Tryptophan degradation enzymes and Angiotensin (1-7) expression in human placenta.

Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO) are key enzymes for tryptophan degradation, regulating immune tolerance during pregnancy. The intrauterine renin-angiotensin system is also involved in the progression of a healthy pregnancy. Angiotensin(1-7) maintains the integrity of fetal membranes via counteracting the pro-inflammatory actions of Angiotensin II. No data are available on placental Angiotensin(1-7) co-expression with TDO. We aimed to characterize TDO mRNA expression and its localization in different areas of the placenta of physiological pregnancies delivered at term; its co-expression with Angiotensin(1-7) and its correlation with the plasma kynurenine/tryptophan (Kyn/Trp) ratio was investigated. This prospective observational study included a nonconsecutive series of 20 singleton uncomplicated pregnancies delivered vaginally. TDO mRNA was expressed in both maternal and fetal sides of the placentas and TDO protein also in the villi and it was co-expressed with IDO1 in almost half of the placental cells at these sites. The percentage of TDO+ and IDO1+ cells appeared to be influenced by maternal pre-gestational smoking and newborn weight. A strong correlation was found between the percentage of TDO+ and IDO1+ cells in the villi. TDO+ cells also expressed Angiotensin(1-7), with a higher percentage on the fetal side and in the villi compared to the maternal one. Kyn/Trp plasma ratio was not correlated with IDO and TDO expression nor with the patient's characteristics. Collectively, our data indicate that TDO is detectable in placental tissue and is co-expressed with IDO and with Angiotensin(1-7)+ on the fetal side and in the villi.

Interleukin-33 Induces the Enzyme Tryptophan Hydroxylase 1 to Promote Inflammatory Group 2 Innate Lymphoid Cell-Mediated Immunity.

Group 2 innate lymphoid cells (ILC2s) regulate immunity, inflammation, and tissue homeostasis. Two distinct subsets of ILC2s have been described: steady-state natural ILC2s and inflammatory ILC2s, which are elicited following helminth infection. However, how tissue-specific cues regulate these two subsets of ILC2s and their effector functions remains elusive. Here, we report that interleukin-33 (IL-33) promotes the generation of inflammatory ILC2s (ILC2INFLAM) via induction of the enzyme tryptophan hydroxylase 1 (Tph1). Tph1 expression was upregulated in ILC2s upon activation with IL-33 or following helminth infection in an IL-33-dependent manner. Conditional deletion of Tph1 in lymphocytes resulted in selective impairment of ILC2INFLAM responses and increased susceptibility to helminth infection. Further, RNA sequencing analysis revealed altered gene expression in Tph1 deficient ILC2s including inducible T cell co-stimulator (Icos). Collectively, these data reveal a previously unrecognized function for IL-33, Tph1, and ICOS in promoting inflammatory ILC2 responses and type 2 immunity at mucosal barriers.

Reduction of Total Brain and Cerebellum Volumes Associated With Neuronal Autoantibodies in Patients With APECED.

Context: In autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), autoantibodies (AutoAbs) labeling brain neurons were reported; conversely, brain MRI alterations associated with these AutoAbs were never reported. Objectives: To describe brain alterations in APECED and to correlate them with AutoAbs against glutamic acid decarboxylase (GAD), tyrosine hydroxylase (TH), and 5-tryptophan hydroxylase (5-HT) neurons. Design and Participants: Fourteen Sardinian patients with APECED and age-matched control subjects were recruited for MRI analysis and blood sampling to detect AutoAbs to GAD, TH, and 5-HT neurons by using rat brain sections. The majority of patients (n = 12) were investigated for AutoAbs a decade earlier, and 7 of 12 were positive for AutoAbs to GAD and TH neurons. Main Outcomes: Patients with APECED had smaller cerebellum and gray matter volumes, with a ventricular enlargement and a total cerebrospinal fluid (CSF) increase, compared with controls (P < 0.01). In 11 of 14 patients, brain abnormalities were associated with AutoAbs to GAD or TH neurons (titer 1:100 to 15,000) that had persisted for 10 years in 7 of 11 patients. AutoAbs to 5-HT neurons were revealed in all patients with AutoAbs to TH neurons. A decrease in whole brain and cerebellum volumes (P = 0.028) was associated with AutoAbs to GAD neurons, and a CSF increase was associated with AutoAbs to GAD and TH/5-HT neurons (P < 0.05). HLA alleles did not appear to be involved in neuronal autoimmunity. Conclusions: Brain alterations and neuronal AutoAbs were observed in 78.6% of Sardinian patients with APECED, suggesting a brain autoimmune reaction. Prolonged clinical follow-up must be conducted for the possible appearance of clinical neurologic consequences.

