Measurement of receptor-mediated functional activation of G proteins in postmortem human brain membranes.
Guanine nucleotide-binding regulatory proteins (G proteins) play a pivotal role in receptor-mediated transmembrane signal transduction, and have been implicated in modes of action of psychotropic drugs as well as in pathogenesis of psychiatric disorders. In the present investigation, functional activation of G proteins coupled with several receptors, in particular with GABAB receptors, was assessed by agonist-induced stimulation of high-affinity GTPase, an enzyme that is intrinsic to alpha subunit of G protein, in postmortem human frontal cortical membranes. High-affinity GTPase activity was stimulated by GABA as well as (+/-)-baclofen, a selective GABAB receptor agonist, with EC50 values of 60-150 and 10-40 microM, respectively, in a Mg(2+)-dependent manner. The (+/-)-baclofen-stimulated response was antagonized by the selective GABAB receptor antagonist, 2-hydroxy-saclofen, in a competitive manner with a KB value of 59 microM. Although the maximal percent increase above basal value (% Emax) for GABAB receptor-mediated high-affinity GTPase activity was varied from subject to subject, % Emax values for both agonists were highly correlated with each other, and replicable and stable in a given subject, indicating that this measure is trustworthy as an index of functional coupling between receptors and G proteins in future studies at the aim of elucidating possible alteration of receptor/G protein interaction in psychiatric disorders. The % Emax values for GABAB receptor-mediated responses were correlated inversely with brain storage duration, which should be critically considered in postmortem studies. The increases in high-affinity GTPase activity stimulated by several agonists other than GABAB receptor agonists seemed too low to quantify for making a comparison in future studies.
Odagaki
Y
Y
Department of Psychiatry, Hokkaido University School of Medicine, Sapporo, Japan.
Nishi
N
N
Ozawa
H
H
Saito
T
T
Takahata
N
N
Riederer
P
P
Koyama
T
T
D000368:Aged; D000369:Aged, 80 and over; D001418:Baclofen / Q000494:pharmacology; D001667:Binding, Competitive / Q000502:physiology; D001921:Brain / Q000378:metabolism*; D002102:Cadaver; D005260:Female; D018755:GABA Agonists / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D015636:Magnesium Chloride / Q000494:pharmacology; D008297:Male; D008566:Membranes / Q000378:metabolism; D008875:Middle Aged; D018080:Receptors, GABA-B / Q000502:physiology*; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D018755:GABA Agonists; D018080:Receptors, GABA-B; D015636:Magnesium Chloride; D005680:gamma-Aminobutyric Acid; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D001418:Baclofen