Expression of CFTR and Cl(-) conductances in cells of pulmonary neuroepithelial bodies.
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The pulmonary neuroendocrine cell system comprises solitary neuroendocrine cells and clusters of innervated cells or neuroepithelial bodies (NEBs). NEBs figure prominently during the perinatal period when they are postulated to be involved in physiological adaptation to air breathing. Previous studies have documented hyperplasia of NEBs in cystic fibrosis (CF) lungs and increased neuropeptide (bombesin) content produced by these cells, possibly secondary to chronic hypoxia related to CF lung disease. However, little is known about the role of NEBs in the pathogenesis of CF lung disease. In the present study, using a panel of cystic fibrosis transmembrane conductance regulator (CFTR)-specific antibodies and confocal microscopy in combination with RT-PCR, we demonstrate expression of CFTR message and protein in NEB cells of rabbit neonatal lungs. NEB cells expressed CFTR along with neuroendocrine markers. Confocal microscopy established apical membrane localization of the CFTR protein in NEB cells. Cl(-) conductances corresponding to functional CFTR were demonstrated in NEB cells in a fresh lung slice preparation. Our findings suggest that NEBs, and related neuroendocrine mechanisms, likely play a role in the pathogenesis of CF lung disease, including the early stages before establishment of chronic infection and chronic lung disease. (+info)
Immunochemical characterization of brain and pineal tryptophan hydroxylase.
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Recombinant mouse tryptophan hydroxylase (TPH) was expressed in Escherichia coli, using a bacterial expression vector and has been purified to homogeneity by sonication followed by Sepharose 4B column chromatography and native slab gel electrophoresis. This purified enzymatically active TPH protein was used for production of a specific antiserum. This antiserum identified the predicted TPH band (molecular weight, 54 kDa) on Western blot of crude extracts from the rat and mouse dorsal raphe, and the rat pineal gland. However, this antiserum recognized an additional protein band of lower molecular weight (48 kDa) in pineal extract. It is not clear whether the 48 kDa TPH band represents an isozyme or a protease cleavage product of TPH. Since the pineal gland contains higher TPH mRNA and lower TPH activity when it is compared with dorsal raphe nucleus enzyme, this lower molecular weight TPH may participate in the reduced TPH specific activity. In addition, there are no specific TPH inhibitors in the pineal gland and this lower molecular weight TPH is inactive or has a very low specific activity. This antiserum specifically immunostained serotonergic cell bodies in the dorsal raphe nuclei, some large caliber serotonergic processes in the dorsal raphe area as well as terminals in the olfactory bulb. It also immunolabeled the pineal gland and immunoprecipitated equally well TPH protein from the dorsal raphe nucleus and the pineal gland in a concentration-dependent manner. (+info)
Influence of tryptophan hydroxylase and serotonin transporter genes on fluvoxamine antidepressant activity.
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The aim of the present study was to test a possible effect of the A218C tryptophan hydroxylase (TPH) gene variant on the antidepressant activity of fluvoxamine in a sample of major and bipolar depressives, with or without psychotic features. Two hundred and seventeen inpatients were treated with fluvoxamine 300 mg and either placebo or pindolol in a double blind design for 6 weeks. The severity of depressive symptoms was weekly assessed with the Hamilton Rating Scale for Depression. TPH allelic variants were determined in each subject by using a PCR-based technique. No significant finding was observed in the overall sample as well as in the pindolol group, while TPH*A/A was associated with a slower response to fluvoxamine treatment in subjects not taking pindolol (P = 0.001). This effect was independent from the previously reported influence of 5-HTTLPR polymorphism. If confirmed, these results may shed further light on the genetically determined component of the response to pharmacological treatments, thus helping the clinician to individualize each patient's therapy according to their genetic pattern. (+info)
Conversion of L-tryptophan to serotonin and melatonin in human melanoma cells.
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We showed in human melanoma cells tryptophan hydroxylase (TPH) and hydroxyindole methyltransferase genes expression with the sequential enzymatic activities of TPH, serotonin (Ser) N-acetyltransferase and hydroxyindole methyltransferase. The presence of the products Ser, 5OH-tryptophan, N-acetylserotonin, melatonin (Mel), 5-methoxytryptamine and 5-methoxytryptophol was documented by liquid chromatography-mass spectrometry. Thus, human melanoma cells can synthesize and metabolize Ser and Mel. (+info)
Up-regulation of tryptophan hydroxylase expression and serotonin synthesis by sertraline.
