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 F2α(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 (pHo) from 7.4 to 7.2, which normalized intracellular pH (pHi) 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.
Footnotes
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Send reprint requests to: Richard J. Paul, Ph.D., Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, 231 Bethesda Ave., Cincinnati, OH 45267-0576. E-mail: richard.paul{at}uc.edu
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↵1 Supported by National Institutes of Health HL23240 and American Heart Association SW-95–40-S (to R.J.P.).
- Abbreviations:
- pHi
- intracellular pH
- SP
- substance P
- SNP
- sodium nitroprusside
- NO
- nitric oxide
- EDHF
- endothelium-derived hyperpolarizing factor
- EDNO
- endothelium-derived nitric oxide
- [Ca2+]i
- intracellular calcium concentration
- l-NNA
- NG-nitro-l-arginine
- TBA
- tetrabutylammonium chloride
- MOPS
- 3-(N-morpholino)propanesulfonic acid
- BCECF-AM
- 2′,7′-biscarboxyethyl-5(6)-carboxyfluorescein tetraacetoxymethylester
- pHo
- extracellular pH
- Received January 21, 1999.
- Accepted June 5, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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