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.

Intracellular inflammatory and antioxidant pathways in postmortem frontal cortex of subjects with major depression: effect of antidepressants.

BACKGROUND: Studies show that Toll-like receptors (TLRs), members of the innate immune system, might participate in the pathogenesis of the major depressive disorder (MDD). However, evidence of this participation in the brain of patients with MDD has been elusive. METHODS: This work explores whether the protein expression by immunodetection assays (Western blot) of elements of TLR-4 pathways controlling inflammation and the oxidative/nitrosative stress are altered in postmortem dorsolateral prefrontal cortex of subjects with MDD. The potential modulation induced by the antidepressant treatment on these parameters was also assessed. Thirty MDD subjects (15 antidepressant-free and 15 under antidepressant treatment) were matched for gender and age to 30 controls in a paired design. RESULTS: No significant changes in TLR-4 expression were detected. An increased expression of the TLR-4 endogenous ligand Hsp70 (+ 33%), but not of Hsp60, and the activated forms of mitogen-activated protein kinases (MAPKs) p38 (+ 47%) and JNK (+ 56%) was observed in MDD. Concomitantly, MDD subjects present a 45% decreased expression of DUSP2 (a regulator of MAPKs) and reduced (- 21%) expression of the antioxidant nuclear factor Nrf2. Antidepressant treatment did not modify the changes detected in the group with MDD and actually increased (+ 25%) the expression of p11, a protein linked with the transport of neurotransmitters and depression. CONCLUSION: Data indicate an altered TLR-4 immune response in the brain of subjects with MDD. Additional research focused on the mechanisms contributing to the antidepressant-induced TLR-4 pathway modulation is warranted and could help to develop new treatment strategies for MDD.

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.

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.

Characterization of regulators of G-protein signaling RGS4 and RGS10 proteins in the postmortem human brain.

Regulator of G-protein signaling (RGS) proteins are a large family of proteins that accelerate GTPase rate of the Gα subunits and therefore, negatively regulate G-protein signaling. Expression of RGS4 and RGS10 proteins was characterized in human prefrontal cortex attending to methodological (subcellular localization, antibody specificity and sensitivity, postmortem delay (PMD) and storage conditions of the samples) and demographic issues (age and gender of the subjects). Anti-RGS4 (N-16) antibody revealed a unique and specific band of 38 kDa that was highly enriched in the plasma membrane. Anti-RGS10 (C-20) antibody revealed two specific bands of 24 and 27kDa, corresponding to two possible isoforms of this protein, which were predominantly localized in the cytosol. Antibody dilution and protein linearity studies confirmed the sensitivity of the signal. A large number of samples from 58 individuals presenting well spread PMD, storage time, age of the subjects at the time of death, and male and female distribution were studied. A positive linear relationship between the age and RGS4 immunoreactivity was observed. There was a negative influence of PMD on the RGS10 27 kDa band immunoreactivity but a positive relationship emerged between the PMD and RGS4 immunoreactivity. Storage time of the samples did not have any influence on RGS4 nor on RGS10 immunoreactivity, showing their stability at -70°C. When studying the RGS4 and RGS10 protein expression density in males and females, no significant difference was found between groups. This study demonstrates that RGS4 and RGS10 proteins can be detected by immunoreactive techniques in postmortem human brain cortex. The study provides important matching conditions that should be taken into account in postmortem brain studies of neuropsychiatric diseases.

Evaluation of a pharmacology educational activity based on a research project: a randomized, controlled and blind analysis of medical students' perceptions.

An experimental model of teaching/learning involving the formulation, execution and presentation of results of a research project has been developed and introduced as part of a Basic Pharmacology course for medical students at the University of the Basque Country (UPV/EHU). The perceptions of the students who participated in the experimental model were evaluated and compared with those who participated in a traditional model of practical activity. An 18-point questionnaire evaluated students' perceptions of aspects of the course itself (such as its duration and organization), personal response characteristics (such as entertainment, interest and effort required) and the current and future utility of the activities which had been carried out. A randomized, double-controlled and double-blind study compared experimental (n = 110) and control groups (n = 63). Students pertaining to the experimental group reported deeper satisfaction and greater participation in the activity. They evaluated more positively the utility of the educational activity for their future profession and more frequently considered that they had acquired useful skills or attitudes. Members of the experimental group recognized that they had invested more time and effort than those of the control group. No differences related to organization, support received and attitudes of teachers were observed between groups. In conclusion, a transitional intervention from traditional models towards PBL-based medical education was favourably evaluated by the participants. The activity was received with deeper satisfaction when compared with a traditional model of practical activity in Pharmacology.

