2 inhibition on cigarette smoke exposure-induced ET receptor upregulation in rat cerebral arteries

Toxicology and Applied Pharmacology 304 (2016) 70–78

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The effects of MEK1/2 inhibition on cigarette smoke exposure-induced ET receptor upregulation in rat cerebral arteries Lei Cao a,b, Na-Na Ping b, Yong-Xiao Cao b, Wei Li c,⁎, Yan Cai b, Karin Warfvinge a, Lars Edvinsson a a b c

Division of Experimental Vascular Research, Institute of Clinical Sciences in Lund, Lund University, Sweden Department of Pharmacology, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi, China Department of Hepatobiliary Surgery, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, China

a r t i c l e

i n f o

Article history: Received 7 October 2015 Revised 11 May 2016 Accepted 18 May 2016 Available online 19 May 2016 Keywords: Cigarette smoke Cerebral artery Endothelin receptor MEK1/2 Rat Receptor upregulation

a b s t r a c t Cigarette smoking, a major stroke risk factor, upregulates endothelin receptors in cerebral arteries. The present study examined the effects of MEK1/2 pathway inhibition on cigarette smoke exposure-induced ET receptor upregulation. Rats were exposed to the secondhand smoke (SHS) for 8 weeks followed by intraperitoneal injection of MEK1/2 inhibitor, U0126 for another 4 weeks. The urine cotinine levels were assessed with high-performance liquid chromatography. Contractile responses of isolated cerebral arteries were recorded by a sensitive wire myograph. The mRNA and protein expression levels of receptor and MEK/ERK1/2 pathway molecules were examined by real-time PCR and Western blotting, respectively. Cerebral artery receptor localization was determined with immunohistochemistry. The results showed the urine cotinine levels from SHS exposure group were significantly higher than those from the fresh group. In addition, the MEK1/2 inhibitor, U0126 significantly reduced SHS exposure-increased ETA receptor mRNA and protein levels as well as contractile responses mediated by ETA receptors. The immunoreactivity of increased ETA receptor expression was primarily cytoplasmic in smooth muscle cells. In contrast, ETB receptor was noted in endothelial cells. However, the SHS-induced decrease in endothelium-dependent relaxation was unchanged after U0126 treatment. Furthermore, SHS increased the phosphorylation of MEK1/2 and ERK1/2 protein in cerebral arteries. By using U0126 could inhibit the phosphorylated ERK1/2 protein but not MEK1/2. Taken together, our data show that treatment with MEK1/2 pathway inhibitor offsets SHS exposure-induced ETA receptor upregulation in rat cerebral arteries. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Cigarette smoking is well-known risk factor for cardiovascular diseases (Huang et al., 2010). In a smoking chamber, the cigarette smoking-derived fine particles occupy 90% of indoor PM2.5 (Xiao et al., 2016). Accordingly, cigarette smoke is an important indoor pollution source (Semple and Latif, 2014). Cigarette smoking, both active smoking and secondhand smoking (SHS) are independent risk factors for cardiovascular diseases like stroke (Bonita et al., 1999). SHS exposure has obtained more attention because it is involuntary smoke exposure, and the sidestream smoke (smoke that goes into the air directly from a burning cigarette) received by secondhand smokers contains higher concentrations of toxic gaseous components than mainstream smoke (smoke that is directly inhaled by smokers) (Ambrose and Barua, 2004). To our knowledge, more than 4000 cigarette smoke components have been Abbreviations: ACh, acetylcholine; DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethylsulfoxide; ET, endothlin; Emax, maximal contraction; GAPDH, glyceraldehyde-3phosphate dehydrogenase; p, phosphorylated; SMC, smooth muscle cell; S6c, sarafotoxin 6c; SHS, secondhand smoke. ⁎ Corresponding author at: Department of Hepatobiliary Surgery, First Affiliated Hospital of Xi'an Jiaotong University, 277 West Yanta Road, Xi'an 710061, China. E-mail address: [email protected] (W. Li).

http://dx.doi.org/10.1016/j.taap.2016.05.013 0041-008X/© 2016 Elsevier Inc. All rights reserved.

identified. Some of these can promote cell proliferation and the release of vasoactive substances. Furthermore, these molecules may induce blood-brain barrier dysfunction and aggravate the disruption of normal endothelial cell function via direct or indirect mechanisms (Edvinsson and Povlsen, 2011). The effects of vasomotor are mediated by vascular receptors located on smooth muscle cells or endothelium. The number and function of these vascular receptors can be affected by many factors. Diseases or environment can induce an alteration of receptor quantity. The increase of the receptor number is called as receptor upregulation which results in enhancement of receptor effects. We have revealed that there is a change of vascular receptor expression such as endothelin type A (ETA) receptor, endothelin type B (ETB) receptor and 5-hydroxytryptamine 1B receptor in different types of stroke, which is a novel aspect of cerebrovascular disorders pathophysiology (Edvinsson and Povlsen, 2011). Contractile receptor upregulation may enhance vasoconstriction, which may be associated with reduced blood flow in late cerebral ischemia after subarachnoid hemorrhage and in the development of the penumbral area after a stroke (Vikman and Edvinsson, 2006; Vikman et al., 2006). Endothelin-1 (ET-1) is the most potent vasoconstrictor and activates both ETA and ETB receptors in the cerebrovascular system (Yanagisawa et al., 1988). ETA receptors are expressed on vascular

