Risk factors of pediatric ocular trauma related to ophthalmic complications.

BACKGROUND: Ocular injury is a leading cause of monocular blindness in children. However, data on the association of injury type with ophthalmological complications are lacking. This study aimed to investigate the risk factors of pediatric ocular injuries related to ophthalmological complications. METHODS: This retrospective, observational study was conducted in a pediatric emergency department (ED) in Japan from March 2010 to March 2021. Patients aged less than 16 years presenting with ocular trauma, diagnosed on the basis of the International Classification of Diseases 10: S05, 0-9, were enrolled. Emergency department follow-up visits for the same complaint were excluded. The patients' sex, age, arrival time, injury mechanism, symptoms, examinations, diagnosis, history of urgent ophthalmological consultation, outcomes, and ophthalmological complications were investigated. The primary endpoints were the odds ratio (OR) and 95% confidence interval (CI) of the incidence of ophthalmological complications, defined as any new acute complaint or worsening/persistence of an initial complaint following or resulting from ocular trauma. RESULTS: In total, 469 patients were analyzed. The median age was 7.3 years; the interquartile range (IQR) was 3.1-11.5 years. The most common diagnosis was contusion (79.3%), followed by lamellar laceration (11.7%). Seven patients (1.5%) had ophthalmological complications during follow up. Bivariate analysis demonstrated that daytime ED visit, impact with a sharp object, animal-related injury, visual impairment, decreased visual acuity, and open globe injury were factors significantly associated with ophthalmological complications. CONCLUSION: Daytime ED visit, impact with a sharp object, animal-related injury, visual impairment, decreased visual acuity, and open globe injury were independent factors of ophthalmological complications.

Characteristics of pediatric ocular trauma in a pediatric emergency department in Japan.

INTRODUCTION: Pediatric ocular trauma is a common complaint in pediatric emergency departments (ED) and is a major cause of acquired monocular blindness. However, data on its epidemiology and management in the ED are lacking. The objective of this study was to describe the characteristics and management of pediatric ocular trauma patients who visited a Japanese pediatric emergency department (ED). METHODS: The present, retrospective, observational study was conducted in a pediatric ED in Japan between March 2010 and March 2021. Children younger than 16 years who visited our pediatric ED and received the diagnosis of ocular trauma were included. ED visits for follow-up examinations for the same complaint were excluded. The patients' sex, age, arrival time, mechanism of injury, signs and symptoms, examinations, diagnosis, history of urgent ophthalmological consultation, outcomes, and ophthalmological complications were extracted from electronic medical records. RESULTS: In total, 469 patients were included; of these, 318 (68%) were male, and the median age was 7.3 years. The incident leading to trauma occurred most frequently at home (26%) and most often involved being struck in the eye (34%). In 20% of the cases, the eye was struck by some body part. Tests performed in the ED included visual acuity testing (44%), fluorescein staining (27%), and computed tomography (19%). Thirty-seven (8%) patients underwent a procedure in the ED. Most patients had a closed globe injury (CGI), with only two (0.4%) having an open globe injury (OGI). Eighty-five (18%) patients required an urgent ophthalmological referral, and 12 (3%) required emergency surgery. Ophthalmological complications occurred in only seven patients (2%). CONCLUSION: Most cases of pediatric ocular trauma seen in the pediatric ED were CGI, with only a few cases leading to emergency surgery or ophthalmological complications. Pediatric ocular trauma can be safely managed by pediatric emergency physicians.

Agonistic properties of a series of psychotropic drugs at 5-HT1A receptors in rat and human brain membranes determined by [35S]GTPγS binding assay.

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.

5-HT2A receptor- and M1 muscarinic acetylcholine receptor-mediated activation of Gαq/11 in postmortem dorsolateral prefrontal cortex of opiate addicts.

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.

5-HT2A receptor-mediated Gαq/11 activation in psychiatric disorders: A postmortem study.

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.

