ABSTRACT
The crosstalk between the opioidergic system and mitogen-activated protein kinases (MAPKs) has a critical role in mediating stress-induced behaviors related to the pathophysiology of anxiety. The present study evaluated the basal status and stress-induced alterations of cortico-thalamic MAPKs and other cell fate-related signaling pathways potentially underlying the anxiogenic endophenotype of PDYN gene-deficient mice. Compared to littermates, PDYN knockout (KO) mice had lower cortical and or thalamic amounts of the phospho-activated MAPKs c-Jun N-terminal kinase (JNK1/2) and extracellular signal-regulated kinase (ERK1/2). Similarly, PDYN-KO animals displayed reduced cortico-thalamic densities of total and phosphorylated (at Ser191) species of the cell fate regulator Fas-associated protein with death domain (FADD) without alterations in the Fas receptor. Exposure to acute restraint and chronic mild stress stimuli induced the robust stimulation of JNK1/2 and ERK1/2 MAPKs, FADD, and Akt-mTOR pathways, without apparent increases in apoptotic rates. Interestingly, PDYN deficiency prevented stress-induced JNK1/2 and FADD but not ERK1/2 or Akt-mTOR hyperactivations. These findings suggest that cortico-thalamic MAPK- and FADD-dependent neuroplasticity might be altered in PDYN-KO mice. In addition, the results also indicate that the PDYN gene (and hence dynorphin release) may be required to stimulate JNK1/2 and FADD (but not ERK1/2 or Akt/mTOR) pathways under environmental stress conditions.
Subject(s)
Proto-Oncogene Proteins c-akt , Signal Transduction , Mice , Animals , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/physiology , Apoptosis/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Mitogen-Activated Protein Kinase Kinases/metabolism , TOR Serine-Threonine Kinases/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
BACKGROUND: Many psychoactive compounds have been developed to have more beneficial clinical efficacy than conventional drugs by adding agonistic action at 5-HT1A receptors. The aim of the present study was to evaluate several psychotropic drugs that had been reported to behave as an agonist at 5-HT1A receptor (aripiprazole, brexpiprazole, asenapine, lurasidone, and vortioxetine) in both rat and postmortem human brain membranes. METHODS: The [35S]GTPγS binding assay for Gi/o proteins coupled with 5-HT1A receptors was performed in rat brain membranes and postmortem human brain membranes. RESULTS: The specific binding was stimulated by brexpiprazole in rat hippocampus, human hippocampus, and human prefrontal cortex. Aripiprazole also behaved as an agonist in the same brain regions. Interestingly, its potency was much higher in rat hippocampal membranes than in human brain membranes, indicating the possibility of species differences. Although vortioxetine was an efficacious stimulator at high concentrations, its potency was undeterminable because of a lack of saturability. In addition to 5-HT1A receptor agonism, involvement of other components, e.g., 5-HT1B receptor agonism, was speculated by the biphasic inhibitory effects of the selective 5-HT1A receptor neutral antagonist. Negligible stimulatory effects were obtained as to lurasidone and asenapine. CONCLUSIONS: Our previous studies have raised the concept of a psychoactive drug group with a common pharmacological mechanism of action, i.e., 5-HT1A receptor agonism, consisting of perospirone, aripiprazole, ziprasidone, clozapine, quetiapine, nemonapride, and trazodone. The present study demonstrates the data indicating that brexpiprazole and probably vortioxetine are included in this drug group. Lurasidone and asenapine are excluded from this group.
Subject(s)
Receptor, Serotonin, 5-HT1A , Serotonin , Rats , Humans , Animals , Aripiprazole/pharmacology , Serotonin/pharmacology , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Vortioxetine/pharmacology , Receptor, Serotonin, 5-HT1A/metabolism , Lurasidone Hydrochloride/pharmacology , Serotonin Receptor Agonists/pharmacology , Brain/metabolism , Psychotropic Drugs/pharmacologyABSTRACT
Recent findings unveil the pharmacological modulation of imidazoline I2 receptors (I2-IR) as a novel strategy to face unmet medical neurodegenerative diseases. In this work, we report the chemical characterization, three-dimensional quantitative structure-activity relationship (3D-QSAR) and ADMET in silico of a family of benzofuranyl-2-imidazoles that exhibit affinity against human brain I2-IR and most of them have been predicted to be brain permeable. Acute treatment in mice with 2-(2-benzofuranyl)-2-imidazole, known as LSL60101 (garsevil), showed non-warning properties in the ADMET studies and an optimal pharmacokinetic profile. Moreover, LSL60101 induced hypothermia in mice while decreased pro-apoptotic FADD protein in the hippocampus. In vivo studies in the familial Alzheimer's disease 5xFAD murine model with the representative compound, revealed significant decreases in the protein expression levels of antioxidant enzymes superoxide dismutase and glutathione peroxidase in hippocampus. Overall, LSL60101 plays a neuroprotective role by reducing apoptosis and modulating oxidative stress.
