1521-0103/358/3/558–568$25.00 http://dx.doi.org/10.1124/jpet.116.234153THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 358:558–568, September 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Endothelium-Dependent Contractions of Isolated Arteries toThymoquinone Require Biased Activity of Soluble GuanylylCyclase with Subsequent Cyclic IMP Production s

Charlotte M. Detremmerie, Zhengju Chen, Zhuoming Li,1 Khalid M. Alkharfy,Susan W.S. Leung, Aimin Xu, Yuansheng Gao, and Paul M. VanhoutteDepartment of Pharmacology and Pharmacy and State Key Laboratory for Pharmaceutical Biotechnology, Li Ka Shing Faculty ofMedicine, University of Hong Kong, Hong Kong S.A.R., China (C.M.D., Z.L., S.W.S.L., A.X., P.M.V.); Department of ClinicalPharmacy, King Saud University, Saudi Arabia (K.M.A.) and Department of Physiology and Pathophysiology, Peking UniversityHealth Science Centre, Beijing, China (Z.C., Y.G.)

Received April 2, 2016; accepted June 15, 2016

ABSTRACTPreliminary experiments on isolated rat arteries demonstratedthat thymoquinone, a compound widely used for its antioxidantproperties and believed to facilitate endothelium-dependentrelaxations, as a matter of fact caused endothelium-dependentcontractions. The present experiments were designed to de-termine the mechanisms underlying this unexpected response.Isometric tension was measured in rings (with and withoutendothelium) of rat mesenteric arteries and aortae and of porcinecoronary arteries. Precontracted preparations were exposed toincreasing concentrations of thymoquinone, which causedconcentration-dependent, sustained further increases in tension(augmentations) that were prevented by endothelium removal,Nv-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). InL-NAME–treated rings, the NO-donor diethylenetriamine NON-Oate 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 com-pound augmented the content (measured with ultra-high perfor-mance liquid chromatography–tandem mass spectrometry) ofcIMP, but not that of cGMP; these increases in cIMP contentwere prevented by endothelium removal, L-NAME, and ODQ. Theaugmentation of contractions caused by thymoquinone wasprevented in porcine arteries, but not in rat arteries, by 1-(5-isoquinolinylsulfonyl)homopiperazine dihydrochloride and trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamidedihydrochloride (Rho-kinase inhibitors); in the latter, but notin the former, itwas reducedby3,5-dichloro-N-[[(1a,5a,6-exo,6a)-3-(3,3-dimethylbutyl)-3-azabicyclo[3.1.0]hex-6-yl]methyl]-benzamidehydrochloride (T-type calcium channel inhibitor), demonstrat-ing species/vascular bed differences in the impact of cIMPon calcium handling. Thymoquinone is the first pharmaco-logical agent that causes endothelium-dependent augmen-tation of contractions of isolated arteries, which requiresendothelium-derived NO and biased sGC activation, resultingin the augmented production of cIMP favoring the contractileprocess.

IntroductionThymoquinone, 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 administrationimproves age-related endothelial dysfunction (Idris-Khodjaand Schini-Kerth, 2012). In preliminary experiments on isolatedrat arteries, aimed at investigating the vasodilator properties ofthymoquinone, a serendipitous finding was that the compoundactually augmented sustained contractions in preparations withendothelium (Fig. 1A). In the present study, the mechanismsunderlying this unexpected, endothelium-dependent responsewere characterized. The first results revealed a novel pharma-cological mode of action because the endothelium-dependentaugmentations evoked by thymoquinone could not be explainedby conventional causes of such responses [decreased production

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

1Current affiliation: Department of Pharmacology, School of PharmaceuticalSciences, Sun Yat-sen University, Guangzhou, China.

dx.doi.org/10.1124./jpet.116.234153.s This 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, Nv-nitro-L-arginine methylester; nifedipine, 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester; NO, nitric oxide; NQO1, NAD(P)H:quinoneacceptor oxidoreductase-1; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; sGC, soluble guanylyl cyclase; UPLC-MS/MS, ultra-highperformance liquid chromatography–tandem mass spectrometry; Y-27632, trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarbox-amide dihydrochloride; YC-1, 5-[1-(phenylmethyl)-1H-indazol-3-yl]-2-furanmethanol.

558

http://jpet.aspetjournals.org/content/suppl/2016/06/22/jpet.116.234153.DC1Supplemental material to this article can be found at:

at ASPE

T Journals on M

arch 9, 2020jpet.aspetjournals.org

Dow

nloaded from

of nitric oxide (NO) or increased release of vasoconstrictor pros-taglandins, endothelin-1] or oxygen-derived free radicals (Tanget al., 2007; Tang and Vanhoutte, 2009; Goel et al., 2010;Vanhoutte, 2011)].The independency of the augmentation by thymoquinone on

known endothelium-dependent mechanisms resembles theone observed in coronary arteries acutely exposed to hypoxia.The latter phenomenon, counterintuitively, depends on endo-thelial NO production and a subsequent activation of solubleguanylyl cyclase (sGC) (Gräser and Vanhoutte, 1991; Chanet al., 2011). Indeed, in isolated coronary arteries, acutehypoxia augments contractions by biased activation of sGC,which produces cyclic IMP (cIMP) rather than its canonicalproduct, cGMP, leading to a pronounced vasoconstriction(Chen et al., 2014). The present experiments show thatthymoquinone-induced augmentations can also be attributedto such biased activity of sGC in the vascular smooth muscle

cells in response to endothelium-derived NO, whereby theproduction of cIMP as second messenger explains the changesin Ca21 handling initiating the contraction. To the best ofthe authors’ knowledge, the present findings provide a firstexample of a pharmacological agent, thymoquinone, inducingvasospasms explained by biased activity of sGC.

Materials and MethodsAnimals and Tissue Preparation

All of the animal experimental procedures were approved by theCommittee on the Use of Live Animals for Teaching and Research ofthe University of Hong Kong, and were carried out in accordancewith theGuide for the Care and Use of Laboratory Animals, publishedby the National Institutes of Health (8th edition, revised 2011).

