Endothelium-Dependent Contractions Of Isolated Arteries To Thymoquinone Require Biased Activation Of Soluble GuanylylCyclase

OBJECTIVES: Preliminary studies showed serendipitously that thymoquinone, a natural compound known for its vasodilator properties, causesa contraction in precontracted blood vessels. Experiments were designed to determine the mechanisms underlying this augmentation caused by thymoquinone in isolated contracted arteries.

METHODS: Ex vivo experiments were designed using isolated mesenteric arteries (n=4-6) and aortae (n=4-7) of twelve to fourteen weeks old Sprague-Dawley rats [sacrificed through exsanguination after anesthesia with 70mg/kg pentobarbital in propylene glycol:saline: ethanol (4:5:1)administered intraperitoneally] and porcine coronary arteries (n=4-8, collected at the local abattoir).

The arteries were cut into rings and used for isometric tension measurement in conventional organ chambers, in the absence and presence of pharmacological inhibitors. In some rings, the endothelium was removed mechanically using a wooden toothpick or a forceps. After obtaining an optimal rest tension, the preparations were contracted with phenylephrine (rat arteries) or serotonin (porcine arteries) and exposed to thymoquinone (10-7 to 10-3 M).

Some of the rings were pooled and used for Western blotting of phosphorylated endothelial nitric oxide synthase (eNOS) and total eNOS or for measurement of cyclic nucleotides through ultra-high performance liquid chromatography coupled with tandem mass spectrometry detection.

RESULTS: In rings with endothelium of the three different arteries, thymoquinone caused a sustained further contraction, which was concentration-dependent and prevented by endothelium-removal, L-NAME [nitric oxide (NO) synthase inhibitor] and ODQ [soluble guanylyl cyclase (sGC) inhibitor]. In L-NAME-treated rings, the NO-donor diethylenetriamine NONOate restored and even increased thymoquinone-induced contractions; YC-1(sGC activator) and inosine 3’,5’-cyclic monophosphate (cyclic IMP) caused a similar restoration. Western blotting showed an increase in phosphorylation of eNOS with thymoquinone.

By contrast, in ODQ-treated preparations, a cell-permeable guanosine 3’,5’-cyclic monophosphate (cyclic GMP) analogue or inorganic pyrophosphate did not restore augmentation. Thymoquinone augmented the content of cyclic IMP, but not that of cyclic GMP or adenosine 3’,5’-cyclic monophosphate. The augmentation of contraction was not significantly affected by inhibitors of cyclooxygenases or endothelin-receptors. It was prevented in porcine coronary arteries, but not in rat arteries, by HA-1077 and Y-27632 (Rho-kinase inhibitors); in the latter, but not in the former it was reduced by ML-218 (T-type calcium channel inhibitor), demonstrating species differences in the impact of cyclic IMP on calcium handling.

CONCLUSIONS: Taken into conjunction, these findings indicate that thymoquinone causes an endothelium-dependent augmentation of contractions of isolated arteries. This facilitation requires endothelium-derived NO and sGC activation, but not the presence of cyclic GMP. It is explained by the production of cyclic IMP favoring the contractile process.