Taurine in toad brain and blood under different conditions of osmolality: an immunohistochemical study.

The concentrations of taurine in blood and brain regions of the toad Bufo boreas have been measured. Most of these values are considerably lower than those found in mammals. Using an antibody prepared against conjugated taurine, the distribution of taurine in three brain regions of the toad has been visualized. The possible osmoregulatory functions of taurine have been investigated by making toads hyper- or hypo-osmotic in vivo. Induction of hypoosmolality is accompanied by a massive taurine tide in blood plasma, but has no immediate effects upon the taurine concentrations in the brain areas studied. However, histochemical visualization indicates a marked redistribution of taurine between cellular components and extracellular space of brain tissues. This may indicate that taurine has an osmoregulatory function in brain tissue under hypo-osmotic conditions. Hyperosmolality results in no elevation of the taurine concentration in blood plasma of toads, but rather in a very gradual decline of total plasma taurine content over a prolonged time period. Histochemical studies reveal little change in frontal cortex after 1 hour but deeper staining of many neurons in optic lobe accompanied by greater staining in the extracellular fluid. By 3 hours there is a depletion of taurine from all compartments of cerebral cortex tissues. No evidence of any prolonged direct osmoregulatory role for taurine is indicated under hyperosmotic conditions. A possible indirect osmoregulatory function of taurine is discussed.

GABA metabolism in the substantia nigra, cortex, and hippocampus during status epilepticus.

The metabolism of GABA and other amino acids was studied in the substantia nigra, the hippocampus and the parietal cortex of rats following microinjections of GAMMA-vinyl-GABA during status epilepticus induced by lithium and pilocarpine. GABA metabolism showed striking regional variations. In controls, both GABA concentration and rate of GABA synthesis were highest in the substantia nigra and lowest in cortex, as expected. In substantia nigra, status epilepticus resulted in a 2 1/2 fold decline in the rate of GABA synthesis and in a 307% increase in the turnover time of the GABA pool. In hippocampus, the rate of GABA synthesis was not altered significantly, but the turnover time of the GABA pool was 284% of controls, and the size of that pool increased to 208% of controls. By contrast, in cortex, where seizure activity is limited in this model, the rate of GABA synthesis increased to 230% of controls while pool size and turnover time did not change. Aspartate concentration decreased in all three brain regions. These data suggest that the observed reduction of the rate of GABA synthesis in substantia nigra could play a key role in seizure spread in this model of status epilepticus.

Regulation of fatty acid beta-oxidation in rat heart mitochondria.

In an attempt to elucidate the mechanism by which the rate of fatty acid oxidation is tuned to the energy demand of the heart, the effects of changing intramitochondrial ratios of [acetyl-CoA]/[CoASH] and [NADH]/[NAD+] on the rate of beta-oxidation were studied. When 10 mM L-carnitine was added to coupled rat heart mitochondria to lower the ratio of [acetyl-CoA]/[CoASH], the rate of palmitoylcarnitine beta-oxidation, as measured by the formation of acid-soluble products, was stimulated more than fourfold at state 4 respiration while beta-oxidation at state 3 respiration was hardly affected. Neither oxaloacetate nor acetoacetate, added to mitochondria to lower the [NADH]/[NAD+] ratio, stimulated beta-oxidation. Rates of respiration at states 3 and 4 were unchanged by additions of L-carnitine, oxaloacetate, or acetoacetate. Determinations of intramitochondrial ratios of [acetyl-CoA]/[CoASH] by high performance liquid chromatography yielded values close to 10 for palmitoylcarnitine-supported respiration at state 4 and 2.5 at state 3 respiration. Addition of 10 mM L-carnitine caused a dramatic decrease of these ratios to less than 0.2 at both respiration states. Studies with purified or partially purified enzymes revealed strong inhibitions of 3-ketoacyl-CoA thiolase by acetyl-CoA and of L-3-hydroxyacyl-CoA dehydrogenase by NADH. Moreover, the activity of 3-ketoacyl-CoA thiolase at concentrations of acetyl-CoA and CoASH prevailing at state 3 respiration was 4 times higher than its activity in the presence of acetyl-CoA and CoASH observed at state 4. Altogether, this study leads to the conclusion that the rate of beta-oxidation in heart can be regulated by the intramitochondrial ratio of [acetyl-CoA]/[CoASH] which reflects the energy demand of the tissue. The thiolytic cleavage catalyzed by 3-ketoacyl-CoA thiolase may be the site at which beta-oxidation is controlled by the [acetyl-CoA]/[CoASH] ratio.

