Measurement of membrane potential and intracellular Ca2+ of arteriolar endothelium and smooth muscle in vivo

We have developed an intensity analysis technique for fluorescence microsco py that allows us to measure, in real time, the diameter and the membrane p otential or intracellular calcium ([Ca2+](i)) of in vivo arteriolar endothe lium 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 th e 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. Me mbrane depolarization of the smooth muscle using KCI caused a decrease in t he ratio of emission, 620 nm/560 nm (similar to6 mV/% ratio). The ratio of excitation, 340 nm/380 nm, increased with increasing free Ca2+. Methacholin e, a muscarinic receptor agonist, caused arteriolar dilation (12.2 +/- 0.9 mum). It produced hyperpolarization of the endothelium and smooth muscle (2 .8 +/- 0.6% and 2.3 +/- 0.3% in ratio). Methacholine also induced an increa se in [Ca2+](i) (11.0 +/- 1.1% in ratio) of the endothelium. In contrast, m ethacholine caused a biphasic change in [Ca2+](i) of the smooth muscle, a r apid 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 an alysis can be used to determine in real time the changes in membrane potent ial or [Ca2+], of in vivo endothelium or smooth muscle. (C) 2001 Academic P ress.