A new mutation site in the AIRE gene causes autoimmune polyendocrine syndrome type 1.

Autoimmune polyendocrine syndrome type 1 (APS-1, OMIM 2403000) is a rare autosomal recessive disease that is caused by autoimmune regulator (AIRE). The main symptoms of APS-1 are chronic mucocutaneous candidiasis, autoimmune adrenocortical insufficiency (Addison's disease) and hypoparathyroidism. We collected APS-1 cases and analysed them. The AIRE genes of the patient and his family members were sequenced to identify whether the APS-1 patient had an AIRE mutation. We discovered a mutation site (c.206A>C) that had never before been reported in the AIRE gene located in exon 2 of the AIRE gene. This homogyzous mutation caused a substitution of the 69th amino acid of the AIRE protein from glutamine to proline (p.Q69P). A yeast two-hybrid assay, which was used to analyse the homodimerization properties of the mutant AIRE protein, showed that the mutant AIRE protein could not interact with the normal AIRE protein. Flow cytometry and RT-qPCR analyses indicated that the new mutation site could decrease the expression levels of the AIRE, glutamic acid decarboxylase 65 (GAD65) and tryptophan hydroxylase-1 (TPH1) proteins to affect central immune tolerance. In conclusion, our research has shown that the new mutation site (c.206A>C) may influence the homodimerization and expression levels and other aspects of the AIRE protein. It may also impact the expression levels of tissue-restricted antigens (TRAs), leading to a series of autoimmune diseases.

Gastrointestinal immunity against tryptophan hydroxylase-1, aromatic L-amino-acid decarboxylase, AIE-75, villin and Paneth cells in APECED.

Gastrointestinal dysfunction is a disabling manifestation of APECED possibly related to an autoimmune intestinal aggression. We evaluated its features in a cohort of 31 Finnish patients. The most frequent manifestations were constipation (48%), diarrhea, dysphagia and retrosternal pain (45%). AADC and TPH-1 autoantibodies were detected in 51% and 45% of the patients, respectively. Forty-three percent displayed a T-cell response to AADC. One third of the patients also had AIE-75 (33%) and villin (29%)-specific autoantibodies while antibodies against brush borders and Paneth cells were detected in 29% and 20%, respectively. Intestinal IL-17 expression was absent/decreased in 77% of the cases. Duodenal CgA and serotonin expression was absent/decreased in 50% and 66% of the patients, respectively. Constipation correlated with lacking serotonin expression and AADC antibodies (p < 0.05).

Tryptophan hydroxylase autoantibodies as markers of a distinct autoimmune gastrointestinal component of autoimmune polyendocrine syndrome type 1.

