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Research ArticleCardiovascular

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

Charlotte M. Detremmerie, Zhengju Chen, Zhuoming Li, Khalid M. Alkharfy, Susan W.S. Leung, Aimin Xu, Yuansheng Gao and Paul M. Vanhoutte
Journal of Pharmacology and Experimental Therapeutics September 2016, 358 (3) 558-568; DOI: https://doi.org/10.1124/jpet.116.234153

Abstract

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.

Introduction

Thymoquinone, a biologically active constituent of Nigella sativa, possesses vasodilator properties (Suddek, 2010). In mice, it protects against sepsis-induced morbidity and mortality (Alkharfy et al., 2011), and in rats, its chronic administration improves age-related endothelial dysfunction (Idris-Khodja and Schini-Kerth, 2012). In preliminary experiments on isolated rat arteries, aimed at investigating the vasodilator properties of thymoquinone, a serendipitous finding was that the compound actually augmented sustained contractions in preparations with endothelium (Fig. 1A). In the present study, the mechanisms underlying this unexpected, endothelium-dependent response were characterized. The first results revealed a novel pharmacological mode of action because the endothelium-dependent augmentations evoked by thymoquinone could not be explained by conventional causes of such responses [decreased production of nitric oxide (NO) or increased release of vasoconstrictor prostaglandins, endothelin-1] or oxygen-derived free radicals (Tang et al., 2007; Tang and Vanhoutte, 2009; Goel et al., 2010; Vanhoutte, 2011)].

Fig. 1.
Fig. 1.

Effect of thymoquinone on isometric tension in isolated arteries. (A) Original recording of the effect of thymoquinone (3 × 10−5 mol/L) in rings with endothelium of rat mesenteric artery precontracted with phenylephrine (10−6 mol/L). (B–D) Effects of increasing concentrations of thymoquinone in rings with or without endothelium of rat aortae (n = 6) (B) and mesenteric arteries (n = 4) (C) during contractions to phenylephrine (10−8 to 10−6 mol/L) and of porcine coronary arteries (n = 9) (D) during contractions to serotonin (10−8 to 10−5 mol/L). Changes in tension are expressed as percentage of the reference contraction to KCl (60 mmol/L) in rat arteries (B and C) or as percentage of the precontraction to serotonin in porcine coronary arteries (D). Insets: Corresponding areas under curve of the contraction phase of the concentration-response graphs (B–D). E(+), with endothelium; E(−), without endothelium. Data shown as means ± S.E.M.; n represents the number of rings of different animals (i.e., individual observations). *Indicates statistically significant differences from controls (P ≤ 0.05).

The independency of the augmentation by thymoquinone on known endothelium-dependent mechanisms resembles the one observed in coronary arteries acutely exposed to hypoxia. The latter phenomenon, counterintuitively, depends on endothelial NO production and a subsequent activation of soluble guanylyl cyclase (sGC) (Gräser and Vanhoutte, 1991; Chan et al., 2011). Indeed, in isolated coronary arteries, acute hypoxia augments contractions by biased activation of sGC, which produces cyclic IMP (cIMP) rather than its canonical product, cGMP, leading to a pronounced vasoconstriction (Chen et al., 2014). The present experiments show that thymoquinone-induced augmentations can also be attributed to such biased activity of sGC in the vascular smooth muscle cells in response to endothelium-derived NO, whereby the production of cIMP as second messenger explains the changes in Ca2+ handling initiating the contraction. To the best of the authors’ knowledge, the present findings provide a first example of a pharmacological agent, thymoquinone, inducing vasospasms explained by biased activity of sGC.

Materials and Methods

Animals and Tissue Preparation

All of the animal experimental procedures were approved by the Committee on the Use of Live Animals for Teaching and Research of the University of Hong Kong, and were carried out in accordance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (8th edition, revised 2011).

Rat Arteries.

The experiments were conducted in 10- to 12-wk-old male Sprague Dawley rats (380–450 g) bred in the animal facility of the institution. The animals were anesthetized with pentobarbital sodium (70 mg/kg, given i.p.) and sacrificed through exsanguination after confirming the absence of lower limb reflexes. The superior mesenteric arteries or the thoracic aortae were dissected free and placed in modified Krebs-Ringer bicarbonate solution of the following composition (in mmol/L): NaCl, 129; potassium chloride (KCl), 4.7; KH2PO4, 1.18; MgSO4, 1.17; NaHCO3, 14.9; glucose, 5.5; calcium disodium EDTA, 0.026; CaCl2, 2.5 (control solution). The arteries were cut into rings (3–4 mm in length). In some preparations, the endothelium was removed by perfusing the blood vessel with 0.5 ml Triton (0.5%, 1 ml/min) prior to cutting the rings; successful removal of the endothelium was confirmed by the loss of relaxation in response to acetylcholine (10−6 mol/L).

Porcine Coronary Arteries.

Pig (of undefined age and gender) hearts were collected from the local abattoir and immersed in ice-cold aerated (95% O2–5% CO2) control solution. The hearts arrived in the laboratory within 2 hours after sacrifice of the pigs. Coronary arteries were isolated and placed in control solution, and the fat and connective tissue of the adventitia were removed. The arteries were cut into rings (3–4 mm in length). In some preparations, the endothelium was removed mechanically by rubbing the luminal surface with a wooden stick (Furchgott and Zawadzki, 1980); successful removal of the endothelium was confirmed by the loss of relaxation in response to bradykinin (10−5 mol/L).

Isometric Tension Measurement

The preparations were suspended in organ chambers; filled with warmed (37°C), aerated (95% O2, 5% CO2) control solution; and connected to force transducers (ADInstruments, Sydney, Australia) for isometric tension recording (PowerLab; ADInstruments). The rings were stretched to an optimal resting tension [1.2–1.8 g for rat mesenteric arteries, 2.5–3.5 g for rat aortae, and 8–10 g for pig arteries (determined from established individual length-tension relationships)]. After an equilibration period of 1 hour, a steady state contraction was obtained with KCl (60 mmol/L) at the optimal resting tension and served as the reference contraction, against which the subsequent measurements were normalized to compensate for the differences in the amount of vascular smooth muscle among different arterial rings. The reference contractions, taken as 100%, were 0.95 ± 0.1 g for rat mesenteric arteries (n = 32), 2.0 ± 0.15 g for rat aortae (n = 48), and 3.5 ± 0.7 g for porcine coronary arteries (n = 24). The rings were then allowed to equilibrate for 1 hour. They were exposed to thymoquinone [either by cumulative addition of increasing concentrations (10−7 to 10−3 mol/L) or by administration of a single concentration (10−5 mol/L or 3 × 10−5 mol/L in both rat and porcine arteries)] under basal conditions (quiescent preparations) or during contractions to either phenylephrine (10−8 to 10−6 mol/L) or serotonin (10−8 to 10−5 mol/L). The concentrations of these precontracting agents were titrated to obtain in all preparations a level corresponding to 50% of the reference contraction to KCl (60 mmol/L) prior to thymoquinone administration (Supplemental Fig. 1). In certain experiments the effect of increasing concentrations (10−7 to 10−3 mol/L) of either thymol or 1,4-benzoquinone was also determined. Where appropriate, rings were incubated with pharmacological inhibitors or agonists for 40 minutes before obtaining contractions to phenylephrine or serotonin. In calcium depletion experiments, the control solution was replaced with an isosmotic calcium-free solution, and 25 mmol/L caffeine was added before precontraction to deplete intracellular calcium stores.

cGMP Immunoassay

Porcine coronary arterial rings (with or without endothelium) were quickly frozen in liquid nitrogen after a plateau contraction was obtained with thymoquinone (3 × 10−5 mol/L) or after incubating with vehicle for the same period of time (control group), and were stored at −80°C until use. cGMP levels were measured as described (Chan et al., 2011).

