Enzymatic features of serotonin biosynthetic enzymes and serotonin biosynthesis in plants

Serotonin, a pineal hormone in mammals, is found in a wide range of plant species at detection levels from a few nanograms to a few milligrams, and has been implicated in several physiological roles, such as flowering, morphogenesis and adaptation to environmental changes. Serotonin synthesis requires two enzymes, tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H), with TDC serving as a rate-limiting step because of its high K_m relation to the substrate tryptophan (690 µM) and its undetectable expression level in control plants. However, T5H and downstream enzymes, such as serotonin N-hydroxycinnamoyl transferase (SHT), have low K_m values with corresponding substrates. This suggests that the biosynthesis of serotonin or serotonin-derived secondary metabolites is restricted to cellular stages when high tryptophan levels are present.Key words: feruloylserotonin, serotonin, tryptamine, tryptamine 5-hydroxylase, tryptophan, tryptophan biosynthesis, tryptophan decarboxylaseSerotonin is found in a broad range of plants and is abundant in reproductive organs, such as fruits and seeds.^1–3 Even though many physiological roles for serotonin in plants have been proposed,^2–7 its actual roles have yet to be examined in detail using molecular, biochemical and genetic approaches. In plants, serotonin is synthesized by two enzymes: tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H). TDC decarboxylates tryptophan into tryptamine, after which T5H hydroxylates tryptamine into serotonin.^8–10 TDC expresses at an undetectable level in rice leaves, whereas T5H expresses constitutively.^11,12     

Characterization of tryptamine 5-hydroxylase and serotonin synthesis in rice plants

Serotonin is a well-known pineal hormone that in mammals plays a key role in mood. In plants, serotonin is implicated in several physiological roles such as flowering, morphogenesis, and adaptation to environmental changes. However, its biosynthetic enzyme in plants has not been characterized. Therefore, we measured the serotonin content and enzyme activity responsible for serotonin biosynthesis in rice seedlings. Tryptamine 5-hydroxylase (T5H), which converts tryptamine into serotonin, was found as a soluble enzyme that had maximal activity in the roots. The maximal activity of T5H was closely associated with the enriched synthesis of serotonin in roots. Tetrahydropterine-dependent T5H activity was inhibited by tyramine, tryptophan, 5-OH-tryptophan, and octopamine, but remained unaltered by dopamine in vitro. The tissues of rice seedlings grown in the presence of tryptamine exhibited a dose-dependent increase in serotonin in parallel with enhanced T5H enzyme activity. However, no significant increase in serotonin was observed in rice tissues grown in the presence of tryptophan, suggesting that tryptamine is a bottleneck intermediate substrate for serotonin synthesis.     

Characterization of the serotoninergic system in the C57BL/6 mouse skin.

We showed expression of the tryptophan hydroxylase gene and of tryptophan hydroxylase protein immunoreactivity in mouse skin and skin cells. Extracts from skin and melanocyte samples acetylated serotonin to N-acetylserotonin and tryptamine to N-acetyltryptamine. A different enzyme from arylalkylamine N-acetyltransferase mediated this reaction, as this gene was defective in the C57BL6 mouse, coding predominantly for a protein without enzymatic activity. Serotonin (but not tryptamine) acetylation varied according to hair cycle phase and anatomic location. Serotonin was also metabolized to 5-hydroxytryptophol and 5-hydroxyindole acetic acid, probably through stepwise transformation catalyzed by monoamine oxidase, aldehyde dehydrogenase and aldehyde reductase. Activity of the melatonin-forming enzyme hydroxyindole-O-methyltransferase was notably below detectable levels in all samples of mouse corporal skin, although it was detectable at low levels in the ears and in Cloudman melanoma (derived from the DBA/2 J mouse strain). In conclusion, mouse skin has the molecular and biochemical apparatus necessary to produce and metabolize serotonin and N-acetylserotonin, and its activity is determined by topography, physiological status of the skin, cell type and mouse strain.     

