Bupivacaine-induced contraction is attenuated by endothelial nitric oxide release modulated by activation of both stimulatory and inhibitory phosphorylation (Ser1177 and Thr495) of endothelial nitric oxide synthase

European Journal of Pharmacology 853 (2019) 121–128

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Cardiovascular pharmacology

Bupivacaine-induced contraction is attenuated by endothelial nitric oxide release modulated by activation of both stimulatory and inhibitory phosphorylation (Ser1177 and Thr495) of endothelial nitric oxide synthase

T

Soo Hee Leea,1, Chang-Shin Parkb,1, Seong-Ho Okc,d, Dana Kimb, Kyung Nam Kimb, ⁎ Jeong-Min Honge, Ji-Yoon Kimf, Sung Il Baef, Seungmin Anf, Ju-Tae Sohna,g, a Department of Anesthesiology and Pain Medicine, Gyeongsang National University College of Medicine, Gyeongsang National University Hospital, 15 Jinju-daero 816 beon-gil, Jinju-si, Gyeongsangnam-do 52727, Republic of Korea b Department of Pharmacology, Hypoxia-Related Disease Research Center, Inha Research Institute for Medical Sciences, Inha University College of Medicine, Inha-ro 100, Incheon 22212, Republic of Korea c Department of Anesthesiology and Pain Medicine, Gyeongsang National University Changwon Hospital, Changwon 51427, Republic of Korea d Department of Anesthesiology and Pain Medicine, Gyeongsang National University College of Medicine, 15 Jinju-daero 816 beon-gil, Jinju-si, Gyeongsangnam-do 52727, Republic of Korea e Department of Anesthesia and Pain Medicine, Pusan National University Hospital, Pusan National University School of Medicine, Busan 49241, Republic of Korea f Department of Anesthesiology and Pain Medicine, Gyeongsang National University Hospital, 15 Jinju-daero 816 beon-gil, Jinju-si, Gyeongsangnam-do 52727, Republic of Korea g Institute of Health Sciences, Gyeongsang National University, Jinju-si 52727, Republic of Korea

A R T I C LE I N FO

A B S T R A C T

Keywords: Bupivacaine Endothelial nitric oxide Endothelial nitric oxide synthase Src kinase Caveolin-1 Vasoconstriction

This study examined the mechanism associated with the endothelium-dependent attenuation of vasoconstriction induced by bupivacaine (BPV), with a particular focus on the upstream cellular signaling pathway of endothelial nitric oxide synthase (eNOS) phosphorylation induced by BPV in human umbilical vein endothelial cells (HUVECs). BPV concentration-response curves were investigated in the isolated rat aorta. The effects of Nωnitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), methylene blue, calmidazolium, the Src kinase inhibitor 4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and the combination of L-arginine and L-NAME on BPV-induced contraction in endothelium-intact aorta preparations were examined. The effects of BPV alone and in combination with PP2 on the phosphorylation of eNOS (at Ser1177 or Thr495), caveolin-1 and Src kinase were examined in HUVECs. BPV-induced contraction was lower in endothelium-intact aortae than in endothelium-denuded aortae. L-NAME, ODQ, methylene blue and calmidazolium increased BPV-induced contraction in endothelium-intact aortae, whereas PP2 alone and combined treatment with L-arginine and L-NAME inhibited BPV-induced contraction. Low-concentration BPV (30 µM) induced both stimulatory (Ser1177) and inhibitory (Thr495) phosphorylation of eNOS in HUVECs. However, high-concentration BPV (150 µM) induced only stimulatory (Ser1177) eNOS phosphorylation. Additionally, phosphorylation of Src kinase, caveolin-1 and inhibitory eNOS (Thr495) induced by low-concentration BPV was inhibited by PP2. These results suggest that contraction induced by low-concentration BPV is attenuated by endothelial nitric oxide release, which is modulated both stimulatory (Ser1177) and inhibitory eNOS phosphorylation (Thr495). BPV-induced phosphorylation of eNOS (Thr495) is indirectly mediated by an upstream cellular signaling pathway involving Src kinase (Tyr416) and caveolin-1 (Tyr14).

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Correspondence to: Department of Anesthesiology and Pain Medicine, Gyeongsang National University Hospital, 79 Gangnam-ro, Jinju-si 52727, Republic of Korea. E-mail address: [email protected] (J.-T. Sohn). 1 These authors contributed equally to this work as first authors. https://doi.org/10.1016/j.ejphar.2019.03.026 Received 12 December 2018; Received in revised form 13 March 2019; Accepted 14 March 2019 Available online 15 March 2019 0014-2999/ © 2019 Elsevier B.V. All rights reserved.