L. Cao et al. / Toxicology and Applied Pharmacology 304 (2016) 70–78

smooth muscle cells (SMCs) and mediate vasoconstriction, while ETB receptors are situated on vascular endothelial cells and mediate vasodilatation. ET-1 and ET receptors may have considerable importance in stroke development. ET-1 levels are increased in stroke (Barone et al., 1994). ET receptors are upregulated at the mRNA and protein levels in cerebral arteries after thromboembolic stroke in humans (Vikman and Edvinsson, 2006). Correlated findings were observed in rats following cerebral ischemia and experimental subarachnoid hemorrhage (Stenman et al., 2002; Hansen-Schwartz et al., 2003). In an early study, we demonstrated that receptor was upregulated in SHS-exposed cerebral vessels (Cao et al., 2013; Huang et al., 2010) and airway (Lei et al., 2008). Among the upregulation phenomenon, ET receptors, especially ETA receptors, displayed increased expression at the protein and mRNA level, and in contractile property after SHS exposure (Cao et al., 2011). However, the mechanism remains poorly understood. We believe that SHS-induced receptor upregulation is closely related to Raf/MEK/ERK pathway activation during SHS exposure (Cao et al., 2012a, 2012b). In the present study, we designed a method to test the hypothesis that treatment with MEK1/2 inhibitor can reverse the SHS exposure-induced receptor upregulation in rat cerebral arteries. In this model, we started inhibitor treatment after the 8-week in vivo SHS exposure. We measured urine cotinine levels to quantify the SHS exposure. We assessed the ETB and ETA receptor-mediated contractile response after 8 weeks exposure to confirm the model. To investigate whether the process can be reversed, we examined receptor contractile functions in addition to their mRNA and protein expression after inhibitor treatment. We performed immunohistochemistry to demonstrate receptor localization.

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10 week time point, 4–5 rats from each group were sacrificed to determine whether 2 weeks of inhibitor administration modified SHS exposure-induced vascular alterations. At 12 week time point, the remaining rats from every group were sacrificed for examination. 2.3. Urine and artery collection After the last day of 8 week exposure, the animals were separately placed in metabolic cages, and the urine samples were collected over a 24 h period. At 8-, 10- and 12-week time points, the rats from each group were anaesthetized with CO2 and decapitated. The brains were immediately removed and chilled in an ice-cold bicarbonate buffer solution (Cao et al., 2012a, 2012b). The basilar arteries, middle cerebral arteries and circle of Willis arteries were carefully removed free from the adhering tissue. Basilar arteries were cut into cylindrical segments with approximately 2 mm in length for in vitro pharmacology studies. In addition, vessels pieces were snap-frozen at − 80 °C for real-time PCR and Western blot. Some of the arteries were fixed in paraformaldehyde for immunohistochemistry. 2.4. Determination of urinary cotinine level

2. Materials and methods

All of the urine samples were frozen and stored until assay. The urine cotinine levels were used as a measure of exposure efficacy and were assessed by high-performance liquid chromatography (Czekaj et al., 2005). For cotinine analysis, a previously reported liquid-liquid method was modified and used throughout (Ceppa et al., 2000). Cotininemethyl-d3 was used as an internal standard for the measurement. Cotinine levels were adjusted using creatinine and presented in ng/mg creatinine.

2.1. Animals

2.5. In vitro pharmacology

Male Sprague-Dawley rats (200–250 g) were obtained from the Animal Center of Xi'an Jiaotong University. All of the rats were maintained on a normal diet and were allowed free access to food and water. This experimental protocol was approved by the Committee at Xi'an Jiaotong University Animal Ethics.

Rat cerebral artery contractile responses were recorded in vitro by mounting cylindrical segments in wire myographs (Danish Myo Technology A/S, Aarhus, Denmark) (Mulvany and Halpern, 1977). Two parallel wires were inserted into the artery lumen; one was connected to a force displacement transducer that was attached to a digital converter unit, and the other was connected to an adjustable micrometer screw that set the distance between the wires and hence the vascular tone. The arteries were initially progressively stretched to 90% of the normal internal circumference, which is the size each vessel would have if it was fully relaxed under a transmural pressure of 100 mm Hg (Huang et al., 2010). The normalization procedure makes certain that all vessel segments are set to a normalized internal circumference to give a maximal response. Subsequently, arterial viability was determined by exposure to high potassium (63.5 mM K+) bicarbonate buffer solution (Cao et al., 2012a, 2012b) that causes smooth muscle cell contraction via membrane depolarization and calcium influx (Ratz et al., 2005). The K+-induced contraction was used as a reference for the contractile capacity. Individual receptor-mediated responses were evaluated by cumulative application of its agonists S6c (ETB receptor) and ET-1 (ETB and ETA receptors). To specifically measure ETA receptor-mediated responses, ETB receptors were desensitized by S6c addition prior to ET-1 application (Cao et al., 2011). Cerebral artery endothelial function was assessed using 10−5 M acetylcholine (ACh) following a pre-contraction induced by 10−7 M ET-1.