Peripheral serotonin dynamics in the rainbow trout (Oncorhynchus mykiss)

Serotonin (5-hydroxytryptamine, 5-HT) occurs in a wide range of tissues throughout the body of the rainbow trout. Results reported here indicate that the main peripheral sources of serotonin are the intestinal tract and the gill epithelium (levels above 1500 ng/g). The high intestinal serotonin concentration is mostly due to serotoninergic nerve fibres, which are present at high density in the intestinal wall. Only about 2% of serotonin is associated with mucosal enterochromaffin cells. In the remaining tissues studied serotonin concentration was below 160 ng/g: the highest concentrations were seen in the anterior and posterior kidneys, followed by the liver, heart, and spleen. 5-Hydroxyindolacetic acid (5-HIAA) levels, except in plasma, were generally lower than serotonin levels, and were below our detection limits in heart, spleen and posterior kidney. Acute d-fenfluramine treatment (5 or 15 mg/kg i.p.) significantly increased 5-HIAA/5-HT ratio in the anterior intestine, pyloric caeca and plasma. Serotonin released from intestinal serotoninergic fibres in response to d-fenfluramine treatment is metabolized locally, and only a small part reaches the blood, from where it can be taken up and metabolized by other peripheral tissues, such as the liver and gill epithelium. The non-metabolized serotonin pool in the blood appears to be located extracellularly, not intracellularly as in mammals. In view of these findings, we present an overview of peripheral serotonin dynamics in rainbow trout.     

Cellular mode of serotonin action on CI− transport in the rabbit corneal epithelium

The present work examines serotonin-induced changes in cell potential difference and barrier resistances in the corneal epithelium in vitro using voltage-measuring microelectrodes and related techniques. Component resistances were determined using voltage and resistance profiles of the epithelium before and during the serotonin response. Serotonin, added to the stromal side of the cornea in the presence of nialamide, markedly reduced transcorneal and apical membrane resistances, while basal barrier resistance increased slightly and shunt resistance was unchanged. The marked drop in apical membrane resistance after serotonin treatment reflects an increase in apical membrane chloride permeability, inasmuch as the serotonin-stimulated short-circuit current is indistinguishable from the increase in net chloride flux. Prolonged (more than 1 h) exposure of corneas to serotonin markedly depolarized the epithelial cells and reduced the voltage divider ratio from 12.3 ± 2.1 to 1.5 ± 0.5, while not significantly affecting the stimulated short-circuit current. These later effects suggest changes in epithelial ion distribution during long periods of stimulation by serotonin.     

Serotonin accumulation in transgenic rice by over-expressing tryptophan decarboxlyase results in a dark brown phenotype and stunted growth

A mutant M47286 with a stunted growth, low fertility and dark-brown phenotype was identified from a T-DNA-tagged rice mutant library. This mutant contained a copy of the T-DNA tag inserted at the location where the expression of two putative tryptophan decarboxlyase genes, TDC-1 and TDC-3, were activated. Enzymatic assays of both recombinant proteins showed tryptophan decarboxlyase activities that converted tryptophan to tryptamine, which could be converted to serotonin by a constitutively expressed tryptamine 5′ hydroxylase (T5H) in rice plants. Over-expression of TDC-1 and TDC-3 in transgenic rice recapitulated the stunted growth, dark-brown phenotype and resulted in a low fertility similar to M47286. The degree of stunted growth and dark-brown color was proportional to the expression levels of TDC-1 and TDC-3. The levels of tryptamine and serotonin accumulation in these transgenic rice lines were also directly correlated with the expression levels of TDC-1 and TDC-3. A mass spectrometry assay demonstrated that the dark-brown leaves and hulls in the TDC-overexpressing transgenic rice were caused by the accumulation of serotonin dimer and that the stunted growth and low fertility were also caused by the accumulation of serotonin and serotonin dimer, but not tryptamine. These results represent the first evidence that over-expression of TDC results in stunted growth, low fertility and the accumulation of serotonin, which when converted to serotonin dimer, leads to a dark brown plant color.     

Intestinal glucose and galactose transport in the cultured gilthead bream (Sparus aurata)

1. Electrical parameters and transepithelial glucose and galactose transport were determined in vitro across anterior and posterior intestine of the culture fish Sparus aurata. 2. Electrical potential difference (PD) and short-circuit current (Isc) were serosa-positive in anterior intestine, while they were serosa-negative or near zero in posterior intestine. 3. Tissue conductance (Gt) was higher in posterior than in anterior intestine. In both parts it was decreased when the Na ion was omitted in mucosal and serosal reservoirs. 4. Addition of glucose or galactose to the mucosal side of intestine caused an increase in PD and Isc in posterior intestine but did not significantly change PD and Isc in anterior intestine. 5. Isotopic flux of glucose and galactose measurements in short-circuit conditions showed a net active glucose and galactose absorption in posterior intestine, while in anterior intestine active transport of glucose or galactose was not observed. 6. The net transport of glucose and galactose in posterior intestine was decreased to zero in the absence of Na in mucosal and serosal reservoirs or in the presence of ouabain (1 mM) in serosal solution.     

