Development of an image-based system for measurement of membrane potential, intracellular Ca(2+) 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(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.
Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri, USA.
Srikanth R
SR
Ella
Yan
Y
Yang
Philip S
PS
Clifford
Jyoti
J
Gulia
Kim A
KA
Dora
Gerald A
GA
Meininger
Michael J
MJ
Davis
Michael A
MA
Hill
D000818:Animals; D001160:Arterioles / Q000166:cytology / Q000502:physiology*; D001463:Barbiturates; D002118:Calcium / Q000378:metabolism; D003374:Coumarins; D004983:Ethanolamines; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes; D057809:HEK293 Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D066298:In Vitro Techniques; D007555:Isoxazoles; D008297:Male; D008564:Membrane Potentials; D009119:Muscle Contraction; D032389:Myocytes, Smooth Muscle / Q000502:physiology*; D018408:Patch-Clamp Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley
D001463:Barbiturates; D003374:Coumarins; D004983:Ethanolamines; D005456:Fluorescent Dyes; D007555:Isoxazoles; C502247:N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidyl-ethanolamine; C052795:bis(1,3-dibutylbarbiturate)trimethine oxonol; D002118:Calcium