The effect of caffeine on some mouse brain free amino acid levels.

Changes in free amino acids were examined in the central nervous system of mice treated with caffeine for three weeks. Caffeine was administered in the drinking water, and at the end of three weeks the level of caffeine in the cerebral cortex was 113 +/- 19 micrograms/g. When amino acid levels in cerebral hemispheres, midbrain, pons and medulla, and cerebellum were measured a significant increase in glutamine levels was found in all four regions. Glycine, alanine, serine, threonine, and GABA were significantly reduced in some regions. Caffeine appears to alter some of the metabolic or transport processes regulating amino acid pools in the brain. The decrease of GABA found in pons and medulla may contribute to the observed increase in reflex excitability after caffeine.

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.

Depolarization of brain cortex slices and synaptosomes by lithium. Determination of K+-equilibrium potential in cortex slices.

K+-equilibrium potential was determined in brain cortex slices of rat by measuring 86Rb+ distribution between the extra- and intracellular space. The ratio of internal to external Rb+ concentration was 39 +/- 1.8, corresponding to a resting membrane potential of 93.8 mV. Li+ (1-126 mM) decreased the membrane potential in both cortex slices and synaptosomes in a concentration-dependent manner. The presence of 1 mM Li+ was enough to cause a slight but distinct depolarization. During incubation in Li+-containing medium slices took up K+; however, for depolarization the presence of extracellular Li+ seemed to be necessary.

Modulation of the serotonin S2-receptor in brain after chronic lithium.

In vivo effects of chronic lithium administration on dopaminergic and serotonergic receptor binding were studied in the striatum and cerebral cortex of the rat. [3H]Domperidone was used as the ligand for the dopaminergic receptor, and [3H]ketanserin for the serotonergic system. Long-term ingestion of lithium (2-3 months) resulted in high levels of lithium in the cerebral cortex and significantly higher potassium levels; the sodium content remained at normal levels. The kinetic constants (Kd and Bmax) of [3H]domperidone binding sites measured in the striatum did not show any deviation from control values, but the receptor concentration (Bmax) of [3H]ketanserin binding sites was significantly reduced in the cerebral cortex of lithium-treated rats. The apparent dissociation constant (Kd) was not changed. The results indicate that the serotonergic component of the [3H]spiperone binding site, which we had previously found to be affected by chronic lithium treatment and which was shown by Peroutka and Snyder to be the 5-HT2 receptor, is selectively affected by lithium.

Dietary lithium during development: changes in amino acid levels, ionic content, and [3H]spiperone binding in the brain of rats.

We found that chronic lithium diet affects the sensitivity of neuroleptic receptors and the content of amino acids in the brain, and that the changes in adult animals differ from those in young rats. Pregnant rats were kept on lithium diet (pellets with 0.21% Li2CO3 and 0.21% NaCl) during the gestation period and the offspring were kept on lithium for six weeks after delivery. Control rats were kept on normal diet under identical conditions. In corpus striatum and cerebral cortex of lithium-treated young rats a reduction in apparent dissociation constant and no change in (3H)spiperone total binding sites were found, suggesting a sensitization of the neuroleptic receptor; this result was unlike that obtained with adult lithium-treated rats, where the total number of binding sites was decreased. The lithium content of brain was very high (2.32 meq/kg of wet weight), whereas in the serum only 0.75 meq/l was recorded. K+ and Na+ levels increased by 20% and 9% respectively in the brain and remained at normal levels in the serum. Analysis of free amino acids in the cerebral cortex, midbrain, and cerebellum showed increases in GABA and glycine levels in all three regions, a significant increase in taurine in midbrain, and an increase in lysine in cerebral cortex and cerebellum. The results indicate that the effect of chronic dietary lithium given during pregnancy on the neuroleptic receptor in young rats is different from that in adult animals. It produces an increase in the number of the neuroleptic receptor sites instead of the decline in the number of binding sites found in adult rats. It remains to be established whether this effect is related more to the age of the animal tested or to the stage of development of the CNS at which the lithium was administered.

Effects of cations and temperature on the binding of [3H]spiperone to sheep caudate nucleus.

The specific [3H]spiperone binding by sheep caudate nucleus homogenate is increased by divalent cations. The effect of Ca2+ or Mn2+ (5 mM) is temperature-dependent, and it is optimal at about 37 degrees, but is relatively low below 15 degrees and above 50 degrees. In the absence of added Ca2+ or Mn2+, the maximal specific [3H]spiperone binding is observed at about 25 degrees, and the cations shift the optimum to about 37 degrees. Under the experimental conditions used, the KD is about 0.6 nM and is not influenced by Ca2+ or Mn2+, or by temperature (25 and 37 degrees). In addition to Ca2+ and Mn2+, Mg2+ and Zn2+ also increase the specific [3H]spiperone binding, but to a smaller extent. At the concentrations of Ca2+, Mn2+, Mg2+ and Zn2+ which produce a maximal increase in the [3H]spiperone binding, the membranes are nearly saturated with the cations which bind about 100 nmoles of Ca2+ or Mg2+/mg of protein, 170 nmoles Zn2+/mg of protein and at least 300 nmoles Mn2+/mg of protein. It is suggested that the cations increase the [3H]spiperone binding by either exposing more binding sites, by preventing denaturation or by increasing the solubility of [3H]spiperone in the membrane phase, or by a combination of these processes.