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1. Introduction

weight 250–300 g). The descending thoracic aorta was dissected and isolated from the thoracic cavity. The isolated rat aorta was bathed in Krebs solution composed of sodium chloride (118 mM), sodium bicarbonate (25 mM), glucose (11 mM), potassium chloride (4.7 mM), calcium chloride (2.4 mM), magnesium sulfate (1.2 mM) and monopotassium phosphate (1.2 mM), and the periaortic tissues, including the fat and connective tissue, were removed under a microscope. The aorta was cut into 2.5-mm aortic rings, and the endothelium of some aortic rings was removed by inserting two 25-gauge needles into the lumen of the aortic rings and rolling the aortic rings back and forth for approximately 15 s. Isolated aortic rings were suspended in a Grass isometric transducer (FT-03, Grass Instrument, Quincy, Massachusetts, USA) with 10 mL Krebs solution maintained at 37 °C. In the preliminary experiment, the optimal resting tension was defined as the amount of resting tension required to achieve the largest contractile response to isotonic 60 mM KCl in a rat aorta ring of the size used in this experiment (Guan et al., 2007; Sung et al., 2012). In addition, based on similar previous experiments, we chose a baseline resting tension of 3.0 g in this experiment (Dodd-o et al., 1997; Kim et al., 1998). This baseline resting tension was maintained for 120 min to reach equilibrium, and the preexisting Krebs solution was replaced with fresh Krebs solution every 30 min (Dodd-o et al., 1997). The Krebs solution in the organ bath was aerated with 95% oxygen and 5% carbon dioxide to maintain a pH of approximately 7.4. To verify the endothelial denudation of the isolated rat aorta, we used the following procedure. When phenylephrine (10−8 M) produced a sustained and stable contraction, acetylcholine (10−5 M) was added to the organ bath to verify endothelial denudation. Aortas with less than 15% acetylcholine-induced relaxation from the phenylephrine-induced contraction were regarded as endothelium denuded. In addition, the integrity of the endothelium-intact aortae was confirmed using the following procedure. A higher concentration of phenylephrine (10−7 M) was used for these aortae than for the endothelium-denuded aortae due to endothelial nitric oxide release. When 10−7 M phenylephrine produced a sustained and stable contraction, acetylcholine was added to the organ bath, and aortas with more than 80% acetylcholine-induced relaxation from the phenylephrine-induced contraction were regarded as endothelium intact. After the endothelial integrity and denudation of isolated rat aortas precontracted with phenylephrine were assessed through acetylcholine-induced relaxation, the isolated aortic rings were washed with fresh Krebs solution to remove the phenylephrine and acetylcholine, and baseline resting tension was recovered. Contraction induced by isotonic 60 mM KCl was measured in all aortic rings with or without endothelium and used as a reference value to express the magnitude of contraction induced by BPV. Then, the contracted aortic rings were washed with fresh Krebs solution to restore baseline resting tension. The following experimental protocols were performed.

Bupivacaine (BPV) is an aminoamide local anesthetic used in regional anesthesia and nerve block (Singh et al., 2018). Aminoamide local anesthetics, which include levobupivacaine, ropivacaine and mepivacaine, cause vasoconstriction at low concentrations and attenuate vasoconstriction (i.e., cause vasodilation) at high concentrations (Baik et al., 2011; Ok et al., 2013a, 2013b; Sung et al., 2012). In addition, the vascular response (vasoconstriction) induced by levobupivacaine, ropivacaine and mepivacaine is reportedly attenuated by endothelial nitric oxide (Baik et al., 2011; Ok et al., 2013a, 2013b; Sung et al., 2012). Caveolin is involved in cellular signal transduction, which is associated with upstream signal components (G protein-coupled receptors, receptor tyrosine kinases, and nonreceptor tyrosine kinases including Src kinase) and downstream signal components such as endothelial nitric oxide synthase (eNOS) and ion channels (Patel et al., 2008). Caveolin-1 (CAV1) phosphorylation induced by Src kinase contributes to cardiac protection and may be involved in signal transduction cascades activated by cellular stressors (Patel et al., 2007; Volonté et al., 2001). The interaction of CAV1 with eNOS via the caveolin scaffolding domain reduces nitric oxide synthase activity due to inhibition of calmodulin binding (Patel et al., 2008). A pathway involving Src kinase, CAV1, and eNOS stimulated by resveratrol produces nitric oxide in human umbilical vein endothelial cells (HUVECs) (Klinge et al., 2008). On the other hand, nitric oxide produced by eNOS inhibits eNOS phosphorylation via the Src kinase-dependent phosphorylation of CAV1 (Chen et al., 2012). In addition, the interaction of CAV1 and G protein-coupled receptor kinase-2 attenuates eNOS in injured sinusoidal endothelial cells (Liu et al., 2017). Phosphorylation of the serine/threonine or tyrosine sites of eNOS is well known as a posttranslational modification mechanism that affects eNOS activity, as reviewed by Heiss and Dirsch (2014). Several factors, such as shear stress, vascular endothelial growth factor and insulinstimulated eNOS activity, induce differential levels of eNOS phosphorylation at Ser1177, a stimulatory site (Burgering and Coffer, 1995; Dimmeler et al., 1999). On the other hand, Thr495 of eNOS is known as an inhibitory phosphorylation site (Sugimoto et al., 2007). Drug-induced changes in the levels of Ser1177- and Thr495-eNOS phosphorylation may be involved in the nitric oxide-mediated modulation of vascular tone. However, the specific upstream cellular signaling pathways associated with BPV-induced eNOS phosphorylation remain unknown. Thus, this study examined the cellular signaling pathway associated with the endothelial nitric oxide-mediated modulation of vasoconstriction induced by BPV in the rat aorta, with a particular focus on the upstream cellular signaling pathway associated with BPV-induced eNOS phosphorylation in HUVECs. In this study, we suggest that the degree of BPV-induced modulation of eNOS activity is based on these two eNOS phosphorylation sites (Ser1177 and Thr495) and propose a possible mechanism associated with the nitric oxide-dependent differential attenuation of vasoconstriction observed after BPV treatment at different concentrations (low and high) in a wire myography study using aortic rings.

