Free 3-nitrotyrosine causes striatal neurodegeneration in vivo.

Peroxynitrite formation has been demonstrated in several neurodegenerative disorders; thus far, protein nitration and consequent alterations in protein function are implicated as mechanistic events. Free 3-nitrotyrosine (free-3NT) is also elevated in these settings; a neurotoxic role for this modified amino acid has not been investigated. We tested the hypothesis that free-3NT is neurotoxic in vivo, using a mouse model of striatal degeneration. The neurodegenerative effects of the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) (unilateral intrastriatal injection, 64 nmol) were compared with free-3NT (32 nmol) or free-tyrosine (free-TYR) (32 nmol). 6-OHDA-treated mice exhibited significant ipsilateral turning behavior after d-amphetamine challenge, indicative of unilateral striatal injury (ipsilateral-contralateral turning differential, 21.1 +/- 6.8). Significant turning behavior was also observed in free-3NT-treated mice but not in free-tyrosine-treated mice (free-3NT, 16.0 +/- 3.9; free-TYR, 1 +/- 2.7; p < 0.01). Immunohistochemistry was used to evaluate striatal tyrosine hydroxylase (TH) content. 6-OHDA or free-3NT treatment caused severe reductions in TH immunoreactivity in injected striata compared with the contralateral hemisphere (injected/contralateral immunoreactivity ratio: 6-OHDA, 0.23 +/- 0.07; free-3NT, 0.49 +/- 0.02). Free-tyrosine treatment had no effect (1.03 +/- 0.09). Turning behavior was correlated with striatal TH ratio (p < 0.01). Furthermore, we observed a striking unilateral reduction in TH-positive cell body counts in the substantia nigra pars compacta of 6-OHDA- and free-3NT-treated mice (injected/contralateral cell count ratio: 6-OHDA, 0.40 +/- 0.04; free-3NT, 0.59 +/- 0.02). Free-tyrosine treatment had no effect (1.05 +/- 0.04). No evidence for increased striatal protein incorporation of 3NT was observed in any treatment group. These data represent the first evidence that free-3NT can elicit neurodegenerative effects in vivo; free-3NT may have a causal role in neurodegenerative conditions.

Does intervention by a nurse improve medication compliance?

A random sample of patients taking two or more drugs, at least one of which was digoxin or methyldopa, was drawn from a medical clinic population. After giving informed consent, the patients were randomized into control and experimental groups. The experimental group was seen by a specially prepared nurse interventionist who attempted to improve medication compliance. The levels of the drugs in the blood were taken as indicators of medication compliance. Our results document that patients exposed to nurse intervention were more compliant than the general clinic population, but were not more compliant than a nonintervention control group. Problems encountered in the collection and interpretation of compliance study data were identified and discussed.

Effects of cocaine on sensory inhibition in rats: preliminary data.

The purpose of this study was to determine the effect of cocaine on inhibitory sensory processing mechanisms in the brain. To accomplish this aim, recording electrodes were surgically placed into the vertex region of 12 rats. After recovery from surgery, rats were injected once daily for 5 days with either cocaine (20 mg/kg, IP) or saline. Immediately and 23 hr after each injection, the rats were tested for sensory gating mechanisms. They were presented with a series of two clicking sounds, a conditioning and testing click, delivered 0.5 sec apart, and the amplitude of the N40 responses to each of these clicks was recorded. The ratios of the amplitude of the N40 response to the testing click over that of the conditioning click (T/C ratio) were calculated for each animal for each testing period. The T/C ratios of the control (Saline-injected) animals were less than one, indicating that the conditioning stimulus was able to activate inhibitory neural pathways, producing a decrease in the response to the testing stimulus. The T/C ratios of the cocaine-treated animals were significantly greater than those of controls when the tests were conducted either immediately after injection or 23 hr later. These observations suggest that cocaine can impair mechanisms involved in the gating of responses to auditory stimuli. The higher T/C ratio found at 23 hr after cocaine injection suggests that an impairment in the gating mechanism may be produced by an arousal response that is associated with the environment in which the animals had been injected with cocaine.

Effects of repeated cocaine administration on sensory inhibition in rats: preliminary data.

