The lathyrus toxin, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (ODAP), and homocysteic acid sensitize CA1 pyramidal neurons to cystine and L-2-amino-6-phosphonohexanoic acid.

A brief exposure of hippocampal slices to L-quisqualic acid (QUIS) sensitizes CA1 pyramidal neurons 30- to 250-fold to depolarization by certain excitatory amino acids analogues, e.g., L-2-amino-6-phosphonohexanoic acid (L-AP6), and by the endogenous compound, L-cystine. This phenomenon has been termed QUIS sensitization. A mechanism similar to that previously described for QUIS neurotoxicity has been proposed to describe QUIS sensitization. Specifically, QUIS has been shown to be sequestered into GABAergic interneurons by the System x(c)(-) and subsequently released by heteroexchange with cystine or L-AP6, resulting in activation of non-NMDA receptors. We now report two additional neurotoxins, the Lathyrus excitotoxin, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (ODAP), and the endogenous compound, L-homocysteic acid (HCA), sensitize CA1 hippocampal neurons >50-fold to L-AP6 and >10-fold to cystine in a manner similar to QUIS. While the cystine- or L-AP6-mediated depolarization can be inhibited by the non-NMDA receptor antagonist CNQX in ODAP- or QUIS-sensitized slices, the NMDA antagonist D-AP5 inhibits depolarization by cystine or L-AP6 in HCA-sensitized slices. Thus, HCA is the first identified NMDA agonist that induces phosphonate or cystine sensitization. Like QUIS sensitization, the sensitization evoked by either ODAP or HCA can be reversed by a subsequent exposure to 2 mM alpha-aminoadipic acid. Finally, we have demonstrated that there is a correlation between the potency of inducers for triggering phosphonate or cystine sensitivity and their affinities for System x(c)(-) and either the non-NMDA or NMDA receptor. Thus, the results of this study support our previous model of QUIS sensitization and have important implications for the mechanisms of neurotoxicity, neurolathyrism and hyperhomocystinemia.

L-Quisqualic acid transport into hippocampal neurons by a cystine-sensitive carrier is required for the induction of quisqualate sensitization.

A brief exposure of hippocampal slices to L-quisqualic acid sensitizes CA1 pyramidal neurons 30-250-fold to depolarization by two classes of excitatory amino acid analogues: (1) those whose depolarizing effects are rapidly terminated following washout, e.g. L-2-amino-4-phosphonobutanoic acid (L-AP4) and L-2-amino-6-phosphonohexanoic acid (L-AP6) and (2) those whose depolarizing effects persist following washout, e.g. L-aspartate-beta-hydroxamate (L-AbetaH). This process has been termed quisqualate sensitization. In this study we directly examine the role of amino acid transport systems in the induction of quisqualate sensitization. We report that L-quisqualate is a low-affinity substrate (K(M)=0.54 mM) for a high capacity (V(max)=0.9 nmol (mg protein)(-1) min(-1)) Na(+)-dependent transport system(s) and a high-affinity substrate (K(M)=0.033 mM) for a low-capacity (V(max)=0.051 nmol (mg protein)(-1) min(-1)) transporter with properties similar to the cystine/glutamate exchange carrier, System x(c-). We present evidence that suggests that System x(c-) participates in quisqualate sensitization. First, simultaneous application of L-quisqualate and inhibitors of System x(c-), but not inhibitors of Na(+)-dependent glutamate transporters, prevents the subsequent sensitization of hippocampal neurons to phosphonates or L-AbetaH. Second, L-quisqualic acid only sensitizes hippocampal neurons to other substrates of System x(c-), including cystine. Third, immunocytochemical analysis of L-quisqualate uptake demonstrates that only inhibitors of System x(c-) inhibit the highly concentrative uptake of L-quisqualate into a widely dispersed group of GABAergic hippocampal interneurons. We conclude that quisqualate sensitization is a direct consequence of the unique interaction of various excitatory amino acids, namely L-quisqualate, cystine, and phosphonates, with the exchange carrier, System x(c-). Therefore, the results of this study have important implications for the mechanism by which L-quisqualate, and other substrates of this transporter which are also excitatory amino acid agonists (such as glutamate and beta-N-oxalyl-L-alpha,beta-diaminopropionic acid, beta-L-ODAP) may trigger neurotoxicity.