2.2. Experimental protocols First, the effect of endothelial denudation on BPV-induced vasoconstriction in isolated rat thoracic aorta preparations was assessed. Vasoconstriction induced by the cumulative addition of BPV (10−6 to 3 ×10−4 M) was produced in isolated rat aorta tissue with or without endothelium. BPV-induced vasoconstriction plateaued approximately 3–5 min after the addition of bupivacaine into the organ bath and was then sustained for approximately 3–4 min depending on the concentration of BPV. Increasing concentrations of BPV were then added to the organ bath to produce a cumulative BPV concentration-response curve. Second, the effect of the nitic oxide synthase (NOS) inhibitor Nωnitro-L-arginine methyl ester (L-NAME; 10−4 M) alone and in combination with L-arginine (10−4 M) on BPV-induced vasoconstriction in isolated endothelium-intact rat aorta preparations was assessed. After isolated endothelium-intact rat aorta preparations were pretreated with L-NAME (10−4 M) alone for 20 min or L-arginine (10−4 M) for 30 min

2. Materials and methods All experimental protocols were approved by the Institutional Animal Care and Use Committee of Gyeongsang National University, and all experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals approved by Gyeongsang National University. 2.1. Preparation of isolated aortic rings and isometric tension measurement The isolated rat aortas used for isometric tension measurement were prepared as described previously (Ok et al., 2018). Carbon dioxide (100%) was used to kill male Sprague-Dawley rats (N = 57, body 122

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followed by L-NAME (10−4 M) for 20 min, vasoconstriction induced by the cumulative addition of BPV (10−6 to 3 × 10−4 M) was produced in the presence or absence of pretreatment drugs (L-NAME alone or in combination with L-arginine). Third, the effects of the nitic oxide-sensitive guanylate cyclase (GC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), the nonspecific GC inhibitor methylene blue and the calmodulin-regulated enzyme inhibitor calmidazolium on BPV-induced vasoconstriction were assessed in endothelium-intact aorta preparations. After endotheliumintact aortae were pretreated with ODQ (10−6 M), methylene blue (10−6 M), or calmidazolium (10−5 and 3 × 10−5 M) for 20 min or left without pretreatment, the vasoconstriction induced by the cumulative addition of BPV (10−6 to 3 × 10−4 M) in isolated endothelium-intact rat aorta tissue was assessed. Fourth, in light of Western blots showing that 4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-amino-5-(4chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2; 20 µM) inhibited the phosphorylation of Src kinase, CAV1 and eNOS (Thr495) induced by a low concentration of BPV (30 µM) in HUVECs (Fig. 7C), the effect of the Src kinase inhibitor PP2 on BPV-induced vasoconstriction in isolated endothelium-intact rat aorta preparations was assessed. As the Src kinase inhibitor PP2 has been reported to attenuate the contraction of vascular smooth muscle induced by phenylephrine, we investigated the effect of PP2 on the contraction induced by BPV in endothelium-intact aorta preparations pretreated with L-NAME (Min et al., 2012). After some endothelium-intact rat aortae were pretreated with PP2 (2 × 10−5 M) alone for 20 min, L-NAME alone for 35 min, and L-NAME (10−4 M) for 15 min followed by PP2 (2 × 10−5 M) for 20 min, the vasoconstriction induced by the cumulative addition of BPV (10−6 to 3 × 10−4 M) in isolated endothelium-intact rat aorta tissue was assessed with or without drug pretreatment. In addition, the effects of L-NAME on the PP2-mediated inhibition of BPV (10−4 M)-induced contraction in endothelium-intact aortae were examined. After endothelium-intact aortae were pretreated with L-NAME (10−4 M) for 15 min, BPV (10−4 M)-induced contraction in the endothelium-intact aorta was generated in the presence or absence of L-NAME, and PP2 (2 × 10−5 M) was then added to the organ bath. PP2-mediated inhibition of BPV (10−4 M)-induced contraction was assessed in the presence or absence of L-NAME. The concentrations of all the inhibitors, including L-NAME, ODQ, methylene blue, calmidazolium and PP2, were chosen based on the concentrations used in previous studies (Chen et al., 2012; de Oliveira et al., 2016; Sung et al., 2012).

SDS-PAGE, were transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were incubated overnight with the corresponding primary antibodies (1:1000 or 1:2000) at 4 °C with gentle agitation after being blocked with 5% nonfat dry milk powder Tris-buffered saline containing 0.1% TWEEN 20. The blots were washed and incubated with a 1:4000 dilution of a corresponding antimouse or anti-rabbit alkaline phosphatase-conjugated secondary antibody and then washed three times with Tris-buffered saline TWEEN buffer. The transferred proteins were visualized with an enhanced chemiluminescence kit (Pierce, Rockford, IL, USA). The signals were quantified using NIH Image 1.62 software. In all of the plots, each point represents the average value from three independent experiments. 2.5. Materials All drugs were commercially available and of the highest purity. LNAME, ODQ, PP2 and calmidazolium were obtained from SigmaAldrich. BPV was purchased from Reyon Pharmaceutical Co., Ltd. (Seoul, Korea). ODQ, PP2 and calmidazolium were dissolved in dimethyl sulfoxide (DMSO; final organ bath concentration: 0.1%). The other drugs were dissolved in distilled water. Antibodies against eNOS (#9572, 1:1000), p-Ser1177-eNOS (#9571, 1:1000), p-Thr495-eNOS (#9574, 1:1000), p-Tyr14-CAV-1 (#3251, 1:1000), Src kinase (#2108, 1:1000) and p-Tyr416-Src kinase (#2101, 1:1000) were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against CAV1 (sc0894, 1:2000) and beta-actin (A1978, 1:10000) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and SigmaAldrich, respectively. 2.6. Data analysis The data are expressed as the mean ± S.D. BPV-induced contraction was expressed as the percentage of maximal contraction induced by 60 mM KCl. The vascular response induced by each concentration of BPV was analyzed using repeated-measures analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test. The effects of endothelial denudation and various inhibitors on BPV-induced contraction in isolated rat aorta tissue were analyzed using two-way repeated-measures ANOVA followed by Bonferroni's multiple comparison test. The effect of L-NAME on the PP2-mediated inhibition of BPV (10−4 M)-induced contraction was analyzed using an unpaired Student's t-test. The effects of BPV alone and in combination with PP2 on the phosphorylation of Src kinase (Tyr416), CAV1 (Tyr14) and eNOS (Ser1177 or Thr495) were analyzed using the Mann-Whitney test or one-way ANOVA followed by Bonferroni's multiple comparison. A P value less than 0.05 was considered statistically significant.