Serotonin phase-shifts the circadian rhythm of locomotor activity in the cockroach

Serotonin, a putative neurotransmitter in insects, was found to cause consistent phase shifts of the circadian rhythm of locomotor activity of the cockroach Leucophaea maderae when administered during the early subjective night as a series of 4-microliters pulses (one every 15 min) for either 3 or 6 hr. Six-hour treatments with dopamine also caused significant phase shifts during the early subjective night, but 3-hr treatments with dopamine had no phase-shifting effect. Other substances tested in early subjective night (norepinephrine, octopamine, gamma-aminobutyric acid, glutamate, carbachol, histamine, tryptophan, tryptamine, N-acetyl serotonin, or 5-hydroxyindole-3-acetic acid) did not consistently cause phase shifts. The phase-shifting effect of serotonin was found to be phase-dependent. The phase response curve (PRC) for serotonin treatments was different from the PRC for light. Like light, serotonin caused phase delays in the late subjective day and early subjective night, but serotonin did not phase-shift rhythms when tested at phases where light causes phase advances.     

Effects of tryptamine on active sodium and chloride transport in the isolated bullfrog cornea

The effects of the serotonin analogue, tryptamine, on the active transepithelial transport of Na⁺ and Cl⁻ in the in vitro bullfrog cornea were studied. Tryptamine, 1 mM, inhibited both the short-circuit current (I_sc) and potential difference (PD) of corneas transporting either Na⁺ alone or both Na⁺ and Cl⁻. The electrical resistance, R, increased in all cases. Both unidirectional Na⁺ and Cl⁻ fluxes were decreased by tryptamine and these changes accounted for the inhibitory effects on the I_sc. The effects of tryptamine were considered along with with those of 2 mM theophylline and 0.1 mM ouabain. Tryptamine inhibited the I_sc and both undirectional Cl⁻ fluxes which were previously stimulated by theophylline. Theophyline addition, after tryptamine preincubation, increased the Cl⁻ undirectional fluxes but did not restore the inhibited I_sc. The inhibitory effects of tryptamine on active Na⁺ and Cl⁻ transport were different from those of ouabain. While both drugs inhibited the forward Na⁺ and Cl⁻ fluxes, their backfluxes decreased with tryptamine and increased with ouabain. The addition to the bathing solution of tryptamine after ouabain preincubation reduced the ouabain-increased backward Cl⁻ flux and further increased the electrical resistance. These results are analyzed in terms of an electrical model from which it appears that tryptamine's mechanism of action was to decrease cellular permeability to the transepithelial movement of Na⁺ and Cl⁻.     

Origins of the transient anterior-posterior asymmetry in the frog lens fiber potential

The origin of the transient asymmetry of intracellular resting potentials between the anterior and posterior lens fibers was investigated in the isolated American bullfrog lens by a conventional microelectrode technique. In high K+, Rb+, Cs+, or NH+4 test solution applied only to the lens anterior or posterior side, anterior fibers depolarized at a slower rate than posterior ones. After a long exposure, however, the transient potential difference disappeared. The magnitude of the depolarizations of the lens fibers was in the order of K+ greater than Rb+ greater than Cs+ greater than NH+4. The resting potentials plotted as a function of external K+ concentrations ([K]0) were in agreement with Nernst equation predictions with a slope of 58 mV/decade ion concentration change. A small Na+ permeability is unmasked at a [K]0 less than 10 mM. It was concluded that the transient difference measured in potentials of anterior and posterior lens fibers on increasing external K+, Rb+, Cs+ or NH+4 depends on the anterior epithelial cell layer, which is a diffusional barrier for ions penetrating into the lens interior.     