BACKGROUND: Autoantibodies to tryptophan hydroxylase (TPHAbs) directed against serotonin-producing enterochromaffin cells (EC) have been reported in autoimmune-polyendocrine-syndrome type 1 (APS-1) patients with gastrointestinal dysfunction (GID). Serotonin plays a critical role in enteric function and its peripheral blood levels reflect serotonin release from the gastrointestinal tract. AIMS: We test the hypothesis that TPHAbs mark a distinct autoimmune component of APS-1 characterized by an autoimmune attack toward EC, which results in clinical GID. METHODS: TPHAbs were measured in 64 APS-1 patients. Endoscopy with gastric (antrum/body) and duodenal biopsy was carried in 16 TPHAbs+ patients (8 with and 8 without GID) and in 2 TPHAbs- patients (without GID). Immunohistochemistry of biopsy specimens was carried out using antibodies to serotonin, chromogranin-A, CD3, CD4, CD8, and CD20. Serotonin serum levels were measured in TPHAbs+ and TPHAbs- patients who had endoscopy. RESULTS: Thirty-seven of 64 patients were TPHAbs+ (11/12 with GID and 26/52 without GID; P < .001). Gastric and duodenal biopsies in all 8 TPHAb+ patients with GID showed lymphocytic infiltration with increased CD3+CD8+ intraepithelial lymphocytes and absence of EC. Furthermore, mean serotonin serum levels were below the normal range in TPHAb+ patients with GID (P < .01). In 8 TPHAb+ patients without GID gastric and duodenal biopsies showed different grades of inflammatory infiltration and reduced number of EC. Mean serotonin serum levels were near the lower limit of the normal range. In all TPHAbs+ patients the biopsies showed a reduced number of chromogranin-A positive cells consistent with enteroendocrine cells depletion. TPHAbs- patients without GID showed normal gastrointestinal mucosa and serotonin serum levels. CONCLUSIONS: TPHAbs appear to be markers of a distinct autoimmune component of APS-1. Progressive involvement of the gastrointestinal EC leads to the transition from preclinical to clinical disease, characterized by GID and reduced serotonin serum levels.

Production of serotonin by tryptophan hydroxylase 1 and release via platelets contribute to allergic airway inflammation.

RATIONALE: 5-Hydroxytryptamine (5-HT) is involved in the pathogenesis of allergic airway inflammation (AAI). It is unclear, however, how 5-HT contributes to AAI and whether this depends on tryptophan hydroxylase (TPH) 1, the critical enzyme for peripheral 5-HT synthesis. OBJECTIVES: To elucidate the role of TPH1 and the peripheral source of 5-HT in asthma pathogenesis. METHODS: TPH1-deficient and TPH1-inhibitor-treated animals were challenged in ovalbumin and house dust mite models of AAI. Experiments with bone marrow chimera, mast cell-deficient animals, platelets transfusion, and bone marrow dendritic cells (BMDC) driven model of AAI were performed. 5-HT levels were measured in bronchoalveolar lavage fluid or serum of animals with AAI and in human asthma. MEASUREMENTS AND MAIN RESULTS: 5-HT levels are increased in bronchoalveolar lavage fluid of mice and people with asthma after allergen provocation. TPH1 deficiency and TPH1 inhibition reduced all cardinal features of AAI. Administration of exogenous 5-HT restored AAI in TPH1-deficient mice. The pivotal role of 5-HT production by structural cells was corroborated by bone marrow chimera experiments. Experiments in mast cell-deficient mice revealed that mast cells are not a source of 5-HT, whereas transfusion of platelets from wild-type and TPH1-deficient mice revealed that only platelets containing 5-HT enhanced AAI. Lack of endogenous 5-HT in vitro and in vivo was associated with an impaired Th2-priming capacity of BMDC. CONCLUSIONS: In summary, TPH1 deficiency or inhibition reduces AAI. Platelet- and not mast cell-derived 5-HT is pivotal in AAI, and lack of 5-HT leads to an impaired Th2-priming capacity of BMDC. Thus, targeting TPH1 could offer novel therapeutic options for asthma.

Autoantibodies against aromatic amino acid hydroxylases in patients with autoimmune polyendocrine syndrome type 1 target multiple antigenic determinants and reveal regulatory regions crucial for enzymatic activity.

Patients with autoimmune polyendocrine syndrome type 1 (APS-1) frequently have autoantibodies directed against the aromatic amino acid hydroxylases tryptophan hydroxylase (TPH) and tyrosine hydroxylase (TH). We aimed to characterize these autoantibodies with regard to their antigenic determinants, their influence on enzymatic activity and their clinical associations. In particular, we wanted to compare autoantibodies against the two different isoforms of TPH, which display different tissue distribution. Using sera from 48 Scandinavian APS-1 patients we identified 36 patients (75%) with antibodies against one or more of these three enzymes. Antibodies against TPH1, but not TPH2, were associated with malabsorption in the whole Scandinavian cohort, while TH antibodies were associated with dental enamel hypoplasia in Norwegian patients. Subsequent experiments with selected patient sera indicated that while the C-terminal domain was the immunodominant part of TPH1, the epitopes of TPH2 and TH were mainly located in the N-terminal regulatory domains. We also identified a TPH1 specific epitope involved in antibody mediated inhibition of enzyme activity, a finding that provides new insight into the enzymatic mechanisms of the aromatic amino acid hydroxylases and knowledge about structural determinants of enzyme autoantigens. In conclusion, TPH1, TPH2 and TH all have unique antigenic properties in spite of their structural similarity.