Subject(s)
Alzheimer Disease/drug therapy , Benzofurans/pharmacology , Imidazoles/pharmacology , Imidazoline Receptors/antagonists & inhibitors , Alzheimer Disease/metabolism , Animals , Apoptosis/drug effects , Benzofurans/chemical synthesis , Benzofurans/chemistry , Cell Line, Tumor , Dose-Response Relationship, Drug , Humans , Imidazoles/chemical synthesis , Imidazoles/chemistry , Imidazoline Receptors/metabolism , Ligands , Male , Mice , Molecular Structure , Oxidative Stress/drug effects , Structure-Activity RelationshipABSTRACT
BACKGROUND: Chronic exposure to opiates causes the development of tolerance and physical dependence as well as persistent brain neuroplasticity. Despite a wealth of postmortem human studies for opiate addicts, little direct information regarding the functional status of serotonergic and cholinergic receptor-mediated signaling pathways in the human brain of opiate addicts is yet available. METHODS: Functional activation of Gαq/11 proteins coupled to 5-HT2A and M1 type muscarinic acetylcholine receptor (mAChR) was assessed by using the method named [35S]GTPγS binding/immunoprecipitation in frontal cortical membrane preparations from postmortem human brains obtained from opiate addicts and matched controls. RESULTS: Concentration-response curves for 5-HT and carbachol in individual subjects were analyzed according to a nonlinear regression model, which generated the values of maximum percent increase (%Emax), negative logarithm of the half-maximal effect (pEC50) and slope factor. As for 5-HT2A receptor-mediated Gαq/11 activation, the %Emax values were reduced significantly and the pEC50 values were decreased significantly in opiate addicts as compared to the control group. Regarding carbachol-induced Gαq/11 activation, no significant difference in %Emax or pEC50 values was detected between the both groups, whereas the slope factor was increased significantly in opiate addicts as compared to the control group. CONCLUSION: Our data demonstrate that the signaling pathways mediated by Gαq/11 proteins coupled with 5-HT2A receptors and M1 mAChRs in prefrontal cortex are functionally altered in opiate addicts in comparison with control subjects. These alterations may underpin some aspects of addictive behavior to opiate as well as neuropsychological consequences or comorbid mental disorders associated with opioid use.
Subject(s)
Analgesics, Opioid/adverse effects , Dorsolateral Prefrontal Cortex/drug effects , Dorsolateral Prefrontal Cortex/metabolism , Opiate Alkaloids/adverse effects , Opioid-Related Disorders/metabolism , Receptor, Muscarinic M1/metabolism , Receptor, Serotonin, 5-HT2A/metabolism , Adult , Brain/drug effects , Brain/metabolism , Female , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Humans , Male , Middle Aged , Neuronal Plasticity/drug effects , Signal Transduction/drug effects , Young AdultABSTRACT
BACKGROUND: Alpha2A-adrenergic (α2A-AR) and cannabinoid CB1 (CB1-R) receptors exert their functions modulating multiple signaling pathways, including MEK-ERK (extracellular signal-regulated kinases) and FADD (Fas-associated protein with death domain) cascades. These molecules are relevant in finding biased agonists with fewer side effects, but the mechanisms involving their modulations by α2A-AR- and CB1-R in vivo are unclear. This study investigated the roles of Gαi2 and Gαz proteins in mediating α2A-AR- and CB1-R-induced alterations of MEK-ERK and FADD phosphorylation (p-) in mouse brain cortex. METHODS: Gαi2 or Gαz protein knockdown was induced in mice with selective antisense oligodeoxinucleotides (ODNs; 3 nmol/day, 5 days) prior to UK-14,304 (UK or brimonidine; 1 mg/kg) or WIN55212-2 (WIN; 8 mg/kg) acute treatments. Inactivated (p-T286) MEK1, activated (p-S217/221) MEK1/2, activated (p-T202/Y204) ERK1/2, p-S191 FADD, and the corresponding total forms of these proteins were quantified by immunoblotting. RESULTS: Increased (+ 88%) p-T286 MEK1 cortical density, with a concomitant reduction (-43%) of activated ERK was observed in UK-treated mice. Both effects were attenuated by Gαi2 or Gαz antisense ODNs. Contrastingly, WIN induced Gαi2- and Gαz-independent upregulations of p-T286 MEK1 (+ 63%), p-S217/221 MEK1/2 (+ 86%), and activated ERK (+ 111%) in brain. Pro-apoptotic FADD was downregulated (- 34 to 39%) following UK and WIN administration, whereas the neuroprotective p-S191 FADD was increased (+ 74%) in WIN-treated mice only. None of these latter effects required from Gαi2 or Gαz protein integrity. CONCLUSION: The results indicate that α2A-AR (UK), but not CB1-R (WIN), agonists use Gαi2 and Gαz proteins to modulate MEK-ERK, but not FADD, pathway in mouse brain cortex.
