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PubMed1991

Auxin transport in suspension-cultured soybean root cells : I. Characterization.

The kinetic parameters of auxin transport in suspension-cultured soybean (Glycine max [L.] Merr.) root cells were investigated. The same processes that are responsible for polar indoleacetic acid (IAA) transport in other plant tissues were found to occur in soybean root cells. These include (a) passive diffusion of the undissociated auxin molecule across the plasma membrane, (b) uptake via a specific, saturable carrier, and (c) phytotropin-sensitive efflux. Metabolism of exogenously added IAA was rapid; at the end of a 15-minute uptake period >80% of the IAA taken up had been converted to other compounds. The time course of [(14)C]IAA uptake in the first 90 seconds revealed two phases, the first corresponding to a rate of uptake approximately twice as large as the second phase. The transition to the second phase was delayed in the presence of the phytotropins triiodobenzoic acid or naphthylphthalamic acid, suggesting that an increase in the efflux of label as IAA accumulates in the cytoplasm is responsible for the transition. Carrier-mediated uptake contributes between 50 and 60% to the total rate of auxin uptake from a 0.28 micromolar IAA solution, with passive diffusion accounting for the remainder. Kinetic analysis of carrier-mediated uptake revealed a pH optimum of 5.0 and a Michaelis-Menten constant of 0.4 micromolar at pH 5.5. Because phytotropins had no effect on the initial rate of uptake, the efflux carrier does not appear to be involved in the uptake process.

Journal Article↗
PubMed1991

Auxin Transport in Suspension-Cultured Soybean Root Cells : II. Anion Effects on Carrier-Mediated Uptake.

To test the hypothesis that the carrier-mediated component of the indoleacetic acid (IAA) influx involves an electrogenic proton/IAA anion symport, the effects on the IAA influx of salts expected to depolarize the membrane potential were examined in suspension-cultured soybean (Glycine max [L.] Merr.) root cells. Although KCl does inhibit carrier-mediated uptake, the effect is specific to the anion at low concentrations and not due to more general processes such as changes in ionic or osmotic strength. Other anions such as bromide, iodide, and fluoride inhibit the carrier more strongly. Because potassium iminodiacetate, which is also expected to depolarize the membrane potential, has no inhibitory effect on the IAA influx, there is no evidence for the involvement of the membrane potential in carrier-mediated uptake. It is therefore most likely that in soybean cells, if carrier-mediated uptake occurs via a proton symport, the H(+):IAA- stoichiometry is 1:1. At concentrations greater than 70 millimolar, sorbitol, a nonionic osmoticum, inhibits carrier-mediated IAA uptake. The effects of specific anions and osmotic potential on the uptake carrier necessitates the reevaluation of other auxin transport studies in which KCl was routinely used as an agent with which to depolarize the membrane potential.

Journal Article↗
PubMed1991

Auxin-enhanced glucan autohydrolysis in maize coleoptile cell walls.

Cell walls isolated from auxin-pretreated maize (Zea mays L.) coleoptile segments were assayed to disclose evidence for the existence of enhanced autolysis. To improve the sensitivity of the measurements and to facilitate kinetic analysis, isolated cell walls were consolidated within a small column, and the autolysis rate was directly determined from the sugar content of the effluent. This protocol revealed that the maximum rate of autohydrolysis of walls prepared from segments occurs within the first 2 hours and a steady decline commences almost immediately. Walls from indoleacetic acid pretreated segments (0.5-4 hours) released sugar at a higher rate initially (110-125% of controls) and the enhanced rate of autolysis continued for 6 to 8 hours, but then it became equivalent to that of the controls. Pretreatment of the segments at acidic pH had no effect on the measurable rates of autolysis. The (1-->3), (1-->4)-beta-d-glucan content of the walls and the extractable glucanase activities support the hypothesis that temporal enhancement of autohydrolysis is a function of auxin on enzyme activity. The progressive decline in autolysis during prolonged incubations is consistent with the decrease in the quantity of the beta-d-glucan in the wall. The relationship between glucan content and autolysis rate is supported by the observation that while glucose pretreatment of segments had only a small effect on initial autolysis rates, the presence of the sugar during pretreatment served to extend the interval over which higher rates of autolysis could be sustained. The results demonstrate that autolysis is related to auxin-induced wall metabolism in maize coleoptiles.

