title: Measurement of membrane potential and intracellular Ca(2+) of arteriolar endothelium and smooth muscle in vivo.
abstract: 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 ([Ca(2+)]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 [Ca(2+)]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 ( approximately 6 mV/% ratio). The ratio of excitation, 340 nm/380 nm, increased with increasing free Ca(2+). Methacholine, a muscarinic receptor agonist, caused arteriolar dilation (12.2 +/- 0.9 &microm). 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 [Ca(2+)]i (11.0 +/- 1.1% in ratio) of the endothelium. In contrast, methacholine caused a biphasic change in [Ca(2+)]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 [Ca(2+)]i of in vivo endothelium or smooth muscle.
authors: Department of Anesthesiology, University of Rochester Medical Center, Rochester, New York 14642, USA.
authors: Y
authors: Y
authors: Chen
authors: R J
authors: RJ
authors: Rivers
mesh_terms: D000818:Animals; D001160:Arterioles / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D004730:Endothelium, Vascular / Q000378:metabolism / Q000502:physiology*; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000031:analogs & derivatives* / Q000378:metabolism; D008564:Membrane Potentials; D016210:Methacholine Chloride / Q000494:pharmacology; D008856:Microscopy, Fluorescence / Q000379:methods*; D018721:Muscarinic Agonists / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000378:metabolism / Q000502:physiology*; D011726:Pyridinium Compounds / Q000378:metabolism
chemical_list: C089669:1-(3-sulfonatopropyl)-4-(beta-(2-(di-n-octylamino)-6-naphthyl)vinyl)pyridinium betaine; D005456:Fluorescent Dyes; D018721:Muscarinic Agonists; D011726:Pyridinium Compounds; C098106:fura-PE3; D016210:Methacholine Chloride; D002118:Calcium; D016257:Fura-2