Conditional Deletions of Hdc Confirm Roles of Histamine in Anaphylaxis and Circadian Activity but Not in Autoimmune Encephalomyelitis.

Histamine is best known for its role in allergies, but it could also be involved in autoimmune diseases such as multiple sclerosis. However, studies using experimental autoimmune encephalomyelitis (EAE), the most widely used animal model for multiple sclerosis, have reported conflicting observations and suggest the implication of a nonclassical source of histamine. In this study, we demonstrate that neutrophils are the main producers of histamine in the spinal cord of EAE mice. To assess the role of histamine by taking into account its different cellular sources, we used CRISPR-Cas9 to generate conditional knockout mice for the histamine-synthesizing enzyme histidine decarboxylase. We found that ubiquitous and cell-specific deletions do not affect the course of EAE. However, neutrophil-specific deletion attenuates hypothermia caused by IgE-mediated anaphylaxis, whereas neuron-specific deletion reduces circadian activity. In summary, this study refutes the role of histamine in EAE, unveils a role for neutrophil-derived histamine in IgE-mediated anaphylaxis, and establishes a new mouse model to re-explore the inflammatory and neurologic roles of histamine.

Evidence that histaminergic neurons are devoid of estrogen receptor alpha in the ewe diencephalon during the breeding season.

In sheep as in rat, it has been highly suggested that neuronal histamine (HA) participates to the estradiol (E2)-induced GnRH and LH surges, through H1 receptor. With the aim of determining if E2 could act directly on HA neurons, we examined here whether HA neurons express estrogen receptor alpha (ERα) in the ewe diencephalon during the breeding season. We first produced a specific polyclonal antibody directed against recombinant ovine histidine decarboxylase (oHDC), the HA synthesizing enzyme. Using both this anti-oHDC antibody and an anti-ERα monoclonal antibody in double label immunohistochemistry, we showed that HA neurons do not express ERα in diencephalon of ewes with different hormonal status. This result diverges from those obtained in rat, in which around three quarters of HA neurons express ERα in their nucleus. This discrepancy between these two mammal species may reflect difference in their neuronal network.

Systemic lack of canonical histamine receptor signaling results in increased resistance to autoimmune encephalomyelitis.

Histamine (HA) is a key regulator of experimental allergic encephalomyelitis (EAE), the autoimmune model of multiple sclerosis. HA exerts its effects through four known G-protein-coupled receptors: H1, H2, H3, and H4 (histamine receptors; H(1-4)R). Using HR-deficient mice, our laboratory has demonstrated that H1R, H2R, H3R, and H4R play important roles in EAE pathogenesis, by regulating encephalitogenic T cell responses, cytokine production by APCs, blood-brain barrier permeability, and T regulatory cell activity, respectively. Histidine decarboxylase-deficient mice (HDCKO), which lack systemic HA, exhibit more severe EAE and increased Th1 effector cytokine production by splenocytes in response to myelin oligodendrocyte gp35-55. In an inverse approach, we tested the effect of depleting systemic canonical HA signaling on susceptibility to EAE by generating mice lacking all four known G-protein-coupled-HRs (H(1-4)RKO mice). In this article, we report that in contrast to HDCKO mice, H(1-4)RKO mice develop less severe EAE compared with wild-type animals. Furthermore, splenocytes from immunized H(1-4)RKO mice, compared with wild-type mice, produce a lower amount of Th1/Th17 effector cytokines. The opposing results seen between HDCKO and H1-4RKO mice suggest that HA may signal independently of H1-4R and support the existence of an alternative HAergic pathway in regulating EAE resistance. Understanding and exploiting this pathway has the potential to lead to new disease-modifying therapies in multiple sclerosis and other autoimmune and allergic diseases.

Histamine production by human neutrophils.

