Endothelium-Dependent Contractions of Isolated Arteries to Thymoquinone Require Biased Activity of Soluble Guanylyl Cyclase with Subsequent Cyclic IMP Production.

Preliminary experiments on isolated rat arteries demonstrated that thymoquinone, a compound widely used for its antioxidant properties and believed to facilitate endothelium-dependent relaxations, as a matter of fact caused endothelium-dependent contractions. The present experiments were designed to determine the mechanisms underlying this unexpected response. Isometric tension was measured in rings (with and without endothelium) of rat mesenteric arteries and aortae and of porcine coronary arteries. Precontracted preparations were exposed to increasing concentrations of thymoquinone, which caused concentration-dependent, sustained further increases in tension (augmentations) that were prevented by endothelium removal, Nω-nitro-L-arginine methyl ester [L-NAME; nitric oxide (NO) synthase inhibitor], and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; soluble guanylyl cyclase [sGC] inhibitor). In L-NAME-treated rings, the NO-donor diethylenetriamine NONOate restored the thymoquinone-induced augmentations; 5-[1-(phenylmethyl)-1H-indazol-3-yl]-2-furanmethanol (sGC activator) and cyclic IMP (cIMP) caused similar restorations. By contrast, in ODQ-treated preparations, the cell-permeable cGMP analog did not restore the augmentation by thymoquinone. The compound augmented the content (measured with ultra-high performance liquid chromatography-tandem mass spectrometry) of cIMP, but not that of cGMP; these increases in cIMP content were prevented by endothelium removal, L-NAME, and ODQ. The augmentation of contractions caused by thymoquinone was prevented in porcine arteries, but not in rat arteries, by 1-(5-isoquinolinylsulfonyl)homopiperazine dihydrochloride and trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride (Rho-kinase inhibitors); in the latter, but not in the former, it was reduced by 3,5-dichloro-N-[[(1α,5α,6-exo,6α)-3-(3,3-dimethylbutyl)-3-azabicyclo[3.1.0]hex-6-yl]methyl]-benzamide hydrochloride (T-type calcium channel inhibitor), demonstrating species/vascular bed differences in the impact of cIMP on calcium handling. Thymoquinone is the first pharmacological agent that causes endothelium-dependent augmentation of contractions of isolated arteries, which requires endothelium-derived NO and biased sGC activation, resulting in the augmented production of cIMP favoring the contractile process.

Activation of NQO-1 mediates the augmented contractions of isolated arteries due to biased activity of soluble guanylyl cyclase in their smooth muscle.

Earlier studies on isolated arteries demonstrated that the para-quinone thymoquinone, like acute hypoxia, induces augmentation of contractions, depending on biased activity of soluble guanylyl cyclase (sGC), generating inosine-3',5'-cyclic monophosphate (cyclic IMP) rather than guanosine-3',5'-cyclic monophosphate (cyclic GMP). NAD(P)H:quinone oxidoreductase 1 (NQO-1), the enzyme responsible for biotransformation of quinones into hydroquinones, was examined for its involvement in these endothelium-dependent augmentations, establishing a link between the metabolism of quinones by NQO-1 and biased sGC activity. Isolated arteries of Sprague-Dawley rats (aortae and mesenteric arteries) and farm pigs (coronary arteries) were studied for measurement of changes in tension and collected to measure NQO-1 activity or its protein level. ß-lapachone, an ortho-quinone and hence substrate of NQO-1, increased the activity of the enzyme and augmented contractions in arteries with endothelium. This augmentation was inhibited by endothelium removal and inhibitors of endothelial NO synthase (eNOS), sGC, or NQO-1; in preparations without endothelium or treated with an eNOS inhibitor, it was restored by the NO donor DETA NONOate and by ITP and cyclic IMP, revealing biased sGC activity as the underlying mechanism, as with thymoquinone. Hydroquinone, the end product of quinone metabolism by NQO-1, augmented contractions depending on sGC activation but in an endothelium-independent manner. In coronary arteries, repeated acute hypoxia caused similar augmentations as those to quinones that were inhibited by the NQO-1 inhibitor dicoumarol. Augmentations of contraction observed with different naturally occurring quinones and with acute hypoxia are initiated by quinone metabolism by NQO-1, in turn interfering with the NO/biased sGC pathway, suggesting a possibly detrimental role of this enzyme in ischemic cardiovascular disorders.