Miriam Banay-Schwartz

EFFECTS OF PRENATAL ADMINISTRATION OF NICOTINE ON AMINO ACID POOLS, PROTEIN METABOLISM, AND NICOTINE BINDING IN THE BRAIN

The effects of nicotine on brain protein metabolism and on the properties of the nicotine binding site were investigated in newborn animals exposed to nicotine during gestation. Brain protein synthesis rates measured in vivo were lower by 18% in newborn of treated animals. Protein degradation rates measured in vitro in the presence of nicotine were lower by 13%. The effect was specific forl-(-) nicotine, sinced-(+)nicotine, nicotinic acid, or nicotinamide had no effect on degradation rates. Newborn brain amino acid levels, mainly nonessential amino acids and amino acids of putative neurotrans nitter function, were changed some-what; an increase in the level of taurine (13%), threonine (21%), serine (35%) and glycine (35%), and a decrease in lysine (14%) was observed in the offspring of nicotine treated animals (0.5 mg/kg, s.c., 2×daily throughout gestation). These changes could not account for the decrease in protein metabolism. Nicotine binding was higher by 25% in the offspring of animals exposed to nicotine during gestation. No such increase was found after treatment of adult rats with nicotine, indicating that the properties of the nicotine binding site change with age.

Relationship of ATP levels to amino acid transport in slices of mouse brain

Abstract Slices of brain tissue upon incubation accumulate amino acids against a concentration gradient. The relationship of this accumulation to the ATP levels in the tissue was investigated. Freshly prepared slices were shown to contain levels of ATP that were decreased from in vivo levels but were restored within 30 min incubation to levels close to that in the living brain. Increasing ATP levels further had no effect on amino acid uptake, while metabolic inhibitors that decreased the content of ATP in the slice also inhibited the uptake of amino acids. In general, inhibitors that caused larger ATP decreases also inhibited uptake more, but there were differences among the amino acids; using the same metabolic inhibitor, or lowering ATP to the same level, did not inhibit the uptake of all amino acids to the same extent. Ouabain also inhibited uptake and decreased ATP and K + levels; restoring ATP and K + levels in the presence of ouabain did not restore amino acid uptake; ouabain inhibition was not reversible. The inhibition by cyanide was reversible if the slices were transferred to CN-free medium; uptake was restored more than ATP. Although under a number of experimental conditions decrease in ATP levels and decrease in the concentrative uptake of amino acids were parallel, it was possible to show a high level of uptake in the presence of low ATP levels and also strong inhibition of uptake in the presence of high ATP levels. These findings suggest that ATP levels do not directly determine uptake, or even that ATP is not the primary energy supply for transport.

The Distribution of Cathepsin D Activity in Adult and Aging Human Brain Regions

Abstract: We measured the activity of cathepsin D, the major cerebral protease, in 50 separate areas of the central nervous system of adult and aged humans, using hemoglobin as the substrate. The activity showed significant regional heterogeneity, with average differences of 50–100% between the lower and higher level areas, and a more than threefold difference between the lowest and highest levels. The forebrain, midbrain, and hindbrain each had areas of high and low activity; cerebellum and cord areas were among those with low activity. Cathepsin levels tended to increase with age in about half of the areas analyzed, and the increases were significant in 14. Statistically significant decreases with aging were observed in two areas. The increases varied between 30 and 60%, and the decreases were 20%. Enzyme activity in thalamus, hypothalamus, pons, medulla, and cerebellum increased with age. In the ventrolateral medulla, which contains the major portion of the cerebral noradrenergic cells, the cathepsin D levels increased with age; in the dorsal raphe area, which contains the major portion of the cerebral serotonergic cells, the enzyme levels decreased. The change with age in human brain seems to be less than what we observed in rat brain, where activity more than doubled in most areas. The changes in enzyme levels need to be tested at more ages to establish a pattern of changes in activity throughout life.

Heterogeneous Distribution of Functionally Important Amino Acids in Brain Areas of Adult and Aging Humans

The regional distribution of seven amino acids thought to have inhibitory neurotransmitter or neurotransmitter precursor function—GABA, glycine, taurine, serine, threonine, phenylalanine, and tyrosine—was determined in 52 discrete areas from brain of adult and old humans. Significant heterogeneity was found, with 3- to 16-fold differences in levels in the various regions analyzed. The patterns of distribution were somewhat different from those in the adult or old rat brain. Relatively few changes were seen in old brain. Heterogeneity in distribution has to be taken into account in assessing physiological changes in amino acid levels and metabolism.

Independence of amino acid uptake from tissue swelling in incubated slices of brain

Abstract Brain slices take up water during incubation, increasing the wet weight of tissue up to 50%. Metabolic inhibitors further increase this swelling process. The presence of 4% Dextran-80 (80,000 mol.wt.) or polyethylene glycol (4000 mol. wt.) in the incubation medium decreases swelling and reduces this effect of metabolic inhibitors. Dextran and polyethyleneglycol (PEG) reduce extracellular marker space to a smaller degree than water uptake. Therefore, intracellular swelling is also inhibited. During incubation of brain slices, sodium levels increase and potassium levels decrease, while ATP does not change. The ion changes are also reduced by Dextran and PEG. Amino acid uptake by the slices is not affected by Dextran or PEG. When amino acid uptake is partially inhibited by lack of glucose or sodium in the medium, swelling is greater than in the control media. This swelling is decreased below that of the uninhibited controls by Dextran, but the inhibition of amino acid uptake is not affected. We conclude that amino acid uptake is independent of intracellular and to some degree also of extracellular swelling. Increased uptake in slices as compared to brain in vivo is probably due to a change in control mechanisms for cerebral homeostasis. Our results indicate that this cannot be explained by shifts in the cellular water compartments. Previous work indicated that possible shifts in ion distribution and ATP levels also cannot be responsible for the increased uptake in slices. These results support hypotheses in which mechanisms that control the composition of the extracellular compartment play key roles in cerebral homeostasis.

