TAURINE IN DEVELOPING RAT BRAIN: SUBCELLULAR DISTRIBUTION AND ASSOCIATION WITH SYNAPTIC VESICLES OF [35S]TAURINE IN MATERNAL, FETAL AND NEONATAL RAT BRAIN

The concentration of taurine in the brain of the fetus in several species is higher than that found in the mature animal. In order to explore the functional significance of this, we have studied the subcellular distribution of taurine and [³⁵S]taurine in the brain of the mother, the fetus and the neonate after [³⁵S]taurine was administered to pregnant rats. In maternal brain, the distribution of taurine and of radioactivity (all of which was recovered from brain as taurine) in the subcellular fractions of maternal brain were essentially identical and were recovered primarily in two fractions (72% taurine, 71% [³⁵S]taurine was soluble, S₃; 16% and 17%, respectively, was in the crude mitochondrial and synaptosomal fraction, P₂). After further fractionation of P₂, most of the taurine and [³⁵S]taurine were in the cytoplasmic, O, and the synaptosomal, B, fractions. In the neonatal brain, shortly after birth there was a decrease in taurine and [³⁵S]taurine recovered in the supernatant fraction, S₃, accompanied by an increase in the percentage of taurine and [³⁵S]taurine recovered in the crude mitochondrial fraction. A small percentage of taurine and [³⁵S]taurine was consistently recovered in the synaptic vesicle fraction. Fractionation of the synaptic vesicles on a gel column separated the vesicle bound taurine completely from the free taurine: approx 1% of the taurine in the synaptic vesicle fraction was eluted with vesicles and could not be released by hypo-osmotic shock. The pattern of development in subcellular fractions of neonatal rat brain labelled with [³⁵S]taurine via intraperitoneal injections of the pregnant mother may be an indication of maturation or protection of putative taurinergic nerve endings.     

Taurine in Developing Rhesus Monkey Brain

The concentrations of taurine in all regions of fetal and neonatal rhesus monkey brain are greater than in the same regions of adult monkey brain. [³⁵S]Taurine injected into pregnant rhesus monkeys is accumulated by the fetus. This process occurs rapidly in most tissues, but occurs slowly in fetal brain. Neonatal rhesus monkey brain also accumulates [³⁵S]taurine slowly compared with other tissues after i.v. injection, and continues to accumulate [³⁵S]taurine for a long period of time. These results suggest that the accumulation and exchange of taurine in developing rhesus monkey brain is slow, as found in neonatal rats, and that if there is a period of development at which rapid exchange of brain taurine occurs in the rhesus monkey, it is before the rapid brain growth spurt.     

Taurine deficiency in the kitten subcellular distribution of taurine and [35S]taurine in brain

Taurine concentration decreases rapidly in the tissues and physiological fluids of kittens fed a diet of partially purified casein which lacks taurine. We have studied the subcellular distribution in cerebrum of taurine and [³⁵S]taurine administered intravenously to these animals. The taurine concentration of all the fractions isolated from the cerebrum of taurine-deficient kittens was approximately sevenfold less than that observed in the fractions of cerebrum isolated from control kittens. The [³⁵S]taurine was approximately twofold greater in all the brain fractions isolated from the taurine-deficient kittens compared with those isolated from the control kittens. The percent distributions of taurine and [³⁵S]taurine in the fractions isolated from the cerebrum of control and deficient kittens were identical. Thus, in the face of a severe diet-induced deficiency of taurine in kitten brain, there appears to be no conservation of taurine by any particular subcellular pool of taurine. These studies provide no evidence for differences in compartmentation of taurine in cerebrum of taurine-deficient kittens compared with control kittens.     

