guanosine-5--o-(3-thiotriphosphate) and Cadaver

The agonist-stimulated guanosine 5'-(gamma-[(35)S]thio)triphosphate binding assay was used to anatomically localize receptor-activated G-proteins by autoradiography in post mortem human brain. The optimal conditions for guanosine 5'-(gamma-[(35)S]thio)triphosphate binding to human brain sections were established in post mortem samples of the prefrontal cortex, hippocampus, basal ganglia, brainstem and cerebellar cortex. An excess of GDP (2mM) was required to decrease basal activity and obtain effective stimulation by specific agonists. guanosine 5'-(gamma-[(35)S]Thio)triphosphate binding was increased after stimulation with specific agonists of different G-protein-coupled receptors. They include cannabinoid (WIN55212-2), mu-opioid ([D-Ala(2),N-Me-Phe(4), Gly(5)-ol]enkephalin), serotonin-1A [(+/-)-8-hydroxy-2-(di-n-propylamino)tetralin] and serotonin-1B/1D (sumatriptan), cholinergic muscarinic receptors (carbachol) and alpha(2)-adrenoceptors (UK14304). Such stimulation reached 1458%, 440%, 188%, 219%, 61% and 339%, respectively, over the basal levels. In tissue sections, the use of the above-mentioned agonists (10(-4)M) showed patterns of anatomical distribution similar to those already described by receptor autoradiography, with high densities over the hippocampus (serotonin-1A receptors), cortex (alpha(2)-adrenoceptors) and striatum (mu-opioid receptors). The highest binding levels were reached with the cannabinoid receptor agonist in most of the analysed brain regions. Carbachol produced only moderate stimulation of those same regions. The blockage of agonist-stimulated guanosine 5'-(gamma-[(35)S]thio)triphosphate binding by selective antagonists verified that the effect was receptor mediated. This technique provides a method to identify modifications of the receptor-mediated activation of G-proteins in post mortem human brain with anatomical resolution. It also provides valuable information on the level of drug efficacy in the human species.

Topics: Adult; Autoradiography; Brain; Cadaver; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Receptors, Cell Surface; Tissue Distribution

We have measured basal, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and forskolin-stimulated adenylyl cyclase activity in samples of frontal and parietal cortex obtained at post-mortem from suicides, with a firm retrospective diagnosis of depression. The suicides were divided into those free of antidepressants and those receiving antidepressants. Each suicide was individually compared to a gender and age-matched control. Although we found no significant differences in adenylyl cyclase activity between controls and either antidepressant-free or antidepressant-treated suicides, there was a trend for lower stimulated adenylyl cyclase activity in suicides.

Topics: Adenylyl Cyclases; Adolescent; Adult; Aged; Antidepressive Agents; Brain; Cadaver; Colforsin; Depression; Female; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Male; Middle Aged; Signal Transduction; Suicide

The effects of agonal status, postmortem delay, and age on human brain adenylyl cyclase activity were determined in membrane preparations of frontal cortex from a series of 18 nondemented subjects who had died with no history of neurological or psychiatric disease. Basal and guanosine 5'-O-(3-thiotriphosphate)-, aluminum fluoride-, and forskolin-stimulated enzyme activities were not significantly reduced over an interval from death to postmortem of between 3 and 37 h and were also not significantly different between individuals dying with a long terminal phase of an illness and those dying suddenly. Basal and aluminum fluoride-stimulated enzyme activities showed a negative correlation with increasing age of the individual. In subsequent experiments, basal and guanosine 5'-O-(3-thiotriphosphate)-, aluminum fluoride-, and forskolin-stimulated enzyme activities were compared in five brain regions from a series of eight Alzheimer's disease and seven matched nondemented control subjects. No significant differences were observed between the groups for either basal activity or activities in response to forskolin stimulation of the catalytic subunit of the enzyme. In contrast, enzyme activities in response to stimulation with guanosine 5'-O-(3-thiotriphosphate) and aluminum fluoride were significantly reduced in preparations of neocortex and cerebellum from the Alzheimer's disease cases compared with the nondemented controls. Lower guanosine 5'-O-(3-thiotriphosphate)-, but not aluminum fluoride-, stimulated activity was also observed in preparations of frontal cortex from a group of four disease controls compared with nondemented control values. The disease control group, which contained Parkinson's disease and progressive supranuclear palsy patients, showed increased forskolin-stimulated activity compared with both the nondemented control and the Alzheimer's disease groups. These findings indicate a widespread impairment of G protein-stimulated adenylyl cyclase activity in Alzheimer's disease brain, which occurs in the absence of altered enzyme catalytic activity and which is unlikely to be the result of non-disease-related factors associated with the nature of terminal illness of individuals.

Topics: Adenylyl Cyclases; Aged; Aged, 80 and over; Aging; Aluminum; Aluminum Compounds; Alzheimer Disease; Brain; Cadaver; Colforsin; Fluorides; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Reference Values; Time Factors