Fundamental features of receptor-mediated Gαi/o activation in human prefrontal cortical membranes: A postmortem study.

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.

Functional coupling of M1 muscarinic acetylcholine receptor to Gαq/11 in dorsolateral prefrontal cortex from patients with psychiatric disorders: a postmortem study.

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.

Dopamine-induced functional activation of Gαq mediated by dopamine D1-like receptor in rat cerebral cortical membranes.

Although multiple roles of dopamine through D1-like (D1 and D5) and D2-like (D2, D3, and D4) receptors are initiated primarily through stimulation or inhibition of adenylyl cyclase via Gs/olf or Gi/o, respectively, there have been many reports indicating diverse signaling mechanisms that involve alternative G protein coupling. In this study, dopamine-induced Gαq activation in rat brain membranes was investigated. Agonist-induced Gαq activation was assessed by increase in guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding to Gαq determined by [35S]GTPγS binding/immunoprecipitation assay in rat brain membranes. Dopamine-stimulated Gαq functionality was highest in cortex as compared to hippocampus or striatum. In cerebral cortical membranes, this effect was mimicked by benzazepine derivatives with agonist properties at dopamine D1-like receptors, that is, SKF83959, SKF83822, R(+)-SKF81297, R(+)-SKF38393, and SKF82958, but not by the compounds with dopamine D2-like receptor agonist properties except for aripiprazole. Against expectation, stimulatory effects were also induced by SKF83566, R(+)-SCH23390, and pergolide. The pharmacological profiling by using a series of antagonists indicated that dopamine-induced response was mediated through dopamine D1-like receptor, which was distinct from the receptor involved in 5-HT-induced response (5-HT2A receptor). Conversely, the responses induced by SKF83566, R(+)-SCH23390, and pergolide were most likely mediated by 5-HT2A receptor, but not by dopamine D1-like receptor. Caution should be paid when interpreting the experimental data, especially in behavioral pharmacological research, in which SKF83566 or R(+)-SCH23390 is used as a standard selective dopamine D1-like receptor antagonist. Also, possible clinical implications of the agonistic effects of pergolide on 5-HT2A receptor has been mentioned.

Functional activation of Gαq/11 protein via α1 -adrenoceptor in rat cerebral cortical membranes.

Although it is recognized that α1 -adrenoceptors are coupled to diverse intracellular signalling pathways, its primary transduction mechanisms are evoked by activating phospholipase C in the cell membrane through Gαq/11 , resulting in production of inositol 1,4,5-trisphosphate and diacylglycerol. However, there have been few studies that indicate directly the involvement of Gαq/11 proteins in this signalling pathway in the central nervous system. In the current study, we tried to pharmacologically characterize (-)-adrenaline-stimulated [35 S]GTPγS binding to Gαq/11 in rat brain membranes. Functional activation of Gαq/11 coupled to α1 -adrenoceptor was investigated by using [35 S]GTPγS binding/immunoprecipitation assay in the membranes prepared from rat cerebral cortex, hippocampus, and striatum. The specific [35 S]GTPγS binding to Gαq/11 was stimulated by (-)-adrenaline in a concentration-dependent and saturable manner in rat cerebral cortical membranes. In hippocampal or striatal membranes, the stimulatory effects of (-)-adrenaline were scarce. The effect of (-)-adrenaline was potently inhibited by prazosin, a potent and selective α1 -adrenoceptor antagonist, but not by yohimbine, a selective α2 -adrenoceptor antagonist. The response was mimicked by cirazoline, but not by R(-)-phenylephrine. Although oxymetazoline also stimulated the specific [35 S]GTPγS binding to Gαq/11 as an apparent "super-agonist", detailed pharmacological characterization revealed that its agonistic properties in this experimental system were derived from off-target effects on 5-HT2A receptors, but not via α1 -adrenoceptors. In conclusion, functional coupling of α1 -adrenoceptors to Gαq/11 proteins are detectable in rat brain membranes by means of [35 S]GTPγS binding/immunoprecipitation assay. It is necessary to interpret the experimental data with caution when oxymetazoline is included as an agonist at α1 -adrenoceptors.

