NO synthase inhibition attenuates EDHF-mediated relaxation induced by TRPV4 channel agonist GSK1016790A in the rat pulmonary artery: Role of TxA2

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Original research article

NO synthase inhibition attenuates EDHF-mediated relaxation induced by TRPV4 channel agonist GSK1016790A in the rat pulmonary artery: Role of TxA2 Pule Addison, Thakur Uttam Singh *, Subhashree Parida, Soumen Choudhury, Jaya Kiran Kasa, Susanth V. Sukumaran, Sajad Ahmad Darzi, Kannan Kandasamy, Vishakha Singh, Dinesh Kumar, Santosh Kumar Mishra

Q1 M.

Division of Pharmacology and Toxicology, Indian Veterinary Research Institute, Izatnagar, India

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A R T I C L E I N F O

Article history: Received 14 August 2015 Received in revised form 1 January 2016 Accepted 4 January 2016 Available online xxx Keywords: EDHF Nitric oxide TRPV4 channels Pulmonary artery Thromboxane

A B S T R A C T

Background: The aim of the present study was to observe the concomitant activation of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) pathways by TRPV4 channel agonist GSK1016790A in the rat pulmonary artery and explore the mechanism by which NO synthase inhibition attenuates EDHF-mediated relaxation in endothelium-intact rat pulmonary artery. Methods: Tension experiments were conducted on the pulmonary artery from male Wistar rats. Results: TRPV4 channel agonist GSK1016790A (GSK) caused concentration-dependent relaxation (Emax 86.9  4.6%; pD2 8.7  0.2) of the endothelium-intact rat pulmonary artery. Combined presence of apamin and TRAM-34 significantly attenuated the relaxation (Emax 61.1  6.0%) to GSK. L-NAME (100 mM) significantly attenuated (8.2  2.9%) the relaxation response to GSK that was resistant to apamin plus TRAM34. However, presence of ICI192605 or furegrelate alongwith L-NAME revealed the GSK-mediated EDHFresponse (Emax of 28.5  5.2%; Emax 24.5  4.3%) in this vessel, respectively. Further, these two TxA2 modulators (ICI/furegrelate) alongwith L-NAME had no effect on SNP-induced endothelium-independent relaxation in comparison to L-NAME alone. This EDHF-mediated relaxation was sensitive to inhibition by K+ channel blockers apamin and TRAM-34 or 60 mM K+ depolarizing solution. Further, combined presence of apamin and TRAM-34 in U46619 pre-contracted pulmonary arterial rings significantly reduced the maximal relaxation (Emax 71.6  6.9%) elicited by GSK, but had no effect on the pD2 (8.1  0.03) of the TRPV4 channel agonist in comparison to controls (Emax, 92.4  4.3% and pD2, 8.3  0.06). Conclusion: The present study suggest that NO and EDHF are released concomitantly and presence of thromboxane modulators (ICI/furegrelate) unmasks the GSK-mediated EDHF response in the rat pulmonary artery. ß 2016 Published by Elsevier Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences.

12 Introduction

Abbreviations: ACh, acetylcholine; ANOVA, analysis of variance; EDHF, endothelium-derived hyperpolarizing factor; GSK, GSK1016790A; ICI192605, [4-(Z)-6-(2-0chlrophenyl-4-Ohydroxyphenyl-1,3-dioxan-cis5-yl) hexenoic acid]; IKCa (KCa3.1), intermediate conductance potassium channel; L-NAME, NG-nitro-L-arginine methyl ester; MKHS, modified Krebs–Henseleit solution; NO, nitric oxide; NOS, nitric oxide synthase; PE, phenylephrine; PKG, protein kinase G; SKCa (KCa2.3), small conductance potassium channel; SNP, sodium nitroprusside; TRAM 34, 1-[(2chlorophenyl)diphenylmethyl]-1H-pyrazole; TRPV4, transient receptor potential vanilloid 4; TP, thromboxane/prostanoid receptor. * Corresponding author. E-mail address: [email protected] (T.U. Singh).

