Ratiometric measurement of endothelial depolarization in arterioles
with a potential-sensitive dye.
Beach, James M., Eugene D. McGahren, Jun Xia, and Brian R. Duling.
Departments of Biomedical Engineering, Surgery and Molecular
Physiology and Biological Physics, University of Virginia Health
Sciences Center, Charlottesville, Virginia 22908
APStracts 2:0494H, 1995.
A fluorescence ratio technique based on the voltage-sensitive dye di
-8-ANEPPS has been developed for recording membrane potential changes
during vascular responses of arterioles. Perfusion of hamster cheek
pouch arterioles with the dye labeled the endothelial cell layer.
Voltage responses from the endothelium of intact arterioles were
determined by analysis of voltage-induced shifts in fluorescence
emission wavelengths from dye spectra imaged from the vessel wall.
Membrane depolarization caused the dye spectrum to shift toward blue
wavelengths with maximal fluorescence changes near 560nm and 620nm.
In isolated non-perfused arterioles, comparison of continuous dual
-wavelength recordings with simultaneous microelectrode recordings
showed that the ratio of fluorescence intensities (F620 / F560)
accurately followed changes in membrane potential (6-21 mV) during
vasoconstriction. The dye response was linear with respect to
potential changes from -56 to -6mV, with a voltage-sensitivity of 9.7
% change in the ratio per 100 mV. Membrane potential responses in
isolated and intact arterioles following potassium stimulation
consisted of rapid ( < 0.5 sec) depolarization followed by slow
repolarization over several seconds. Potassium-induced
depolarizations were conducted along arterioles and the values of the
electrical length constant for conducted depolarization determined by
optical and microelectrode methods were in agreement. We conclude
that ratio analysis of di-8-ANEPPS fluorescence emission can be used
to accurately record membrane potential changes on the time scale of
seconds during vasomotor activity from arterioles.
Received 17 February 1995; accepted in final form 10 October
1995.
APS Manuscript Number H154-5.
Article publication pending Am. J. Physiol. (Heart Circ. Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 30 November 95