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We previously demonstrated that metaphit (a phencyclidine analogue with an acylating isothiocyanate group) induces occurrence of audiogenic seizures in mice exposed to audio stimulation 24 h after metaphit administration. We have studied various receptor systems associated with excitatory and inhibitory networks: sites for competitive and noncompetitive antagonists of the N-methyl D-aspartic acid (NMDA) receptor complex, for [3H]muscimol on the gamma-aminobutyric acid (GABA) receptor complex, and for [3H]batrachotoxinin A20-alpha-benzoate on the voltage-dependent sodium channel. Mice were examined for neurochemical changes at 24 h after pretreatment with metaphit, when susceptibility to audiogenic seizures is greatest. Ex vivo receptor binding studies detected no changes; in vivo labeling of the phencyclidine site in the NMDA receptor complex was reduced by 20% in cortical and midbrain regions. A separate group of experiments was aimed at measuring brain levels of metaphit. One minute after retroorbital administration of [3H]metaphit at a dose sufficient to produce susceptibility to audiogenic seizures 24 h later, the brain level of [3H]metaphit (determined by high-performance liquid chromatography, HPLC) was 49 pmol/mg tissue; at 1, 4, and 24 h, the level was 12, 6, and 1.4 pmol/mg tissue or microM if metaphit was evenly distributed throughout the brain. Although the observed metaphit concentrations during the first 4 h are high enough to acylate receptors, no firm evidence for acylation was found for most of the examined receptors. Finally, the time course of the brain level of metaphit showing a continuous decrease is entirely different from that of development of the seizure susceptibility, which peaks at 18-24 h.

Metaphit [an analogue of phencyclidine (PCP) with an acylating isothiocyanate group] induced audiogenic clonic to clonic-tonic seizures in mice exposed to audio stimulation 24 h after metaphit administration. The incidence of seizures was reduced by treatment 30 min before audio stimulation with specific PCP-like compounds [5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine maleate (MK-801), and PCP itself], competitive N-methyl-D-aspartate antagonists 2-amino-5-phosphonopentanoic acid (AP-5 and NPC-12626), antiepileptic drugs [phenobarbital (PB), phenytoin (PHT)], and gamma-aminobutyric acid (GABA) agonists (muscimol and diazepam). In contrast, when given in conjunction with metaphit, most of these drugs were ineffective in protecting animals from audiogenic seizures 24 h later. Only compounds with long half-lives (t1/2) such as MK-801, PB, and PHT had a protective effect. High-performance liquid chromatography (HPLC) determination of [3H]MK-801 showed its long-term presence in the brain after intraperitoneal (i.p.) administration of [3H]MK-801. Audiogenic seizures observed 24 h after metaphit administration were potentiated by administration of the GABA antagonist picrotoxin 15 min before audio stimulation, and picrotoxin-induced spontaneous seizures were enhanced by pretreatment (24 h earlier) with a dose of metaphit that in itself did not produce spontaneous seizures at the time of the picrotoxin test. Similar observations were made with N-methyl D-aspartic acid (NMDA) instead of picrotoxin. Thus, an interplay exists between excitatory glutaminergic and inhibitory GABAergic circuitries in the metaphit seizure model.

The carrier-mediated efflux of [3H]1-methyl-4-phenylpyridine (MPP+) and [3H]dopamine was examined in mouse striatal synaptosomal P2 fractions. Although the two compounds are transported by the same carrier, the translocation of the carrier-ligand complex is more rapid with MPP+ than with dopamine. With dopamine-stimulated efflux of preloaded [3H]dopamine, externally present dopamine at a concentration of 1.3 microM reduced the intrasynaptosomal concentration of [3H]dopamine by 50% (the ECR value) with 8 min of incubation. The ECR value of dopamine in promoting the efflux of [3H]MPP+, however, was only 0.15 microM. Similarly, ascorbic acid was weaker in enhancing the efflux of [3H]dopamine (ECR greater than 2000 microM) than that of [3H]MPP+ (ECR = 567 microM). This effect of ascorbic acid on the efflux of [3H]MPP+ was attenuated by mazindol, a blocker of dopamine uptake. It is proposed that ascorbic acid has a neuromodulatory role involving changes at the level of carrier-membrane translocation and/or orientation.