Journal Article↗
PubMed1992

Changes in molecular size of previously deposited and newly synthesized pea cell wall matrix polysaccharides : effects of auxin and turgor.

Effects of indoleacetic acid (IAA) and of turgor changes on the apparent molecular mass (M(r)) distributions of cell wall matrix polysaccharides from etiolated pea (Pisum sativum L.) epicotyl segments were determined by gel filtration chromatography. IAA causes a two- to threefold decline in the peak M(r) of xyloglucan, relative to minus-auxin controls, to occur within 0.5 hour. IAA causes an even larger decrease in the peak M(r) concurrently biosynthesized xyloglucan, as determined by [(3)H]fucose labeling, but this effect begins only after 1 hour. In contrast, IAA does not appreciably affect the M(r) distributions of pectic polyuronides or hemicellulosic arabinose/galactose polysaccharides within 1.5 hours. However, after epicotyl segments are cut, their peak polyuronide M(r) increases and later decreases, possibly as part of a wound response. Xyloglucan also undergoes IAA-independent changes in its M(r) distribution after cutting segments. In addition, the peak M(r) of newly deposited xyloglucan increases from about 9 kilodaltons shortly after deposition to about 30 kilodaltons within 0.5 hour. This may represent a process of integration into the cell wall. A step increase in turgor causes the peak M(r) of previously deposited xyloglucan (but not of the other major polymers) to increase about 10-fold within 0.5 hour, returning to its initial value by 1.5 hours. This upshift may comprise a feedback mechanism that decreases wall extensibility when the rate of wall extension suddenly increases. IAA-induced reduction of xyloglucan M(r) might cause wall loosening that leads to cell enlargement, as has been suggested previously, but the lack of a simple relation between xyloglucan M(r) and elongation rate indicates that loosening must also involve other wall factors, one of which might be the deposition of new xyloglucan of much smaller size. Although the M(r) shifts in polyuronides may represent changes in noncovalent association, and for xyloglucan this cannot be completely excluded, xyloglucan seems to participate in a dynamic process that can both decrease and increase its chain length, possible mechanisms for which are suggested.

Journal Article↗
PubMed1992

Comparison of the outer and inner epidermis : inhibition of auxin-induced elongation of maize coleoptiles by glucan antibodies.

Polyclonal antibodies, raised against beta-d-glucans prepared from oat (Avena sativa L.) caryopses, cross-reacted specifically with (1-->3),(1-->4)-beta-d-glucans when challenged in a dot blot analysis of related polymers bound to a cellulose thin layer chromatography plate. The antibodies suppressed indoleacetic acid (IAA)-induced elongation of segments from maize (Zea mays L.) coleoptiles when the outer surface was abraded. However, IAA-induced elongation of nonabraded segments or segments with abrasion restricted to the interior of the cylinder was not influenced by the antibodies. Fab fragments prepared from the antibodies gave similar results. The capacity for IAA to overcome outward curvature of split coleoptile segments was partially reversed by treatment of the segments with the antibodies. Fluorescence microscopy revealed that antibody penetration was largely restricted to the epidermal cell wall region. These results support the view that the degradation of (1-->3),(1-->4)-beta-d-glucans in the outer epidermal cell wall serves an essential role in auxin-induced elongation of Poaceae coleoptiles.

Journal Article↗
PubMed1994

Monoamine oxidase-A: pharmacodynamics in humans of moclobemide, a reversible and selective inhibitor.