Histamine is an important mediator in the development of allergic reactions. Only a small subset of human cell types is able to produce histamine. No previous studies have shown that human neutrophils are among them. The present work was undertaken to analyze whether human neutrophils produce histamine, and to determine what agonists are involved in histamine production by human neutrophils. The expression of histidine decarboxylase in human neutrophils was established by quantitative PCR, Western blotting, and flow cytometry analysis. The activity of the enzyme was determined by ELISA, which measured histamine in the culture supernatant of neutrophils stimulated with a set of classical agonists. Human neutrophils are bona fide histamine-producing cells. Neutrophils store ∼0.29 pg/cell and release ∼50% of the histamine content in an antigen-dependent manner and on stimulation with other neutrophil agonists. Basal expression of histidine decarboxylase, the rate-limiting enzyme in histamine production, is higher in neutrophils from patients with allergies than from healthy donors. Our results cannot be ascribed to cell contamination for several reasons. LPS failed to induce histamine release by basophils, whereas it induced histamine release by neutrophils; and we did not detect basophils, monocytes, or lymphocytes in our neutrophil preparations. Eosinophils, albeit detected, were only 0.001-0.004% of the final cell population, and they did not store or release histamine on antigen or LPS stimulation. Antigens to which patients with allergies were sensitized stimulated release of histamine from neutrophils. These observations represent a novel view of neutrophils as possible source of histamine in the allergic diseases.

Inhibition of histamine-mediated signaling confers significant protection against severe malaria in mouse models of disease.

From the inoculation of Plasmodium sporozoites via Anopheles mosquito bites to the development of blood-stage parasites, a hallmark of the host response is an inflammatory reaction characterized by elevated histamine levels in the serum and tissues. Given the proinflammatory and immunosuppressive activities associated with histamine, we postulated that this vasoactive amine participates in malaria pathogenesis. Combined genetic and pharmacologic approaches demonstrated that histamine binding to H1R and H2R but not H3R and H4R increases the susceptibility of mice to infection with Plasmodium. To further understand the role of histamine in malaria pathogenesis, we used histidine decarboxylase-deficient (HDC(-/-)) mice, which are free of histamine. HDC(-/-) mice were highly resistant to severe malaria whether infected by mosquito bites or via injection of infected erythrocytes. HDC(-/-) mice displayed resistance to two lethal strains: Plasmodium berghei (Pb) ANKA, which triggers cerebral malaria (CM), and Pb NK65, which causes death without neurological symptoms. The resistance of HDC(-/-) mice to CM was associated with preserved blood-brain barrier integrity, the absence of infected erythrocyte aggregation in the brain vessels, and a lack of sequestration of CD4 and CD8 T cells. We demonstrate that histamine-mediated signaling contributes to malaria pathogenesis. Understanding the basis for these biological effects of histamine during infection may lead to novel therapeutic strategies to alleviate the severity of malaria.

Nitric oxide mediates T cell cytokine production and signal transduction in histidine decarboxylase knockout mice.

Histamine is a key regulator of the immune system. Several lines of evidence suggest the role of histamine in T cell activation and accelerated Th1 immune response is a hallmark of histidine decarboxylase knockout (HDC-KO) mice, with a complete lack of endogenously produced histamine. According to our previous work, T lymphocytes produce NO upon activation, and NO is necessary for effective T cell activation. To study the role of histamine in T cell activation, we investigated cytokine production and T cell signal transduction in HDC-KO and wild-type (WT) mice. In the absence of histamine, an elevated IFN-gamma mRNA and protein levels of splenocytes (p < 0.001; p = 0.001, respectively) were associated with a markedly increased (2.5-fold, p = 0.0009) NO production, compared with WT animals. Furthermore, histamine treatment decreased the NO production of splenocytes from both WT and HDC-KO mice (p = 0.001; p = 0.0004, respectively). NO precursor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1,2-diolate-diethylenetriamine elicited IFN-gamma production (p = 0.0002), whereas NO synthase inhibitors N(G)-monomethyl-L-arginine and nitronidazole both inhibited IFN-gamma production (p = 0.002 and p = 0.01, respectively), suggesting the role of NO in regulating IFN-gamma synthesis. Cytoplasmic Ca(2+) concentration of unstimulated T cells was increased in the HDC-KO mice (p = 0.02), whereas T cell activation-induced delta Ca(2+)-signal was similar in both HDC-KO and WT animals. Our present data indicate that, in addition to its direct effects on T lymphocyte function, histamine regulates cytokine production and T cell signal transduction through regulating NO production.