Temperature alters ethanol-induced fluidization of C57 mouse brain membranes.

The interaction between temperature and ethanol-induced fluidization was investigated in brain synaptic plasma membranes from C57BL/6 mice. Changes in fluidity were measured using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. Fluorescence polarization was tested in the presence and absence of ethanol at 25, 32 and 37 degrees C. An increase in temperature resulted in a significant increase in the baseline fluidity of the membranes and an increase in the magnitude of ethanol-induced fluidization of brain membranes. The combined effect of temperature on baseline fluidity and the magnitude of the response to ethanol resulted in a significant temperature-related increase in the relative response to ethanol (% change in polarization). The minimum concentration of ethanol required to cause a significant increase in the fluidity of the membranes was 170.7 mM at 25 degrees C and 85.3 mM at both 32 and 37 degrees C. The present results indicate that temperature-related changes in the effects of ethanol on membrane properties may underlie the effects of temperature on ethanol sensitivity in C57 mice.

Alterations of GABA metabolism and seizure susceptibility in the substantia nigra of the kindled rat acclimating to changes in osmotic state.

Seizure susceptibility and GABA metabolism were altered in the substantia nigra [SN] of adult male Sprague Dawley rats when these animals were acclimating to an altered plasma osmolality. Changes in GABA metabolism were measured in vivo in SN of the freely moving rat. Suitable precautions were taken to avoid any post-mortem flux of glutamate to GABA and to correct for the underestimation of GABA build up in SN due to the finite diffusion rate of gamma-vinyl GABA [GVG] after stereotaxic injection of small amounts into one side of the brain. Control experiments provided evidence that changes in osmolality, within a normal physiological range, did not affect significantly gamma-aminobutyric acid transaminase [GABA-T]. Also kindling via the medial septum [MS], in the absence of electrical stimulation did not alter GABA metabolism in SN, thus providing a stable baseline for studies of osmotic effects. Hyperosmolality was associated with a rise in seizure thresholds, with a marked reduction of the rate of GABA synthesis in SN, and with a substantial increase in turnover time of the GABA pool. Hypoosmolality, of a degree known to be associated with mild cerebral edema and swelling localized to astrocytes, markedly reduced seizure threshold, and reduced GABA pool size in SN, but did not alter the rate of GABA synthesis significantly. These results demonstrate by new and independent means the relationship between GABA metabolism in the SN and seizure susceptibility in vivo.

Changes of amino acid gradients in brain tissues induced by microwave irradiation and other means.

Focused microwave irradiation to the head (FMI) has been used extensively by neurochemists for rapid inactivation of enzymatic activity in brain tissues and the preservation, for in vitro analysis, of in vivo substrate concentrations. Periodically the suitability of this technique for regional studies has been questioned. Evidence has now been obtained, on the basis of altered concentration gradients for GABA and taurine from the Substantia Nigra (SN) to an Adjacent Dorsal Area (ADJ), that FMI not only inactivates enzymes, but also facilitates rapid diffusion of small molecules from areas of high concentrations to adjacent areas of lower concentration. To a lesser extent, the implantation of plastic injection cannulas also decreased these concentration gradients. These results offer clear evidence that FMI is ill suited and unreliable for studies designed to map and compare the "in vivo" regional concentrations of diffusible organic molecules (such as amino acids) in brain tissues. Any invasive technique that compromises membrane barriers is likely to produce smaller similar effects.

Long-term oral administration of memory-enhancing doses of tacrine in mice: a study of potential toxicity and side effects.

Recently, tacrine (1, 2, 3, 4-tetrahydro-9-aminoacridine; THA; TAC) has received international attention as an oral agent capable of relieving some of the cognitive symptoms accompanying Alzheimer's disease (AD). When given acutely and parenterally (by injection), tacrine has also enhanced memory retention in animals and man. This study evaluates the clinical potential of this agent by assessing toxicity and major side effects of a memory-enhancing dose of tacrine in mice. Groups of mice received either tacrine or vehicle (placebo) orally for 4 to 6 months. A lack of toxicity after this prolonged treatment with TAC was indicated by: (a) no significant impairment on a battery of behavioral toxicity tests; (b) improved memory retention; (c) a significant but only slight elevation of ornithine transcarbamylase activity in blood serum; (d) no abnormality as revealed with light microscopy of liver tissue; and (e) no gross organ pathology in visceral organs.

