Biochemical studies on the receptor mediated effects of L-2 amino-4-phosphonobutyrate receptors in the mammalian central nervous system

Gibbons, Simon James (1990) Biochemical studies on the receptor mediated effects of L-2 amino-4-phosphonobutyrate receptors in the mammalian central nervous system. University of Southampton, Doctoral Thesis.

Abstract

A number of biochemical approaches have been used to examine the receptor mediated effects of the glutamate analogue, L-2-amino-4-phosphonobutyrate (L-AP4). Changes in intrasynaptosomal calcium concentrations were measured as ⁴⁵calcium uptake and using the fluorescent indicator dyes, fura-2 and quin-2. L-AP4 caused a small, non-significant reduction in depolarisation induced calcium mobilisation, as measured by both techniques. N-methyl-D-aspartate (NMDA) stimulated ⁴⁵calcium uptake by synaptosomes but had no effect on intrasynaptosomal calcium as measured using fura-2 and quin-2. The binding of DL-[³H]-AP4 to crude synaptic membranes showed the characteristics of the previously reported chloride/calcium binding site. No evidence was found for the presence of a cryptic site that might represent the substrate for the synaptic depressant effects of AP4. Thin slice autoradiography of L-[³H]-glutamate was performed in a number of buffers. The displacement of the ligand by L-AP4 was consistent with an interaction with the NMDA, kainate and α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptors. Therefore there was no clear indication of a specific AP4 binding site in these studies. DL-[^3H]-AP4 did not bind to brain slices in autoradiographic experiments. [^3H]-kainate and [^3H]-AMPA autoradiography supported the observations made using L-[^3H]-glutamate as the ligand. Phosphoinositide hydrolysis in guinea pig brain slices was demonstrated to be linked to excitatory amino acid receptors. The recognition site for this effect was pharmacoligcally similar to that reported by other workers for rat brain. Ibotenate and quisqualate stimulated phosphoinositide turnover but NMDA had no effect. AP4 inhibited the effects of ibotenate and quisqualate in a stereospecific manner and had a weak non-specific stimulatory effect. The effects of L-AP4 on phosphoinositide turnover do not appear to mediated by either the chloride/calcium dependent binding site or the site which mediates synaptic depression. These studies indicate L-AP4 may cause synaptic depression by reducing depolarisation induced calcium entry and consequently transmitter release via an action at an as yet unidentified receptor.

This record has no associated files available for download.

Contributors

Download statistics