Tryptophan hydroxylase-1 regulates immune tolerance and inflammation.

Nutrient deprivation based on the loss of essential amino acids by catabolic enzymes in the microenvironment is a critical means to control inflammatory responses and immune tolerance. Here we report the novel finding that Tph-1 (tryptophan hydroxylase-1), a synthase which catalyses the conversion of tryptophan to serotonin and exhausts tryptophan, is a potent regulator of immunity. In models of skin allograft tolerance, tumor growth, and experimental autoimmune encephalomyelitis, Tph-1 deficiency breaks allograft tolerance, induces tumor remission, and intensifies neuroinflammation, respectively. All of these effects of Tph-1 deficiency are independent of its downstream product serotonin. Because mast cells (MCs) appear to be the major source of Tph-1 and restoration of Tph-1 in the MC compartment in vivo compensates for the defect, these experiments introduce a fundamentally new mechanism of MC-mediated immune suppression that broadly impacts multiple arms of immunity.

Autoantibodies in autoimmune polyglandular syndrome type I patients react with major brain neurotransmitter systems.

Patients with autoimmune polyglandular syndrome type I (APS1) often display high titers of autoantibodies (autoAbs) directed against aromatic L-amino acid decarboxylase (AADC), tyrosine hydroxylase (TH), tryptophan hydroxylase (TPH), and glutamic acid decarboxylase (GAD). Neurological symptoms, including stiff-man syndrome and cerebellar ataxia, can occur in subjects with high levels of GAD autoAbs, particularly when patient sera can immunohistochemically stain gamma-aminobutyric acid (GABA) neurons. However, it was not known if APS1 sera can also stain major monoamine systems in the brain. Therefore, in this work we applied sera from 17 APS1 patients known to contain autoAbs against AADC, TH, TPH, and/or GAD to rat brain sections and processed the sections according to the sensitive immunohistochemical tyramide signal amplification method. We found that autoAbs in sera from 11 patients were able to stain AADC-containing dopaminergic, serotonergic, and noradrenergic as well as AADC only (D-group) neurons and fibers in the rat brain, in several cases with a remarkably high quality and sensitivity (dilution up to 1:1,000,000); and, since they are human antibodies, they offer a good opportunity for performing multiple-labeling experiments using antibodies from other species. Six APS1 sera also stained GABAergic neuronal circuitries. Similar results were obtained in the mouse and primate brain. Our data demonstrate that many APS1 sera can immunostain the major monoamine and GABA systems in the brain. Only in a few cases, however, there was evidence that these autoAbs can be associated with neurological manifestations in APS1 patients, as, e.g., shown in previous studies in stiff-man syndrome.

Immunohistochemical evidence for the presence of tryptophan hydroxylase in the brains of insects as revealed by sheep anti-tryptophan hydroxylase polyclonal antibody.

Immediately following the discovery of tryptophan hydroxylase in Drosophila, we demonstrated the presence of tryptophan hydroxylase in the brain of the beetle Harmonia axyridis (Coleoptera: Coccinellidae). However, whether tryptophan hydroxylase is present in the brains of other insects is still a matter of discussion. In the current study, sheep anti-tryptophan hydroxylase polyclonal antibody has been applied to test for tryptophan hydroxylase immunoreactivity in a broader taxonomic range of insect brains, including holometabolous and hemimetabolous insects: one species each of Coleoptera, Hymenoptera, Diptera, and Blattaria, and two species of Lepidoptera. All species show consistent tryptophan hydroxylase immunoreactivity with distribution patterns matching that of serotonin. The immuno-positive results of such an antibody in brains from diverse orders of insects suggest that specific tryptophan hydroxylase responsible for central serotonin synthesis is probably present in the brains of all insects.

Sicilian family with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) and lethal lung disease in one of the affected brothers.