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The neurotransmitter serotonin is involved in a variety of brain functions, and abnormal changes in serotonin neurotransmission are associated with an array of psychiatric disorders, including depression. Sertraline is a selective serotonin reuptake inhibitor (SSRI) and an effective antidepressant. Sertraline increases the serotonin concentration in the synaptic cleft by a short-term action; however, clinical improvement is observed only after several weeks, suggesting that the therapeutic effect may be caused by long-term alterations in serotonin transmission. We determined the effects of sertraline on serotonin synthesis in vivo and in vitro. Long-term treatment of rats with sertraline up-regulated mRNA and protein levels of the serotonin-synthesizing enzyme tryptophan hydroxylase (TPH), as determined by in situ hybridization and immunocytochemistry, respectively. In vitro studies using RBL-2H3 cells also showed an increase in mRNA and protein levels of TPH by sertraline, as determined by Northern blot and immunoblot analyses, respectively. This was accompanied by increases in the levels of TPH enzymatic activity and total serotonin. These data demonstrate that in addition to the known short-term action as an uptake blocker, sertraline also exerts a long-term effect on the serotonin neurotransmission by enhancing serotonin synthesis. A similar effect was observed with another SSRI, fluoxetine, but not with the non-SSRI chlorpromazine. The up-regulation of TPH gene expression by sertraline was attenuated by the protein kinase A (PKA) inhibitor N-[2-(p-bromocinnamylamine)-ethyl]-5-isoquinolinesulfonamine, suggesting that a mechanism involving the PKA signaling pathway might at least in part mediate the long-term therapeutic action. (+info)
Serotoninergic and melatoninergic systems are fully expressed in human skin.
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We investigated the cutaneous expression of genes and enzymes responsible for the multistep conversion of tryptophan to serotonin and further to melatonin. Samples tested were human skin, normal and pathologic (basal cell carcinoma and melanoma), cultured normal epidermal and follicular melanocytes, melanoma cell lines, normal neonatal and adult epidermal and follicular keratinocytes, squamous cell carcinoma cells, and fibroblasts from dermis and follicular papilla. The majority of the samples showed simultaneous expression of the genes for tryptophan hydroxylase, arylalkylamine N-acetyltransferase (AANAT), and hydroxyindole-O-methyltransferase (HIOMT). The products of AANAT activity were identified by RP-HPLC with fluorimetric detection in human skin and in cultured normal and malignant melanocytes and immortalized keratinocytes; HIOMT activity was detected in human skin, keratinocytes, and melanoma cells. N-acetylserotonin (NAS) was detected by RP-HPLC in human skin extracts. NAS identity was confirmed further by LC/MS in keratinocytes. In conclusion, we provide evidence that the human skin expresses intrinsic serotonin and melatonin biosynthetic pathways. (+info)
Postural modifications and neuronal excitability changes induced by a short-term serotonin depletion during neonatal development in the rat.
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Serotonin (5-HT) plays an important role both in the development and in the recovery of locomotion after spinalization in vertebrates. We investigated the contribution of the serotonergic system to the maturation of the lumbar motoneurons and networks in the neonatal rat. A 5-HT synthesis inhibitor, p-chlorophenylalanine (PCPA), was administered daily from the first postnatal day (P0) onward. This protocol depleted serotonin in the spinal cord within 3-4 d, as demonstrated by immunohistochemistry. PCPA-treated rats exhibited postural changes characterized by lesser flexion at the knee and ankle levels and lesser extension of the hip. Posture was asymmetric, suggesting possible deficits in the interlimb coordination. Intracellular recordings were made at P3-5 from motoneurons innervating different hindlimb muscles, using the in vitro brainstem-spinal cord-nerve-attached preparation. In PCPA-treated rats, the conduction velocity of motoneurons was increased, and their excitability was decreased (because of higher rehobase and input conductance) compared with sham animals. In accordance with postural observations, changes were more pronounced in hip extensor/knee flexor than in ankle extensor motoneurons. The maturation of repetitive firing properties was stopped by PCPA treatment, although PCPA, applied in vitro, had no effect on membrane properties. The spontaneous endogenously generated activity, which is a characteristic of immature networks, was increased in PCPA-treated rats, suggesting that developing lumbar networks are sensitive to 5-HT levels. Serotonin may play a critical role during development in regulating the balance between the excitability of motoneurons and that of interneurons. Interneuronal excitability is crucial for the activity-dependent development of spinal cord networks. (+info)
7-Hydroxytryptophan, a novel, specific, cytotoxic agent for carcinoids and other serotonin-producing tumors.
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BACKGROUND: Carcinoids and small cell lung carcinomas stimulate their growth in an autocrine manner by releasing serotonin, an effect that is blocked by selective serotonergic receptor antagonists that, unfortunately, exert undesirable side effects on serotonergic central nervous function. Moreover, conventional chemotherapeutic agents, such as streptozocin, fluorouracil, cyclophosphamide, and doxorubicin, which target tumor cells directly, have produced disappointing results in the treatment of patients with these tumors in the advanced stage. Therefore, there is still a need for more specific and potent chemotherapeutic agents in the fight against serotonin-producing tumors. METHODS: The authors synthesized 7-hydroxytryptophan to test its chemotherapeutic value in cell culture, using a system consisting of serotonin-producing and nonproducing cell lines. RESULTS: The authors chose tryptophan hydroxylase, the rate-limiting enzyme of serotonin biosynthesis, which is expressed highly in small cell lung carcinomas and carcinoids, as a target for the induction of cellular suicide by chemotherapy. They found that this otherwise substrate specific enzyme was capable of metabolizing in situ a harmless tryptophan analogue, 7-hydroxytryptophan, to a potent toxin, 5,7-dihydroxytryptamine, a conversion blocked by the specific tryptophan hydroxylase inhibitor parachlorophenylalanine. CONCLUSIONS: These data suggest that 7-hydroxytryptophan may be a highly specific chemotherapeutic compound against serotonin-producing tumors that also interferes with the autocrine capabilities of serotonin synthesis. (+info)