2.3. Cell lines and cell culture HUVECs were grown in M199 supplemented with growth factors under 5% CO2 as commercially described (Ok et al., 2011). Cells from passages 3–6 were used. HUVECs grown to 80% confluence were treated with BPV in serum-free M199 for various durations (5, 10, 30 and 60 min) or at various concentrations (10, 30, 90 and 150 µM).

3. Results 3.1. Effects of BPV alone and in combination with inhibitors on the isolated rat aorta

2.4. Western blot analysis BPV produced vasoconstriction at low concentrations (3 × 10−6 M to 3 ×10−5 M; Fig. 1; P < 0.05 versus drug-free control) and vasodilation (attenuated vasoconstriction) at high concentrations (10−4 and 3 × 10−4 M; Fig. 1; P < 0.01 versus 3 × 10−5 M) in isolated endothelium-denuded rat aorta preparations. Similarly, BPV produced vasoconstriction at low concentrations (3 × 10−5 M and 10−4 M; Fig. 1; P < 0.001 versus drug-free control) and vasodilation (attenuated vasoconstriction) at high concentrations (3 × 10−4 M; Fig. 1; P < 0.001 versus 10−4 M) in endothelium-intact rat aorta preparations. BPV-induced vasoconstriction was more intense in endotheliumdenuded aortae than in endothelium-intact aortae (Fig. 1, P < 0.05 at 3 × 10−6 to 10−4 M BPV). Pretreatment with L-NAME (10−4 M) enhanced BPV-induced contraction in endothelium-intact rat aortae (Fig. 2, P < 0.001 at 10−5 to 3 × 10−4 M BPV). However, combined

Extracts of commercial HUVECs (ATCC CRL-1730, American Type Culture Collection, Manassas, VA, USA) were prepared in ice-cold lysis buffer (1% NP-40, 0.1 M phenylmethylsulfonyl fluoride, 320 mM sucrose, 200 mM HEPES and 1 mM EDTA, pH 7.2) containing protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and phosphatase inhibitor cocktail (Roche, Germany). The cells were disrupted three times by sonication at 3 W on ice for 5 s. Protein concentrations in cell lysates were determined using a commercial bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA). Total protein (10–20 µg) was mixed with sample buffer (250 mM Tris-HCl, pH 6.8, 4% SDS, 40% glycerol, 0.02% bromophenol blue, 20% dithiothreitol) and heated at 100 °C for 10 min. The protein mixtures were electrophoresed on a gradient SDS-polyacrylamide gel, and the samples, once separated by 123

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increased at various concentrations of BPV (10, 30, 90 and 150 µM; P < 0.05 versus drug-free control). However, p-Thr495-eNOS expression was significantly higher in cells treated with BPV than in drug-free controls, with peak expression at 30 µM BPV (P < 0.01; Fig. 5A and C), but there was no significant difference in expression between drug-free controls and those treated with high concentrations of BPV (90 and 150 µM; Fig. 5A and C). No significant changes in the expression of total eNOS were observed. The levels of p-Tyr14-CAV1 and CAV1 were examined. Interestingly, the highest expression of p-Tyr14-CAV1 was observed after treatment with 30 µM BPV (P < 0.05; Fig. 5B and C), and pTyr14-CAV1 expression in cells treated with 90 µM and 150 µM BPV was not significantly different from that in drug-free controls (Fig. 5B and C). There was no significant change in the expression of total CAV1 with BPV treatment.

Fig. 1. Effect of endothelial denudation (N = 10) on the contraction induced by bupivacaine in the isolated rat aorta. Data are shown as the mean ± S.D. and expressed as the percentage of isotonic 60-mM KCl-induced contraction. N indicates the number of rats from which the descending thoracic aortae were derived. * P < 0.05 and #P < 0.001 versus endothelium-intact aortae.