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Endothelial dysfunction is considered to play an important role Q214 in pulmonary hypertension [1]. Therefore, it is of considerable 15 interest to understand the mechanisms underlying endothelium- 16 dependent regulation of pulmonary arterial tone in health and 17 diseases. The endothelium in pulmonary blood vessels regulates 18 the vascular tone by release of several vasoactive factors that 19 include both vasodilators such as nitric oxide, endothelium- 20 derived hyperpolarizing factors (EDHF) and prostacyclin as well as 21 vasoconstrictors like thromboxane A2, endothelin, leukotrienes 22 and superoxide anions [2]. In the rat pulmonary artery, it has been 23 shown that nitric oxide and EDHF are the two predominant 24

http://dx.doi.org/10.1016/j.pharep.2016.01.003 1734-1140/ß 2016 Published by Elsevier Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences.

Please cite this article in press as: Addison MP, et al. NO synthase inhibition attenuates EDHF-mediated relaxation induced by TRPV4 channel agonist GSK1016790A in the rat pulmonary artery: Role of TxA2. Pharmacol Rep (2016), http://dx.doi.org/10.1016/ j.pharep.2016.01.003

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vasodilators mediating endothelium-dependent relaxation [3–6]. Nitric oxide (NO) and EDHF operate simultaneously in middle cerebral artery and various other vasculatures [7–9]. There are various substances which are responsible for release of NO and EDHF like acetylcholine in the canine [10] and rat pulmonary arteries [3,4] bradykinin in bovine pulmonary supernumerary arteries [11] and pulmonary resistance arteries of the newborn piglet [12]. However, concomitant release of NO and EDHF by TRPV4 channel agonist GSK1016790A is not clear in the rat pulmonary vasculature. IKCa and SKCa channels are responsible for EDHF- mediated relaxation in endothelium of pulmonary arteries and bronchiolar epithelium of the rat [13]. The loss of the EDHF response may be primarily responsible for the endothelial dysfunction in sepsis, and its restoration by a selective iNOS inhibitor may improve pulmonary vasodilation [5]. Previously, it has been reported that GSK-induced relaxation was negligible in presence of L-NAME in the rat pulmonary artery [6]. However, an amplified relaxation response was observed in presence of indomethacin along with L-NAME which had been defined as EDHF [6]. A previous report demonstrated that exogenously applied NO attenuated the EDHF response in rabbit carotid and porcine coronary arteries [14] On the other hand, one component, SKCa channel of EDHF response was inhibited by blockage of nitric oxide synthase (NOS) in middle cerebral artery of rat [15]. A previous report demonstrated that TxA2 receptors stimulation leads to progressive loss of smooth muscle hyperpolarization due to EDHF which occurs due to inactivation of the endothelial SKCa in the rat isolated mesenteric artery [16]. TxA2 synthesis could be inhibited with NO by interacting with heme active site of thromboxane synthase enzyme [17] and further a report stated that NO desensitizes thromboxane receptors through PKG signaling pathway [18]. Activation of endothelial and epithelial KCa2.3 channels leads to relaxation in small pulmonary arteries and bronchioles in human [19]. However, in current study we have tried to explore the interaction between NO and EDHF as it is poorly understood. Some reports also suggest that NO inhibits EDHF-response in various blood vessels but it is not studied in rat pulmonary artery, moreover, its mechanism of action still remains unclear. Therefore, the first objective of the present study was to study the concomitant activation of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) pathways by TRPV4 channel agonist GSK1016790A in rat pulmonary artery. The second objective was to explore the mechanism by which NO synthase inhibition attenuates EDHF-mediated relaxation in endothelium-intact rat pulmonary artery.

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Materias and methods

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Animals

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Healthy adult male Wistar rats (150–200 g) were procured from the Laboratory Animal Resource Section, Indian Veterinary Research Institute, Izatnagar, India. Animals were kept for acclimatization for a period of seven days before conduction of experiments. All protocols, employed in this study, were approved by the Institutional Animal Ethics Committee, Indian Veterinary Research Institute, Izatnagar.

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Tension recording

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The animals were killed by excising the abdominal aorta under urethane (1.2 g/kg body weight intraperitoneally) anesthesia. Heart and lungs enbloc were removed and transferred to ice cold Modified Krebs–Henseleit solution (MKHS) of the following composition (in mM): NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2,