Subject(s)
Cerebral Cortex/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Fas-Associated Death Domain Protein/metabolism , GTP-Binding Protein alpha Subunit, Gi2/metabolism , Mitogen-Activated Protein Kinase Kinases/metabolism , Receptor, Cannabinoid, CB1/metabolism , Signal Transduction/physiology , Animals , Down-Regulation/physiology , Male , Mice , Phosphorylation/physiology , Up-Regulation/physiologyABSTRACT
OBJECTIVES: Serotonin-2A (5-HT2A) receptors play an important role in the regulation of many brain functions that are disturbed in patients with such psychiatric diseases as mood disorders and schizophrenia. The objective of this study was to evaluate 5-HT2A receptor-mediated signalling pathway through Gαq/11 activation in psychiatric patients by using post-mortem brain samples. METHODS: Functional activation of Gαq/11 proteins coupled to 5-HT2A receptors was determined by means of [35S]GTPγS binding/immunoprecipitation assay in post-mortem prefrontal cortex of psychiatric patients diagnosed as bipolar disorder (BP), major depressive disorder (MDD), and schizophrenia, and individually matched controls. The effects of antipsychotic treatment as well as suicide were also analysed. RESULTS: There was no significant difference in maximum percent increase (%Emax) or slope factor among the four groups. The negative logarithm of concentration eliciting the half-maximal effect (pEC50) was significantly reduced in BP and schizophrenia patients as compared to controls. These alterations were attributable to antipsychotic medication. The pEC50 values in 'non-suicide' group of schizophrenia, but not in 'suicide' group, were significantly reduced as compared with controls. CONCLUSIONS: Altered 5-HT2A receptor-mediated signalling pathway through Gαq/11 proteins in prefrontal cortex might be apparently involved in pathophysiology and pharmacotherapy of BP and schizophrenia. In schizophrenic patients, these alterations as a result of successful treatment with antipsychotic agents may help in prevention of suicidal behaviour.
Subject(s)
Depressive Disorder, Major , Mental Disorders , Schizophrenia , Depressive Disorder, Major/drug therapy , Humans , Prefrontal Cortex , Receptor, Serotonin, 5-HT2A , Schizophrenia/drug therapy , SerotoninABSTRACT
BACKGROUND: While the alteration of I2 receptors has been associated with neurodegenerative and psychiatric disorders, among other brain dysfunctions, I2 selective agonists are also capable of inducing analgesia in models of chronic pain, improving cognition and inducing hypothermia and neuroprotection. However, the literature evaluating the antidepressant-like potential of I2 ligands is scarce and showed mixed results, whereas some studies reported antidepressant-like effects for certain I2 ligands others denied them. In this context, we evaluated the antidepressant-like potential of a highly selective I2-receptor ligand, LSL 60101 ([2-(2-benzofuranyl)-2-imidazole]). METHODS: LSL 60101 was administered in adult male Sprague-Dawley rats daily during 16 days (doses of 10 and 20 mg/kg, ip) and its antidepressant-like potential was assessed through the course of treatment in the forced-swim test, novelty-suppressed feeding test and two-bottle choice test (sucrose preference). The regulation of several key neuroplasticity markers (i.e., FADD, p-ERK1/2, ERK1/2, p-JNK1/2, JNK1/2, mBDNF) was evaluated 24-h post-treatment by western blot analysis in the right hippocampus and the proliferation of neural progenitors was quantified in the left hippocampus by immunohistochemistry. RESULTS: The results showed that LSL 60101 did not induce an antidepressant-like effect over the course of treatment in any of the behavioral tests conducted, and it did not alter any of the hippocampal neuroplasticity markers evaluated. CONCLUSION: These results add to the existing literature by suggesting that not all I2 ligands might be capable of displaying an antidepressant-like potential, and that particularities in the chemical structure of each compound might help explain these discrepancies and deserve future studies.
