Real-time monitoring of histamine released from rat basophilic leukemia (RBL-2H3) cells with a histamine microsensor using recombinant histamine oxidase.
To detect low levels of histamine, we developed a histamine microsensor using recombinant histamine oxidase. Histamine oxidase with a histidine tag 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(tau)-methylhistamine. The sensor had three carbon film electrodes modified with osmium-polyvinylpyridine-based gel containing horseradish peroxidase, histamine oxidase, and Ag. When a standard solution of histamine was aspirated at a flow rate of 2 microl/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 x 10(6)) were stimulated by various concentrations of antigen (2, 20, and 200 ng/ml), the histamine concentrations were 0.32, 2.7, and 1.3 microM, respectively, and 20 ng/ml of antigen was found to be the optimal concentration for the antigen-antibody reaction. In contrast, when thapsigargin, an inhibitor of Ca-ATPase in the endoplasmic reticulum, was added (50, 100, and 500 nM), the detected current increased with thapsigargin concentrations and the measured histamine concentrations were 28 nM, 1.3 microM, and 2.7 microM, respectively. These results indicate that the microsensor is useful for the analysis of histamine release from mast cells.
Iwaki
Shoko
S
Department of Pharmacology, Ehime University School of Medicine, Ehime 791-0295, Japan.
Ogasawara
Masahito
M
Kurita
Ryoji
R
Niwa
Osamu
O
Tanizawa
Katsuyuki
K
Ohashi
Yuichi
Y
Maeyama
Kazutaka
K
D006631:Amine Oxidase (Copper-Containing) / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006632:Histamine / Q000032:analysis* / Q000378:metabolism; D006636:Histamine Release / Q000502:physiology*; D006861:Hydrogen Peroxide / Q000737:chemistry; D015471:Leukemia, Basophilic, Acute / Q000503:physiopathology; D008991:Monitoring, Physiologic / Q000295:instrumentation / Q000379:methods; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D019284:Thapsigargin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D019284:Thapsigargin; D006632:Histamine; D006861:Hydrogen Peroxide; D006631:Amine Oxidase (Copper-Containing); D000252:Calcium-Transporting ATPases