Baczkó, István

Bedreag, Ovidiu

Duicu, Oana

Jost, Norbert László

Kiss, Loránd

Muntean, Danina

Petruș, Alexandra

Sturza, Adrian

Utu, Diana

Petruș Alexandra

Sturza Adrian

Utu Diana

Duicu Oana

Bedreag Ovidiu

Kiss Loránd

Baczkó István

Muntean Danina

Jost Norbert László

SZTE Publicatio Repozitórium - SZTE - Repository of Publications

http://www.revistadechimie.ro REV.CHIM.(Bucharest)♦67♦No. 5 ♦2016908Modulation of Vascular Reactivity by Novel SyntheticBenzopyran Analogues in Rat AortasALEXANDRA PETRUS1,2#, ADRIAN STURZA1,2#, DIANA UTU1, OANA DUICU1,2, OVIDIU BEDREAG3, LORAND KISS4,ISTVAN BACZKO1, DANINA MUNTEAN1,2*,  NORBERT JOST1,21 Victor Babes  University of Medicine and Pharmacy Timisoara, Faculty of Medicine, Department of Pathophysiology, 14 SplaiulTudor Vladimirescu, 300173,Timisoara, Romania2 Victor Babes University of Medicine and Pharmacy Timisoara, Center for Translational Research and Systems Medicine,14 Splaiul Tudor Vladimirescu, 300173, Timisoara,  Romania3 Victor Babes University of Medicine and Pharmacy Timisoara, Faculty of Medicine, Department of Anesthesiology andIntensive Care, 156 Liviu Rebreanu Blvd., 300723, Timisoara, Romania4 Institute of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Szeged, Eotvos u. 6, H 6720, Szeged, HungaryA substantial body of research described ATP-dependent potassium (KATP) channels, sarcolemmal andmitochondrial,  with different physiological functions and pharmacological behaviour in various tissues(pancreatic â-cells, brain, skeletal muscle and vascular smooth muscle). The present study was purportedto assess the effects of three novel benzopyran derivatives, KL-1487, KL-1492 and KL-1507, analogues ofBMS191095 (a selective opener of mitoKATP channels) on vascular function and reactive oxygen species(ROS) production. To this aim, we performed organ bath experiments on rat aortic rings in the presence vs.absence of each of the mentioned compounds (10 µmol/L). Incubation of vascular rings with each benzopyrananalogue elicited a significant decrease of the contractile response to phenylephrine vs. control, thussuggesting a vasodilator action. However, only two of the benzopyran derivatives, KL-1492 and KL-1507,significantly improved the endothelial-dependent relaxation. Interestingly, these effects were present alsoin endothelium denuded vessels. None of these compounds modified vascular ROS production. In conclusion,in murine aortic segments, KL-1492 and KL-1507, two novel benzopyran analogues elicited an endothelial-independent modulation of vascular reactivity. Further studies will elucidate whether these compoundsmay be useful in restoring the vascular response in pathological conditions associated with endothelialdysfunction.Keywords: benzopyran derivatives, endothelial function, organ bath, vascular murine rings* email: daninamuntean@umft.ro                                                      # Authors with equally contributionThe ATP-sensitive potassium channels (KATP) are highlyinvestigated due to their unique property which is thecoupling of membrane excitability with cellular energeticsin several metabolic active tissues,  such as heart, brain,pancreas, smooth and skeletal muscle, respectively[1, 2,3]. The channels are closed under normal metabolicconditions and become activated in the setting of varioustypes of metabolic stress, including conditions associatedwith tissue ischemia[4]. In the heart, studies havedemonstrated the existence of two subtypes ofKATPchannels, sarcolemmal (sarcKATP) and mitochondrialKATP channels (mitoKATP); the channels are activated duringischemia/hypoxia due to an decrease in ATP concentrationand protect the ischemic myocardium by severalmechanisms [5]. Subsequently, a theory has emerged thatthe selective pharmacological modulation of KATP channelsin the heart can have beneficial effects in cardiovascularpathology. The inhibition of sarc KATP channels was shownto decrease the incidence of ischemia-related arrhythmias(malignant tachyarrhythmia and atrial fibrillation) bydecreasing the action potential duration [6], whereasmitoKATP opening was associated with the preservation ofmitochondrial structure [7], increased ATP production andpreservation during ischemia, increased functionalrecovery at reperfusion [8] and infarct size reduction [9].The major limitation regarding the available compoundsis represented by the lack of selectivity. Thus, there areseveral unselective agents that open both types of KATPchannels such as: pinacidil, cromakalim, minoxidil,levosimendan, aprikalim, P-1060, EMD 60480. Morerecently,  a selective mito KATP channel opener withbenzopyran structure: BMS 191095 (fig.1), has beensynthesized; the compound elicited cytoprotection in celllines via the inhibition of Ca2+ influx from extracellularenvironment [10], the reduction of basal ROS production,and the improvement in ATP homeostasis [11, 12].Fig. 1.The chemicalstructure of BMS191095The aim of the present study was to assess the effectsof novel benzopyran derivatives, analogues of BMS191095(KL-1487, KL-1492 and KL-1507) on vascular function andreactive oxygen species (ROS) production in a murineexperimental model.Experimental partMaterial and methodsSprague-Dawley female rats (weighing 250-350 g) werepurchased from Cantacuzino Institute (Bucharest,Romania) and were acclimated for 2 weeks prior to thestudy. Animals were housed under standard conditions(constant temperature and humidity of 22.5 ± 2° C and 55+ 5%, 12-h light/dark cycle). Twenty-four hours prior to theREV.CHIM.