Plague as a biological weapon: medical and public health management. Working Group on Civilian Biodefense.

OBJECTIVE: The Working Group on Civilian Biodefense has developed consensus-based recommendations for measures to be taken by medical and public health professionals following the use of plague as a biological weapon against a civilian population. PARTICIPANTS: The working group included 25 representatives from major academic medical centers and research, government, military, public health, and emergency management institutions and agencies. EVIDENCE: MEDLINE databases were searched from January 1966 to June 1998 for the Medical Subject Headings plague, Yersinia pestis, biological weapon, biological terrorism, biological warfare, and biowarfare. Review of the bibliographies of the references identified by this search led to subsequent identification of relevant references published prior to 1966. In addition, participants identified other unpublished references and sources. Additional MEDLINE searches were conducted through January 2000. CONSENSUS PROCESS: The first draft of the consensus statement was a synthesis of information obtained in the formal evidence-gathering process. The working group was convened to review drafts of the document in October 1998 and May 1999. The final statement incorporates all relevant evidence obtained by the literature search in conjunction with final consensus recommendations supported by all working group members. CONCLUSIONS: An aerosolized plague weapon could cause fever, cough, chest pain, and hemoptysis with signs consistent with severe pneumonia 1 to 6 days after exposure. Rapid evolution of disease would occur in the 2 to 4 days after symptom onset and would lead to septic shock with high mortality without early treatment. Early treatment and prophylaxis with streptomycin or gentamicin or the tetracycline or fluoroquinolone classes of antimicrobials would be advised.

Cyclobutane quisqualic acid analogues as selective mGluR5a metabotropic glutamic acid receptor ligands.

The conformationally constrained cyclobutane analogues of quisqualic acid (Z)- and (E)-1-amino-3-[2'-(3',5'-dioxo-1',2', 4'-oxadiazolidinyl)]cyclobutane-1-carboxylic acid, compounds 2 and 3, respectively, were synthesized. Both 2 and 3 stimulated phosphoinositide (PI) hydrolysis in the hippocampus with EC50 values of 18 +/- 6 and 53 +/- 19 microM, respectively. Neither analogue stimulated PI hydrolysis in the cerebellum. The effects of 2 and 3 were also examined in BHK cells which expressed either mGluR1a or mGluR5a receptors. Compounds 2 and 3 stimulated PI hydrolysis in cells expressing mGluR5a but not in those cells expressing mGluR1a. The EC50 value for 2 was 11 +/- 4 microM, while that for 3 was 49 +/- 25 microM. Both 2 and 3 did not show any significant effect on cells expressing the mGluR2 and mGluR4a receptors. In addition, neither compound blocked [3H]glutamic acid uptake into synaptosomal membranes, and neither compound was able to produce the QUIS effect as does quisqualic acid. This pharmacological profile indicates that 2 and 3 are selective ligands for the mGluR5a metabotropic glutamic acid receptor.

Persistent depression of synaptic responses occurs in quisqualate sensitized hippocampal slices after exposure to L-aspartate-beta-hydroxamate.