Influence of chronic lithium administration on binding to benzodiazepine- and histamine H1-receptors in rat brain.

In rat, chronic lithium treatment lowered the Kd of [3H]-mepyramine in midbrain, and reduced the Bmax in midbrain and pons-medulla. Binding of [3H]flunitrazepam in cerebellum and midbrain was not altered. The suggestion that some actions of lithium may occur by way of central nervous system receptors is further supported by these observations that lithium's effects on neurotransmitter systems are regional and specific.

Lithium: effect on [3H]spiperone binding, ionic content, and amino acid levels in the brain of rats.

After prolonged treatment of rats with lithium (pellets, 0.21% lithium carbonate, or 0.5 mg/ml lithium chloride in drinking water) for three months, the level of lithium in plasma was 0.87 meq/liter; in several brain regions, between 1.06-1.39 mueq/g wet weight. The content of sodium and potassium inthe plasma was normal. The level of potassium in the brain regions tested increased by 13-30% and that of sodium by about 10%. Glycine levels increased significantly in all the regions (cerebral cortex, midbrain, cerebellum, and spinal cord). In the cerebellum GABA was also increased, while glutamine was decreased. In midbrain, apart from increases in glycine levels, alanine, valine, GABA and lysine were also increased. In the spinal cord, glutamic acid was also increased. Changes were largely in the putative neurotransmitters. Long-term treatment with lithium also influenced the high-affinity binding of [3H] spiperone in the cerebral cortex and corpus striatum. Two specific binding sites were found in both brain regions; the main change was the reduction in the lower affinity binding site (B max 2).

Effect of lithium and sodium ions on opiate- and dopamine-receptor binding.

The effects of lithium and sodium were studied in the corpus striatum and cerebral cortex of rats. Lithium was inhibitory at low concentrations but at 20 mM it increased the binding of [G-3H]naloxone (specific activity 15.6 Ci/mmol). Sodium stimulated the high-affinity binding of this compound. Membranes obtained from the rats treated with lithium showed lower specific binding of both [3H]naloxone and [3H]DHM. Binding of [3H]d-alanine Leu-enkephalin was not changed in the brains of lithium-treated rats, but that of [3H]-spiroperidol was lowered. Cerebral cortex and striatum of lithium-treated rats had a decreased apparent dissociation constant and a lower receptor concentration of naloxone binding sites.

Distribution of opiate-like substances in rat tissues.

Rat tissues were tested for their ability to inhibit the binding of [3H]dihydromorphine or [3H]naloxone to membrane-bound opiate receptors. By this criterion, morphine-like substances were found in lung, heart, liver, and kidney as well as in brain. The relative activity of the extracts, based on initial tissue weight, differed with the radioactive lignand employed. With dihydromorphine, the order was as above. With naloxone, lung was most active, followed by heart, brain, liver, and kidney. The ability of all tissue extracts to inhibit opiate binding was reduced by 100 mM NaC1 and slightly reduced by 1 mM MnC1(2). Gel filtration using Sephadex G-25 indicated that the inhibitory substances were heterogeneous in molecular weight. Only with brain and kidney extracts was there significant activity at the elution volume where enkephalins would be expected. Fractionation using Amberlite XAD-2, a resin which selectively absorbs hydrophobic materials, again indicated that the major protion of activity in all tissue extracts was due to substances other than enkephalins.

The effects of morphine on the accumulation of homovanillic and 5-hydroxyindoleacetic acids in the choroid plexus of rats.

1 Choroid plexus obtained from the lateral ventricles of the rat actively accumulated homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA). 2 Morphine 5 X 10(-6) M to 5 X 10(-4)M potentiated 5-HIAA accumulation but did not affect HVA accumulation. Levorphanol and dextorphan had little effect. 3 Naloxone at high concentrations inhibited both HVA and 5-HIAA accumulation. 4 Glutamic acid, glycine, and arginine also decreased 5-HIAA accumulation, but lysine, tryptophan, and aspartic acid had no effect. 5 Probenecid, naloxone, arginine, glycine, and tryptophan blocked the increase of 5-HIAA accumulation induced by morphine. 6 Acute or chronic morphine treatment did not increase the accumulation of 5-HIAA. 7 These results suggest that the increase of 5-HIAA or HVA in brain by morphine is not due to the inhibition of the elimination of these metabolites from the choroid plexus.