3.3. Time-dependent effects of BPV on the phosphorylation of eNOS and CAV1 Based on the phosphorylation of eNOS and CAV1 measured at various concentrations of BPV, the time-dependent (5–60 min) levels of phosphorylation activity at 30 and 150 µM BPV were compared because these concentrations corresponded to the highest and lowest expression of p-Tyr14-CAV1 and p-Thr495-eNOS induced by BPV. Cells treated with a low concentration of BPV (30 µM) showed a significant timedependent increase in both p-Ser1177-eNOS and p-Thr495-eNOS compared with drug-free controls (P < 0.05; Fig. 6A). However, BPV at a high concentration (150 µM) merely increased p-Ser1177-eNOS expression and did not significantly alter p-Thr495-eNOS expression (P < 0.05; Fig. 6A). In addition, the highest expression of p-Tyr14CAV1 was observed at 30 min after treatment with 30 µM BPV (P < 0.01; Fig. 6B), and p-Tyr14-CAV1 expression after 150 µM BPV treatment, although significantly higher than that in drug-free controls, did not show a time-dependent change (P < 0.05; Fig. 6B).

Fig. 2. Effect of Nω-nitro-L-arginine methyl ester (L-NAME) alone and in combination with L-arginine on the contraction induced by bupivacaine in the isolated endothelium-intact rat aorta. Data (N = 8) are shown as the mean ± S.D. and expressed as the percentage of isotonic 60-mM KCl-induced contraction. N indicates the number of rats from which descending thoracic aortae were derived. * P < 0.01 and †P < 0.001 versus control. #P < 0.001 versus L-NAME alone.

3.4. Effects of BPV on Src kinase phosphorylation The levels of p-Tyr416-Src kinase, which indicate the activity of Src kinase (known as an upstream kinase of p-Tyr14-CAV1), were examined. Similar to the expression of p-Tyr14-CAV1, the highest expression of p-Tyr416-Src kinase after treatment with 30 or 150 µM BPV was found at 30 min after 30 µM BPV (P < 0.01, Figs. 6B and 7A).

treatment with L-arginine (10−4 M) and L-NAME (10−4 M) attenuated BPV-induced contraction compared with L-NAME alone (Fig. 2, P < 0.001 at 3 × 10−5 M BPV). Pretreatment with the GC inhibitor ODQ (10−6 M) or methylene blue (10−6 M) increased BPV-induced contraction (Fig. 3A and B, P < 0.01 at 3 × 10−5 to 3 × 10−4 M BPV). The calmodulin-regulated enzyme inhibitor calmidazolium (10−5 and 3 × 10−5 M) increased BPV-induced contraction (Fig. 3C, P < 0.05 at 10−4 M BPV). The Src kinase inhibitor PP2 (2 × 10−5 M) attenuated BPV-induced contraction in endothelium-intact rat aortae pretreated with or without L-NAME (Fig. 4A and B, P < 0.05 at 3 × 10−5 to 3 × 10−4 M BPV). Posttreatment with PP2 (2 × 10−4 M) attenuated BPV (10−4 M)-induced contraction by 72 ± 8% in endothelium-intact aorta without L-NAME pretreatment, whereas posttreatment with PP2 (2 ×10−4 M) attenuated BPV (10−4 M)-induced contraction by 25 ± 8% in endothelium-intact aorta pretreated with L-NAME (Fig. 4C). Thus, pretreatment with L-NAME (10−4 M) attenuated the PP2 (2 × 10−5 M)-mediated inhibition of BPV (10−4 M)-induced contraction in endothelium-intact aortae (Fig. 4C, P < 0.001 versus no LNAME).

3.5. Effects of Src kinase inhibition on BPV-induced phosphorylation of eNOS and CAV1 The effects of the Src-CAV1-eNOS pathway on the phosphorylation of eNOS at Ser1177 and Thr495 were examined by pretreatment with PP2 (20 µM), a selective inhibitor of the Src kinase family. Low-concentration BPV (30 µM) treatment for 30 min significantly increased pTyr416-Src and p-Tyr14-CAV1 expression (P < 0.001; Fig. 7B and C). The increase in the phosphorylation of these two proteins was completely inhibited after PP2 pretreatment (P < 0.001 versus BPV alone; Fig. 7B and C). The expression levels of p-Ser1177-eNOS and p-Thr495eNOS were significantly increased by 30 µM BPV (P < 0.05; Fig. 7B and C), but interestingly, PP2 pretreatment significantly suppressed only the phosphorylation of eNOS at Thr495 induced by low-concentration BPV (30 µM) (P < 0.01 versus BPV alone, Fig. 7B and C). 4. Discussion

3.2. Concentration-dependent effects of BPV on the phosphorylation of eNOS and CAV1

This study suggests that the endothelial nitric oxide-mediated attenuation of low-concentration BPV-induced contraction is regulated by both stimulatory (Ser1177) and inhibitory (Thr495) phosphorylation of eNOS (Fig. 8). The major findings of this in vitro study are as follows: 1) BPV-induced contraction was lower in endothelium-intact aortae than

The phosphorylation of eNOS at Ser1177, which increases its activity, and at Thr495, which inhibits its activity, was measured. As shown in Fig. 5A and C, p-Ser1177-eNOS expression was significantly 124

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Fig. 3. A and B: Effect of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, N = 7) and methylene blue (N = 7) on the contraction induced by bupivacaine in the isolated endothelium-intact rat aorta. Data are shown as the mean ± S.D. and expressed as the percentage of isotonic 60-mM KCl-induced contraction. N indicates the number of rats from which descending thoracic aortae were derived. * P < 0.05, †P < 0.01 and #P < 0.001 versus control. C: Effect of calmidazolium (10−5 and 3 ×10−5 M, N = 6) on the contraction induced by bupivacaine in the isolated endothelium-intact rat aorta. Data are shown as the mean ± S.D. and expressed as the percentage of isotonic 60-mM KCl-induced contraction. N indicates the number of rats from which descending thoracic aortae were derived. * P < 0.05 and †P < 0.001 versus control.