Subject(s)
Antidepressive Agents/pharmacology , Benzofurans/pharmacology , Imidazoles/pharmacology , Imidazoline Receptors/drug effects , Anhedonia , Animals , Feeding Behavior/drug effects , Hippocampus/cytology , Hippocampus/drug effects , Male , Neural Stem Cells/drug effects , Neuronal Plasticity/drug effects , Rats , Rats, Sprague-Dawley , Swimming/psychology , Weight Gain/drug effectsABSTRACT
BACKGROUND: Dysregulations of endocannabinoids and/or cannabinoid (CB) receptors have been implicated in the pathophysiology and treatment of major depressive disorder (MDD). METHODS: CB1 and CB2 receptors, neuroprotective mTOR (mechanistic target of rapamycin) and pro-apoptotic JNK1/2 (c-Jun-N-terminal kinases) were quantified by immunoblotting in postmortem prefrontal cortex of MDD and controls, and further compared in antidepressant (AD)-free and AD-treated subjects. Neuroplastic proteins (PSD-95, Arc, spinophilin) were quantified in MDD brains. RESULTS: Total cortical CB1 glycosylated (≈54/64 kDa) receptor was increased in MDD (+20%, n=23, p=0.02) when compared with controls (100%, n=19). This CB1 receptor upregulation was quantified in AD-treated (+23%, n=14, p=0.02) but not in AD-free (+14%, n=9, p=0.34) MDD subjects. In the same MDD cortical samples, CB2 glycosylated (≈45 kDa) receptor was unaltered (all MDD: +11%, n=23, p=0.10; AD-free: +12%, n=9, p=0.31; AD-treated: +10%, n=14, p=0.23). In MDD, mTOR activity (p-Ser2448 TOR/t-TOR) was increased (all MDD: +29%, n=18, p=0.002; AD-free: +33%, n=8, p=0.03; AD-treated: +25%, n=10, p=0.04). In contrast, JNK1/2 activity (p-Thr183/Tyr185/t-JNK) was unaltered in MDD subjects. Cortical PSD-95, Arc, and spinophilin contents were unchanged in MDD. LIMITATIONS: A relative limited sample size. Some MDD subjects had been treated with a variety of ADs. The results must be understood in the context of suicide victims with MDD. CONCLUSIONS: The upregulation of CB1 receptor density, but not that of CB2 receptor, as well as the increased mTOR activity in PFC/BA9 of subjects with MDD (AD-free/treated) support their contributions in the complex pathophysiology of MDD and in the molecular mechanisms of antidepressant drugs.
Subject(s)
Cannabinoids , Depressive Disorder, Major , Depressive Disorder, Major/drug therapy , Humans , Prefrontal Cortex , Receptors, Cannabinoid , TOR Serine-Threonine KinasesABSTRACT
To elucidate possible abnormalities in transmembrane signal transduction in psychiatric diseases, use of autopsy brain is a feasible approach. However, postmortem studies should be interpreted with caution concerning such factors as age, gender, psychotropic drug history, agonal state, postmortem delay (PMD), and storage period. In this study, agonist-induced [35S]GTPγS binding was performed in postmortem dorsolateral prefrontal cortical membranes of 40 control subjects. In addition to the previously reported G protein-coupled receptor (GPCR)-mediated Gi/o activation, κ-opioid receptor-mediated [35S]GTPγS binding was detected by using U-50,448. The responses elicited by 16 different agonists were determined, and the effects of several factors were investigated. Gender difference was negligible. Concentration-response curve of histamine H3 receptor-mediated [35S]GTPγS binding was shifted rightward in the subjects with some drugs detected at toxicological screening. Age-related alterations were minimal, except for the age-dependent supersensitivity of µ-opioid receptor-mediated Gαi/o activation, revealed by endomorphin-1- and DAMGO-stimulated [35S]GTPγS binding. Age-related increase in %Emax values was also detected as to DPDPE-induced [35S]GTPγS binding through δ-opioid receptors. With an exception of NOP receptor/G-protein coupling, GPCR-mediated [35S]GTPγS binding is relatively stable irrespective of PMD or storage period. There were many positive correlations among the %Emax values for different receptor subtypes, which might reflect formation of heterodimer complex of such GPCRs coupled to the same Gi/o proteins. These results provide us with important fundamental data in the future project using human postmortem brains from patients with psychiatric disorders.
Subject(s)
GTP-Binding Proteins/metabolism , Prefrontal Cortex/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Female , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Humans , Male , Middle Aged , Prefrontal Cortex/pathology , Receptors, Opioid, kappa/metabolism , Receptors, Opioid, mu/metabolism , Signal Transduction/physiology , Young AdultABSTRACT
Imidazoline I2 receptors (I2-IR), widely distributed in the CNS and altered in patients that suffer from neurodegenerative disorders, are orphans from a structural point of view, and new I2-IR ligands are urgently required for improving their pharmacological characterization. We report the synthesis and three-dimensional quantitative structure-activity relationship (3D-QSAR) studies of a new family of bicyclic α-iminophosphonates endowed with relevant affinities for human brain I2-IR. Acute treatment in mice with a selected compound significantly decreased Fas-associated protein with death domain (FADD) in the hippocampus, a key signaling mediator of neuroprotective actions. Additionally, in vivo studies in the familial Alzheimer's disease 5xFAD murine model revealed beneficial effects in behavior and cognition. These results are supported by changes in molecular pathways related to cognitive decline and Alzheimer's disease. Therefore, bicyclic α-iminophosphonates are tools that may open new therapeutic avenues for I2-IR, particularly for unmet neurodegenerative conditions.