Rat Arteries. The experiments were conducted in 10- to 12-wk-oldmale Sprague Dawley rats (380–450 g) bred in the animal facility of

Fig. 1. Effect of thymoquinone on isometric tension in isolated arteries. (A) Original recording of the effect of thymoquinone (3 � 1025 mol/L) in ringswith endothelium of rat mesenteric artery precontracted with phenylephrine (1026 mol/L). (B–D) Effects of increasing concentrations of thymoquinone inrings with or without endothelium of rat aortae (n = 6) (B) and mesenteric arteries (n = 4) (C) during contractions to phenylephrine (1028 to 1026 mol/L)and of porcine coronary arteries (n = 9) (D) during contractions to serotonin (1028 to 1025 mol/L). Changes in tension are expressed as percentage of thereference 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(2), withoutendothelium. Data shown as means 6 S.E.M.; n represents the number of rings of different animals (i.e., individual observations). *Indicatesstatistically significant differences from controls (P # 0.05).

Vasoconstrictions Requiring Biased Activity of sGC 559

at ASPE

T Journals on M

arch 9, 2020jpet.aspetjournals.org

Dow

nloaded from

the institution. The animals were anesthetized with pentobarbitalsodium (70 mg/kg, given i.p.) and sacrificed through exsanguinationafter confirming the absence of lower limb reflexes. The superiormesenteric arteries or the thoracic aortae were dissected free and placedin modified Krebs-Ringer bicarbonate solution of the following composi-tion (inmmol/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(3–4mm in length). In some preparations, the endotheliumwas removedby perfusing the blood vessel with 0.5 ml Triton (0.5%, 1 ml/min) prior tocutting the rings; successful removal of the endothelium was confirmedby the loss of relaxation in response to acetylcholine (1026 mol/L).

Porcine Coronary Arteries. Pig (of undefined age and gender)hearts were collected from the local abattoir and immersed in ice-coldaerated (95% O2–5% CO2) control solution. The hearts arrived in thelaboratorywithin2hoursafter sacrifice of thepigs.Coronaryarterieswereisolated and placed in control solution, and the fat and connective tissue ofthe adventitia were removed. The arteries were cut into rings (3–4mm inlength). In some preparations, the endothelium was removed mechan-ically by rubbing the luminal surface with a wooden stick (Furchgott andZawadzki, 1980); successful removal of the endotheliumwas confirmed bythe loss of relaxation in response to bradykinin (1025 mol/L).

Isometric Tension Measurement

The preparations were suspended in organ chambers; filled withwarmed (37°C), aerated (95%O2, 5%CO2) control solution; and connectedto force transducers (ADInstruments, Sydney, Australia) for isometrictension recording (PowerLab; ADInstruments). The rings were stretchedto 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 fromestablished individual length-tension relationships)]. After an equilibra-tion period of 1 hour, a steady state contraction was obtained with KCl(60 mmol/L) at the optimal resting tension and served as the referencecontraction, against which the subsequent measurements were normal-ized to compensate for the differences in the amount of vascular smoothmuscle amongdifferent arterial rings.The reference contractions, takenas100%,were 0.9560.1 g for ratmesenteric arteries (n532), 2.060.15 g forrat aortae (n5 48), and 3.56 0.7 g for porcine coronary arteries (n5 24).The rings were then allowed to equilibrate for 1 hour. They were exposedto thymoquinone [either by cumulative addition of increasing concentra-tions (1027 to 1023 mol/L) or by administration of a single concentration(1025 mol/L or 3 � 1025 mol/L in both rat and porcine arteries)] underbasal conditions (quiescent preparations) or during contractions to eitherphenylephrine (1028 to 1026 mol/L) or serotonin (1028 to 1025 mol/L). Theconcentrations of these precontracting agents were titrated to obtain in allpreparations a level corresponding to 50% of the reference contraction toKCl (60mmol/L)prior to thymoquinoneadministration (SupplementalFig.1). In certain experiments the effect of increasing concentrations (1027 to1023 mol/L) of either thymol or 1,4-benzoquinone was also determined.Where appropriate, rings were incubated with pharmacological inhibitorsor agonists for 40 minutes before obtaining contractions to phenylephrineor serotonin. In calcium depletion experiments, the control solution wasreplaced with an isosmotic calcium-free solution, and 25 mmol/L caffeinewas added before precontraction to deplete intracellular calcium stores.

cGMP Immunoassay

Porcine coronary arterial rings (with or without endothelium) werequickly frozen in liquidnitrogen after a plateau contractionwas obtainedwith thymoquinone (3 � 1025 mol/L) or after incubating with vehicle forthe same period of time (control group), and were stored at 280°C untiluse. cGMP levels were measured as described (Chan et al., 2011).

Ultra-High Performance Liquid Chromatography–TandemMass Spectrometry

Rings of porcine coronary arteries and rat aortae (with endothe-lium) 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 � 1025 mol/L)was also included. Thirty minutes later, prostaglandin F2a (porcinearteries, 2 � 1026 mol/L) or phenylephrine (rat arteries, 1026

mol/L) was added. After 30minutes, the preparations were exposedto thymoquinone (1026 to 1024 mol/L or a single dose of 3 � 1025

mol/L) for 3 minutes and quickly frozen in liquid nitrogen. Thefrozen tissues (200–400 mg) from at least four individual animalswere pooled (because a large quantity of tissue is needed for themeasurement) and homogenized in 100% methanol (containing5–10 ng/ml tenofovir as an internal standard) and centrifuged(12,000 rpm, 20 minutes, 4°C); the supernatant was dried usingTermovap Sample Concentrator (Model DC-12; Anpel LaboratoryTechnologies, Shanghai, China), dissolved in 120 ml H2O, andfiltered with a 0.22-mm filter for ultra-high performance liquidchromatography–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 atemperature-controlled autosampler, as described (Chen et al., 2014).Briefly, after the injection of 10 ml of the sample, analytes were separatedusinga columnsaver [0.2mmfilter,ASSY,FRIT (Waters)] andaBEH-C18column (50 mm� 2.1 mm, 1.7 mm;Waters) at 25°C. Eluent A wasMilliQpurewater containing 10mmol/L ammoniumacetate and 0.1% (v/v) aceticacid, and eluent B consisted of methanol containing 0.1% (v/v) acetic acid.The gradient startedwith 97% of A and 3% of B for 1minute, and then thefraction of Bwas raised to 15% in 1minute, held for 1.5minutes, and thenrestored to starting conditions in 1 minute and held for 30 seconds. Theflow rate was 0.3 ml/min. The total runtime was 5 minutes.

