Lack of Tryptophan Hydroxylase-1 in Mice Results in Gait Abnormalities

The role of peripheral serotonin in nervous system development is poorly understood. Tryptophan hydroxylase-1 (TPH1) is expressed by non-neuronal cells including enterochromaffin cells of the gut, mast cells and the pineal gland and is the rate-limiting enzyme involved in the biosynthesis of peripheral serotonin. Serotonin released into circulation is taken up by platelets via the serotonin transporter and stored in dense granules. It has been previously reported that mouse embryos removed from Tph1-deficient mothers present abnormal nervous system morphology. The goal of this study was to assess whether Tph1-deficiency results in behavioral abnormalities. We did not find any differences between Tph1-deficient and wild-type mice in general motor behavior as tested by rotarod, grip-strength test, open field and beam walk. However, here we report that Tph1 (−/−) mice display altered gait dynamics and deficits in rearing behavior compared to wild-type (WT) suggesting that tryptophan hydroxylase-1 expression has an impact on the nervous system.     

Effect of serotonin on cyclic nucleotides of human platelets.

Serotonin produced a 6 to 10 fold increase of cyclic GMP over baseline levels of this nucleotide in platelets. Maximum stimulation was reached within 30 sec to 1 min after addition of serotonin and was dependent upon its concentration in the medium. Inhibition of serotonin uptake by methysergide, dihydroergotamine and chloroimipramine did not influence the serotonin-induced stimulation of cyclic GMP but glutaraldehyde and formaldehyde blocked it completely. Cyclic AMP levels in platelets were not affected by serotonin. The serotonin-induced stimulation of cyclic GMP is independent of the uptake of this biogenic amine by platelets and is not due to platelet aggregation.     

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.     

Recent advances in understanding serotonin regulation of cardiovascular function

Serotonin is an important neurohormonal factor that has been implicated in cardiovascular function. It can regulate vascular tone, act directly on cardiomyocytes and stimulate chemosensitive nerves in the heart. Cardiovascular dysfunction is observed when serotonin signaling is altered or when variation in serotonin concentration occurs. Recent studies have provided evidence that, in the absence of peripheral serotonin synthesis, blood serotonin (which is almost exclusively stored in platelets) is markedly reduced, and that this drop leads to heart failure. This implies that the level of circulating serotonin is a key factor in maintaining normal cardiovascular activity. These findings offer new prospects for the use of serotonin in therapies for cardiovascular diseases.     

Paracrine modulation of cholangiocyte serotonin synthesis orchestrates biliary remodeling in adults

Paracrine signaling between cholangiocytes and stromal cells regulates biliary remodeling. Cholangiocytes have neuroepithelial characteristics and serotonin receptor agonists inhibit their growth, but whether they are capable of serotonin biosynthesis is unknown. We hypothesized that cholangiocytes synthesize serotonin and that cross talk between liver myofibroblasts (MF) and cholangiocytes regulates this process to influence biliary remodeling. Transwell cultures of cholangiocytes ± MF, and tryptophan hydroxylase-2 knockin (TPH2KI) mice with an inactivating mutation of the neuronal tryptophan hydroxylase (TPH) isoform, TPH2, were evaluated. Results in the cell culture models confirm that cholangiocytes have serotonin receptors and demonstrate for the first time that these cells express TPH2 and produce serotonin, which autoinhibits their growth but stimulates MF production of TGF-β(1). Increased TGF-β(1), in turn, counteracts autocrine inhibition of cholangiocyte growth by repressing cholangiocyte TPH2 expression. Studies of TPH2KI mice confirm that TPH2-mediated production of serotonin plays an important role in remodeling damaged bile ducts because mice with decreased TPH2 function have reduced biliary serotonin levels and exhibit excessive cholangiocyte proliferation, accumulation of aberrant ductules and liver progenitors, and increased liver fibrosis after bile duct ligation. This new evidence that cholangiocytes express the so-called neuronal isoform of TPH, synthesize serotonin de novo, and deploy serotonin as an autocrine/paracrine signal to regulate regeneration of the biliary tree complements earlier work that revealed that passive release of serotonin from platelets stimulates hepatocyte proliferation. Given the prevalent use of serotonin-modulating drugs, these findings have potentially important implications for recovery from various types of liver damage.     