Optimization and pharmacological characterization of receptor-mediated Gi/o activation in postmortem human prefrontal cortex.

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.

The influence of isoflavone for denervation-induced muscle atrophy.

PURPOSE: Decrease in activity stress induces skeletal muscle atrophy. A previous study showed that treatment with a high level (20%) of isoflavone inhibits muscle atrophy after short-term denervation (at 4 days) in mice. The present study was designed to elucidate whether the dietary isoflavone aglycone (AglyMax) at a 0.6% prevents denervation-mediated muscle atrophy, based on the modulation of atrogin-1- or apoptosis-dependent signaling. METHODS: Mice were fed either a normal diet or 0.6% AglyMax diet. One week later, the right sciatic nerve was cut. The wet weight, mean fiber area, amount of atrogin-1 and cleaved caspase-3 proteins, and the percentages of apoptotic nuclei were examined in the gastrocnemius muscle at 14 days after denervation. RESULTS: The 0.6% AglyMax diet significantly attenuated denervation-induced decreases in fiber atrophy but not the muscle wet weight. In addition, dietary isoflavone suppressed the denervation-induced apoptosis in spite of there being no significant changes in the amount of cleaved caspase-3 protein. In contrast, the 0.6% AglyMax diet did not significantly modulate the protein expression of atrogin-1 in the denervated muscle of mice. CONCLUSIONS: The isoflavone aglycone (AglyMax) at a 0.6% significantly would modulate muscle atrophy after denervation in mice, probably due to the decrease in apoptosis-dependent signaling.

Resveratrol attenuates denervation-induced muscle atrophy due to the blockade of atrogin-1 and p62 accumulation.

Decrease in activity stress induces skeletal muscle atrophy. A previous study showed that treatment with resveratrol inhibits muscular atrophy in mdx mice, a model of DMD. However, almost all studies using resveratrol supplementation have only looked at adaptive changes in the muscle weight. The present study was designed to elucidate whether the resveratrol-inducing attenuation of skeletal muscle actually reflects the adaptation of muscle fibers themselves, based on the modulation of atrogin-1- or p62-dependent signaling. Mice were fed either a normal diet or 0.5% resveratrol diet. One week later, the right sciatic nerve was cut. The wet weight, mean fiber area, and amount of atrogin-1 and p62 proteins were examined in the gastrocnemius muscle at 14 days after denervation. The 0.5% resveratrol diet significantly prevented denervation-induced decreases in both the muscle weight and fiber atrophy. In addition, dietary resveratrol suppressed the denervation-induced atrogin-1 and p62 immunoreactivity. In contrast, 0.5% resveratrol supplementation did not significantly modulate the total protein amount of atrogin-1 or p62 in the denervated muscle of mice. Resveratrol supplementation significantly prevents muscle atrophy after denervation in mice, possibly due to the decrease in atrogin-1 and p62-dependent signaling.

Functional coupling between adenosine A1 receptors and G-proteins in rat and postmortem human brain membranes determined with conventional guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding or [35S]GTPγS/immunoprecipitation assay.

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.

Functional activation of Gαq coupled to 5-HT2A receptor and M1 muscarinic acetylcholine receptor in postmortem human cortical membranes.

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.

p62/SQSTM1 but not LC3 is accumulated in sarcopenic muscle of mice.

