FACTORS INFLUENCING BRAIN AND TISSUE LEVELS OF TRYPTAMINE: SPECIES, DRUGS AND LESIONS

With a modification of the spectrophotofluorometric (SPF) method of HESS & UDENFRIEND (1959) (J. Pharmac. exp. Ther. 127 , 175-177), brain tryptamine levels in the rat (20.9 ng/g) and guinea-pig (20.7 ng/g) were found to be less than those in the dog (32.1 ng/g) and cat (52.2 ng/g). Regional distribution studies in the dog and cat showed that tryptamine was present in all major brain regions with highest concentrations in the spinal cord. Blood levels of tryptamine in the guinea-pig, dog and cat (6-7 ng/ml) were lower than brain levels. Pargyline significantly increased brain tryptamine in both the dog and cat; whereas, isocarboxazid (after 4 h) increased brain tryptamine levels in the dog but decreased brain levels in the cat. Reserpine (0.5-1.0 mg/kg per day for 1-4 days) did not significantly decrease brain, spinal cord or blood tryptamine levels in the dog. Spinal cord transection did not decrease tryptamine levels below the lesion in the chronic spinal dog.     

Comparative Ontogenesis of Brain Tryptamine, Serotonin, and Tryptophan

The pattern of ontogenetic development of tryptophan (TP), tryptamine (T), indole-3-acetic acid (IAA), 5-hydroxytryptamine (5-HT; serotonin), and 5-hydroxyindole-3-acetic acid (5-HIAA) in the brains of rats aged 1-45 days is presented. Analysis of the five components in each brain allows the calculation of the acid/amine and amine/amino acid ratios. These metabolic indexes are a useful tool to study and compare the metabolic origins and fates of both amines. The ontogenetic patterns of TP, T, and IAA are very similar, especially during the first week postpartum. The highest and lowest levels found for T were 2.2 ng/g and 0.1 ng/g at the 1st and 5th day, respectively. The temporal relationship between the T/TP and IAA/T ratios suggests the existence of mechanisms protecting T against monoamine oxidase (MAO) which develop in parallel to synaptogenesis. Significant correlations were found between TP and IAA during the whole period studied and between TP and T during the first week after birth. The 5-HT peak found during the first postpartum week could be due to a non-neuronal pool of 5-HT protected against MAO and possibly contained in mast cells. Preliminary determinations on leptomeningeal membranes suggest the existence of such a pool.     

Characterization of auxin conjugates in Arabidopsis. Low steady-state levels of indole-3-acetyl-aspartate, indole-3-acetyl-glutamate, and indole-3-acetyl-glucose

Amide-linked indole-3-acetic acid (IAA) conjugates constitute approximately 90% of the IAA pool in the dicot Arabidopsis, whereas ester-linked conjugates and free IAA account for approximately 10% and 1%, respectively when whole seedlings are measured. We show here that IAA-aspartate Asp, IAA-glutamate (Glu), and IAA-glucose (Glc) are present at low levels in Arabidopsis. Nine-day-old wild-type Arabidopsis seedlings yielded 17.4 +/- 4.6 ng g(-1) fresh weight IAA-Asp and 3.5 +/- 1.6 ng g(-1) fresh weight IAA-Glu, and IAA-Glc was present at 7 to 17 ng g(-1) fresh weight in 12-d-old wild-type seedlings. Total IAA content in 9-d-old Arabidopsis seedlings was 1, 200 +/- 178 ng g(-1) fresh weight, so these three IAA conjugates together made up only 3% of the conjugate pool throughout the whole plant. We detected less than wild-type levels of IAA-Asp and IAA-Glu (7.8 +/- 0.4 ng g(-1) fresh weight and 1.8 +/- 0.3 ng g(-1) fresh weight, respectively) in an Arabidopsis mutant that accumulates conjugated IAA. Our results are consistent with IAA-Asp, IAA-Glu, and IAA-Glc being either minor, transient, or specifically localized IAA metabolites under normal growth conditions and bring into question the physiological relevance of IAA-Asp accumulation in response to high concentrations of exogenous IAA.     

