RATIOMETRIC MEASUREMENT OF ENDOTHELIAL DEPOLARIZATION IN ARTERIOLES WITH A POTENTIAL-SENSITIVE DYE

A fluorescence ratio technique based on the voltage-sensitive dye yl)- 8-[beta-[2-(di-n-butylamino)-6-naphthyl]vinyl] pyridinium betaine (di- 8-ANEPPS) has been developed for recording membrane potential changes during vascular responses of arterioles. Perfusion of hamster cheek po uch arterioles with the dye labeled the endothelial cell layer. Voltag e responses from the endothelium of intact arterioles were determined by analysis of voltage-induced shifts in fluorescence emission wavelen gths from dye spectra imaged from the vessel wall. Membrane depolariza tion caused the dye spectrum to shift toward blue wavelengths, with ma ximal fluorescence changes near 560 and 620 nm. In isolated nonperfuse d arterioles, comparison of continuous dual-wavelength recordings with simultaneous microelectrode recordings showed that the ratio of fluor escence intensities (fluorescence at 620 nm to fluorescence at 560 nm) accurately followed changes in membrane potential (6-21 mV) during va soconstriction. The dye response was linear with respect to potential changes from -56 to -6 mV, with a voltage sensitivity of 9.7% change i n the ratio per 100 mV. Membrane potential responses from in vitro and in vivo arterioles after potassium stimulation consisted of rapid (<0 .5 -s) depolarization followed by slow repolarization over several sec onds. Potassium-induced depolarizations were conducted along arteriole s, and the values of the electrical length constant for conducted depo larization determined by optical and microelectrode methods were in ag reement. We conclude that ratio analysis of di-8-ANEPPS fluorescence e mission can be used to accurately record membrane potential changes on the time scale of seconds during vasomotor activity from arterioles.