AIM: We investigated the pathway of autophagy signaling linked to sarcopenia of mice. METHODS: Young adult (3-month) and aged (24- month) C57BL/6J mice were used. Using real-time PCR, Western blotting, and immunohistochemical microscopy, we evaluated the amounts of p62/SQSTM1, LC3, and Beclin-1 in the quadriceps muscle change with aging in mice. RESULTS: Marked fiber atrophy (30%) and many fibers with central nuclei were observed in the aged mice. Western blotting using homogenate of the cytosolic fraction clearly showed that the amounts of p62/SQSTM1 and Beclin-1 proteins were significantly increased in the aged skeletal muscle. The amounts of these proteins in both nuclear and membrane fractions did not change significantly with age. Immunofluorescence labeling indicated that aged mice more frequently possessed p62/SQSTM1-positive fibers in the cytosol in quadriceps muscle than young ones (aged: 14% vs. young: 1%). In aged muscle, p62/SQSTM1-positive fibers were significantly smaller than the surrounding p62/SQSTM1-negative fibers. Aging did not elicit significant changes in the mRNA levels of p62/SQSTM1 and Beclin-1, but decreased LC3 mRNA level. In aged muscle, the location of p62/SQSTM1 immunoreactivity was similar to that of Beclin-1 protein, but not LC3. CONCLUSION: Sarcopenia in mice appears to include a marked defect of autophagy signaling.

Comparative analysis of pharmacological properties of xanomeline and N-desmethylclozapine in rat brain membranes.

BACKGROUND: 3(3-Hexyloxy-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (xanomeline) and N-desmethylclozapine are of special interest as promising antipsychotics with better efficacy, especially for negative symptoms and/or cognitive/affective impairment. METHODS: The guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding experiments were performed using (1) conventional filtration technique, (2) antibody-capture scintillation proximity assay, and (3) immunoprecipitation method, in brain membranes prepared from rat cerebral cortex, hippocampus, and striatum. RESULTS: Xanomeline had agonistic activity at the M1 muscarinic acetylcholine receptor (mAChR) in all brain regions, as well as at the 5-HT1A receptor in the cerebral cortex and hippocampus. On the other hand, N-desmethylclozapine exhibited slight agonistic effects on the M1 mAChR, and agonistic properties at the 5-HT1A receptor in the cerebral cortex and hippocampus. This compound also behaved as an agonist at the δ-opioid receptor in the cerebral cortex and striatum. In addition, the stimulatory effects of N-desmethylclozapine on [(35)S]GTPγS binding to Gαi/o were partially mediated through mAChRs (most likely M4 mAChR subtype), at least in striatum. CONCLUSIONS: The agonistic effects on the mAChRs (particularly M1 subtype, and also probably M4 subtype), the 5-HT1A receptor and the δ-opioid receptor expressed in native brain tissues, some of which are common to both compounds and others specific to either, likely shape the unique beneficial effectiveness of both compounds in the treatment for schizophrenic patients. These characteristics provide us with a clue to develop newer antipsychotics, beyond the framework of dopamine D2 receptor antagonism, that are effective not only on positive symptoms but also on negative symptoms and/or cognitive/affective impairment.

Adenosine A1( )receptors are selectively coupled to Gα(i-3) in postmortem human brain cortex: Guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding/immunoprecipitation study.

By means of guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding assay combined with immunoprecipitation using anti-Gα subunit antibody, we recently reported 5-HT2A receptor- and M1 muscarinic acetylcholine receptor-mediated Gαq activation in rat cerebral cortical membranes (Odagaki et al., 2014). In the present study, this method has been applied to postmortem human brains, with focusing on adenosine receptor-mediated G-protein activation. In the exploratory experiments using a series of agonists and the antibodies specific to each Gα subtypes in the presence of low (10 nM) or high (50 µM) concentration of GDP, the most prominent increases in specific [(35)S]GTPγS binding in the membranes prepared from human prefrontal cortex were obtained for the combinations of adenosine (1mM)/anti-Gαi-3 in the presence of 50 µM GDP as well as 5-HT (100 µM)/anti-Gαq and carbachol (1mM)/anti-Gαq in the presence of 10nM GDP. Adenosine-induced activation of Gαi-3 emerged only when GDP concentrations were increased higher than 10 µM, and the following experiments were performed in the presence of 300 µM GDP. Adenosine increased specific [(35)S]GTPγS binding to Gαi-3 in a concentration-dependent manner to 251.4% of the basal unstimulated binding, with an EC50 of 1.77 µM. The involvement of adenosine A1 receptor was verified by the experiments using selective agonists and antagonists at adenosine A1 or A3 receptor. Among the α subunits of Gi/o class (Gαi-1, Gαi-2, Gαi-3, and Gαo.), only Gαi-3 was activated by 1mM adenosine, indicating that human brain adenosine A1 receptor is coupled preferentially, if not exclusively, to Gαi-3.