Ultra-High Performance Liquid Chromatography–Tandem Mass Spectrometry

Rings of porcine coronary arteries and rat aortae (with endothelium) were equilibrated for 60 minutes in control solution aerated with 95% O2–5% CO2 (pH 7.4, 37°C). In some experiments, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 3 × 10−5 mol/L) was also included. Thirty minutes later, prostaglandin F2α (porcine arteries, 2 × 10−6 mol/L) or phenylephrine (rat arteries, 10−6 mol/L) was added. After 30 minutes, the preparations were exposed to thymoquinone (10−6 to 10−4 mol/L or a single dose of 3 × 10−5 mol/L) for 3 minutes and quickly frozen in liquid nitrogen. The frozen tissues (200–400 mg) from at least four individual animals were pooled (because a large quantity of tissue is needed for the measurement) and homogenized in 100% methanol (containing 5–10 ng/ml tenofovir as an internal standard) and centrifuged (12,000 rpm, 20 minutes, 4°C); the supernatant was dried using Termovap Sample Concentrator (Model DC-12; Anpel Laboratory Technologies, Shanghai, China), dissolved in 120 μl H2O, and filtered with a 0.22-μm filter for ultra-high performance liquid chromatography–tandem mass spectrometry detection (UPLC-MS/MS) analysis.

Cyclic nucleotides were separated using an ACQUITY UPLC system (Waters, Milford, MA) equipped with a binary pump, a degasser, and a temperature-controlled autosampler, as described (Chen et al., 2014). Briefly, after the injection of 10 μl of the sample, analytes were separated using a column saver [0.2 μm filter, ASSY, FRIT (Waters)] and a BEH-C18 column (50 mm × 2.1 mm, 1.7 μm; Waters) at 25°C. Eluent A was MilliQ pure water containing 10 mmol/L ammonium acetate and 0.1% (v/v) acetic acid, and eluent B consisted of methanol containing 0.1% (v/v) acetic acid. The gradient started with 97% of A and 3% of B for 1 minute, and then the fraction of B was raised to 15% in 1 minute, held for 1.5 minutes, and then restored to starting conditions in 1 minute and held for 30 seconds. The flow rate was 0.3 ml/min. The total runtime was 5 minutes.

For quantification of the cyclic nucleotide, mass detection was performed on a Qtrap 5500 mass spectrometer (AB Sciex, Foster City, CA) using multiple selected ion monitoring analysis in positive ionization mode. The multiple selected ion monitoring transitions were detected with a 50-ms dwell time. Parameters of tandem mass spectrometry fragments are listed in Supplemental Table 1. Ion source settings and collision gas pressure were optimized manually (current [CUR] = 40 psi, nebulizing gas (pressure) [GS1] = 30 psi, drying gas (pressure) [GS2] = 30 psi, ionspray (voltage) [IS] = 5500 V, collision gas [CAD] = MEDIUM, temperature of ion source [TEMP] = 400°C). Data were acquired and analyzed with Analyst version 1.5.1 (AB Sciex) (Beste et al., 2012; Chen et al., 2014).

Drugs

Acetylcholine, 1,4-benzoquinone, bradykinin, caffeine, 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester (nifedipine), 1-(5-isoquinolinylsulfonyl) homopiperazine dihydrochloride (HA-1077), ODQ, 3,5-dichloro-N-[[(1α,5α,6-exo,6α)-3-(3,3-dimethylbutyl)-3-azabicyclo[3.1.0]hex-6-yl]methyl]-benzamide hydrochloride (ML-218), 5-[1-(phenylmethyl)-1H-indazol-3-yl]-2-furanmethanol (YC-1), (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4 i][1,6]benzodiazocine-10-carboxylic acid methyl ester, indomethacin, inorganic pyrophosphate, Nω-nitro-L-arginine methyl ester (L-NAME), phenylephrine, serotonin, thymol, and thymoquinone were purchased from Sigma-Aldrich (St. Louis, MO). (9S,10S,12R)-2,3,9,10,11,12-Hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester and apocynin were obtained from Calbiochem Biochemicals (San Diego, CA). 8-Bromo-cGMP (8-Br-cGMP) was purchased from Biolog (Bremen, Germany). Diethylenetriamine (DETA) NONOate and prostaglandin F2α were obtained from Cayman Chemicals. Trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride (Y-27632) and (Z)-7-[(1S,4R,5R,6S)-5-[(E,3S)-3-hydroxyoct-1-enyl]-3-oxabicyclo[2.2.1]heptan-6-yl]hept-5-enoic acid were obtained from Tocris Bioscience (Bristol, UK). The concentrations of pharmacological inhibitors used were selected from earlier work in the laboratory and/or from the literature.

Data Analysis

Data are presented as means ± S.E.M. Contractions were expressed as percentage of the reference contraction to KCl (60 mmol/L) in rat arteries and as percentage of the precontraction to serotonin in porcine coronary arteries due to higher variability in precontraction levels in the latter.

To clarify the presentation of the results obtained with increasing concentrations of thymoquinone, areas under the curve were calculated from the concentration-response curves using computer software (Prism version 4; GraphPad Software, San Diego, CA). For those areas under the curve, only the contraction phase of the response was considered (examples are depicted in Fig. 1), as this is the scope of the current research. In the legends, n refers to the number of individual observations in preparations from different rats or pigs, except for the UPLC-MS/MS measurement of cyclic nucleotides, where it refers to individual assays of samples pooled from at least four different rats or pigs. Statistical analysis was performed using Student t test (when comparing single results for two independent groups) or one/two-way analysis of variance (when comparing more than two independent groups or more than one result per group, i.e., more than one independent variable), followed by the Bonferroni post hoc test (Prism; GraphPad Software). P values equal to or less than 0.05 were considered to indicate statistically significant differences.

Results

Quiescent Arteries.

In quiescent preparations (with or without endothelium) of rat aortae, rat mesenteric arteries, and porcine coronary arteries, increasing concentrations (10−7 to 10−3 mol/L) of thymoquinone did not cause significant changes in tension (data not shown). These experiments demonstrate that thymoquinone is not a vasoconstrictor per se.