NaHCO3 11.9, KH2PO4 1.2 and D-glucose 11.1 (pH 7.4). Both the branches of the main pulmonary artery (right and left) were dissected carefully and cleaned of adhering connective tissues under a dissecting microscope. These pulmonary artery rings were used for further study. The rat pulmonary arterial rings were mounted on two stainless steel hooks and suspended in 10 ml organ baths containing MKHS, maintained at 37 8C. Further, pulmonary artery rings were continuously aerated with medical gas (21% O2 + 5% CO2 + 74% N2) mixture. 1.0 g passive tension was applied during the equilibration period of 90 min and the bath solution was changed every 15 min. Tension was recorded using a high sensitivity isometric force transducer and stored in a computer using Chart version 5.4.1 software program (Powerlab, AD Instruments, Bella Vista, NSW, Australia) for further analysis. After the completion of equilibration period, tissue viability was checked by recording the contraction to 80 mM K+ depolarizing solution. Concentration–response curves to relaxants (GSK and SNP) were elicited in arterial segments pre-contracted with a submaximal concentration of phenylephrine (0.01–1 mM) or U46619 (10–100 nM) to achieve near identical pre-contraction levels. 1 mM of ACh was added at the plateau of phenylephrine contraction to examine the endothelial integrity. If the relaxant response to 1 mM ACh was more than 80%, it was considered endothelium-intact pulmonary artery. L-NAME pretreatment sensitized the pre-contractions to phenylephrine before eliciting relaxation responses to different vasodilators. Hence, an appropriate concentration of phenylephrine, as stated above, was used to achieve matching contraction level of the arterial segments before eliciting relaxations to vasodilators [6].

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Experimental protocols

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Concomitant activation of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) pathways by TRPV4 channel agonist GSK1016790A in the rat pulmonary artery To rule out the concomitant release of NO and EDHF by TRPV4 channel agonist GSK1016790A (GSK), a concentration response curve to GSK was developed in combined presence of 100 nM apamin and 1 mM TRAM-34. GSK (10 10–10 7 M), added cumulatively at an increment of 0.5 log unit, caused concentrationdependent relaxation in endothelium-intact pulmonary artery rings pre-contracted with 0.01–1 mM phenylephrine. Apart from this, a dose-response to GSK was elicited in combined presence of L-NAME, apamin and TRAM-34.

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Effect of L-NAME and ICI192605 (ICI) (a thromboxane receptor antagonist) or furegrelate (thromboxane synthase blocker) on SNPinduced relaxation in the pulmonary artery To confirm the specificity of EDHF-mediated GSK-induced relaxation, a concentration-response curve to SNP (10 11–10 5 M) was elicited in pulmonary arterial rings pretreated for 30 min with 100 mM L-NAME and 100 mM L-NAME plus 10 mM ICI/10 mM furegrelate.

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To elucidate the L-NAME resistant GSK-induced relaxation modulation by ICI192605 or furegrelate in the rat pulmonary artery In order to assess the modulatory effect of vasocontractile substances in endothelium like prostanoids, the pulmonary arterial rings were pretreated with either ICI (a thromboxane receptor blocker) or furegrelate (a thromboxane synthase inhibitor). The contribution of EDHF pathway to GSK (10 10– 10 7 M)-induced relaxation was evaluated by eliciting concentration-dependent relaxation to the TRPV4 channel agonist on phenylephrine-contracted pulmonary artery rings pretreated with 100 mM L-NAME plus 10 mM ICI/10 mM furegrelate for 30 min.

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Please cite this article in press as: Addison MP, et al. NO synthase inhibition attenuates EDHF-mediated relaxation induced by TRPV4 channel agonist GSK1016790A in the rat pulmonary artery: Role of TxA2. Pharmacol Rep (2016), http://dx.doi.org/10.1016/ j.pharep.2016.01.003

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Effect of the apamin, SKCa channel blocker, and TRAM-34, IKCa channel blocker, or 60 mM K+ in EDHF response elicited by GSK in the rat pulmonary arterial rings To confirm that EDHF contributes to the relaxant response elicited by GSK through vascular smooth muscle hyperpolarization, the arterial rings were incubated with 100 mM L-NAME plus 10 mM ICI/furegrelate for 30 min and a concentration-response curve to GSK (10 10–10 7 M) was elicited in 60 mM K+ precontracted rings. To find out the role of SKCa and IKCa channels in EDHF-mediated relaxation response to GSK (10 10–10 7 M), the pulmonary arterial rings were incubated with specific blockers of SKCa (100 nM apamin) plus IKCa channels (1 mM TRAM-34) for 30 min before eliciting the concentration-response curve to GSK on phenylephrine pre-contracted tissues.