The bicycloannulation reaction between cyclohexenone and indolyl enamines yields trans-3-(cyclic amino)-2-(3'-indolyl)bicyclo[2.2.2]octan-5-ones, and these adducts are conformationally restricted analogues of indolylethylamine (tryptamine) which exhibit structure-dependent affinity for the serotonin 5HT2 and 5HT1a receptors. The stereochemistry of the isomeric endo and exo adducts obtained is assigned from the 1H NMR spectra of the specifically deuterated alkenes prepared from the ketones by the Bamford-Stevens reaction. Molecular mechanics calculations indicate that the conformational flexibility of the amino and indolyl groups is restricted through van der Waals interactions with the bridges of the bicyclic unit. These compounds inhibit the binding of [3H]ketanserin to 5HT2 sites in mouse cerebrocortical membranes, and the binding of [3H]-8-hydroxy-2-(di-n-propylamino)tetralin ([3H]-8-OH-DPAT) to 5HT1a sites in mouse hippocampal membranes. The endo compounds are the most potent, and molecular mechanics calculations indicate that these isomers have a less bulky bicyclo bridge proximate to the amine group and more conformational freedom about the C alpha-C beta-N(+)-H dihedral angle (tau 3). In the 5HT2 assay, endo-trans-3-(N-piperidinyl)-2-(3'-indolyl)bicyclo[2.2.2]octan-5-one (10a) is the most potent, and endo-trans-3-(N-pyrrolidinyl)-2-(3'-indolyl)bicyclo[2.2.2]oct-5-ene (12a) is the most potent in the 5HT1a assay. A phenyl-substituted adduct shows the least affinity in these two assays. These data provide insight into the structural differences between the 5HT1a and 5HT2 receptor sites.

Chronic administration of caffeine to mice did not alter cellular (low-affinity) transport as measured in brain slices of the amino acids ?-aminobutyric acid, glutamic acid, and glycine. Chronic caffeine administration did, however, increase the long-term (60-min) uptake of ?-aminoisobutyric acid and valine into brain slices. A similar tendency, although not statistically significant, towards increased amino acid uptake was also seen in the transport of phenylalanine and lysine across the blood-brain barrier in chronically treated rats. The increase in neutral amino acid uptake seems unrelated to changes in brain protein metabolism, since chronic caffeine administration did not significantly alter brain protein synthesis rates. In vitro, caffeine (0.01-1 mM) did not alter brain slice transport of the amino acids ?-aminobutyric acid, glutamic acid, glycine, valine, and the amino acid analog ?-aminoisobutyric acid. Caffeine (1-100 ?M) was also ineffective in altering the synaptosomal (high-affinity) transport of the neurotransmitter amino acids ?-aminobutyric acid, glutamic acid, and glycine. These data support the idea that caffeine is a mildly stimulating drug with no significant effect on neurotransmitter amino acid transport systems in rodent brain.

The inhibition of uptake of [3H] dopamine and [3H] 1-methyl-4-phenylpyridine (MPP+) was examined in mouse striatal synaptosomal preparations. Kinetic analysis indicated that ascorbic acid is a noncompetitive inhibitor of [3H] MPP+ uptake. No inhibition of [3H] dopamine uptake is observed. The dopamine uptake blockers, GBR-12909, cocaine, and mazindol strongly inhibit (IC50 less than 1 uM) both [3H] dopamine and [3H] MPP+ transport. Nicotine, its metabolites, and other tobacco alkaloids are weak inhibitors (IC50 greater than 1 mM) except 4-phenylpyridine and lobeline, which are moderate inhibitors (IC50 = 3 to 40 uM) of both [3H] dopamine and [3H] MPP+ uptake. These similarities in potencies are in agreement with the suggestion that [3H] MPP+ and [3H] dopamine are transported by the same carrier. The differences observed in the alteration of dopaminergic transport and mazindol binding by ascorbic acid suggest that ascorbic acid's effects on [3H] MPP+ transport are related to translocation and/or dissociation processes occurring subsequent to the initial binding event.