We have recently reported that acute administration of cocaine to rats alters their sensory inhibitory capacity as tested in a paired click paradigm (S1/S2). Whether such acutely induced changes are persistent, is not known. In order to shed some light on the degree of spontaneous reversibility of cocaine-induced decreased sensory inhibition, rats were tested immediately after cocaine administration and 9 days after cessation of cocaine exposure. Six rats received cocaine HCl 20 mg/kg intraperitoneally and six rats received normal saline for 5 consecutive days. The amplitudes of the S1 responses were significantly decreased in the cocaine animals during the injection days only, but not 9 days later. Two measures of sensory inhibition were employed, S2/S1 x 100 amplitude ratio and S1-S2 amplitude difference. The ratio measure indicated a significant decrease in inhibitory capacity in the cocaine group during the injection days, and remained significantly decreased 9 days after cessation of cocaine administration. The data suggest that repeated cocaine administration can induce persistent deficit in the ability of the rat's brain to inhibit incoming irrelevant sensory stimuli.

The importance of dopaminergic neurotransmission in the hypermotility response produced by the administration of N-methyl-D-aspartic acid into the nucleus accumbens.

The bilateral injection of N-methyl-D-aspartic acid (NMA) into the nucleus accumbens of rats has been shown to stimulate locomotor activity. This response is antagonized by drugs that interfere with dopaminergic neurotransmission, such as reserpine, alpha-methyl-p-tyrosine (AMPT) and haloperidol, suggesting that NMA may exert its effects by stimulating the release of dopamine (DA) from nerve terminals. To test this hypothesis, the ability of NMA to release endogenous DA from slices of nucleus accumbens, which were incubated in magnesium-free medium was evaluated. It was found that NMA, at concentrations of 0.1 and 1 mM, did not stimulate the release of endogenous DA from slices in magnesium-free normal medium, medium containing pargyline (to inhibit monoamine oxidase) or medium containing methylphenidate (to block the reuptake of released DA). In contrast, both amphetamine (10(-5) M) and a high potassium (20 and 40 mM) stimulated the release of endogenous dopamine. The lack of effect of NMA on the release of endogenous DA was supported by in vivo studies which showed that the injection of NMA into the nucleus accumbens, in a dose that stimulated locomotor activity, did not increase the turnover of dopamine as reflected by an increase in the concentration of DOPAC. In contrast, the direct administration of haloperidol (13 nmol) into the nucleus accumbens produced a marked increase in the concentration of DOPAC. To determine the role of activation of DA receptors in the hypermotility response to NMA, NMA was administered together with subthreshold doses of either DA or apomorphine into the nucleus accumbens of rats pretreated with AMPT.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of GABAergic transmission in the subpallidal region on the hypermotility response to the administration of excitatory amino acids and picrotoxin into the nucleus accumbens.

Excitatory amino acids and picrotoxin have been shown to produce an intense stimulation of co-ordinated locomotor activity after bilateral administration into the nucleus accumbens of rats. The objective of this study was to determine the role of GABAergic neurotransmission in t he substantia innominata/lateral preoptic area in the hypermotility responses to excitatory amino acids and picrotoxin. It was found that the bilateral administration of muscimol into the substantia innominata/lateral preoptic area almost completely inhibited the stimulation of locomotor activity induced by the administration of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), kainic acid, N-methyl-D-aspartic acid and picrotoxin into the n. accumbens. In contrast, muscimol did not inhibit locomotor activity or induce catalepsy in control animals injected with saline into the nucleus accumbens. The inhibitory effect of muscimol on the hypermotility responses to excitatory amino acids and picrotoxin was not due to diffusion to the nucleus accumbens and the activation of GABAergic receptors at this site. This conclusion is preoptic area, which effectively inhibited the responses to AMPA and picrotoxin, were either much less effective or not effective in inhibiting these responses when injected into the nucleus accumbens. These observations suggest that the stimulation of locomotor activity produced by the injection of excitatory amino acids or picrotoxin into the nucleus accumbens may be mediated by the inhibition of a GABAergic neuronal pathway projecting from the nucleus accumbens to the substantia innominata/lateral preoptic area.

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.

Enhanced stereotyped response to amphetamine after pretreatment with small doses of molindone.

