Biased Activation of Soluble Guanylyl Cyclase Causes Contraction of Isolated Arteries: Role of Cyclic IMP

Abstract

To identify the mechanisms underlying the unexpected endothelium-dependent contraction caused by thymoquinone, a natural compound with vasodilator properties.

Isometric tension was measured in rings of Sprague-Dawley rat aortae and mesenteric arteries and porcine coronary arteries. Precontracted preparations were exposed to increasing concentrations of thymoquinone (TQ), in the absence/presence of endothelium and pharmacological inhibitors/activators. Rings were pooled for Western blotting of endothelial nitric oxide (NO) synthase (eNOS) or measurement of cyclic nucleotide levels with ultra-high performance liquid chromatography coupled with mass spectrometry (UPLC-MS:MS).

TQ caused concentration-dependent augmentations of contractions in the three arteries, prevented by endothelium-removal. The augmentation was not prevented by anti-oxidants, inhibitors of cyclooxygenases or endothelin-antagonists. However, it was prevented by inhibitors of eNOS, NADPH oxidoreductase (NQO-1), and soluble guanylyl cyclase (sGC). Western blotting revealed that TQ increased phosphorylation of eNOS.

In L-NAME treated rings, the NO-donor DETA NONOate and the sGC-activator YC-1 restored the TQ-induced augmentation; by contrast, in ODQ- treated rings, the cell permeable cyclic GMP did not. UPLC-MS:MS measurements revealed that TQ increased intracellular levels of cyclic IMP. The dependency of the TQ-induced augmentation on biased sGC-activation was confirmed by the restoration obtained with cyclic IMP in L- NAME- /ODQ-treated rings. Rho-kinase and L-type voltage-dependent calcium channel (VDCC) blockers prevented the augmentation in porcine coronary arteries, but not in rat arteries, while T-type VDCC-blockers did so in the latter, but not in the former, implying heterogeneity in impact of cyclic IMP on calcium handling.

The present study shows that TQ causes an endothelium-dependent contraction that counterintuitively depends on a biased sGC-activation by NO (following NQO-1 activation) to produce cyclic IMP instead of cyclic GMP. Cyclic IMP causes contraction of the vascular smooth muscle cells through alteration of calcium handling. TQ is the first pharmacological agent described to cause endothelium-dependent contractions of isolated arteries with similar characteristics as hypoxic vasoconstrictions; depending on the presence of NO causing biased sGC-activation.

Supported by the General Research Fund, Hong Kong Research Grant Council.