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Effect of U46619 on GSK-induced relaxation in the rat pulmonary artery

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To find out the involvement of thromboxane receptor on GSKinduced relaxation, a dose-response curve to GSK (10 10–10 7 M) was elicited in 10 nM U46619 (thromboxane receptor agonist) precontracted pulmonary arterial rings. A dose-response curve to GSK was developed in presence of 100 nM apamin (SKCa blocker) plus 1 mM TRAM-34 (IKCa Blocker) to rule out the EDHF pathway in U46619 precontracted rings. Further a dose-response to GSK was elicited in combined presence of L-NAME, apamin and TRAM-34 in U46619 precontracted rings.

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Drugs Acetylcholine chloride, L-NAME, SNP, phenylephrine, apamin, TRAM 34, GSK1016790A were purchased from Sigma Aldrich Chemicals (St Louis, MO). ICI192605 was purchased from TOCRIS, Bristol, U.K. Furegrelate and U46619 were obtained from Cayman

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chemicals, USA. GSK1016790A and TRAM 34 were dissolved in dimethyl sulphoxide (DMSO). All other drugs were dissolved in distilled water.

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Data analysis and statistics Results are expressed as mean  SEM with n equal to number of animals. Relaxation responses of the arterial rings were expressed as the percentage reversal of the phenylephrine/U46619 contraction. Alteration in basal tone to GSK was expressed as percentage of 80 mM K+-induced contraction. Emax (the maximal response) and EC50 (the concentration producing 50% of the maximal response) to vasoactive agonists were determined by nonlinear regression analysis (sigmoidal dose-response with variable slope) using Graph Pad Prism version 4 (San Diego, CA, USA). Sensitivity/potency was expressed as pD2 = log EC50. Data were analyzed by two-way ANOVA for multiple comparisons followed by Bonferroni post hoc test.

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Results

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Concomitant activation of NO and EDHF pathways by TRPV4 channel agonist GSK1016790A (GSK) in the rat pulmonary artery

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The representative tracing in Fig. 1A shows that GSK (10 10– 10 7 M), added cumulatively at an increment of 0.5 log unit, caused concentration-dependent relaxation in endothelium-intact pulmonary artery rings pre-contracted with 1 mM phenylephrine. The precontraction amplitude with PE was 0.40  0.02 g before eliciting GSK-induced relaxation. Line diagram in Fig. 1D illustrates that GSK elicited concentration-dependent relaxation with Emax of 86.9  4.6% and pD2 of 8.7  0.24 (n = 11). In order to assess the concomitant activation of EDHF and NO mechanisms in GSK-induced relaxation, EDHF blockers apamin and TRAM-34 and NO synthase inhibitor L-NAME were used. SKCa channel blocker apamin (100 nM)

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Fig. 1. The representative tracings show the concentration-response curves to GSK1016790A (GSK) (10 10–10 7 M) as control (A), in combined presence of apamin (SKCa blocker) and TRAM-34 (IKCainhibitor) (B) and in L-NAME, apamin and TRAM-34 treated (C) endothelium-intact rat pulmonary arterial rings pre-contracted with phenylephrine. (D) The line diagram depicts the inhibition of GSK-induced relaxation in the presence of TRAM-34 (1 mM) plus apamin (100 nM) and in combined presence of L-NAME, apamin and TRAM-34 in phenylephrine pre-contracted arterial rings. The line diagram depicts the mean values of relaxation to GSK. The vertical bars represent SEM. The data were analyzed by two-way ANOVA, followed by Bonferroni post hoc test. #p < 0.001 in comparison to control. n = 7–12 and refers to number of animals.

Please cite this article in press as: Addison MP, et al. NO synthase inhibition attenuates EDHF-mediated relaxation induced by TRPV4 channel agonist GSK1016790A in the rat pulmonary artery: Role of TxA2. Pharmacol Rep (2016), http://dx.doi.org/10.1016/ j.pharep.2016.01.003

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or IKCa channel blocker TRAM-34 (1 mM) alone had no significant effect on GSK-induced relaxation (Emax 75.3  12.3%; pD2 9.1  0.47; n = 4 and Emax 75.1  9.7%; pD2 8.4  0.24; n = 6; respectively). However, these two potassium channel blockers in combination significantly (p < 0.05) reduced the maximal relaxation (Emax 61.1  6.0%; n = 12) elicited by GSK (Fig. 1B and D), but had no effect on the pD2 (8.5  0.24; n = 12) of the TRPV4 channel agonist in comparison to controls. L-NAME (100 mM) abolished the residual relaxation to GSK that was resistant to apamin plus TRAM-34 (Fig. 1C and D). The pre-contractions levels of PE with different pretreatments, such as apamin plus TRAM-34 (0.36  0.02 g) or L-NAME together with apamin and TRAM-34 (0.39  0.03 g), were not significantly different from those of controls (0.40  0.02 g).