Exposure of slices of rat hippocampus to quisqualic acid produces an enhanced sensitivity of neurons to depolarization by other excitatory amino acid analogues, particularly amino acid phosphonates. The phosphonates may act at extracellular sites, since their depolarizing effects are rapidly reversed by washout with phosphonate-free incubation medium. We now wish to report a novel class of excitatory amino acid analogues that induce a persistent depolarization that is not reversed by washout. Exposure of quisqualate-sensitized slices of rat hippocampus to 400 microM L-aspartate-beta-hydroxamate for 8 min results in the complete depression of extracellular synaptic field potentials. This depression persists for at least 1 h after washout of the hydroxamate compound. Analogous compounds L-glutamate-gamma-hydroxamate, D-aspartate-beta-hydroxamate and the phosphonate derivative L-2-amino-3-phosphonopropanoic acid (L-AP3) induce a similar but weaker persistent depression of the field potentials. Previous studies also demonstrated that exposure of hippocampal slices to L-alpha-aminoadipate blocks or reverses quisqualate sensitization, making the neurons unresponsive to depolarization by phosphonate compounds. We now report that L-alpha-aminoadipate also blocks or reverses the persistent depolarization of quisqualate-sensitized neurons which is induced by exposure to the hydroxamates or L-AP3.

A 3-amino-4-hydroxy-3-cyclobutene-1,2-dione-containing glutamate analogue exhibiting high affinity to excitatory amino acid receptors.

The syntheses of several novel N-(hydroxydioxocyclobutenyl)-containing analogues of gamma-amino-butyric acid and L-glutamate were undertaken to test the hypothesis that derivatives of 3,4-dihydroxy-3-cyclobutene-1,2-dione (squaric acid), such as 3-amino-4-hydroxy-3-cyclobutene-1,2-dione, could serve as a replacement for the carboxylate moiety in neurochemically interesting molecules. The syntheses were successfully accomplished by preparation of a suitably protected diamine or diamino acid followed by reaction with diethyl squarate. Subsequent deprotection resulted in the isolation of the corresponding N-(hydroxydioxocyclobutenyl)-containing analogues 13, 14, and 18. These analogues were screened as displacers in various neurochemical binding site assays. The L-glutamate analogue 18, which showed high affinity as a displacer for kainate and AMPA binding, was also examined for agonist potency for CA1 pyramidal neurons of the rat hippocampal slice preparation. It rivaled AMPA as one of the most potent agonists for depolarizing pyramidal neurons in medium containing 2.4 mM Mg+2 ions in which kainate/AMPA receptors are active but NMDA receptors are inhibited (IC50 = 1.1 microM). It was 1 order of magnitude less potent for depolarizing pyramidal neurons under conditions in which kainate/AMPA receptors were inhibited by 10 microM CNQX but NMDA receptors were active in 0.1 mM Mg(+2)-containing medium (IC50 = 10 microM). Compound 18 did not induce sensitization of CA1 pyramidal cells to depolarization by phosphonate analogues of glutamate (the QUIS-effect).

Effects of quisqualic acid analogs on metabotropic glutamate receptors coupled to phosphoinositide hydrolysis in rat hippocampus.

L-Glutamic acid (L-Glu) and L-aspartic acid (L-Asp) activate several receptor subtypes, including metabotropic Glu receptors coupled to phosphoinositide (PI) hydrolysis. Quisqualic acid (Quis) is the most potent agonist of these receptors. There is evidence that activation of these receptors may cause a long lasting sensitization of neurons to depolarization, a phenomenon called the Quis effect. The purpose of the current studies was to use Quis analogs and the recently identified metabotropic receptor antagonist, (+)-alpha-methyl-4-carboxy-phenylglycine((+)-MCPG), to define the structural properties required for interaction with the metabotropic receptors coupled to PI hydrolysis and to determine if the Quis effect is mediated by these receptors. The effects of Quis analogs on PI hydrolysis were studied in the absence or presence of the metabotropic receptor-specific agonist 1SR,3RS-1-amino-1,3-cyclopentanedicarboxylic acid (1SR,3RS-ACPD) in neonatal rat hippocampus. Some of the compounds that induce the Quis effect also stimulate PI hydrolysis, including Quis itself and 9 (homoquisqualic acid). Not all of the Quis analogs that stimulate PI hydrolysis, however, induce the Quis effect, including 7A (EC50 = 750 +/- 150 microM) and (RS)-4-bromohomoibotenic acid (BrHI) (EC50 = 130 +/- 40 microM). Although (+)-MCPG blocked PI hydrolysis stimulated by Quis (IC50 = 370 +/- 70 microM), it had no effect on the induction of the Quis effect. Other Quis analogs did not stimulate PI hydrolysis but rather blocked the effects of 1SR,3RS-ACPD. The IC50 values were 240 +/- 70 microM for 2, 250 +/- 90 microM for 3, and 640 +/- 200 microM for 4. Data for inhibition by 2 and 3 were consistent with non-competitive mechanisms of action. These studies provide new information about the structural features of Quis required for interaction with metabotropic receptors coupled to PI hydrolysis and provide evidence that the Quis effect is not mediated by (+)-MCPG sensitive subtypes of these receptors.

