 to induce transphosphorylation of the full-length TrkC receptor (TrkC-FL) and the phosphorylation of downstream signalling molecules, including PLCγ1, Akt, GSK-3β and ERK1/2. NT3 was effective in protecting the cells against apoptosis triggered by serum withdrawal or thapsigargin but not IFN-β. Prolonged exposure to the cytokine had little effects on TrkC-FL levels but markedly enhanced the messenger RNA (mRNA) and protein levels of the truncated isoform TrkC-T1, a dominant-negative receptor that inhibits TrkC-FL activity. Cell depletion of TrkC-T1 by small interfering RNA (siRNA) treatment enhanced NT3 signalling through TrkC-FL and allowed the neurotrophin to counteract IFN-β-induced apoptosis. Furthermore, the upregulation of TrkC-T1 by IFN-β was associated with the inhibition of NT3-induced recruitment of the scaffold protein tamalin to TrkC-T1 and tamalin tyrosine phosphorylation. These data indicate that IFN-β exerts a negative control on NT3 pro-survival signalling through a novel mechanism involving the upregulation of TrkC-T1.
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. stably expressed in CHO-K1 cells. ERK1/2 stimulation by antidepressants and LPA was suppressed by pertussis toxin and inhibition of Src, phosphatidylinositol-3 kinase and insulin-like growth factor-I receptor (IGF-IR) activities. Antidepressants and LPA induced tyrosine phosphorylation of IGF-IR and insulin receptor-substrate-1 through LPA1 and Src. Prolonged exposure of CHO-K1 fibroblasts to either mianserin, mirtazapine or LPA enhanced cell proliferation as indicated by increased [3H]-thymidine incorporation and Ki-67 immunofluorescence. This effect was inhibited by blockade of LPA1- and ERK1/2 activity. These data provide evidence that different antidepressants induce LPA1 activation, leading to receptor tyrosine kinase transactivation, stimulation of ERK1/2 signaling and enhanced cell proliferation.
Gq/11-coupled muscarinic acetylcholine receptors (mAChRs) belonging to M1, M3 and M5 subtypes hav... more Gq/11-coupled muscarinic acetylcholine receptors (mAChRs) belonging to M1, M3 and M5 subtypes have been shown to activate the metabolic sensor AMP-activated protein kinase (AMPK) through Ca(2+)/calmodulin-dependent protein kinase kinase-β (CaMKKβ)-mediated phosphorylation at Thr172. However, the source of Ca(2+) required for this response has not been yet elucidated. Here, we investigated the involvement of store-operated Ca(2+) entry (SOCE) in AMPK activation by pharmacologically defined M3 mAChRs in human SH-SY5Y neuroblastoma cells. In Ca(2+)-free medium the cholinergic agonist carbachol (CCh) caused a transient increase of phospho-Thr172 AMPK that rapidly ceased within 2min. Conversely, in the presence of extracellular Ca(2+) CCh-induced AMPK phosphorylation lasted for at least 180min. The SOCE modulator 2-aminoethoxydiphephenyl borate (2-APB), at a concentration (50μM) that suppressed CCh-induced intracellular Ca(2+) ([Ca(2+)]i) plateau, inhibited CCh-induced AMPK phosphorylation. CCh triggered the activation of the endoplasmic reticulum Ca(2+) sensor stromal interaction molecule (STIM) 1, as indicated by redistribution of STIM1 immunofluorescence into puncta, and promoted the association of STIM1 with the SOCE channel component Orai1. Cell depletion of STIM1 by siRNA treatment reduced both CCh-induced [Ca(2+)]i plateau and AMPK activation. M3 mAChRs increased glucose uptake and this response required extracellular Ca(2+) and was inhibited by 2-APB, STIM1 knockdown, CaMKKβ and AMPK inhibitors, and adenovirus infection with dominant negative AMPK. Thus, the study provides evidence that SOCE is required for sustained activation of AMPK and stimulation of downstream glucose uptake by M3 mAChRs and suggests that SOCE is a critical process connecting M3 mAChRs to the control of neuronal energy metabolism.
Type I interferons (IFNs) induce apoptosis of neuroblastoma cells, but the molecular mechanisms r... more Type I interferons (IFNs) induce apoptosis of neuroblastoma cells, but the molecular mechanisms regulating this event have not been completely elucidated. Here, we investigated the role of p38 mitogen activated protein kinase (MAPK) activity, a key regulator of apoptosis and a known modulator of IFN-induced responses in non-neuronal cells. We show that in SH-SY5Y human neuroblastoma cells IFN-β induced a delayed and sustained increase of p38 MAPK activity through a novel mechanism involving the sequential activation of Janus kinase-signal transducer and activator of transcription-1 signalling, enhanced expression of the NADPH oxidase catalytic subunit gp91(phox), increased reactive oxygen species production and stimulation of the MAPK kinase kinase transforming growth factor-β-activated kinase 1. Either blockade of p38 MAPK by the second generation inhibitors BIRB0796 and VX745 or siRNA knockdown of p38α MAPK enhanced IFN-β-induced apoptosis of neuroblastoma cells. Exposure to IFN-β increased the phosphorylation of the small heat shock protein HSP27 at Ser15, Ser78 and Ser82 with a time course similar to p38 MAPK activation and this response was suppressed by either p38α MAPK depletion or pharmacological inhibition of p38 MAPK and MAPK-activated protein kinase 2 (MK2). Either silencing of HSP27 expression by siRNA or MK2 inhibition potentiated IFN-β-induced apoptotic death. These results indicate that IFN-β-induced apoptosis of human SH-SY5Y neuroblastoma cells is associated with a long-lasting up-regulation of p38 MAPK activity, stimulation of MK2 and phosphorylation of the pro-survival protein HSP27. Moreover, the data show that inhibition of p38 MAPK signalling potentiates the anti-neuroblastoma activity of the cytokine, indicating that this pathway mediates a counter-regulatory response.
