Vol. 291, Issue 1, 335-344, October 1999

Hypoxia and Alkalinization Inhibit Endothelium-Derived Nitric Oxide But Not Endothelium-Derived Hyperpolarizing Factor Responses in Porcine Coronary Artery¹

Shunichi Shimizu and Richard J. Paul

Department of Molecular and Cellular Physiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio

We investigated the mechanisms by which hypoxia and alkalinization inhibit the endothelium-dependent relaxation to Substance P (SP) in porcine coronary artery. In a KCl contracture, the major component of the SP response is endothelium-derived nitric oxide (EDNO), whereas with receptor-mediated 9,11-dideoxy-ll,9-epoxymethanoprostaglandin F₂ (U46619) stimulation, the SP response is dependent on both EDNO and endothelium-derived hyperpolarization factor. Intracellular alkalinization by NH₄Cl reduced the peak of SP responses when arteries were contracted with KCl, whereas with U46619 stimulation, the peak was little effected but the duration was shortened. In endothelial cell-denuded arteries, alkalinization with NH₄Cl shifted the sodium nitroprusside concentration-relaxation relations rightward. The effects of NH₄Cl in SP- and sodium nitroprusside-induced relaxations were attenuated by decreasing extracellular pH (pH_o) from 7.4 to 7.2, which normalized intracellular pH (pH_i) to control levels. In contrast, in U46619 contractures, the SP response in the presence of a NO synthase inhibitor was unaffected by NH₄Cl. Moreover, hypoxia blunted but did not abolish the responses to SP for U46619 contractures; addition of KCl, however, abolished the SP response under hypoxia. Endothelial [Ca²⁺]_i was measured with fura-2 differentially loaded only into endothelial cells on intact arteries. Despite the attenuation of the SP response in KCl contractures by NH₄Cl or hypoxia, endothelial [Ca²⁺]_i responses were unchanged. Our results suggest that hypoxia and alkalinization inhibit EDNO but not endothelium-derived hyperpolarization factor relaxations through a mechanism(s) not involving endothelial cell [Ca²⁺]_i. Inhibition of EDNO relaxation by alkalinization with NH₄Cl is likely to occur at the level of activation of guanylate cyclase and/or at a step downstream in smooth muscle.