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.     

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.     

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.     

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.     

Sulfur Amino Acid Metabolism in the Developing Rhesus Monkey Brain: Subcellular Studies of Taurine, Cysteinesulfinic Acid Decarboxylase, γ-Aminobutyric Acid, and Glutamic Acid Decarboxylase

Abstract: Taurine, cysteinesulfinic acid decarboxylase (CSAD), glutamate, γ-aminobutyric acid (GABA), and glutamic acid decarboxylase (GAD) were measured in subcellular fractions prepared from occipital lobe of fetal and neonatal rhesus monkeys. In addition, the distribution of [³⁵S]taurine in subcellular fractions was determined after administration to the fetus via the mother, to the neonate via administration to the mother prior to birth, and directly to the neonate at various times after birth. CSAD, glutamate, GABA, and GAD all were found to be low or unmeasurable in early fetal life and to increase during late fetal and early neonatal life to reach values found in the mother. Taurine was present in large amounts in early fetal life and decreased slowly during neonatal life, arriving at amounts found in the mother not until after 150 days of age. Significant amounts of taurine, CSAD, GABA, and GAD were associated with nerve ending components with some indication that the proportion of brain taurine found in these organelles increases during development. All subcellular pools of taurine were rapidly labeled by exogenously administered [³⁵S]taurine. The subcellular distribution of all the components measured was compatible with the neurotransmitter or putative neuro-transmitter functions of glutamate, GABA, and taurine. The large amount of these three amino acids exceeds that required for such function. The excess of glutamate and GABA may be used as a source of energy. The function of the excess of taurine is still not clear, although circumstantial evidence favors an important role in the development and maturation of the CNS.     

TAURINE IN THE BRAIN AND LIVER OF THE DEVELOPING HUMAN AND MONKEY

—The concentrations of taurine in brain and liver from human fetuses (2nd trimester) and adults and from Rhesus monkeys from mid-gestation, through birth and neonatal life to maturity have been measured. The concentration of taurine in human and monkey fetal brain was 4-5-fold higher than that in adult monkey brain. In human fetal brain the concentration of taurine decreased linearly with increasing crown-rump length (r=−0·75; P < 0·001). In fetal monkey brain, no correlation with gestational age was found. After birth, the concentration of taurine in monkey brain decreased linearly with increasing age (r=−0·96; P < 0·001) until values comparable to those found in the adult were reached 8-9 months after birth, approximately the end of weaning. The concentration of taurine in liver both from fetal humans and from fetal monkeys was approximately twice that in mature liver. Concentrations of taurine similar to those found in adult liver were reached within a few days of birth, compared to several months for brain. These results suggest that taurine may be associated with brain development in addition to any functional role it may play in the mature brain.     

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.     

Evidence for specific synaptosomal localization of exogenous accumulated taurine

Abstract— Equilibrium or incomplete equilibrium density gradient centrifugation was used to characterize the subcellular localization of exogenous [^{3 5}S]taurine which was taken up by minces or homogenates of rat cerebral cortex. [^{3 5}S]Taurine is accumulated in synaptosomes, which sediment more slowly than l -[³H]norepinephrine-accumulating particles. When [^{3 5}S]taurine and [³H]GABA are accumulated by minces, a small difference in the sedimentation profile of taurine and GABA was observed, but no difference was found when taurine and intrasynaptosomal potassium were compared. However, potassium sedimented more slowly after incubation of homogenates than of minces. These data give evidence for the accumulation of [^{3 5}S]taurine by a specific synaptosomal population.     