For quantification of the cyclic nucleotide, mass detection wasperformed on a Qtrap 5500 mass spectrometer (AB Sciex, Foster City,CA) using multiple selected ion monitoring analysis in positiveionization mode. The multiple selected ion monitoring transitionswere detected with a 50-ms dwell time. Parameters of tandem massspectrometry fragments are listed in Supplemental Table 1. Ion sourcesettings and collision gas pressure were optimized manually (current[CUR] 5 40 psi, nebulizing gas (pressure) [GS1] 5 30 psi, drying gas(pressure) [GS2] 5 30 psi, ionspray (voltage) [IS] 5 5500 V, collisiongas [CAD] 5 MEDIUM, temperature of ion source [TEMP] 5 400°C).Data were acquired and analyzed with Analyst version 1.5.1 (ABSciex) (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 dimethylester (nifedipine), 1-(5-isoquinolinylsulfonyl) homopiperazine dihy-drochloride (HA-1077), ODQ, 3,5-dichloro-N-[[(1a,5a,6-exo,6a)-3-(3,3-dimethylbutyl)-3-azabicyclo[3.1.0]hex-6-yl]methyl]-benzamidehydrochloride (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:39,29,19-kl]pyrrolo[3,4 i][1,6]benzodiazocine-10-carboxylic acid methyl ester, indomethacin,inorganic pyrophosphate, Nv-nitro-L-arginine methyl ester (L-NAME), phenylephrine, serotonin, thymol, and thymoquinone werepurchased 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:39,29,19-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acidhexyl ester and apocynin were obtained from Calbiochem Biochemi-cals (San Diego, CA). 8-Bromo-cGMP (8-Br-cGMP) was purchasedfrom Biolog (Bremen, Germany). Diethylenetriamine (DETA) NON-Oate and prostaglandin F2a were obtained from Cayman Chemicals.Trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamidedihydrochloride (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 acidwere obtained from Tocris Bioscience (Bristol, UK). The concentrationsof pharmacological inhibitors used were selected from earlier work inthe laboratory and/or from the literature.

560 Detremmerie et al.

at ASPE

T Journals on M

arch 9, 2020jpet.aspetjournals.org

Dow

nloaded from

Data Analysis

Data are presented as means6 S.E.M. Contractions were expressedas percentage of the reference contraction to KCl (60 mmol/L) in ratarteries and as percentage of the precontraction to serotonin in porcinecoronary arteries due to higher variability in precontraction levels inthe latter.

To clarify the presentation of the results obtained with increasingconcentrations of thymoquinone, areas under the curve were calcu-lated from the concentration-response curves using computer soft-ware (Prism version 4; GraphPad Software, San Diego, CA). Forthose areas under the curve, only the contraction phase of theresponse was considered (examples are depicted in Fig. 1), as thisis the scope of the current research. In the legends, n refers to thenumber of individual observations in preparations from differentrats or pigs, except for the UPLC-MS/MS measurement of cyclicnucleotides, where it refers to individual assays of samples pooledfrom at least four different rats or pigs. Statistical analysis wasperformed using Student t test (when comparing single results fortwo independent groups) or one/two-way analysis of variance (whencomparing more than two independent groups or more than oneresult per group, i.e., more than one independent variable), followedby the Bonferroni post hoc test (Prism; GraphPad Software). P valuesequal to or less than 0.05 were considered to indicate statisticallysignificant differences.

ResultsQuiescent Arteries. In quiescent preparations (with or

without endothelium) of rat aortae, ratmesenteric arteries, andporcine coronary arteries, increasing concentrations (1027 to1023 mol/L) of thymoquinone did not cause significant changesin tension (data not shown). These experiments demonstratethat thymoquinone is not a vasoconstrictor per se.Contracted Arteries. In contracted [with phenylephrine

(1028 to 1026 mol/L)] aortic rings with endothelium (Fig. 1B),the cumulative addition of thymoquinone [1026 to 3 � 1024

mol/L] caused significant further increases in tension (aug-mentation; maximal at 1024 mol/L), whereas concentrationshigher than 3 � 1024 mol/L induced relaxations. In prepara-tions without endothelium (Fig. 1B), the compound onlyevoked relaxations, which were not affected significantly bythe pharmacological inhibitors used in the further study of thecontraction phase of the response to thymoquinone (Supple-mental Table 2). Areas under the curve calculated for thecontraction phase of the concentration-response curves of theresponses to thymoquinone were comparable during contrac-tions of aortic rings to phenylephrine (58.2 6 23.2 arbitraryunits), prostaglandin F2a (68.5 6 16.5 arbitrary units), or thethromboxane-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 6 15.3 arbitrary units). Comparableresults were obtained also in rat mesenteric rings withendothelium, in which 1026 to 1024 mol/L thymoquinonecaused concentration-dependent, significant augmentations(maximal at 1024 mol/L) of the contractions to phenylephrine(Fig. 1C), demonstrating that the augmenting effect of thecompound can occur in both small arteries and conduitvessels. Likewise, thymoquinone caused moderate but signif-icant further increases in tension in rings with endothelium ofporcine coronary arteries contracted with serotonin (1028 to1025 mol/L); the augmentation by thymoquinone was maxi-mal at 1025 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 clearendothelium dependency in the different vascular beds (insets ofFig. 1, B–D). Taken in conjunction, these findings demonstratethat at concentrations compatible with plasma levels of thecompound after oral administration in vivo (Pathan et al., 2011),thymoquinone facilitates contractions, that this augmentationis endothelium-dependent, and that it is not species-specific.The further experiments focused on the endothelium-dependentaugmentations caused by the compound.Endothelium-dependent contractions can be caused by

endothelium-derived prostanoids and/or the generation ofoxygen-derived free radicals (Tang et al., 2007; Tang andVanhoutte, 2009; Vanhoutte, 2011). However, this is unlikelyto explain the endothelium-dependent further increases intension caused by thymoquinone because neither indometha-cin (1025 mol/L; inhibitor of cyclooxygenases) nor apocynin(1024 mol/L; antioxidant) significantly decreased the responsein rat aortae or in porcine preparations (Supplemental Fig. 2).The used concentrations of these compounds reduce or abolishendothelium-dependent contractions to acetylcholine in ratarteries (Shi et al., 2007; Tang et al., 2007). Likewise, theproduction or release of endothelin-1, which can mediateendothelium-dependent contractions (Goel et al., 2010), isnot likely to explain the thymoquinone-induced augmenta-tion, as it was not affected by the endothelin-1 receptorsubtype A/endothelin-1 receptor subtype B antagonist bosen-tan (1026 mol/L) in either rat aortae or porcine coronaryarteries (Supplemental Fig. 2).By contrast, the augmentation by thymoquinone of contrac-