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Effects of TxA2 receptor blocker ICI or thromboxane synthase inhibitor furegrelate on L-NAME-resistant GSK-induced relaxation of the rat pulmonary artery

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In the presence of L-NAME (100 mM), GSK (10 10–10 7 M)induced maximal relaxation was 8.2  2.9% (n = 11) in PE (0.1 mM) pre-contracted (0.44  0.04 g) endothelium-intact pulmonary arterial rings (Fig. 2A) in comparison to 25% contribution of EDHF (sensitive to apamin + TRAM-34) to GSK-induced endotheliumdependent relaxation in the absence of NO synthase inhibitor. In order to examine the role of endothelium-derived contractile factor such as TxA2 to the attenuation of EDHF response in the absence of NO synthesis, we have used ICI or furegrelate in the presence of L-NAME. Arterial rings pre-treated with L-NAME (100 mM) and ICI (10 mM) and pre-contracted with PE 0.1 mM (0.42  0.03 g), GSK (10 10–10 7 M) produced concentration-dependent relaxation with the Emax of 28.5  5.2% (n = 8) versus L-NAME alone 8.2  2.9% (n = 11). Similarly, in the combined presence of L-NAME (100 mM) and furegrelate (10 mM), the relaxation response to GSK was significantly (p < 0.05) greater (Emax 24.5  4.3% (n = 10) than that observed with L-NAME alone (8.2  2.9%; n = 11). The pre-contraction tone with L-NAME plus furegrelate was 0.42  0.03 g. To study that ICI or furegrelate specifically potentiated GSKinduced EDHF-mediated relaxation, we assessed the effects of these two TxA2 modulators on the relaxation response to endothelium-independent vasodilator SNP in endothelium-intact pulmonary artery rings. Fig. 2B shows that SNP (10 11–10 5 M)induced concentration-dependent relaxations in the presence of 100 mM L-NAME (pD2, 8.2  0.04 and Emax, 107.4  1.5%; n = 6) were not significantly different from that elicited in the presence of either 100 mM L-NAME plus 10 mM ICI (pD2, 8.0  0.04 and Emax,

108.2  1.6%; n = 6) or 100 mM L-NAME plus 10 mM furegrelate (pD2, 8.1  0.05 and Emax, 110.1  1.7%; n = 6).

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Effect of apamin and TRAM-34 combination or 60 mM K+ on EDHFmediated GSK-induced relaxations in the pulmonary arterial rings

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To illustrate that GSK-induced relaxations in the presence of either L-NAME plus ICI or L-NAME plus furegrelate are mediated by the EDHF pathway, two experimental approaches were made. First approach was to block the EDHF mechanism with the combination of apamin and TRAM-34 and the second approach was to elicit GSK-induced relaxation in pulmonary artery rings pre-contracted with 60 mM K+-depolarizing solution (a protocol that prevents EDHF relaxation resulting from membrane hyperpolarization). Fig. 3A depicts that in the presence of 100 mM L-NAME plus 10 mM ICI for 30 min, GSK (10 10–10 7 M) elicited concentrationdependent relaxation with Emax of 26.9  4.8% (n = 10) in PE precontracted (0.42  0.03 g) pulmonary arterial rings. However, in the combined presence of 100 mM L-NAME, 10 mM ICI, 0.1 mM apamin and 10 mM TRAM-34 for 30 min, GSK-induced relaxation was significantly (p < 0.05) reduced (Emax 7.6  3.8%, n = 4) in the arterial rings pre-contracted with 0.1 mM PE (0.41  0.08 g). Fig. 3B shows that in the presence of 100 mM L-NAME plus 10 mM furegrelate for 30 min, GSK (10 10–10 7 M) produced concentration-dependent relaxation (Emax 25.9  4.9%; n = 8) in 0.1 mM PE pre-contracted (0.42  0.03 g) tissues. Additional treatment of the arterial rings with 0.1 mM apamin and 10 mM TRAM-34 significantly (p < 0.05) reduced the relaxation response (10.7  3.0%; n = 6) to GSK. Fig. 3C and D depicts the effect of high K+ (60 mM) depolarizing solution on EDHF mediated GSK-induced relaxation. GSK-induced relaxations in arterial segments pre-treated either 100 mM LNAME plus 10 mM ICI (Emax 26.9  4.8%; n = 10) or 100 mM L-NAME plus 10 mM furegrelate (Emax 25.9  4.9%; n = 8) were nearly abolished in 60 mM K+ pre-contracted tissues (n = 6 for each group).