(Bucharest)♦67♦No. 5 ♦2016 http://www.revistadechimie.ro 909Fig. 2. Phenylephrine-induced contraction(A) andacetylcholine-inducedrelaxation(B) in rat aorticrings with normalendothelium (E+) in thepresence of  theinvestigated compoundsvs.the non-treated rings(n=5-6/group, *<0.05)Fig. 3. Phenylephrine-induced contraction(A) andacetylcholine-inducedrelaxation (B) in rat aorticrings with denudedendothelium (E-) in thepresence of  theinvestigated compoundsvs.the non-treated rings(n=4-6/group, *<0.05).Fig. 4.Rat aortic H2O2generation measured byFOX assay in thepresence of KL-1478, KL-1492, KL-1507 vs. thenon-treated rings(n=6/group)ABABexperiment solid food was withdrawn with no limitationin water supply.All experimental procedures used in this study wereconducted in accordance with the Directive 2010/63/EUand the Romanian Law no. 43/May 2014 concerning theprotection of animals used for scientific purposes. Theexperimental protocol was approved by the Committeefor Research Ethics of Victor Babes  University for Medicineand Pharmacy of Timisoara, Romania. Vascular segmentswere obtained after animal anaesthesia with anintraperitoneal injection of ketamine (30 mg/kg body mass)and xylazine (5 mg/kg body mass).Organ Bath StudiesOrgan bath experiments were performed on rat aorticrings in the presence of diclofenac (10 µmol/L) in order toinhibit the synthesis of prostaglandins. The concentrationof phenylephrine, used for preconstriction, was adjustedto obtain a preconstriction level of 80% of the contractionelicited by KCl(80 mmol/L). Endothelium-dependentrelaxation to cumulative concentrations of acetylcholine(Ach) was recorded in the presence vs. the absence ofbenzopyran derivatives, analogues of BMS 191095 (KL-1487, KL-1492 and KL-1507; 10µmol/L) and compared withthe effect of HMR 1098 (the cardioselective inhibitor ofsarcKATP channels, 10 µmol/L) according to a protocolstandardized in ref [13]. In a separate group of experiments,endothelium was removed by a treatment with CHAPS (5mg/mL dissolved in glucose solution 50g/L, exposure for40 s).ROS Measurements Hydrogen peroxide production was assessed in rataortic samples,  in the presence vs. the absence ofbenzopyran analogues  (30 min preincubation with KL1478,KL1492, KL1507, 10 µM) by Ferrous iron xylenol orangeOXidation (FOX) assay, as previously described [14,15]using the PeroxiDetect Kit (Sigma Aldrich). The principleof the assay is that peroxides oxidize Fe2+ to  Fe3+ ions atacidic pH. The Fe3+ ion will form a colored adduct withxylenol orange (XO,3,32 -bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephthalein, sodium salt),which is measured spectrofotometrically at 560 nm.StatisticsAll values are presented as mean ± SEM. Relaxationswere calculated from individual dose-response curves. Thevalues were analysed using the GraphPad Prism 5.Todetermine the global vascular reactivity the followingvariables of the Hill equation for a sigmoidal relationship:maximal response, concentration required to achieve 50%of the maximal response – EC50 (-log[M]) and the Hill slope,were computed. Values of p<0.05 were consideredstatistically significant.ReagentsAll reagents used were of the highest quality availableand were purchased from Sigma-Aldrich, Invitrogen,Applichem and Abcam.The novel compounds weresynthesized and kindly provided by Dr. L.Kiss from theDepartment of Pharmaceutical Chemistry, University ofSzeged, Hungary.Results and discussionsEx Vivo Stimulation With The Novel BenzopyranDerivativesReduced Contractility And Improved Relaxationin Rat Aortic RingsAll three benzopyran derivatives determined asignificant decrease of the contraction to PHE vs.control(fig. 2A), as well as an important improvement ofthe endothelial-dependent relaxation (fig.2B), observationsthat are relevant to a vasodilator action of these novelcompounds.The Presence Of An Intact Endothelium Is Not NecessaryForThe Vascular Effects Of The Benzopyran DerivativesIn order to investigate whether the above mentionedvascular effects are dependent on a functionalendothelium, we repeated the experiments in endothelium-denuded aortic rings. Interestingly, both the decrease incontractility (fig. 3A) and the improvement in relaxation(fig. 3B), were still present, strongly suggesting that thevascular effects of these compounds are endothelial-independent.Benzopyran Derivatives Do Not Interfere With BasalProduction of Vascular Hydrogen PeroxideSubsequently, we have assessed the effects of the threecompounds(10 µM) on the basal H2O2 production in intactaortic rings. However, none of the investigated compounds(KL-1487; KL-1492, KL-1507) interferedwith ROSgeneration in the normal murine vessels (fig 4).http://www.revistadechimie.ro REV.CHIM.