Subject(s)
Alzheimer Disease/drug therapy , Imidazoles/therapeutic use , Imidazoline Receptors/metabolism , Nootropic Agents/therapeutic use , Organophosphonates/therapeutic use , Animals , Chlorocebus aethiops , Cycloaddition Reaction , Dogs , Female , HeLa Cells , Hippocampus/drug effects , Humans , Imidazoles/chemical synthesis , Imidazoles/metabolism , Imidazoles/pharmacokinetics , Ligands , Madin Darby Canine Kidney Cells , Mice , Molecular Structure , Nootropic Agents/chemical synthesis , Nootropic Agents/metabolism , Nootropic Agents/pharmacokinetics , Organophosphonates/chemical synthesis , Organophosphonates/metabolism , Organophosphonates/pharmacokinetics , Quantitative Structure-Activity Relationship , Vero CellsABSTRACT
Accumulating studies have implicated intracellular signaling through muscarinic acetylcholine receptors (mAChRs) in psychiatric illness. In the present study, carbamylcholine chloride (carbachol)-induced Gαi/o and Gαq/11 activation was identified in postmortem human prefrontal cortical membranes. The following two sample cohorts were used: subjects [1], consisting of 40 controls without neuropsychiatric disorders, and subjects [2], consisting of 20 with bipolar disorder (BP), 20 major depressive disorder (MDD), 20 schizophrenia, and 20 controls, strictly sex- and age-matched. Carbachol-stimulated [35S]GTPγS binding to human brain membranes was assessed by the two methods, i.e., conventional method using filtration techniques (Gαi/o activation coupled to M2/M4 mAChRs) applied to subjects [1], and [35S]GTPγS binding/immuno precipitation assay (Gαq/11 activation coupled to M1 mAChR) applied to subjects [1] and [2]. The concentration eliciting the half-maximal effect (EC50), maximum percent increase (%Emax), and slope factor were obtained from concentration-response curve of carbachol-induced Gαi/o and Gαq/11 activation. The pEC50 values of both carbachol-induced Gαi/o and Gαq/11 activations in subjects [1] were significantly correlated, though its implications or underlying molecular processes are unclear. The results of M1 mAChR-mediated Gαq/11 activation in subjects [2] indicated no significant disorder-specific alterations. However, the distribution patterns of the pEC50 values showed unequal variances among the groups. There was a significant inverse correlation between the %Emax values and the pEC50 values in subjects with schizophrenia, but not in those with BP or MDD, or controls. These data support the notion that schizophrenia patients consist of biologically heterogeneous subgroups with respect to M1 mAChR-mediated signaling pathways.
Subject(s)
Bipolar Disorder/metabolism , Depressive Disorder, Major/metabolism , GTP-Binding Protein alpha Subunits/metabolism , Prefrontal Cortex/metabolism , Receptor, Muscarinic M1/metabolism , Schizophrenia/metabolism , Signal Transduction , Adult , Aged , Autopsy , Carbachol/pharmacology , Cohort Studies , Female , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Humans , Male , Middle AgedABSTRACT
Imidazoline receptors historically referred to a family of nonadrenergic binding sites that recognize compounds with an imidazoline moiety, although this has proven to be an oversimplification. For example, none of the proposed endogenous ligands for imidazoline receptors contain an imidazoline moiety but they are diverse in their chemical structure. Three receptor subtypes (I1, I2, and I3) have been proposed and the understanding of each has seen differing progress over the decades. I1 receptors partially mediate the central hypotensive effects of clonidine-like drugs. Moxonidine and rilmenidine have better therapeutic profiles (fewer side effects) than clonidine as antihypertensive drugs, thought to be due to their higher I1/α 2-adrenoceptor selectivity. Newer I1 receptor agonists such as LNP599 [3-chloro-2-methyl-phenyl)-(4-methyl-4,5-dihydro-3H-pyrrol-2-yl)-amine hydrochloride] have little to no activity on α 2-adrenoceptors and demonstrate promising therapeutic potential for hypertension and metabolic syndrome. I2 receptors associate with several distinct proteins, but the identities of these proteins remain elusive. I2 receptor agonists have demonstrated various centrally mediated effects including antinociception and neuroprotection. A new I2 receptor agonist, CR4056 [2-phenyl-6-(1H-imidazol-1yl) quinazoline], demonstrated clear analgesic activity in a recently completed phase II clinical trial and holds great promise as a novel I2 receptor-based first-in-class nonopioid analgesic. The understanding of I3 receptors is relatively limited. Existing data suggest that I3 receptors may represent a binding site at the Kir6.2-subtype ATP-sensitive potassium channels in pancreatic ß-cells and may be involved in insulin secretion. Despite the elusive nature of their molecular identities, recent progress on drug discovery targeting imidazoline receptors (I1 and I2) demonstrates the exciting potential of these compounds to elicit neuroprotection and to treat various disorders such as hypertension, metabolic syndrome, and chronic pain.