The effects of ascorbic acid on the synaptosomal uptake of [3H]MPP+, [3H]dopamine, and [14C]GABA were examined in attempts to understand the mechanism of ascorbic acid attenuation of MPTP neurotoxicity. [3H]Dopamine uptake was increased at lower levels (0.01 and 0.1 mM) and decreased at higher levels (10 mM) of ascorbic acid. Ascorbic acid inhibited [3H]MPP+ uptake (IC50 = 0.1 mM) and [14C]GABA uptake (IC50 = 10 mM). Washout of ascorbic acid restored uptake of [3H]dopamine and [3H]MPP+, suggesting that ascorbic-acid-induced lipid peroxidation was not involved in the effect on uptake. In addition to the possible involvement of antioxidant mechanisms in the in vivo attenuation of the neurotoxicity of MPTP by ascorbic acid, the present results indicate a direct effect of ascorbic acid in inhibiting the uptake of MPP+ into dopaminergic nerve terminals.

Presynaptic nerve terminals when depolarized are sensitive to morphological and functional alteration by horseradish peroxidase. Mouse brain slices, 0.1 mm, depolarized by a K(+)-HEPES buffer and exposed to horseradish peroxidase exhibited alterations in both synaptic vesicle membrane structure and in high-affinity [(14)C]?-aminobutyric acid uptake. The post stimulatory retrieval of synaptic vesicles from the nerve terminal plasma membrane in the presence of horseradish peroxidase resulted in a decrease in the synaptic vesicle population with a concurrent increase in non-synaptic vesicle membrane structures. High-affinity [(14)C]?-aminobutyric acid uptake into 0.1-mm slices of mouse cerebral cortex and ponsmedulla-spinal cord was inhibited by 31% and 24%, respectively, after incubation for 60 min in K(+)-HEPES buffer containing horseradish peroxidase. Superoxide dismutase protected both the synaptic vesicle membrane and the high-affinity uptake system from the deleterious effects of horseradish peroxidase, pointing to the possible involvement of superoxide anion radicals in the horseradish peroxidase-related effects. These horseradish peroxidase induced alterations appear to be directed towards the exposed synaptic vesicle membrane, since non-stimulated brain slices exposed to horseradish peroxidase do not exhibit a reduction in either high- or low-affinity [(14)C]?-aminobutyric acid uptake. Low-affinity uptake of [(14)C]?-aminobutyric acid and [(14)C]?-aminoisobutyric acid into cortical slices was not affected after incubation in K(+)-HEPES with horseradish peroxidase. Low-affinity uptake, however, is reduced by the high-K(+)/Na(+)-free stimulatory incubation prior to uptake. It appears, thus, that high- and low-affinity uptake are distinct and different systems, with the high-affinity transport system structurally associated with synaptic vesicle membrane.

In a cortical P2 fraction, [14C]gamma-aminobutyric acid ([14C]GABA), [14C]glycine, [14C]taurine, and [14C]glutamic and [14C]aspartic acids are transported by four separate high-affinity transport systems with L-glutamic acid and L-aspartic acid transported by a common system. GABA transport in cortical synaptosomal tissue occurs by one high-affinity system, with no second, low-affinity, transport system detectable. Only one high-affinity system is observed for the transport of aspartic/glutamic acids; as with GABA transport, no low-affinity transport is detectable. In the uptake of taurine and glycine (cerebral cortex and pons-medulla-spinal cord) both high- and low-affinity transport processes could be detected. The high-affinity GABA and high-affinity taurine transport classes exhibit some overlap, with the GABA transport system being more specific and having a much higher Vmax value. High-affinity GABA transport exhibits no overlap with either the high-affinity glycine or the high-affinity aspartic/glutamic acid transport class, and in fact they demonstrate somewhat negative correlations in inhibition profiles. The inhibition profiles of high-affinity cortical glycine transport and those of high-affinity cortical taurine and aspartic/glutamic acid transport also show no significant positive relationship. The inhibition profiles of high-affinity glycine transport in the cerebral cortex and in the pons-medulla-spinal cord show a significant positive correlation with each other; however, high-affinity glycine uptake in the pons-medulla-spinal cord is more specific than that in the cerebral cortex. The inhibition profile of high-affinity taurine transport exhibits a nonsignificant negative correlation with that of the aspartic/glutamic acid transport class.