Pretreatment of rats with small doses of the antipsychotic drug, molindone, enhanced the stereotyped behavioral response to amphetamine. In order to determine whether molindone enhanced amphetamine-induced stereotypy by the same mechanism as chronic administration of amphetamine or drugs that inhibit central noradrenergic transmission, the effect of these drugs on the stereotyped behavior produced by beta-phenethylamine (PEA) was compared. Following the administration of phenoxybenzamine, reserpine and diethyldithiocarbamate, the stereotyped response produced by beta-phenethylamine was intensified. In contrast, neither molindone nor chronic pretreatment with amphetamine altered beta-phenethylamine-induced stereotypy. As shown previously with chronic amphetamine pretreatment, molindone also failed to enhance the stereotyped response produced by apomorphine. However, in contrast to the effects of chronic administration of amphetamine, molindone both increased the striatal concentration of dihydroxyphenylacetic acid (DOPAC) and blocked the ability of small doses of apomorphine to decrease this dopamine (DA) metabolite. The doses of molindone that blocked the apomorphine-induced reduction in the concentration of DOPAC in the striatum correlated with the doses that enhanced amphetamine-induced stereotypy. Since the decrease in DOPAC in the striatum produced by apomorphine is thought to be mediated through the stimulation of striatal DA autoreceptors, these results suggest that molindone enhances amphetamine-induced stereotypy by selectively inhibiting DA autoreceptors.

Folate induced-hypermotility response after bilateral injection into the nucleus accumbens of the rat. Possible mediation through dopaminergic mechanisms.

Folic acid (FA) and certain of its reduced congeners produce excitatory effects when applied to neuronal tissue. Recent evidence has suggested that folates have other biological properties in common with the excitatory amino acids. The purpose of this study was to determine the activity of folate compounds in a system sensitive to excitatory amino acids. Bilateral injection of folic acid into the nucleus accumbens resulted in a marked increase in locomotor activity at doses of 2.5 and 5 micrograms. Larger doses resulted in behavioral responses, such as body tremor and labored breathing, which interfered with the locomotor response. Similarly, 5-formyltetrahydrofolic acid (FTHF) produced a marked hypermotility response after bilateral injection into the nucleus accumbens (2.5-25 micrograms), while dihydrofolic acid, tetrahydrofolic acid, and 5-methyltetrahydrofolic acid were ineffective. Pretreatment with reserpine (10 mg/kg, i.p.) markedly reduced the hypermotility response elicited by folic acid and FTHF as did pretreatment with haloperidol in both peripheral (0.8 mg/kg) and direct (5 micrograms) injection into the nucleus accumbens. In addition, injection of muscimol (30 ng), which depresses hypermotility induced by dopamine and amphetamine, produced a significant decrease in the hypermotility response produced by folic acid. In contrast, pretreatment with phentolamine (5 mg/kg, i.p.) or propranolol (4 mg/kg, i.p.) did not decrease folic acid or FTHF-induced responses. These results suggest that folic acid and FTHF produce an increase in locomotor activity by facilitating dopaminergic neurotransmission in the nucleus accumbens, possibly by inducing the release of dopamine from the nerve terminals. Thus, these folates have effects similar to those of the excitatory amino acids when injected into the nucleus accumbens.

BehavioraL and biochemical changes caused by muscimol in mice withdrawN from haloperidol administration.

This study was designed to determine the behavioral and biochemical effects of the gamma-aminobutyric acid (GABA) agonist, muscimol, in mice withdrawn from chronic haloperidol administration. Mice received either haloperidol or vehicle In their drinking water for 35 days, after which the haloperidol was replaced with vehicle. Seven days after withdrawal from chronic treatment with haloperidol, the effect of apomorphine on the climbing behavior of haloperidol-withdrawn mice was markedly enhanced as compared to control mice that received only vehicle. Muscimol produced a dose-related reduction in the intensity of climbing behavior induced by apomorphine in both control and haloperidol-withdrawn mice. However, the inhibition of climbing behavior induced by muscimol was significantly greater in haloperidol-withdrawn animals. In addition, in haloperidol-withdrawn mice, muscimol produced a significant reduction in striatal homovanillic (HVA) levels with no change in striatal dopamine (DA) levels, suggesting a decrease in DA turnover. In support of this conclusion, muscimol decreased the disappearance of dopamine in haloperidol-withdrawn mice that were injected with alpha-methyl-p-tyrosine. Both the behavioral and biochemical effects of muscimol were blocked by the GABA antagonist, picrotoxin. These results indicate that after chronic haloperidol administration there is not only an enhanced response to dopaminergic agonists but also to GABAergic agonists.