Amino acid analysis using 1-naphthylisocyanate as a precolumn high performance liquid chromatography derivatization reagent

A method which uses 1-naphthylisocyanate as an HPLC precolumn derivatization reagent for amino acid analysis is described. Derivatization is carried out by adding the isocyanate dissolved in dry acetone to a buffered amino acid solution followed by extraction of the excess reagent with cyclohexane. The resulting naphthylcarbamoyl amino acids are stable and highly fluorescent, with excitation maxima at 238 and 305 nm and an emission maximum at 385 nm, for most amino acids. Ultraviolet detection near 222 nm, the absorption maximum, can also be employed. HPLC procedures permitting the analysis of protein hydrolysates, brain extract, cerebrospinal fluid, and blood plasma are presented. The method is particularly suitable for auto-sampler procedures since samples can be derivatized and diluted in advance and stored at room temperature in the sampler while awaiting injection. Other advantages include high sensitivity, the possibility of recovering the derivatives from the column effluent, and the absence of a reagent peak in the chromatograms.

Calpain activity in adult and aged human brain regions

We assayed calpain activity in 27 human brain regions from adult (43–65 years of age) and aged (66–83 years of age) postmortem tissue samples. Calpain I (μM Ca-requiring) activity was 10% or less of the total activity; it was below detectable levels in a number of areas, and so data are are expressed as total (μM+mM Ca-dependent) calpain activity. The distribution of the enzyme was regionally heterogeneous. Highest activity was found in the spinal cord, followed by the amygdala, and levels in mesencephalic areas and in cerebellar grey matter were also high. Levels in cerebellar white matter, tegmentum, pons, and putamen were low, and activity in cortical areas was also relatively low. Although in some areas activity seemed higher with aging, the differences were not statistically significant. We previously found that the regional distribution of cathepsin D in human and in rat brain is similar, this seems to be true for calpain activity as well. The increase of protease activity with age found in rat brain is not found in human areas, as was shown previously with cathepsin D, and in the present study with calpain.

Nucleoside Uptake by Slices of Mouse Brain

: The properties of the uptake of nucleosides and nucleotides by brain cells were examined in slices of mouse brain. Of the compounds tested, adenine and adenosine had the most rapid uptake and reached the highest levels. Uptake was mediated, as shown by saturability and strong inhibition, by low temperature, or by cyanide, and was only partially sodium‐ or calcium‐dependent. The inhibition pattern by analogues indicated the presence of several uptake systems (possibly four), as shown by differences between adenine and guanine uptake, between adenine and adenosine uptake, and between adenosine and cytidine uptake. The properties of uptake systems for nucleotides and nucleosides were somewhat different from those for amino acids.

Changes in Brain Protease Activity in Aging

Abstract: We measured changes in protease activity with aging, conducting assays of cathepsin D and calpain II activities and the rate of degradation of cytoskeletal proteins, preparing the enzymes and substrates from young and aged brains. Calpain preparations added to the young and to the aged substrates were standardized with casein as substrate so that age‐related changes in calpain specificity and substrate susceptibility were measured. Several age‐related differences were observed in substrate susceptibility and in enzyme activity. With respect to substrate, the neurofilament protein from young animals was somewhat more susceptible to calpain action than that from older animals. With respect to enzyme activity, calpain from aged brain cleaved neurofilament protein at a faster rate than did calpain from young. With neurofilaments, the most rapid breakdown usually occurred when enzyme from aged tissue was incubated with substrate from young. Kidney enzyme of aged rats incubated with neurofilament substrate of aged rats resulted in a more rapid breakdown than enzyme of young kidney incubated with substrate of young. The age dependence of tubulin breakdown was somewhat different from that of neurofilament breakdown. The most rapid breakdown usually occurred when using enzyme from young with tubulin from young. Incubation of neurofilament protein or tubulin with cathepsin D did not reveal any differences with aging. These studies suggest that an increase in enzyme activity observed previously during aging may also include changes in the properties of the enzyme (substrate specificity) and/or in the properties of their endogenous substrates (susceptibility to breakdown).

Effect of food deprivation on glutathione and amino acid levels in brain and liver of young and aged rats

The effect of short-term food deprivation on glutathione (GSH) and amino acid levels in brain regions of young and aged rats was compared with changes observed in liver. Animals aged 3 months and 24 months were deprived of food for 48 h. GSH and amino acid levels from cerebral cortex, cerebellum, pons medulla, and liver were assayed and compared with levels in animals of the same age fed normal diets. In liver in both young and old rats, GSH levels fell 30%, from 13 mumol/g tissue to 8.7 mumol/g tissue. Significant changes were observed in other amino acids, including an increase of 30-50% in methionine, glycine, and glutamine, and a decrease of 30-50% in alanine in liver of both young and aged rats, and a 4-fold increase in taurine in young. In brain, little change was observed upon food deprivation. No decrease was observed in GSH, and only small changes were observed in other amino acids. In the aged animal aspartate, glutamate, and alanine levels were slightly lower; tyrosine in cerebellum was reduced by 30%, and both glycine and tyrosine in the pons medulla were reduced by 20-30%. In the brain areas examined, levels of GSH ranged from 1-2 mumol/g in young and 0.8-1.4 mumol/g in old; with levels in pons medulla being lower than those in cerebral cortex. In brain, in contrast to liver, levels were scarcely affected by short-term food deprivation.(ABSTRACT TRUNCATED AT 250 WORDS)