Corticoid formation by the monkey fetal adrenal: Evaluation of 17-, 21- and 11-hydroxylase activities

There is indirect evidence that cortisol synthesis in the fetal rhesus monkey adrenal gland is limited at Day 135 of gestation but increases thereafter. This study was conducted to ascertain whether a reduced synthetic capacity is caused by a deficiency in 17-, 21- or 11-hydroxylase activity. For the sake of comparison 11- and 21-hydroxylases were also estimated in adult adrenals. 11-, 21-Hydroxylases were measured in the entire adrenal by the oxidation of NADPH by mitochondria and microsomes, respectively. 17-Hydroxylase was evaluated in outer and inner regions of the fetal gland by the formation of [³H]17-hydroxyprogesterone, -11-deoxycortisol, -cortisol and -androstenedione from [³H]progesterone. The maximum velocity of both the 11- and 21-hydroxylase was similar in fetal and adult glands indicating that corticoid formation in the fetus is not constrained by levels of these enzymes.[³H]Progesterone was extensively metabolized to -17-hydroxyprogesterone, -androstenedione, -11-deoxycortisol and -cortisol by homogenates from both regions of the fetal adrenal. The ratio of [³H]-cortisol to [³H]11-deoxycortisol was consistently higher in incubations of the inner glandular area. Together, these findings indicate that 17-hydroxylase is also active at Day 135 and that the 11-hydroxylase may be more concentrated in the fetal cortex. These data suggest in addition that the restriction in cortisol formation occurs at a step prior to the metabolism of progesterone to cortisol.     

Fate of axonally transported taurine and proteins in the developing rabbit visual system following optic nerve section

These experiments were performed to characterize the axonally transported taurine in the visual system of developing rabbits. [³⁵S]Taurine, transported axonally after intravitreal injection, disappeared from the components of the visual system more rapidly after nerve section than it did with intact nerves. The decrease was most rapid in the youngest animals, and tended to be most pronounced in the elements nearest to the section (optic nerve, optic tract).³H-labeled proteins present in the visual system changed less markedly than [³⁵S]taurine after nerve section; only in the youngest rabbits was there a marked decrease. These results suggest that a greater proportion of the intraaxonal taurine is labile in young rabbits than in mature rabbits.     

Stable isotope study of plasma taurine kinetics in rhesus monkey

Taurine is one of the most abundant amino acids in mammals and there is increasing evidence for the importance of taurine during development. Plasma taurine kinetics in a rhesus monkey was studied using [1,2-13C2]taurine. Taurine in plasma was derivatized to its dimethylaminomethylene methyl ester, separated on a gas chromatographic column, and the [M+2+H]+/[M+H]+ ion ratio was measured by ammonia chemical ionization mass spectrometry. The results were comparable to those obtained from the simultaneous radioisotope tracer study using [35S]taurine. This stable isotope method requires only 200 microliters of plasma for precise and accurate determination and is suitable for taurine kinetic studies in human infants.     

Plasmodium knowlesi: In vitro biosynthesis of methionine

Methionine biosynthesis was studied in rhesus monkey erythrocytes infected with Plasmodium knowlesi malaria which were cultured in vitro with l-[3-¹⁴C]serine, methyl-[¹⁴C]tetrahydrofolic acid, and l-[³⁵S]homocysteine. Radioactivity derived from [3-¹⁴C]serine was detected in approximately equivalent amounts in methionine and thymidylic acid by thin-layer chromatography of acid-hydrolysates of washed erythrocytes. The results with methyl-[¹⁴C]tetrahydrofolic acid were inconclusive. Radioactivity from l-[³⁵S]homocysteine also appeared in methionine but the level of homocysteine required for maximal activity was tenfold that of serine. The results indicate that the serine: 5,10-methylenetetrahydrofolic acid: 5-methyl-tetrahydrofolic acid: methionine biosynthetic pathway is present in the P. knowlesi malaria parasite.     

Taurine in the developing rabbit visual system: Changes in concentration and axonal transport including a comparison with axonally transported proteins

[³⁵S]Taurine injected intravitreally into rabbits was transported axonally to the optic nerve terminals. Considerably more [³⁵S]taurine was transported in young rabbits than in mature rabbits. The time course of taurine transport did not parallel that of proteins labeled with [³H]proline in the same system. The concentration of taurine in all components of the visual system, except retina, was greater in young animals than in mature animals, and was especially high in optic nerve. The possible functions of the high concentrations of taurine and the greater amount of axonally transported taurine in developing mammalian CNS are discussed.     