(Bucharest)♦67♦No. 5 ♦2016910In the present study,performed in rat aortic rings, weinvestigated the effects of three novel benzopyranderivatives (KL-1487, KL-1492 and KL-1507) on vascularreactivityand ROS production, respectively.We havepreviously described in isolated rat heart mitochondria thatthese compounds elicited an uncoupling effect andalso,respiratory inhibition (in high doses) in a K+-independent manner. Moreover, we found that, whenapplied in the highest concentrationtested (150 microM)all compounds were able to decrease the H2O2 release[16]. All these effects have been reported to becardioprotective, in the settings of I/R injury.Since their discovery in cardiac myocytes, KATP channelshave been described in numerous other cell types, includingvascular smooth muscle cells [17], where theiropeningcaused membrane hyperpolarization, a decrease inintracellular calcium concentration and also vasodilation[4].Numerous KATP openers like pinacidil, nicorandil andcromakalim have vasorelaxant effects on systemic andcoronary blood vessels and also increase blood flow [4],thus being involved in reactive coronary hyperemia,metabolic coronary vasodilation and local control ofcoronary blood flow [18,19]. Conversely, glibenclamide(the classic KATP inhibitor) was reported to causedepolarization in vascular smooth muscle cells, asignificant increase in vascular resistance and thus, adecrease in arterial diameter [20, 21].However, controversial results are available in theliterature with respect to the effects of the selective channelopener, BMS 191095. Accordingly, both no effects on thevascular activity [22] and a vasodilator action of thecompound (at 50 and 100 microM) on cerebral arteries,have been reported[23]. Also, it has been reported thatvasodilatation can be elicited by two different mechanisms,depending on the molecular structure of the compounds.Studies conducted on mesenteric arteries with pinacidiland diazoxide were able to demonstrate that vasodilationelicited by pinacidil required subunit SUR2B, whereasdiazoxide-induced vasodilation occurred independently ofSUR-containing KATP channels and involved inhibition ofmitochondrial ETC [24]. In the case of BMS 191095, studiesshowed that, unlike diazoxide [23], the mitochondria-mediated vasodilation was independent of ROS production,but with a common pathway for both agents, namely thephosphoinositide-3 kinase/endothelial nitric oxide synthasepathway [25].Several studies demonstrated that activation of KATPchannels expressed in endothelial cells can trigger NOrelease [26] by increasing the transmembrane Ca2+ influx[27]; the production of other autacoids,such asprostaglandins, is also regulated via intracellular calcium[28, 29].Other studies have demonstrated that KATP openingis not involved in the endothelial-dependent relaxationmediated by acetylcholine (ACh) [30, 31].Our study indicates that only two of the testedcompounds, KL-1492 and KL-1507 (but not KL-1487),significantly improved relaxation in the presence ofcumulative doses of ACh (fig. 2B). Interestingly, the effectwas independent on the presence of a functionalendothelium, an intriguing observation that clearly requiresfurther investigation for the elucidation of the underlyingsignal transduction. Moreover, at variance from the resultsobtained in isolated mitocondria, no effect on ROSgeneration could be detected.ConclusionsWe have demonstrated here that in vitro application oftwo novel benzopyran analoguues, KL-1492 and KL-1507,modulate vascular reactivity in healthy murine aorticsegments without interfering with ROS generation.Whether the beneficial effects can be recapitulated inconditions associated endothelial dysfunction, such asdiabetes mellitus, are currently under investigation.Acknowledgements: This work was supported by a grant of the Ministryof National Education, CNCS – UEFISCDI, project number PN-II-ID-PCE-2012-4-0512.References1. MUNTEAN,D.M., KISS,L., JOST,N., BACZKÓ, I. Curr Pharm Des,21(8),2015, p. 1091.2. 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CORREA, D.S., RABETTI, A.C., BRAZ R., J. Med. Biol. Res., 24, nr.7, 1991, p. 729.Manuscript received: 9.03.2016

Modulation of Vascular Reactivity by Novel Synthetic Benzopyran Analogues in Rat Aortas

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Modulation of Vascular Reactivity by Novel Synthetic Benzopyran Analogues in Rat Aortas

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