Probing the role of tryptophan-derived secondary metabolism in defense responses against Bipolaris oryzae infection in rice leaves by a suicide substrate of tryptophan decarboxylase

Tryptophan-derived secondary metabolites, including serotonin and its hydroxycinnamic acid amides, markedly accumulate in rice leaves in response to pathogen attack. These compounds have been implicated in the physical defense system against pathogen invasion by being deposited in cell walls. Serotonin is biosynthesized from tryptophan via tryptamine, and tryptophan decarboxylase (TDC) catalyzes the first committed reaction. In this study, (S)-α-(fluoromethyl)tryptophan (S-αFMT) was utilized to investigate the effects of the inhibition of TDC on the defense responses of rice leaves. S-αFMT, enantiospecifically synthesized from l-tryptophan, effectively inhibited TDC activity extracted from rice leaves infected by Bipolaris oryzae. The inhibition rate increased dependently on the incubation time, indicating that S-αFMT served as a suicide substrate. Treatment of rice seedlings with S-αFMT suppressed accumulation of serotonin, tryptamine, and hydroxycinnamic acid amides of serotonin in a dose-dependent manner in B. oryzae-inoculated leaves. The lesions formed on seedlings treated with S-αFMT lacked deposition of brown materials, and those leaves were severely damaged in comparison with leaves without S-αFMT treatment. Administrating tryptamine to S-αFMT-treated leaves restored accumulation of tryptophan-derived secondary metabolites as well as deposition of brown material. In addition, tryptamine administration reduced damage caused by fungal infection. Accordingly, the accumulation of tryptophan-derived secondary metabolites was suggested to be part of the effective defense mechanism of rice.     

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.     

Characterization of rice tryptophan decarboxylases and their direct involvement in serotonin biosynthesis in transgenic rice

l-Tryptophan decarboxylase (TDC) and l-tyrosine decarboxylase (TYDC) belong to a family of aromatic l-amino acid decarboxylases and catalyze the conversion of tryptophan and tyrosine into tryptamine and tyramine, respectively. The rice genome has been shown to contain seven TDC or TYDC-like genes. Three of these genes for which cDNA clones were available were characterized to assign their functions using heterologous expression in Escherichia coli and rice (Oryza sativa cv. Dongjin). The purified products of two of the genes were expressed in E. coli and exhibited TDC activity, whereas the remaining gene could not be expressed in E. coli. The recombinant TDC protein with the greatest TDC activity showed a K _m of 0.69 mM for tryptophan, and its activity was not inhibited by phenylalanine or tyrosine, indicating a high level of substrate specificity toward tryptophan. The ectopic expression of the three cDNA clones in rice led to the abundant production of the products of the encoded enzymes, tyramine and tryptamine. The overproduction of TYDC resulted in stunted growth and a lack of seed production due to tyramine accumulation, which increased as the plant aged. In contrast, transgenic plants that produced TDC showed a normal phenotype and contained 25-fold and 11-fold higher serotonin in the leaves and seeds, respectively, than the wild-type plants. The overproduction of either tyramine or serotonin was not strongly related to the enhanced synthesis of tyramine or serotonin derivatives, such as feruloyltyramine and feruloylserotonin, which are secondary metabolites that act as phytoalexins in plants.     

Serotonin regulates mammary gland development via an autocrine-paracrine loop

Mammary gland development is controlled by a dynamic interplay between endocrine hormones and locally produced factors. Biogenic monoamines (serotonin, dopamine, norepinephrine, and others) are an important class of bioregulatory molecules that have not been shown to participate in mammary development. Here we show that mammary glands stimulated by prolactin (PRL) express genes essential for serotonin biosynthesis (tryptophan hydroxylase [TPH] and aromatic amine decarboxylase). TPH mRNA was elevated during pregnancy and lactation, and serotonin was detected in the mammary epithelium and in milk. TPH was induced by PRL in mammosphere cultures and by milk stasis in nursing dams, suggesting that the gene is controlled by milk filling in the alveoli. Serotonin suppressed beta-casein gene expression and caused shrinkage of mammary alveoli. Conversely, TPH1 gene disruption or antiserotonergic drugs resulted in enhanced secretory features and alveolar dilation. Thus, autocrine-paracrine serotonin signaling is an important regulator of mammary homeostasis and early involution.     

Evidence for the Presence of Serotonin Receptors Negatively Coupled to Adenylate Cyclase in the Rabbit Iris-Ciliary Body

