Measurement of membrane potential and intracellular ca²⁺ of arteriolar endothelium and smooth muscle in vivo

We have developed an intensity analysis technique for fluorescence microscopy that allows us to measure, in real time, the diameter and the membrane potential or intracellular calcium ([Ca²⁺]_i) of in vivo arteriolar endothelium or smooth muscle. Cheek pouch arterioles of anesthetized hamsters were luminally or abluminally labeled with Di-8-ANEPPS, a voltage-sensitive dye, or Fura PE3, a calcium indicator. The peak fluorescence intensities of the images were used to locate the endothelium or smooth muscle. The changes in membrane potential or [Ca²⁺]_i were determined based on the ratiometric analysis of fluorescence intensity of the endothelium or smooth muscle. Membrane depolarization of the smooth muscle using KCl caused a decrease in the ratio of emission, 620 nm/560 nm (∼6 mV/% ratio). The ratio of excitation, 340 nm/380 nm, increased with increasing free Ca²⁺. Methacholine, a muscarinic receptor agonist, caused arteriolar dilation (12.2 ± 0.9 μm). It produced hyperpolarization of the endothelium and smooth muscle (2.8 ± 0.6% and 2.3 ± 0.3% in ratio). Methacholine also induced an increase in [Ca²⁺]_i (11.0 ± 1.1% in ratio) of the endothelium. In contrast, methacholine caused a biphasic change in [Ca²⁺]_i of the smooth muscle, a rapid reduction (-3.4 ± 0.2% in ratio) followed by a prolonged increase (2.4 ± 0.2% in ratio). These results demonstrate that the peak intensity analysis can be used to determine in real time the changes in membrane potential or [Ca²⁺]_i of in vivo endothelium or smooth muscle.