Effect of pretreatment with amphetamine on the interaction between amphetamine and dopamine neurons in the nucleus accumbens.

The present study was designed to examine the effect of pretreatment with amphetamine on the ability of amphetamine to release dopamine from slices of the nucleus accumbens and striatum and to stimulate locomotor activity or stereotyped behavior, after direct injection into either the nucleus accumbens or the striatum. Rats were injected twice daily for 5 days with either amphetamine (5 mg/kg, i.p.) or saline. At 33 days after this pretreatment, the release of endogenous dopamine from both regions of the brain in vitro by amphetamine and the changes in behavioral responses to the direct injection of amphetamine into either region were examined. Amphetamine at both 1 and 10 microM stimulated the release of endogenous dopamine from slices prepared from both of the brain areas. The release of dopamine by amphetamine was increased in rats pretreated with amphetamine. Consistent with its ability to stimulate endogenous release of dopamine, amphetamine, when injected into the nucleus accumbens, stimulated locomotor activity, while stereotyped behavior was enhanced when amphetamine was injected into the striatum. However, the locomotor activity and stereotyped behavioral responses to small doses of amphetamine (5, 10 or 25 micrograms) were not significantly greater in amphetamine-pretreated rats, compared to saline-pretreated animals. A greater stimulation of both responses in amphetamine-pretreated rats was only observed when a large dose (50 micrograms) of amphetamine was administered into either the nucleus accumbens or striatum.(ABSTRACT TRUNCATED AT 250 WORDS)

Time course of the development of the enhanced behavioral and biochemical responses to amphetamine after pretreatment with amphetamine.

These experiments were designed to examine the time course of development of the enhanced stereotyped behavioral response to amphetamine after withdrawal from chronic pretreatment with amphetamine and to determine whether this time course correlates with that of the enhancement in the amphetamine-induced stimulation of the release of dopamine (DA) from striatal slices. Rats were pretreated with amphetamine (5 mg/kg, i.p.) or saline, twice daily for 5 consecutive days. At 3, 15 and 30 days after withdrawal of the drug the stereotyped behavioral response and the release of endogenous DA from slices of striatum in response to a challenge dose of amphetamine were measured. At all 3 times tested, the stereotyped behavioral response to the challenge dose of amphetamine was enhanced in the rats pretreated with amphetamine, with the greatest degree of enhancement seen at 15 and 30 days after withdrawal of the drug. At these times, the responses were associated with a significant attenuation in the stimulation of locomotor activity produced by the challenge dose of amphetamine, which was probably related to the enhanced stereotyped behavioral response. Amphetamine stimulated the release of endogenous DA from slices of striatum in rats pretreated with saline and amphetamine. However, the release of endogenous DA from slices of rats pretreated with amphetamine was significantly greater than that of saline-pretreated rats at 15 and 30 days after withdrawal of the drug, but not at 3 days after withdrawal. Thus, pretreatment with amphetamine resulted in enhanced behavioral and biochemical responses to amphetamine which increased over time after withdrawal of the drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Dopaminergic agonists: comparative actions of amine and sulfonium analogues of dopamine.

We have investigated the possibility that structural modifications of the sulfonium analogue of dopamine (4) would produce the same pattern of biological activity as structural modifications of dopamine. A series of methyl- tetralinyl -, and naphthalenylsulfonium analogues 5-7 were prepared and tested for their ability to inhibit the potassium-evoked release of [3H]acetylcholine from striatal slices. All compounds were tested under normal conditions and after depletion of dopamine stores with reserpine and alpha-methyl-p-tyrosine. The amine and sulfonium analogues 2-6 all showed direct agonist activity. The sulfonium analogue 7 produced, predominantly, indirect activity. In contrast to the amine analogues, chemical modifications of the sulfonium compounds produced little change in their dopamine agonist activity.

Synthesis and pharmacological evaluation of sulfonium analogues of dopamine: nonclassical dopamine agonists.