1. Single oral doses of 300, 450 and 600 mg moclobemide, a monoamine oxidase type A inhibitor, were administered in a cross-over design to eight healthy male volunteers. Plasma concentrations of the parent drug and of two monoamine metabolites (3,4-dihydroxyphenylglycol DHPG from noradrenaline; 5-hydroxy-indoleacetic acid 5HIAA from serotonin) were measured over time. 2. A physiological pharmacokinetic-pharmacodynamic model was used to describe MAO-A inhibition as reflected in the alterations of monoamine metabolites. Population values for the model parameters were obtained by a two-stage method allowing for repeated dosing per subject. 3. Even at the lowest dose an effect of moclobemide on plasma DHPG and 5HIAA concentrations was detectable in most subjects for up to 24 h. In contrast to DHPG, 5HIAA formation was only partially suppressed by moclobemide (maximum fractional extent of enzyme inhibition Imax: 0.57, CV 26%) suggesting the existence of 5HIAA formation pathways independent of those inhibitable by moclobemide. 4. Plasma moclobemide concentrations associated with 50% of maximum enzyme inhibition (IC50) were in the range of 100 (IC50,5HIAA at 300 mg) to 400 micrograms l-1 (IC50,DHPG at 600 mg).

Administration, Oral↗
PubMed1985

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.

Animals↗
PubMed1989

Formation of thiazolidine-4-carboxylic acid represents a main metabolic pathway of 5-hydroxytryptamine in rat brain.

Incubation of 5-hydroxytryptamine (5-HT) with rat brain homogenate resulted in the formation of (4R)-2-[3'-(5'-hydroxyindolyl)-methyl]-1,3-thiazolidine-4-carboxyl ic acid (5'-HITCA) as the major metabolite. The substance represents the condensation product of 5-hydroxyindole-3-acetaldehyde with L-cysteine. The chemical structure was confirmed by chromatographic and chemical methods as well as by fast atom bombardment mass spectrometry. Incubation of 5-HT in the presence of L-cysteine yielded the thiazolidine as the main metabolite up to 4 h. Under these conditions, the concentration of 5-hydroxyindole-3-acetic acid (5-HIAA) amounted to about 20% and 57% of 5'-HITCA (0.5 h and 4 h, respectively). In contrast to these findings, indole-3-acetic acid (IAA) was identified as the major metabolite when tryptamine was incubated under similar conditions. (4R)-2-(3'-Indolylmethyl)-1,3-thiazolidine-4-carboxylic acid (ITCA) was found to be the main conversion product of tryptamine only during the first 30 min. To investigate the fate of the thiazolidines, radiolabelled and unlabelled ITCA was incubated with rat brain homogenate. The compound was degraded enzymatically and rapidly. Subcellular fractionation revealed that the enzyme activity was present mainly in the cytosolic fraction whereas the preparation of mitochondria showed less activity. The responsible enzyme is presumably a carbon-sulfur lyase (EC 4.4.1.-). The major metabolite was isolated by HPLC and identified by mass spectrometry as well as by comparison with reference compounds to be IAA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1961

Effect of L-tryptophan on diuresis and 5-hydroxyindoleacetic acid excretion in the rat.

Oral administration of L-tryptophan to rats produced two main biochemical and pharmacological effects: a marked increase in urinary 5-hydroxyindoleacetic acid excretion, and a significant reduction in the urine flow after a water load. Urinary 5-hydroxyindoleacetic acid excretion reached its maximum 2 to 6 hr after the administration of tryptophan, and it increased with the dose of the amino acid. Antidiuresis was seen after the administration of L-tryptophan, 200 mg/kg, or more. The effect appeared promptly and it was roughly proportional to the dose of the amino acid administered. Both antidiuretic effect and increase in urinary 5-hydroxyindoleacetic acid excretion were more intense after oral than after parenteral administration of L-tryptophan. D-Tryptophan, in oral doses up to 1,000 mg/kg, produced neither an increase in urinary 5-hydroxyindoleacetic acid nor a reduction of diuresis. Available evidence suggests that reduction of urine flow is a consequence of biosynthesis and release of 5-hydroxytryptamine by the gastrointestinal mucosa. Tryptamine produced by direct decarboxylation of L-tryptophan does not seem to play any important role.