tions of rat aortae (Fig. 2A) andmesenteric arteries (Fig. 2C) tophenylephrine was abolished by either L-NAME [1024 mol/L;inhibitor of nitric oxide (NO) synthases] or ODQ (1025 mol/L;inhibitor of sGC). Likewise, the augmentation evoked bythymoquinone of contractions of porcine coronary arteries toserotonin was prevented by both L-NAME and ODQ (Fig. 2E).These results indicate a dependency of the response onendothelium-dependent NO, causing activation of sGC.The inhibition by L-NAME andODQ is reminiscent of their

inhibitory effect on the endothelium-dependent augmenta-tion caused by hypoxia in coronary arteries, which can becircumvented by NO donors or synthetic activators of sGC(Gräser and Vanhoutte, 1991; Pearson et al., 1996; Chanet al., 2011; Chen et al., 2014). Therefore, DETA NONOate[1025 mol/L; optimal concentration of the NO donor de-termined in previous experiments (Chan et al., 2011)] orYC-1 (1025 mol/L; sGC activator) was administered to ringswith endothelium of either rat aortae (Fig. 2A), mesentericarteries (Fig. 2C), or porcine coronary arteries (Fig. 2E),incubated with L-NAME; the two compounds restored orfurther increased the augmentations by thymoquinone of thecontractions to phenylephrine/serotonin despite the presenceof the endothelial NO synthase (eNOS) inhibitor. Similarly,DETA NONOate and YC-1 restored the augmentations tothymoquinone in rings of rat arteries (Fig. 2, B and D) andporcine arteries (Fig. 2F) without endothelium. These observa-tions imply a key role for activation of sGC in the phenomenon,similar to hypoxic vasospasm.Importance of the Quinone Moiety. To verify the im-

portance of the quinone moiety of thymoquinone, the effect ofthe latter (Fig. 3A) was compared with those of thymol (1027

to 3 � 1024 mol/L; Fig. 3B) and 1,4-benzoquinone (1027 to 1023

mol/L; Fig. 3C) in preparations with or without endothelium

Vasoconstrictions Requiring Biased Activity of sGC 561

at ASPE

T Journals on M

arch 9, 2020jpet.aspetjournals.org

Dow

nloaded from

Fig. 2. Effects of eNOS and sGC inhibitors and activators on the augmentation caused by thymoquinone in isolated arteries. (A, C, and E) Effects ofL-NAME (1024 mol/L), ODQ (1025 mol/L), DETA NONOate (1025 mol/L, in rings treated with L-NAME), and YC-1 (1025 mol/L, in rings treated withL-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 ofporcine coronary arteries (n = 4–8) (E), contracted with phenylephrine (rat arteries, 1028 to 1026 mol/L) or with serotonin (porcine arteries, 1028 to 1025

mol/L). (B, D, and F) Effects of DETA NONOate (1025 mol/L) and YC-1 (1025 mol/L) on thymoquinone-induced augmentations in rings without

562 Detremmerie et al.

at ASPE

T Journals on M

arch 9, 2020jpet.aspetjournals.org

Dow

nloaded from

contracted with phenylephrine. Thymol caused concentration-dependent relaxations of aortic rings; the concentration-relaxationcurve was not affected significantly by endothelium removal or byincubation with L-NAME, but augmented significantly by ODQ

(Fig. 3B).By contrast, the cumulativeadditionof 1,4-benzoquinonecaused significant further increases in tension, whereas concen-trations higher than 3 � 1024 mol/L induced relaxations. Thefurther increases in tension caused by 1,4-benzoquinone were

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 (1028 to 1026 mol/L), in the absence or presence of L-NAME (1024 mol/L) or ODQ(1025 mol/L). The control group includes untreated preparations with endothelium. Changes in tension are presented as percentage of the referencecontraction to KCl (60 mmol/L) and shown as means 6 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).

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 withphenylephrine (rat arteries, 1028 to 1026 mol/L) or with serotonin (porcine arteries, 1028 to 1025 mol/L). In all graphs, the control group includesuntreated preparations with endothelium. The augmentations are shown as areas under curve of the contraction phase of the correspondingconcentration-response graphs. Data shown as means 6 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).

Vasoconstrictions Requiring Biased Activity of sGC 563

at ASPE

T Journals on M

arch 9, 2020jpet.aspetjournals.org

Dow

nloaded from

decreased by L-NAME and abolished by endothelium removal orODQ (Fig. 3C). These findings indicate that its quinone moietyplays an essential role in the augmenting effect of thymoquinone.cIMP. By contrast to DETA NONOate, the administration

of 8-Br-cGMP (1025 mol/L) to porcine coronary artery prepa-rations with endothelium treated with ODQ (1025 mol/L) didnot restore the contraction to 3 � 1025 mol/L thymoquinone(Supplemental Fig. 3A). Likewise, the immunoassay assess-ment of the cGMP level showed no significant differencebetween porcine coronary arterial control rings and prepara-tions incubated with 3 � 1025 mol/L thymoquinone (Supple-mental Fig. 4). This observation prompts the interpretationthat increases in cGMP production resulting from sGCactivation cannot be held responsible for the facilitation ofcontraction 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 vascularsmoothmuscle can reachmicromolar levels and help to controlCa21 homeostasis (Prosdocimo et al., 2010). This byproductcould thus account for the phenomenon seen with thymoqui-none. To test that possibility, rings with endothelium ofporcine coronary arteries incubated with ODQ (1025 mol/L)were exposed to pyrophosphate (1025 mol/L). This compoundwas not able to restore the augmentation of contraction by 3�1025 mol/L thymoquinone (Supplemental Fig. 3A).Hypoxic augmentations in coronary arteries are most likely

due to the transformation of inosine triphosphate (ITP) to cIMPby activated sGC (Beste and Seifert, 2013; Chen et al., 2014).UPLC-MS/MS revealed that, in rings of coronary arteries withendothelium, thymoquinone (3 � 1025 mol/L) caused a signifi-cant increase in the production of cIMP during contractions toprostaglandin F2a (Fig. 4A); endothelium removal, L-NAME (Fig.4C), and ODQ (Fig. 4E) prevented the effect of thymoquinone.