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Characteristics of GSK-induced endothelium-dependent relaxation in the pulmonary arterial rings pre-contracted with thromboxane analogue U46619

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The representative tracing in Fig. 4A shows that GSK (10 10– 10 7 M), added cumulatively at an increment of 0.5 log unit, caused concentration-dependent relaxation in endotheliumintact pulmonary artery rings pre-contracted with 10 nM U46619. The precontraction tone with U46619 was 0.38  0.02 g before eliciting GSK-induced relaxation. Fig. 4A and

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Fig. 2. (A) The line diagram depicts the mean values of concentration-dependent relaxation to cumulatively added GSK1016790A in endothelium-intact pulmonary artery in presence of either 100 mM L-NAME (n = 11) or 100 mM L-NAME plus 10 mM ICI (n = 8) or 100 mM L-NAME plus 10 mM furegrelate (n = 10). (B) The line diagram shows the mean values of concentration-dependent cumulatively added SNP-induced endothelium-independent relaxation in endothelium intact rat pulmonary artery in presence of either 100 mM L-NAME (n = 6) or 100 mM L-NAME plus 10 mM ICI (n = 6) or 100 mM L-NAME plus 10 mM furegrelate (n = 6).The vertical bars represent SEM. The data were analyzed by two-way ANOVA, followed by Bonferroni post hoc test. * p < 0.05; $p < 0.01 and #p < 0.001 in comparison to control.

Please cite this article in press as: Addison MP, et al. NO synthase inhibition attenuates EDHF-mediated relaxation induced by TRPV4 channel agonist GSK1016790A in the rat pulmonary artery: Role of TxA2. Pharmacol Rep (2016), http://dx.doi.org/10.1016/ j.pharep.2016.01.003

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Fig. 3. (A) The line diagram depicts the inhibition of L-NAME/ICI192605 (EDHF)-resistant relaxation to GSK in the presence of IKCa inhibitor TRAM-34 (1 mM) plus SKCa blocker apamin (100 nM) in phenylephrine pre-contracted arterial rings (n = 4–10). (B) The line diagram depicts the inhibition of L-NAME/furegrelate (EDHF)-resistant relaxation to GSK in the presence of TRAM-34 (1 mM) plus apamin (100 nM) in phenylephrine pre-contracted arterial rings (n = 6–8). (C) Line diagram shows concentration-response curve to GSK (10 10–10 7) elicited in 60 mM K+/phenylephrine-pre-contracted rings in the presence of 100 mM L-NAME plus 10 mM ICI (n = 6–10). (D) Line diagram shows concentration-response curve to GSK (10 10–10 7) elicited in 60 mM K+/phenylephrine-pre-contracted rings in the presence of 100 mM L-NAME plus 10 mM furegrelate (n = 6–8).The data were analyzed by two-way ANOVA, followed by Bonferroni post hoc test. * p < 0.05; $p < 0.01 and #p < 0.001 in comparison to control.

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E illustrates that GSK (10 10–10 7 M) caused concentrationdependent relaxations (pD2, 8.3  0.06 and Emax, 92.4  4.3%; n = 10) in U46619 pre-contracted endo (+) pulmonary arterial rings. To find out the concomitant activation of EDHF and NO mechanisms in GSK-induced relaxation in thromboxane analogue U46619 precontracted tissue, EDHF blockers apamin and TRAM-34 and NO synthase inhibitor L-NAME were used. Combined presence of SKCa channel blocker apamin (100 nM) or IKCa channel blocker TRAM-34 (1 mM) significantly (p < 0.05) reduced the maximal relaxation (Emax 71.6  6.9%; n = 6) elicited by GSK (Fig. 4B and E), but had no effect on the pD2 (8.1  0.03; n = 6) of the TRPV4 channel agonist in comparison to controls. L-NAME (100 mM) reduced (Emax 14.1  3.3%; n = 4) the residual relaxation to GSK that was resistant to apamin plus Tram-34 (Fig. 4C and E). Further, in combined presence of L-NAME, apamin and TRAM-34, GSK-induced relaxation was absent (11.4  4.5% contraction; n = 6) in comparison to control (Fig. 4D and E). The pre-contraction levels of U46619 with different pretreatments, such as apamin plus TRAM-34 (0.45  0.02 g) or L-NAME (0.49  0.03 g), were not significantly different from those of controls (0.38  0.02 g).