TAURINE IN DEVELOPING RAT BRAIN: MATERNAL-FETAL TRANSFER OF [35S]TAURINE AND ITS FATE IN THE NEONATE

The transfer of [³⁵] taunne, injected intrapentoneally into pregnant rats (near term), to fetal tissues has been measured. Taurine can enter fetal brain as easily as it can fetal liver. In contrast, it cannot enter mature brain as easily as it can enter mature liver. After birth, [³⁵S] taurine, which had been injected into the dam before birth of the pups, continues to accumulate in the brain of the pups for some days. During the neonatal period, the concentration of taurine is decreasing, but the total pool of taurine in the brain is increasing rapidly. In order to help supply this increasing pool, the taurine present in the brain at birth appears to be conserved and an increasing amount of taurine is synthesized in situ. The net result during the neonatal period of development is that brain taurine specific radioactivity decreases and brain taurine has a very slow rate of turnover.     

Cyclic AMP-induced release of [14C]taurine from pinealocytes.

Pinealocytes were prelabelled with [¹⁴C]taurine. Twenty-four hours later they were treated with derivatives of cyclic AMP. It was found that dibutyryl cyclic AMP and ?-chloro-phenyl-thio cyclic AMP treatment caused a large increase in the release of [¹⁴C]taurine. The effect of dibutryl cyclic AMP on [¹⁴C]taurine release was near maximal fifteen minutes after treatment started. In view of the known stimulatory effects of norepinephrine on pineal cyclic AMP and the recent discovery that norepinephrine causes the release of taurine from pinealocytes, one can conclude that norepinephrine stimulates [¹⁴C]taurine release from pinealocytes by acting through a cyclic AMP mechanism.     

COMPARATIVE STUDIES ON THE DEGRADATION OF GABA and TAURINE IN THE BRAIN

Abstract— The degradation of taurine and GABA in mammalian brain was studied in vivo and in vitro. Small amounts of [³⁵S]isethionate (10–20 pmol/g brain wet weight) and [³⁵S]sulphate (about 2 pmol/g) were detected in mouse brain after intramuscular injection of [³⁵S]taurine. Taurine also produced isethionate in rat brain homogenates (about 20 nmol/h/g protein) and subcellular fractions (about 40 nmol/h/g protein in synaptosomes and about 300 nmol/h/g in mitochondria), but the reaction was not stimulated either by external electrical pulses or by the addition of various cofactors (NAD and NADP in both oxidized and reduced forms, riboflavin, glutathione. pyridoxal-5'-phosphate, ATP) to the incubation medium. [¹⁴C]GABA was readily metabolized to [¹⁴C]succinate both in vivo and in vitro. Isethionate formation activity was concentrated in the mitochondrial fraction, as was also GABA-T activity. Partially purified GABA-T from calf brain also slightly catalysed the formation of [³⁵S]isethionate (about 1.3 μmol/min/g protein) from [³⁵S]taurine. It appears that the slight formation of isethionate from taurine is coupled to GABA-T activity. The formation of isethionate from taurine is so small, that it apparently has no role in the control of the brain taurine pool.     

LABELLING OF BRAIN PROTEINS AT EARLY PERIODS AFTER SUBCUTANEOUS INJECTION OF A MIXTURE OF [U-14C]GLUCOSE AND [3H]GLUTAMATE

To obtain evidence of the site of conversion of [U-¹⁴C]glucose into glutamate and related amino acids of the brain, a mixture of [U-¹⁴C]glucose and [³H]glutamate was injected subcutaneously into rats. [³H]Glutamate gave rise to several ³H-labelled amino acids in rat liver and blood; only ³H-labelled glutamate, glutamine or γ-aminobutyrate were found in the brain. The specific radioactivity of [³H]glutamine in the brain was higher than that of [³H]glutamate indicating the entry of [³H]glutamate mainly in the ‘small glutamate compartment’. The ¹⁴C-labelling pattern of amino acids in the brain and liver after injection of [U-¹⁴C]glucose was similar to that previously reported (Gaitonde et al., 1965). The specific radioactivity of [¹⁴C]glutamine in the blood and liver after injection of both precursors was greater than that of glutamate between 10 and 60 min after the injection of the precursors. The extent of labelling of alanine and aspartate was greater than that of other amino acids in the blood after injection of [U-¹⁴C]glucose. There was no labelling of brain protein with [³H]glutamate during the 10 min period, but significant label was found at 30 and 60 min. The highest relative incorporation of [¹⁴C]glutamate and [¹⁴C]aspartate in rat brain protein was observed at 5 min after the injection of [U-¹⁴C]glucose. The results have been discussed in the context of transport of glutamine synthesized in the brain and the site of metabolism of [U-¹⁴C]glucose in the brain.     