Histidine decarboxylase-stimulating and inflammatory effects of alendronate in mice: involvement of mevalonate pathway, TNFalpha, macrophages, and T-cells.

Nitrogen-containing bisphosphonates (NBPs) are powerful anti-bone-resorptive drugs, but they frequently induce various inflammatory side effects. Recent clinical applications have disclosed an unexpected new side effect, jaw-bone necrosis and exposure. In vitro studies suggest that the inflammatory effects of NBPs are due to Vgamma2Vdelta2 T-cells, stimulated directly and/or indirectly [the latter via isopentenylpyrophosphate (IPP) in the mevalonate pathway]. Rats and mice, however, lack Vgamma2Vdelta2 T-cells, yet NBPs still induce necrotic and inflammatory reactions. In mice, NBPs induce IL-1-dependent inflammatory reactions, such as inductions of histidine decarboxylase (HDC, the histamine-forming enzyme) in the liver, lung, spleen, and bone marrow, an increase in granulocytic cells in the peritoneal cavity, pleural exudation, and splenomegaly. Here, we examined the involvement of IPP, TNF, macrophages, and T-cells in the inflammatory actions of alendronate (a typical NBP) in mice. Various statins (mevalonate-synthesis inhibitors) suppressed the alendronate-induced HDC inductions, while mevalonate itself augmented such inductions. IPP injection also induced HDC. Like IL-1-deficient mice, TNF-deficient mice were resistant to alendronate-stimulated HDC induction. Alendronate-stimulated HDC inductions were significantly weaker in macrophage-depleted mice and in nude mice than in control mice. Similar, though generally less clear-cut, results were obtained when other alendronate-induced inflammatory reactions were examined. These results suggest that (i) inhibition of the mevalonate pathway causes and/or modifies at least some inflammatory actions of alendronate in mice, (ii) in addition to IL-1, TNF is also involved in the inflammatory actions of alendronate, and (iii) alendronate may act on a variety of cells, including macrophages and T-cells.

Histidine decarboxylase, DOPA decarboxylase, and vesicular monoamine transporter 2 expression in neuroendocrine tumors: immunohistochemical study and gene expression analysis.

Histidine decarboxylase (HDC) and vesicular monoamine transporter 2 (v-MAT2) are involved in the biosynthesis and storage of histamine. DOPA decarboxylase (DDC) is involved in the biosynthesis of a variety of amines and shares a high degree of homology with HDC. HDC and v-MAT2 immunoreactivities (IR) have recently been detected in well-differentiated neuroendocrine tumors (WDNETs) and poorly differentiated neuroendocrine carcinomas (PDNECs) of various sites and have been proposed as general endocrine markers. We evaluated HDC and v-MAT2 IR in a series of 117 WDNETs and PDNECs from different sites. Western blotting analysis was performed to verify the specificity of anti-DDC and anti-HDC antibodies. Real-time RT-PCR was performed using specific probes for HDC and DDC on 42 cases, examined also for DDC IR. HDC and v-MAT2 IR were observed in the majority of WDNETs and PDNECs of all sites and HDC-IR cases were always also DDC-IR. In contrast, high levels of HDC mRNA were detected only in the gastroenteropancreatic WDNETs, which did not show increased DDC mRNA levels. On the other hand, bronchial carcinoids and lung PDNECs showed high DDC mRNA levels, but nearly undetectable HDC mRNA levels. Western blotting analysis showed a cross-reaction between anti-HDC and anti-DDC antibodies. HDC should not be considered as a general endocrine marker and HDC IR in bronchial carcinoids and PDNECs of the lung can probably be attributed to a cross-reaction with DDC.

Inducible histamine protects mice from P. acnes-primed and LPS-induced hepatitis through H2-receptor stimulation.