Alterations in the olfactory system and impairment of olfactory function have been reported in se... more Alterations in the olfactory system and impairment of olfactory function have been reported in several neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases (1, 2). The participation of proteinase activated receptors (PARs) in these neuropathological ...
Acetylcholine inhibits, by 30-40%, the basal adenylate cyclase activity of purified synaptic plas... more Acetylcholine inhibits, by 30-40%, the basal adenylate cyclase activity of purified synaptic plasma membranes prepared from rat striatum (EC50 = 3 microM). Cholinergic receptor agonists inhibit this cyclase activity with the following rank order of potency: oxtremorine greater than acetylcholine greater than arecoline greater than methacholine greater than or equal to muscarine greater than or equal to carbachol greater than bethanechol. Nicotine fails to inhibit the cyclase, and d-tubocurarine fails to inhibit the action of cholinergic drugs. In contrast, atropine and scopolamine antagonize the effect of acetylcholine. The enzyme inhibition elicited by acetylcholine requires the presence of GTP, and disappears after intrastriatal injection of kainic acid. From these results, we infer that striatal adenylate cyclase can be modulated by muscarinic receptors.
Opioid receptors located in the ventral tegmental area are known to regulate dopamine (DA) releas... more Opioid receptors located in the ventral tegmental area are known to regulate dopamine (DA) release from mesocortical afferents to medial prefrontal cortex (mPFC) but little is known on whether in this cortical region activation of opioid receptors affect DA receptor signaling. In the present study we show that in mouse mPFC concomitant activation of either d-or l-opioid receptors, but not j-opioid receptors, potentiated DA D1-like receptor-induced stimulation of adenylyl cyclase activity through a G protein bc subunit-dependent mechanism. In tissue slices of mPFC, the combined addition of the opioid agonist leu-enkephalin and the DA D1-like receptor agonist SKF 81297 produced more than additive increase in the phosphorylation state of AMPA and NMDA receptor subunits GluR1 and NR1, respectively. Moreover, in primary cultures of mouse frontal cortex neurons, DA D1-like receptor-induced Ser133 phosphorylation of the transcription factor cyclic AMP responsive element binding protein was potentiated by concurrent stimulation of opioid receptors. Double immunofluorescence analysis of cultured cortical cells indicated that a large percentage of DA D1 receptor positive cells expressed either d-or l-opioid receptor immunoreactivity. These data indicate that in mouse mPFC activation of land d-opioid receptors enhances DA D1-like receptor signaling likely through converging regulatory inputs on bc-stimulated adenylyl cyclase isoforms. This previously unrecognized synergistic interaction may selectively affect DA D1 transmission at specific postsynaptic sites where the receptors are co-localized and may play a role in prefrontal DA D1 regulation of opioid addiction.
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 and α-amino-3-hydroxy-5-methylisoxazole-4-propionate glutamate receptors, whereas in primary cultures of accumbal cells, which were found to coexpress NOP and DA D1 receptors, N/OFQ curtailed DA D1 receptor-induced cAMP-response element-binding protein phosphorylation. Thus, in accumbens and striatum N/OFQ exerts an inhibitory constraint on DA transmission by acting on either pre-synaptic NOP receptors inhibiting TH phosphorylation and DA synthesis or post-synaptic NOP receptors selectively down-regulating DA D1 receptor signaling.
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 /-isoproterenol, in increasing cyclic AMP formation. The synergistic interaction consists of an increase in the maximal a0denylyl cyclase activation without a significant change in the potency of each agonist. CCh also fails to affect 125ICRH binding to olfactory bulb membranes. The synergism requires micromolar concentrations of GTP. Substitution of the stable GTP analog guanosine 5′-O-(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 muscarinic 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.
1 In rat striatal membranes, muscarinic toxin 3 (MT3), a selective ligand of the cloned m4 recept... more 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 DI receptorstimulated adenylyl cyclase activities with pA2 values of 8.09 and 8.15, respectively. 2 In radioligand binding experiments, MT3 increased the Kd but did not change the Bm value of [3H]-N-methylscopolamine (3H]-NMS) binding to rat striatal muscarinic receptors. The toxin displaced the major portion of the [3HJ-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.
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. Key Words: Phorbol esters-Adenylate cyclase-Protein kinase C-Rat striatum. Olianas M. C. and Onali P. Phorbol esters increase GTP-dependent adenylate cyclase activity in rat brain striatal membranes. J . Neurochem. 47, 890-897 (1986). Ahhreviutions used; AMPP(NH)P, adenyl-5'-yl-imidodiphos-phate; DA or dopamine, 3,4-dihydroxyphenylethylamine; Gpp(NH)p, guanyl-5'-yl-imidodiphosphate; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; N, and Ni, stimulatory and inhibitory guanine nucleotide-binding protein of adenylate cyclase, respectively; R( -)PIA, R( -)-N6-(2-phenyliso-propy1)adenosine; PMA, 4pphorbol 12-myristate 13-acetate.
The intraperitoneal administration of SCH 23390, a selective D l receptor antagonist, produced ca... more The intraperitoneal administration of SCH 23390, a selective D l receptor antagonist, produced catalepsy and increased the firing rate of dopamine (DA) neurons in the substantia nigra (SN-DA neurons). Maximal increase (110%) was of the same magnitude as that produced by a supramaximal dose of haloperidol, which produced no additional activation over that produced by SCH 23390. Contrary to haloperidol, SCH 23390 failed to increase the affinity of tyrosine hydroxylase (TH) for the pteridine cofactor and produced only a modest increase (30%) in DA synthesis. Moreover, SCH 23390 did not prevent haloperidol-induced activation of either TH or DA synthesis. It is concluded that blockade of postsynaptic D~ receptors results in the stimulation of SN-DA neurons, but this effect fails to activate TH and DA synthesis unless presynaptic DA autoreceptors are blocked.