Modulation of serotonin receptors by specific phosphatidylcholines.

Treatment of mouse cortical brain membranes with dioleoylphosphatidylcholine produced a large (50%) decrease in serotonin binding sites. The time course for this effect paralleled an increase in oleic acid in membrane phosphatidycholine and an increase in membrane fluidity. "Active Lipid" produced a similar decrease in serotonin binding sites, while fluidizing the membranes even more strongly. Distearoylphosphatidylcholine had no effect on serotonin binding sites or membrane fluidity by itself, but was capable of counteracting both the reduction in binding sites and membrane fluidity produced by "Active Lipid". The data indicate that specific phosphatidylcholines can have profound effects on serotonin receptors, but a clear picture of the relative importance of membrane fluidity per se versus more specific phospholipid effects will require further investigation.

Effect of altered blood plasma osmolalities on regional brain amino acid concentrations and focal seizure susceptibility in the rat.

Blood plasma hypo- or hyperosmolality alters significantly the concentration of some amino acids in brain tissues of the medial septum and hippocampus of adult Sprague-Dawley rats. With some notable exceptions, brain amino acid concentrations decreased under hypoosmotic conditions and increased under hyperosmotic conditions. Osmotic changes and brain amino acid changes appear to be related to each other in an almost linear fashion. A comparison of rats and toads indicates that the patterns of changes in brain amino acid concentrations in response to a hypoosmotic plasma osmolality were almost identical for both species. Changes achievable under hyperosmotic conditions were considerably greater in toads. When rats with kindled epileptogenic foci were made hypoosmotic by water-loading, seizure thresholds decreased dramatically. Our data suggest a possible relationship between the hypoosmotically induced biochemical changes in brain tissues (especially some amino acid neurotransmitters and neurotransmitter precursors) and the hypoosmotically induced increase in seizure susceptibility.

Abnormal feeding behavior of western toads (Bufo boreas) kept in a hyperosmotic environment. I. A quantitative behavioral analysis as related to cerebral amino acid concentrations.

Discriminant analysis of eleven behavioral variables associated with feeding permitted the assignment of hyperosmotically-acclimating (HOA) toads (Bufo boreas) to six different behavioral states. These behavioral states could be correlated with specific alterations in the level of select amino acids in three regions of the toad's central nervous system. By considering only those amino acids that showed equivalent levels in corresponding brain regions of normally-behaving, freshwater-acclimating (FWA) and HOA toads, it was possible to focus upon just nine amino acids as possible modulators of feeding behavior. Four of these amino acids were markedly elevated in two or more abnormal behavioral states: gamma-aminobutyric acid (GABA) in all three brain areas; glutamate in the cerebral hemispheres; aspartate in the cerebral hemispheres and olfactory bulbs; and phenylalanine in the olfactory bulbs and optic lobes. Other possible behavior modulators identified were: lysine and threonine in the cerebral hemispheres; carnosine in the olfactory bulbs; and valine and alanine in the optic lobes.

High proline levels in the brains of mice as related to specific learning deficits.

Hyperprolinemic PRO/Re mice have been studied as potential models for hyperprolinemia in man. In addition to high proline levels, some heretofore unreported amino acid abnormalities in the brains of PRO/Re mice are described. The T-maze and shuttlebox learning abilities of PRO/Re mice were compared with those of CD-1 mice having normal proline levels. PRO/Re mice had a significant deficit for T-maze learning, but a significantly greater aptitude for shuttlebox learning when compared to CD-1 mice. By studying the F3 progency of the PRO/Re X CD-1 cross, these strain-specific differences in learning ability for different tasks were shown to be unrelated to the differences in brain proline levels. F3 mice could be subdivided into two distinct groups: those with high proline (HP+) and low proline (HP-) titers. Other amino acids in brain tissues were essentially identical in both groups. A comparison of learning abilities of these HP+ mice with their HP- littermates showed no meaningful differences. However, the slightly slower rate at which HP+ mice acquired shuttlebox learning was sufficiently consistent over the 8 day training period so that it became significant. These results do not support the hypothesis that high levels of proline in brain tissues and blood are necessarily accompanied by impaired learning and memory, but are in agreement with those studies of hyperprolinemia in man that suggest no consistent learning deficits in hyperprolinemic subjects. The results seem to validate the suitability of the PRO/Re mouse as a model for hyperprolinemia in man. The data suggest also that the altered amino acid pattern in brains of PRO/Re mice has multiple etiologies.(ABSTRACT TRUNCATED AT 250 WORDS)

Proline in the cerebrospinal fluid of normal subjects and Alzheimer's-disease patients, as determined with a new double-labeling assay technique.