Serotonin activates angiogenic phosphorylation signaling in human endothelial cells

Serotonin, a known neurotransmitter, also functions as an angiokine to promote angiogenesis. The majority of serotonin in the human body is stored in platelets, and platelet aggregation leads to significant release of serotonin in thrombotic tumor environment. We have investigated serotonin signaling in human endothelial cells. Through G-protein-coupled receptors, serotonin at physiologically relevant concentrations activated Src/PI3K/AKT/mTOR/p70S6K phosphorylation signaling, and this activation was similar to that seen with VEGF. This finding provides insight into the overlapping angiogenic signaling pathways stimulated by serotonin in tumor environment, and suggests one of the mechanisms underlying resistance to current VEGF-targeting antiangiogenic therapy against cancer.     

Type I Interferon-Mediated Skewing of the Serotonin Synthesis Is Associated with Severe Disease in Systemic Lupus Erythematosus

Serotonin, a highly pro-inflammatory molecule released by activated platelets, is formed by tryptophan. Tryptophan is also needed in the production of kynurenine, a process mediated by the type I interferon (IFN)-regulated rate-limiting enzyme indoleamine 2,3-dioxygenase (IDO). The aim of this study was to investigate levels of serotonin in patients with the autoimmune disease systemic lupus erythematosus (SLE), association to clinical phenotype and possible involvement of IDO in regulation of serotonin synthesis. Serotonin levels were measured in serum and plasma from patients with SLE (n=148) and healthy volunteers (n=79) by liquid chromatography and ELISA, as well as intracellularly in platelets by flow cytometry. We found that SLE patients had decreased serotonin levels in serum (p=0.01) and platelets (p<0.0001) as compared to healthy individuals. SLE patients with ongoing type I IFN activity, as determined by an in-house reporter assay, had decreased serum levels of serotonin (p=0.0008) as well as increased IDO activity (p<0.0001), as determined by the kynurenine/tryptophan ratio measured by liquid chromatography. Furthermore, SLE sera induced IDO expression in WISH cells in a type I IFN-dependent manner (p=0.008). Also platelet activation contributed to reduce overall availability of serotonin levels in platelets and serum (p<0.05). Decreased serum serotonin levels were associated with severe SLE with presence of anti-dsDNA antibodies and nephritis. In all, reduced serum serotonin levels in SLE patients were related to severe disease phenotype, including nephritis, suggesting involvement of important immunopathological processes. Further, our data suggest that type I IFNs, present in SLE sera, are able to up-regulate IDO expression, which may lead to decreased serum serotonin levels.     

A regulatory domain in the N terminus of tryptophan hydroxylase 2 controls enzyme expression

Serotonin is involved in a variety of physiological processes in the central nervous system and the periphery. As the rate-limiting enzyme in serotonin synthesis, tryptophan hydroxylase plays an important role in modulating these processes. Of the two variants of tryptophan hydroxylase, tryptophan hydroxylase 2 (TPH2) is expressed predominantly in the central nervous system, whereas tryptophan hydroxylase 1 (TPH1) is expressed mostly in peripheral tissues. Although the two enzymes share considerable sequence homology, the regulatory domain of TPH2 contains an additional 41 amino acids at the N terminus that TPH1 lacks. Here we show that the extended TPH2 N-terminal domain contains a unique sequence involved in the regulation of enzyme expression. When expressed in cultured mammalian cells, TPH2 is synthesized less efficiently and is also less stable than TPH1. Removal of the unique portion of the N terminus of TPH2 results in expression of the enzyme at a level similar to that of TPH1, whereas protein chimeras containing this fragment are expressed at lower levels than their wild-type counterparts. We identify a region centered on amino acids 10-20 that mediates the bulk of this effect. We also demonstrate that phosphorylation of serine 19, a protein kinase A consensus site located in this N-terminal domain, results in increased TPH2 stability and consequent increases in enzyme output in cell culture systems. Because this domain is unique to TPH2, these data provide evidence for selective regulation of brain serotonin synthesis.     