Proteinase-activated receptors (PARs) are a family
of four G protein–coupled receptors that are ... more Proteinase-activated receptors (PARs) are a family
of four G protein–coupled receptors that are widely distributed
in the CNS and involved in neural cell proliferation,
differentiation and survival. The olfactory system undergoes
continuous neurogenesis throughout life and may represent
a critical target of PAR cellular actions. In the present study
we investigated the functional activity of PAR1 and PAR2 in
microdissected tissue preparations of olfactory nerve–glomerular
layer (ON–GL), external plexiform layer (EPL) and
granule cell layer (GRL) of the rat main olfactory bulb and in
primary cultures of olfactory neuroepithelial cells. Activation
of either PAR1 or PAR2 regulated multiple signaling pathways,
including activation of pertussis-toxin sensitive Gi/o
proteins, inhibition of cyclic AMP formation, stimulation of
Gq/11-mediated phosphoinositide (PI) hydrolysis, phosphorylation
of Ca2/calmodulin-dependent protein kinase II and
activation of the monomeric G protein Rho, predominantly in
ON–GL, whereas only activation of Rho was detected in the
deeper layers. Olfactory nerve lesion by nasal irrigation with
ZnSO4 induced a marked decrease of PAR signaling in ON–GL.
In primary cultures of olfactory neurons, double immunofluorescence
analysis showed the localization of PAR1 and PAR2 in
cells positive for olfactory-marker protein and neuron-specific
enolase. Cell exposure to either nanomolar concentrations of
thrombin and trypsin or PAR-activating peptides caused rapid
neurite retraction. This study provides the first characterization
of the laminar distribution of PAR1 and PAR2 signaling in rat
olfactory bulb, demonstrates the presence of the receptors
in olfactory sensory neurons and suggests a role of PARs
in olfactory sensory neuron neuritogenesis.
Type I interferons (IFNs) are known to cause neuropsychiatric side effects, which have been propo... more Type I interferons (IFNs) are known to cause neuropsychiatric side effects, which have been proposed to be mediated by either peripheral actions or activation of glial cells. In the present study, we have investigated whether these cytokines could act directly on neuronal cells and regulate signaling pathways involved in cell death. In human SH-SY5Y neuroblastoma cells, type I IFNs rapidly stimulated tyrosine phosphorylation of Janus kinase and signal transducer and activator of transcription (STAT) through type I IFN receptor. Prolonged exposure to IFN-β induced apoptotic cell death accompanied by cytochrome C release, cleavage of caspases 9, 7, 3 and poly-(ADP ribose) polymerase and DNA fragmentation. Janus kinase inhibition reduced IFN-β-stimulated TyK2 and STAT1 phosphorylation, STAT1 transcriptional activity, induction of double-stranded RNA-activated protein kinase (PKR) and caspase cleavage. PKR induction was associated with enhanced PKR activity and chemical inhibition of PKR reduced IFN-stimulated caspase activation. Moreover, long-term IFN-β treatment led to down-regulation of phosphatidylinositol 3-kinase/Akt signaling and IFN-β-induced apoptosis was attenuated in cells expressing constitutively active Akt. Similarly, in mouse primary neurons IFN-β induced STAT phosphorylation, caspase 3 cleavage and inhibition of Akt signaling. Thus, type I IFNs can directly impair neuronal survival by regulating multiple signaling molecules promoting the intrinsic apoptotic pathway. This effect may contribute to the cytokine neurotoxicity.
Specific receptors for pituitary adenylate cyclase-activating polypeptide (PACAP), a novel peptid... more Specific receptors for pituitary adenylate cyclase-activating polypeptide (PACAP), a novel peptide with neuroregulatory and neurotrophic functions, have recently been identified in the retinas of different mammalian species. In the present study, expression of PACAP receptors and PACAP was investigated in the retinas of 12-18-week human embryos. Radioligand binding studies showed that the two forms of PACAP with 38 and 27 amino acids (PACAP 38 and PACAP 27, respectively) displaced the binding of 1251-PACAP 27 with lC5o values in the picomolar range, whereas functional receptor assays demonstrated that the two peptides were potent and effective stimulators of adenylyl cyclase activity. In contrast, vasoactive intestinal peptide (VIP) and human peptide histidine-isoleucine, which are homologous to PA-CAP, displayed lower affinities for the 1251-PACAP 27 binding site and were much less potent stimulators of cyclic AMP formation. Glucagon and secretin were inactive in both receptor assays. The expression of specific PACAP receptors was further investigated by reverse transcription-polymerase chain reaction technique, which showed the presence of mRNAs coding for PACAP type I and for nonselective PACAP type II (both VIP 1 and VIP2) receptors. By the same technique, expression of PACAP mRNA was also detected. These data indicate that the developing human retina synthesizes PACAP and that the peptide may act on retinal cells by predominantly stimulating PACAP type I receptors coupled to cyclic AMP formation. Key Words: Pituitary adenylate cyclaseactivating polypeptide-Vasoactive intestinal peptide-Receptor binding assay-Adenylyl cyclase-Reverse transcriptionpolymerase chain reaction -Human fetal retina.