The effect of acute and chronic nicotine on the metabolism of specific brain proteins was examined by measuring incorporation of labeled valine into protein, with densitometric scanning of proteins resolved by gel electrophoresis. Acute and chronic administration of nicotine (0.4 mg/kg per 30 min for 2 hours, s.c., or 0.5 mg/kg per 30 min for 5 days (Alzet mini-pump implanted subcutaneously] reduced incorporation of [14C]valine administered by approximately 6-7%. The results with chronic nicotine administration indicated a lack of tolerance for this effect of nicotine. Mecamylamine, a nicotinic ganglionic antagonist, does not seem to block the inhibition of protein synthesis. Small increases in protein content were observed in a high- and a low-molecular-weight region of SDS-polyacrylamide gel, used to separate proteins from newborn brain. In adult brain after chronic nicotine administration, selective increases and a decrease were seen in selective bands. Results are consonant with selective effects of nicotine on the synthesis or degradation of specific brain proteins.

Mouse cortical synaptosomal structure and function are altered when exposed to hypoxanthine/xanthine oxidase (HPX/XOD)-generated active oxygen/free radical species. The structure of both the synaptic vesicle and plasma membrane systems are altered by HPX/XOD treatment. The alteration of synaptic vesicle structure is exhibited by a significant increase in the cumulative length of nonsynaptic vesicle membrane per nerve terminal. With respect to the nerve terminal plasma membrane, the length of the perimeter of the synaptosome is increased as the membrane pulls away from portions of the terminal in blebs. The functional lesion generated by HPX/XOD treatment results in a reduction in selective high-affinity gamma-[14C]aminobutyric acid (GABA) uptake. Kinetic analysis of the reduction in high-affinity uptake reveals that the Vmax is significantly altered whereas the Km is not. Preincubation with specific active oxygen/free radical scavengers indicates that the super-oxide radical is directly involved. This radical, most probably in the protonated perhydroxyl form, initiates lipid peroxidative damage of the synaptosomal membrane systems. Low-affinity [14C]GABA transport is unaltered by the HPX/XOD treatment. The apparent ineffectiveness of free radical exposure on low-affinity [14C]GABA transport coupled with its effectiveness in reducing high-affinity transport supports the idea that two separate and different amino acid uptake systems exist in CNS tissue, with the high-affinity being more sensitive (lipid-dependent) and/or more energy-dependent (Na+,K+-ATPase) than the low-affinity system.

Analysis in mouse brain slices of the uptake of acetyl-L-[N-methyl-14C]carnitine with time showed it to be concentrative, and kinetic analysis gave a Km of 1.92 mM and a Vmax of 1.96 mumol/min per ml, indicating the presence of a low-affinity carrier system. The uptake was energy-requiring and sodium-dependent, being inhibited in the presence of nitrogen (absence of O2), sodium cyanide, low temperature (4 degrees C), and ouabain, and in the absence of Na+. The uptake of acetyl-L-carnitine was not strictly substrate-specific; gamma-butyrobetaine, L-carnitine, L-DABA, and GABA were potent inhibitors, hypotaurine and L-glutamate were moderate inhibitors, and glycine and beta-alanine were only weakly inhibitory. In vivo, acetyl-L-carnitine transport across the blood-brain barrier had a brain uptake index of 2.4 +/- 0.2, which was similar to that of GABA. These results indicate an affinity of acetyl-L-carnitine to the GABA transport system.

Metaphit induces audiogenic seizures in mice. The most severe clonic/tonic seizures occur 18-24 h after metaphit administration. After 48 h the incidence of the seizure episodes begin to diminish. These audiogenic seizures can be prevented by the administration of either PCP or MK-801 24 h after metaphit and 30 min prior to audio stimulation. These seizures may be due to a modulation of the PCP recognition site by metaphit which results in an enhanced probability that the NMDA/PCP ion channels are open.

In an effort to determine the specificity of MPTP/MPP+ toxicity with respect to the dopaminergic systems, the effect of prior MPTP treatment on [3H]MPP+ uptake in the striatum and olfactory tubercle of BALB/cBy mice was examined. Kinetic analysis of [3H]MPP+ uptake indicated a reduction of Vmax values in both striatum (49%, P less than 0.05) and olfactory tubercule (26%, P less than 0.05). MPTP treatment did not significantly alter the Km in either region, although MPP+ accumulates in both olfactory tubercle and striatum, these dopaminergic systems show different sensitivity to the neurotoxicity of MPTP/MPP+.