Type 4a metabotropic glutamate receptor: identification of new potent agonists and differentiation from the L-(+)-2-amino-4-phosphonobutanoic acid-sensitive receptor in the lateral perforant pathway in rats.

Before the discovery of the metabotropic glutamate receptors (mGluRs), the glutamate analogue L-2-amino-4-phosphonobutanoic acid (L-AP4) was identified as a potent presynaptic inhibitor of evoked synaptic transmission in the lateral perforant pathway in rats. The localization and L-AP4 sensitivity of the mGluR4a subtype of mGluRs were consistent with the hypothesis that this receptor mediates the synaptic depressant effects of L-AP4 in the lateral perforant pathway. In the present study, the pharmacology of mGluR4a expressed in baby hamster kidney 570 cells was characterized and compared with that previously reported for the lateral perforant pathway responses. The endogenous excitatory amino acid L-aspartate was inactive at mGluR4a, whereas L-homocysteic acid was only 5-fold less potent than L-glutamate. These data suggest that L-homocysteic acid may be an endogenous agonist at mGluR4a. Of the 30 L-AP4 analogues examined, several compounds were identified as agonists at mGluR4a. The cyclopropyl-AP4 analogue (Z)-(+/-)-2-amino-2,3-methano-4-phosphonobutanoic acid inhibited forskolin-stimulated cAMP production with an EC50 of 0.58 microM, which is comparable to that of L-AP4 (EC50 = 0.43 microM). Two other cyclic analogues of L-AP4 were approximately 10-fold less potent as agonists at mGluR4a, i.e., (+/-)-1-amino-3-(phosphonomethylene)cyclobutanecarboxylic acid (EC50 = 4.4 microM) and (E)-(+/-)-2-amino-2,3-methano-4-phosphonobutanoic acid (EC50 = 7.9 microM). Comparison of the potencies of the compounds for activation of mGluR4a with their potencies for inhibition of lateral perforant pathway responses demonstrates that some compounds have comparable activities in the two systems, whereas several compounds are at least 10-fold more potent in one of the systems. In addition, although the mGluR antagonist (+)-alpha-methyl-4-carboxyphenylglycine blocked the effects of L-AP4 in the lateral perforant pathway, it did not block the effects of L-AP4 at the cloned receptor. These data provide evidence that mGluR4a does not mediate the effects of L-AP4 in the lateral perforant pathway, they provide new tools to identify the function of these receptors in the mammalian central nervous system, and they indicate that the effects of L-AP4 in the lateral perforant pathway are mediated by a (+)-alpha-methyl-4-carboxyphenylglycine-sensitive receptor.

Synthesis of oxadiazolidinedione derivatives as quisqualic acid analogues and their evaluation at a quisqualate-sensitized site in the rat hippocampus.

The ability of quisqualic acid (1) to sensitize neurons to depolarization by omega-phosphono alpha-amino acid analogues of excitatory amino acids is a highly specific phenomenon and is termed the QUIS effect. In an attempt to elucidate the structure-activity relationships for this sensitization, analogues 2-6 of quisqualic acid have been synthesized. Compounds 4, 5, and 6 showed no quisqualate sensitization with respect to L-2-amino-6-phosphonohexanoic acid (L-AP6), while compounds 2 and 3 were 1/10 and 1/1000, respectively, as active as quisqualic acid in sensitizing neurons toward L-AP6.