2.2. Experimental Protocol The experiment lasted 12 weeks. Rats were exposed to either SHS or fresh air. The rats were placed in a closed exposure chamber (115 × 50 × 65 cm) and exposed to cigarette smoke generated from commercially available filtered cigarettes (Marlboro, 1.0 mg of nicotine and 12 mg of tar content). To mimic the SHS exposure, 5 cigarettes were successively lit in the chamber and allowed to burn freely for 90 min (75 min for cigarette burning and another 15 min for the smoke diffusion in the chamber). Afterwards, fresh air inhalation was permitted in the chamber. The rats were exposed to SHS twice per day (morning and afternoon). 70 rats were divided into 7 experimental groups: A) Rats exposed to cigarette smoke or fresh air for 12 weeks (12 rats for each group). B) Rats exposed to cigarette smoke for 8 weeks followed by daily ip administration of U0126 (5 mg/kg and 15 mg/kg) dissolved in DMSO or DMSO alone for another 4 weeks (10 rats for each group). C) Rats exposed to fresh air for 8 weeks followed by daily ip administration of U0126 (15 mg/kg, 8 rats) dissolved in DMSO or DMSO alone (8 rats) for another 4 weeks.

At 8 week time point, 3 rats respectively from 12-week SHS and 12week fresh air exposure group were sacrificed to confirm the model. At

2.6. Real-time PCR RNA extraction and real-time PCR was performed as described earlier (Cao et al., 2011). Briefly, total RNA was isolated using an RNeasy mini kit according to the manufacturer's protocol (Qiagen GmbH, Hilden, Germany). Total RNA was reverse transcribed to cDNA using TaqMan reverse transcription reagents (Applied Biosystems, Foster city, CA, USA). Quantitative real-time PCR was performed using ETB and ETA

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receptors primers with the SYBR® Green kit in a GeneAmp 7300 sequence detection system (Applied Biosystems) and the data were normalized to the housekeeping gene Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primers used were as follows: ETB receptor, forward, 5′-GAT ACG ACA ACT TCC GCT CCA-3′, reverse, 5′-GTC CAC GAT GAG GAC AAT GAG-3′; ETA receptor, forward, 5′-GTC GAG AGG TGG CAA AGA CC-3′, reverse, 5′-ACA GGG CGA AGA TGA CAA CC-3′; GADPH, forward, 5′-GGC CTT CCG TGT TCC TAC C-3′, reverse, 5′-CGG CAT GTC AGA TCC ACA AC-3′. The data were analyzed using the comparative cycle threshold (CT) method (Ahnstedt et al., 2012).

contractile response. Two-way analysis of variance with/without Bonferroni's post-test was performed to compare the two corresponding data points at each concentration on the two or more curves. The ACh-induced relaxation is expressed as a percentage of the contractile response elicited by 10− 7 M ET-1. Target gene mRNA levels were expressed relative to the housekeeping gene GAPDH. Target protein expression levels were either represented relation to β-actin or total ERK1/2. One-way ANOVA with Bonferroni's post-test was used for multiple comparisons. The calculations and statistical analysis were performed using Graph Pad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). P b 0.05 was considered to be statistically significant.