AMP-activated protein kinase (AMPK) and δ-opioid receptors (DORs) are both involved in controllin... more AMP-activated protein kinase (AMPK) and δ-opioid receptors (DORs) are both involved in controlling cell survival, energy metabolism, and food intake, but little is known on the interaction between these two signaling molecules. Here we show that activation of human DORs stably expressed in Chinese hamster ovary (CHO) cells increased AMPK activity and AMPK phosphorylation on Thr172. DOR-induced AMPK phosphorylation was prevented by pertussis toxin, reduced by protein kinase A (PKA) activators, and unaffected by PKA, transforming growth factor-β-activated kinase 1, mitogen-activated protein kinase, and protein kinase C inhibitors. Conversely, the DOR effect was reduced by Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) inhibition, apyrase treatment, G(q/11) antagonism, and blockade of P2 purinergic receptors. Apyrase treatment also depressed DOR stimulation of intracellular Ca(2+) concentration, whereas P2 receptor antagonism blocked DOR stimulation of inositol phosphate acc...
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, 2010
Tricyclic antidepressants (TCAs) have been reported to interact with the opioid system, but their... more 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 -opioid receptors (CHO/DOR), TCAs displaced [3H]naltrindole binding and stimulated guanosine 5-O- (3-[35S]thio)triphosphate ([35S]GTPS) 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 -opioid receptors in rat dorsal striatum and nucleus accumbens and human frontal cortex. In CHOcells expressing -opioid recep...
Background and purpose
Although clinically useful for their immunomodulatory, antiproliferative ... more Background and purpose
Although clinically useful for their immunomodulatory, antiproliferative and antiviral properties, type I interferons (IFNs) are involved in the pathogenesis of several neurodegenerative/neuroinflammatory diseases. In the present study, we investigated the ability of cholinergic stimulation to protect from IFN-β-induced neuronal apoptosis. Experimental approach
The effects of the cholinergic agonist carbachol (CCh) on IFN-β-induced apoptosis of human SH-SY5Y neuroblastoma cells were examined by using Western blot, immunofluorescence and cytofluorimetry. The involvement of muscarinic acetylcholine receptors (mAChRs) was assessed by using selective antagonists and siRNA transfection. Pharmacological inhibitors and over-expression of ERK2 and an ERK2 constitutively active form (ERK2-CA) were employed to study ERK1/2 signalling. The effects of oxotremorine-M (Oxo-M) on IFN-β-induced apoptosis of mouse hippocampal neurons were examined by measuring cleaved caspase 3 expression. Key results
In SH-SY5Y cells, CCh inhibited IFN-β-induced mitochondrial cytochrome c release, activation of caspases 9, 7 and 3, PARP cleavage and DNA fragmentation. The anti-apoptotic effect of CCh was mediated by M3 mAChRs, blocked by the Gq/11 antagonist YM254890 and the protein kinase C inhibitor Go 6983, impaired by inhibition of ERK1/2 pathway, potentiated by over-expression of ERK2, and mimicked by ERK2-CA. Blockade of JNK activation enhanced the CCh anti-apoptotic response. IFN-β inhibited JNK activation and up-regulated CCh-induced ERK1/2 signalling. In hippocampal neurons, IFN-β-induced apoptosis was reduced by Oxo-M and the effect was antagonized by blockade of M1/M3 mAChRs and ERK1/2. Conclusions and implications
Stimulation of mAChRs counteracted IFN-β-induced neuronal apoptosis through the activation of ERK1/2 signalling. The data indicate that activation of ERK1/2-coupled mAChRs may be an effective strategy for preventing IFNs neurotoxicity.
Although clinically useful for the treatment of various diseases, type I interferons (IFNs) have ... more Although clinically useful for the treatment of various diseases, type I interferons (IFNs) have been implicated as causative factors of a number of neuroinflammatory disorders characterized by neuronal damage and altered CNS functions. As neurotrophin 3 (NT3) plays a critical role in neuroprotection, we examined the effects of IFN-β on the signalling and functional activity of the NT3/TrkC system. We found that prolonged exposure of differentiated human SH-SY5Y neuroblastoma cells to IFN-β impaired the ability of NT3 to induce transphosphorylation of the full-length TrkC receptor (TrkC-FL) and the phosphorylation of downstream signalling molecules, including PLCγ1, Akt, GSK-3β and ERK1/2. NT3 was effective in protecting the cells against apoptosis triggered by serum withdrawal or thapsigargin but not IFN-β. Prolonged exposure to the cytokine had little effects on TrkC-FL levels but markedly enhanced the messenger RNA (mRNA) and protein levels of the truncated isoform TrkC-T1, a dominant-negative receptor that inhibits TrkC-FL activity. Cell depletion of TrkC-T1 by small interfering RNA (siRNA) treatment enhanced NT3 signalling through TrkC-FL and allowed the neurotrophin to counteract IFN-β-induced apoptosis. Furthermore, the upregulation of TrkC-T1 by IFN-β was associated with the inhibition of NT3-induced recruitment of the scaffold protein tamalin to TrkC-T1 and tamalin tyrosine phosphorylation. These data indicate that IFN-β exerts a negative control on NT3 pro-survival signalling through a novel mechanism involving the upregulation of TrkC-T1.
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. stably expressed in CHO-K1 cells. ERK1/2 stimulation by antidepressants and LPA was suppressed by pertussis toxin and inhibition of Src, phosphatidylinositol-3 kinase and insulin-like growth factor-I receptor (IGF-IR) activities. Antidepressants and LPA induced tyrosine phosphorylation of IGF-IR and insulin receptor-substrate-1 through LPA1 and Src. Prolonged exposure of CHO-K1 fibroblasts to either mianserin, mirtazapine or LPA enhanced cell proliferation as indicated by increased [3H]-thymidine incorporation and Ki-67 immunofluorescence. This effect was inhibited by blockade of LPA1- and ERK1/2 activity. These data provide evidence that different antidepressants induce LPA1 activation, leading to receptor tyrosine kinase transactivation, stimulation of ERK1/2 signaling and enhanced cell proliferation.