Contracted Arteries.

In contracted [with phenylephrine (10−8 to 10−6 mol/L)] aortic rings with endothelium (Fig. 1B), the cumulative addition of thymoquinone [10−6 to 3 × 10−4 mol/L] caused significant further increases in tension (augmentation; maximal at 10−4 mol/L), whereas concentrations higher than 3 × 10−4 mol/L induced relaxations. In preparations without endothelium (Fig. 1B), the compound only evoked relaxations, which were not affected significantly by the pharmacological inhibitors used in the further study of the contraction phase of the response to thymoquinone (Supplemental Table 2). Areas under the curve calculated for the contraction phase of the concentration-response curves of the responses to thymoquinone were comparable during contractions of aortic rings to phenylephrine (58.2 ± 23.2 arbitrary units), prostaglandin F2α (68.5 ± 16.5 arbitrary units), or the thromboxane-prostanoid receptor agonist (Z)-7-[(1S,4R,5R,6S)-5-[(E,3S)-3-hydroxyoct-1-enyl]-3-oxabicyclo[2.2.1]heptan-6-yl]hept-5-enoic acid (59.2 ± 15.3 arbitrary units). Comparable results were obtained also in rat mesenteric rings with endothelium, in which 10−6 to 10−4 mol/L thymoquinone caused concentration-dependent, significant augmentations (maximal at 10−4 mol/L) of the contractions to phenylephrine (Fig. 1C), demonstrating that the augmenting effect of the compound can occur in both small arteries and conduit vessels. Likewise, thymoquinone caused moderate but significant further increases in tension in rings with endothelium of porcine coronary arteries contracted with serotonin (10−8 to 10−5 mol/L); the augmentation by thymoquinone was maximal at 10−5 mol/L and abrogated by endothelium removal (Fig. 1D). Areas under the curve calculated for the contraction phase of the concentration-response curves confirm a clear endothelium dependency in the different vascular beds (insets of Fig. 1, B–D). Taken in conjunction, these findings demonstrate that at concentrations compatible with plasma levels of the compound after oral administration in vivo (Pathan et al., 2011), thymoquinone facilitates contractions, that this augmentation is endothelium-dependent, and that it is not species-specific. The further experiments focused on the endothelium-dependent augmentations caused by the compound.

Endothelium-dependent contractions can be caused by endothelium-derived prostanoids and/or the generation of oxygen-derived free radicals (Tang et al., 2007; Tang and Vanhoutte, 2009; Vanhoutte, 2011). However, this is unlikely to explain the endothelium-dependent further increases in tension caused by thymoquinone because neither indomethacin (10−5 mol/L; inhibitor of cyclooxygenases) nor apocynin (10−4 mol/L; antioxidant) significantly decreased the response in rat aortae or in porcine preparations (Supplemental Fig. 2). The used concentrations of these compounds reduce or abolish endothelium-dependent contractions to acetylcholine in rat arteries (Shi et al., 2007; Tang et al., 2007). Likewise, the production or release of endothelin-1, which can mediate endothelium-dependent contractions (Goel et al., 2010), is not likely to explain the thymoquinone-induced augmentation, as it was not affected by the endothelin-1 receptor subtype A/endothelin-1 receptor subtype B antagonist bosentan (10−6 mol/L) in either rat aortae or porcine coronary arteries (Supplemental Fig. 2).

By contrast, the augmentation by thymoquinone of contractions of rat aortae (Fig. 2A) and mesenteric arteries (Fig. 2C) to phenylephrine was abolished by either L-NAME [10−4 mol/L; inhibitor of nitric oxide (NO) synthases] or ODQ (10−5 mol/L; inhibitor of sGC). Likewise, the augmentation evoked by thymoquinone of contractions of porcine coronary arteries to serotonin was prevented by both L-NAME and ODQ (Fig. 2E). These results indicate a dependency of the response on endothelium-dependent NO, causing activation of sGC.

Fig. 2.
Fig. 2.

Effects of eNOS and sGC inhibitors and activators on the augmentation caused by thymoquinone in isolated arteries. (A, C, and E) Effects of L-NAME (10−4 mol/L), ODQ (10−5 mol/L), DETA NONOate (10−5 mol/L, in rings treated with L-NAME), and YC-1 (10−5 mol/L, in rings treated with L-NAME) on thymoquinone-induced augmentations in rings with endothelium of rat aortae (n = 4–7) (A), of rat mesenteric arteries (n = 4–6) (C), and of porcine coronary arteries (n = 4–8) (E), contracted with phenylephrine (rat arteries, 10−8 to 10−6 mol/L) or with serotonin (porcine arteries, 10−8 to 10−5 mol/L). (B, D, and F) Effects of DETA NONOate (10−5 mol/L) and YC-1 (10−5 mol/L) on thymoquinone-induced augmentations in rings without endothelium of rat aortae (n = 3–6) (B), of rat mesenteric arteries (n = 3–6) (D), and of porcine coronary arteries (n = 3–7) (F), contracted with phenylephrine (rat arteries, 10−8 to 10−6 mol/L) or with serotonin (porcine arteries, 10−8 to 10−5 mol/L). In all graphs, the control group includes untreated preparations with endothelium. The augmentations are shown as areas under curve of the contraction phase of the corresponding concentration-response graphs. Data shown as means ± S.E.M.; n represents the number of rings of different animals (i.e., individual observations). *Indicates statistically significant differences from controls; †indicates statistically significant differences between the following groups: “L-NAME” and “L-NAME + DETA,” “L-NAME” and “L-NAME + YC-1,” “no E” (E, endothelium) and “no E + DETA,” or “no E” and “no E + YC-1” (P ≤ 0.05).

The inhibition by L-NAME and ODQ is reminiscent of their inhibitory effect on the endothelium-dependent augmentation caused by hypoxia in coronary arteries, which can be circumvented by NO donors or synthetic activators of sGC (Gräser and Vanhoutte, 1991; Pearson et al., 1996; Chan et al., 2011; Chen et al., 2014). Therefore, DETA NONOate [10−5 mol/L; optimal concentration of the NO donor determined in previous experiments (Chan et al., 2011)] or YC-1 (10−5 mol/L; sGC activator) was administered to rings with endothelium of either rat aortae (Fig. 2A), mesenteric arteries (Fig. 2C), or porcine coronary arteries (Fig. 2E), incubated with L-NAME; the two compounds restored or further increased the augmentations by thymoquinone of the contractions to phenylephrine/serotonin despite the presence of the endothelial NO synthase (eNOS) inhibitor. Similarly, DETA NONOate and YC-1 restored the augmentations to thymoquinone in rings of rat arteries (Fig. 2, B and D) and porcine arteries (Fig. 2F) without endothelium. These observations imply a key role for activation of sGC in the phenomenon, similar to hypoxic vasospasm.