Isoform-specific roles of the GTPase activating protein Nadrin in cytoskeletal reorganization of platelets

Cytoskeletal reorganization of activated platelets plays a crucial role in hemostasis and thrombosis and implies activation of Rho GTPases. Rho GTPases are important regulators of cytoskeletal dynamics and function as molecular switches that cycle between an inactive and an active state. They are regulated by GTPase activating proteins (GAPs) that stimulate GTP hydrolysis to terminate Rho signaling. The regulation of Rho GTPases in platelets is not explored. A detailed characterization of Rho regulation is necessary to understand activation and inactivation of Rho GTPases critical for platelet activation and aggregation. Nadrin is a RhoGAP regulating cytoplasmic protein explored in the central nervous system. Five Nadrin isoforms are known that share a unique GAP domain, a serine/threonine/proline-rich domain, a SH3-binding motif and an N-terminal BAR domain but differ in their C-terminus. Here we identified Nadrin in platelets where it co-localizes to actin-rich regions and Rho GTPases. Different Nadrin isoforms selectively regulate Rho GTPases (RhoA, Cdc42 and Rac1) and cytoskeletal reorganization suggesting that – beside the GAP domain – the C-terminus of Nadrin determines Rho specificity and influences cell physiology. Furthermore, Nadrin controls RhoA-mediated stress fibre and focal adhesion formation. Spreading experiments on fibrinogen revealed strongly reduced cell adhesion upon Nadrin overexpression. Unexpectedly, the Nadrin BAR domain controls Nadrin-GAP activity and acts as a guidance domain to direct this GAP to its substrate at the plasma membrane. Our results suggest a critical role for Nadrin in the regulation of RhoA, Cdc42 and Rac1 in platelets and thus for platelet adhesion and aggregation.     

Stress Upregulates TPH1 but not TPH2 mRNA in the Rat Dorsal Raphe Nucleus: Identification of Two TPH2 mRNA Splice Variants

Serotonin is implicated in stress-related psychopathologies. Two isoforms of the rate-limiting enzyme of serotonin biosynthesis, tryptophan hydroxylase, TPH1 and TPH2, are known. We show here that in the rat dorsal raphe nucleus (DRN), the nucleus that contains the highest number of 5-HT neurons in the brain, TPH1 mRNA reveals a low level of expression but is detectable both by quantitative real-time PCR and in situ hybridization whereas in the pineal gland (PiG), TPH1 mRNA is strongly expressed. To examine effects of stress on TPH expression we exposed male Wistar rats to daily restraint stress for 1 week. As shown by quantitative real-time PCR, TPH1 mRNA is 2.5-fold upregulated by the stress in DRN but not in PiG. Using 3′-RACE, we identified two TPH2 mRNA splice variants in the rat DRN which differ in the length of their 3′-untranslated regions (UTRs). TPH2b (with a short 3′-UTR) is the predominant variant in the DRN, whereas TPH2a (with a longer 3′-UTR) shows a low abundance in this nucleus. In the PiG, only TPH2b is detectable revealing a low level of expression. Expression of both TPH2 splice variants is not affected by stress, neither in DRN nor in the PiG. These data indicate that TPH1 in the serotonergic neurons of the DRN might be relevant for stress-induced psychopathologies.     

Serotonin metabolism in thrombocytes and microvascular hemostasis in spontaneous arterial hypertension

Serotonin content and accumulation in platelets and its release from them, as well as changes in thrombus formation in mesenteric arterioles and venules of the small intestine have been investigated in control rats and rats with spontaneous hypertension (SHR). Serotonin accumulation in platelets was determined upon its incubation with platelets. Disodium ADP salt was used as an inductor of release. Laser-induced thrombosis was caused by microvessels exposure to impulse laser irradiation. The control animals revealed a significant difference between the initial serotonin platelet level and serotonin level upon incubation and release; in values, the values of basic thrombus-forming parameters were higher than in arterioles. In SHR there is a decrease in biogenic amine content in platelets, a depression in its accumulation and release, an increase in the time of thrombus growth, its size up to the separation of the first embolus and its length along the vascular wall. It is concluded that spontaneous hypertension is characterized by decreased functional activity of platelets and depressed resistance of arterioles and venules to thrombus formation.     