Gq/11-coupled muscarinic acetylcholine receptors (mAChRs) belonging to M1, M3 and M5 subtypes hav... more Gq/11-coupled muscarinic acetylcholine receptors (mAChRs) belonging to M1, M3 and M5 subtypes have been shown to activate the metabolic sensor AMP-activated protein kinase (AMPK) through Ca(2+)/calmodulin-dependent protein kinase kinase-β (CaMKKβ)-mediated phosphorylation at Thr172. However, the source of Ca(2+) required for this response has not been yet elucidated. Here, we investigated the involvement of store-operated Ca(2+) entry (SOCE) in AMPK activation by pharmacologically defined M3 mAChRs in human SH-SY5Y neuroblastoma cells. In Ca(2+)-free medium the cholinergic agonist carbachol (CCh) caused a transient increase of phospho-Thr172 AMPK that rapidly ceased within 2min. Conversely, in the presence of extracellular Ca(2+) CCh-induced AMPK phosphorylation lasted for at least 180min. The SOCE modulator 2-aminoethoxydiphephenyl borate (2-APB), at a concentration (50μM) that suppressed CCh-induced intracellular Ca(2+) ([Ca(2+)]i) plateau, inhibited CCh-induced AMPK phosphorylation. CCh triggered the activation of the endoplasmic reticulum Ca(2+) sensor stromal interaction molecule (STIM) 1, as indicated by redistribution of STIM1 immunofluorescence into puncta, and promoted the association of STIM1 with the SOCE channel component Orai1. Cell depletion of STIM1 by siRNA treatment reduced both CCh-induced [Ca(2+)]i plateau and AMPK activation. M3 mAChRs increased glucose uptake and this response required extracellular Ca(2+) and was inhibited by 2-APB, STIM1 knockdown, CaMKKβ and AMPK inhibitors, and adenovirus infection with dominant negative AMPK. Thus, the study provides evidence that SOCE is required for sustained activation of AMPK and stimulation of downstream glucose uptake by M3 mAChRs and suggests that SOCE is a critical process connecting M3 mAChRs to the control of neuronal energy metabolism.
Type I interferons (IFNs) induce apoptosis of neuroblastoma cells, but the molecular mechanisms r... more Type I interferons (IFNs) induce apoptosis of neuroblastoma cells, but the molecular mechanisms regulating this event have not been completely elucidated. Here, we investigated the role of p38 mitogen activated protein kinase (MAPK) activity, a key regulator of apoptosis and a known modulator of IFN-induced responses in non-neuronal cells. We show that in SH-SY5Y human neuroblastoma cells IFN-β induced a delayed and sustained increase of p38 MAPK activity through a novel mechanism involving the sequential activation of Janus kinase-signal transducer and activator of transcription-1 signalling, enhanced expression of the NADPH oxidase catalytic subunit gp91(phox), increased reactive oxygen species production and stimulation of the MAPK kinase kinase transforming growth factor-β-activated kinase 1. Either blockade of p38 MAPK by the second generation inhibitors BIRB0796 and VX745 or siRNA knockdown of p38α MAPK enhanced IFN-β-induced apoptosis of neuroblastoma cells. Exposure to IFN-β increased the phosphorylation of the small heat shock protein HSP27 at Ser15, Ser78 and Ser82 with a time course similar to p38 MAPK activation and this response was suppressed by either p38α MAPK depletion or pharmacological inhibition of p38 MAPK and MAPK-activated protein kinase 2 (MK2). Either silencing of HSP27 expression by siRNA or MK2 inhibition potentiated IFN-β-induced apoptotic death. These results indicate that IFN-β-induced apoptosis of human SH-SY5Y neuroblastoma cells is associated with a long-lasting up-regulation of p38 MAPK activity, stimulation of MK2 and phosphorylation of the pro-survival protein HSP27. Moreover, the data show that inhibition of p38 MAPK signalling potentiates the anti-neuroblastoma activity of the cytokine, indicating that this pathway mediates a counter-regulatory response.
Alterations in the olfactory system and impairment of olfactory function have been reported in se... more Alterations in the olfactory system and impairment of olfactory function have been reported in several neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases (1, 2). The participation of proteinase activated receptors (PARs) in these neuropathological ...
Acetylcholine inhibits, by 30-40%, the basal adenylate cyclase activity of purified synaptic plas... more Acetylcholine inhibits, by 30-40%, the basal adenylate cyclase activity of purified synaptic plasma membranes prepared from rat striatum (EC50 = 3 microM). Cholinergic receptor agonists inhibit this cyclase activity with the following rank order of potency: oxtremorine greater than acetylcholine greater than arecoline greater than methacholine greater than or equal to muscarine greater than or equal to carbachol greater than bethanechol. Nicotine fails to inhibit the cyclase, and d-tubocurarine fails to inhibit the action of cholinergic drugs. In contrast, atropine and scopolamine antagonize the effect of acetylcholine. The enzyme inhibition elicited by acetylcholine requires the presence of GTP, and disappears after intrastriatal injection of kainic acid. From these results, we infer that striatal adenylate cyclase can be modulated by muscarinic receptors.