Presence and Measurement of Imidazoleacetic Acid, a γ-Aminobutyric Acid Agonist, in Rat Brain and Human Cerebrospinal Fluid

Imidazoleacetic acid (IAA) was unequivocally demonstrated in rat brain, human CSF, and human plasma by a gas chromatographic-mass spectrometric method that can reliably quantify as little as 8 pmol, i.e., 1 ng. Owing to tautomerism of the imidazole ring, IAA and [15N, 15N]IAA, the internal standard, each formed two chromatographically distinct isomers after derivatization of the ring nitrogens with either ethyl chloroformate or methyl chloroformate. The isomers of n-butyl(N-ethoxycarbonyl)imidazole acetate and n-butyl(N-methoxycarbonyl)imidazole acetate were identified by analysis with methane chemical ionization and electron impact ionization of molecular and fragment ions. The levels (mean +/- SEM) of free IAA were 140 +/- 14 pmol/g and 2.7 +/- 0.2 pmol/ml in brains of untreated rats and human lumbar CSF, respectively. Mean levels of IAA in brains of anesthetized rats, perfused free of blood, did not differ significantly from mean levels of anesthetized, nonperfused controls or from untreated rats. The source or sources of IAA in brain and CSF are unknown. Because IAA is a potent agonist at gamma-aminobutyrate receptors, it merits examination as a regulator in brain.     

Biosynthesis of indole-3-acetic acid in tomato shoots: Measurement,mass-spectral identification and incorporation of −2H from −2H2O into indole-3-acetic acid,d- and l-tryptophan,indole-3-pyruvate and tryptamine

Indole-3-acetic acid (IAA) and its putative precursors, l- and d-tryptophan, indole-3-pyruvate, and tryptamine were isolated from tomato (Lycopersicon esculentum (L.) Mill.) shoots, identified by mass spectrometry, and measured using capillary gas chromatography with an electron capture detector and radioactive internal standards. Average amounts present were 7.9ng · (g FW)^–-1 IAA, 5.7ng · (g FW)^–-1 indole-3-pyruvate, 132 ng · (g FW)^–-1 tryptamine, 103 ng · (g FW)^–-1 d-tryptophan, and 2250 ng · (g FW)^–-1 l-tryptophan. Indole-3-acetaldoxime was not found; detection limits were less than 1ng · (g FW)^–-1. When tomato shoots were incubated for 6, 10 and 21 h in 30% ^–2H₂O, up to four positions in IAA, l- and d-tryptophan, tryptamine and indole-3-pyruvate became labelled with ^–2H. Compounds became labelled rapidly with 10% of IAA molecules containing ^–2H after 6 h. The percentage of labelled molecules of IAA and l-tryptophan increased up to 10 h but then decreased again, correlating with an increase in the total shoot tryptophan and presumably a result of protein hydrolysis in the excised, slowly senescing tissue. The amount of ^–2H in d-tryptophan also showed an increase followed by a decrease, but the proportion of labelled molecules was much less than in l-tryptophan and IAA. Tryptamine became labelled initially at a similar rate to IAA but continued to accumulate ^–2H up to 21 h. We conclude that tryptamine is synthesized from a different pool of tryptophan from that used in IAA synthesis, and is not a major endogenous precursor of IAA in tomato shoots. Indole-3-pyruvate was the most heavily labelled compound after 6 and 10 h incubation (21-h data not available). Furthermore, the proportion of ^–2H-labelled indole-3-pyruvate molecules was quantitatively consistent with the amount of label in IAA. On the other hand, a quantitative comparison of the IAA turnover rate and the rate of ^–2H incorporation into both l- and d-tryptophan indicates that IAA is not made from the total shoot pool of either l- or d-tryptophan. Instead IAA appears to be synthesized from a restricted pool which is turning over rapidly and which has access to both newly synthesized tryptophan and that from protein hydrolysis.Abbreviations GC-ecd gas chromatography with electroncapture detector - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - IAOX indole-3-acetaldoxime - IPyA indole-3-pyruvate - PFB pentafluorobenzyl - R_T retention time - TNH₂ tryptamine - Trp tryptophan - SIM selected ion monitoring We wish to thank Ms. Sue Alford for running the mass spectra and Dr Harry Young for advice with the mass spectrometry. The work was supported by grants from the University of Auckland Research Committee and the C. Alma Baker Trust fund. The mass spectrometer was supported jointly by the University Grants Commitee (NZ) and the DSIR Division of Horticulture and Processing.     