Serotonin has no obvious effect on basal cyclic AMP levels but reduces the forskolin-, isoproterenol-, and vasoactive intestinal peptide-induced stimulation of cyclic AMP levels in a dose-dependent manner. Serotonergic, cholinergic, muscarinic, alpha-adrenergic, and dopaminergic antagonists have no effect on the serotonin response. Topical application of a serotonin/pargyline solution to the living eye causes desensitisation of the serotonin response in the iris-ciliary body, an observation confirming the presence of specific serotonergic receptors linked to adenylate cyclase. The 5-HT1A [5-hydroxytryptamine (serotonin) type 1A] receptor agonists 8-hydroxy-2-(di-n-propylamino)tetralin and buspirone mimic the serotonin response in reducing the forskolin-stimulated cyclic AMP levels, as do the indole derivatives 5-methoxytryptamine, 5-hydroxtryptophan, and tryptamine. However, the ineffectiveness of the 5-HT1A agonist ipsapirone and the inability of spiroxatrine to block the serotonin response show that classical 5-HT1A receptors are not involved. The serotonin response is blocked by pertussis toxin and is insensitive to the phosphodiesterase inhibitor theophylline, which indicates the involvement of an inhibitory guanine regulatory protein in the coupling of the serotonin receptor to the adenylate cyclase catalytic unit.     

Tryptamine and Some Related Molecules Block the Accumulation of a Light-Sensitive Pool of Cyclic AMP in the Dark-Adapted, Dark-Incubated Mouse Retina

Dark-adapted retinas of mice (C57BL/6J) incubated in the dark in media containing 1 mM 3-isobutylmethylxanthine (IBMX) or 5 mM Co2+ accumulate cyclic AMP (cAMP). A portion of this pool is light sensitive, as light can prevent or reverse its accumulation. Similarly, tryptamine, serotonin, 5-methoxytryptamine, bufotenine, and 5-methoxydimethyltryptamine can block the accumulation of the light-sensitive pool of cAMP, whereas tryptophan, melatonin, N-acetylserotonin, 5-methoxytryptophol, and tetrahydro-beta-carbolines are inactive. The phenomenon is not seen with mutant mouse retinas (rd/rd), which lack most photoreceptors, but persists in abnormal retinas containing photoreceptors but with extensive neuronal depletion in the inner retina. Tryptamine also inhibits cAMP accumulation in either dark or light-adapted retinas exposed to forskolin alone but not in media containing high levels of forskolin plus 1 mM IBMX. There is some suggestion that serotonin 5-HT-2 antagonists can partially reverse the action of the tryptamines, but hitherto undescribed receptors may be involved. Current data suggest that photoreceptors are the target for the action of the tryptamines.     

Effect of 5-hydroxytryptamine on sodium transport across bullfrog skin

Summary 5-hydroxytryptamine, when present in the solution bathing the inside surface of bullfrog skin at concentrations of 0.25–25.0 mM, reduced both electrical potential difference and short-circuit current across the skin. The magnitude of reduction in potential difference and short-circuit current was dependent on 5 HT concentration. Reduction in sodium influx entirely accounted for the reduction in short-circuit current. Preliminary evidence suggested a competition between 5 HT and vasopressin in the production of their effects on sodium transport across the skin, while high Ca⁺⁺ concentrations and 5 HT seemed to act independently of each other.Dr. Henry C. and Bertha H. Buswell Fellow.     

Function of serotonin in seeds of walnuts

In plants serotonin (5-hydroxytryptamine) may function as a hormone and as a protective agent against predation. A role for serotonin as a secondary plant product involved in ammonia detoxification in seeds of walnuts (Juglans regia) is now also proposed. Serotonin is formed from tryptophan synthesized via the constitutive enzymes of the shikimate pathway localized in the plastids, and is stored in protein bodies developed in the cotyledons during maturation. By the accumulation of serotonin in these protein bodies, the seeds, which have no vacuoles for storage or excretion of hydrophilic secondary plant products, are able to detoxify ammonia by the synthesis of serotonin.     

Chronic amphetamine administration decreases brain tryptophan hydroxylase activity in cats

Chronic administration of d-amphetamine sulfate (7.5 mg/kg, i.p. every 12 hrs. for 6 days) to cats produced significant decreases in the Vmax of brain-stem and forebrain tryptophan hydroxylase when measured 1 day (?34 and ?46%) and 10 days (?17 and ?30%) after the final amphetamine injection. Serotonin and 5-hydroxyindoleacetic acid (5HIAA) levels were decreased by a similar magnitude. A single injection of amphetamine (7.5 mg/kg) produced no significant changes in tryptophan hydroxylase activity, serotonin, or 5HIAA when measured 1 day after the injection. Neither acute nor chronic amphetamine treatment produced any significant changes in the Km of tryptophan hydroxylase for either tryptophan or the natural co-factor, tetrahydrobiopterin. These data suggest that chronic amphetamine treatment decreases central serotonergic neurotransmission by an action on the rate-limiting enzyme in serotonin biosynthesis.