Multiple Mechanisms for Inhibition of Excitatory Amino Acid Receptors Coupled to Phosphoinositide Hydrolysis

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Abstract
Excitatory amino acid (EAA) analogues activate receptors that are coupled to the increased hydrolysis of phosphoinositides (Pis). In these studies, hippocampal slices were prepared from neonatal rats (6–11 days old) to characterize the effects of EAA analogues on these receptors. The concentrations of ibotenate and trans-(pmn)-1-amino-1,3-cyclopentanedicarboxylate (trans-ACPD) required to evoke half-maximal stimulation (EC50 values) were 28 and 51 μM, respectively. Although the data for stimulation of PI hydrolysis by ibotenate and trans-ACPD were best fit to theoretical curves that had Hill slopes of 1, data for stimulation of PI hydrolysis by quisqualate were best fit to two sites. The EC50 values were 0.43 and 44 μM. The high-affinity sites were 70% of the total. A number of EAA analogues were tested for inhibition of PI metabolism. One of these, L-aspartate-β-hydroxamate (L-AβHA), was identified as a novel inhibitor of this response. L-AβHA was equipotent as an inhibitor of PI metabolism stimulated by ibotenate, quisqualate, and trans-ACPD. The data for this inhibition were best fit to two sites. Between 32 and 48% of the total sites had high affinity with IC50 values in the range of 1.2–6.3 μM. The low-affinity sites had IC50 values between 610 and 2,700 μM. DL-2-Amino-3-phosphonopro-pionate (DL-AP3) was also equipotent as an inhibitor of PI hydrolysis stimulated by ibotenate, quisqualate, and trans-ACPD (IC50 values were 480–850 μM). In contrast to the data for L-AβHA, the data for DL-AP3 were best fit to a single site. Both of these inhibitors reduced the maximal response caused by the agonists, consistent with noncompetitive mechanisms of action. Several experiments were designed to examine potential mechanisms for these noncompetitive effects. These studies suggest that either L-AβHA and DL-AP3 bind to a site on the receptor and irreversibly block activation of the receptor, or that these inhibitors act via a distinct site that specifically regulates EAA receptors coupled to PI hydrolysis.

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