Subject(s)
Imidazoline Receptors/metabolism , Imidazolines/metabolism , Imidazolines/pharmacology , Animals , Antihypertensive Agents/pharmacology , Antihypertensive Agents/therapeutic use , Clonidine/pharmacology , Clonidine/therapeutic use , Humans , Imidazoles/pharmacology , Imidazoles/therapeutic use , Ligands , Quinazolines/pharmacology , Quinazolines/therapeutic use , Randomized Controlled Trials as TopicABSTRACT
The biochemical abnormalities in transmembrane signal transduction mediated through G protein-coupled receptors (GPCRs) have been postulated as underlying pathophysiology of psychiatric diseases such as schizophrenia and mood disorders. In the present study, the experimental conditions of agonist-induced [35 S]GTPγS binding in postmortem human brain membranes were optimized, and the responses induced by a series of agonists were pharmacologically characterized. The [35 S]GTPγS binding assay was performed in postmortem human prefrontal cortical membranes by means of filtration techniques, and standardized as to GDP concentration, membrane protein content, MgCl2 and NaCl concentrations in assay buffer, incubation period and effect of white matter contamination. Under the standard assay conditions, the specific [35 S]GTPγS binding was stimulated by the addition of 15 compounds in a concentration-dependent manner. Of these agonists, R(+)-8-OH-DPAT, UK-14,304, DAMGO and DPDPE showed apparently biphasic concentration-response curves. As for these four responses, only higher-potency site was pharmacologically characterized. The receptors involved in the responses investigated were 5-HT1A receptor (probed with R(+)-8-OH-DPAT or 5-HT), α2A -adrenoceptor (UK-14,304 or (-)-epinephrine), M2 /M4 mAChRs (carbachol), adenosine A1 receptor (adenosine), histamine H3 receptor (histamine), group II mGlu (l-glutamate), GABAB receptor (baclofen), µ-opioid receptor (DAMGO or endomophin-1), δ-opioid receptor (DPDPE or SNC-80) and NOP (nociceptin). Although dopamine also activated specific [35 S]GTPγS binding, this response was likely mediated via α2A -adrenoceptor, but not dopamine receptor subtypes. The present study provides us with fundamental aspects of the strategy for elucidation of probable abnormalities of neural signalling mediated by G proteins activated through multiple GPCRs in the brain of psychiatric patients.
Subject(s)
GTP-Binding Proteins/agonists , GTP-Binding Proteins/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Prefrontal Cortex/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Binding, Competitive , Female , Humans , Male , Middle Aged , Protein Binding , Receptor, Adenosine A1/metabolism , Receptor, Serotonin, 5-HT2C/metabolism , Receptors, Adrenergic, alpha-2/metabolism , Receptors, GABA-B/metabolism , Receptors, Histamine H3/metabolism , Receptors, Metabotropic Glutamate/metabolism , Receptors, Opioid, mu/metabolism , Receptors, sigma/metabolism , Young Adult , Sigma-1 ReceptorABSTRACT
Ketamine (KET) is an antidepressant and hypnotic drug acting as an antagonist at excitatory NMDA glutamate receptors. The working hypothesis postulated that KET-induced sleep in mice results in dysregulation of mitogen-activated protein kinases (MAPK) MEK-ERK sequential phosphorylation and upregulation of survival p-FADD and other neuroplastic markers in brain. Low (5-15â¯mg/kg) and high (150â¯mg/kg) doses of KET on target proteins were assessed by Western immunoblot in mouse brain cortex. During the time course of KET (150â¯mg/kg)-induced sleep (up to 50â¯min) p-MEK was increased (up to +79%) and p-ERK decreased (up to -46%) indicating disruption of MEK to ERK signal. Subhypnotic KET (5-15â¯mg/kg) also revealed uncoupling of p-MEK (+13-81%) to p-ERK (unchanged content). KET did not alter contraregulatory MAPK mechanisms such as inactivated p-MEK1 (ERK dampening) and phosphatases MKP1/2/3 (ERK dephosphorylation). As other relevant findings, KET (5, 15 and 150â¯mg/kg) upregulated p-FADD in a dose-dependent manner, and for the hypnotic dose the effect paralleled the time course of sleep which resulted in increased p-FADD/FADD ratios. KET (150â¯mg/kg) also increased NF-κΒ and PSD-95 neuroplastic markers. Flumazenil (a neutral allosteric antagonist at GABAA receptor) prolonged KET sleep and blocked p-MEK upregulation, indicating the involvement of this receptor as a negative modulator. SL-327 (a MEK inhibitor) augmented KET sleep, further indicating the relevance of reduced p-ERK1/2 in KET-induced hypnosis. These findings suggest that hypnotic and subhypnotic doses of KET inducing uncoupling of p-MEK to p-ERK signal and regulation of p-ERK (downregulation) and p-FADD (upregulation) may participate in the expression of some of its adverse effects (e.g. amnesia, dissociative effects).