BACKGROUND & AIMS: Inducible histamine and histamine H2-receptors have been suggested to be involved in innate immune response. METHODS: We examined a functional role of inducible histamine in the protection against hepatic injury and lethality in Propionibacterium acnes -primed and lipopolysaccharide-induced hepatitis, using histidine decarboxylase knockout and H2-receptor knockout mice. RESULTS: Lipopolysaccharide challenge after Propionibacterium acnes priming increased histidine decarboxylase activity in the liver of wild-type mice, associated with a marked elevation of histamine turnover. Histidine decarboxylase-like immunoreactivity was observed in CD68-positive Kupffer cells/macrophages. Treatment of wild-type mice with famotidine or ranitidine but not d -chlorpheniramine augmented hepatic injury and inhibited the survival rate significantly. The same dose of Propionibacterium acnes and lipopolysaccharide induced severe hepatitis and high lethality in histidine decarboxylase knockout and H2-receptor knockout mice; the former were rescued by the subcutaneous injection of histamine. Immunohistochemical study supported the protective role of histamine against the apoptosis of hepatocytes. Histamine suppressed the expression of IL-18 and tumor necrosis factor alpha in the liver, leading to the reduced plasma levels of cytokines including IL-18, TNF-alpha, IL-12, IFN-gamma, and IL-6. CONCLUSIONS: These findings as a whole indicated that endogenously produced histamine in Kupffer cells/macrophages plays a very important role in preventing excessive innate immune response in endotoxin-induced fulminant hepatitis through the stimulation of H2-receptors.

Knock-out of the histidine decarboxylase gene modifies the repertoire of natural autoantibodies.

Natural antibodies (NA) are antibodies produced in the absence of known immunization with specific antigens. NA are found in the blood of healthy humans and mice. Histamine influences many aspects of the immune response, including antibody production. However, the role of histamine in the generation of NA has not yet been studied. In this work, we used an ELISA assay to characterize the self-antigen binding repertoires of NA in wild type (WT) mice and in histidine decarboxylase knock-out (HDC-KO) mice, unable to synthesize histamine. We now report that HDC-KO and WT mice differed in the patterns of autoreactivity of their IgM and IgG NA. The NA in HDC-KO sera manifested a larger repertoire of IgM autoantibodies than did the WT sera. The self-antigens bound by IgM from HDC-KO mice included structural proteins, enzymes associated with cellular metabolism, double-stranded and single-stranded DNA, and tissue-specific antigens like insulin. There were relatively fewer differences in the NA repertoire of IgG autoantibodies of the mice: notably, the HDC-KO sera reacted with glutamic acid decarboxylase (GAD), an antigen associated with autoimmune diabetes. These results demonstrate that endogenous histamine can influence the self-reactivity of the NA repertoire.

Analysis of antibody reactivity against cysteine sulfinic acid decarboxylase, a pyridoxal phosphate-dependent enzyme, in endocrine autoimmune disease.

The structurally related group II pyridoxal phosphate (PLP)-dependent amino acid decarboxylases glutamic acid decarboxylase (GAD), aromatic L-amino acid decarboxylase (AADC), and histidine decarboxylase (HDC) are known autoantigens in endocrine disorders. We report, for the first time, the prevalence of serum autoantibody reactivity against cysteine sulfinic acid decarboxylase (CSAD), an enzyme that shares 50% amino acid identity with the 65- and 67-kDa isoforms of GAD (GAD-65 and GAD-67), in endocrine autoimmune disease. Three of 83 patients (3.6%) with autoimmune polyendocrine syndrome type 1 (APS1) were anti-CSAD positive in a radioimmunoprecipitation assay. Anti-CSAD antibodies cross-reacted with GAD-65, and the anti-CSAD-positive sera were also reactive with AADC and HDC. The low frequency of anti-CSAD reactivity is in striking contrast to the prevalence of antibodies against GAD-65, AADC, and HDC in APS1 patients, suggesting that different mechanisms control the immunological tolerance toward CSAD and the other group II decarboxylases. Moreover, CSAD may be a useful mold for the construction of recombinant chimerical antigens in attempts to map conformational epitopes on other group II PLP-dependent amino acid decarboxylases.

Histamine and histidine decarboxylase are hallmark features of ECL cells but not G cells in rat stomach.