Fig. 4. A and B: Effect of 4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d] pyrimidine, 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2) on the contraction induced by bupivacaine in the isolated endothelium-intact rat aorta without (A, N = 6) or with (B, N = 6) Nω-nitro-L-arginine methyl ester (L-NAME). Data are shown as the mean ± S.D. and expressed as the percentage of isotonic 60-mM KCl-induced contraction. N indicates the number of rats from which descending thoracic aortae were derived. A: * P < 0.01 and #P < 0.001 versus control. B: * P < 0.001 versus control. †P < 0.05, #P < 0.01 and §P < 0.001 versus L-NAME alone. C: Effect of L-NAME (N = 6) on the PP2-mediated inhibition of bupivacaine-induced contraction in the isolated endotheliumintact rat aorta. Data are shown as the mean ± S.D. and expressed as the percentage of bupivacaine (10−4 M)-induced maximal contraction. N indicates the number of rats from which descending thoracic aortae were derived. * P < 0.001 versus PP2 alone.

(30 µM) of BPV in HUVECs (Fig. 7C). Nitric oxide is produced from L-arginine by eNOS, which is activated by calmodulin and increased intracellular calcium in the endothelium (Gielen et al., 2011; Sohn et al., 2004). Nitric oxide in the endothelium diffuses to vascular smooth muscle and activates GC, which leads to vasodilation via the production of cyclic guanosine monophosphate

in endothelium-denuded aortae. 2) Pretreatment with L-NAME, ODQ, methylene blue and calmidazolium increased BPV-induced contraction in endothelium-intact aortae, whereas pretreatment with L-NAME plus L-arginine or PP2 alone decreased BPV-induced contraction. 3) Pretreatment with PP2 attenuated the phosphorylation of Tyr416-Src kinase, Tyr14-CAV1 and Thr495-eNOS induced by a low concentration 125

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eNOS phosphorylation induced by increased endothelial calcium and calmodulin. Consistent with the PP2-mediated inhibition of the phosphorylation of Tyr416-Src kinase, Tyr14-CAV1 and Thr495-eNOS by a low concentration of BPV (30 µM) (Fig. 7C), PP2 attenuated BPV (3 × 10−5 M)-induced contraction (Fig. 4A), suggesting that the Src kinase-mediated pathway additively contributes to BPV-induced contraction in the endothelium-intact aorta. The magnitude of the PP2mediated inhibition of BPV-induced contraction was greater in endothelium-intact aorta preparations without L-NAME than in those with L-NAME (Fig. 4C). Thus, taken together with the results from the Western blot analysis, our observations indicate that the PP2-mediated inhibition of BPV-induced contraction is mediated primarily by the attenuation of the BPV-induced activation of inhibitory eNOS (Thr495) and partially by the direct inhibition of vascular smooth muscle contraction induced by BPV. Furthermore, in endothelium-intact aortae with and without L-NAME, PP2 inhibited contraction induced by highconcentration BPV (3 × 10−4 M) (Fig. 4A and B). This PP2-mediated, nitric oxide-independent inhibition may be associated with the direct inhibition by PP2 of Src kinase-mediated contraction or focal-adhesionmediated contraction induced by a high concentration of BPV in vascular smooth muscle, supporting the hypothesis that a high concentration of BPV (150 µM) induces only stimulatory phosphorylation of eNOS (Ser1177) (Min et al., 2012; Yayama et al., 2014). Treatment with low-concentration (30 µM) BPV not only increased the levels of p-Ser1177-eNOS phosphorylation but also increased the levels of p-Thr495-eNOS phosphorylation (Figs. 5 and 6). In addition, the levels of p-Tyr14-CAV1 (Fig. 6B) and p-Tyr416-Src kinase (Fig. 7A), one of the tyrosine kinases upstream of CAV1, were effectively increased 30 min after 30 µM BPV treatment. Interestingly, these increases induced by BPV (30 µM) were confirmed in parallel with increases in the expression of both p-Ser1177-eNOS and p-Thr495-eNOS. In particular, the BPV (30 µM)-induced increase in p-Thr495-eNOS expression was indirectly mediated by the phosphorylation of proteins in the tyrosine kinase pathway (p-Tyr416-Src kinase/p-Tyr14-CAV1), as demonstrated by pretreatment with the Src kinase family inhibitor PP2 (Fig. 7B and C). PP2 pretreatment had no significant effect on the increase in BPV (30 µM)-induced p-Ser1177-eNOS expression (Fig. 7B and C); however, the increased levels of BPV (30 µM)-induced p-Thr495eNOS expression were lower than the drug-free basal levels (Fig. 7B and C). The PP2-induced dephosphorylation of Thr495-eNOS phosphorylation induced by BPV (30 µM) without a concomitant decrease in Ser1177-eNOS phosphorylation induced by BPV (30 µM) may also promote eNOS activity, producing a relatively large amount of nitric oxide, which is consistent with the PP2-mediated inhibition of BPV (3 × 10−5 M)-induced contraction of endothelium-intact aortae (Fig. 4A). Similarly, increased eNOS activity after dephosphorylation of Thr495-eNOS has been reported in endothelial cells treated with a cooked food ingredient such as norfuraneol (Schmitt et al., 2009). In contrast to low-concentration BPV, high-concentration BPV (150 µM) induced phosphorylation of Ser1177 of eNOS (Fig. 6A) and induced