Importance of the Quinone Moiety.

To verify the importance of the quinone moiety of thymoquinone, the effect of the latter (Fig. 3A) was compared with those of thymol (10−7 to 3 × 10−4 mol/L; Fig. 3B) and 1,4-benzoquinone (10−7 to 10−3 mol/L; Fig. 3C) in preparations with or without endothelium contracted with phenylephrine. Thymol caused concentration-dependent relaxations of aortic rings; the concentration-relaxation curve was not affected significantly by endothelium removal or by incubation with L-NAME, but augmented significantly by ODQ (Fig. 3B). By contrast, the cumulative addition of 1,4-benzoquinone caused significant further increases in tension, whereas concentrations higher than 3 × 10−4 mol/L induced relaxations. The further increases in tension caused by 1,4-benzoquinone were decreased by L-NAME and abolished by endothelium removal or ODQ (Fig. 3C). These findings indicate that its quinone moiety plays an essential role in the augmenting effect of thymoquinone.

Fig. 3.
Fig. 3.

Effects of quinones on isometric tension in isolated rat arteries. Effect of thymoquinone (A), thymol (B), and 1,4-benzoquinone (C) on rat aortae (n = 4), with or without endothelium, contracted with phenylephrine (10−8 to 10−6 mol/L), in the absence or presence of L-NAME (10−4 mol/L) or ODQ (10−5 mol/L). The control group includes untreated preparations with endothelium. Changes in tension are presented as percentage of the reference contraction to KCl (60 mmol/L) and shown as means ± S.E.M.; n represents the number of rings of different animals (i.e., individual observations). *Indicates statistically significant differences from control (P ≤ 0.05).

cIMP.

By contrast to DETA NONOate, the administration of 8-Br-cGMP (10−5 mol/L) to porcine coronary artery preparations with endothelium treated with ODQ (10−5 mol/L) did not restore the contraction to 3 × 10−5 mol/L thymoquinone (Supplemental Fig. 3A). Likewise, the immunoassay assessment of the cGMP level showed no significant difference between porcine coronary arterial control rings and preparations incubated with 3 × 10−5 mol/L thymoquinone (Supplemental Fig. 4). This observation prompts the interpretation that increases in cGMP production resulting from sGC activation cannot be held responsible for the facilitation of contraction with thymoquinone.

Inorganic pyrophosphate is a known by-product of the transformation of GTP to cGMP by sGC (Potter, 2011). Extracellular levels of pyrophosphate generated by vascular smooth muscle can reach micromolar levels and help to control Ca2+ homeostasis (Prosdocimo et al., 2010). This byproduct could thus account for the phenomenon seen with thymoquinone. To test that possibility, rings with endothelium of porcine coronary arteries incubated with ODQ (10−5 mol/L) were exposed to pyrophosphate (10−5 mol/L). This compound was not able to restore the augmentation of contraction by 3 × 10−5 mol/L thymoquinone (Supplemental Fig. 3A).

Hypoxic augmentations in coronary arteries are most likely due to the transformation of inosine triphosphate (ITP) to cIMP by activated sGC (Beste and Seifert, 2013; Chen et al., 2014). UPLC-MS/MS revealed that, in rings of coronary arteries with endothelium, thymoquinone (3 × 10−5 mol/L) caused a significant increase in the production of cIMP during contractions to prostaglandin F2α (Fig. 4A); endothelium removal, L-NAME (Fig. 4C), and ODQ (Fig. 4E) prevented the effect of thymoquinone. Similar results were obtained in rings of rat aortae (Fig. 4, B, D, and F). The levels of cAMP measured with UPLC-MS/MS were not affected by thymoquinone or ODQ, whereas 3 × 10−5 mol/L of the compound and ODQ reduced those of cGMP (Supplemental Fig. 5). In isolated rat arteries with endothelium exposed to L-NAME (10−4 mol/L), exogenously administered cIMP (3 × 10−4 mol/L) was able to restore the augmentation to thymoquinone, and this to the same extent as the NO-donor DETA NONOate (Fig. 5). However, the results showed a certain variability between groups of preparations, as DETA NONOate did not increase the response to thymoquinone compared with the control group to the same extent as observed in the first group of rat aortae (Fig. 2).

Fig. 4.
Fig. 4.

Effect of thymoquinone on cIMP levels in isolated arteries. (A–F) Effect of thymoquinone on the intracellular level of cIMP measured with UPLC-MS/MS in rings of porcine coronary arteries (n = 4–6) (A, C, and E) precontracted with prostaglandin F2α (porcine arteries, 2 × 10−6 mol/L) and of rat aortae (n = 4–5) (B, D, and F) precontracted with phenylephrine (rat arteries, 10−6 mol/L). (A and B) Original tracings of cIMP measurements and effect of thymoquinone (3 × 10−5 mol/L). The blue line depicts the signal for the internal standard (IS) tenofovir, and the red line the signal for cIMP. (C and D) Effect of thymoquinone (3 × 10−5 mol/L) on the intracellular level of cIMP in rings of porcine coronary arteries (n = 4–6) (C), and of rat aortae (n = 4–5) (D) with endothelium [E(+)], with endothelium treated with L-NAME (10−4 mol/L) [E(+) & L-NAME], and without endothelium [E(−)]. (E and F) Effect of thymoquinone (10−6 to 10−4 mol/L in porcine and 3 × 10−5 mol/L in rat arteries) on the intracellular level of cIMP in rings of porcine coronary arteries (n = 4–6) (E), and of rat aortae (n = 4–5) (F) with endothelium and with endothelium treated with ODQ (3 × 10−5 mol/L). The control group includes untreated preparations with endothelium. Data shown as concentration in pmol/mg protein tissue and presented as means ± S.E.M.; n represents the number of experiments for which at least four rings were pooled from four different animals (i.e., pooled observations). *Indicates statistically significant differences from the control group with endothelium [E(+), black bar]; †indicates statistically significant differences from the thymoquinone group with endothelium [E(+), white bar] (P ≤ 0.05). Note: The first peak observed in the signal for cIMP (red line) has not been identified yet; however, as there seems to be no correlation/relationship between this unidentified peak and the changes in cIMP signal, the former is not likely to be a metabolite of the latter.

Fig. 5.
Fig. 5.

Effect of exogenously administered cIMP on augmentation with thymoquinone in isolated rat aortae. Effects of L-NAME (10−4 mol/L), DETA NONOate (10−5 mol/L, in rings treated with L-NAME), and cIMP (3 × 10−4 mol/L, in rings treated with L-NAME) on thymoquinone-induced augmentations in rings with endothelium of rat aortae (n = 6) during contractions to phenylephrine (10−8 to 10−6 mol/L). The augmentations are shown as areas under curve of the contraction phase of the corresponding concentration-response graphs. The control group includes untreated preparations with endothelium. Data shown as means ± S.E.M.; n represents the number of rings of different animals (i.e., individual observations). *Indicates statistically significant differences from controls; †indicates statistically significant differences between the groups “L-NAME” and “L-NAME + DETA,” and “L-NAME” and “L-NAME + cIMP” (P ≤ 0.05).