A new mass fragmentographic method for the simultaneous analysis of tryptophan, tryptamine, indole-3-acetic acid, serotonin, and 5-hydroxyindole-3-acetic acid in the same sample of rat brain.

A new method for the concurrent extraction and quantification of tryptophan (Trp), tryptamine (T), indole-3-acetic acid (IAA), serotonin (5-HT), and 5-hydroxyindole-3-acetic acid (5-HIAA) in samples of rat brain is presented. Homogenization is carried out in 0.1 n HCl containing 1 n KCl and 0.2% NaHSO₃. After centrifugation at 100,000g, the supernatant is percolated through a column of XAD-2 resin, eluted with distilled methanol, and the resulting eluate is evaporated to dryness. The dry residue is then derivatized to yield the pentafluoropropionated (PFP) and methylpentafluoropropionated (Me-PFP) derivatives. Identification and quantification is readily achieved by gas chromatography-mass fragmentographic analysis on a OV-17 or Dexsil 300 column. Endogenous levels in whole rat brain established by this method are IAA, 13,1 ± 2.0 ng/g (n = 6); T, less than 380 pg/g (n = 6); Trp, 4.16 ± 0.23 μg/g (n = 6); 5-HIAA, 442 ± 24 ng/g (n = 6); and 5-HT, 526 ± 81 ng/g (n = 5).     

The effects of some antipsychotic drugs, D-amphetamine, and reserpine on the concentration and rate of accumulation of tryptamine and 5-hydroxytryptamine in the mouse striatum

The mouse striatum contains about 2 ng/g of tryptamine and 600 ng/g of 5-hydroxytryptamine. No significant changes in mouse striatal tryptamine were observed after the administration of chlorpromazine, haloperidol, spiperone, or alpha-flupenthixol. The levels of 5-hydroxytryptamine were moderately reduced by chlorpromazine, spiperone, and alpha-flupenthixol but not by haloperidol. The administration of antipsychotic drugs to mice pretreated with a monoamine oxidase inhibitor (pargyline) produced an increase in the rate of accumulation of striatal tryptamine compared with that of pargyline-treated mice. In contrast, the rate of accumulation of 5-hydroxytryptamine after monoamine oxidase inhibition was reduced by chlorpromazine, spiperone, and alpha-flupenthixol but not haloperidol. D-Amphetamine administration did not change either tryptamine or its 5-hydroxyderivative while reserpine increased tryptamine and reduced 5-hydroxytryptamine. The results suggest that changes in striatal tryptamine may be controlled by the availability of tryptophan, the amino acid precursor of tryptamine.     

Comparison of the biodistribution of free or liposome-entrapped Crotalus durissus terrificus (South American rattlesnake) venom in mice

The local absorption rate, clearance and tissue distribution of Crotalus durissus terrificus venom, (Cdt) were examined using a two-antibody sandwich ELISA assay. We compared the biodistribution of both free or encapsulated Cdt in mice. Following subcutaneous injection of 10 microg/mouse of free Cdt (0.8 LD50), venom was detected in serum after 15 min, showed its highest level at 30 min (45+/-5 ng/ml) and was cleared from the circulation after 6 h. After 2 h of inoculation, venom was detected in the kidney (57+/-9 ng/g of tissue), spleen (18+/-4 ng/g of tissue) and brain (14+/-6 ng/g of tissue). For both subcutaneous or intravenous injection of free Cdt, venom was firstly detected in the kidney. No Cdt appeared either in the kidney, spleen, brain, or other tissues after subcutaneous inoculation of encapsulated venom even though a higher dose was used, 25 microg/mouse (2 LD50). Venom remained at the site of injection for a period of 1 week. Following intravenous injection of encapsulated venom (5 microg/mouse, 2 LD50), venom was detected in liver and spleen tissues. The biodistribution of encapsulated venom is discussed in relation to the effects of reduction of toxicity and increase of adjuvanticity.     

