Characterization of [3H]thiocolchicoside binding sites in rat spinal cord and cerebral cortex.
Thiocolchicoside, a semi-synthetic derivative of the naturally occurring compound colchicoside with a relaxant effect on skeletal muscle, has been found to displace both [3H]gamma-aminobutyric acid ([3H]GABA) and [3H]strychnine binding, suggesting an interaction with both GABA and strychnine-sensitive glycine receptors. In order to gain further insight into the interaction of thiocolchicoside with these receptors, the binding of [3H]thiocolchicoside in rat spinal cord-brainstem and cortical synaptic membranes was characterized. [3H]Thiocolchicoside binding was saturable in both tissues examined. In spinal cord-brainstem membranes, we found a K(D) of 254 +/- 47 nM and a Bmax of 2.39 +/- 0.36 pmol/mg protein, whereas in cortical membranes, a K(D) of 176 nM and a Bmax of 4.20 pmol/mg protein was observed. A similar K(D) value was found in kinetic experiments performed in spinal cord-brainstem membranes. Heterologous displacement experiments showed that GABA and strychnine displaced the binding in a dose-dependent manner, whereas glycine was ineffective. [3H]Thiocolchicoside binding was also displaced by several GABA(A) receptor agonists and antagonists, but not by baclofen, flunitrazepam, guvacine, picrotoxin or by other drugs unrelated to GABA transmission. In spinal cord-brainstem, and to a lower extent, in cortical membranes, GABA and its analogs were not able to completely displace [3H]thiocolchicoside specific binding indicating that, besides GABA(A) receptors, thiocolchicoside can bind to another unidentified site. Unlabelled thiocolchicoside, however, completely displaced [3H]muscimol binding both in cortical and in spinal cord-brainstem synaptic membranes with an IC50 in the low microM range. Neurosteroids were found to modulate the binding in cortical but not in spinal cord-brainstem synaptic membranes. We conclude that [3H]thiocolchicoside binding shows a pharmacological profile indicating an interaction with the GABA(A) receptor. The different affinities for the GABA(A) receptor agonists and antagonists and sensitivity to neurosteroids obtained in the cerebral cortex and in the spinal cord may indicate a preferential interaction of the compound with a subtype of the GABA(A) receptor. The data also indicate that [3H]thiocolchicoside binds to another site(s), whose nature remains to be elucidated.
Balduini
W
W
Istituto di Farmacologia e Farmacognosia, Università di Urbino, Italy. balduini@fis.uniurb.it
Cimino
M
M
Depoortere
H
H
Cattabeni
F
F
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001933:Brain Stem / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism*; D003078:Colchicine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D018755:GABA Agonists / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D009118:Muscimol / Q000378:metabolism; D009465:Neuromuscular Agents / Q000378:metabolism* / Q000494:pharmacology; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D011963:Receptors, GABA-A / Q000378:metabolism; D018080:Receptors, GABA-B / Q000378:metabolism; D018009:Receptors, Glycine / Q000378:metabolism; D013116:Spinal Cord / Q000378:metabolism*; D013256:Steroids / Q000494:pharmacology; D013570:Synaptic Membranes / Q000378:metabolism
D000596:Amino Acids; D018755:GABA Agonists; D009465:Neuromuscular Agents; D011963:Receptors, GABA-A; D018080:Receptors, GABA-B; D018009:Receptors, Glycine; D013256:Steroids; D009118:Muscimol; D003078:Colchicine; C004280:thiocolchicoside