Studies of a glutamate receptor linked to phosphoinositide metabolism

Jones, Philip Glyn (1991) Studies of a glutamate receptor linked to phosphoinositide metabolism. University of Southampton, Doctoral Thesis.

Abstract

In addition to the four receptors coupled to ion-channels, there exists a receptor for L-glutamate, which is linked to the hydrolysis of membrane phosphoinositides. In the guinea pig cerebral cortex, this receptor can be activated by trans-1-aminocyclopentane-1,3-dicarboxylate (trans-ACPD), ibotenate, quisqualate and glutamate. The response was seen throughout the brain but little evidence for multiple receptor subtypes was obtained. The highest receptor densities were found in the cortex and hippocampus. Trans-ACPD and ibotenate were equipotent full agonists having EC₅₀ values of 48±6μM and 63±14μM respectively and they evoked the same maximal effect of approximately 400% of basal inositol monophosphate formation. Both the 1S,3R and 1R,3S isomers of trans-ACPD were effective agonists, having EC₅₀ values of 16±7μM and 143±19μM respectively. Cis-ACPD was also a full agonist albeit weaker than the trans (EC_{50 =} 166±21μM), but only the 1S,3S isomer was able to stimulate inositol phosphate formation (EC_{50 =} 100±29μM). Quisqualate, the most potent compound used (EC_{50 =} 13±7 μM), was a partial agonist, eliciting a maximal response of 260% of basal and it significantly inhibited the response to trans-ACPD. L-glutamate was the least potent agonist (EC_50> 1000μM). NMDA and kainate, were however, ineffective in stimulating the formation of inositol phosphate. 1mM kainate significantly inhibited the response to ACPD but NMDA was unable to do so. The effects of ibotenate and trans-ACPD were insensitive to NMDA antagonists but could be non-competitively antagonised by L-2-amino-3-phosphonopropionate (L-AP3) and L-2-amino-4-phosphonobutyrate (L-AP4). L-AP3 was more potent than L-AP4 (IC_50 values = 497±27μM and 997±103μM respectively) but neither compound could fully inhibit the response due to their weak agonist actions. Activation of protein kinase C by phorbol 12, 13 dibutyrate (1 and 10μM) dramatically reduced the EAA stimulated formation of inositol phosphates but the receptor independent stimulation by 10mM NaF, was not affected. Immunoprecipitation of G-protein α-subunits following phorbol ester treatment of brain slices, failed to provide evidence for their phosphorylation and since the site of PKC action is neither at, or downstream of the G-protein, it is likely to be the receptor itself. Ibotenate and trans-ACPD, in an L-AP4 sensitive manner, stimulated the calcium dependent release of endogenous glutamate from guinea pig cerebrocortical synaptosomes. The pharmacology of this release would suggest involvement of the PI-linked receptor but neither phospholipase C inhibitors or GDPBS, following permeabilisation of the synaptosomes, were able to inhibit the release. The congruence of these two responses remains to be established.

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