Development of an Image-Based System for Measurement of Membrane Potential, Intracellular Ca2+ and Contraction in Arteriolar Smooth Muscle Cells

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-i(2+) 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-i(2+) and contraction.