Development of an image-based system for measurement of membrane potential, intracellular Ca(2+) and contraction in arteriolar smooth muscle cells.

Ella, SR; Yang, Y; Clifford, P; Gulia, J; Dora, K; Meininger, G; Davis, M; Hill, M

Journal article

Development of an image-based system for measurement of membrane potential, intracellular Ca(2+) and contraction in arteriolar smooth muscle cells.

Abstract:: 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.

Publication status:: Published

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APA Style

Ella, S. R., Yang, Y., Clifford, P., Gulia, J., Dora, K., Meininger, G., Davis, M., & Hill, M. (2010). Development of an image-based system for measurement of membrane potential, intracellular Ca(2+) and contraction in arteriolar smooth muscle cells. Microcirculation (New York, N.Y. : 1994), 17(8), 629–640.

MLA Style

Ella, SR, et al. “Development of an Image-Based System for Measurement of Membrane Potential, Intracellular Ca(2+) and Contraction in Arteriolar Smooth Muscle Cells.” Microcirculation (New York, N.Y. : 1994), vol. 17, no. 8, 2010, pp. 629–40.

Chicago Style

Ella, SR, Y Yang, P Clifford, et al. 2010. “Development of an Image-Based System for Measurement of Membrane Potential, Intracellular Ca(2+) and Contraction in Arteriolar Smooth Muscle Cells.” Microcirculation (New York, N.Y. : 1994) 17 (8): 629–40.
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