Edmund A. Debler

Uptake of acetyl-L-carnitine in the brain.

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 aKm of 1.92 mM and aVmax of 1.96 μmol/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°C), and ouabain, and in the absence of Na+. The uptake of acetyl-l-carnitine was not strictly substrate-specific; γ-butyrobetaine,l-carnitine,l-DABA, and GABA were potent inhibitors, hypotaurine andl-glutamate were moderate inhibitors, and glycine and β-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, an isothiocyanate analog of PCP, induces audiogenic seizures in mice

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.

Ascorbic acid and striatal transport of [3H]1-methyl-4-phenylpyridine (MPP+) and [3H]dopamine

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 acids effects on [3H] MPP+ transport are related to translocation and/or dissociation processes occurring subsequent to the initial binding event.

Superoxide Radical-Mediated Alteration of Synaptosome Membrane Structure and High-Affinity γ-[14C]Aminobutyric Acid Uptake

Abstract: 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 γ‐[14C]aminobutyric acid (GABA) uptake. Kinetic analysis of the reduction in high‐affinity uptake reveals that the V.max is significantly altered whereas the Km is not. Preincubation with specific active oxygen/free radical scavengers indicates that the superoxide 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.

Kinetics of [3H]MPP+ uptake in dopaminergic neurons of mouse: regional effects of MPTP neurotoxicity.

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+.

Nicotine-induced changes in the metabolism of specific brain proteins

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.

Effects of caffeine on amino acid transport in the brain.

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.

Anticonvulsant Effects of Phencyclidine and PCP-like Drugs on Audiogenic Seizures Induced by Metaphit in Mice

Phencyclidine (PCP), at low doses (0.3–3 mg/kg), selectively delayed the onset or prevented the occurrence of pentylenetetrazol (PTZ)-induced tonic, but not clonic seizures. However, at a dose of 10 mg/kg it acted as a convulsant agent (Hayes and Balster, 1985). PCP-like drugs, when evaluated with respect to potential anti/pro-convulsant actions, demonstrated only anticonvulsant properties. Modulation of the N-methyl-D-aspartate (NMDA) receptor site by PCP and PCP-like compounds as a mechanism of anticonvulsant activity has been suggested by Leander et al. (1988), in which a strong correlation was demonstrated between the anticonvulsant activity of PCP/PCP-like compounds and the minimal effective dose which blocks NMDA receptor-mediated NMDA-induced lethality.

Damage to the high-affinity γ-aminobutyric acid (GABA) uptake system in mouse brain by horseradish peroxidase (HRP)

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.