Opioid receptors located in the ventral tegmental area are known to regulate dopamine (DA) releas... more Opioid receptors located in the ventral tegmental area are known to regulate dopamine (DA) release from mesocortical afferents to medial prefrontal cortex (mPFC) but little is known on whether in this cortical region activation of opioid receptors affect DA receptor signaling. In the present study we show that in mouse mPFC concomitant activation of either d-or l-opioid receptors, but not j-opioid receptors, potentiated DA D1-like receptor-induced stimulation of adenylyl cyclase activity through a G protein bc subunit-dependent mechanism. In tissue slices of mPFC, the combined addition of the opioid agonist leu-enkephalin and the DA D1-like receptor agonist SKF 81297 produced more than additive increase in the phosphorylation state of AMPA and NMDA receptor subunits GluR1 and NR1, respectively. Moreover, in primary cultures of mouse frontal cortex neurons, DA D1-like receptor-induced Ser133 phosphorylation of the transcription factor cyclic AMP responsive element binding protein was potentiated by concurrent stimulation of opioid receptors. Double immunofluorescence analysis of cultured cortical cells indicated that a large percentage of DA D1 receptor positive cells expressed either d-or l-opioid receptor immunoreactivity. These data indicate that in mouse mPFC activation of land d-opioid receptors enhances DA D1-like receptor signaling likely through converging regulatory inputs on bc-stimulated adenylyl cyclase isoforms. This previously unrecognized synergistic interaction may selectively affect DA D1 transmission at specific postsynaptic sites where the receptors are co-localized and may play a role in prefrontal DA D1 regulation of opioid addiction.
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 and α-amino-3-hydroxy-5-methylisoxazole-4-propionate glutamate receptors, whereas in primary cultures of accumbal cells, which were found to coexpress NOP and DA D1 receptors, N/OFQ curtailed DA D1 receptor-induced cAMP-response element-binding protein phosphorylation. Thus, in accumbens and striatum N/OFQ exerts an inhibitory constraint on DA transmission by acting on either pre-synaptic NOP receptors inhibiting TH phosphorylation and DA synthesis or post-synaptic NOP receptors selectively down-regulating DA D1 receptor signaling.
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 /-isoproterenol, in increasing cyclic AMP formation. The synergistic interaction consists of an increase in the maximal a0denylyl cyclase activation without a significant change in the potency of each agonist. CCh also fails to affect 125ICRH binding to olfactory bulb membranes. The synergism requires micromolar concentrations of GTP. Substitution of the stable GTP analog guanosine 5′-O-(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 muscarinic 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.
1 In rat striatal membranes, muscarinic toxin 3 (MT3), a selective ligand of the cloned m4 recept... more 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 DI receptorstimulated adenylyl cyclase activities with pA2 values of 8.09 and 8.15, respectively. 2 In radioligand binding experiments, MT3 increased the Kd but did not change the Bm value of [3H]-N-methylscopolamine (3H]-NMS) binding to rat striatal muscarinic receptors. The toxin displaced the major portion of the [3HJ-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.
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. Key Words: Phorbol esters-Adenylate cyclase-Protein kinase C-Rat striatum. Olianas M. C. and Onali P. Phorbol esters increase GTP-dependent adenylate cyclase activity in rat brain striatal membranes. J . Neurochem. 47, 890-897 (1986). Ahhreviutions used; AMPP(NH)P, adenyl-5'-yl-imidodiphos-phate; DA or dopamine, 3,4-dihydroxyphenylethylamine; Gpp(NH)p, guanyl-5'-yl-imidodiphosphate; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; N, and Ni, stimulatory and inhibitory guanine nucleotide-binding protein of adenylate cyclase, respectively; R( -)PIA, R( -)-N6-(2-phenyliso-propy1)adenosine; PMA, 4pphorbol 12-myristate 13-acetate.
The intraperitoneal administration of SCH 23390, a selective D l receptor antagonist, produced ca... more The intraperitoneal administration of SCH 23390, a selective D l receptor antagonist, produced catalepsy and increased the firing rate of dopamine (DA) neurons in the substantia nigra (SN-DA neurons). Maximal increase (110%) was of the same magnitude as that produced by a supramaximal dose of haloperidol, which produced no additional activation over that produced by SCH 23390. Contrary to haloperidol, SCH 23390 failed to increase the affinity of tyrosine hydroxylase (TH) for the pteridine cofactor and produced only a modest increase (30%) in DA synthesis. Moreover, SCH 23390 did not prevent haloperidol-induced activation of either TH or DA synthesis. It is concluded that blockade of postsynaptic D~ receptors results in the stimulation of SN-DA neurons, but this effect fails to activate TH and DA synthesis unless presynaptic DA autoreceptors are blocked.
Proteinase-activated receptors (PARs) are a family
of four G protein–coupled receptors that are ... more Proteinase-activated receptors (PARs) are a family
of four G protein–coupled receptors that are widely distributed
in the CNS and involved in neural cell proliferation,
differentiation and survival. The olfactory system undergoes
continuous neurogenesis throughout life and may represent
a critical target of PAR cellular actions. In the present study
we investigated the functional activity of PAR1 and PAR2 in
microdissected tissue preparations of olfactory nerve–glomerular
layer (ON–GL), external plexiform layer (EPL) and
granule cell layer (GRL) of the rat main olfactory bulb and in
primary cultures of olfactory neuroepithelial cells. Activation
of either PAR1 or PAR2 regulated multiple signaling pathways,
including activation of pertussis-toxin sensitive Gi/o
proteins, inhibition of cyclic AMP formation, stimulation of
Gq/11-mediated phosphoinositide (PI) hydrolysis, phosphorylation
of Ca2/calmodulin-dependent protein kinase II and
activation of the monomeric G protein Rho, predominantly in
ON–GL, whereas only activation of Rho was detected in the
deeper layers. Olfactory nerve lesion by nasal irrigation with
ZnSO4 induced a marked decrease of PAR signaling in ON–GL.