2.7. Western blotting Cerebral artery proteins were extracted as described previously (Cao et al., 2011). Afterwards, equal protein amounts (40 μg) were loaded on a 4–15% ready gel precast gel (Bio-Rad Laboratories, Hercules, CA, USA) for protein separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the separated proteins were transferred to a nitrocellulose membrane. The membrane was then blocked with 5% non-fat milk or bovine serum albumin, and incubated with ETB receptor (diluted 1:200, sc-21196, Santa Cruz Biotechnology, Santa Cruz, CA, USA), ETA receptor (diluted 1:100, sc-33536, Santa Cruz Biotechnology), phospho-MEK1/2 (diluted 1:1000, #9121, Cell Signaling Technology, Beverly, MA, USA), phospho-ERK1/2 (diluted 1:2000, #4370, Cell Signaling Technology) or β-actin (diluted 1:200, sc47778, Santa Cruz Biotechnology), MEK1/2 (diluted 1:1000, #9122, Cell Signaling Technology) and ERK1/2 (diluted 1:2000, #4696, Cell Signaling Technology) primary antibodies at 4 °C overnight. The next day, the blots were incubated with the respective secondary antibodies conjugated with horseradish peroxidase for 1 h at room temperature (antigoat diluted 1:5000, sc-2020, Santa Cruz Biotechnology; anti-rabbit diluted 1:2000, #7074, Cell Signaling Technology; anti-mouse diluted 1:2000, #7076, Cell Signaling Technology). Finally, proteins were visualized using a Fujifilm LAS-1000 Luminescent Image Analyzer (Fujifilm, Stanford, CT, USA), and the band intensity was quantified using Image J software (http://rsb.info.nih.gov/ij/). 2.8. Immunohistochemistry Cerebral arteries were fixed in 4% paraformaldehyde. Vessel tissues underwent dehydration, clearing and infiltration and then were embedded with paraffin wax. The specimens were cut into 4 μm sections. Subsequently, the slides were deparaffinized, rehydrated and then boiled in microwave oven for 10 min for antigen retrieval. Immunostaining was performed overnight with the following primary antibodies: sheepanti rat ETB 1:250 (#ALX-210-506A, Enzo Life Science Inc., Farmingdale, NY, USA) and sheep-anti rat ETA 1:100 (#ALX-210-507A, Enzo Life Science Inc.). Secondary antibody was applied for 1 h (1:50 Texas Red dye-conjugated donkey-anti sheep, #713-076-147, Jackson ImmunoResearch, West Grove, PA, USA). Sections were then mounted in anti-fading medium containing DAPI that stains nuclei (Vectashield, Vector Laboratories Inc., Burlingame, CA, USA). Omission of primary antibody served as negative control. Immunoreactivity was visualized at the appropriate wavelength with an epifluorescence microscope (Nikon 80i; Tokyo, Japan) and photographed with an attached Nikon DS-2Mv camera. 2.9. Calculations and statistics The data are represented as the means ± SE, and n refers to the number of rats. The urine cotinine levels were normalized by creatinine and represented in ng/mg creatinine. The Emax values represent maximal contraction. The pEC50 values represent the negative logarithm of the concentrations that produced 50% Emax. Unpaired student's t-test was applied to compare two data sets. The vasomotor responses to the receptor agonists are expressed as a percentage of the K+-induced

3. Results 3.1. General condition In the course of the experiment, the fresh air-exposed rats behaved normally. In contrast, the SHS-exposed animals demonstrated anxiety and excessive sweating in the beginning of SHS inhalation exposure. During SHS exposure, the animals became calm and fell asleep. After exposure, the rats demonstrated abnormal mobility. 3.2. Urine cotinine levels To determine the degree of SHS inhalation exposure in the rats, cotinine levels were measured. The result showed that urine cotinine levels in the SHS exposure group (672.5 ± 98.3 ng/mg) were much higher than that in the fresh air group (52.4 ± 11.5 ng/mg, P b 0.01). 3.3. ET receptor-mediated constriction of cerebral arteries in SHS-exposed rats Cerebral artery contractile responses in 8 week SHS-exposed rats were examined in a wire myograph. Initially, cerebral arteries were exposed to 63.5 mmol/L K+ to elicit a contractile response, which was used as a reference for contractile capacity (=100%). K+-induced contractions did not differ significantly between fresh air (13.7 ± 1.8 mN) and SHS-exposed cerebral arteries (14.5 ± 2.1 mN). Therefore, the receptor-mediated contractile responses were demonstrated as the percentage of K+-elicited contraction. The selective ETB receptor agonist, sarafotoxin 6c (S6c) was used to assess ETB receptor-mediated vascular contraction (Moller et al., 1997). The cumulative application of increasing S6c concentration induced little response in fresh cerebral arteries (Emax: 5.8 ± 1.0%). After 8 week SHS exposure, the ETB receptor-mediated contraction was the same as that of the fresh vessels, and the Emax value was only 6.5 ± 1.1%. After ETB receptor desensitization was performed, ET-1 was cumulatively added to baths to induce ETA receptor-mediated contraction. The characterization of ETB and ETA receptors has previously been verified using their specific antagonists IRL2500 and FR139317 (Hansen-Schwartz and Edvinsson, 2000). ETA receptormediated vasoconstriction was enhanced after SHS exposure with increased Emax and higher pEC50 values compared with fresh arteries. The Emax was 136.2% ± 9.2% in fresh arteries and 182.7% ± 9.6% in SHS-exposed arteries (P b 0.05). There was a leftward shift of the concentration-response curve in SHS-exposed arteries, in which the pEC50 value was increased from 8.38 ± 0.11 (fresh) to 8.70 ± 0.10 (SHS, P b 0.05). 3.4. Effect of U0126 on ET receptor-mediated contractions in cerebral arteries subjected to SHS exposure Following 8 weeks of SHS or fresh air exposure, the rats were administered with different doses of U0126 or DMSO for 4 weeks. The cerebral artery contractile function was examined at both 10 and 12 week time point. The K+-induced cerebral artery contraction values are provided