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Discussion

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The present study showed that (1) Concomitant activation of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) by TRPV4 channel agonist GSK1016790A (GSK) in the rat pulmonary artery; (2) L-NAME inhibited EDHF response in the rat pulmonary vasculature; (3) Further, ICI192605 and furegrelate revealed the EDHF response in presence of L-NAME in this vasculature; (4) EDHF-response was absent in high K (60 mM);

(5) EDHF-response was specific to endothelium of pulmonary vasculature. Combined presence of SKCa channel blocker (apamin) and IKCa channel blockers (TRAM-34) significantly attenuated GSK-induced relaxation in endothelium intact rat pulmonary artery. These results showed that there was simultaneous release of NO and EDHF in this vasculature. The findings of the present study is in agreement with earlier reports which showed simultaneous operation of both the pathways in rat hepatic artery [7] and middle cerebral artery [9]. Further, it is substantiated that AChinduced relaxation in the canine [10] and rat pulmonary artery [3,4] is mediated by NO and EDHF. TRPV4 channel agonist GSKinduced relaxation through NO and EDHF in the rat pulmonary artery was observed recently [6]. GSK-induced relaxation was barely 10% in presence of nitric oxide synthase inhibitor (LNAME) in previous findings published from the laboratory [6]. However, GSK-induced relaxation was enhanced in presence of ICI192605 or furegrelate. This amplified relaxation to GSK was termed as EDHF in this vasculature which was appeared through inhibition of thromboxane receptor or thromboxane synthase. Finding of the present study is in complete agreement with the earlier report which showed that either antagonism of TP receptors or blockade of thromboxane synthase restored an input through SKCa channel in the rat middle cerebral artery as SKCa is the one of the essential components of EDHF [15]. The present study is also supported with the report that showed NO inhibits 20-HETE, a metabolite of arachidonic acid, and 20-HETE is responsible for inhibition of EDHF response in coronary artery of porcine [20]. TxA2 receptor activation leads to inhibitory effect on EDHF response in the rat mesenteric artery. It inhibits SKCa channel

Please cite this article in press as: Addison MP, et al. NO synthase inhibition attenuates EDHF-mediated relaxation induced by TRPV4 channel agonist GSK1016790A in the rat pulmonary artery: Role of TxA2. Pharmacol Rep (2016), http://dx.doi.org/10.1016/ j.pharep.2016.01.003

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Fig. 4. The representative raw tracings show the concentrations-response curves to GSK1016790A (GSK) (10 10–10 7 M) in U46619 pre-contracted pulmonary arterial rings as control (A), in combined presence of apamin (SKCa blocker) and TRAM-34 (IKCa inhibitor) (B) L-NAME (C) and L-NAME + apamin + TRAM-34-treated (D) in endotheliumintact rat pulmonary arterial rings pre-contracted with U46619. (E) The line diagram depicts the inhibition of GSK-induced relaxation in the presence of TRAM-34 (1 mM) plus apamin (100 nM) and L-NAME alone in U46619 pre-contracted arterial rings (n = 7–12). The line diagram depicts the mean values of relaxation to GSK. The vertical bars represent SEM. The data were analyzed by two-way ANOVA, followed by Bonferroni post hoc test. *p < 0.05 in comparison to control; #p < 0.001 in comparison to control. n = 4–10 and refers to number of animals.

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activity of EDHF [16]. Other studies suggested that inhibition of nitric oxide synthesis activates thromboxane A2 and shows contractile effect in the rat isolated middle cerebral arteries [21,22]. Further, a recent study suggested that NOS modulates pulmonary vascular responses to TRPV4 activation in the rat [23]. EDHF appears in combined presence of L-NAME and indomethacin [5,6,24]. In the present study, an enhanced relaxation response to GSK was observed in combined presence of L-NAME and ICI/furegrelate. The finding suggested that inhibition of thromboxane/prostanoid receptor (TP) receptor or TxA2 synthase leads to unmasking of L-NAME induced inhibition. The current findings are in agreement with a previous report which showed that in presence of ICI/furegrelate along with L-NAME, a marked EDHF response was visible in the rat middle cerebral artery [15]. Further, present study may be substantiated with the report where the presence of L-NAME and indomethacin leads to a sufficient EDHF relaxation in rat pulmonary artery [5,6].

