Simultaneous Monitoring of Intra‐ and Extracellular O 2 ·− Production Associated with NAD(P)H Oxidase in Single Coronary Arterial Smooth Muscle Cells

Abstract

The present study was designed to develop a novel method to simultaneously monitor extra- and intracellular O2·− 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 O2·− 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 (O2·− production) were simultaneously monitored inside and outside CASMCs. It was found that oxotremorine (OXO) induced increase in extracellular O2·− production was earlier and more rapid than intracellular one. A similar pattern was also observed when CASMCs were challenged by another M1-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 O2·− production inside and outside CASMCs was substantially blocked. However, angiotensin II induced a different pattern showing a parallel increase in O2·− Eth-DNA signal both outside and inside cells. In conclusion, a simultaneous monitoring assay of intra- and extracellular O2·− 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 O2·− production from single CASMCs (Supported by NIH Grants HL57244, HL70726, and HL075316).