L. Cao et al. / Toxicology and Applied Pharmacology 304 (2016) 70–78 Table 1 Effect of 2 week U0126 administration on the Emax and pEC50 values of the concentrationcontractile response curves induced by endothelin receptor agonists ET-1 in cerebral arteries. Group 10 w SHS 8 w SHS + 2 w DMSO 8 w SHS + 2 w U0126 (5 mg/kg) 8 w SHS + 2 w U0126 (15 mg/kg) 10 w fresh 8 w fresh + 2 w DMSO 8 w fresh + 2 w U0126 (15 mg/kg)

n 4 5 5 5 4 4 4

K+ (mN) 12.8 ± 1.8 13.7 ± 2.0 14.8 ± 2.8 13.2 ± 2.8 11.8 ± 1.6 12.3 ± 3.0 14.5 ± 2.7

Emax (% of K+) #

183.4 ± 7.9 191.8 ± 14.1 180.6 ± 10.8 143.5 ± 8.2* 130.6 ± 8.3 128.4 ± 11.8 134.1 ± 9.3

pEC50 8.86 ± 0.08 8.54 ± 0.13 8.76 ± 0.12 9.05 ± 0.12 8.60 ± 0.11 8.67 ± 0.17 8.65 ± 0.13

The data are expressed as the means ± SEM and n refers to the number of animals. K+-induced responses are expressed as absolute values of contraction (mN). ET-1-induced responses are expressed as percentage of K + -induced contraction.*P b 0.05: SHS + DMSO vs. SHS + U0126 (10 mg/kg); #P b 0.05: Fresh vs. SHS. ET-1, endothelin-1; SHS, secondhand smoke; w, weeks.

in Table 1, and the results did not differ significantly among the 7 groups (P N 0.05). At 10 week time point, there were no significant differences in S6cinduced artery contraction responses between treated and untreated

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animals (Fig. 1A). ET-1 induced potent vasoconstriction on cerebral arteries in all of the groups. There were no significant differences in the Emax and pEC50 values of the response curves among the fresh air, fresh air + U0126 and fresh air + DMSO groups (Table 1), which suggests that U0126 per se did not affect the normal cerebral artery contractility mediated by ETA receptors. However, the ET-1-induced contractile responses were higher in 10 week SHS-exposed arteries and in 8 week SHS + 2 week DMSO-administered cerebral arteries, compared with fresh air-exposed arteries (Fig. 1B). Fig. 1B demonstrates that exposure to 10 week SHS did not differ from 8 week SHS + 2 week DMSO in ET-1-induced cerebral artery contractions. The Emax of response curves was at the same level in the two groups (Table 1, P N 0.05), suggesting that ceasing SHS exposure for 2 weeks did not recover the increased response of ET-1 to normal activity. The maximal contraction of the response curves was 130.6% ± 8.3% in fresh air-exposed arteries. However, the Emax values were increased to over 180% ± 8% in 10 week SHS-exposed cerebral arteries (Table 1). U0126 (5 mg/kg) administration did not alter the ET-1-induced contractile response with unchanged Emax versus 8 week SHS + 2 week DMSO group (Table 1). In contrast, the higher U0126 dose (15 mg/kg) reduced the 8 week SHS exposure-increased contraction with significantly

Fig. 1. Effect of U0126 on ET receptor-mediated contractions in rat cerebral arteries subjected to SHS exposure. The rats were exposed to fresh air or SHS for 8 weeks, which was followed by U0126 injection (i.p., 5 mg/kg or 15 mg/kg) for 2 weeks (A and B) or 4 weeks (C and D). The rat basilar arteries were isolated. The contractions were induced by S6c, a specific ETB receptor agonist and ETB receptor-meditated concentration-contractile curves were constructed (A and C). After ETB receptor desensitization, ET-1 was cumulatively added to baths to induce ETA receptor-mediated contraction (B and D). The contractile responses are shown in the percentage of K+-induced contraction. Each data point is derived from 4 to 5 animals and data are expressed as the means ± SE. ⁎P b 0.05, ⁎⁎P b 0.01 vs. fresh 10 w or 12 w; #P b 0.05, ##P b 0.01 vs. SHS + DMSO.

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decreased Emax of 143.5% ± 8.2%, compared with SHS + DMSO group (Emax: 191.8% ± 14.1%, P b 0.05). At 12 week time point, S6c-induced contractions still remained unchanged in rat cerebral arteries among the 7 groups (Fig. 1C). However, there were obvious alterations in ETA receptor-mediated vasoconstriction. There was a significant reduction in several data points of the response curve in 8 week SHS + 4 week DMSO group, compared with 12 week SHS exposure group (Fig. 1D). A rightward shift of the contractile curve was noted by 8 week SHS + 4 week DMSO administration. The pEC50 was significantly decreased but the Emax was unchanged (Table 2). The Emax in the 8 week SHS + 4 week DMSO group was still elevated versus the 12-week fresh air exposed arteries (Table 2), indicating that the SHS exposure-induced enhanced vasoconstriction still existed and did not return to the normal condition of fresh controls (Fig. 1D). After 4 week U0126 administration, a significant decrease of ETA receptor-mediated contractile responses was observed. Two different doses of U0126 reduced the increase of SHS exposure-mediated contraction. There was a statistical decrease of Emax values in the U0126 groups compared with the SHS + DMSO group (Table 2). In addition, the pEC50 value in the higher dose U0126 group was reduced more than that in the lower dose group (Table 2, P b 0.05), although there were no significant differences in Emax. 3.5. Effect of U0126 on ACh-induced endothelium-dependent relaxation in cerebral arteries subjected to SHS exposure

