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

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-HT_1A receptor (probed with R(+)-8-OH-DPAT or 5-HT), α_2A -adrenoceptor (UK-14,304 or (-)-epinephrine), M₂ /M₄ mAChRs (carbachol), adenosine A₁ receptor (adenosine), histamine H₃ receptor (histamine), group II mGlu (l-glutamate), GABA_B 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 α_2A -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.