Design, synthesis, and biological activity of a potent inhibitor of the neuropeptidase N-acetylated alpha-linked acidic dipeptidase.

A series of substituted phosphonate derivatives were designed and synthesized in order to study the ability of these compounds to inhibit the neuropeptidase N-acetylated alpha-linked acidic dipeptidase (NAALADase). The molecules were shown to act as inhibitors of the enzyme, with the most potent (compound 3) having a Ki of 0.275 nM. The potency of this compound is more than 1000 times greater than that of previously reported inhibitors of the enzyme. NAALADase is responsible for the catabolism of the abundant neuropeptide N-acetyl-aspartylglutamate (NAAG) into N-acetylaspartate and glutamate. NAAG has been proposed to be a neurotransmitter at a subpopulation of glutamate receptors; alternatively, NAAG has been suggested to act as a storage form of synaptic glutamate. As a result, inhibition of NAALADase may show utility as a therapeutic intervention in diseases in which altered levels of glutamate are thought to be involved.

Comparative actions of synthetic omega-grammotoxin SIA and synthetic omega-Aga-IVA on neuronal calcium entry and evoked release of neurotransmitters in vitro and in vivo.

The effects of synthetic omega-grammotoxin SIA (omega-GsTxSIA) and synthetic omega-Aga-IVA were tested in in vitro and in vivo neurochemical assays that are reflective of voltage-sensitive calcium channel function. Synthetic omega-GsTx SIA inhibited K(+)-evoked rat and chick synaptosomal 45Ca2+ flux, K(+)-evoked release of [3H]D-aspartate and [3H]norepinephrine from rat hippocampal brain slices and K(+)-evoked release of [3H]norepinephrine from chick cortical brain slices with potency values that were comparable to those found previously with omega-GsTx SIA purified from the venom of the tarantula spider Grammostola spatulata. These results indicate that trace contaminants do not account for the pharmacology of purified omega-GsTx SIA. omega-GsTx SIA caused a complete inhibition of rat synaptosomal 45Ca2+ flux and hippocampal slice [3H]D-aspartate release, whereas omega-Aga-IVA caused a maximal inhibition of approx 75%. omega-GsTx SIA and omega-Aga-IVA caused an identical partial inhibition of K(+)-evoked increases of intracellular calcium in cortical neurons in primary culture. The addition of nitrendipine to either omega-GsTx SIA or omega-Aga-IVA resulted in an additive and virtually complete inhibition of the cortical neuron intracellular calcium response. In in vivo microdialysis studies, the K(+)-evoked release of glutamate from hippocampus of awake freely moving rats was inhibited with the following rank order of potency: omega-conotoxin GVIA > omega-GsTx SIA > omega-Aga-IVA. Complete inhibition of K(+)-evoked hippocampal glutamate release was observed with 300 nM omega-conotoxin GVIA and 3 microM omega-GsTx SIA. In urethane anesthetized rats, omega-CgTx GVIA caused a partial inhibition, whereas omega-GsTx SIA caused a concentration-dependent and complete inhibition, of basal serotonin release in the hippocampus. Therefore, omega-GsTx SIA was shown to inhibit responses that are sensitive to omega-conotoxin GVIA, omega-Aga-IVA and omega-conotoxin MVIIC, consistent with the notion that omega-GsTx SIA inhibits N-, P- and Q-type high threshold voltage-sensitive calcium channels.

Acute cardiotoxicity of the Anti-HIV dideoxynucleoside, F-ddA, in the rat.