Table 2 Effect of 4 week U0126 administration on the Emax and pEC50 values of the concentrationcontractile response curves induced by endothelin receptor agonists ET-1 in cerebral arteries.

Group 12 w SHS 8 w SHS + 4w DMSO 8 w SHS + 4w U0126 (5 mg/kg) 8 w SHS + 4w U0126 (15 mg/kg) 12 w Fresh 8 w Fresh + 4 w DMSO 8 w Fresh + 4 w U0126 (15

n K+ (mN) 5 5 5 5 5 4 4

14.3 ± 2.70 11.7 ± 2.17 15.7 ± 4.50 12.3 ± 2.79 13.7 ± 2.84 13.4 ± 2.84 13.4 ± 2.01

Emax (% of K +)

pEC50 #

180.2 ± 5.3 181.8 ± 12.9 137.3 ± 5.4* 127.2 ± 4.7* 127.1 ± 10.6 132.6 ± 10.5 147.1 ± 13.5

8.87 ± 0.05** 8.42 ± 0.12 8.70 ± 0.07 8.46 ± 0.06$ 8.59 ± 0.15 8.67 ± 0.15 8.38 ± 0.16

mg/kg) The data are expressed as the means ± SEM and n refers to the number of animals. K+-induced responses are expressed as absolute values of contraction (mN). ET-1-induced responses are expressed as percentage of K + -induced contraction. *P b 0.05, **P b 0.01: SHS + DMSO vs. SHS, SHS + U0126 (5 and 10 mg/kg); #P b 0.05: Fresh vs. SHS; $P b 0.05: SHS + U0126 (5 mg/kg) vs. SHS + U0126 (10 mg/kg). ET-1, endothelin-1; SHS, secondhand smoke; w, weeks.

decreased by treatment with 15 mg/kg U0126. However, cerebral arteries that were exposed to 5 mg/kg U0126 still displayed high intensity of ETA receptor fluorescence staining and did not display notable differences to SHS-exposed arteries. 3.8. Effect of U0126 on MEK/ERK1/2 signaling pathway in cerebral arteries

ET-1 (10−7 M) was added to baths to induce a potent and sustained contraction in cerebral arteries. Endothelium-dependent relaxation was obtained by addition of 10−5 M ACh on pre-contracted arteries. The precontraction was relaxed to different degrees in different groups. The relaxation is presented as the percentage of the ET-1-induced contraction. The ACh-induced relaxation was decreased significantly in SHS-exposed arteries (Fig. 2). The relaxation was reduced from 57.1% ± 7.2% in fresh arteries to approximately 30.2% ± 5.4% in 12 week SHS-exposed ones. However, we did not find significant difference in relaxation between 8 week SHS + 4 week U0126 treatment and 8 week SHS + 4 week DMSO group (Fig. 2), suggesting that inhibitor administration did not alter endothelium injury caused by SHS exposure. 3.6. Effect of U0126 on ET receptor expression in cerebral arteries subjected to SHS exposure

The protein expression levels of total MEK1/2, phosphorylated (p)MEK1/2, total ERK1/2 and p-ERK1/2 were detected by Western blotting in cerebral arteries. Fig. 5 showed that the protein expressions of total MEK1/2 and ERK1/2 were unaltered among all the groups. However, the protein expression bands of p-MEK1/2 and p-ERK1/2 exhibited notably increased density in the SHS groups (both 12 week SHS group and 8 week SHS + 4 week DMSO group) as compared to the fresh groups, demonstrating that the phosphorylation of MEK1/2 and ERK1/2 were increased by SHS exposure. The data suggested that SHS activates MEK/ERK1/2 signaling pathway in cerebral arteries. The p-MEK1/2 protein showed similar expression levels between the SHS + U0126 and SHS groups, indicating that the MEK1/2 inhibitor U0126 did not change the phosphorylation of MEK1/2 per se. However, the phosphorylated protein levels of ERK1/2 was obviously deceased in the SHS + U0126 groups as compared to the SHS groups (Fig. 5), which