These findings are compatible with the hypothesis that cIMP mediates the endothelium-dependent augmentation by thymoquinone.

Downstream Signaling.

Protein kinases A and G are major downstream effectors of the responses to cyclic nucleotides (Rubin and Rosen, 1975), but apparently are not involved in the augmentation caused by thymoquinone. This conclusion is based on the observations that incubation with (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester (3 × 10−7 mol/L), an established inhibitor of protein kinase A (Papapetropoulos et al., 1995), did not significantly affect the augmentation to thymoquinone (10−5 mol/L) in rat aortic rings with endothelium (Supplemental Fig. 3B). Likewise, (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester [10−6 mol/L; selective protein kinase G inhibitor (Smolenski et al., 1998)] did not significantly alter the thymoquinone-induced response (Supplemental Fig. 3B).

Rho-associated protein kinase has been proposed to be responsible for sGC-dependent hypoxic contractions (Chan et al., 2011; Chen et al., 2014). Incubation with HA-1077 (10−5 mol/L) or Y-27632 (10−5 mol/L), two established Rho-associated protein kinase inhibitors (Davies et al., 2000), significantly reduced the thymoquinone-induced augmentation in porcine coronary arteries (Fig. 6A), but not in rat aortae (Fig. 6C) with endothelium. An increase in extracellular calcium influx could also explain the augmentation of contraction with thymoquinone. Indeed, exogenous calcium depletion, using an isosmotic calcium-free control solution, reduced the augmentation in both porcine coronary arteries (Fig. 6B) and rat aortae (Fig. 6D). The inhibitor of voltage-dependent calcium-channel nifedipine (10−5 mol/L) reduced the thymoquinone-induced augmentation in the porcine coronary arteries (Fig. 6B), but did not significantly affect the response in rat aortae (Fig. 6D). In addition to the L-type voltage-dependent calcium channels, opening of T-type calcium channels can contribute to contractions of the isolated rat aorta (Duggan and Tabrizchi, 2000). The 3,5-dichloro-N-[[(1α,5α,6-exo,6α)-3-(3,3-dimethylbutyl)-3-azabicyclo[3.1.0]hex-6-yl]methyl]-benzamide hydrochloride [3 × 10−5 mol/L; selective T-type calcium-channel inhibitor (Xiang et al., 2011)] decreased the augmentation to thymoquinone significantly in the rat aorta (Fig. 6D), but not in porcine coronary arteries (Fig. 6B), suggesting the involvement of T-type calcium channels in the augmentation caused by the compound in rat arteries.

Fig. 6.
Fig. 6.

Calcium handling and augmentation with thymoquinone in isolated arteries. (A–C) Effects of HA-1077 (10−5 mol/L) and Y-27632 (10−5 mol/L) on thymoquinone-induced augmentations in rings with endothelium of porcine coronary arteries (n = 5–7) (A) and of rat aortae (n = 4–7) (C) during contractions to serotonin (porcine arteries, 10−8 to 10−5 mol/L) or to phenylephrine (rat arteries, 10−8 to 10−6 mol/L). (B–D) Effects of nifedipine (10−5 mol/L), ML-218 (3 × 10−5 mol/L), and Ca2+ depletion (in the presence of 25 mmol/L caffeine) on thymoquinone-induced augmentations in rings with endothelium of porcine coronary arteries (n = 5–7) (B) and of rat aortae (n = 4–7) (D) precontracted with serotonin (porcine arteries, 10−8 to 10−5 mol/L) or with phenylephrine (rat arteries, 10−8 to 10−6 mol/L). The control group includes untreated preparations with endothelium. The augmentations are shown as areas under curve of the contraction phase of the corresponding concentration-response graphs. Data shown as means ± S.E.M.; n represents the number of rings of different animals (i.e., individual observations). *Indicates statistically significant differences from controls (P ≤ 0.05).

Discussion

The major finding of the present experiments is that thymoquinone induces endothelium-dependent increases in tension of isolated arteries. The endothelium-dependent augmentation by thymoquinone requires previous activation of the contractile process, as it is not observed in quiescent preparations. The vasoconstrictor response to the quinone is abolished by an eNOS inhibitor but reinstalled by an exogenous NO donor after endothelium removal or eNOS inhibition, implying an obligatory role of NO in its endothelium dependency.

NO under most circumstances is a powerful endogenous vasodilator. Hence, its involvement in an endothelium-dependent contractile response seems paradoxical, to say the least. The contractions evoked by thymoquinone show a striking similarity in pharmacological characteristics with hypoxic contractions observed in earlier work in coronary arteries of dogs and pigs (Gräser and Vanhoutte, 1991; Pearson et al., 1996; Chan et al., 2011; Chen et al., 2014). The latter, like the contractions evoked by thymoquinone, are endothelium-dependent, as demonstrated ex vivo in pulmonary, femoral, and coronary arteries of the dog (De Mey and Vanhoutte, 1982, 1983; Rubanyi and Vanhoutte, 1985; Gräser and Vanhoutte, 1991; Pearson et al., 1996) and in coronary arteries of the pig (Chan et al., 2011; Chen et al., 2014). As for the response to thymoquinone (present study), inhibitors of eNOS abolish the hypoxic response in isolated coronary arteries, implying the involvement of NO. However, in the presence of L-NAME, the addition of NO donors restores the hypoxic augmentation (Gräser and Vanhoutte, 1991; Pearson et al., 1996; Chan et al., 2011; Chen et al., 2014), as it does for the contractions to thymoquinone (present experiments), confirming the involvement of the NO pathway. Inhibitors of sGC prevent both hypoxic augmentation (Gräser and Vanhoutte, 1991; Chan et al., 2011; Chen et al., 2014) and contractions to thymoquinone (present findings), but stimulators of this enzyme restore them in preparations treated with L-NAME or in rings without endothelium, implying a role of activated sGC in those responses. The classic end product resulting from the activity of this enzyme is cGMP (Potter, 2011). However, no restoration of hypoxic responses (Chan et al., 2011; Chen et al., 2014) or augmentation by thymoquinone (present findings) was observed after incubation with 8-Br-cGMP (cell-permeable form of cGMP). Measurements of cGMP levels showed no increase upon exposure to hypoxia (Chan et al., 2011) or to thymoquinone (present findings) in concentrations causing sustained endothelium-dependent augmentation. Inhibition of protein kinase G, the key enzyme targeted by cGMP (Rubin and Rosen, 1975; Lucas et al., 2000), did not prevent augmentation by hypoxia (Chan et al., 2011) or thymoquinone (present findings). Thus, these responses require activation of sGC, but not the presence of cGMP. Likewise, the absence of changes in cAMP levels and the lack of effect of a protein kinase A inhibitor on the thymoquinone-induced augmentation make a contribution of this cyclic nucleotide most unlikely.