2'-beta-Fluoro-2',3'-dideoxyadenosine (F-ddA), an acid-stable, purine dideoxynucleoside with in vitro anti-HIV activity, has been selected by the NCI as a clinical trial candidate. A recent report that high, single doses of F-ddA produce cardiotoxicity in rats prompted the present investigation whose objective was to quantitate this effect and establish a relationship between this toxicity and F-ddA plasma concentrations. Microscopic examination of cardiac tissues for degenerative lesions established the effects of F-ddA and ddA on three iv schedules [daily x 1(2.5-250 mg/kg); daily x 5(125, 250 mg/kg), and BID x 1 (250 mg/kg)] as well as one oral schedule [BID x 1 (500 mg/kg) using 8- to 12-week old female Sprague-Dawley rats. For both F-ddA and ddA, the group mean severity of the cardiac lesions was dose-dependent and proportional to the measured plasma concentrations of the undeaminated parent drugs. F-ddI and ddI, were essentially nontoxic in this study (iv, 250 mg/kg, daily x 1 and daily x 5), since plasma concentrations exceeding 2 mM produced only minimal cardiac lesions. The cardiomyopathy of F-ddA was minimal to mild for all iv doses except 250 mg/kg (daily x 1) and usually was greater than that of ddA at any given dose. This is a consequence of the fact that F-ddA is deaminated 20 times more slowly than ddA, resulting in higher plasma concentrations of F-ddA relative to ddA at any given time for any given dose. Neither F-ddA nor ddA was more cardiotoxic on a repeated iv schedule (daily x 5) than when administered only once, suggesting that rat cardiotoxicity is related Cmax rather than total exposure. In this most sensitive species, the formation of cardiac lesions above the background level is associated with i.v. F-ddA administration when the F-ddA plasma concentration approaches 300 microM, 30-50 times the anticipated therapeutic level in humans.

Extracellular glutamate levels increase with age in the lateral striatum: potential involvement of presynaptic D-2 receptors.

In the lateral striatum of aged rats, dopamine D-2 receptor density is reduced and glutamate tissue content is elevated. D-2 receptor agonists have been shown to inhibit stimulated glutamate release. In the present study, microdialysis was used to investigate a potential role for D-2 receptors in the modulation of striatal glutamate efflux from 4-, 12-, 18-, and 24-26-month-old Fischer 344 rats. Extracellular basal glutamate concentrations significantly increased as a function of age in the lateral, but not medial, striatum. Neither the D-2 agonist, LY 163502, nor the D-2 antagonist, sulpiride, influenced basal glutamate efflux, suggesting that the dopaminergic system is not involved in the observed age-related increase in extracellular basal glutamate levels. In contrast to basal efflux, potassium-evoked glutamate release was not altered with age. However, LY 163502 significantly inhibited stimulated glutamate release in 4-month-old rats. This inhibitory action was not observed at any other age. Sulpiride alone did not alter stimulated glutamate release, but it did block the inhibitory effect of LY 163502 in the 4-month-old rats. These results provide in vivo evidence for an age-related functional loss in the modulation of striatal glutamate release by dopamine D-2 receptors in addition to increased basal glutamate efflux, which is not related to D-2 receptor modulation. Such mechanisms could be important in the pathophysiology of striatal cell death during aging and age-related neurodegenerative diseases.

Kappa agonist-induced reduction in dopamine release: site of action and tolerance.

Kappa opioid agonists are known to inhibit dopamine release. We sought to determine the site of this action and the relationship of tolerance to this effect. Microdialysis perfusion of the nucleus accumbens in unanesthetized rats was used to monitor dopamine release, as well as DOPAC, HVA, and 5-HIAA efflux. Administration of the kappa agonist U-50488H (0.5-10 mg/kg, s.c.) resulted in a dose-related inhibition of basal dopamine release and a delayed reduction in HVA efflux. When added directly to the perfusion medium, U-50488H (10 microM) similarly reduced dopamine release and HVA efflux; however, a much higher concentration (1 mM) produced a transient increase in dopamine release. The more potent kappa agonist, spiradoline mesylate (5 mg/kg, s.c.) caused a more profound and long lasting reduction in dopamine release than that observed with U-50488H. Repeated injections of spiradoline (7 injections over 3 days at 5 and 10 mg/kg, s.c.) resulted in a persistent reduction in dopamine release with no further reduction in release being observed following an acute injection of spiradoline (1 mg/kg, s.c.). We conclude that kappa agonists act to inhibit dopamine release from the nucleus accumbens via a direct effect in that region, and that tolerance does not occur to this neurochemical effect. Thus, kappa agonists may prove useful in chronic conditions resulting from excessive dopamine release.