Compartmentalization of neuronal and peripheral serotonin synthesis in Drosophila melanogaster

In Drosophila, one enzyme (Drosophila tryptophan-phenylalanine hydroxylase, DTPHu) hydroxylates both tryptophan to yield 5-hydroxytryptophan, the first step in serotonin synthesis, and phenylalanine, to generate tyrosine. Analysis of the sequenced Drosophila genome identified an additional enzyme with extensive homology to mammalian tryptophan hydroxylase (TPH), which we have termed DTRHn. We have shown that DTRHn can hydroxylate tryptophan in vitro but displays differential activity relative to DTPHu when using tryptophan as a substrate. Recent studies in mice identified the presence of two TPH genes, Tph1 and Tph2, from distinct genetic loci. Tph1 represents the non-neuronal TPH gene, and Tph2 is expressed exclusively in the brain. In this article, we show that DTRHn is neuronal in expression and function and thus represents the Drosophila homologue of Tph2. Using a DTRHn-null mutation, we show that diminished neuronal serotonin affects locomotor, olfactory and feeding behaviors, as well as heart rate. We also show that DTPHu functions in vivo as a phenylalanine hydroxylase in addition to its role as the peripheral TPH in Drosophila, and is critical for non-neuronal developmental events.     

Primary dysfunction in aggregation and secretion of SHRSP platelets: not secondary to the circulation of "exhausted" platelets

The thrombin-induced secretion of [14C]-serotonin and adenine nucleotides from stroke-prone spontaneously hypertensive rats (SHRSP) platelets was markedly reduced with the development of hypertension accompanying hypo-aggregability compared with that from age-matched Wistar Kyoto rats (WKY) platelets. Calcium Ionophore A23187-induced secretion and aggregation were also attenuated in SHRSP platelets. Additionally, an enhancement of platelet secretion as well as aggregation by extracellular Ca2+ was less in SHRSP platelets than in WKY platelets. The platelet contents of adenine nucleotides and serotonin were not different between SHRSP and WKY at 5-16 weeks of age whereas they became significantly lower in SHRSP beginning at 22 weeks. The serotonin content in SHRSP platelets at 36 weeks of age was only 55% of that in WKY platelets. It is suggested that the reduced platelet aggregation and secretion observed in SHRSP platelets at ages lower than approximately 20 weeks are not secondary phenomena to the circulation of degranulated platelets, but the primary defect of SHRSP platelets appears to be an impaired function of Ca2+.     

Hyperserotonemia in autism: the potential role of 5HT-related gene variants

Increased platelet serotonin level (PSL) has been consistently found in a portion of autistic patients. Suggested mechanisms for hyperserotonemia in autism have been increased synthesis of serotonin (5HT) by tryptophan hydroxylase (TPH), increased uptake into platelets through 5HT transporter (5HTt), diminished release from platelets through 5HT2A receptor (5HT2Ar) and decreased metabolism by monoamine oxydase (MAOA). The allelic influence of genes, encoding the mentioned 5HT elements, on PSL was investigated in 63 autistic subjects. Our study shows that 5HTt-LPR and -1438AG 5HT(2Ar) genotypes did not significantly affect PSL. However, significantly higher PSLs were observed in subjects with "cc" genotype of a218c TPH and subjects with "4" genotype of uVNTR MAOA. In addition, when TPH-cc and MAOA-4 were combined as "high 5HT" genotypes, a correlative increase in PSL was observed with the increase in the number of "high 5HT" genotypes. These results suggest a possible synergistic effect of genes regulating 5HT synthesis/degradation in dysregulation of the peripheral 5HT homeostasis of autistic patients.     

Abnormal cardiac activity in mice in the absence of peripheral serotonin synthesis