Although cGMP is regarded the sole second messenger synthesized by sGC in response to NO (Waldman and Murad, 1987; Friebe and Koesling, 2009; Stasch and Evgenov, 2013), the enzyme can synthesize several other cyclic nucleotides, in particular cIMP, using ITP as substrate (Beste et al., 2012; Beste and Seifert, 2013). The production of this cyclic nucleotide underlies hypoxic augmentation in porcine coronary arteries (Chen et al., 2014). Likewise, the present UPLC-MS/MS measurements demonstrate that thymoquinone, like hypoxia, augments cIMP synthesis, not only in the porcine coronary artery, but also in the rat aorta. Thus, it would seem reasonable to conclude that cIMP acts as the second messenger mediating the NO-dependent, sGC-dependent augmentation of vasoconstriction caused by thymoquinone. Therefore, thymoquinone appears to be a pharmacological tool permitting the exploration of vasoconstrictor signals that require the biased activity of sGC (Chen et al., 2014; Gao and Vanhoutte, 2014; Gao et al., 2015). However, the present observations do not rule out the possibility that the cIMP produced by a biased sGC activation in response to thymoquinone is actively transported out of the vascular smooth muscle cells and acts extracellularly. In addition, it is not impossible that cIMP is transformed intracellularly into a metabolite responsible for the observed effect, although earlier experiments indicate that, under hypoxic conditions, the contraction is caused by cIMP itself, as it is enhanced by phosphodiesterase inhibitors (Gräser and Vanhoutte, 1991; Chen et al., 2014).

The augmentation by hypoxia is reduced by inhibitors of Rho-kinase (HA-1077 or Y-27632), indicating the involvement of calcium sensitization in the phenomenon (Chan et al., 2011; Chen et al., 2014). To judge from the experiments under hypoxic conditions in porcine coronary arteries, the activation of Rho-kinase reduces the activity of myosin light chain phosphatase, thereby resulting in increased phosphorylation of myosin light chain and thus augmented contractility of vascular smooth muscle (Somlyo and Somlyo, 2003). In the present study, a similar effect of Rho-kinase inhibitors on the thymoquinone-induced augmentation was observed in the porcine coronary artery, but surprisingly not in the rat aorta. A similar dissociation was obtained with the L-type calcium-channel inhibitor nifedipine. Thus, in porcine coronary arterial smooth muscle, thymoquinone depends on sensitization by Rho-kinase of the action of calcium ions entering the cells through L-type calcium channels. By contrast, in rat vascular smooth muscle, T-type, rather than L-type, calcium channels appear to mediate the effect of thymoquinone, to judge from the inhibition of the response observed in the present experiments with a selective inhibitor of the former, but not of the latter. These results imply different expression patterns of voltage-dependent calcium channels in different species or vascular beds. In line with this interpretation, L-type voltage-dependent calcium channels seem to be the major contributors to voltage-gated calcium entry in coronary myocytes (Quignard et al., 1997), whereas in rat aortae, both L-type and T-type voltage-dependent calcium channels are equally expressed (Ball et al., 2009). The present findings do not permit further speculation yet as to the mechanisms underlying the observed species/vascular bed difference, although they indicate that in the studied rat arteries the impact of cIMP on Rho-kinase is less pronounced than its effect on T-type calcium channels. Nonetheless, the conjunction of the present observations and the earlier experiments with hypoxia (Chen et al., 2014) suggests that augmented production of cIMP by sGC results in facilitation of the contractile process in vascular smooth muscle.

The present experiments in rat arteries imply that the quinone moiety is essential for the action of thymoquinone in evoking endothelium-dependent contractions. The enzyme NAD(P)H:quinone acceptor oxidoreductase-1 (NQO1; EC 1.6.99.2) is highly expressed in the vascular wall (Zhu et al., 2007; Han et al., 2009). It plays an important role in the body’s defense against oxidative stress in part by detoxifying quinones and their derivatives, thereby preventing their participation in redox cycling (Riley and Workman, 1992; Ross et al., 2000; Han et al., 2009). NQO1 metabolizes thymoquinone, presumably because of its structural similarity to ubiquinone, the natural electron carrier in mitochondria (Sutton et al., 2012). Indeed, thymoquinone can act as an electron acceptor during the oxidation of NADH to NAD+ (Staniek and Gille, 2010). NQO1 regulates the NAD+/NADH ratio, which when increased in turn initiates a signaling cascade involving CD38 (adenosine diphosphate-ribose cyclase) and resulting in Ca2+ mobilization. An increase in NAD+ concentration generated by NQO1 as a result of the action of the enzyme on thymoquinone thus could explain the facilitation of contractions caused by the latter. The present experiments do not permit further speculation as to the possible molecular link(s) between activation of NQO1 and the substrate switch from GTP to ITP by sGC. However, they confirm the obligatory role of the presence of NO and of the activation of this enzyme in certain endothelium-dependent contractions (Fig. 7).

Fig. 7.
Fig. 7.

Schematic overview of mechanisms underlying the augmentation by thymoquinone in isolated arteries. Effect of thymoquinone on sGC, biasing its activity upon stimulation by endothelial NO toward cIMP rather than cGMP production, leading to contraction rather than relaxation. The subsequent effects of cIMP on calcium homeostasis in the vascular smooth muscle cells are shown also. Right: In porcine coronary arteries, cIMP causes L-type voltage-dependent calcium influx and intracellular calcium sensitization (by activation of Rho-kinase); left: in rat arteries, cIMP causes T-type voltage-dependent calcium influx, eventually augmenting contraction.

In conclusion, the present pharmacological experiments reveal for thymoquinone a novel, and to date unique mechanism of action, which favors the occurrence of deleterious endothelium-dependent, sGC-mediated contractions. Contractions requiring biased activation (by endogenous NO or synthetic activators) of sGC with the production of cIMP have been observed with hypoxia (Chen et al., 2014; Gao and Vanhoutte, 2014; Gao et al., 2015) and occur preferentially in parts of the coronary arteries that have previously been exposed to ischemia followed by reperfusion (Pearson et al., 1996). Such hypoxic contractions may help to understand the greater occurrence of cardiovascular complications, in cardiac patients with sleep apnea (Butt et al., 2011; Lee et al., 2011). By determining the mechanisms underlying the endothelium-dependent augmentation caused by thymoquinone and possibly other agents causing biased activity of sGC, the cellular target(s) involved in cardiovascular complications due to hypoxia can be verified, thus facilitating the development of novel strategies to prevent coronary hypoxic vasospasm.

Acknowledgments

The authors thank Dr. Bernard Marchand for insightful discussion, Godfrey Man and Yee Har Chung for excellent technical assistance, and Ivy Wong for superb editorial assistance.

Authorship Contributions

Participated in research design: A.K.M., X.A, C.M.D., Z.L., S.W.S.L., P.M.V.