Past studies have implicated proline involvement in the function of memory and learning. A new micromethod has been developed that is suitable for measuring proline accurately in as little as 0.1 ml of CSF. In normal human CSF, the average proline level was found to be consistently about 1.3 microM. In the CSF of patients with Alzheimer's disease and mixed dementias, the levels of proline showed no statistically significant difference from proline levels in the CSF of normal controls. Furthermore, the proline levels in the CSF of the Alzheimer's disease patients did not reflect, consistently, the cognitive deficits or the symptomatic severity of the disease. Proline levels in CSF showed no statistically significant change with the age of individuals tested.

Inhibitors of crayfish glutamic acid decarboxylase.

Crayfish glutamic acid decarboxylase (GAD), like the homologous enzymes from other species, is inhibited by carbonyl-trapping agents (e.g. aminooxyacetic acid; AOAA) and sulfhydryl reagents (e.g. 5,5'-dithiobis-(2-nitrobenzoic acid); DTNB). It also is inhibited by the product GABA, many anions (e.g. SCN- and Cl-), and some cations (e.g. Zn+2). The inhibition by AOAA, but not that by DTNB, was prevented by increasing the concentration of the pyridoxal phosphate (PLP) coenzyme. GABA blocked the effects of PLP on enzyme activity. The inhibition by AOAA, DTNB, GABA, and chloride all were competitive with substrate. The effect of GABA occurs at physiological concentrations and may contribute to the regulation of GAD activity in vivo. The quantitative effect of anions is dependent on the cation with which they are administered. ATP stimulated GAD activity in homogenates prepared with potassium phosphate or Tris-acetate buffer, even when no exogenous PLP was provided.

Assay and properties of glutamic acid decarboxylase in homogenates of crayfish nervous tissue.

The activity of glutamic acid decarboxylase (GAD) was measured in homogenates of crayfish nervous tissue. Radioactive GABA and CO2 were formed from radioactive glutamic acid in approximately equimolar amounts. Product formation was linear for 9.5 hr at 11-32 degrees C with about 1-30 micrograms homogenate protein. Enzyme activity remained high at pH 7-10 but declined steeply above pH 10.5 and below pH 7. Enzyme activity was stimulated by pyridoxal phosphate, 2-mercaptoethanol, and potassium phosphate; at higher than optimal concentrations of each the activity was reduced. Sodium phosphate altered the stimulatory effect of potassium phosphate. Crayfish GAD behaves like a typical neural GAD but is distinguishable biochemically from GAD of other species.

Rapid visualization of proteins in isoelectric focusing gels: isolation of brain tubulin subspecies.

A method for visualization of unmodified proteins in polyacrylamide isoelectric focusing (IEF) gels is described. The proteins appear as white, translucent bands when disc IEF gels are placed in water. The gel can be scanned with a scanning spectrophotometer, or the protein bands can be excised using a simple apparatus, which is described, and the protein eluted. The method is fast, selective for proteins, sensitive, and quantitative. It has been used to isolate tubulin species separated by as little as 0.5 mm in disc IEF gels.

L-Proline inhibition of glutamate release.

The influence of 1 mM-L-proline on electrically stimulated release of endogenous glutamate from slices of rat frontal cortex was measured. One mM-L-proline approximates the in vitro concentration that inhibits glutamate-induced spreading depression. Also, this L-proline level has been reported to be present in brain after an intraperitoneal injection that induced amnesia in chicks. L-Proline but not D-proline exhibits an inhibitory effect on glutamate release, thus supporting the suggestion that this mechanism may play a role in memory retention and/or formation.

Brain tubulin microheterogeneity in the mouse during development and aging.

Mouse brain tubulin was analyzed on isoelectric focusing gels. High-resolution gels utilizing Bio-Rad ampholytes (pH 4--6) revealed 5--6 bands in the region corresponding to the alpha-subunit of tubulin and 10 or more for the beta-subunit. The same general banding pattern was observed regardless of the method of preparation of the tubulin. Two species prominent in the brains of immature mice, alpha 6 and beta 2, virtually disappeared during maturation, while species beta 6 to beta 10 appeared. No significant changes from the mature pattern were seen during aging (examined at 12, 23, and 30 months of age).