Functional activation of G-proteins coupled with muscarinic acetylcholine receptors in rat brain membranes.

The functional activation of Gi/o proteins coupled to muscarinic acetylcholine receptors (mAChRs) was investigated with the conventional guanosine-5'-O-(3-[(35)S]thio) triphosphate ([(35)S]GTPγS) binding assay in rat brain membranes. The most efficacious stimulation elicited by acetylcholine or carbachol (CCh) was obtained in striatal membranes. The pharmacological properties of mAChR-mediated [(35)S]GTPγS binding determined with a series of muscarinic agonists and antagonists were almost identical among the three brain regions investigated, i.e., cerebral cortex, hippocampus, and striatum, except for the apparent partial agonist effects of (αR)-α-cyclopentyl-α-hydroxy-N-[1-(4-methyl-3-pentenyl)-4-piperidinyl]benzeneacetamide fumarate (J 104129) observed only in the hippocampus, but not in the other two regions. Among the muscarinic toxins investigated, only MT3 attenuated CCh-stimulated [(35)S] GTPγS binding. The highly selective allosteric potentiator at the M4 mAChR subtype, 3-amino-N-[(4-chlorophenyl)methyl]-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide (VU 10010), shifted the concentration-response curve for CCh leftwards as well as upwards. On the other hand, neither thiochrome nor brucine N-oxide was effective. The increases induced by CCh and 5-HT were essentially additive, though not completely, indicating that the mAChRs and 5-HT1A receptors were coupled independently to distinct pools of Gi/o proteins. Collectively, all of the data suggest that functional activation of Gi/o proteins coupled to mAChRs, especially the M4 subtype, is detectable by means of CCh-stimulated [(35)S]GTPγS binding assay in rat discrete brain regions.

Functional activation of Gαq via serotonin2A (5-HT2A) and muscarinic acetylcholine M1 receptors assessed by guanosine-5׳-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding/immunoprecipitation in rat brain membranes.

Functional coupling between serotonin2A (5-HT2A) receptors and Gαq proteins in native brain membranes has been sparsely reported thus far. In the present study, the guanosine-5׳-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding assay combined with immunoprecipitation using magnetic beads (Dynabeads Protein A) coated with anti-Gαq antibody was developed. Under experimental conditions optimised for assay constituents (GDP, MgCl2, and NaCl), for contents of membrane protein, anti-Gαq antibody, and Dynabeads Protein A, and for the incubation period, 5-HT stimulated specific [35S]GTPγS binding to Gαq in rat cerebral cortical membranes in a concentration-dependent and saturable manner, with a signal/noise ratio that was sufficiently high for further detailed pharmacological characterisation. This characterisation revealed an involvement of 5-HT2A receptors. Activation of Gαq proteins was also detectable by the addition of carbachol via muscarinic acetylcholine M1 receptors, (-)-epinephrine, and dopamine, but not by L-glutamate or (±)-baclofen. When 5-HT2A receptors and M1 receptors were stimulated simultaneously, there were non-additive effects, indicating that the two receptors were coupled to the same components of Gαq proteins in the rat cerebral cortex. This method will serve as an efficacious strategy for neurobiological investigations aimed at elucidating the physiological and pathological implications of signal transduction systems mediated via Gαq proteins coupled with 5-HT2A receptors and muscarinic acetylcholine M1 receptors.