In primary cultures of olfactory neurons, double immunofluorescence
analysis showed the localization of PAR1 and PAR2 in
cells positive for olfactory-marker protein and neuron-specific
enolase. Cell exposure to either nanomolar concentrations of
thrombin and trypsin or PAR-activating peptides caused rapid
neurite retraction. This study provides the first characterization
of the laminar distribution of PAR1 and PAR2 signaling in rat
olfactory bulb, demonstrates the presence of the receptors
in olfactory sensory neurons and suggests a role of PARs
in olfactory sensory neuron neuritogenesis.
Type I interferons (IFNs) are known to cause neuropsychiatric side effects, which have been propo... more Type I interferons (IFNs) are known to cause neuropsychiatric side effects, which have been proposed to be mediated by either peripheral actions or activation of glial cells. In the present study, we have investigated whether these cytokines could act directly on neuronal cells and regulate signaling pathways involved in cell death. In human SH-SY5Y neuroblastoma cells, type I IFNs rapidly stimulated tyrosine phosphorylation of Janus kinase and signal transducer and activator of transcription (STAT) through type I IFN receptor. Prolonged exposure to IFN-β induced apoptotic cell death accompanied by cytochrome C release, cleavage of caspases 9, 7, 3 and poly-(ADP ribose) polymerase and DNA fragmentation. Janus kinase inhibition reduced IFN-β-stimulated TyK2 and STAT1 phosphorylation, STAT1 transcriptional activity, induction of double-stranded RNA-activated protein kinase (PKR) and caspase cleavage. PKR induction was associated with enhanced PKR activity and chemical inhibition of PKR reduced IFN-stimulated caspase activation. Moreover, long-term IFN-β treatment led to down-regulation of phosphatidylinositol 3-kinase/Akt signaling and IFN-β-induced apoptosis was attenuated in cells expressing constitutively active Akt. Similarly, in mouse primary neurons IFN-β induced STAT phosphorylation, caspase 3 cleavage and inhibition of Akt signaling. Thus, type I IFNs can directly impair neuronal survival by regulating multiple signaling molecules promoting the intrinsic apoptotic pathway. This effect may contribute to the cytokine neurotoxicity.
Specific receptors for pituitary adenylate cyclase-activating polypeptide (PACAP), a novel peptid... more Specific receptors for pituitary adenylate cyclase-activating polypeptide (PACAP), a novel peptide with neuroregulatory and neurotrophic functions, have recently been identified in the retinas of different mammalian species. In the present study, expression of PACAP receptors and PACAP was investigated in the retinas of 12-18-week human embryos. Radioligand binding studies showed that the two forms of PACAP with 38 and 27 amino acids (PACAP 38 and PACAP 27, respectively) displaced the binding of 1251-PACAP 27 with lC5o values in the picomolar range, whereas functional receptor assays demonstrated that the two peptides were potent and effective stimulators of adenylyl cyclase activity. In contrast, vasoactive intestinal peptide (VIP) and human peptide histidine-isoleucine, which are homologous to PA-CAP, displayed lower affinities for the 1251-PACAP 27 binding site and were much less potent stimulators of cyclic AMP formation. Glucagon and secretin were inactive in both receptor assays. The expression of specific PACAP receptors was further investigated by reverse transcription-polymerase chain reaction technique, which showed the presence of mRNAs coding for PACAP type I and for nonselective PACAP type II (both VIP 1 and VIP2) receptors. By the same technique, expression of PACAP mRNA was also detected. These data indicate that the developing human retina synthesizes PACAP and that the peptide may act on retinal cells by predominantly stimulating PACAP type I receptors coupled to cyclic AMP formation. Key Words: Pituitary adenylate cyclaseactivating polypeptide-Vasoactive intestinal peptide-Receptor binding assay-Adenylyl cyclase-Reverse transcriptionpolymerase chain reaction -Human fetal retina.
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Papers by MC Olianas
Although clinically useful for their immunomodulatory, antiproliferative and antiviral properties, type I interferons (IFNs) are involved in the pathogenesis of several neurodegenerative/neuroinflammatory diseases. In the present study, we investigated the ability of cholinergic stimulation to protect from IFN-β-induced neuronal apoptosis.
Experimental approach
The effects of the cholinergic agonist carbachol (CCh) on IFN-β-induced apoptosis of human SH-SY5Y neuroblastoma cells were examined by using Western blot, immunofluorescence and cytofluorimetry. The involvement of muscarinic acetylcholine receptors (mAChRs) was assessed by using selective antagonists and siRNA transfection. Pharmacological inhibitors and over-expression of ERK2 and an ERK2 constitutively active form (ERK2-CA) were employed to study ERK1/2 signalling. The effects of oxotremorine-M (Oxo-M) on IFN-β-induced apoptosis of mouse hippocampal neurons were examined by measuring cleaved caspase 3 expression.
Key results
In SH-SY5Y cells, CCh inhibited IFN-β-induced mitochondrial cytochrome c release, activation of caspases 9, 7 and 3, PARP cleavage and DNA fragmentation. The anti-apoptotic effect of CCh was mediated by M3 mAChRs, blocked by the Gq/11 antagonist YM254890 and the protein kinase C inhibitor Go 6983, impaired by inhibition of ERK1/2 pathway, potentiated by over-expression of ERK2, and mimicked by ERK2-CA. Blockade of JNK activation enhanced the CCh anti-apoptotic response. IFN-β inhibited JNK activation and up-regulated CCh-induced ERK1/2 signalling. In hippocampal neurons, IFN-β-induced apoptosis was reduced by Oxo-M and the effect was antagonized by blockade of M1/M3 mAChRs and ERK1/2.