Diffusion of intracerebrally injected [1-14C]arachidonic acid and [2-3H]Glycerol in the mouse brain

[2-³H]Glycerol and [1-¹⁴C]arachidonic acid were injected into the region of the frontal horn of the left ventricle of mice and were distributed rapidly throughout the brain. After 10 sec, most of the radioactive fatty acid was found in the hemisphere near the injection site; after 10 min, it was recovered in similar proportions in the cerebellum and brain stem. [2-³H]Glycerol showed a heterogeneous distribution, with most of the label remaining in the left hemisphere even after 10 min. On a fresh weight basis, cerebrum, cerebellum, and brain stem were found to contain similar amounts of labeled glycerol. However, the amount of [1-¹⁴C]arachidonate in cerebrum was only 50% of that recovered from cerebellum or brain stem. Brain ischemia or a single electroconvulsive shock reduced the spread of the label, producing an accumulation of radioactivity in the injected hemisphere, except for an increase in [2-³H]glycerol in the brain stem during ischemia. Despite the significant decrease in available precursor in the cerebellum and brain stem after electroshock, the amount of label incorporated into lipids was not altered in these areas and only slightly diminished in the cerebrum.     

The metabolism of [14C]nicotine in the cat

The metabolism of [2'-(14)C]nicotine given as an intravenous injection in small doses to anaesthetized and unanaesthetized cats has been studied. A method is described for the quantitative determination of [(14)C]nicotine and [(14)C]cotinine in tissues and body fluids. Nanogram amounts of these compounds have been detected. After a single dose of 40mug. of [(14)C]nicotine/kg., 55% of the injected radioactivity was excreted in the urine within 24hr., but only 1% of this radioactivity was unchanged nicotine. [(14)C]Nicotine is metabolized extremely rapidly, [(14)C]cotinine appearing in the blood within 2.5min. of intravenous injection. [(14)C]Nicotine accumulates rapidly in the brain and 15min. after injection 90% of the radioactivity still represents [(14)C]nicotine. Metabolites of [(14)C]nicotine have been identified in liver and urine extracts. [(14)C]Nicotine-1'-oxide has been detected in both liver and urine.     

Synthesis and radiobiological applications of [35S]l-homocysteine thiolactone

[³⁵S]l-Homocysteine thiolactone ([³⁵S]l-HCTL) was synthesized and its biodistribution evaluated as a potential brain radioprotective agent and as a tissue hypoxia marker. Drug uptake in mouse brain exceeded that in s.c. tumor 3 h post injection only. Multiple indicator dilution experiments in the rabbit heart indicate that membrane permeability of [³⁵S]l-HCTL does not limit its usefulness as a hypoxia marker. In addition, a positive correlation was observed between regional coronary blood flow and myocardial content of [³⁵S]adenosylhomocysteine formed from [³⁵S]homocysteine and adenosine.     

UPTAKE AND RELEASE OF TAURINE FROM RAT BRAIN SLICES

Abstract— Rapid efflux of [³⁵S]taurine from rat brain slices was observed on electrical stimulation. Slower release resulted when the Ca²⁺ content of the perfusion medium was replaced with Mg²⁺. Uptake of [³⁵S]taurine into rat cortical slices was unaffected by GABA, glutamic acid, glycine and leucine but was inhibited by alanine, ouabain, KCN and 2,4-dinitrophenol. Of a number of analogues of taurine, 2-aminoethylsulphinic acid was the most potent in inhibiting the uptake of [³⁵S]taurine. The rate of uptake was found to be decreased by lowering the incubation temperature. The possibility that taurine may be a neurotransmitter is discussed.     