ETB receptor mRNA and protein levels were unaffected by SHS exposure or U0126 treatment in cerebral arteries from all of the groups (Fig. 3A, C). SHS exposure (12 week SHS or 8 week SHS + 4 week DMSO) significantly increased ETA receptor mRNA and protein expression levels compared with fresh controls (Fig. 3B, D). In addition, 8 week SHS + 4 week DMSO did not decrease receptor expression from the 12 week SHS exposure group. Treatment with low dose U0126 showed unchanged ETA receptor mRNA levels compared with 8 week SHS + 4 week DMSO. However, both low and high dose U0126 significantly reduced the ETA receptor mRNA and protein levels in cerebral arteries compared with the 8 week SHS + 4 week DMSO-treated groups (Fig. 3B, D). 3.7. ET receptor immunohistochemistry in SHS-exposure cerebral arteries The immunoreactivity of ETB receptor was almost exclusively found in the endothelial cells (Fig. 4A). In contrast, ETA receptor immunoreactivity was abundant in SMCs. Staining with 4′, 6-diamidino-2phenylindole (DAPI) found that the ETA receptor expression was localized in cytoplasm, but not in the nuclei (Fig. 4A). The fluorescence intensity of ETB receptor was similar in all 7 groups (Fig. 4B). An enhanced ETA receptor immunoreactivity was detected in cerebral arteries after 12 week SHS exposure and 8 week SHS + 4 week DMSO compared with fresh controls (Fig. 4B). This increased staining intensity was

Fig. 2. Acetylcholine (ACh)-induced relaxation response in rat cerebral arteries. The rats were exposed to fresh air or SHS for 8 weeks and then injected (i.p.) with U0126 (15 mg/kg) for 4 weeks. After pre-contraction was induced by ET-1 (10−7 M). ACh (10−5 M) was added into the baths to induce a relaxation. Relaxation responses are shown as the percentage of ET-1-induced contraction. Each data is derived from 4 to 5 animals and data are expressed as the means ± SE. ⁎P b 0.01 vs. fresh group; ns, no significance vs. SHS + DMSO group.

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suggested that U0126 could inhibit the activation of ERK1/2, the downstream kinase of MEK1/2, in cerebral arteries. 4. Discussion This is the first demonstration that treatment with the MEK1/2 inhibitor, U0126 reduces SHS exposure-induced ETA receptor upregulation in cerebral arteries. The increased cerebral artery contractile response, receptor mRNA and protein levels were significantly decreased by 4 week inhibitor treatment. ETA receptors localization was detected in the cytoplasm of cerebral artery SMCs. In addition, the rat body weight increased more slowly during the SHS exposure period. Rat urine cotinine levels were notably elevated after SHS exposure compared with fresh air-exposed rats. Cigarette smoking is a strong risk factor that is associated with the increased incidence of different types of stroke (Allen and Bayraktutan, 2008). The combustion products of cigarette smoke

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include over 4000 different substances. Approximately 400 appear in the gaseous phase and about 3500 in the particulate phase of cigarette smoke. The components of cigarette smoke are present in inhaled smoke and may be detected in active and passive smokers (Witschi et al., 1997). Among the components of cigarette smoke, cotinine is a major nicotine metabolite with a relatively long half-life (15–20 h) and stable present in plasma and urine, and has been widely used as the best indicator of cigarette smoke exposure (Kuo et al., 2002). Therefore, we measured the cotinine level in the urine of rats that were exposed to SHS exposure for 8 weeks. We determined that in these rats, the urine cotinine level was significantly increased to over 600 ng/mg CREA. Other authors reported similar results (Zhang et al., 2009). In addition, it was reported that both the quantity of tobacco smoke and time of exposure influence cotinine excretion in urine (Florek et al., 2003; Zhang et al., 2009). When smoke concentration was constant, extended exposure significantly increased the urinary cotinine levels. The concentration of cotinine in rat urine was correlated with both the exposure

Fig. 3. Effect of U0126 on ETB and ETA receptor mRNA and protein expressions in rat cerebral arteries subjected to SHS exposure. The rats were exposed to fresh air or SHS for 8 weeks and then injected (i.p.) with U0126 (5 mg/kg or 15 mg/kg) for 4 weeks. The mRNA levels (A and B) for ETB and ETA receptors are expressed relative to GAPDH. The protein levels (C and D) for the receptors are expressed relative to β-actin. Each data is derived from 4 to 5 animals and data are expressed as the means ± SE. Statistical analysis was performed using one-way ANOVA with Bonferroni's post-test. ⁎P b 0.05, ⁎⁎P b 0.01 vs. fresh controls, #P b 0.05, ##P b 0.01 vs. SHS + DMSO group.