The influence of 3,4-dihydroxyphenylacetic acid on the accumulation of 5-hydroxyindoleacetic acid in the choroid plexus and kidney cortex slices of rats.

In vitro, the choroid plexus of rats accumulates 5-hydroxyindoleacetic acid (5-HIAA) by active transport. In the experiments presented here, the kidney cortex slices also showed active accumulation of this organic acid, which proved to be inhibited by most of the organic acids tested. In the choroid plexus 3,4-dihydroxyphenylacetic acid (DOPAC), a metabolite of dopamine, stimulated the accumulation of 5-HIAA, whereas in the kidney slices DOPAC was inhibitory. This stimulating effect of DOPAC was blocked by homovanillic acid and probenecid. Metabolic inhibitors such as 2,4-dinitrophenol and N-ethylmaleimide also blocked the stimulation, but sodium fluoride was ineffective. Omission of calcium, but not magnesium ion in the incubation medium depressed the accumulation of 5-HIAA. DOPAC still produced the stimulating action in calcium-free medium. The release of 5-HIAA from choroid plexus was retarded by DOPAC. These results suggest that the stimulating action of DOPAC may be due to a calcium-dependent active transport system and delay of 5-HIAA release.

The amphetamine-induced inhibition of dopamine biosynthesis in rat striatum.

The dopamine biosynthetic machinery of intact synaptosomes of rat striatum showed a 5-fold increase in development from 3-day-old neonates to adults, and it was fully developed between 2-3 weeks after birth. Concurring with this development was the appearance 2 weeks after birth of a regulatory mechanism(s) through which amphetamine in vivo induced an inhibition of dopamine biosynthesis. The inhibition was not appreciably reversed when haloperidol, in addition to amphetamine, was administered.

Isotopic labeling of synaptosomes that accumulate choline and the effect of narcotic drugs.

Subcellular studies of choline uptake of rat striatum indicated a correspondence between the Na(+)-dependent uptake and choline acetyltransferase (ChAc), whereas there was a lack of correspondence between the Na(+)-independent uptake and ChAc. Subcellular studies also showed a correspondence between the Na(+)-dependent uptake and hemicholinium-3 inhibition, and more important, particles that accumulate choline were shown to consist of at least two subcellular populations. A comparison was made of kinetic data from three areas of the rat brain: corpus striatum, cerebral cortex, and hypothalamus. Taken together, our data on choline uptake give added support to the idea that the Na(+)-dependent choline transport is concentrated in the striatum and specifically related to cholinergic nerve endings. Morphine and methadone in vitro inhibited the Na(+)-dependent choline uptake. In vivo morphine induced a significant lowering of theV max in the rat cerebral cortex, but not in the striatum. This finding is consistent with the known action of morphine on acetylcholine turnover.

Morphine-induced kinetic alterations of choline acetyltransferase of the rat caudate nucleus.

1. In order to explain the decrease of choline acetyltransferase (2.3.1.6.) activity observed in the caudate nucleus of morphine-treated rats, partially purified preparations of the enzyme were used in kinetic studies, with choline as substrate.2. The apparent Michaelis constant for the enzyme obtained from normal rats was found to be 0.9 mM choline; this value doubled when the animals were killed one hour after a single injection of morphine (30 mg/kg). When the rats were injected daily for 4 or 15 days, and killed one hour after the last injection, the apparent Km value was 2.1 mM in each case. Prolonged daily treatment with morphine, followed by 48 h withdrawal, or by administration of 4 mg/kg of naloxone (given half an hour after the last injection of morphine) resulted in apparent Km values of 1.3-1.5 mM of choline, suggesting a gradual return to the lower, normal substrate requirement. Vmax changes were insignificant.3. The effect of morphine added in vitro to different enzyme preparations was also studied. The Km values of 0.9 mM, in the enzyme isolated from normal rats, increased to 2.0 after incubation in vitro with 12.5 mM morphine. Similar increases were found in enzymes obtained from rats 48 h after the withdrawal of morphine or from rats injected with naloxone after prolonged morphine treatment. The high apparent Km values, found in enzyme obtained from animals killed one hour after the last dose of morphine, did not change upon incubation with 12.5 mM morphine. A similar pattern of Km changes was noticed after incubation with 25 mM acetylcholine.4. An increase of 32% in acetylcholine (ACh) level was found in the caudate nucleus one hour after subcutaneous injection of 30 mg/kg of morphine. Return to normal values was observed when morphine was administered daily. After two to three weeks of daily treatment and subsequent withdrawal from morphine for 48 h, the levels of ACh were normal. If the daily treated rats were given naloxone within half an hour of the last injection of morphine, and killed 30 min later, the levels of ACh remained normal.5. Fifty per cent inhibition of enzyme activity was observed upon in vitro incubation with 75 mM acetylcholine, or with 25 mM morphine. The same degree of inhibition was noticed when the enzyme was obtained from normal or from morphine-treated rats.