Conversion of [U-14C]threonine into 14C-labelled amino acids in the brain of thiamin-deficient rats.

In confirmation of the findings of Gaitonde et al. (1974), a decrease in the brain concentration of threonine and serine, and an increase in glycine, were observed in rats maintained on a thiamin-deficient diet. Similar changes were found in the blood, and the concentration of several other amino acids in the blood decreased significantly. There was a correlation between the concentrations of threonine, serine, aspartate and asparagine in the brain and blood. In experiments in which [U-14C]threonine was injected into rats most of the radioactivity in the brain and blood of control rats was, as expected, in threonine in the acid soluble metabolites. In contrast, a considerable proportion of radioactivity was also found in other amino acids, namely glutamate, glutamine, aspartate, gamma-aminobutyrate and alanine, in the brain of thiamin-deficient rats. [U-14C]Threonine was also converted into 14C-labelled lactate and glucose, but the extent of this conversion was severalfold higher in thiamin-deficient than in control rats. This finding gave evidence of the stimulation in thiamin-deficient rats of the catabolism of [U-14C]threonine to [14C]lactate by the aminoacetone pathway catalysed by threonine dehydrogenase, and into succinate via propionate by the alpha-oxobutyrate pathway catalysed by threonine dehydratase (deaminase). The measurement of specific radioactivities of glutamate, aspartate and glutamine after injection of [U-14C]threonine, indicated a stimulation of the activities of threonine dehydrogenase and threonine dehydratase (deaminase) in the brain of thiamin-deficient rats. The specific radioactivities of glutamate, asparatate and glutamine int he brain were consistent with an alteration in the metabolism of threonine, mainly in the 'large' compartment of the brain of thiamin-deficient rats. The measurement of relative specific radioactivity of proteins after injection of [U-14C]threonine indicated a marked decrease in the synthesis of proteins, mainly in the liver of thiamin-deficient rats.     

Transport, nutritional and metabolic studies of taurine in staphylococci

A specific, Na+-dependent, energy-requiring transport system for taurine has been reported recently in the Staphylococcus aureus M strain. Taurine was taken up vigorously by all S. aureus strains tested. The system was Na+-dependent, and Na+ decreased the Km but had no effect on the Vmax of the transport system. Among coagulase-negative staphylococci, the Staphylococcus epidermidis group (a taxonomically related group of species associated with humans or other primates) and the free-living, wide-ranging species Staphylococcus sciuri showed vigorous taurine uptake. Somewhat lower rates were found in the Staphylococcus saprophyticus group. Low or barely detectable uptake rates were noted in other staphylococcal species that were primarily of animal origin. No taurine uptake was detected in a variety of other bacterial species tested. Taurine uptake, which was not Na+-dependent, occurred in a Pseudomonas aeruginosa strain grown on taurine as sole energy, carbon, nitrogen, and sulphur source, but not when it was grown in a gluconate/salts medium. In nutritional studies we were unable to demonstrate a role for taurine as a sulphur source for S. aureus. [1,2-14C]- and [35S]taurine were taken up during overnight growth of cells, and radioactivity was distributed similarly among cellular fractions, indicating that the carbon and sulphur atoms of taurine were not cleaved and had the same fate. We were unable to demonstrate any catabolism of taurine in radiorespirometric experiments to detect evolution of 14CO2 by cells incubated with [1,2-14C]taurine. Thus, we found no evidence for a role of taurine in the energy, carbon and sulphur metabolism of S. aureus.     