The oxyntic mucosa of the rat stomach is rich in ECL cells which produce and secrete histamine in response to gastrin. Histamine and the histamine-forming enzyme histidine decarboxylase (HDC) have been claimed to occur also in the gastrin-secreting G cells in the antrum. In the present study, we used a panel of five HDC antisera and one histamine antiserum to investigate whether histamine and HDC are exclusive to the ECL cells. By immunocytochemistry, we could show that the ECL cells were stained with the histamine antiserum and all five HDC antisera. The G cells, however, were not stained with the histamine antiserum, but with three of the five HDC antisera. Thus, histamine and HDC coexist in the ECL cells (oxyntic mucosa) but not in G cells (antral mucosa). Western blot analysis revealed a typical pattern of HDC-immunoreactive bands (74, 63 and 54 kDa) in oxyntic mucosa extracts with all five antisera. In antral extracts, immunoreactive bands were detected with three of the five HDC antisera (same as above); the pattern of immunoreactivity differed from that in oxyntic mucosa. Food intake of fasted rats or treatment with the proton pump inhibitor omeprazole raised the HDC activity and the HDC protein content of the oxyntic mucosa but not of the antral mucosa; the HDC activity in the antrum was barely detectable. We suggest that the HDC-like immunoreactivity in the antrum represents a cross-reaction with non-HDC proteins and conclude that histamine and HDC are hallmark features of ECL cells but not of G cells.

Histidine decarboxylase, a pyridoxal phosphate-dependent enzyme, is an autoantigen of gastric enterochromaffin-like cells.

Patients with autoimmune polyendocrine syndrome type 1 often have autoantibodies against neurotransmitter synthesizing enzymes, including the pyridoxal phosphate-dependent enzymes glutamic acid decarboxylase and aromatic L-amino acid decarboxylase. Using a candidate approach, we have identified the histamine-synthesizing enzyme histidine decarboxylase, also pyridoxal phosphate dependent, as an autoantigen in this disorder. Anti-histidine decarboxylase antibodies reacting with in vitro translated antigen were found in 36/97 (37%) of autoimmune polyendocrine syndrome type 1 patients studied. The antibodies also reacted with the native enzyme in HMC-1 cell lysates and did not cross-react with the highly homologous aromatic L-amino acid decarboxylase. Anti-histidine decarboxylase antibodies were associated with a history of intestinal dysfunction (P = 0.017). Gastric and duodenal biopsies from a patient with anti-histidine decarboxylase antibodies were studied by immunohistochemistry. The oxyntic mucosa was found to lack the histamine producing enterochromaffin-like cells, suggestive of an autoimmune destruction. To our knowledge, this is the first report of autoantibodies against histidine decarboxylase and absence of gastric enterochromaffin-like cells.

L-histidine decarboxylase as a probe in studies on histamine.

Because the Falck-Hillarp formaldehyde fluorescence method, which was superbly applied to identify catecholaminergic and serotonergic neurons, is not applicable to histamine, the first author (T.W.) developed an antibody to L-histidine decarboxylase (HDC) for identification of the histaminergic neuron system in the brain. The anti-HDC antibody was of great use for mapping the location and distribution of this histaminergic neuron system. (S)-alpha-fluoromethylhistidine, a specific and potent irreversible inhibitor of HDC, was also very useful in studies on functions of the neuron system. The activity of HDC is increased by various agents, treatments, and physiological conditions. We found new compounds that increased HDC activity (i.e., tetradecanoylphobol acetate (TPA), other tumor promoters, and staphylococcal enterotoxin A); and using mast cell-deficient mutant (W/W(v)) mice, we obtained evidence that this increase occurred in macrophages. To further characterize the mechanism of increases in HDC activity, the second author (H.O.) cloned human HDC cDNA and a human HDC gene. In studies on the regulation mechanism of the HDC gene, which is expressed only in limited types of cells such as mast cells, enterochromaffin-like cells in the stomach, cells in the tuberomammillary nucleus of the brain, and macrophages, CpG islands in the promoter region of the HDC gene were found to be demethylated in cells expressing the gene, whereas they are methylated in other cells that do not express the HDC gene. In collaboration with many other researchers, we developed HDC knockout mice. The resulting research is producing a lot of interesting findings in our laboratory as well as in others. In summary, HDC has been and will be useful in studies on functions of histamine.