Administration, Oral↗
PubMed1991

Long-term pinealectomy alters hypothalamic serotonin metabolism in the rat.

In the present study, the effects of long-term pinealectomy on tryptophan, 5-hydroxytryptamine (5-HT or serotonin), 5-hydroxy-3-indoleacetic acid (5-HIAA), and tryptophan hydroxylase and monoamine oxidase activities were studied in preoptic area-anterior hypothalamus (POA-AH) and in the medial and posterior hypothalamus of the rat. After pinealectomy, 5-HT levels decreased significantly in medial hypothalamus but increased in the POA-AH. The levels of 5-HIAA decreased significantly in the POA-AH and medial hypothalamus. Tryptophan levels remained unchanged while tryptophan hydroxylase activity diminished significantly in POA-AH and medial hypothalamus. Monoamine oxidase activity remained unchanged in the hypothalamic regions. These results suggest that pinealectomy induces differential inhibitory actions on the serotoninergic terminal regions, mainly in anterior and medial hypothalamic areas.

Animals↗
PubMed1993

Hydroxyindole-O-methyltransferase activity in ocular and brain structures of rabbit and hen.

Relative activities of hydroxyindole-O-methyltransferase (HIOMT) of some brain and ocular structures of the rabbit and hen were analyzed using different 5-hydroxyindoles, i.e., N-acetylserotonin (NAS), 5-hydroxytryptophol (HTOL), 5-hydroxytryptophan (HTP), 5-hydroxytryptamine (HT), and 5-hydroxy-3-indoleacetic acid (HIAA), as enzyme substrates. Pineal glands of both species, as well as hen retina, are capable of producing, to varying degrees, melatonin, 5-methoxytryptophol, and 5-methoxytryptamine. Hen choroid and iris-ciliary body O-methylated NAS and HTOL, whereas rabbit choroid and, to a much lesser extent, hypothalamus and cerebral cortex all O-methylated only NAS. No measurable HIOMT activity was found in hen brain. NAS was a preferred substrate for HIOMT in the hen tissues, whereas in the rabbit pineal gland NAS and HTOL were equally good substrates for HIOMT. Other tested 5-hydroxyindoles, i.e., HTP, HT, and HIAA, were poor methyl acceptors. Of the tissues examined, the highest HIOMT activity was found in the hen pineal gland, followed by the rabbit pineal gland and hen retina. No significant differences between day and nighttime enzyme activities were observed in the pineal gland and retina of either species. The data suggest that in vertebrates some nervous and ocular tissues possess the potential to produce 5-methoxyindole compounds; however, the HIOMT-catalyzed process shows remarkable substrate-, tissue- and species-dependent variations.

Acetylserotonin O-Methyltransferase↗
PubMed1991

Analysis of monoamines in the cerebrospinal fluid of Chinese patients with Alzheimer's disease.

We have established HPLC assay conditions that could measure the levels of 14 monoamines and their metabolites simultaneously. Monoamine levels in cerebrospinal fluids of 12 Chinese patients with Alzheimer's disease were compared with those in samples from patients with benign prostate hyperplasia. Of the 14 monoamines and metabolites, only three were found to be present in all samples. Although the levels of 5-hydroxy-3-indoleacetic acid (HIAA) and homovanillic acid (HVA) in cerebrospinal fluid of patients with Alzheimer's disease were lower, and the levels of 3-methoxy-4-hydroxyphenylglycol (MHPG) higher, as compared to control patients, no significant differences were found between these two groups.

3,4-Dihydroxyphenylacetic Acid↗