INTRODUCTION: We report the clinical and immunological features of the autoimmune regulator gene (AIRE) in two Sicilian brothers with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). They were compound heterozygotes with R203X/R257X. Both had oral candidiasis since the first year of life and later developed hypoparathyroidism and Addison disease. The elder brother had experienced recurrent lower respiratory infections since 5 years of age and over the years developed severe obstructive lung disease with bronchiectasis, which led to death at 18 years of age. Both brothers had circulating autoantibodies against tryptophan hydroxylase and serotonin-producing cells were absent in the duodenal mucosa. This was associated with intestinal dysfunction in only the elder brother. CONCLUSION: (1) In the first Sicilian family with APECED reported up to now we found a heterozygous mutation that had been previously reported only once. (2) In the older brother of this family we observed a severe and lethal lung disease; this case adds to a growing literature describing this association between APECED and respiratory illnesses. (3) Tryptophan hydroxylase antibodies might be hypothesized to be the marker of an autoimmune gastrointestinal illness possibly associated with APECED.

Role of a serotonin precursor in development of gut microvilli.

Monoamines exert diverse functions in various cells in peripheral organs as well as in the central nervous system. 5-Hydroxy-l-tryptophan (5-HTP) has been simply regarded as a precursor of serotonin, and it is believed that the biological significance of 5-HTP is essentially ascribable to the production of serotonin. Systemic treatment with 5-HTP is often applied to patients with low serotonin levels in the brain. Here we show that endogenous and exogenous 5-HTP but not serotonin induced the development of microvilli in the gut villi epithelium. In contrast, serotonin but not 5-HTP regulated phagocytosis by macrophages. 5-HTP specifically induced actin remodeling and decreased phosphorylation of extracellular signal-regulated kinase (ERK) in the gut, whereas serotonin stimulated actin remodeling and increased ERK phosphorylation in macrophages. Functionally, inhibition of ERK activity promoted the development of microvilli in the gut and ameliorated phagocytosis by macrophages. Thus, 5-HTP and serotonin contribute to distinct cell-type-specific functions via common mediators. Our study might create an opportunity to explore the effects of exogenously applied 5-HTP in humans.

Glucocorticoid modulation of tryptophan hydroxylase-2 protein in raphe nuclei and 5-hydroxytryptophan concentrations in frontal cortex of C57/Bl6 mice.

Considerable attention has focused on regulation of central tryptophan hydroxylase (TPH) activity and protein expression. At the time of these earlier studies, it was thought that there was a single central TPH isoform. However, with the recent identification of TPH2, it becomes important to distinguish between regulatory effects on the protein expression and activity of the two isoforms. We have generated a TPH2-specific polyclonal antiserum (TPH2-6361) to study regulation of TPH2 at the protein level and to examine the distribution of TPH2 expression in rodent and human brain. TPH2 immunoreactivity (IR) was detected throughout the raphe nuclei, in lateral hypothalamic nuclei and in the pineal body of rodent and human brain. In addition, a prominent TPH2-IR fiber network was found in the human median eminence. We recently reported that glucocorticoid treatment of C57/Bl6 mice for 4 days markedly decreased TPH2 messenger RNA levels in the raphe nuclei, whereas TPH1 mRNA was unaffected. The glucocorticoid-elicited inhibition of TPH2 gene expression was blocked by co-administration of the glucocorticoid receptor antagonist mifepristone (RU-486). Using TPH2-6361, we have extended these findings to show a dose-dependent decrease in raphe TPH2 protein levels in response to 4 days of treatment with dexamethasone; this effect was blocked by co-administration of mifepristone. Moreover, the glucocorticoid-elicited inhibition of TPH2 was functionally significant: serotonin synthesis was significantly reduced in the frontal cortex of glucocorticoid-treated mice, an effect that was blocked by mifepristone co-administration. This study provides further evidence for the glucocorticoid regulation of serotonin biosynthesis via inhibition of TPH2 expression, and suggest that elevated glucocorticoid levels may be relevant to the etiology of psychiatric diseases, such as depression, where hypothalamic-pituitary-adrenal axis dysregulation has been documented.

Differential tissue distribution of tryptophan hydroxylase isoforms 1 and 2 as revealed with monospecific antibodies.