Defective angiogenesis in the inflammatory granulation tissue in histidine decarboxylase-deficient mice but not in mast cell-deficient mice.

We have analyzed the role of histamine in the angiogenesis of the granulation tissue in histidine decarboxylase-deficient (HDC(-/-)) mice, mast cell-deficient mice (WBB6F1-W/W(V)), and their corresponding wild-type mice (HDC(+/+) and WBB6F(1)(+/+)). In HDC(+/+) mice, subcutaneous implantation of a cotton thread in the dorsum induced granulation tissue formation with angiogenesis, while the topical injection of anti-vascular endothelial growth factor (VEGF) IgG strongly suppressed them. In HDC(-/-) mice which showed lower VEGF levels in the granulation tissue, there was notably less angiogenesis and granulation tissue formation than in HDC(+/+) mice. The topical injection of histamine or the H(2) agonist dimaprit rescued the defective angiogenesis and granulation tissue formation in HDC(-/-) mice. There was no significant difference in the granulation tissue formation and angiogenesis between WBB6F1-W/W(V) and WBB6F1(+/+) mice. In addition, macrophages in the granulation tissue were found to express HDC. Our findings indicate that histamine derived from non-mast cells plays a significant role in the angiogenesis of the inflammatory granulation tissue.

Distribution of amylin-immunoreactive neurons in the monkey hypothalamus and their relationships with the histaminergic system.

Amylin (AMY) is a 37 amino acid peptide of pancreatic origin that has been localized in peripheral and central nervous structures. Both peripheral and central injection of the peptide causes various effects, including anorectic behavior in rats. Prompted by previous reports showing that the anorectic effect of AMY is mediated by histamine release, we immunohistochemically investigated possible relationships between these two systems at the light microscopical level. Monkey (Macaca fuscata japonica) hypothalamus specimens were submitted to immunohistochemical double staining procedures using AMY and histidine decarboxylase (HDC) antisera. AMY-immunoreactive neurons were found widely distributed in several nuclei of the monkey hypothalamus including the supraoptic, paraventricular, perifornical, periventricular, ventromedial, arcuate, and tuberomammillary nuclei. We detected AMY-immunoreactive nerve fibers throughout the hypothalamus, the median eminence and hypothalamus-neurohypophysial tract. Although AMY- and HDC-immunoreactive neuronal cell bodies occupied distinct hypothalamic zones, many HDC-immunoreactive cell bodies and dendrites, particularly those in the periventricular, arcuate, and rostral tuberomammillary regions, were surrounded by numerous AMY-immunoreactive nerve fiber varicosities. These findings demonstrate for the first time the presence of a discrete number of AMY-immunoreactive neurons in the monkey hypothalamus and add morphological support to the experimental data demonstrating that AMY probably exerts its influence on food intake via the histaminergic system.

Histidine decarboxylase expression in human melanoma.

Histamine has been implicated as one of the mediators involved in regulation of proliferation in both normal and neoplastic tissues. Histidine decarboxylase, the only enzyme that catalyzes the formation of histamine from L-histidine, is an essential regulator of histamine levels. In this study, we investigated the gene and protein expression of histidine decarboxylase in melanoma. Reverse transcriptase polymerase chain reaction and in situ hybridization studies of WM-35, WM-983/B, HT-168, and M1 human melanoma cell lines both resulted in positive signals for histidine decarboxylase messenger RNA. A polyclonal chicken antibody was developed against human histidine decarboxylase and protein expression was confirmed by western blot analysis of the cell lysates, revealing a predominant immunoreactive band at approximately 54 kDa corresponding to monomeric histidine decarboxylase. Protein expression of histidine decarboxylase was also shown by flow cytometric analysis and strong punctate cytoplasmic staining of melanoma cell lines. Moreover, both primary and metastatic human melanoma tissues were brightly stained for histidine decarboxylase. When compared with the very weak or no reactions on cultivated human melanocytes both western blot and immunohistochemical studies showed much stronger histidine decarboxylase expression in melanoma cells. These findings suggest that expression of histidine decarboxylase is elevated in human melanoma.