Conducted experiments: C.M.D., Z.C., Z.L.

Performed data analysis: C.M.D., Z.C., Z.L.

Wrote or contributed to the writing of the manuscript: C.M.D., S.W.S.L., Y.G., P.M.V.

Footnotes

    • Received April 2, 2016.
    • Accepted June 15, 2016.

  • ↵1 Current affiliation: Department of Pharmacology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China.

  • This work was supported in part by the Distinguished Scientific Fellowship Program of King Saud University (Riyadh, Saudi Arabia) and by the General Research Fund [17112914] of the Hong Kong Research Grant Council.

  • dx.doi.org/10.1124./jpet.116.234153.

  • ↵Embedded ImageThis article has supplemental material available at jpet.aspetjournals.org.

Abbreviations

cIMP
cyclic IMP
DETA
diethylenetriamine
8-Br-cGMP
8-bromo-cGMP
eNOS
endothelial NO synthase
HA-1077
1-(5-isoquinolinylsulfonyl)homopiperazine dihydrochloride
ITP
inosine triphosphate
KCl
potassium chloride
L-NAME
Nω-nitro-L-arginine methyl ester
nifedipine
1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester
NO
nitric oxide
NQO1
NAD(P)H:quinone acceptor oxidoreductase-1
ODQ
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one
sGC
soluble guanylyl cyclase
UPLC-MS/MS
ultra-high performance liquid chromatography–tandem mass spectrometry
Y-27632
trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride
YC-1
5-[1-(phenylmethyl)-1H-indazol-3-yl]-2-furanmethanol

