Abstract

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-llα,9α-epoxymethanoprostaglandin F_2α(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.