2002 Fiscal Year Final Research Report Summary
Real-time monitoring of histamine released from a single histamine neuron with a histamine microsensor using recombinant histamine oxidase
| Project/Area Number |
13557008
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
General pharmacology
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| Research Institution | Ehime University |
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Principal Investigator |
MAEYAMA Kazutaka Ehime University School of Medicine, Dept.of Pharmacology, Professor, 医学部, 教授 (00157158)
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| Co-Investigator(Kenkyū-buntansha) |
OGASAWARA Masahito Ehime University School of Medicine, Dept.of Pharmacology, Assistant Professor, 医学部, 講師 (00325367)
NIWA Osamu NTT Lifestyle and Environmental Technology, Laboratories, Senior Researcher, 生活環境研究所・環境情報研究部, 主幹研究員
TANIZAWA Katsuyuki Institute of Scientific and Industrial Research, Osaka University, Professor, 産業科学研究所, 教授 (20133134)
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| Project Period (FY) |
2001 – 2002
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| Keywords | histamine / histamine oxidase / microsensor / rat basophilic leukemia cells (RBL-2H3) / electrochemical detection / real-time monitoring / osmium-gel / mast cells |
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| Research Abstract |
Histamine, a bioactive amine, plays roles in allergic reaction, gastric secretion and neuronal transmission. For real-time monitoring of histamine release from histamine containing cells such as mast cells, enterochromaffin-like cells and histamine neurons, we developed a histamine microsensor using recombinant histamine oxidase(HAO). The histamine sensor is based on the determination of the reducing current amperometrically resulted from the redox reaction with horseradish peroxidase and hydrogen peroxide which is one of the products of histamine catalyzed by HAO. HAO is a copper/topa quinone-containing amine oxidase found in Arthrobacter globiformis and has already sequenced. HAO with a histidine tag, which was overexpressed in E.coli, was readily purified using a histidine affinity column. The enzyme showed higher catalytic activity on histamine than diamines (e.g.putrescine and cadaverine) or N-methylhistamine. The sensor had three carbon film electrodes modified with osmium-polyvinylpiridine-based gel containing horseradish peroxidase, histamine oxidase and Ag. When a standard solution of histamine was aspirated at a flow rate of 2 μl/min, the detected current was proportional to the histamine concentration and the lower detection limit was 11.3 nM. When rat basophilic leukemia cells (1×10^6) were stimulated by 20 ng/ml of DNP-BSA as an antigen, the histamine concentrations were 2.7μM.
These results indicate that the microsensor is useful for the analysis of histamine release from cultured cells such as mast cells.
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