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of the neurotransmitter serotonin. Once thought to be a single-gene product, TPH is now known to exist in two isoforms-TPH1 is found in the pineal and gut, and TPH2 is selectively expressed in brain. Heretofore, probes used for localization of TPH protein or mRNA could not distinguish between the TPH isoforms because of extensive homology shared by them at the nucleotide and amino acid level. We have produced monospecific polyclonal antibodies against TPH1 and TPH2 using peptide antigens from nonoverlapping sequences in the respective proteins. These antibodies allow the differentiation of TPH1 and TPH2 upon immunoblotting, immunoprecipitation, and immunocytochemical staining of tissue sections from brain and gut. TPH1 and TPH2 antibodies do not cross-react with either tyrosine hydroxylase or phenylalanine hydroxylase. Analysis of mouse tissues confirms that TPH1 is the predominant form expressed in pineal gland and in P815 mastocytoma cells with a molecular weight of 51 kDa. TPH2 is the predominant enzyme form expressed in brain extracts from mesencephalic tegmentum, striatum, and hippocampus with a molecular weight of 56 kDa. Antibody specificity against TPH1 and TPH2 is retained across mouse, rat, rabbit, primate, and human tissues. Antibodies that distinguish between the isoforms of TPH will allow studies of the differential regulation of their expression in brain and periphery.

Autoantibodies to human tryptophan hydroxylase and aromatic L-amino acid decarboxylase.

OBJECTIVE: To assess the prevalence of autoantibodies (Abs) to tryptophan hydroxylase (TPH) and aromatic l-amino acid decarboxylase (AADC) in patients with different autoimmune diseases and to analyse their respective epitopes. DESIGN: TPH and AADC Abs were measured in an immunoprecipitation assay using (35)S-labelled full-length and fragments of TPH and AADC. METHODS: Patients with different autoimmune adrenal diseases (n=84), non-adrenal autoimmune diseases (n=37), idiopathic vitiligo (n=8) and 56 healthy blood donors were studied. RESULTS: Fourteen of twenty-three (61%) of patients with autoimmune polyglandular syndrome (APS) type I and 1/34 (3%) of patients with isolated Addison's disease (AD) were positive for TPH Abs. None of the patients with APS type II (n=27), coeliac disease (n=10), autoimmune thyroid disease (AITD) (n=11), type 1 diabetes mellitus (DM) (n=16) or idiopathic vitiligo (n=8) was positive for TPH Abs. AADC Abs were detected in 12/23 (52%) patients with APS type I, in 1/29 (3%) patients with APS type II and 1/34 (3%) patients with isolated AD. None of the patients with coeliac disease, type 1 DM, AITD or idiopathic vitiligo was positive for AADC Abs. TPH Abs were found to interact with the C-terminal amino acids (aa) 308-423, central aa 164-205 and N-terminal aa 1-105 of the TPH molecule. AADC Ab binding epitopes were within the C-terminal aa 382-483, the central aa 243-381 and the N-terminal aa 1-167. CONCLUSIONS: Our study suggests that TPH Abs and AADC Abs react with several different epitopes and that different epitopes are recognized by different sera. The prevalence of TPH Abs and AADC Abs in patients with APS type I in our study is in agreement with previous reports. TPH Abs and AADC Abs were found very rarely in patients with other forms of autoimmune adrenal disease and were not detected in patients with non-adrenal autoimmune diseases.

Activation of mu-opioid receptors inhibits synaptic inputs to spinally projecting rostral ventromedial medulla neurons.