Alterations in neurotransmitter amino acid content in the aging rat striatum are subregion dependent.

The topographical distribution of putative neurotransmitter amino acids in both 6- and 20-month-old Fischer 344 rats was studied in eight striatal subregions. Tissue levels of glutamate, aspartate, GABA, and taurine in the 20-month-old rats were elevated in virtually all of the anterior striatal subregions examined. In addition, aspartate levels were higher in all dorsomedial subregions, while glutamate and taurine levels were elevated in all lateral and ventrolateral subregions, respectively. Other amino acids such as glutamate, serine, and alanine did not display any specific subregional changes. These findings demonstrate specific striatal subregion changes in neurotransmitter amino acid content as a function of aging.

A rapid and simple HPLC microassay for biogenic amines in discrete brain regions.

A rapid microassay is described for the measurement of biogenic amines using an isocratic HPLC system with electrochemical detection. Catecholamines, indoleamines and their major metabolites were extracted with 150 microliters of perchloric acid from brain tissue punches (less than 250 micrograms) using a simple one-step sample preparation method. These compounds were separated on a short (80 mm) column with 3 microns particle size packing, and electrochemically detected within a total run time of less than 6 minutes. Detection limit sensitivity was approximately 2-5 pg. This method, detailed in an easy-to-follow description, reduces assay time, minimizes the possibility for errors, maximizes efficiency, and requires only standard HPLC equipment and supplies.

An improved and rapid HPLC-EC method for the isocratic separation of amino acid neurotransmitters from brain tissue and microdialysis perfusates.

An improved, HPLC with electrochemical detection method for the isocratic separation and determination of amino acids from post-mortem brain tissue and from microdialysates of awake-behaving animals is described. Optimal conditions that maximize stability, resolution, and sensitivity were determined for the pre-column derivatization of amino acids using o-phthalaldehyde and B-mercaptoethanol. Ten different amino acids including aspartate, glutamate, taurine, tyrosine and GABA were effectively resolved within 18 min. Tissue measurements from caudate, globus pallidus and substantia nigra showed regional variations in amino acid content. Microdialysis of the striatum yielded significant amounts of all amino acids examined, including GABA, from only 25 microliter of perfusate.

Atropine sulfate and 2-pyridine aldoxime methylchloride elicit stress-induced convulsions and lethality in mice and guinea pigs.

The present study demonstrates that dose combinations of atropine sulfate and 2-pyridine aldoxime methylchloride (2-PAM), which do not produce any overt toxic effects on the behavior of mice or guinea pigs in a stable environment, elicit clonic-tonic convulsions and death when the animals are physically stressed by cold water swimming. Phenoxybenzamine (1-6 mg/kg), diazepam (0.625 and 1.25 mg/kg) and pilocarpine (2.5 and 5 mg/kg) significantly decreased (or abolished) the occurrence of atropine and 2-PAM stressed-induced convulsions and/or lethality. In contrast, propranolol (20 mg/kg), was ineffective in preventing either convulsions or lethality. Changes in plasma glucose levels and internal body temperature did not appear to explain the precipitation of convulsions or ensuing death. These results suggest that during acute physical stress, relatively low doses of atropine and 2-PAM produce toxic and lethal effects due to the activation of alpha-adrenergic mechanisms along with a concomitant inactivation of cholinergic mechanisms.

Magnesium selectively inhibits N-methyl-aspartic acid-induced hypermotility after intra-accumbens injection.

The excitatory amino acids, N-methyl-aspartic acid, kainic acid and quisqualic acid have been shown to produce a marked increase in locomotor activity after bilateral injection into the rat nucleus accumbens. The intra-accumbens injection of magnesium inhibited the hypermotility response produced by N-methyl-aspartic acid in a dose-dependent manner. However, magnesium had no significant inhibitory effect on the increase in motility produced by either kainic acid or quisqualic acid. In contrast to magnesium, calcium produced a weak inhibitory action on N-methyl-aspartic acid-induced hypermotility. These data suggest that in the nucleus accumbens, at least two receptor types (N-methyl-aspartic acid/magnesium-sensitive and non-N-methyl-aspartic acid/magnesium-insensitive receptors) are present which can mediate the stimulation of locomotor activity produced by excitatory amino acids.