Subject(s)
Cerebral Cortex/drug effects , Fas-Associated Death Domain Protein/metabolism , Immobility Response, Tonic/drug effects , Ketamine/pharmacology , MAP Kinase Signaling System/drug effects , Neuronal Plasticity/drug effects , Receptors, GABA-A/metabolism , Analgesics/pharmacokinetics , Animals , Cerebral Cortex/physiology , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Flumazenil/pharmacology , GABA Modulators/pharmacology , Male , Mice , Nerve Tissue Proteins/metabolism , Reflex, Righting/drug effects , Time FactorsABSTRACT
As populations increase their life expectancy, age-related neurodegenerative disorders such as Alzheimer's disease have become more common. I2-Imidazoline receptors (I2-IR) are widely distributed in the central nervous system, and dysregulation of I2-IR in patients with neurodegenerative diseases has been reported, suggesting their implication in cognitive impairment. This evidence indicates that high-affinity selective I2-IR ligands potentially contribute to the delay of neurodegeneration. In vivo studies in the female senescence accelerated mouse-prone 8 mice have shown that treatment with I2-IR ligands, MCR5 and MCR9, produce beneficial effects in behavior and cognition. Changes in molecular pathways implicated in oxidative stress, inflammation, synaptic plasticity, and apoptotic cell death were also studied. Furthermore, treatments with these I2-IR ligands diminished the amyloid precursor protein processing pathway and increased Aß degrading enzymes in the hippocampus of SAMP8 mice. These results collectively demonstrate the neuroprotective role of these new I2-IR ligands in a mouse model of brain aging through specific pathways and suggest their potential as therapeutic agents in brain disorders and age-related neurodegenerative diseases.
Subject(s)
Aging/drug effects , Behavior, Animal/drug effects , Cognition/drug effects , Hippocampus/drug effects , Imidazoline Receptors/agonists , Aging/metabolism , Amyloid beta-Protein Precursor/metabolism , Animals , Apoptosis/drug effects , Disease Models, Animal , Female , Hippocampus/metabolism , Mice , Oxidative Stress/drug effects , Recognition, Psychology/drug effectsABSTRACT
Midazolam and ketamine-induced anesthesia were recently shown to induce a disruption of MEK/ERK sequential phosphorylation with parallel upregulation of p-FADD in the mouse brain. The present study was designed to assess whether other structurally diverse anesthetic agents (pentobarbital, ethanol, chloral hydrate, isoflurane) also impair brain p-MEK to p-ERK signal and increase p-FADD during the particular time course of 'sleep' in mice. Pentobarbital (50â¯mg/kg)-, ethanol (4000â¯mg/kg)-, chloral hydrate (400â¯mg/kg)-, and isoflurane (2% in O2)-induced anesthesia (range: 24-60 min) were associated with unaltered or increased p-MEK1/2 (up to +155%) and decreased p-ERK1/2 (up to -60%) contents, revealing disruption of MEK to ERK activation in mouse brain cortex. These anesthetic agents also upregulated cortical p-FADD (up to +110%), but not total FADD (moderately decreased), which resulted in increased neuroplastic/survival p-FADD/FADD ratios (up to +2.8 fold). The inhibition of pentobarbital metabolism with SKF525-A (a cytochrome P450 inhibitor) augmented barbiturate anesthesia (2.6 times) and induced a greater and sustained upregulation of p-MEK with p-ERK downregulation, as well as prolonged increases of p-FADD content and p-FADD/FADD ratio (effects lasting for more than 240 min). Pentobarbital also upregulated significantly the cortical contents of other markers of neuroplasticity such as the ERK inhibitor p-PEA-15 (up to +46%), the transcription factor NF-κB (up to +27%) and the synaptic density protein PSD-95 (up to +20%) during 'sleep'. The results reveal a paradoxical stimulation of p-MEK without the concomitant (canonical) activation of p-ERK (e.g. with pentobarbital and isoflurane), for which various molecular mechanisms are discussed. The downregulation of brain p-ERK may participate in the manifestations of adverse effects displayed by most hypnotic/anesthetic agents in clinical use (e.g. amnesia).