Synthesis of taurine in rat liver and brain in vivo

The in vivo formation of taurine and the analysis of labeled taurine precursors was examined in rat brain and liver at different times after an intracisternal injection of [³⁵S]cysteine and an intraperitoneal injection of [³H]cysteine, simultaneously administered. The distribution pattern of radioactivity was similar in liver and brain. Most of the labeling in both organs (85% in brain and 80% in liver) was recovered in glutathione (oxidized and reduced), cysteic acid, cysteine sulfinic acid, hypotaurine, cystathionine, and a mixed disulfide of cysteine and glutathione. The relative rates of labeling of cysteine sulfinic acid and taurine in liver and brain suggest than in vivo, liver possesses a higher capacity for taurine synthesis than brain. A small amount of [³H]taurine was detected in brain after intraperitoneal injection of [³H]cysteine. The time of appearance of this [³H]taurine as well as the fact that it occurs when [³H]cysteine is not detectable in brain or plasma suggests that it was probably not synthesized in brain from labeled precursors but formed elsewhere and transported into the brain through an exchange process.     

Brain cholesterol XVIII: EFFECt of methylphenidate (Ritalin) on [U-14C] glucose and [2-3H] acetate incorporation.

The effect of a single injection of methylphenidate (Ritalin, 4 mg/kg) on precursor ([2-3H]acetate and [U-14C]glucose) incorporation into brain cholesterol was studied. The drug caused a steady decrease in the concentration of brain cholesterol during the 24-hr period examined. Incorporation studies during this time with [U-14C]glucose indicated higher than normal incorporation for all time periods studied. The most significant incorporation increases took place 2 and 4 hr after drug injection. Experiments using [2-3H]acetate as the sterol precursor gave incorporation values which tended (not significantly) to be lower than control values at 2 and 4 h. The values after 12 hr were less than normal, while the 24-hr group indicated an increase to or slightly higher than normal values. These data suggest that the pharmacological effect of methylphenidate may be due to lowering of brain cholesterol levels directly or on some more basic metabolic process leading to a decreased level of membrane sterols.     

Characteristics of nitric oxide-evoked [H]taurine release from cerebral cortical neurons

Pharmacological characteristics of [³H]taurine release evoked by nitric oxide (NO) were investigated using mouse cerebral cortical neurons in primary culture. NO generators such as S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) dose-dependently increased [³H]taurine release from neurons. Such stimulatory effects of NO generators were completely abolished by hemoglobin, a NO radical scavenger, indicating that these [³H]taurine releases might be due to NO liberated from SNAP and SNP. Sodium withdrawal from incubation buffer significantly inhibited the SNAP- and SNP-induced [³H]taurine releases, whereas the removal of calcium showed no alterations in the [³H]taurine release evoked by NO generators. β-Alanine and guanidinoethane sulfonate, inhibitors of carrier-mediated taurine transport system, inhibited the SNAP- and SNP-evoked releases of [³H]taurine in a dose-dependent manner. These results indicate that the NO-evoked [³H]taurine release from cerebral cortical neurons is mediated by the reverse process of sodium-dependent carrier-mediated taurine transport system.     

TAURINE IN DEVELOPING RAT BRAIN: CHANGES IN BLOOD-BRAIN BARRIER

Abstract— The fate of [³⁵S]taurine injected intraperitoneally or intracranially has been compared in rats throughout neonatal development. The amount of [³⁵S]taurine present in the whole rat pup 24 h after intraperitoneal injection increases during development to a maximum 15 days after birth, and declines thereafter, whereas the amount of [³⁵S]taurine reaching the brain 24 h after intraperitoneal injection was greatest in the first 5 days after birth. The amount of [³⁵S]taurine remaining in the brain 24 h after intracranial injection does not vary throughout the period of neonatal development. These results suggest that the 'blood-brain' barrier is more accessible to taurine in the first few days after birth than later in neonatal development, and that factors other than simple exchange are involved.     