Fig. 4. Effect of thymoquinone on cIMP levels inisolated arteries. (A–F) Effect of thymoquinoneon the intracellular level of cIMP measured withUPLC-MS/MS in rings of porcine coronaryarteries (n = 4–6) (A, C, and E) precontractedwith prostaglandin F2a (porcine arteries, 2 �1026 mol/L) and of rat aortae (n = 4–5) (B, D, and F)precontracted with phenylephrine (rat arteries,1026 mol/L). (A and B) Original tracings of cIMPmeasurements and effect of thymoquinone (3 �1025 mol/L). The blue line depicts the signal forthe internal standard (IS) tenofovir, and the redline the signal for cIMP. (C and D) Effect ofthymoquinone (3 � 1025 mol/L) on the intracel-lular level of cIMP in rings of porcine coronaryarteries (n = 4–6) (C), and of rat aortae (n = 4–5)(D) with endothelium [E(+)], with endotheliumtreated with L-NAME (1024 mol/L) [E(+) &L-NAME], and without endothelium [E(2)]. (Eand F) Effect of thymoquinone (1026 to 1024

mol/L in porcine and 3 � 1025 mol/L in ratarteries) on the intracellular level of cIMP inrings of porcine coronary arteries (n = 4–6) (E),and of rat aortae (n = 4–5) (F) with endotheliumand with endothelium treated with ODQ (3 �1025 mol/L). The control group includes un-treated preparations with endothelium. Datashown as concentration in pmol/mg proteintissue and presented as means 6 S.E.M.; nrepresents the number of experiments forwhich at least four rings were pooled from fourdifferent animals (i.e., pooled observations).*Indicates statistically significant differencesfrom the control group with endothelium [E(+),black bar]; †indicates statistically significantdifferences from the thymoquinone group withendothelium [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/relation-ship between this unidentified peak and thechanges in cIMP signal, the former is not likelyto be a metabolite of the latter.

564 Detremmerie et al.

at ASPE

T Journals on M

arch 9, 2020jpet.aspetjournals.org

Dow

nloaded from

Similar results were obtained in rings of rat aortae (Fig. 4, B, D,and F). The levels of cAMP measured with UPLC-MS/MS werenot affected by thymoquinone or ODQ, whereas 3� 1025 mol/Lof the compound and ODQ reduced those of cGMP (Supple-mental Fig. 5). In isolated rat arteries with endotheliumexposed to L-NAME (1024 mol/L), exogenously administeredcIMP (3� 1024 mol/L) was able to restore the augmentation tothymoquinone, and this to the same extent as the NO-donorDETA NONOate (Fig. 5). However, the results showed acertain variability between groups of preparations, as DETANONOate did not increase the response to thymoquinonecomparedwith the control group to the same extent as observedin the first group of rat aortae (Fig. 2).These findings are compatible with the hypothesis that

cIMP mediates the endothelium-dependent augmentationby thymoquinone.Downstream Signaling. Protein kinases A and G are

major downstream effectors of the responses to cyclicnucleotides (Rubin and Rosen, 1975), but apparentlyare not involved in the augmentation caused by thymoqui-none. This conclusion is based on the observations thatincubation 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:39,29,19-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester(3 � 1027 mol/L), an established inhibitor of protein kinaseA (Papapetropoulos et al., 1995), did not significantlyaffect the augmentation to thymoquinone (1025 mol/L) inrat 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:39,29,19-kl]-pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methylester [1026 mol/L; selective protein kinase G inhibitor

(Smolenski et al., 1998)] did not significantly alter thethymoquinone-induced response (Supplemental Fig. 3B).Rho-associated protein kinase has been proposed to be re-

sponsible for sGC-dependent hypoxic contractions (Chan et al.,2011; Chen et al., 2014). Incubation with HA-1077 (1025 mol/L)or Y-27632 (1025 mol/L), two established Rho-associated pro-tein kinase inhibitors (Davies et al., 2000), significantly reducedthe thymoquinone-induced augmentation in porcine coronaryarteries (Fig. 6A), but not in rat aortae (Fig. 6C) with endothe-lium. An increase in extracellular calcium influx could alsoexplain the augmentation of contraction with thymoquinone.Indeed, exogenous calcium depletion, using an isosmoticcalcium-free control solution, reduced the augmentation inboth porcine coronary arteries (Fig. 6B) and rat aortae (Fig.6D). The inhibitor of voltage-dependent calcium-channelnifedipine (1025 mol/L) reduced the thymoquinone-inducedaugmentation in the porcine coronary arteries (Fig. 6B), butdid not significantly affect the response in rat aortae (Fig.6D). In addition to the L-type voltage-dependent calciumchannels, opening of T-type calcium channels can contributeto contractions of the isolated rat aorta (Duggan andTabrizchi, 2000). The 3,5-dichloro-N-[[(1a,5a,6-exo,6a)-3-(3,3-dimethylbutyl)-3-azabicyclo[3.1.0]hex-6-yl]methyl]-benzamidehydrochloride [3 � 1025 mol/L; selective T-type calcium-channelinhibitor (Xiang et al., 2011)] decreased the augmentation tothymoquinone significantly in the rat aorta (Fig. 6D), but notin porcine coronary arteries (Fig. 6B), suggesting the in-volvement of T-type calcium channels in the augmentationcaused by the compound in rat arteries.

DiscussionThe major finding of the present experiments is that

thymoquinone induces endothelium-dependent increases intension of isolated arteries. The endothelium-dependent aug-mentation by thymoquinone requires previous activation of thecontractile process, as it is not observed in quiescent prepara-tions. The vasoconstrictor response to the quinone is abolishedby an eNOS inhibitor but reinstalled by an exogenousNOdonorafter endothelium removal or eNOS inhibition, implying anobligatory 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 theleast. The contractions evoked by thymoquinone show astriking similarity in pharmacological characteristics withhypoxic contractions observed in earlier work in coronaryarteries 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, areendothelium-dependent, as demonstrated ex vivo in pulmo-nary, femoral, and coronary arteries of the dog (De Mey andVanhoutte, 1982, 1983; Rubanyi and Vanhoutte, 1985; Gräserand Vanhoutte, 1991; Pearson et al., 1996) and in coronaryarteries of the pig (Chan et al., 2011; Chen et al., 2014). As forthe response to thymoquinone (present study), inhibitors ofeNOS abolish the hypoxic response in isolated coronaryarteries, implying the involvement of NO. However, in thepresence of L-NAME, the addition of NO donors restores thehypoxic augmentation (Gräser and Vanhoutte, 1991; Pearsonet al., 1996; Chan et al., 2011; Chen et al., 2014), as it doesfor the contractions to thymoquinone (present experiments),

Fig. 5. Effect of exogenously administered cIMP on augmentation withthymoquinone in isolated rat aortae. Effects of L-NAME (1024 mol/L),DETA NONOate (1025 mol/L, in rings treated with L-NAME), and cIMP(3� 1024 mol/L, in rings treated with L-NAME) on thymoquinone-inducedaugmentations in rings with endothelium of rat aortae (n = 6) duringcontractions to phenylephrine (1028 to 1026 mol/L). The augmentationsare shown as areas under curve of the contraction phase of thecorresponding concentration-response graphs. The control group in-cludes untreated preparations with endothelium. Data shown as means 6S.E.M.; n represents the number of rings of different animals (i.e., in-dividual observations). *Indicates statistically significant differences fromcontrols; †indicates statistically significant differences between the groups“L-NAME” and “L-NAME + DETA,” and “L-NAME” and “L-NAME + cIMP”(P # 0.05).