Effects of pentylenetetrazol on GABA-A/benzodiazepine/picrotoxinin receptor complexes in rat brain regions

The effects of acute convulsive doses of pentylentetrazol (PTZ) on [³⁵S]t-butyl-bicyclophosphorothionate (TBPS), [³H]flunitrazepam (FNP), [³H]muscimol, and [³H]-aminobutyric acid (GABA) binding sites were examined in well-washed homogenates of various brain regions of rat. Except for a significant increase in the number of striatal [³⁵S]TBPS binding sites, no significant change in [³⁵S]TBPS, [³H]FNP, [³H]muscimol, and [³H]GABA binding was found in various brain regions 30 min after subcutaneous injection of PTZ at 90 or 100 mg/kg. Similarly there were no significant changes in [³⁵S]TBPS and [³H]FNP binding to unwashed P₂ membranes of cerebral cortices 30 min following administration of convulsive doses of PTZ. These experiments failed to demonstrate acute modulation of GABA-A/benzodiazepine/picrotoxinin receptor complex by PTZ in the various brain regions examined except striatum. The significance of the increased [³⁵S]TBPS binding in striatum caused by PTZ remains unclear.     

UPTAKE AND RELEASE OF TAURINE FROM CEREBRAL CORTEX SLICES AND THEIR SUBCELLULAR COMPARTMENTS

—Cortex slices, synaptosomes and C-6 glioma cells were used to study [³⁵S]taurine uptake and its electrically-stimulated release. After exposure to taurine at two concentrations, the synaptosome preparation subsequently derived from the slices contained 41% of the particle-bound taurine and 16% of the total in the tissue. The uptake of [¹⁴C]GABA by C-6 glioma cells was inhibited 3-fold more by β-alanine than by l -DABA, whilst synaptosome preparations showed the opposite pattern, l -DABA being 2 or 3 times more effective than β-alanine. [³⁵S]Taurine uptake inhibition by l -DABA was low for synaptosomes and C-6 glioma, whereas β-alanine showed considerable effect on C-6 glioma (41%) and slices of white matter (ependyma; 50%). Synaptosome preparations showed little effect with β-alanine. When 30 min rather than 5 min incubations were employed, β-alanine depressed [³⁵S]taurine uptake by cortex slices by 30%. Taurine was taken up by a calcium-dependent mechanism and subcellular fractionation indicated that the synaptosome fraction showed losses commensurate with the net taurine release when low stimulation currents were used.     

Sulfur amino acid metabolism in the developing rhesus monkey brain: Interrelationship of taurine and glutamate

The relationship of taurine to glutamate, and to other amino acids, has been examined in the occipital lobe of the developing rhesus monkey. During development taurine decreases in concentration (4.96 mol/g in fetus to 1.52 mol/g in adult) while glutamate increases (7.92 mol/g in fetus to 11.26 mol/g in adult). When the concentration of taurine is plotted against that of glutamate in fetal, neonatal and adult animals there is a significant correlation in the fetal (p<0.01) and adult (p<0.01) but not in the neonatal occipital lobe samples. This correlation in both fetal and adult brain is specific for these two amino acids. Subcellular fractionation studies further indicate that this relationship may be of special importance in nerve endings.This paper is dedicated to Dr. Derek Richter on his seventy-fifth birthday.     

Formation and accumulating of hypotaurine in rat liver regenerating after partial hepatectomy

Hypotaurine is considered to be an intermediate in the major pathway for the biosynthesis of taurine in mammals yet is rarely detected in mammalian tissue. The activity of cysteinesulfinic acid decarboxylase, the enzyme presumably responsible for the biosynthesis of hypotaurine, is frequently present in great amounts in tissue, whereas the mechanism for the conversion of hypotaurine to taurine is poorly understood, there being some doubt at present if an enzyme exists for such a purpose. This paper reports the accumulation of hypotaurine in the liver of rats regenerating after partial hepatectomy. Further, the formation and accumulation of [³⁵S]hypotaurine from [³⁵S]methionine under the same conditions was observed. No hypotaurine was detected in liver of sham-operated control animals, even after the intraperitoneal injection of authentic hypotaurine. These observations suggest that rat liver normally possesses a mechanism for the rapid conversion of hypotaurine to taurine and that this mechanism is impeded in liver regenerating after partial hepatectomy.     