Conclusions and implications
Stimulation of mAChRs counteracted IFN-β-induced neuronal apoptosis through the activation of ERK1/2 signalling. The data indicate that activation of ERK1/2-coupled mAChRs may be an effective strategy for preventing IFNs neurotoxicity.
of four G protein–coupled receptors that are widely distributed
in the CNS and involved in neural cell proliferation,
differentiation and survival. The olfactory system undergoes
continuous neurogenesis throughout life and may represent
a critical target of PAR cellular actions. In the present study
we investigated the functional activity of PAR1 and PAR2 in
microdissected tissue preparations of olfactory nerve–glomerular
layer (ON–GL), external plexiform layer (EPL) and
granule cell layer (GRL) of the rat main olfactory bulb and in
primary cultures of olfactory neuroepithelial cells. Activation
of either PAR1 or PAR2 regulated multiple signaling pathways,
including activation of pertussis-toxin sensitive Gi/o
proteins, inhibition of cyclic AMP formation, stimulation of
Gq/11-mediated phosphoinositide (PI) hydrolysis, phosphorylation
of Ca2/calmodulin-dependent protein kinase II and
activation of the monomeric G protein Rho, predominantly in
ON–GL, whereas only activation of Rho was detected in the
deeper layers. Olfactory nerve lesion by nasal irrigation with
ZnSO4 induced a marked decrease of PAR signaling in ON–GL.
In primary cultures of olfactory neurons, double immunofluorescence
analysis showed the localization of PAR1 and PAR2 in
cells positive for olfactory-marker protein and neuron-specific
enolase. Cell exposure to either nanomolar concentrations of
thrombin and trypsin or PAR-activating peptides caused rapid
neurite retraction. This study provides the first characterization
of the laminar distribution of PAR1 and PAR2 signaling in rat
olfactory bulb, demonstrates the presence of the receptors
in olfactory sensory neurons and suggests a role of PARs
in olfactory sensory neuron neuritogenesis.
Although clinically useful for their immunomodulatory, antiproliferative and antiviral properties, type I interferons (IFNs) are involved in the pathogenesis of several neurodegenerative/neuroinflammatory diseases. In the present study, we investigated the ability of cholinergic stimulation to protect from IFN-β-induced neuronal apoptosis.
Experimental approach
The effects of the cholinergic agonist carbachol (CCh) on IFN-β-induced apoptosis of human SH-SY5Y neuroblastoma cells were examined by using Western blot, immunofluorescence and cytofluorimetry. The involvement of muscarinic acetylcholine receptors (mAChRs) was assessed by using selective antagonists and siRNA transfection. Pharmacological inhibitors and over-expression of ERK2 and an ERK2 constitutively active form (ERK2-CA) were employed to study ERK1/2 signalling. The effects of oxotremorine-M (Oxo-M) on IFN-β-induced apoptosis of mouse hippocampal neurons were examined by measuring cleaved caspase 3 expression.
Key results
In SH-SY5Y cells, CCh inhibited IFN-β-induced mitochondrial cytochrome c release, activation of caspases 9, 7 and 3, PARP cleavage and DNA fragmentation. The anti-apoptotic effect of CCh was mediated by M3 mAChRs, blocked by the Gq/11 antagonist YM254890 and the protein kinase C inhibitor Go 6983, impaired by inhibition of ERK1/2 pathway, potentiated by over-expression of ERK2, and mimicked by ERK2-CA. Blockade of JNK activation enhanced the CCh anti-apoptotic response. IFN-β inhibited JNK activation and up-regulated CCh-induced ERK1/2 signalling. In hippocampal neurons, IFN-β-induced apoptosis was reduced by Oxo-M and the effect was antagonized by blockade of M1/M3 mAChRs and ERK1/2.
Conclusions and implications
Stimulation of mAChRs counteracted IFN-β-induced neuronal apoptosis through the activation of ERK1/2 signalling. The data indicate that activation of ERK1/2-coupled mAChRs may be an effective strategy for preventing IFNs neurotoxicity.
of four G protein–coupled receptors that are widely distributed
in the CNS and involved in neural cell proliferation,
differentiation and survival. The olfactory system undergoes
continuous neurogenesis throughout life and may represent
a critical target of PAR cellular actions. In the present study
we investigated the functional activity of PAR1 and PAR2 in
microdissected tissue preparations of olfactory nerve–glomerular
layer (ON–GL), external plexiform layer (EPL) and
granule cell layer (GRL) of the rat main olfactory bulb and in
primary cultures of olfactory neuroepithelial cells. Activation
of either PAR1 or PAR2 regulated multiple signaling pathways,
including activation of pertussis-toxin sensitive Gi/o
proteins, inhibition of cyclic AMP formation, stimulation of
Gq/11-mediated phosphoinositide (PI) hydrolysis, phosphorylation
of Ca2/calmodulin-dependent protein kinase II and
activation of the monomeric G protein Rho, predominantly in
ON–GL, whereas only activation of Rho was detected in the
deeper layers. Olfactory nerve lesion by nasal irrigation with
ZnSO4 induced a marked decrease of PAR signaling in ON–GL.
In primary cultures of olfactory neurons, double immunofluorescence
analysis showed the localization of PAR1 and PAR2 in
cells positive for olfactory-marker protein and neuron-specific
enolase. Cell exposure to either nanomolar concentrations of
thrombin and trypsin or PAR-activating peptides caused rapid
neurite retraction. This study provides the first characterization
of the laminar distribution of PAR1 and PAR2 signaling in rat
olfactory bulb, demonstrates the presence of the receptors
in olfactory sensory neurons and suggests a role of PARs
in olfactory sensory neuron neuritogenesis.