Serotonin (5-HT) controls a wide range of biological functions. In the brain, its implication as a neurotransmitter and in the control of behavioral traits has been largely documented. At the periphery, its modulatory role in physiological processes, such as the cardiovascular function, is still poorly understood. The rate limiting enzyme of 5-HT synthesis, tryptophan hydroxylase (TPH), is encoded by two genes: the well characterized TPH1 gene and a recently identified TPH2 gene. Based on the study of a mutant mouse in which the TPH1 gene has been inactivated by replacement of the beta-galactosidase gene, we established that the neuronal TPH2 is expressed in neurons of the raphe nuclei and of the myenteric plexus, whereas the non-neuronal TPH1, as detected by beta-galactosidase expression, is expressed in the pineal gland and the enterochromaffin cells. Anatomic examination of the mutant mice revealed larger heart sizes as compared to wild-type. Histologic investigations indicated that the primary structure of the heart muscle is not affected. Hemodynamic analyses in mutant animals demonstrated abnormal cardiac activity which ultimately leads to heart failure. This is the first report linking loss of TPH1 gene expression, and thus of peripheral 5-HT, to a cardiac dysfunction phenotype. The TPH1 -/- mutant may be a valuable model for investigating cardiovascular dysfunction such as those observed in human heart failure.     

Sulphinpyrazone and the platelet serotoninergic mechanism in ischaemic heart disease.

A double blind study in 25 patients with ischaemic heart disease and 20 matched healthy controls examined the effect of sulphinpyrazone on the uptake of serotonin by platelets and the basal concentrations of serotonin in platelets. Uptake was measured using tritium labelled serotonin and basal concentrations estimated spectrophotofluorometrically. Serotonin uptake was significantly increased both in the patients with chronic stable angina of effort and in those with a history of myocardial infarction six months or more previously. Sulphinpyrazone reduced serotonin uptake from 94.25 (SE 8.65) to 57.86 (5.37) cpm/10(8) platelets after 24 weeks of treatment in the group with stable angina and from 137.45 (16.26) to 68.08 (8.38) cpm/10(8) platelets in the myocardial infarction group. Raised basal concentrations in the two groups were also reduced by sulphinpyrazone. Placebo had no effect on serotonin uptake or basal concentrations in either group of patients. The ability of sulphinpyrazone to inhibit uptake and reduce basal concentrations of serotonin in patients with ischaemic heart disease may be yet another mechanism through which this drug exerts its beneficial antiplatelet effect.     

Daily Variations of Functional Parameters and Density Distribution in Human Blood Platelets

Blood platelets play a critical role in the onset of myocardial infarction, which has been shown to have a circadian rhythmicity with a peak incidence in the morning. In an attempt to correlate platelet parameters with the outcome of cardiovascular diseases, we studied the daily (24-h) variation of the following platelet parameters: distribution pattern of functional heterogeneous platelet subpopulations; serotonin uptake; ketanserin binding; aggregation upon thrombin, serotonin, and ADP stimulation; and platelet count. Furthermore, we analyzed the tryptophan and serotonin concentrations in the blood samples. The percentage of less dense platelets, which represent the subpopulation with the highest preactivation, showed a rhythmicity period of 24 h and an acrophase at 21:18 h. The time course of intermediate and high density platelets was inverse to that of low density platelets. The serotonin uptake exhibited also a rhythmicity with a 24-h period. The acrophase was at 13:50 h. The aggregation curves were inverse to the ketanserin binding curves. The serotonin concentration exhibited a 12-h rhythmicity. The results obtained suggest that (a) changes in platelet activity are reflected by several parameters of platelet function that underlie daily variations; (b) the aggregation curves show a peak in the morning, with an additional peak in the afternoon; and (c) changes in the distribution pattern occur independently from variations in platelet functions like aggregation and serotonin binding.     

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.     

A comparative analysis of serotonin level in rat platelets,serum, and brain during aging

Serotonin functions as a neurotransmitter in the central nervous system and takes part in vascular tone, gastrointestinal motility, and blood coagulation at the periphery. New data on a correlation between serotonin level in platelets and cerebrospinal fluid (Audhya et al., 2012) have renewed interest in the hypothesis that considers a platelet as a model of serotoninergic neuron. In this study, using high performance liquid chromatography, we compared the serotonin level in platelets, serum and different brain regions in 6- and 24-month-old rats. It was found that serotonin level decreased from 0.768 to 0.359 μg per 10⁹ cells in platelets and increased in midbrain from 0.260 to 0.439 μg per 1 g of wet weight during the animal aging. The differences between young and old animals in the serotonin level in serum and other brain regions were statistically not significant. Hence, despite the attractiveness of the hypothesis considering the platelet as a neuron model the data on the platelet serotonin transport should be extrapolated on the neuronal transport with caution, especially for the aging process.