Vasoconstrictions Requiring Biased Activity of sGC 565

at ASPE

T Journals on M

arch 9, 2020jpet.aspetjournals.org

Dow

nloaded from

confirming the involvement of the NO pathway. Inhibitorsof sGC prevent both hypoxic augmentation (Gräser andVanhoutte, 1991; Chan et al., 2011; Chen et al., 2014) andcontractions to thymoquinone (present findings), but stimu-lators of this enzyme restore them in preparations treatedwith L-NAME or in rings without endothelium, implying arole of activated sGC in those responses. The classic endproduct 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 bythymoquinone (present findings) was observed after incuba-tion with 8-Br-cGMP (cell-permeable form of cGMP). Mea-surements of cGMP levels showed no increase upon exposureto hypoxia (Chan et al., 2011) or to thymoquinone (presentfindings) in concentrations causing sustained endothelium-dependent augmentation. Inhibition of protein kinase G, thekey enzyme targeted by cGMP (Rubin and Rosen, 1975; Lucaset al., 2000), did not prevent augmentation by hypoxia (Chanet al., 2011) or thymoquinone (present findings). Thus, theseresponses require activation of sGC, but not the presence ofcGMP. Likewise, the absence of changes in cAMP levels andthe lack of effect of a protein kinase A inhibitor on thethymoquinone-induced augmentation make a contribution ofthis 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, inparticular cIMP, using ITP as substrate (Beste et al., 2012;Beste and Seifert, 2013). The production of this cyclic nucle-otide underlies hypoxic augmentation in porcine coronaryarteries (Chen et al., 2014). Likewise, the present UPLC-MS/MS measurements demonstrate that thymoquinone, likehypoxia, augments cIMP synthesis, not only in the porcinecoronary artery, but also in the rat aorta. Thus, it would seemreasonable to conclude that cIMP acts as the second messen-ger mediating the NO-dependent, sGC-dependent augmenta-tion of vasoconstriction caused by thymoquinone. Therefore,thymoquinone appears to be a pharmacological tool permit-ting the exploration of vasoconstrictor signals that requirethe biased activity of sGC (Chen et al., 2014; Gao andVanhoutte, 2014; Gao et al., 2015). However, the presentobservations do not rule out the possibility that the cIMPproduced by a biased sGC activation in response to thymoqui-none is actively transported out of the vascular smoothmuscle cells and acts extracellularly. In addition, it is notimpossible that cIMP is transformed intracellularly into ametabolite responsible for the observed effect, althoughearlier experiments indicate that, under hypoxic conditions,the contraction is caused by cIMP itself, as it is enhanced byphosphodiesterase inhibitors (Gräser and Vanhoutte, 1991;Chen et al., 2014).

Fig. 6. Calcium handling and augmentationwith thymoquinone in isolated arteries. (A–C)Effects of HA-1077 (1025 mol/L) and Y-27632(1025 mol/L) on thymoquinone-induced augmen-tations in rings with endothelium of porcinecoronary arteries (n = 5–7) (A) and of rat aortae(n = 4–7) (C) during contractions to serotonin(porcine arteries, 1028 to 1025 mol/L) or tophenylephrine (rat arteries, 1028 to 1026 mol/L).(B–D) Effects of nifedipine (1025 mol/L), ML-218(3 � 1025 mol/L), and Ca2+ depletion (in thepresence of 25 mmol/L caffeine) on thymoqui-none-induced augmentations in rings with endo-thelium of porcine coronary arteries (n = 5–7) (B)and of rat aortae (n = 4–7) (D) precontracted withserotonin (porcine arteries, 1028 to 1025 mol/L)or with phenylephrine (rat arteries, 1028 to 1026

mol/L). The control group includes untreatedpreparations with endothelium. The augmentationsare shown as areas under curve of the contrac-tion phase of the corresponding concentration-response graphs. Data shown as means6 S.E.M.;n represents the number of rings of differentanimals (i.e., individual observations). *Indicatesstatistically significant differences from controls(P # 0.05).

566 Detremmerie et al.

at ASPE

T Journals on M

arch 9, 2020jpet.aspetjournals.org

Dow

nloaded from

The augmentation by hypoxia is reduced by inhibitors ofRho-kinase (HA-1077 or Y-27632), indicating the involvementof calcium sensitization in the phenomenon (Chan et al., 2011;Chen et al., 2014). To judge from the experiments underhypoxic conditions in porcine coronary arteries, the activationof Rho-kinase reduces the activity of myosin light chainphosphatase, thereby resulting in increased phosphorylationof myosin light chain and thus augmented contractility ofvascular smooth muscle (Somlyo and Somlyo, 2003). In thepresent study, a similar effect of Rho-kinase inhibitors onthe thymoquinone-induced augmentation was observed in theporcine 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 arte-rial smooth muscle, thymoquinone depends on sensitizationby Rho-kinase of the action of calcium ions entering the cellsthrough L-type calcium channels. By contrast, in rat vascularsmooth muscle, T-type, rather than L-type, calcium channelsappear to mediate the effect of thymoquinone, to judge fromthe inhibition of the response observed in the presentexperiments with a selective inhibitor of the former, but not ofthe latter. These results imply different expression patterns ofvoltage-dependent calcium channels in different species orvascular beds. In line with this interpretation, L-type voltage-dependent calcium channels seem to be the major contribu-tors to voltage-gated calcium entry in coronary myocytes(Quignard et al., 1997), whereas in rat aortae, both L-typeand T-type voltage-dependent calcium channels are equallyexpressed (Ball et al., 2009). The present findings do notpermit further speculation yet as to the mechanisms un-derlying the observed species/vascular bed difference, al-though they indicate that in the studied rat arteries theimpact of cIMP on Rho-kinase is less pronounced than itseffect on T-type calcium channels. Nonetheless, the conjunc-tion of the present observations and the earlier experimentswith hypoxia (Chen et al., 2014) suggests that augmentedproduction of cIMP by sGC results in facilitation of thecontractile process in vascular smooth muscle.The present experiments in rat arteries imply that the