Antagonism of the hypermotility response induced by excitatory amino acids in the rat nucleus accumbens.

Several compounds have been shown to antagonize the excitation of single neurons produced by excitatory amino acids. This study was designed to determine the effectiveness of these compounds in antagonizing the hypermotility response to excitatory amino acids after intra-accumbens administration. Of the putative antagonists tested, D-aminoadipic acid, diaminopimelic acid and glutamic acid diethyl ester all showed significant inhibitory effects on excitatory amino acid-induced hypermotility while 2-amino-5-phosphonovaleric acid, gamma-D-glutamylglycine, 2-amino-4-phosphonobutyric acid and cis-2,3-piperidine dicarboxylic acid were ineffective. D-Aminoadipic acid decreased N-methyl-aspartic acid-induced hypermotility while having no significant effect on the hypermotility responses induced by kainic or quisqualic acids. Diaminopimelic acid markedly decreased N-methyl-aspartic acid- and kainic acid-induced hypermotility but was totally ineffective on quisqualic acid-induced hypermotility. In contrast to D-aminoadipic acid, glutamic acid diethyl ester antagonized the increase in motility produced by kainic and quisqualic acids but not that produced by N-methyl-aspartic acid. The above data suggests that N-methyl-aspartic acid and quisqualic acid may produce their motor effects through the activation of two different receptors in the nucleus accumbens while kainic acid may mediate its hypermotility response through both N-methyl-aspartic acid and quisqualic acid receptors. However, a third receptor type activated solely by kainic acid cannot be excluded at this time.

Effects of excitatory amino acids on locomotor activity after bilateral microinjection into the rat nucleus accumbens: possible dependence on dopaminergic mechanisms.

In order to study the role of excitatory amino acids on motor function, the effects of kainic, quisqualic, and N-methyl-DL-aspartic acids on locomotor activity were determined after their direct injection into the nucleus accumbens. These three amino acids have been used in previous studies to classify receptors for excitatory amino acids in the mammalian spinal cord. After injection into the nucleus accumbens all three amino acids stimulated locomotor activity, with kainic acid being the most potent and N-methyl-DL-aspartic acid the least potent. The maximum intensity of the stimulation produced by kainic and quisqualic acids was greater than that produced by N-methyl-DL-aspartic acid. These results suggest that receptors in the nucleus accumbens, sensitive to kainic and quisqualic acids, play a more important role in the stimulation of locomotor activity than those sensitive to N-methyl-DL-aspartic acid. In addition to the above amino acids, the administration of large doses of L-aspartic and D-glutamic acids also produced hyperactivity, while L-glutamic acid had no effect. To determine whether endogenous dopamine mediates the hypermotility produced by the excitatory amino acids, the response to these amino acids was studied after treatment with reserpine or dopamine receptor blocking agents. Reserpine (5 mg/kg, i.p.), haloperidol (0.8 mg/kg, i.p.) or fluphenazine [5.0 micrograms (total dose) injected into the nucleus accumbens] markedly attenuated the hypermotility induced by excitatory amino acids. These results suggest that the hypermotility produced by excitatory amino acids is mediated through release of dopamine and the subsequent stimulation of dopamine receptors in the nucleus accumbens.

Effect of methadone and morphine on serotonin uptake in rat periaqueductal gray slices.

In vitro effects of d,l-methadone and morphine on [3H]serotonin (3H-5-HT) uptake in rat periaqueductal gray (PAG) slices were investigated. Only methadone had a significant inhibitory effect on 3H-5-HT uptake which was significantly enhanced by naloxone. The systemic administration of methadone did not affect 3H-5-HT uptake in vitro. These data further weaken the possible relationship between narcotic analgesia and blockade of the 5-HT reuptake mechanism.