GAS CHROMATOGRAPHY-MASS FRAGMENTOGRAPHIC DETERMINATION OF INDOLE-3-ACETIC ACID IN RAT BRAIN

Abstract— A gas chromatography-mass fragmentographic procedure is described for the quantitation of indole-3-acetic acid (IAA) in rat brain. IAA was determined as its IV-1-pentafluoropropionyl-methyl ester. Deuterium labeled IAA was employed as an internal standard. Assay sensitivities of at least 500 pg and 2.5 ng could be achieved by single and double ion monitoring respectively. Whole brain IAA concentrations were determined to be 11.6 + 0.75 ng/g (n = 6). IAA concentrations in brain areas ranged from 10 to 64 ng/g with highest levels in the striatum. Rat brain IAA accumulated curvilinearly with respect to time over a 90 min interval following probenecid (200 mg/kg, i.p.). The efflux of IAA from brain appears mediated in part through a probenecid-sensitive active efflex process.     

Quantitation of total MHPG in the rat brain using a non enzymatic hydrolysis procedure. Effects of drugs

An acid-catalyzed procedure has been used to hydrolyze MHPG-sulfate in homogenates of rat brain. The samples (in 0.4 mol/L perchloric acid) are treated for 3 min. at 100 degrees C in a water bath and aliquots are injected into a reversed phase HPLC system. Detection is achieved fluorimetrically. The absolute detection limit for MHPG is 150 pg, which allows the reliable determination of either free or total MHPG in rat brain in concentrations down to 15 ng/g, using the described procedure. The concentration of total MHPG found in the brains of saline-treated rats are 101 +/- 21 ng/g (mean +/- S.D.) which is in a good accordance with the concentration value for the same samples obtained using a GC-MS method (115 +/- 19 ng/g). Rats treated with clonidine (300 micrograms/Kg, i.p.) or yohimbine (10 mg/Kg, i.p.) showed brain concentrations of total MHPG of 68 +/- 22 ng/g and 299 +/- 85 ng/g, respectively. The utility of this method for the analysis of brain regions or brain nuclei (e.g. locus coeruleus) is also shown.     

Pregnenolone, dehydroepiandrosterone, and their sulfate and fatty acid esters in the rat brain

The rat brain contains large amounts of pregnenolone (P) and dehydroepiandrosterone (D) arising from local biosynthetic pathways. We have devised a procedure for the measurement of both "neurosteroids" either unconjugated or released from their sulfate (S) or fatty acid (L) esters. The measurements were performed at the acrophase of the circadian variation of neurosteroids, and confirmed the large accumulation of P (25 +/- 8 ng/g, mean +/- SD) and of PS (19 +/- 6 ng/g) and DS (2.1 +/- 0.5 ng/g) in the brain of adult male rats. We found that fatty acid esters constitute the major species of neurosteroids in brain (PL 46 +/- 14, and DL 36 +/- 7 ng/g, in adult males). The levels of P and DS were increased by daily injection of vehicle to intact males, whereas castration, without or with testosterone or estradiol supplementation (2 mg daily for 7 days), did not produce a significant change of neurosteroids concentrations. Measurements of neurosteroids had not been previously reported in cyclic females. The levels of P, PL, and DS were identical in proestrous females and in intact males, whereas PS (26 +/- 6 ng/g) and DL (50 +/- 16 ng/g) were increased in females. Compared to proestrous females, diestrous females had lower levels of PS (19 +/- 6 ng/g), DS (1.7 +/- 0.4 ng/g), and PL (43 +/- 19 ng/g). These differences suggested a modulatory role of ovarian secretions on the metabolism of neurosteroids.     

Lipoxygenase Metabolism of Arachidonic Acid in Brain

When blood-free mouse brain slices were incubated with exogenous radiolabeled arachidonic acid, gas chromatography/mass spectrometry confirmed that the major radioactive lipoxygenase enzyme product of arachidonic acid was 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), with lesser amounts of 5-hydroxy-5,6,8,11,14-eicosatetraenoic acid and 15-hydroxy-5,8,11,13-eicosatetraenoic acid. When 12-[2H]HETE was used to measure endogenous 12-HETE in brain tissue frozen with liquid nitrogen, the level of 12-HETE was 41 +/- 6 ng/g of wet weight tissue. This frozen tissue level was not due to the presence of blood. When brain slices were incubated in vitro for 20 min, the 12-HETE level increased to 964 +/- 35 ng/g of wet weight tissue. Elimination of residual intravascular blood before tissue incubation reduced the brain slice 12-HETE concentration by one-half.     

