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(Circulation Research. 1997;80:21-27.)
© 1997 American Heart Association, Inc.

Articles

The Effects of Hypoxia on pH_i in Porcine Coronary Artery Endothelium and Smooth Muscle

A Novel Method for Measurements in Endothelial Cells In Situ

Robyn A. Foy, Shunichi Shimizu, Richard J. Paul

the Department of Physiology and Biophysics, University of Cincinnati (Ohio) College of Medicine.

Correspondence to Richard J. Paul, Department of Physiology and Biophysics, University of Cincinnati College of Medicine, 231 Bethesda Ave, Cincinnati, OH 45267-0576. E-mail Richard.Paul@UC.EDU

Endothelium-dependent relaxation of porcine coronary arteries is attenuated under hypoxic conditions. Recent evidence also indicates that pH_i may modulate the release of the endothelium-derived relaxing factor. We tested the hypothesis that hypoxia-induced attenuation of endothelium-dependent relaxation is mediated by alterations in pH_i. We developed a novel method for loading surface cells, whereby endothelial cell pH_i could be measured in situ on the intact porcine coronary artery. Endothelial cells of arterial ring segments were selectively loaded with the fluorescent indicator BCECF-AM. Differential loading of the endothelial cell layer was verified by confocal microscopy. pH_i of the endothelial cells in situ and of endothelium-denuded arteries was measured with a Photon Technology International spectrofluorimeter. The functional integrity of the endothelium was assessed by the endothelium-dependent relaxation to substance P in a paired adjacent ring. In the experimental protocol for pH_i measurements, preparations were perfused with a physiological bicarbonate buffer (pH 7.4), stimulated with KCl (29 mmol/L), and then subjected to hypoxia and reoxygenation. The mean basal pH_i in endothelial cells on the intact six arteries was 6.92±0.07. Addition of KCl to the perfusion medium decreased (P=.025) pH_i to 6.79±0.07. Subsequent bubbling with N₂ increased (P=.009) pH_i to 7.00±0.06, which was reversed by reoxygenation. In contrast to the in situ endothelium, pH_i of the smooth muscle was not significantly altered from its basal value of 7.24±0.06 (n=5) by either KCl or hypoxia. This differential behavior corroborated the confocal data indicating differential dye loading. These data thus suggest that oxygen-sensitive alterations in pH_i may be an important mechanism of signal transduction in endothelial cells.

Key Words: hypoxia • pH • endothelium • smooth muscle • coronary artery

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