Characterization of noradrenaline release in the locus coeruleus of freely moving awake rats by in vivo microdialysis.

RATIONALE: The origin and regulation of noradrenaline (NA) in the locus coeruleus (LC) is unknown. OBJECTIVES: The neurochemical features of NA overflow (nerve impulse dependence, neurotransmitter synthesis, vesicle storage, reuptake, alpha2-adrenoceptor-mediated regulation) were characterized in the LC. METHODS: Brain microdialysis was performed in awake rats. Dialysates were analyzed for NA. RESULTS: NA in the LC decreased via local infusion of Ca2+-free medium (-42+/-5%) or the sodium channel blocker tetrodotoxine (TTX) (-47+/-8%) but increased (333+/-40%) via KCl-induced depolarization. The tyrosine hydroxylase (TH) inhibitor alpha-methyl-p-tyrosine (250 mg kg(-1), i.p.) and the vesicle depletory drug reserpine (5 mg kg(-1), i.p.) decreased NA. Therefore, extracellular NA in the LC satisfies the criteria for an impulse flow-dependent vesicular exocytosis of neuronal origin. Local perfusion of the alpha2-adrenoceptor agonist clonidine (0.1-100 microM) decreased NA (E(max)=-79+/-5%) in the LC, whereas the opposite effect (E(max)=268+/-53%) was observed with the alpha2A-adrenoceptor antagonist BRL44408 (0.1-100 microM). This suggests a tonic modulation of NA release through local alpha2A-adrenoceptors. The selective NA reuptake inhibitor desipramine (DMI) (0.1-100 microM) administered into the LC increased NA in the LC (E(max)=223+/-40%) and simultaneously decreased NA in the cingulate cortex, confirming the modulation exerted by NA in the LC on firing activity of noradrenergic cells and on the subsequent NA release in noradrenergic terminals. CONCLUSION: Synaptic processes underlying NA release in the LC are similar to those in noradrenergic terminal areas. NA in the LC could represent local somatodendritic release, but also the presence of neurotransmitter release from collateral axon terminals.

Alpha 2-adrenoceptor subtypes in the human brain: a pharmacological delineation of [3H]RX-821002 binding to membranes and tissue sections.

In order to study the characterization and localization of [3H]RX-821002 (2-methoxy-idazoxan) binding to alpha 2-adrenoceptor subtypes in several regions of the human brain, we have carried out competition studies using both autoradiography and membrane binding assays. The alpha 2A-adrenoceptor subtype was found to be predominant in the different layers of the frontal cortex, cerebellum and hippocampal formation, while in the neostriatum it was the non-alpha 2A- (alpha 2B- and alpha 2C-) adrenoceptor subtype. In the frontal cortex, in addition to binding to the alpha 2A-adrenoceptor subtype, [3H]RX-821002 bound also to a small portion of alpha 2B- and alpha 2C-adrenoceptors in layer III, and to an unidentified binding site in the external layers. In the hippocampus, both alpha 2A- and non-alpha 2A- (alpha 2B- and alpha 2C-) adrenoceptors were labelled in the dentate gyrus and the CA1 field, together with 5-HT1A receptors. 5-HT1A receptors were labelled predominantly in the stratum pyramidale layer. These results, in addition to delineate the relative presence of alpha 2-adrenoceptor subtypes, indicate that caution is needed when analyzing RX 821002 binding to human brain tissue.

Increased density of I2-imidazoline receptors in human glioblastomas.

A glial location has been proposed for the non-adrenoceptor [3H]idazoxan binding site termed the I2-imidazoline receptor. The specific binding of [3H]idazoxan in the presence of (-)adrenaline was measured in membranes from excised human glioblastomas (n = 6), meningiomas (n = 6) and normal brains (n = 6). The pharmacological profile of the [3H]idazoxan binding in astrocytic tumours was similar to that in normal brain, compatible with the presence of I2-imidazoline receptors. There was a higher density of I2-imidazoline receptors in astrocytic tumours (Bmax = 266 +/- 18 fmol mg-1 protein; p < 0.001) than in normal brain (Bmax = 54 +/- 4 fmol mg-1 protein), with no differences in affinity values. Almost no [3H]idazoxan-specific binding was shown in meningiomas. The results suggest that I2-imidazoline receptors may be a selective marker for glial tumours in the evaluation of intracranial neoplasms.