Immunocytochemical evidence that quisqualate is selectively internalized into a subset of hippocampal neurons.

Quisqualic acid (QUIS) has been shown to interact with several glutamate receptor subtypes and uptake sites. We have previously demonstrated that a brief exposure of hippocampal cells to QUIS sensitizes them to depolarization by the alpha-amino-omega-phosphonate analogues of glutamate, AP4, AP5, and AP6. This QUIS-induced sensitization is accompanied by the active uptake of QUIS into hippocampal slices. In order to localize the sites of QUIS uptake into rat hippocampal slices, a polyclonal antibody against QUIS was raised in rabbits. Utilizing immunocytochemical techniques, we have identified immunoreactive axons and dendrites after brief exposure times to QUIS, and perikarya after longer exposure times to QUIS. The intensity of the QUIS immunoreactivity increased as the exposure time to QUIS increased. QUIS immunoreactivity was primarily found in stratum oriens and stratum radiatum, of regions CA1, CA2, and CA3 of the hippocampus as well as in the hilus and molecular layer of the dentate gyrus. The distribution and morphology of QUIS immunoreactive cells appeared to be similar to those of GABAergic interneurons. Glial fibrillary acidic protein (GFAP) did not co-localize with the QUIS-internalizing cells suggesting that they are not glia. Ultrastructural analysis revealed QUIS immunoreactive profiles within the stratum radiatum. Immunostained profiles at both the light and EM levels appeared, in many cases, to be swollen and showed signs of degeneration. Such changes were only evident in tissue exposed to QUIS. These data demonstrate that QUIS is taken up by a select group of neurons in the rat hippocampus.

Utilization of the resolved L-isomer of 2-amino-6-phosphonohexanoic acid (L-AP6) as a selective agonist for a quisqualate-sensitized site in hippocampal CA1 pyramidal neurons.

Brief exposure of rat hippocampal slices to quisqualic acid (QUIS) sensitizes neurons to depolarization by the alpha-amino-omega-phosphonate excitatory amino acid (EAA) analogues AP4, AP5 and AP6. These phosphonates interact with a novel QUIS-sensitized site. Whereas L-AP4 and D-AP5 cross-react with other EAA receptors, DL-AP6 has been shown to be relatively selective for the QUIS-sensitized site. This specificity of DL-AP6, in conjunction with the apparent preference of this site for L-isomers, suggested that the hitherto unavailable L-isomer of AP6 would be a potent and specific agonist. We report the resolution of the D- and L-enantiomers of AP6 by fractional crystallization of the L-lysine salt of DL-AP6. We also report the pharmacological responses of kainate/AMPA, NMDA, lateral perforant path L-AP4 receptors and the CA1 QUIS-sensitized site to D- and L-AP6, and compare these responses to the D- and L-isomers of AP3, AP4, AP5 and AP7. The D-isomers of AP4, AP5 and AP6 were 5-, 3- and 14-fold less potent for the QUIS-sensitized site than their respective L-isomers. While L-AP4 and L-AP5 cross-reacted with NMDA and L-AP4 receptors, L-AP6 was found to be highly potent and specific for the QUIS-sensitized site (IC50 = 40 microM). Its IC50 values for kainate/AMPA, NMDA and L-AP4 receptors were > 10, 3 and 0.8 mM, respectively. As with AP4 and AP5, sensitization to L-AP6 was reversed by L-alpha-aminoadipate.

Quisqualic acid induced sensitization and the active uptake of L-quisqualic acid by hippocampal slices.

