α2A-Adrenoceptor-specific Stimulation of [35S]GTPγS Binding to Membrane Preparations of Rat Frontal Cortex

Abstract

Functional activation of alpha 2A adrenergic receptors in the crude membranes from rat frontal cortex was studied by a [35S]-guanosine 5'-O-(gamma-thiotriphosphate) ([35S]GTP gamma S) binding assay. alpha 2A agonists UK14304 and guanfacine decreased the ability of GDP to compete with [35S]GTP gamma S binding to the membranes and 0.1 mM GDP was found to be optimal for the following functional experiments. However, even after careful optimization of experimental conditions the specificity of ligands for rat alpha 2 adrenoceptors were not sufficient, as agonists as well as antagonists became activators of other signal transduction systems before achieving their maximal effect in the alpha 2A-adrenergic system. Only using compromising concentration of agonist (up to 1 microM UK14304) and antagonist (up to 1 microM RS79948) to inhibit agonist's effect, allowed us to filtrate out alpha 2A specific effect for characterization of signal transduction in rat frontal cortex membranes for the comparison efficacies of this system for different animals from behavioral experiments.

Modulation of Adenylyl Cyclase Activity in Rat Striatal Homogenate by Dopaminergic Receptors

“…Bound radioactivity was determined by rapid filtration through GF / B glass-fibre filters (Whatman Int., Ltd., Maidstone, UK) using a Brandell cell harvester and three washes of 3 ml of ice-cold washing buffer containing 100 mM NaCl and 20 mM phosphate buffer (pH 7.5) as described previously (14). Nonspecific binding of [ …”