Fig. 5. Concentration-dependent effects of bupivacaine (BPV) on the phosphorylation levels of caveolin-1 (CAV1) and endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells. Western blots prepared with cell lysates were carried out to measure expression of p-Ser1177-eNOS, p-Thr495eNOS, and p-Tyr14-CAV1 and total levels of CAV1 and eNOS in cells treated with different concentrations of BPV (0, 10, 30, 90 and 150 μM) for 30 min. βActin expression was determined as a loading control. A representative blot is shown, and the p-eNOS/eNOS (A: Ser1177, closed circle; Thr495, open circle) and p-CAV1/CAV1 ratios (B) were assessed by densitometric analysis to quantify the immunoblots from three independent experiments (C). Data are expressed as the mean ± S.D. * P < 0.05 and ** P < 0.01 compared to untreated cells.

(cGMP) (Gielen et al., 2011; Sohn et al., 2004). Pretreatment with the NOS inhibitor L-NAME increased BPV-induced contraction in endothelium-intact aortae (Fig. 2). However, combined treatment with Larginine and L-NAME increased BPV-induced contraction less than pretreatment with L-NAME alone (Fig. 2). In addition, the nitric oxidesensitive GC inhibitor ODQ and the nonspecific GC inhibitor methylene blue increased BPV-induced contraction in endothelium-intact aortae (Fig. 3A and B). Finally, the calmodulin-regulated enzyme inhibitor calmidazolium enhanced BPV-induced contraction in endothelium-intact aortae (Fig. 3C). Taken together, the results obtained from this tension study suggest that BPV-induced contraction is inhibited by a pathway involving nitric oxide, GC and cGMP, which is activated by

Fig. 6. Time-dependent effects of bupivacaine (BPV) on the phosphorylation levels of endothelial nitric oxide synthase (eNOS) and caveolin-1 (CAV1) in human umbilical vein endothelial cells. Western blots prepared with cell lysates were carried out to measure pSer1177-eNOS, p-Thr495-eNOS and p-Tyr14CAV1 and total levels of eNOS and CAV1 at different time points (0, 5, 10, 30 and 60 min) in cells treated with 30 or 150 μM BPV. β-Actin expression was determined as a loading control. A representative blot is shown, and the peNOS/eNOS (A: Ser1177, closed circle; Thr495, open circle) and p-CAV1/CAV1 ratios (B) were assessed by densitometric analysis to quantify the immunoblots from three independent experiments. Data are expressed as the mean ± S.D. * P < 0.05 and ** P < 0.01 compared to untreated cells (0 min). 126

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Fig. 7. Time-dependent effects of bupivacaine (BPV) on the expression levels of p-Tyr416-Src and total Src (A) and the effects of BPV in human umbilical vein endothelial cells pretreated with the Src kinase family inhibitor PP2 (B and C). Serial phospho-activation of Src-CAV1-eNOS (both p-Ser1177 and p-Thr495) was detected in BPV (30 mM)-treated cells, and Src-CAV1-p-Thr495-eNOS but not p-Thr1177-eNOS was completely blocked by pretreatment with PP2. β-Actin expression was determined as a loading control. A representative blot is shown, and the p-Src/Src, p-CAV1/CAV1 and p-eNOS/eNOS ratios [Ser1177, closed circle; Thr495, open circle] were determined by densitometric analysis to quantify the immunoblots from three independent experiments. Data are expressed as the mean ± S.D. * P < 0.05, ** P < 0.01 and *** P < 0.001 compared to the untreated control group. ##P < 0.01 and ###P < 0.001 compared to the BPV (30 µM)-treated group.

antagonistic effect that leads to the relative attenuation of nitric oxide production. The attenuated vasoconstriction of endothelium-intact aortae induced by high-concentration BPV (3 × 10−4 M) (Fig. 1) in the current study may be partially associated with the relatively enhanced nitric oxide production due to the exclusively stimulatory phosphorylation of eNOS (Ser1177) induced by high-concentration BPV (150 µM) in HUVECs. Whether phosphorylation of eNOS by tyrosine kinases such as Src or Fyn affects eNOS enzyme activity is not yet clear. Although not presented in this study, we examined the expression of pTyr657-eNOS induced by proline-rich tyrosine kinase 2 (PYK2, pTyr402-PYK2), which is known to inhibit eNOS activity (Bibli et al., 2017; Fisslthaler et al., 2008; Loot et al., 2009). However, there was no change in p-Tyr402-PYK2 expression after BPV treatment (30 µM and 150 µM), and furthermore, the level of Tyr657 phosphorylation of eNOS did not change (data not shown). Taking into consideration both the protein binding (95%) of BPV and its dilution by interstitial fluid after local infiltration, a low concentration (0.008–0.031%) of intradermally administered BPV, which may result in less than approximately 1.16–4.5 × 10−5 M BPV, decreases skin blood flow, whereas a high concentration of BPV (0.25–0.75%), which may result in less than approximately 3.6 × 10−4 to 1 × 10−3 M BPV, increases skin blood flow (Casati and Putzu, 2005; Newton et al., 2005). Intradermal injection of BPV (0.125–0.75%) initially increases and later decreases skin perfusion; this pattern seems to be associated with an initial increase in the concentration of BPV followed by a later reduction (Newton et al., 2000). A high concentration of BPV administered by topical application produces a smaller reduction in sciatic nerve blood flow than a low concentration of BPV, and washout of BPV restores sciatic nerve blood flow, suggesting that a high concentration of BPV is less vasoconstrictive than a low concentration (Partridge, 1991). Although racemic BPV is less vasoconstrictive than