The rostral ventromedial medulla (RVM) is a major locus for the descending control of nociception and opioid analgesia. However, it is not clear how opioids affect synaptic inputs to RVM neurons. In this study, we determined the effect of mu-opioid receptor activation on excitatory and inhibitory synaptic transmission in spinally projecting RVM neurons. RVM neurons were retrogradely labeled with a fluorescent tracer injected into the dorsal horn of the spinal cord in rats. Whole-cell voltage-clamp recordings were performed on labeled RVM neurons in brain slices in vitro. The mu-receptor agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO, 1 microM) significantly decreased the amplitude of evoked excitatory postsynaptic currents (EPSCs) in 52% (9 of 17) of labeled cells. DAMGO also significantly reduced the amplitude of evoked inhibitory postsynaptic currents (IPSCs) in 69% (11 of 16) of cells examined. Furthermore, DAMGO significantly decreased the frequency of miniature EPSCs in 55% (15 of 27) of cells and significantly decreased the frequency of miniature IPSCs in all 12 cells studied. Although most EPSCs and IPSCs were mediated by glutamate and GABA, the nicotinic and glycine receptor antagonists attenuated EPSCs and IPSCs, respectively, in some labeled RVM neurons. Immunocytochemical labeling revealed that only 35% of recorded RVM neurons were tryptophan hydroxylase-positive, and 15% cells had GABA immunoreactivity. Thus, this study provides important functional evidence that activation of mu-opioid receptors decreases the release of both excitatory and inhibitory neurotransmitters onto most spinally projecting RVM neurons.

Rostral ventromedial medulla neurons that project to the spinal cord express multiple opioid receptor phenotypes.

The rostral ventromedial medulla (RVM) forms part of a descending pathway that modulates nociceptive neurotransmission at the level of the spinal cord dorsal horn. However, the involvement of descending RVM systems in opioid analgesia are a matter of some debate. In the present study, patch-clamp recordings of RVM neurons were made from rats that had received retrograde tracer injections into the spinal cord. More than 90% of identified spinally projecting RVM neurons responded to opioid agonists. Of these neurons, 53% responded only to the mu-opioid agonist D-Ala2, N-Me-Phe4, Gly-ol5 enkephalin, 14% responded only to the kappa-opioid agonist U-69593, and another group responded to both mu and kappa opioids (23%). In unidentified RVM neurons, a larger proportion of neurons responded only to mu opioids (75%), with smaller proportions of kappa- (4%) and mu/kappa-opioid (13%) responders. These RVM slices were then immunostained for tryptophan hydroxylase (TPH), a marker of serotonergic neurons. Forty-percent of spinally projecting neurons and 11% of unidentified neurons were TPH positive. Of the TPH-positive spinally projecting neurons, there were similar proportions of mu- (33%), kappa- (25%), and mu/kappa-opioid (33%) responders. Most of the TPH-negative spinally projecting neurons were mu-opioid responders (67%). These findings indicate that functional opioid receptor subtypes exist on spinally projecting serotonergic and nonserotonergic RVM neurons. The proportions of mu- and kappa-opioid receptors expressed differ between serotonergic and nonserotonergic neurons and between retrogradely labeled and unlabeled RVM neurons. We conclude that important roles exist for both serotonergic and nonserotonergic RVM neurons in the mediation of opioid effects.

A monoclonal antibody to tryptophan hydroxylase: applications and identification of the epitope.

Recombinant rabbit tryptophan hydroxylase (TPH) was expressed in Escherichia coli and purified from inclusion bodies by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A mouse monoclonal antibody and rabbit and sheep polyclonal antibodies were generated. In immunohistochemical studies of formaldehyde-fixed primate brain, the monoclonal strongly labeled not only cell bodies in the raphe nuclei but also fibers in the cerebral cortex. Truncation mutants and peptide pre-competition were used to localize the epitope to E103SVPWFP109. Although the primary sequences of TPH encoded by mRNAs from brain and pineal gland are identical, differences in the immunoreactivity of TPH protein from these two sources were observed in blot immunolabeling studies. TPH immunoreactivity migrated as an M(r) approximately equal 56000 band in each of the tissues except human pineal glands, in which the TPH reactivity was approximately 3 kDa lower. In addition, the relative intensities of TPH immunolabeling across the four tissues differed among these antibodies and a previously described monoclonal antibody against phenylalanine hydroxylase (PH8), which cross-reacts with TPH. Whereas PH8 exhibited roughly equivalent TPH reactivity per protein in both tissues from both species, TPH from human and rat raphe nuclei was preferentially recognized by the present monoclonal. By contrast, the affinity-purified sheep polyclonal antibody reacted preferentially with TPH from human and rat pineal gland, and the affinity-purified rabbit polyclonal antibody appeared to selectively recognize TPH from human pineal gland.