Hippocampal CA1 pyramidal cell neurons are sensitized to depolarization by L-2-amino-4-phosphonobutanoic acid (L-AP4) following exposure to L-quisqualic acid (QUIS). It has been proposed that induction of this 'QUIS-effect' involves uptake of L-QUIS by hippocampal cells. We have used o-phthaldialdehyde (OPA) derivatization and high performance liquid chromatographic (HPLC) separation of extracts from hippocampal slices which have been exposed to varied concentrations of L-QUIS to investigate L-QUIS uptake into hippocampal slices. We observe uptake rates such that the internal concentration of L-QUIS exceeds the bath concentration within 7 min. The fact that this uptake is concentrative indicates that it is mediated by an active transport system. In addition, uptake of L-QUIS may be linked to the induction of the QUIS-effect. At low concentrations of L-QUIS (< 4 microM), the QUIS-effect is only partially induced within the 4 min incubation time which maximally induces the effect when 16 microM L-QUIS is used. However, repeated 4 min exposure periods of slices to low L-QUIS concentrations will eventually induce the QUIS-effect even when each exposure is separated by extensive washout periods. Hence induction is dependent on both concentration and total exposure time. We also examined the effects of L-alpha-aminoadipic acid and L-serine-O-sulfate on the rate of L-QUIS uptake. Exposure of slices to these compounds prior to treatment with L-QUIS will block the physiological effects of L-QUIS. We found that these 'pre-blocking' compounds did not decrease the rate of L-QUIS uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Quisqualic acid analogues: synthesis of beta-heterocyclic 2-aminopropanoic acid derivatives and their activity at a novel quisqualate-sensitized site.

Hippocampal CA1 pyramidal cell neurons are sensitized over 30-fold to depolarization by L-2-amino-4-phosphonobutanoic acid (L-AP4) following exposure to L-quisqualic acid. This phenomenon has been termed the QUIS effect. In the present study several novel L-quisqualic acid analogues have been synthesized and tested for their interaction with the different components of the QUIS-effect system. Replacement of the oxadiazolidinedione ring of L-quisqualic acid with several other types of heterocyclic rings yielded the following quisqualic acid analogues: maleimide 2, N-methylmaleimide 3, N-(carboxymethyl)maleimide 4, succinimides 5A and 5B, and imidazolidinedione 6. None of these analogues were able to mimic the effects of L-quisqualic acid and sensitize hippocampal CA1 neurons to depolarization by L-AP4. Also, unlike L-serine O-sulfate, L-homocysteinesulfinic acid, or L-alpha-aminoadipic acid, none of the analogues were able to preblock or reverse the QUIS effect. However, when the IC50 values for inhibition of the CA1 synaptic field potential of analogues 2-6 were determined both before and after hippocampal slices were exposed to L-quisqualic acid, the IC50 values of analogues 3 and 4 were found to decrease more than 7-fold. Thus, these two compounds behave like L-AP4 rather than L-quisqualic acid in this system in that they exhibit increased potencies in slices that have been pretreated with L-quisqualic acid even though they cannot themselves induce this sensitization. Compounds 3 and 4, therefore, represent the first non-phosphorus-containing compounds to which hippocampal neurons become sensitized following exposure to L-quisqualic acid. No change in the IC50 values was observed for 5A or 5B. Analogues 2 and 6, on the other hand, displayed a high potency for inhibition of the evoked field potential even prior to treatment of the slices with L-quisqualic acid.

Structure-function relationships for analogues of L-2-amino-4-phosphonobutanoic acid on the quisqualic acid-sensitive AP4 receptor of the rat hippocampus.

