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  • MG 149 Under most circumstances NO is a potent

    2022-08-04

    Under most circumstances, NO is a potent endogenous vasodilator. Therefore, its involvement in an endothelium-dependent contraction seems paradoxical, to say the least. Soluble guanylyl cyclase (sGC) is the major downstream target of NO; its inhibition results in the lack of vasoconstrictor response to thymoquinone (Fig. 8B) and hence this enzyme must play a central role in the phenomenon. The canonical end-product of the enzymatic activity of sGC is guanosine 3′,5′-cyclic monophosphate (cyclic GMP). Production of the latter cannot explain the augmentations observed with thymoquinone, as its cell-permeable analogue, 8-bromo-cyclic GMP cannot restore the augmentations in preparations of either pig or rat MG 149 treated with a sGC-inhibitor. Soluble GC can synthetize cyclic nucleotides other than cyclic GMP, in particular inosine 3ʹ,5ʹ-cyclic monophosphate (cyclic IMP), using inosine triphosphate (ITP) as a substrate 21, 22. With the use of ultra-high performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS/MS), thymoquinone was found to increase the production of cyclic IMP (Fig. 9), but not that of cyclic GMP. The increase in intracellular cyclic IMP levels caused by the compound depends on prior eNOS- and sGC-activation (Figure 9, Figure 10) Several mechanisms appear to be involved in the cyclic IMP-mediated thymoquinone-induced augmentation; they differ depending on the blood vessel/species studied. In porcine coronary arterial smooth muscle, thymoquinone causes the activation of Rho-associated protein kinase (ROCK; an enzyme MG 149 playing a key role in the phenomenon of calcium sensitization through inhibition of myosin light chain phosphatase), therefore increasing the action of calcium ions on the contractile proteins; it also opens L-type calcium channels thereby increasing calcium influx for the augmentation only. In the rat arterial smooth muscle, on the other hand, opening of T-type calcium channels contributes to calcium influx and hence contraction; thymoquinone causes augmentation by favoring this mechanism while activation of L-type calcium channels and ROCK do not seem to be involved (Fig. 11). The difference in dependency on L-type (porcine coronary arteries) and T-type (rat aortae) calcium channels of the augmentation with thymoquinone confirms the different expression patterns of voltage-dependent calcium channels depending on the species or the vascular bed studied: L-type voltage-dependent calcium channels seem the major contributors to voltage-gated calcium entry in coronary myocytes, while in the rat aortae, both L-type and T-type voltage-dependent calcium channels are equally expressed
    Parallelism with hypoxic vasoconstriction In addition to the pharmacological agent thymoquinone, an endothelium-dependent, NO-mediated augmentation of contraction has been demonstrated with acute hypoxia in isolated canine pulmonary, femoral and coronary arteries27, 28, 29, 30, 31 and in isolated porcine coronary arteries32, 33. There are several similarities in the characteristics of the augmentation by thymoquinone and of that in response to acute hypoxia (Table 1): augmentations to both stimuli (1) can be restored by exogenous NO and by stimulators of sGC in the absence of endothelium or eNOS activation12, 30, 31, 32, 33; (2) do not necessitate the presence of the classical product of sGC, cyclic GMP; and (3) are associated with increased production of cyclic IMP12, 32, 33, which is dependent on eNOS- and sGC-activation12, 33. It thus seems reasonable to conclude that cyclic IMP acts as the second messenger mediating the NO-dependent, biased sGC-dependent augmentation of vasoconstriction caused by both thymoquinone and acute hypoxia. Although endogenous levels of ITP are lower than those of guanosine triphosphate under normal conditions, acute hypoxia increases intracellular levels of ITP in smooth muscle cells of isolated porcine coronary arteries, as measured by UPLC–MS/MS. Therefore, as a mechanistic similarity exists between thymoquinone- and hypoxia-induced augmentations of contractions, thymoquinone also may increase the bioavailability of ITP in vascular smooth muscle cells, although actual measurements of the levels of the substrate remain to be performed.