Antisera against rat recombinant histidine decarboxylase: immunocytochemical studies in different species.

Histamine is involved in many important biological processes such as allergic reactions, gastric acid secretion and neurotransmission. The formation of histamine is catalysed by the enzyme histidine decarboxylase. In order to understand the role of histamine in different tissues, information about the cellular localisation of the decarboxylase is important. However, the availability of antisera against the enzyme, which can be used in immunocytochemical techniques, has so far been limited, mainly due to the difficulties in purifying sufficient amounts of histidine decarboxylase from various tissues. In the present study we describe the use of antisera raised against rat recombinant histidine decarboxylase to localise the enzyme immunocytochemically in the gastric mucosa of different mammals and submammalian vertebrates. The antisera specifically stained histidine decarboxylase-immunoreactive cells in the gastric mucosa of not only rat, but also of species like frog, chicken, mouse and dog. This is the first report describing the immunocytochemical distribution of the decarboxylase in the gastric mucosa of species other than rat. These antisera are likely to become valuable tools for further studies of the immunocytochemical localisation of histidine decarboxylase.

Monoamine- and histamine-synthesizing enzymes and neurotransmitters within neurons of adult human cardiac ganglia.

BACKGROUND: Cardiac ganglia were originally thought to contain only cholinergic neurons relaying parasympathetic information from preganglionic brain stem neurons to the heart. Accumulating evidence, however, suggests that cardiac ganglia contain a heterogeneous population of neurons that synthesize or respond to several different neurotransmitters and neuropeptides. Reports regarding monoamine and histamine synthesis and neurotransmission within cardiac ganglia, however, present conflicting information or are limited in number. Furthermore, very few studies have examined the neurochemistry of adult human cardiac ganglia. The purpose of this study was, therefore, to determine whether monoamine- and histamine-synthesizing enzymes and neurotransmitters exist within neurons of adult human cardiac ganglia. METHODS AND RESULTS: Human heart tissue containing cardiac ganglia was obtained during autopsies of patients without cardiovascular pathology. Avidin-biotin complex immunohistochemistry was used to demonstrate tyrosine hydroxylase, L-dopa decarboxylase, dopamine beta-hydroxylase, phenylethanolamine-N-methyltransferase, tryptophan hydroxylase, and histidine decarboxylase immunoreactivity within neurons of cardiac ganglia. Dopamine, norepinephrine, serotonin, and histamine immunoreactivity was also found in ganglionic neurons. Omission or preadsorption of primary antibodies from the antisera and subsequent incubation with cardiac ganglia abolished specific staining in all cases examined. CONCLUSIONS: Our results suggest that neurons within cardiac ganglia contain enzymes involved in the synthesis of monoamines and histamine and that they contain dopamine, norepinephrine, serotonin, and histamine immunoreactivity. Our findings suggest a putative role for monoamine and histamine neurotransmission within adult human cardiac ganglia. Additional, functional evidence will be necessary to evaluate what the physiological role of monoamines and histamine may be in neural control of the adult human heart.

Recombinant expression of rat histidine decarboxylase: generation of antibodies useful for western blot analysis.

Histidine decarboxylase catalyses the formation of histamine, an important biological messenger. In spite of the essential biological functions exerted by histamine the knowledge about the mechanisms involved in the regulation of histidine decarboxylase is rather limited. This is most likely due to the limited supply of suitable tools, including highly specific antibodies. In the present study we describe the production and characterisation of specific antisera against rat histidine decarboxylase using recombinant protein synthesised in a bacterial expression system. The antisera were shown to effectively immunoprecipitate histidine decarboxylase activity in extracts of fetal rat liver as well as to detect the histidine decarboxylase protein by Western blot analysis of COS-7 cells expressing recombinant rat histidine decarboxylase. The results demonstrate the successful production of highly specific antisera to histidine decarboxylase which may become valuable tools in future studies of the structure and function of this enzyme.