The concerns of the present study were to determine 1) the conditions
necessary to measure displaceable [³H] L-aspartate binding to membrane fractions of the rat brain, 2) whether the binding demonstrated the charcteristics of the site which is active in vivo, and 3) whether the acidic amino acid neurotransmitters aspartate and glutamate bind to
identical or different sites by comparing the pharmacological specificities
of the [³H] L-aspartate binding with that of [³H] L-glutamate.
The conditions of the [³H] L-aspartate binding assay were determined
in synaptosomal and total particulate fractions of whole rat brain. The
reaction mixture which included the membrane fraction suspended in Tris-HCl
buffer (pH 7.4) in the presence or absence of the compound under test, was
incubated at 37°C for 30 minutes. The reaction was stopped by
centrifugation and the radioactivity in the pellet counted by liquid
scintillation spectrometry.
The [³H] L-aspartate binding was characterized in total particulate fractions of rat cerebellum. The apparent dissociation constant (K[sub=D]) and maximum binding (Bmax), as determined by Scatchard analysis, are 1.64 ± 0.34 μM and 7711 ± fmol/mg protein respectively. The displaceable binding is reversible, saturable, independent of the presence of NA⁺, has an affinity in the range where the neurotransmitter is active in vivo, and demonstrates a pharmacological specificity which includes
stereospecificity. The compounds tested to demonstrate the pharmacological specificity were L-aspartate (IC[sub=50] = 1.81 μM), D-aspartate (IC[sub=50] = 46.6 μM), L-glutamate (IC[sub=50] = 1.24 μM), N-methyl-DL-aspartate (inactive), kainate (inactive), D-alpha-aminoadipate (inactive), and
L-alpha-aminoadipate (IC[sub=50] =7.12 μM). The pharmacological specificity
of [³H] L-aspartate binding was different from that of [³H]
L-glutamate. When the binding data only are considered, therefore,
separate receptors for aspartate and glutamate are indicated.
The pharmacological specificity of the [³H] L-aspartate binding,
that is the affinity of the binding site for N-methyl-DL-aspartate, D- and
L-alpha-aminoadipate, however, does not correlate with the potency of these
compounds derived from iontophoretic studies. L-alpha-aminoadipate is very
effective while N-methyl-DL-aspartate and D-alpha-aminoadipate do not
displace the [³H] L-aspartate binding. In iontophoretic studies,
N-methyl-D-aspartate and D-alpha-aminoadipate are very potent as compared
to aspartate while L-alpha-aminoadipate Is inactive. The
[³H] L-aspartate binding then may not represent the site which is active in vivo. The characteristics of the aspartate site in vivo, however, may not be truely represented in iontophoretic studies because of, for example, uptake of the compounds. The aspartate binding site, therefore, must be identified as that which is activated in vivo. The question of separate receptors for aspartate and glutamate then must still be resolved. / Science, Faculty of / Zoology, Department of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/23229 |
| Date | January 1982 |
| Creators | Stammers, Anthea Mary Tench |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
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