Subject(s)
Brain/drug effects , Fas-Associated Death Domain Protein/metabolism , MAP Kinase Kinase Kinases/drug effects , Pentobarbital/pharmacology , Anesthetics/pharmacology , Animals , Brain/metabolism , Hypnotics and Sedatives/pharmacology , Ketamine/pharmacology , Male , Mice , Neuronal Plasticity/drug effects , Transcriptional Activation/drug effects , Up-Regulation/drug effectsABSTRACT
Adenosine signaling plays a complex role in multiple physiological processes in the brain, and its dysfunction has been implicated in pathophysiology of neuropsychiatric diseases such as schizophrenia and affective disorders. In the present study, the coupling between adenosine A1 receptor and G-protein was assessed by means of two [35S]GTPγS binding assays, i.e., conventional filtration method and [35S]GTPγS binding/immunoprecipitation in rat and human brain membranes. The latter method provides information about adenosine A1 receptor-mediated Gαi-3 activation in rat as well as human brain membranes. On the other hand, adenosine-stimulated [35S]GTPγS binding determined with conventional assay derives from functional activation of Gαi/o proteins (not restricted only to Gαi-3) coupled to adenosine A1 receptors. The determination of adenosine concentrations in the samples used in the present study indicates the possibility that the assay mixture under our experimental conditions contains residual endogenous adenosine at nanomolar concentrations, which was also suggested by the results on the effects of adenosine receptor antagonists on basal [35S]GTPγS binding level. The effects of adenosine deaminase (ADA) on basal binding also support the presence of adenosine. Nevertheless, the varied patterns of ADA discouraged us from adding ADA into assay medium routinely. The concentration-dependent increases elicited by adenosine were determined in 40 subjects without any neuropsychiatric disorders. The increases in %Emax values determined by conventional assay according to aging and postmortem delay should be taken into account in future studies focusing on the effects of psychiatric disorders on adenosine A1 receptor/G-protein interaction in postmortem human brain tissue.
Subject(s)
Brain/metabolism , GTP-Binding Proteins/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Immunoprecipitation/methods , Receptor, Adenosine A1/metabolism , Animals , Binding, Competitive , Female , Humans , Purinergic P1 Receptor Antagonists , Rats , Sulfur Radioisotopes/metabolismABSTRACT
Heterotrimeric guanine nucleotide-binding proteins (G-proteins) play a pivotal role in a wide range of signal transduction pathways, and receptor/G-protein coupling has been implicated in the pathophysiology of mental disorders. In this study, guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding/immunoprecipitation assay for Gαq was applied to postmortem human brains. After its optimization for human prefrontal cortical membranes, we selected 5-hydroxytryptamine (5-HT) and carbachol as efficient agonists for subsequent experiments. The concentration-response curve of 5-HT shifted towards the right by the addition of increasing concentrations of ketanserin (with a pA 2 value of 9.18), indicating the involvement of the 5-HT2A receptor. Besides, the carbachol-stimulated [35S]GTPγS binding to Gαq was competitively antagonized by telenzepine (with a pA 2 value of 8.81), indicating the involvement of the M1 muscarinic acetylcholine receptor (mAChR). Concentration-response curves of 5-HT2A receptor- and M1 mAChR-mediated Gαq activation were determined in 40 subjects. The mean maximum percentage increase (%E max) was 155 and 470%, respectively, and the mean half-maximal effect concentration (EC50) was 131 nM and 15.2 µM, respectively. When the pharmacological parameters were correlated with age, postmortem delay, freezing storage period, and tissue pH, no statistically significant correlation was observed except for the negative correlation between age and %E max value of carbachol-stimulated [35S]GTPγS binding to Gαq. The %E max values for 5-HT2A receptor- and M1 mAChR-mediated Gαq activation also tended to correlate with each other. These results provide fundamental information of Gαq-coupled 5-HT2A receptor and M1 mAChR in native human brains, and lay the foundation for future studies in mental disorder patients.
Subject(s)
Cerebral Cortex/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Receptor, Muscarinic M1/metabolism , Receptor, Serotonin, 5-HT2A/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Cerebral Cortex/drug effects , Dose-Response Relationship, Drug , Female , Humans , Male , Membranes/drug effects , Membranes/metabolism , Middle Aged , Neurotransmitter Agents/pharmacology , Young AdultABSTRACT
Imidazoline receptor antisera-selected (IRAS)/nischarin, a putative I1-imidazoline receptor, has recently been shown to regulate µ-opioid receptor (OR) trafficking and resensitisation. To study a possible involvement of this µ-OR regulator in opiate dependence, the present study assessed by Western blot analysis the contents of IRAS/nischarin and µ-OR in total homogenates and subcellular preparations of postmortem human prefrontal cortex (PFC/BA9) of long-term opiate and mixed opiate/cocaine abusers as well as of matched healthy control subjects. In the PFC/BA9 of long-term opiate/cocaine abusers (all subjects together) IRAS/nischarin content was increased (+67%, p < 0.01, n = 11) when compared with matched controls (n = 10). Similar increases were found for the subgroups of opiate (+72%, n = 6) and mixed opiate/cocaine (+61%, n = 5) abusers. IRAS/nischarin immunocontents were also found increased in subcellular membrane preparations (+61%, p < 0.05, n = 10) of PFC/BA9 from opiate addicts. In the same brain samples, the levels of µ-OR were not different to those in control subjects. Based on the increased contents in brains of opiate abusers and the reported function as µ-OR regulator, IRAS/nischarin could represent a new promising target for treatment of opiate use disorder.