Studies on the neurochemical properties of cystathionine

Following the intracerebral administration of [³⁵S]cystathionine, the synaptosome fraction of rat brain was labelled, the greatest uptake of amino acid being associated with hypothalamus.The uptake of [³⁵S]cystathionine by synaptosome preparations isolated from different regions of brain, was typical of that exhibited by amino acids which are not neurotransmitters.Depolarization of the synaptic membrane had no effect on the efflux of [³⁵S]cystathionine from preloaded synaptosomes.The intracerebral administration of cystathionine resulted in an elevation of the levels of brain cyclic AMP, the effect being particularly evident in the cerebellum. Attempts to reproduce this effect in vitro were unsuccessful.     

Tissue and regional distribution of cysteic acid decarboxylase

A sensitive and rapid assay method for cysteic acid decarboxylase was developed which combined the selectivity of ion exchange resin (a complete retention of the substrate, cysteic acid, and exclusion of the product, taurine) with the speed of a vacuum filtration. The synthesis and purification of³⁵S-labeled cysteic acid were described. The validity of the assay was established by the identification of the reaction product as taurine. With this new method, the decarboxylase activity was measured in discrete regions of bovine brain. Putamen had the highest activity, 172 pmol taurine formed/min/mg protein (100%), followed by caudate nucleus, 90%; cerebral cortex, 82%; hypothalamus, 81%; cerebellar cortex, 79%; cerebellar peduncle, 59%; thalamus, 42%; brain stem, 25%; pons, 10%; and corpus callosum, 3%. The decarboxylase activity in various mouse tissues was also determined as follows: liver, 403; brain, 145; kidney, 143; spinal cord, 59; lung, 21; and spleen, 10 pmol taurine formed/min/mg. No activity could be detected in skeleton muscle and heart, suggesting a different biosynthetic pathway for taurine synthesis in these tissues. The advantages and disadvantages of the new assay method are also discussed.This work was supported in part by grant NS 13224 from the National Institutes of Health, U.S.A., and grant from Huntington's Chorea Foundation in memory of Mrs. Ruth Berman.This work was presented at the Eighth Annual Meeting of American Society for Neurochemistry in Denver, Colorado, March 1977.     

Effects of the ionophores X537A and A23187 on GABA,glycine, and taurine release from chick retinal subcellular fractions

The ionophore X537A at concentrations of 5–20 M stimulated the release of [³H]GABA and [³⁵S]taurine, from retinal subcellular crude nuclear (P₁) and crude synaptosomal (P₂) fractions. The release of [³H]GABA increased 114% and 136% over control values in P₁ and P₂ fractions, respectively. The efflux of [³⁵S]taurine from P₁ was increased by 45% and that from P₂ by 21%. X537A increased⁴⁵Ca²⁺ uptake in the P₂ fraction but not in the P₁ fraction. The effect of X537A on the amino acid release was not dependent on the presence of exogenous calcium. X537A did not affect [³H]GABA or [³⁵S]taurine uptake by the retinal fractions. A23187 enhanced [³H]GABA release from P₁ and P₂ by 52% and 105%, respectively. The ionophore also increased [¹⁴C]glycine liberation in both P₁ (35%) and P₂ (50%) but failed to stimulate [³⁵S]taurine release. A23187 produced a transient increase of⁴⁵Ca²⁺ uptake of 38% in P₁ and 30% in P₂. The effects of A23187 on the release of amino acids were calcium dependent. The amino acid uptake was not affected by the ionophore. These results are consisent with the suggested neurotransmitter role for GABA at the outer synaptic layer and for GABA and glycine at the inner synaptic layer of the retina. A neurotransmitter role for taurine is not supported by the present results.