Hippocampal CA1 pyramidal cell neurons are sensitized to depolarization by L-2-amino-4-phosphonobutanoic acid (L-AP4) following exposure to L-quisqualic acid (QUIS). We have examined the interaction of 43 structural analogues of L-AP4 with both the 'induction' site and the QUIS-sensitive AP4 site in rat hippocampus. The synthesis of cis- and trans-4-phosphonoxy-L-proline, 3-(RS)-amino-5-phosphonopentanoic acid and 2(RS)-amino-5-phenyl-4(RS)-phosphonopentanoic acid (gamma-benzyl AP4) are described. None of the test compounds interact with the induction site; thus L-QUIS remains the only compound known to induce this effect. However, one compound (L-2-amino-3-(5-tetrazolyl)-propanoic acid (L-aspartate tetrazole) 'pre-blocked' and reversed the effects of QUIS. In addition, the potency of 16 analogues increased more than 4-fold following exposure of slices to L-QUIS. Among these, L-AP4, L-AP5, 2-amino-4-(methylphosphino)butanoic acid (AMPB), and E-1(RS)-amino-3(RS)-phosphonocyclopentanecarboxylic acid (E-cyclopentyl AP4) displayed IC50 values of less than 0.100 mM after QUIS. The results presented here suggest that the QUIS-sensitive AP4 site requires a spatial configuration of functional groups similar to that present in E-cyclopentyl AP4. The presence of a primary amino group and a phosphorus-containing group (either monoanionic or dianionic) appear to be required, however, a carboxyl group is not essential for interaction. The pharmacology of the QUIS-sensitive AP4 site suggests that it is distinct from other known binding sites for L-AP4 in the central nervous system (CNS).

Activity of the conformationally rigid 2-amino-4-phosphonobutanoic acid (AP4) analogue (RS)-1-amino-3-(phosphonomethylene)cyclobutane-1-carboxylic acid (cyclobutylene AP5) on evoked responses in the perforant pathway of rat hippocampus.

The highly rigid and conformationally extended 2-amino-4-phosphonobutanoic acid (AP4) analogue (RS)-1-amino-3-(phosphonomethylene)-cyclobutane-1-carboxylic acid (cyclobutylene AP5) was synthesized and found to inhibit evoked responses in the rat lateral perforant path (LPP) with an IC50 of 41 (+/- 1.5 S.E.M.) microM and the medial perforant pathway with an IC50 of 218 (+/- 3.7 S.E.M.) microM. Furthermore, paired pulse potentiation experiments suggest that cyclobutylene AP5 acts, in part, at a presynaptic site in the LPP. Thus, cyclobutylene AP5 appears to act in a similar manner to L-AP4 in the perforant pathway. These data support the hypothesis that L-AP4 assumes an extended conformation at the L-AP4 receptor of the LPP.

Characterization of retinal and hippocampal L-AP4 receptors using conformationally constrained AP4 analogues.

In the past, the absence of useful 2-amino-4-phosphonobutanoic acid (AP4) analogues has hampered the pharmacological study and comparison of different systems which are sensitive to L-AP4. Several conformationally constrained AP4 analogues have now been synthesized: (E)- and (Z)-1-amino-3-phosphonocyclopentanecarboxylic acid [(E)- and (Z)-cyclopentyl AP4], and (E)- and (Z)-1-amino-3-phosphonocyclohexanecarboxylic acid [(E)- and (Z)-cyclohexyl AP4], and the recently synthesized cyclopropyl analogues (E)- and (Z)-2-amino-2,3-methano-4-phosphonobutanoic acid [(E)- and (Z)-cyclopropyl AP4]. Therefore, we have examined and report here the pharmacology of two retinal and two hippocampal L-AP4 sensitive systems using these analogues. In addition, the pharmacology of two kainic acid/alpha-amino-3-hydroxy-5-methylisoxazole-4- propionic acid (KAIN/AMPA) pathways and one N-methyl-D-aspartate (NMDA) hippocampal pathway was examined. We found that the rank order potency of the L-AP4 sensitive systems were similar though not identical. The KAIN/AMPA and NMDA systems had a quite different rank order of potencies than the L-AP4 systems. These data suggest that the L-AP4 receptors in these different systems are structurally similar to each other and differ from both KAIN/AMPA and NMDA receptors.

NMDA-, kainate- and quisqualate-stimulated release of taurine from electrophysiologically monitored rat hippocampal slices.