In order to test whether the nitrogen/ammonium moiety in the dopamine molecule is required for dopaminergic activity, we have synthesized two sulfonium analogues of dopamine and tested them for biological activity in an in vivo and in an in vitro system. These analogues have provided a means of investigating (1) the ability of the sulfonium function to serve as a bioisostere for the dopamine amino group and (2) whether charged molecules have the ability to bind to dopamine receptors. Both sulfonium analogues, 1 and 2, as well as dopamine, when injected directly into the striatum of rats, previously lesioned unilaterally with 6-hydroxydopamine (6-OHDA), produced circling behavior. The potency of the sulfonium analogues was approximately one-tenth that of dopamine. The effects of all three compounds on circling were inhibited by the dopamine antagonist haloperidol. In addition, both sulfonium analogues inhibited the high affinity binding of radiolabeled dopamine to a crude membrane fraction prepared from the striatum. This study suggests that the nitrogen atom found in th dopamine molecule is not essential for dopaminergic activity, since the nitrogen can be replaced by a sulfonium functional group for this activity.

The interaction of ammonium, sulfonium, and sulfide analogues of metoclopramide with the dopamine D2 receptor.

A series of permanently charged ammonium and sulfonium analogues of metoclopramide as well as a permanently uncharged sulfide analogue were synthesized and evaluated for their ability to inhibit apomorphine-induced responses on mouse striatal slices. Three of the four permanently charged analogues were found to inhibit apomorphine's effects, although at higher concentrations than either metoclopramide or its dimethyl analogue. In contrast, the sulfide analogue was inactive at concentrations up to 100 microM. These findings are consistent with earlier studies of chlorpromazine and sulpiride analogues and provide further evidence that dopamine antagonists bind in their charged molecular forms to anionic sites on the D2 receptor. Further, the results of this study in conjunction with those of our earlier sulpiride study would seem to indicate that differences in the biological profiles of metoclopramide, a type 1 benzamide useful as a gastric prokinetic agent, and sulpiride, a type 2 benzamide useful for its antipsychotic effects, are not due to any appreciable differences in the binding of the basic nitrogen atom of these molecules.

Synthesis and D2 dopaminergic activity of pyrrolidinium, tetrahydrothiophenium, and tetrahydrothiophene analogues of sulpiride.

All of the existing dopamine receptor models recognize the amine nitrogen of agonist and antagonist drugs as playing a crucial role in receptor interactions. However, there has been some controversy as to which molecular form of the amine, charged or uncharged, is most important in these interactions. We have synthesized and examined the biological activity of permanently charged and permanently uncharged analogues of the dopaminergic antagonist, sulpiride. Sulpiride and the permanently charged pyrrolidinium (6,7) and tetrahydrothiophenium (9) analogues were able to antagonize the inhibitory effect of apomorphine on the K+-induced release of [3H]acetylcholine from striatal slices. In contrast, the permanently uncharged tetrahydrothiophene analogue 8 was inactive at concentrations up to 100 microM. Additionally, both sulpiride and the tetrahydrothiophenium analogue were able to displace [3H]spiperone from D2 binding sites, while the tetrahydrothiophene analogue was unable to produce any significant displacement. These results are consistent with our previous observations on permanently charged chlorpromazine analogues and provide further evidence that dopaminergic antagonists bind in their charged molecular forms to anionic sites on the D2 receptor.

Synthesis of chiral 1-(2'-amino-2'-carboxyethyl)-1,4-dihydro-6,7-quinoxaline-2,3-diones: alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor agonists and antagonists.

Recently discovered 6,7-disubstituted quinoxaline-2,3-diones, 1, have been found to antagonize specific binding and functional responses to both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainic acid. Although a variety of studies have analyzed the activity of quinoxaline-2,3-diones with various substitutions at positions 6 and 7, there is little information regarding the effects of N-substitution. A racemic mixture of 1-(2'-amino-2'-carboxyethyl)-1,4-dihydroquinoxaline-2, 3-dione (QXAA, 2, R1 = R2 = H) has been synthesized from 1 (R1 = R2 = H). This compound inhibited specific [3H]AMPA binding but not [3H]kainate binding. IC50 values for QXAA, AMPA, and DNQX were 0.69, 0.012, and 0.74 microM, respectively. The R- and S-enantiomers were prepared by asymmetric synthesis. The S-isomer (2b) was 160-fold more potent in binding assays than the R-isomer (2d), with IC50 values of 0.23 and 38 microM, respectively. Both enantiomers were agonists in a functional assay, with the S-isomer having an EC50 value of 3 microM while that for the R-isomer was greater than 1 mM. Methyl substitutions at positions 6 and 7 (2a and 2c) resulted in antagonist compounds characterized by the S- and R-isomers being nearly equipotent, with IC50 values of 51 and 22 microM in the binding assay and EC50 values of 290 and 300 microM in the functional assay. AMPA had an EC50 value of 11 microM and DNQX an EC50 value of 30 microM in the functional assay. Analogs of quinoxalinediones with side chains other than an amino acid moiety on the nitrogen did not show good binding activities.