Identification of prostaglandin D2 as a major prostaglandin in homogenates of rat brain

Prostaglandin (PG) E2, D2, F2alpha and thromboxane B2 (TxB2) were determined in homogenates of rat brain by gas-chromatography--mass spectrometry. The level of PGD2 was 735 +/- 19 ng/g, of PGF2alpha 150 +/- 13 ng/g, of TxB2 112 ng/g and of PGE2 86 +/- 8 ng/g. The same relative proportions of cyclooxygenase products were found in incubates of unstimulated sliced rat brain. 14C-PGH2 was converted in high yield into PGD2 by enzyme(s) present in the soluble fraction of the homogenate. These results indicate that PGD2 is the major cyclooxygenase product in the central nervous system of the rat.     

Sialic acid concentration of brain gangliosides: variation among eight mammalian species

Sialic acid is a vital component of brain gangliosides which play an essential role in the transmission and storage of information in the brain. The concentration of bound sialic acid in gangliosides and free sialic acid in the brain cortex of eight different mammals [human, chimpanzee (Pan troglodytes), rat (Rattus norvegicus), mouse (Mus musculus), rabbit (Oryctolagus cuniculus), sheep (Ovis aries), cow (Bos indicus) and pig (Sus scrofa)] were compared. Total sialic acid concentration (890+/-103 microg/g wet weight tissue, mean+/-SE, n = 6) was 2-4 times higher in the human brain compared with the other species studied (0.001 < p < 0.05). There was no significant difference between human males and females. The rank order of adult brain sialic acid after humans (in microg/g) was rat (493+/-23, n = 12), mouse (445+/-29, n = 16), rabbit (380+/-18, n = 6), sheep (323+/-43, n = 6), cow (304+/-14, n = 6) and pig (252+/-14, n = 6). Apart from the cow vs the sheep, the differences between species were statistically significant (p < 0.05). In the mouse, cow and sheep, total sialic acid concentration increased during maturation by 18-32% (p < 0.05). In a 2-year-old chimpanzee, the sialic acid concentration in the left lobe of the brain cortex was 25% higher than that of right lobe at 6 weeks of age (p < 0.05). Free sialic acid was higher in the human brain cortex (41+/-3 microg/g) than that of the rat and mouse (32+/-3 and 25+/-5 microg/g respectively) and absent from other species. Variation in brain sialic acid concentration among different animals has implications for the evolution of the brain and may affect learning ability in animals.     

Formation of novel D-ring and E-ring isoprostane-like compounds (D4/E4-neuroprostanes) in vivo from docosahexaenoic acid

Free radical-mediated oxidant injury and lipid peroxidation have been implicated in a number of neural disorders. We have reported that bioactive prostaglandin D2/E2-like compounds, termed D2/E2-isoprostanes, are produced in vivo by the free radical-catalyzed peroxidation of arachidonic acid. Docosahexaenoic acid, in contrast to arachidonic acid, is the most abundant unsaturated fatty acid in brain. We therefore questioned whether D/E-isoprostane-like compounds (D4/E4-neuroprostanes) are formed from the oxidation of docosahexaenoic acid. Levels of putative D4/E4-neuroprostanes increased 380-fold after oxidation of docosahexaenoic acid in vitro from 15.2 +/- 6.3 to 5773 +/- 1024 ng/mg of docosahexaenoic acid. Subsequently, chemical approaches and liquid chromatography electrospray ionization tandem mass spectrometry definitively identified these compounds as D4/E4-neuroprostanes. We then explored the formation of D4/E4-neuroprostanes from a biological source, rat brain synaptosomes. Basal levels of D4/E4-neuroprostanes were 3.8 +/- 0.6 ng/mg of protein and increased 54-fold after oxidation (n = 4). We also detected these compounds in fresh brain tissue from rats at levels of 12.1 +/- 2.4 ng/g of brain tissue (n = 3) and in human brain tissue at levels of 9.2 +/- 4.1 ng/g of brain tissue (n = 4). Thus, these studies have identified novel D/E-ring isoprostane-like compounds that are derived from docosahexaenoic acid and that are formed in brain in vivo. The fact that they are readily detectable suggests that ongoing oxidative stress is present in the central nervous system of humans and animals. Further, identification of these compounds provides a rationale for examining their role in neurological disorders associated with oxidant stress.     

