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Simultaneous Monitoring of Intra‐ and Extracellular O 2 ·− Production Associated with NAD(P)H Oxidase in Single Coronary Arterial Smooth Muscle Cells
Author(s) -
ZHANG GUO,
LI PINLAN
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a723-b
Subject(s) - chemistry , nad(p)h oxidase , nad+ kinase , extracellular , biophysics , superoxide , apocynin , intracellular , oxidase test , biochemistry , microbiology and biotechnology , enzyme , biology
The present study was designed to develop a novel method to simultaneously monitor extra‐ and intracellular O 2 ·− production in single bovine coronary arterial smooth muscle cells (CASMCs). Based on the fluorescent oxidation of dihydroethidium (DHE) to ethidium (Eth) and Eth‐DNA binding inside and outside cells, salmon DNA (5 mg/ml) was added on surface of isolated CASMCs and then Matrigel solution was loaded on the top as supportive matrix to trap DNA when it was polymerized at 37°C. Before monitoring O 2 ·− production, CASMCs were loaded with 25 μM DHE for 60 min. Using a high speed wavelength switching fluorescence imaging system, the dynamic changes in Eth‐DNA fluorescence (O 2 ·− production) were simultaneously monitored inside and outside CASMCs. It was found that oxotremorine (OXO) induced increase in extracellular O 2 ·− production was earlier and more rapid than intracellular one. A similar pattern was also observed when CASMCs were challenged by another M 1 ‐receptor agonist, acetylcholine. In the presence of superoxide dismutase (500 U/ml), NAD(P)H oxidase inhibitor, diphenylene iodonium (50 μM) or apocynin (30 μM), or in CASMCs transfected with siRNA of Nox1, OXO‐induced O 2 ·− production inside and outside CASMCs was substantially blocked. However, angiotensin II induced a different pattern showing a parallel increase in O 2 ·− Eth‐DNA signal both outside and inside cells. In conclusion, a simultaneous monitoring assay of intra‐ and extracellular O 2 ·− production is established, which can be used for studies of dynamic changes in NAD(P)H oxidase activity and for clarification of the orientation of O 2 ·− production from single CASMCs (Supported by NIH Grants HL57244, HL70726, and HL075316).