Inhibition by excitatory sulphur amino acids of the high-affinity L-glutamate transporter in synaptosomes and in primary cultures of cortical astrocytes and cerebellar neurons.
A detailed kinetic study of the inhibitory effects of L- and D-enantiomers of cysteate, cysteine sulphinate, homocysteine sulphinate, homocysteate, and S-sulpho-cysteine on the neuronal, astroglial and synaptosomal high-affinity glutamate transport system was undertaken. D-[3H] Aspartate was used as the transport substrate. Kinetic characterisation of uptake in the absence of sulphur compounds confirmed the high-affinity nature of the transport systems, the Michaelis constant (Km) for D-aspartate uptake being 6 microM, 21 microM and 84 microM, respectively, in rat brain cortical synaptosomes and primary cultures of mouse cerebellar granule cells and cortical astrocytes. In those cases where significant effects could be demonstrated, the nature of the inhibition was competitive irrespective of the neuronal versus glial systems. The rank order of inhibition was essentially similar in synaptosomes, neurons and astrocytes. Potent inhibition (Ki approximately Km) of transport in each system was exhibited by L-cysteate, and L- and D-cysteine sulphinate whereas substantially weaker inhibitory effects (Ki greater than 10-1000 times the appropriate Km value) were exhibited by the remaining sulphur amino acids. In general, inhibition: (i) was markedly stereospecific in favor of the L-enantiomers (except for cysteine sulphinate) and (ii) was found to decrease with increasing chain length. Computer-assisted molecular modelling studies, in which volume contour maps of the sulphur compounds were superimposed on those of D-aspartate and L-glutamate, demonstrated an order of inhibitory potency which was, qualitatively, in agreement with that obtained quantitatively by in vitro kinetic studies.
Griffiths
R
R
Department of Biochemistry and Microbiology, University of St. Andrews, Fife, Scotland, U.K.
Grieve
A
A
Dunlop
J
J
Damgaard
I
I
Fosmark
H
H
Schousboe
A
A
D000596:Amino Acids / Q000494:pharmacology*; D000818:Animals; D001224:Aspartic Acid / Q000493:pharmacokinetics; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D003545:Cysteine / Q000031:analogs & derivatives / Q000494:pharmacology*; D005971:Glutamates / Q000493:pharmacokinetics*; D018698:Glutamic Acid; D051379:Mice; D008968:Molecular Conformation; D014179:Neurotransmitter Uptake Inhibitors / Q000494:pharmacology*; D013237:Stereoisomerism; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism
D000596:Amino Acids; D005971:Glutamates; D014179:Neurotransmitter Uptake Inhibitors; D001224:Aspartic Acid; D018698:Glutamic Acid; D003545:Cysteine