While excitatory amino acids (EAAs) are known to evoke the release of taurine in the hippocampus, we have found that taurine is localized primarily in dendrites and only to a lesser extent in terminals in this region. To determine whether taurine is released as a neurotransmitter by non-toxic concentrations of EAAs, or exclusively as a neuroprotectant in response to excitotoxicity, we monitored the release of amino acids from hippocampal slices during simultaneous electrophysiological recording in the CA1 region to assess tissue viability. N-methyl-D-aspartate (NMDA) was the most potent of the EAA agonists tested for stimulating release of taurine. Exposure of slices to 120 microM NMDA increased the concentration of taurine in the perfusate to 1325% of its basal value. Kainate (KA) at a concentration of 128 microM increased taurine to 543% of baseline while quisqualate (Quis) at a concentration of 120 microM increase taurine to only 202% of its baseline value. Release of taurine in response to NMDA and KA peaked during the period when the concentration of the agonist was declining in the bath and did not return to its baseline value until 20 min after removal of the agonist. Increases in release of taurine were associated with concentrations of NMDA, KA, and Quis that caused an incomplete recovery of the CA1 field potential. These results suggest that taurine is primarily released by concentrations of glutamate receptor agonists that exhibit evidence of excitotoxicity in the CA1 region.

Synthesis of the 2-amino-4-phosphonobutanoic acid analogues (E)- and (Z)-2-amino-2,3-methano-4-phosphonobutanoic acid and their evaluation as inhibitors of hippocampal excitatory neurotransmission.

The cyclopropyl compounds (Z)- and (E)-2-amino-2,3-methano-4-phosphonobutanoic acid, 5 and 6, respectively, were prepared as constrained analogues of 2-amino-4-phosphonobutanoic acid (AP4), a selective glutamate receptor ligand. A Horner-Emmons reaction of trimethyl N-(benzyloxycarbonyl)phosphonoglycinate with 2-(diethoxyphosphinyl)acetaldehyde gave the protected dehydroamino acids 9 and 10, which were individually subjected to the following sequence of reactions: cycloaddition of diazomethane, photoelimination of N2, and acid hydrolysis, to give 5 and 6, respectively. Extracellular recording techniques were used to evaluate the abilities of 5 and 6 to block evoked synaptic transmission in specific neuronal pathways of the rat hippocampal slice. In the lateral perforant path (LPP) 5 and 6 were equipotent and possessed IC50 values of 18 and 17 microM, respectively. In the medial perforant path (MPP), 6 (IC50 = 81 microM) was much more potent than 5 (IC50 = 1580 microM). In paired pulse experiments which differentiate presynaptic and postsynaptic inhibition, 5 and 6 enhanced the second response to the same extent as L-AP4, suggesting a presynaptic site of action for these compounds. In contrast, the cyclopentyl AP4 analogues 3 and 4 enhanced the second response to a lesser extent. It was concluded that the biologically active conformation of AP4 in the LPP is different than in the MPP. In order to explain the same potency of 5 and 6 in the LPP, it was postulated that the two analogues assume a conformation that allows their functional groups to occupy the same relative place in space. Molecular modeling showed that the best overlap was achieved when the alpha C-beta C-gamma C-P dihedral angle for 5 was in the range of 130 degrees to 180 degrees and that of 6 was in the range of -130 degrees to -180 degrees. The results suggest that the bioactive conformation of AP4 in the LPP is an extended one.

Pre-exposure to L-homocysteinesulfinic acid blocks quisqualate-induced sensitization to L-2-amino-4-phosphonobutanoic acid.

Quisqualic acid sensitizes hippocampal CA1 neurons to depolarization by L-2-amino-4-phosphonobutanoic acid (L-AP4). This sensitization to L-AP4 is known to be blocked by simultaneous exposure to L-homocysteinesulfinic acid, L-alpha-aminoadipic acid and L-serine-O-sulfate during exposure to quisqualate. We report here that these compounds also act as 'pre-blockers' which, when added and removed from the medium prior to exposure to quisqualate, prevent subsequent induction of sensitization to L-AP4 by quisqualate. This pre-blockade suggests that simple competitive inhibition of extracellular receptor or uptake sites may not be the mechanism by which these compounds attenuate the action of quisqualate in this 'Quis-effect'.