The effects of hypoxia on pHi in porcine coronary artery endothelium and smooth muscle. A novel method for measurements in endothelial cells in situ.

Endothelium-dependent relaxation of porcine coronary arteries is attenuated under hypoxic conditions. Recent evidence also indicates that pHi 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 pHi. We developed a novel method for loading surface cells, whereby endothelial cell pHi 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. pHi 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 pHi measurements, preparations were perfused with a physiological bicarbonate buffer (pH 7.4), stimulated with KCI (29 mmol/L), and then subjected to hypoxia and reoxygenation. The mean basal pHi in endothelial cells on the intact six arteries was 6.92 +/- 0.07. Addition of KCI to the perfusion medium decreased (P = .025) pHi to 6.79 +/- 0.07. Subsequent bubbling with N2 increased (P = .009) pHi to 7.00 +/- 0.06, which was reversed by reoxygenation. In contrast to the in situ endothelium, pHi of the smooth muscle was not significantly altered from its basal value of 7.24 +/- 0.06 (n = 5) by either KCI or hypoxia. This differential behavior corroborated the confocal data indicating differential dye loading. These data thus suggest that oxygen-sensitive alterations in pHi may be an important mechanism of signal transduction in endothelial cells.