Measurement of membrane potential of endothelial cells in single perfused microvessels

The membrane potential is an important modulator of calcium ion flux into endothelial cells of venular microvessels. We developed a method to measure the membrane potential of endothelial cells forming the walls of individually perfused microvessels under the same experimental conditions as those used to measure cytoplasmic calcium concentration and microvessel permeability. The membrane potential-sensitive fluorescent dye, bis-oxonol (1 microM), was added to the perfusate and the changes in bis-oxonol fluorescence intensity (FI) were calibrated in terms of changes in membrane potential using the cationic ionophore, gramicidin. FI changes an average of 0.625% per millivolt. The resting membrane potential of endothelial cells measured in single perfused microvessels, each calibrated individually, was 51.6 +/- 4.9 mV (n = 9). In the presence of high potassium Ringer's solutions (57.9 and 100 mM, [K+]o), the membrane depolarized 25 +/- 3 and 40 +/- 5 mV, respectively. Conversely, low potassium solutions (0.1 mM [K+]o) hyperpolarized the membrane by 23 +/- 4 mV. The endothelial membrane was also depolarized when the Na-K-ATPase was inhibited with ouabain. This method provides new data to test current hypotheses describing the role of the endothelial cell membrane potential as a modulator of microvessel permeability.