Reassessing the role of N-hydroxytryptamine in auxin biosynthesis

The tryptamine pathway is one of five proposed pathways for the biosynthesis of indole-3-acetic acid (IAA), the primary auxin in plants. The enzymes AtYUC1 (Arabidopsis thaliana), FZY (Solanum lycopersicum), and ZmYUC (Zea mays) are reported to catalyze the conversion of tryptamine to N-hydroxytryptamine, putatively a rate-limiting step of the tryptamine pathway for IAA biosynthesis. This conclusion was based on in vitro assays followed by mass spectrometry or HPLC analyses. However, there are major inconsistencies between the mass spectra reported for the reaction products. Here, we present mass spectral data for authentic N-hydroxytryptamine, 5-hydroxytryptamine (serotonin), and tryptamine to demonstrate that at least some of the published mass spectral data for the YUC in vitro product are not consistent with N-hydroxytryptamine. We also show that tryptamine is not metabolized to IAA in pea (Pisum sativum) seeds, even though a PsYUC-like gene is strongly expressed in these organs. Combining these findings, we propose that at present there is insufficient evidence to consider N-hydroxytryptamine an intermediate for IAA biosynthesis.     

DDT降解菌株Chryseobacterium sp. PYR2对小麦的促生作用及其机理

【背景】前期结果表明,DDT降解菌株Chryseobacterium sp. PYR2可高效去除土壤中的DDT等污染物,具有潜在的应用价值,但该菌对植物的影响尚不清楚。【目的】探讨菌株Chryseobacterium sp. PYR2对植物的促生作用及其机理,为后续开发DDT降解及植物促生双效功能菌剂提供理论依据。【方法】配制该菌株的不同梯度稀释菌悬液,用纸卷发芽法和盆栽法研究菌悬液对小麦种子萌发和植株生长的影响;Salkowski法测定PYR2合成吲哚-3-乙酸(Indole-3-acetic acid,IAA)量;单因素实验研究不同培养条件对菌株生长及IAA合成的影响;液相色谱-串联质谱-多反应监测(LC-MS/MS-MRM)方法分析IAA在PYR2菌体内的生物合成途径。【结果】PYR2菌悬液可明显提高小麦种子萌发率并促进小麦植株的生长,小麦的侧根数、株高、鲜重、干重等指标均明显提高。该作用是由于菌株PYR2可以合成植物生长激素IAA。最适IAA合成条件:温度30°C,pH 7.0-8.0,盐浓度0.5%,L-色氨酸50mg/L。代谢液中检测到色醇、色胺和吲哚-3-乙酰胺3种中间代谢产物,推测PYR2体内存在3条IAA合成途径,分别为吲哚-3-丙酮酸(IPy A)、TAM和IAM途径。【结论】菌株PYR2对小麦具有明显的促生效果,是由于其具有多条高效合成IAA的代谢途径,表明其在农药污染土壤的生物修复及作物种植中具有潜在的应用前景。     

Effect of barbitone sodium and thiopental sodium on brain dopamine, noradrenaline, serotonin and 5-hydroxyindoleacetic acid content in Arvicanthis niloticus

