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OBJECTIVE: Changes in smooth muscle cell (SMC) membrane potential (Em) are critical to vasomotor responses. As a fluorescent indicator approach would lessen limitations of glass electrodes in contracting preparations, we aimed to develop a Forster (or fluorescence) resonance energy transfer (FRET)-based measurement for Em. METHODS: The FRET pair used in this study (donor CC2-DMPE [excitation 405 nm] and acceptor DisBAC(4) (3)) provide rapid measurements at a sensitivity not achievable with many ratiometric indicators. The method also combined measurement of changes in Ca(2+) (i) using fluo-4 and excitation at 490 nm. RESULTS: After establishing loading conditions, a linear relationship was demonstrated between Em and fluorescence signal in FRET dye-loaded HEK cells held under voltage clamp. Over the voltage range from -70 to +30 mV, slope (of FRET signal vs. voltage, m) = 0.49 ± 0.07, r(2)  = 0.96 ± 0.025. Similar data were obtained in cerebral artery SMCs, slope (m) = 0.30 ± 0.02, r(2)  = 0.98 ± 0.02. Change in FRET emission ratio over the holding potential of -70 to +30 mV was 41.7 ± 4.9% for HEK cells and 30.0 ± 2.3% for arterial SMCs. The FRET signal was also shown to be modulated by KCl-induced depolarization in a concentration-dependent manner. Further, in isolated arterial SMCs, KCl-induced depolarization (60 mM) measurements occurred with increased fluo-4 fluorescence emission (62 ± 9%) and contraction (-27 ± 4.2%). CONCLUSIONS: The data support the FRET-based approach for measuring changes in Em in arterial SMCs. Further, image-based measurements of Em can be combined with analysis of temporal changes in Ca(2+) (i) and contraction.

Original publication

DOI

10.1111/j.1549-8719.2010.00059.x

Type

Journal article

Journal

Microcirculation

Publication Date

11/2010

Volume

17

Pages

629 - 640

Keywords

Animals, Arterioles, Barbiturates, Calcium, Coumarins, Ethanolamines, Fluorescence Resonance Energy Transfer, Fluorescent Dyes, HEK293 Cells, Humans, Image Processing, Computer-Assisted, In Vitro Techniques, Isoxazoles, Male, Membrane Potentials, Muscle Contraction, Myocytes, Smooth Muscle, Patch-Clamp Techniques, Rats, Rats, Sprague-Dawley