quinone moiety is essential for the action of thymoquinone inevoking endothelium-dependent contractions. The enzymeNAD(P)H:quinone acceptor oxidoreductase-1 (NQO1; EC 1.6.99.2) ishighly expressed in the vascularwall (Zhu et al., 2007;Han et al.,2009). It plays an important role in the body’s defense againstoxidative stress in part by detoxifying quinones and theirderivatives, thereby preventing their participation in redoxcycling (Riley and Workman, 1992; Ross et al., 2000; Hanet al., 2009). NQO1 metabolizes thymoquinone, presumablybecause of its structural similarity to ubiquinone, the naturalelectron carrier in mitochondria (Sutton et al., 2012). Indeed,thymoquinone can act as an electron acceptor during theoxidation of NADH to NAD1 (Staniek and Gille, 2010). NQO1regulates the NAD1/NADH ratio, which when increased in turninitiates a signaling cascade involving CD38 (adenosinediphosphate-ribose cyclase) and resulting in Ca21 mobilization.An increase in NAD1 concentration generated by NQO1 as aresult of the action of the enzyme on thymoquinone thus couldexplain the facilitation of contractions caused by the latter.The present experiments do not permit further speculationas to the possible molecular link(s) between activation ofNQO1 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 certainendothelium-dependent contractions (Fig. 7).In conclusion, the present pharmacological experiments re-

veal for thymoquinone a novel, and to date uniquemechanism ofaction, which favors the occurrence of deleterious endothelium-dependent, sGC-mediated contractions. Contractions requiringbiased activation (by endogenous NO or synthetic activators) ofsGC with the production of cIMP have been observed withhypoxia (Chen et al., 2014; Gao and Vanhoutte, 2014; Gao et al.,2015) and occur preferentially in parts of the coronary arteriesthat have previously been exposed to ischemia followed byreperfusion (Pearson et al., 1996). Such hypoxic contractionsmay help to understand the greater occurrence of cardiovascularcomplications, in cardiac patients with sleep apnea (Butt et al.,2011; Lee et al., 2011). By determining the mechanisms un-derlying the endothelium-dependent augmentation caused bythymoquinone and possibly other agents causing biased activityof sGC, the cellular target(s) involved in cardiovascular compli-cations due to hypoxia can be verified, thus facilitating thedevelopment of novel strategies to prevent coronary hypoxicvasospasm.

Acknowledgments

The authors thank Dr. BernardMarchand 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.

Fig. 7. Schematic overview of mechanisms underlying the augmentationby thymoquinone in isolated arteries. Effect of thymoquinone on sGC,biasing its activity upon stimulation by endothelial NO toward cIMPrather than cGMP production, leading to contraction rather thanrelaxation. The subsequent effects of cIMP on calcium homeostasis inthe vascular smooth muscle cells are shown also. Right: In porcinecoronary arteries, cIMP causes L-type voltage-dependent calcium influxand intracellular calcium sensitization (by activation of Rho-kinase); left:in rat arteries, cIMP causes T-type voltage-dependent calcium influx,eventually augmenting contraction.

Vasoconstrictions Requiring Biased Activity of sGC 567

at ASPE

T Journals on M

arch 9, 2020jpet.aspetjournals.org

Dow

nloaded from

References

Alkharfy KM, Al-Daghri NM, Al-Attas OS, and Alokail MS (2011) The protectiveeffect of thymoquinone against sepsis syndrome morbidity and mortality in mice.Int Immunopharmacol 11:250–254.

Ball CJ, Wilson DP, Turner SP, Saint DA, and Beltrame JF (2009) Heterogeneity ofL- and T-channels in the vasculature: rationale for the efficacy of combined L- andT-blockade. Hypertension 53:654–660.

Beste KY, Burhenne H, Kaever V, Stasch JP, and Seifert R (2012) Nucleotidyl cyclaseactivity of soluble guanylyl cyclase a1b1. Biochemistry 51:194–204.

Beste KY and Seifert R (2013) cCMP, cUMP, cTMP, cIMP and cXMP as possiblesecond messengers: development of a hypothesis based on studies with solubleguanylyl cyclase a(1)b(1). Biol Chem 394:261–270.

Butt M, Khair OA, Dwivedi G, Shantsila A, Shantsila E, and Lip GY (2011) Myo-cardial perfusion by myocardial contrast echocardiography and endothelial dys-function in obstructive sleep apnea. Hypertension 58:417–424.

Chan CKY, Mak J, Gao Y, Man RYK, and Vanhoutte PM (2011) Endothelium-derivedNO, but not cyclic GMP, is required for hypoxic augmentation in isolated porcinecoronary arteries. Am J Physiol Heart Circ Physiol 301:H2313–H2321.

Chen Z, Zhang X, Ying L, Dou D, Li Y, Bai Y, Liu J, Liu L, Feng H, Yu X, 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.

Davies SP, Reddy H, Caivano M, and Cohen P (2000) Specificity and mechanismof action of some commonly used protein kinase inhibitors. Biochem J 351:95–105.

De Mey JG and Vanhoutte PM (1982) Heterogeneous behavior of the canine arterialand venous wall: importance of the endothelium. Circ Res 51:439–447.

De Mey JG and Vanhoutte PM (1983) Anoxia and endothelium-dependent reactivityof the canine femoral artery. J Physiol 335:65–74.

Duggan JA and Tabrizchi R (2000) Influence of T-type Ca21 (mibefradil) and Cl-

(indanyloxyacetic acid 94) channel antagonists on alpha1-adrenoceptor mediatedcontractions in rat aorta. Can J Physiol Pharmacol 78:714–720.

Friebe A and Koesling D (2009) The function of NO-sensitive guanylyl cyclase: whatwe can learn from genetic mouse models. Nitric Oxide 21:149–156.

Furchgott RF and Zawadzki JV (1980) The obligatory role of endothelial cells in therelaxation of arterial smooth muscle by acetylcholine. Nature 288:373–376.

Gao Y, Chen Z, Leung SWS, and Vanhoutte PM (2015) Hypoxic vasospasm mediatedby cIMP: when soluble guanylyl cyclase turns bad. J Cardiovasc Pharmacol 65:545–548.

Gao Y and Vanhoutte PM (2014) Tissues cIMPly do not lie. Naunyn SchmiedebergsArch Pharmacol 387:901–903.

Goel A, Su B, Flavahan S, Lowenstein CJ, Berkowitz DE, and Flavahan NA (2010)Increased endothelial exocytosis and generation of endothelin-1 contributes toconstriction of aged arteries. Circ Res 107:242–251.

