A novel role for antizyme inhibitor 2 as a regulator of serotonin and histamine biosynthesis and content in mouse mast cells.

Antizymes and antizyme inhibitors are key regulatory proteins of polyamine levels by affecting ornithine decarboxylase and polyamine uptake. Our previous studies indicated a metabolic interplay among polyamines, histamine and serotonin in mast cells, and demonstrated that polyamines are present in mast cell secretory granules, being important for histamine storage and serotonin levels. Recently, the novel antizyme inhibitor-2 (AZIN2) was proposed as a local regulator of polyamine biosynthesis in association with mast cell serotonin-containing granules. To gain insight into the role of AZIN2 in the biosynthesis and storage of serotonin and histamine, we have generated bone marrow derived mast cells (BMMCs) from both wild-type and transgenic Azin2 hypomorphic mice, and have analyzed polyamines, serotonin and histamine contents, and some elements of their metabolisms. Azin2 hypomorphic BMMCs did not show major mast cell phenotypic alterations as judged by morphology and specific mast cell proteases. However, compared to wild-type controls, these cells showed reduced spermidine and spermine levels, and diminished growth rate. Serotonin levels were also reduced, whereas histamine levels tended to increase. Accordingly, tryptophan hydroxylase-1 (TPH1; the key enzyme for serotonin biosynthesis) mRNA expression and protein levels were reduced, whereas histidine decarboxylase (the enzyme responsible for histamine biosynthesis) enzymatic activity was increased. Furthermore, microphtalmia-associated transcription factor, an element involved in the regulation of Tph1 expression, was reduced. Taken together, our results show, for the first time, an element of polyamine metabolism -AZIN2-, so far described as exclusively devoted to the control of polyamine concentrations, involved in regulating the biosynthesis and content of other amines like serotonin and histamine.

Histamine transport and metabolism are deranged in salivary glands in Sjogren's syndrome.

OBJECTIVE: To study histamine transport and metabolism of salivary gland (SG) epithelial cells in healthy controls and SS patients. METHODS: Enzymes and transporters involved in histamine metabolism were analysed in cultured human submandibular salivary gland (HSG) epithelial cells and tissue sections using quantitative real-time PCR and immunostaining. HSG cells were used to study [(3)H]histamine uptake [(±1-methyl-4-phenylpyridinium (MPP)] and efflux by liquid scintillation counting. RESULTS: mRNA levels of l-histidine decarboxylase (HDC) and histamine-N-methyltransferase (HNMT) were similar in the control and SS glands, but diamine oxidase was not expressed at all. Organic cation transporter 3 (OCT3) in healthy SG was localized in the acinar and ductal cells, whereas OCT2 was restricted to the myoepithelial cells. Both transporters were significantly decreased in SS at mRNA and protein levels. OCT3-mRNA levels in HSG cells were significantly higher than those of the other studied transporters. Uptake of [(3)H]histamine was inhibited by MPP in a time-dependent manner, whereas [(3)H]histamine-preloaded HSG cells released it. CONCLUSION: Ductal epithelial cells are non-professional histamine-producing cells able to release histamine via OCTs at the resting state up to ∼100 nM, enough to excite H3R/H4R(+) epithelial cells, but not H1R, which requires burst release from mast cells. At the stimulated phase, 50-60 µM histamine passes from the interstitial fluid through the acinar cells to saliva, whereas uptake by ductal cells leads to intracellular degradation by HNMT. OCT3/histamine/H4R-mediated cell maintenance and down-regulation of high histamine levels fail in SS SGs.

Polyamines affect histamine synthesis during early stages of IL-3-induced bone marrow cell differentiation.

Mast cells synthesize and store histamine, a key immunomodulatory mediator. Polyamines are essential for every living cell. Previously, we detected an antagonistic relationship between the metabolisms of these amines in established mast cell and basophilic cell lines. Here, we used the IL-3-driven mouse bone marrow-derived mast cell (BMMC) culture system to further investigate this antagonism in a mast cell model of deeper physiological significance. Polyamines and histamine levels followed opposite profiles along the bone marrow cell cultures leading to BMMCs. alpha-Difluoromethylornithine (DFMO)-induced polyamine depletion resulted in an upregulation of histidine decarboxylase (HDC, the histamine-synthesizing enzyme) expression and activity, accompanied by increased histamine levels, specifically during early stages of these cell cultures, where an active histamine synthesis process occurs. In contrast, DFMO did not induce any effect in either HDC activity or histamine levels of differentiated BMMCs or C57.1 mast cells, that exhibit a nearly inactive histamine synthesis rate. Sequence-specific DNA methylation analysis revealed that the DFMO-induced HDC mRNA upregulation observed in early bone marrow cell cultures is not attributable to a demethylation of the gene promoter caused by the pharmacological polyamine depletion. Taken together, the results support an inverse relationship between histamine and polyamine metabolisms during the bone marrow cell cultures leading to BMMCs and, moreover, suggest that the regulation of the histamine synthesis occurring during the early stages of these cultures depends on the concentrations of polyamines.

Polyamines are present in mast cell secretory granules and are important for granule homeostasis.

BACKGROUND: Mast cell secretory granules accommodate a large number of components, many of which interact with highly sulfated serglycin proteoglycan (PG) present within the granules. Polyamines (putrescine, spermidine and spermine) are absolutely required for the survival of the vast majority of living cells. Given the reported ability of polyamines to interact with PGs, we investigated the possibility that polyamines may be components of mast cell secretory granules. METHODOLOGY/PRINCIPAL FINDINGS: Spermidine was released by mouse bone marrow derived mast cells (BMMCs) after degranulation induced by IgE/anti-IgE or calcium ionophore A23187. Additionally, both spermidine and spermine were detected in isolated mouse mast cell granules. Further, depletion of polyamines by culturing BMMCs with α-difluoromethylornithine (DFMO) caused aberrant secretory granule ultrastructure, impaired histamine storage, reduced serotonin levels and increased ß-hexosaminidase content. A proteomic approach revealed that DFMO-induced polyamine depletion caused an alteration in the levels of a number of proteins, many of which are connected either with the regulated exocytosis or with the endocytic system. CONCLUSIONS/SIGNIFICANCE: Taken together, our results show evidence that polyamines are present in mast cell secretory granules and, furthermore, indicate an essential role of these polycations during the biogenesis and homeostasis of these organelles.