References

  1. ↵
    1. Alkharfy KM,
    2. Al-Daghri NM,
    3. Al-Attas OS, and
    4. Alokail MS
    (2011) The protective effect of thymoquinone against sepsis syndrome morbidity and mortality in mice. Int Immunopharmacol 11:250–254.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Ball CJ,
    2. Wilson DP,
    3. Turner SP,
    4. Saint DA, and
    5. Beltrame JF
    (2009) Heterogeneity of L- and T-channels in the vasculature: rationale for the efficacy of combined L- and T-blockade. Hypertension 53:654–660.
    OpenUrlCrossRef
  3. ↵
    1. Beste KY,
    2. Burhenne H,
    3. Kaever V,
    4. Stasch JP, and
    5. Seifert R
    (2012) Nucleotidyl cyclase activity of soluble guanylyl cyclase α1β1. Biochemistry 51:194–204.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Beste KY and
    2. Seifert R
    (2013) cCMP, cUMP, cTMP, cIMP and cXMP as possible second messengers: development of a hypothesis based on studies with soluble guanylyl cyclase α(1)β(1). Biol Chem 394:261–270.
    OpenUrlPubMed
  5. ↵
    1. Butt M,
    2. Khair OA,
    3. Dwivedi G,
    4. Shantsila A,
    5. Shantsila E, and
    6. Lip GY
    (2011) Myocardial perfusion by myocardial contrast echocardiography and endothelial dysfunction in obstructive sleep apnea. Hypertension 58:417–424.
    OpenUrlCrossRef
  6. ↵
    1. Chan CKY,
    2. Mak J,
    3. Gao Y,
    4. Man RYK, and
    5. Vanhoutte PM
    (2011) Endothelium-derived NO, but not cyclic GMP, is required for hypoxic augmentation in isolated porcine coronary arteries. Am J Physiol Heart Circ Physiol 301:H2313–H2321.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Chen Z,
    2. Zhang X,
    3. Ying L,
    4. Dou D,
    5. Li Y,
    6. Bai Y,
    7. Liu J,
    8. Liu L,
    9. Feng H,
    10. Yu X,
    11. et al.
    (2014) cIMP synthesized by sGC as a mediator of hypoxic contraction of coronary arteries. Am J Physiol Heart Circ Physiol 307:H328–H336.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Davies SP,
    2. Reddy H,
    3. Caivano M, and
    4. Cohen P
    (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351:95–105.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. De Mey JG and
    2. Vanhoutte PM
    (1982) Heterogeneous behavior of the canine arterial and venous wall: importance of the endothelium. Circ Res 51:439–447.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. De Mey JG and
    2. Vanhoutte PM
    (1983) Anoxia and endothelium-dependent reactivity of the canine femoral artery. J Physiol 335:65–74.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Duggan JA and
    2. Tabrizchi R
    (2000) Influence of T-type Ca2+ (mibefradil) and Cl- (indanyloxyacetic acid 94) channel antagonists on alpha1-adrenoceptor mediated contractions in rat aorta. Can J Physiol Pharmacol 78:714–720.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Friebe A and
    2. Koesling D
    (2009) The function of NO-sensitive guanylyl cyclase: what we can learn from genetic mouse models. Nitric Oxide 21:149–156.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Furchgott RF and
    2. Zawadzki JV
    (1980) The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288:373–376.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Gao Y,
    2. Chen Z,
    3. Leung SWS, and
    4. Vanhoutte PM
    (2015) Hypoxic vasospasm mediated by cIMP: when soluble guanylyl cyclase turns bad. J Cardiovasc Pharmacol 65:545–548.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Gao Y and
    2. Vanhoutte PM
    (2014) Tissues cIMPly do not lie. Naunyn Schmiedebergs Arch Pharmacol 387:901–903.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Goel A,
    2. Su B,
    3. Flavahan S,
    4. Lowenstein CJ,
    5. Berkowitz DE, and
    6. Flavahan NA
    (2010) Increased endothelial exocytosis and generation of endothelin-1 contributes to constriction of aged arteries. Circ Res 107:242–251.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Gräser T and
    2. Vanhoutte PM
    (1991) Hypoxic contraction of canine coronary arteries: role of endothelium and cGMP. Am J Physiol 261:H1769–H1777.
    OpenUrl
  18. ↵
    1. Han SJ,
    2. Kang ES,
    3. Kim HJ,
    4. Kim SH,
    5. Chun SW,
    6. Ahn CW,
    7. Cha BS,
    8. Nam M, and
    9. Lee HC
    (2009) The C609T variant of NQO1 is associated with carotid artery plaques in patients with type 2 diabetes. Mol Genet Metab 97:85–90.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Idris-Khodja N and
    2. Schini-Kerth V
    (2012) Thymoquinone improves aging-related endothelial dysfunction in the rat mesenteric artery. Naunyn Schmiedebergs Arch Pharmacol 385:749–758.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Lee CH,
    2. Khoo SM,
    3. Chan MY,
    4. Wong HB,
    5. Low AF,
    6. Phua QH,
    7. Richards AM,
    8. Tan HC, and
    9. Yeo TC
    (2011) Severe obstructive sleep apnea and outcomes following myocardial infarction. J Clin Sleep Med 7:616–621.
    OpenUrlPubMed
  21. ↵
    1. Lucas KA,
    2. Pitari GM,
    3. Kazerounian S,
    4. Ruiz-Stewart I,
    5. Park J,
    6. Schulz S,
    7. Chepenik KP, and
    8. Waldman SA
    (2000) Guanylyl cyclases and signaling by cyclic GMP. Pharmacol Rev 52:375–414.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Papapetropoulos A,
    2. Marczin N,
    3. Mora G,
    4. Milici A,
    5. Murad F, and
    6. Catravas JD
    (1995) Regulation of vascular smooth muscle soluble guanylate cyclase activity, mRNA, and protein levels by cAMP-elevating agents. Hypertension 26:696–704.
    OpenUrlCrossRef
  23. ↵
    1. Pathan SA,
    2. Jain GK,
    3. Zaidi SM,
    4. Akhter S,
    5. Vohora D,
    6. Chander P,
    7. Kole PL,
    8. Ahmad FJ, and
    9. Khar RK
    (2011) Stability-indicating ultra-performance liquid chromatography method for the estimation of thymoquinone and its application in biopharmaceutical studies. Biomed Chromatogr 25:613–620.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Pearson PJ,
    2. Lin PJ,
    3. Schaff HV, and
    4. Vanhoutte PM
    (1996) Augmented endothelium-dependent constriction to hypoxia early and late following reperfusion of the canine coronary artery. Clin Exp Pharmacol Physiol 23:634–641.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Potter LR
    (2011) Guanylyl cyclase structure, function and regulation. Cell Signal 23:1921–1926.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Prosdocimo DA,
    2. Wyler SC,
    3. Romani AM,
    4. O’Neill WC, and
    5. Dubyak GR
    (2010) Regulation of vascular smooth muscle cell calcification by extracellular pyrophosphate homeostasis: synergistic modulation by cyclic AMP and hyperphosphatemia. Am J Physiol Cell Physiol 298:C702–C713.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Quignard JF,
    2. Frapier JM,
    3. Harricane MC,
    4. Albat B,
    5. Nargeot J, and
    6. Richard S
    (1997) Voltage-gated calcium channel currents in human coronary myocytes: regulation by cyclic GMP and nitric oxide. J Clin Invest 99:185–193.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Riley RJ and
    2. Workman P
    (1992) DT-diaphorase and cancer chemotherapy. Biochem Pharmacol 43:1657–1669.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Ross D,
    2. Kepa JK,
    3. Winski SL,
    4. Beall HD,
    5. Anwar A, and
    6. Siegel D
    (2000) NAD(P)H:quinone oxidoreductase 1 (NQO1): chemoprotection, bioactivation, gene regulation and genetic polymorphisms. Chem Biol Interact 129:77–97.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Rubanyi GM and
    2. Vanhoutte PM
    (1985) Hypoxia releases a vasoconstrictor substance from the canine vascular endothelium. J Physiol 364:45–56.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Rubin CS and
    2. Rosen OM
    (1975) Protein phosphorylation. Annu Rev Biochem 44:831–887.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Shi Y,
    2. So KF,
    3. Man RYK, and
    4. Vanhoutte PM
    (2007) Oxygen-derived free radicals mediate endothelium-dependent contractions in femoral arteries of rats with streptozotocin-induced diabetes. Br J Pharmacol 152:1033–1041.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Smolenski A,
    2. Burkhardt AM,
    3. Eigenthaler M,
    4. Butt E,
    5. Gambaryan S,
    6. Lohmann SM, and
    7. Walter U
    (1998) Functional analysis of cGMP-dependent protein kinases I and II as mediators of NO/cGMP effects. Naunyn Schmiedebergs Arch Pharmacol 358:134–139.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Somlyo AP and
    2. Somlyo AV
    (2003) Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev 83:1325–1358.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    1. Staniek K and
    2. Gille L
    (2010) Is thymoquinone an antioxidant? BMC Pharmacol 10 (Suppl 1):A9.
    OpenUrlCrossRef
  36. ↵
    1. Stasch JP and
    2. Evgenov OV
    (2013) Soluble guanylate cyclase stimulators in pulmonary hypertension. Handbook Exp Pharmacol 218:279–313.
    OpenUrlCrossRef
  37. ↵
    1. Suddek GM
    (2010) Thymoquinone-induced relaxation of isolated rat pulmonary artery. J Ethnopharmacol 127:210–214.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Sutton KM,
    2. Doucette CD, and
    3. Hoskin DW
    (2012) NADPH quinone oxidoreductase 1 mediates breast cancer cell resistance to thymoquinone-induced apoptosis. Biochem Biophys Res Commun 426:421–426.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Tang EHC,
    2. Leung FP,
    3. Huang Y,
    4. Félétou M,
    5. So KF,
    6. Man RYK, and
    7. Vanhoutte PM
    (2007) Calcium and reactive oxygen species increase in endothelial cells in response to releasers of endothelium-derived contracting factor. Br J Pharmacol 151:15–23.
    OpenUrlCrossRefPubMed
  40. ↵
    1. Tang EHC and
    2. Vanhoutte PM
    (2009) Prostanoids and reactive oxygen species: team players in endothelium-dependent contractions. Pharmacol Ther 122:140–149.
    OpenUrlCrossRefPubMed
  41. ↵
    1. Vanhoutte PM
    (2011) Endothelium-dependent contractions in hypertension: when prostacyclin becomes ugly. Hypertension 57:526–531.
    OpenUrlCrossRef
  42. ↵
    1. Waldman SA and
    2. Murad F
    (1987) Cyclic GMP synthesis and function. Pharmacol Rev 39:163–196.
    OpenUrlPubMed
  43. ↵
    1. Xiang Z,
    2. Thompson AD,
    3. Brogan JT,
    4. Schulte ML,
    5. Melancon BJ,
    6. Mi D,
    7. Lewis LM,
    8. Zou B,
    9. Yang L,
    10. Morrison R,
    11. et al.
    (2011) The discovery and characterization of ML218: a novel, centrally active T-type calcium channel inhibitor with robust effects in STN neurons and in a rodent model of Parkinson’s disease. ACS Chem Neurosci 2:730–742.
    OpenUrlCrossRefPubMed
  44. ↵
    1. Zhu H,
    2. Jia Z,
    3. Mahaney JE,
    4. Ross D,
    5. Misra HP,
    6. Trush MA, and
    7. Li Y
    (2007) The highly expressed and inducible endogenous NAD(P)H:quinone oxidoreductase 1 in cardiovascular cells acts as a potential superoxide scavenger. Cardiovasc Toxicol 7:202–211.
    OpenUrlCrossRefPubMed
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Vasoconstrictions Requiring Biased Activity of sGC

Charlotte M. Detremmerie, Zhengju Chen, Zhuoming Li, Khalid M. Alkharfy, Susan W.S. Leung, Aimin Xu, Yuansheng Gao and Paul M. Vanhoutte
Journal of Pharmacology and Experimental Therapeutics September 1, 2016, 358 (3) 558-568; DOI: https://doi.org/10.1124/jpet.116.234153
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