Further, the selectivity of TRPV4 channel agonist GSK-induced EDHF mediated relaxation was confirmed by the observation that the L-NAME and ICI/furegrelate did not alter the endotheliumindependent relaxation produced by sodium nitroprusside in comparison to L-NAME alone. In our previous study, it was shown that TRPV4 channel antagonist HC067047 did not change the SNPinduced relaxation which was considered as endotheliumindependent relaxation [6]. However, L-NAME enhanced SNPinduced relaxation in rat pulmonary artery which suggested the mechanism of supersensitivity in vascular smooth muscle to nitrovasodilators in acute NO deficiency [25]. It has been reported that activation of thromboxane receptor leads to loss in the activity of KCa2.3 channel in peripheral artery of the rats as it is essential component of EDHF and ultimately impairs the relaxant response [16]. However present study suggests that activation of both the channels (SKCa and IKCa) release EDHF in this artery. The amplified relaxation response to

Please cite this article in press as: Addison MP, et al. NO synthase inhibition attenuates EDHF-mediated relaxation induced by TRPV4 channel agonist GSK1016790A in the rat pulmonary artery: Role of TxA2. Pharmacol Rep (2016), http://dx.doi.org/10.1016/ j.pharep.2016.01.003

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G Model

PHAREP 448 1–7 M.P. Addison et al. / Pharmacological Reports xxx (2016) xxx–xxx

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GSK in presence of ICI-192,605/furegrelate plus L-NAME was confirmed as EDHF-mediated relaxation by the observation that it was completely absent in tissues pre-contracted with 60 mM K+ depolarizing solution. In vitro depolarization of arterial rings by more extracellular potassium prevents the relaxation response to EDHF due to depolarizing solution creates a K+ gradient that does not favor outward movement of K+ and hyperpolarization [26]. Further, the observation was supported with the findings that apamin plus TRAM-34 significantly reduced GSK-induced EDHF responses in the rat pulmonary artery. The role of SKCa and IKCa channels in mediating EDHF-induced relaxation of rat pulmonary artery has been demonstrated earlier [5,6,13]. Further to confirm the modulation of EDHF by thromboxane in GSK-induced endothelium-dependent relaxation in pulmonary artery rings, the arterial rings were pre-contracted with thrmboxane analogue U46619 and a dose-dependent relaxation was elicited. We blocked the SKCa and IKCa channels with specific blockers (apamin and TRAM-34), a significant inhibition was observed in GSK-induced relaxation in U46619 pre-contracted rings. This indicated that NO and EDHF are simultaneously intact and are released simultaneously like in PE-precontracted rings. In a previous study, it was demonstrated that stimulation of thromboxane receptor with U46619 diminishes the part of SKCa channel in EDHF in middle cerebral artery but synthesis of NO is present [15]. GSK greater than 100 nM causes cell destruction particularly that of the endothelial cells [27]. In the present study, it is looking that higher concentration of GSK may lead to destruction of endothelial cells and produced contraction after certain concentration of GSK. GSK increased the basal tone in endothelium-denuded rat pulmonary arterial rings, it may involve Ca2+ influx through vascular cell TRPV4 channels which results in increased intracellular Ca2+ concentration and leads to smooth muscle contraction [6]. Further, a recent report suggested TRPV4-triggered vascular contraction is a TxA2-mediated response in mouse aorta [28].

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In conclusion, the present study suggests that NO synthesis must be intact for the expression of the EDHF in the rat pulmonary artery. Once NOS inhibits by L-NAME, it leads to attenuation of EDHF response but presence of blockage of thromboxane unmasks the EDHF pathway. Further, a mechanistic study is required to rule out the appearance of EDHF in presence of thromboxane synthesis inhibition.

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Funding

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Q3

Author(s) received financial support from ICAR-Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India for this research. Conflict of interest There is no conflict of interest in this manuscript.

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Authors are thankful to the Director, Indian Veterinary Research Institute, Izatnagar for providing the necessary facilities to complete this work.

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References

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Please cite this article in press as: Addison MP, et al. NO synthase inhibition attenuates EDHF-mediated relaxation induced by TRPV4 channel agonist GSK1016790A in the rat pulmonary artery: Role of TxA2. Pharmacol Rep (2016), http://dx.doi.org/10.1016/ j.pharep.2016.01.003

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