Fig. 8. The putative cellular signaling pathway associated with bupivacaine (BPV)-induced endothelial nitric oxide synthase (eNOS) phosphorylation (at Ser1177 and Thr495) contributing to modulation of nitric oxide (NO) release in human umbilical vein endothelial cells.?: unknown kinase.

phosphorylation of both Tyr416 of Src kinase (Fig. 7A) and Tyr14 of CAV1 (Fig. 6B). The upstream cellular signaling pathway associated with the stimulatory eNOS phosphorylation (Ser1177) induced by lowconcentration (30 µM) and high-concentration (150 µM) BPV remains to be determined (Fig. 8). Given these results obtained from Western blotting in HUVECs, the vasoconstriction of the endothelium-intact aorta induced by a low concentration of BPV (3 × 10−5 M) (Fig. 1) as observed in the current study seems to involve both activation and inhibition of nitric oxide production, with phosphorylation of stimulatory (Ser1177) and inhibitory (Thr495) sites on eNOS, which is an 127

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levobupivacaine, these previous in vivo studies support the observations in the current in vitro study that low-concentration BPV induces vasoconstriction, contributing to the increased duration of anesthesia, whereas high-concentration BPV produces vasodilation (attenuated vasoconstriction) (Newton et al., 2000, 2005; Patridge, 1991). As 0.125–0.75% BPV, which is within the concentration range used in clinical practice, approximately corresponds to 1.8 × 10−4 to 1 × 10−3 M BPV in an in vivo setting, the BPV concentration used in the current study seems to be similar to that encountered in peripheral nerve block using BPV (Casati and Putzu, 2005). However, this study has the following limitations. First, peripheral nerve blood flow is regulated by extrinsic blood vessels, which are small arterioles that communicate with intrinsic blood vessels to supply blood flow to peripheral nerves (Myers and Heckman, 1989; Vadhanan et al., 2015). In contrast, the rat aorta tissue, which is considered a conduit vessel, was used in the present study. Second, in the current study, isolated rat aorta preparations were used for the isometric tension measurements, whereas HUVECs were used to examine the upstream signaling pathway associated with eNOS phosphorylation. Differences between rat aortae and HUVECs should be considered in the interpretation of these results. In conclusion, these results suggest that contraction induced by a low concentration (30 µM) of BPV is attenuated by a calmodulin-nitric oxide-GC pathway, which is modulated through both stimulatory (Ser1177) and inhibitory (Thr495) eNOS phosphorylation (Fig. 8). In addition, the phosphorylation of inhibitory eNOS (Thr495) induced by a low concentration (30 µM) of BPV is mediated by an upstream cellular signaling pathway involving Src kinase (Tyr416) and CAV1 (Tyr14) in HUVECs (Fig. 8).

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Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1D1A1B03930451). This work was supported by Biomedical Research Institute Fund (GNUHBRIF2017-0010) from the Gyeongsang National University Hospital. This work was financially supported by Medical Research Center (2014009392) through the National Research Foundation of Korea (NRF) funded by Ministry of Science, ICT and Future Planning for Chang-Shin Park. Declaration of interest None. References Baik, J.S., Sohn, J.T., Ok, S.H., Kim, J.G., Sung, H.J., Park, S.S., Park, J.Y., Hwang, E.M., Chung, Y.K., 2011. Levobupivacaine-induced contraction of isolated rat aorta is calcium dependent. Can. J. Physiol. Pharmacol. 89, 467–476. Bibli, S.I., Zhou, Z., Zukunft, S., Fisslthaler, B., Andreadou, I., Szabo, C., Brouckaert, P., Fleming, I., Papapetropoulos, A., 2017. Tyrosine phosphorylation of eNOS regulates myocardial survival after an ischaemic insult: role of PYK2. Cardiovasc. Res. 113, 926–937. Burgering, B.M., Coffer, P.J., 1995. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature 376, 599–602. Casati, A., Putzu, M., 2005. Bupivacaine, levobupivacaine and ropivacaine: are they clinically different? Best Pract. Res. Clin. Anaesthesiol. 19, 247–268. Chen, Z., Bakhshi, F.R., Shajahan, A.N., Sharma, T., Mao, M., Trane, A., Bernatchez, P., van Nieuw Amerongen, G.P., Bonini, M.G., Skidgel, R.A., Malik, A.B., Minshall, R.D., 2012. Nitric oxide-dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition. Mol. Biol. Cell 23, 1388–1398. de Oliveira, L.M., de Oliveira, T.S., da Costa, R.M., de Souza Gil, E., Costa, E.A., Passaglia Rde, C., Filgueira, F.P., Ghedini, P.C., 2016. The vasorelaxant effect of gallic acid involves endothelium-dependent and -independent mechanisms. Vasc. Pharmacol. 81, 69–74. Dimmeler, S., Fleming, I., Fisslthaler, B., Hermann, C., Busse, R., Zeiher, A.M., 1999. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399, 601–605. Dodd-o, J.M., Zheng, G., Silverman, H.S., Lakatta, E.G., Ziegelstein, R.C., 1997. Endothelium-independent relaxation of aortic rings by the nitric oxide synthase

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