Gräser T and Vanhoutte PM (1991) Hypoxic contraction of canine coronary arteries:role of endothelium and cGMP. Am J Physiol 261:H1769–H1777.

Han SJ, Kang ES, Kim HJ, Kim SH, Chun SW, Ahn CW, Cha BS, Nam M, and LeeHC (2009) The C609T variant of NQO1 is associated with carotid artery plaques inpatients with type 2 diabetes. Mol Genet Metab 97:85–90.

Idris-Khodja N and Schini-Kerth V (2012) Thymoquinone improves aging-relatedendothelial dysfunction in the rat mesenteric artery. Naunyn Schmiedebergs ArchPharmacol 385:749–758.

Lee CH, Khoo SM, Chan MY, Wong HB, Low AF, Phua QH, Richards AM, Tan HC,and Yeo TC (2011) Severe obstructive sleep apnea and outcomes following myo-cardial infarction. J Clin Sleep Med 7:616–621.

Lucas KA, Pitari GM, Kazerounian S, Ruiz-Stewart I, Park J, Schulz S, ChepenikKP, and Waldman SA (2000) Guanylyl cyclases and signaling by cyclic GMP.Pharmacol Rev 52:375–414.

Papapetropoulos A, Marczin N, Mora G, Milici A, Murad F, and Catravas JD (1995)Regulation of vascular smooth muscle soluble guanylate cyclase activity, mRNA,and protein levels by cAMP-elevating agents. Hypertension 26:696–704.

Pathan SA, Jain GK, Zaidi SM, Akhter S, Vohora D, Chander P, Kole PL, Ahmad FJ,and Khar RK (2011) Stability-indicating ultra-performance liquid chromatographymethod for the estimation of thymoquinone and its application in bio-pharmaceutical studies. Biomed Chromatogr 25:613–620.

Pearson PJ, Lin PJ, Schaff HV, and Vanhoutte PM (1996) Augmented endothelium-dependent constriction to hypoxia early and late following reperfusion of the caninecoronary artery. Clin Exp Pharmacol Physiol 23:634–641.

Potter LR (2011) Guanylyl cyclase structure, function and regulation. Cell Signal 23:1921–1926.

Prosdocimo DA, Wyler SC, Romani AM, O’Neill WC, and Dubyak GR (2010) Regu-lation of vascular smooth muscle cell calcification by extracellular pyrophosphatehomeostasis: synergistic modulation by cyclic AMP and hyperphosphatemia. Am JPhysiol Cell Physiol 298:C702–C713.

Quignard JF, Frapier JM, Harricane MC, Albat B, Nargeot J, and Richard S (1997)Voltage-gated calcium channel currents in human coronary myocytes: regulationby cyclic GMP and nitric oxide. J Clin Invest 99:185–193.

Riley RJ and Workman P (1992) DT-diaphorase and cancer chemotherapy. BiochemPharmacol 43:1657–1669.

Ross D, Kepa JK, Winski SL, Beall HD, Anwar A, and Siegel D (2000) NAD(P)H:quinone oxidoreductase 1 (NQO1): chemoprotection, bioactivation, gene regulationand genetic polymorphisms. Chem Biol Interact 129:77–97.

Rubanyi GM and Vanhoutte PM (1985) Hypoxia releases a vasoconstrictor substancefrom the canine vascular endothelium. J Physiol 364:45–56.

Rubin CS and Rosen OM (1975) Protein phosphorylation. Annu Rev Biochem 44:831–887.

Shi Y, So KF, Man RYK, and Vanhoutte PM (2007) Oxygen-derived free radicalsmediate endothelium-dependent contractions in femoral arteries of rats withstreptozotocin-induced diabetes. Br J Pharmacol 152:1033–1041.

Smolenski A, Burkhardt AM, Eigenthaler M, Butt E, Gambaryan S, Lohmann SM,and Walter U (1998) Functional analysis of cGMP-dependent protein kinases I andII as mediators of NO/cGMP effects. Naunyn Schmiedebergs Arch Pharmacol 358:134–139.

Somlyo AP and Somlyo AV (2003) Ca21 sensitivity of smooth muscle and nonmusclemyosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev83:1325–1358.

Staniek K and Gille L (2010) Is thymoquinone an antioxidant? BMC Pharmacol 10(Suppl 1):A9.

Stasch JP and Evgenov OV (2013) Soluble guanylate cyclase stimulators in pulmo-nary hypertension. Handbook Exp Pharmacol 218:279–313.

Suddek GM (2010) Thymoquinone-induced relaxation of isolated rat pulmonary ar-tery. J Ethnopharmacol 127:210–214.

Sutton KM, Doucette CD, and Hoskin DW (2012) NADPH quinone oxidoreductase1 mediates breast cancer cell resistance to thymoquinone-induced apoptosis. Bio-chem Biophys Res Commun 426:421–426.

Tang EHC, Leung FP, Huang Y, Félétou M, So KF, Man RYK, and Vanhoutte PM(2007) Calcium and reactive oxygen species increase in endothelial cells in responseto releasers of endothelium-derived contracting factor. Br J Pharmacol 151:15–23.

Tang EHC and Vanhoutte PM (2009) Prostanoids and reactive oxygen species: teamplayers in endothelium-dependent contractions. Pharmacol Ther 122:140–149.

Vanhoutte PM (2011) Endothelium-dependent contractions in hypertension: whenprostacyclin becomes ugly. Hypertension 57:526–531.

Waldman SA and Murad F (1987) Cyclic GMP synthesis and function. PharmacolRev 39:163–196.

Xiang Z, Thompson AD, Brogan JT, Schulte ML, Melancon BJ, Mi D, Lewis LM, ZouB, Yang L, Morrison R, et al. (2011) The discovery and characterization of ML218: anovel, centrally active T-type calcium channel inhibitor with robust effects in STNneurons and in a rodent model of Parkinson’s disease. ACS Chem Neurosci 2:730–742.

Zhu H, Jia Z, Mahaney JE, Ross D, Misra HP, Trush MA, and Li Y (2007) The highlyexpressed and inducible endogenous NAD(P)H:quinone oxidoreductase 1 in car-diovascular cells acts as a potential superoxide scavenger. Cardiovasc Toxicol 7:202–211.

Address correspondence to: Dr. Charlotte M. Detremmerie, Department ofPharmacology and Pharmacy, University of Hong Kong, 2/F Laboratory Block,Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong SAR,China. E-mail: cdetremm@hku.hk

568 Detremmerie et al.

at ASPE

T Journals on M

arch 9, 2020jpet.aspetjournals.org

Dow

nloaded from