The quantitative estimation of total dopamine (DA), noradrenaline (NE), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) content in the whole brain tissue of normal Nile grass rat, Arvicanthis niloticus, gives and average of 631 +/- 12 ng DA/g, 366 +/- 12 ng NE/g, 617 +/- 15 ng 5-HT/g and 431 +/- 10 ng 5-HIAA/g fresh brain tissue. The effect of barbitone sodium and thiopental sodium on the total DA, NE, 5-HT and 5-HIAA content in the brain tissue of the Nile grass rat, Arvicanthis niloticus, was studied. The total DA, NE, 5-HT and 5-HIAA contents were determined 5 hr after i.p. injection of different doses of barbitone sodium (20, 40 and 80 mg/ml/100 g body wt) and thiopental sodium (5, 10 and 20 mg/ml/100 g body wt). The effect of different time intervals (1, 10, 30 min, 1, 2.5, 5, 8, 16, 24 and 48 hr) on the total brain DA, NE, 5-HT and 5-HIAA content was investigated after i.p. injection of 40 mg of barbitone sodium and 10 mg of thiopental sodium/ml/100 g body wt. Both barbitone sodium and thiopental sodium caused an increase in DA, NE and 5-HT content and a decrease in 5-HIAA content in the brain tissue of Arvicanthis niloticus. The increase in the whole brain contents of DA, NE and 5-HT after the administration of barbitone sodium and thiopental sodium may be due either to inhibition of transmitter release by an action at the monoamine nerve terminal or to effects causing a decrease in nerve impulse flow. On the other hand, the decrease in 5-HIAA may be due to the decrease in the turnover of 5-HT.     

Biosynthesis and metabolism of indole-3-ethanol and indole-3-acetic acid by Pinus sylvestris L. needles

Combined gas chromatography-mass spectrometry has been used to identify indole-3-ethanol (IEt) in a purified extract from needles of Pinus sylvestris L. Quantitative estimates obtained by high-performance liquid chromatography with fluorescence detection, corrected for samples losses occurring during purification, indicate that Pinus needles contain 46±4 ng g^-1 IEt. This compares with 24.5±6.5 ng g^-1 indole-3-acetic acid (IAA) and 2.3±0.4 ng g^-1 indole-3-carboxylic acid (ICA) (Sandberg et al. 1984, Phytochemistry, 23, 99–102). Metabolism studies with needles incubated in a culture medium in darkness revealed that both [3-¹⁴C]-tryptophan and [2-¹⁴C]tryptamine mine are converted to [¹⁴C]IEt. It was also shown that [3-¹⁴C]IEt acted as a precursor of [¹⁴C]IAA. The observed metabolism appears to be enzymic in nature. The [2-¹⁴C]IAA was not catabolised to [¹⁴C]ICA in detectable quantities implying that, at best, only a minor portion of the endogenous ICA pool in the Pinus needles originates from IAA.Abbreviations DEAE diethylaminoethyl - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - ICA indole-3-carboxylic acid - IEt indole-3-ethanol - PVP polyvinylpyrrolidone     

Accumulation of Indole‐3‐Acetic Acid in Rice sl Mutant Leaves Infected with Bipolaris oryzae

Rice leaves accumulate serotonin in response to infection by Bipolaris oryzae. The leaves of the sl mutant, which is deficient in the gene encoding tryptamine 5‐hydroxylase, accumulate tryptamine instead of serotonin upon infection by B. oryzae. Because tryptamine is a possible precursor of indole‐3‐acetic acid (IAA), we investigated the accumulation of IAA in sl leaves infected with B. oryzae. Liquid chromatography coupled with tandem mass spectrometry analysis indicated that IAA accumulated at approximately 1.5 μmol/gFW in the leaves of sl mutant. This accumulation was suppressed by 95% by the treatment with the tryptamine decarboxylase inhibitor, (S)‐α‐(fluoromethyl)tryptophan, at 100 μm , indicating that tryptamine served as the precursor of IAA. The accumulation of IAA was not reproduced by treatment with CuCl₂ or by exogenous feeding of tryptamine. Furthermore, inoculation of Magnaporthe grisea induced only a lower level of IAA accumulation. On the other hand, B. oryzae produced IAA in culture media containing tryptamine. These findings strongly suggested that the metabolism of tryptamine by B. oryzae was responsible for IAA accumulation in the leaves of the sl mutant. Serotonin added to the culture media was also converted into 5‐hydroxyindole‐3‐acetic acid (5HIAA) at a rate similar to that of tryptamine. Considering that wild‐type rice leaves accumulate serotonin for defensive purposes, reducing the concentration of serotonin by conversion into 5HIAA may be significant as a detoxification process in the interaction between B. oryzae and rice.