Dopamine agonists: effects of charged and uncharged analogues of dopamine.

Dopamine, at physiological pH, may exist as either an uncharged amine or a charged ammonium species. In order to gain insight as to which species is better suited for interaction with the dopamine receptor, we have synthesized dopamine analogues in which the nitrogen atom is replaced with a neutral methyl sulfide, a neutral methyl selenide, a charged dimethylsulfonium iodide, and a charged dimethylselenonium iodide. These analogues were tested for their ability to inhibit the K+-stimulated release of [3H]acetylcholine from striatal slices. At 30 microM concentration, the charged sulfonium and selenonium salts possessed significant agonist activity while the corresponding neutral species were inactive, suggesting that a charged species is optimal for dopamine agonist activity. In addition, the methyl sulfide was converted into the corresponding sulfoxide and sulfone; however, neither of these oxidation products possessed significant activity as dopaminergic agonists.

Charged analogues of chlorpromazine as dopamine antagonists.

Chlorpromazine (1, CPZ) is a potent dopamine antagonist that has been used widely as an antipsychotic agent. Since dopaminergic antagonists, like dopaminergic agonists, exist in solution as the charged and uncharged molecular species, it is not clear which form of the amine is most important for interaction with the dopamine receptor. Previous work from our laboratory has indicated that a variety of permanently charged species could replace the amine/ammonium moiety of dopamine and retain dopamine agonist activity. This paper describes the synthesis and dopamine antagonist activity of both the trimethylammonium iodide and the dimethylsulfonium iodide analogues of chlorpromazine. The permanently uncharged methyl sulfide analogue was also synthesized; however, due to its lack of aqueous solubility, its pharmacological activity could not be evaluated. Binding of both the dimethylsulfonium iodide and the trimethylammonium iodide analogues to D-2 dopamine receptors of rat striatal tissue was observed. The observed relative order of binding was CPZ greater than CPZ sulfonium analogue greater than CPZ ammonium analogue. These compounds had a similar order of activity in antagonizing the apomorphine-induced inhibition of potassium-induced release of [3H]acetylcholine from mouse striatal slices.

Design and synthesis of enantiomers of 3,5-dinitro-o-tyrosine: alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropanoic acid (AMPA) receptor antagonists.

The R- and S-isomers of 3,5-dinitro-o-tyrosine (6a,b) have been synthesized through the use of chemoenzymatic synthesis and shown to bind differentially with the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropanoic acid (AMPA, 3) receptors. The phenolic functional group of these o-tyrosine analogues was designed to act as a bioisostere of the gamma-carboxyl group of glutamate. The S-isomer of 3,5-dinitro-o-tyrosine (6b) was 6.5 times more potent than the R-isomer (6a) in inhibiting [3H]AMPA binding with IC50 values of 13 +/- 7 and 84 +/- 26 microM, respectively. The phenolic group was important for binding affinity since the methoxy compound 7 was less potent than the phenolic compound 6 in inhibiting the binding of AMPA. The free amino group was also shown to be important since the N-acetyl analogue 15 and the N-t-BOC compounds 16 and 17 exhibited very low affinity for the AMPA receptors. AMPA receptor functional tests showed that the o-tyrosine analogues are antagonists and that the S-isomer 6b (IC50 = 630 +/- 140 microM) was more potent than the racemate 6 (IC50 = 730 +/- 88 microM) while the R-isomer 6a was inactive up to 1 mM concentration, which is consistent with the S-isomer having higher binding affinity than the R-isomer.