Modulation of [3H]Diazepam Binding in Rat Cortical Membranes by GABAA Agonists

GABAA receptor agonists modulate [3H]diazepam binding in rat cortical membranes with different efficacies. At 23 degrees C, the relative potencies for enhancement of [3H]diazepam binding by agonists parallel their potencies in inhibiting [3H]gamma-aminobutyric acid [( 3H]GABA) binding. The agonist concentrations needed for enhancement of [3H]diazepam binding are up to 35 times higher than for [3H]GABA binding and correspond closely to the concentrations required for displacement of [3H]bicuculline methochloride (BMC) binding. The maximum enhancement of [3H]diazepam varied among agonists: muscimol = GABA greater than isoguvacine greater than 3-aminopropane sulphonic acid (3APS) = imidazoleacetic acid (IAA) greater than 4,5,6,7-tetrahydroisoxazolo (4,5,6)-pyridin-3-ol (THIP) = taurine greater than piperidine 4-sulphonic acid (P4S). At 37 degrees C, the potencies of agonists remained unchanged, but isoguvacine, 3 APS, and THIP acquired efficacies similar to GABA, whereas IAA, taurine, and P4S maintained their partial agonist profiles. At both temperatures the agonist-induced enhancement of [3H]diazepam binding was reversible by bicuculline methobromide and by the steroid GABA antagonist RU 5135. These results stress the importance of studying receptor-receptor interaction under near-physiological conditions and offer an in vitro assay that may predict the agonist status of putative GABA receptor ligands.     

Dynamics of the conjugate pattern during the infusion of bile acids into isolated rat liver

The conjugate pattern of biliary [14C]bile acids was investigated in isolated perfused rat livers, which were infused with either [24-14C]cholic acid or [24-14C]chenodeoxycholic acid (40 mumol/h) together with or without taurine or cysteine (80 mumol/h). [14C]Bile acids were chromatographed on a thin-layer plate and the distribution of radioactivity on the plate was measured by radioscanning. The biliary excretion of [14C]bile acids was greater in the infusion with [14C]cholic acid than in the infusion with [14C]chenodeoxycholic acid. Biliary unconjugated [14C]bile acids amounted to about 50% of the total after the infusion with [14C]cholic acid, while only about 10% with [14C]chenodeoxycholic acid. In the initial period of infusion, biliary conjugated [14C]bile acids consisted mostly of the taurine conjugate, which decreased with time and the glycine conjugate increased complementarily. When taurine was simultaneously infused, the decrease in the taurine conjugate was suppressed to some extent. Cysteine infused in place of taurine had a similar influence but was less effective than taurine. The taurine content of liver after the infusion with either of the [14C]bile acids decreased greatly compared with that before the infusion, even when taurine or cysteine was infused simultaneously. The glycine content also decreased after the infusion, but the decrease in glycine was smaller than that in taurine. The results suggest that the conjugate pattern of biliary bile acids in rats depends mainly on the amount of taurine which is supplied to hepatic cells either exogenously from plasma or endogenously within themselves.     

Distribution, metabolism and excretion of [1-14C]dolichol injected intravenously into rats.

[1-14C]Dolichol mixed in vitro with rat serum and injected intravenously into rats was rapidly cleared from the circulation in a manner consistent with a two-compartment model. About 80% of the radioactivity recovered from animals killed after 1 day was in the liver, with smaller amounts being found in lung, carcass (internal organs removed), gastrointestinal tract and contents, and spleen. The kidneys, testes and heart contained little radioactivity, and the brain did not appear to take up any [1-14C]dolichol. The half-life for the turnover of radioactivity from [1-14C]dolichol in tissues varied considerably, being 2 days for the lung, 17 for liver and about 50 days for the carcass. After 1 day, and also after 4 and 21 days, most of the radioactivity in all tissues was as [1-14C]dolichol and as [1-14C]dolichyl fatty acyl ester, although a small amount of incorporation of [1-14C]dolichol radioactivity into phospholipids was also observed. Faeces collected over the first 4 days after injection contained 13% of the [1-14C]dolichol dose, but urine and expired air contained only small amounts of radioactivity. Radioactivity in faeces was nearly all as unchanged [1-14C]dolichol and as [1-14C]dolichyl fatty acyl ester. The [1-14C]dolichol remaining in liver after 21 days appeared to be in a pool (possibly lysosomes) where most of it was not subject to excretion.