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Fig. 4. ET receptor immunohistochemistry in SHS-exposure cerebral arteries. Double immunofluorescence staining for ETB/ETA receptors and cell nuclei (DAPI) in cerebral arteries following SHS (A). Photographs in the first lane demonstrate high immunoreactivity of ETB receptors in endothelial cells (white arrow); photographs in the second lane show a strong ETA receptor fluorescence intensity in smooth muscle cell layer. The co-localization of DAPA staining revealed that ETA receptors were localized in the cytoplasm, not in the nuclei (white arrows). Immunofluorescence staining of ETB and ETA receptors in cerebral arteries after U0126 treatment following SHS exposure (B). Photographs in the first lane show immunoreactivity of ETB receptors in endothelium layer and there is no difference of fluorescence intensity among the groups. Photographs in the second lane demonstrate ETA receptor immunofluorescence staining. The immunoreactivity was found in smooth muscle cells. There was stronger intensity of immunofluorescence staining in the SHS and SHS + DMSO groups versus the fresh group. U0126 (5 mg/kg) treatment did not alter the intensity. However, U0126 (15 mg/kg) treatment weakened the immunoreactivity of ETA receptors versus the SHS and SHS + DMSO groups.

time and the number of smoked cigarettes (Florek et al., 2003; Zhang et al., 2009). In the present study, SHS exposure was performed for 8 weeks in the first step. To mimic real treatment, the exposure was stopped in some groups and replaced with U0126 injection or vehicle administration for 4 weeks. In a previous study, the animals received inhibitor administration concomitant with 8 week SHS exposure (Cao et al., 2011). It is a method of prevention rather than treatment; however, it was useful to understand the principle mechanisms involved (Cao et al., 2011, 2013). Therefore, we applied SHS exposure to the rats for 8 weeks

Fig. 5. Effect of U0126 on MEK/ERK1/2 signaling pathway in rat cerebral arteries. The rats were exposed to fresh air or SHS for 8 weeks and then injected (i.p.) with U0126 (5 mg/kg or 15 mg/kg) for 4 weeks. The protein expression levels of total MEK1/2, phosphorylated (p)-MEK1/2, total ERK1/2 and p-ERK1/2 were detected by Western blotting. The protein levels of p-MEK1/2 and p-ERK1/2 are expressed relative to total MEK1/2 and ERK1/2, respectively.

first, which was followed by U0126 administration. In the earlier study, we demonstrated that SHS exposure upregulated ETA receptor by increasing receptor mRNA, protein levels and higher contractile response to receptor agonist (Cao et al., 2011). To confirm the model, the ET receptor-mediated contractile response curve was assessed after the 8 week SHS exposure in the present study. The data showed a similar response curve pattern as what previously described (Cao et al., 2011). The ETB receptor agonist-induced vasoconstriction was unaltered, but the ETA receptor agonist-induced contractile response was significantly increased after 8 SHS exposure. There are increased ET-1 levels in plasma and cerebrospinal fluid after stoke (Barone et al., 1994). The contractile receptors like ETA, ETB receptor, 5hydroxytryptamine1B receptor, thromboxane A2 receptor in cerebral arteries exhibit expressional plasticity (upregulation) in ischemic and hemorrhagic stroke (Edvinsson and Povlsen, 2011). The receptor upregulation can cause increased sensitivity of artery contractility, decreased brain blood supply and increased neurological deficits (Edvinsson and Povlsen, 2011). Because both agonist and receptor are upregulated, the role of ET in stroke may be quite important for disease development and therapy. Selective ET receptor antagonism has shown potential therapeutic utility for experimental stroke (Patel et al., 1995, 1996). The present study observed that SHS results in an upregulation of contractile ETA receptors. It is likely that stroke may exacerbate tissue damage in smokers or individuals exposed to SHS. ETA receptor mRNA and protein expression levels, in addition to the corresponding receptormediated contraction, were increased after 8 weeks of SHS exposure. The 12 week SHS exposure group demonstrated the highest ETA receptor expression among all of the groups. Exposure to 8 week SHS + 4 week DMSO also exhibited upregulated ETA receptors

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compared with 12 week fresh air group. U0126 administration (15 mg/ kg) for 4 weeks significantly reduced the increased ETA receptor expression compared to exposure to 12 week SHS or 8 week SHS + 4 week DMSO. In the present study, we found no significant difference in ETA receptor expression (mRNA and protein level) between 12 week SHS and 8 week SHS + 4 week DMSO groups, which demonstrated that the increased ETA receptor expression did not come down to the 12week fresh air exposure level with 4 week SHS cessation. The Emax of ETA receptor-mediated curves in the two groups were similar; however, they presented significantly different pEC50 values. These data suggested that the efficacy of ETA receptor-mediated response was unaltered, but the potency was significantly changed. Our results demonstrated a significant reduction in several data points of the response curve in the 8 week SHS + 4 week DMSO group, compared with 12 week fresh air group. A rightward shift of the contractile curve was noted by 4 week SHS exposure cessation with a significantly decreased pEC50, indicating that the potency of ETA receptor-mediated response was significantly lowered by ceasing SHS exposure for 4 weeks. However, the Emax of ETA receptor-mediated curve was not altered by SHS cessation. Furthermore, the ETA receptor was still upregulated in the 12 week SHS exposure group compared with the non-SHS exposure group. The endothelium maintains vascular homeostasis via the release of active vasodilators (Luksha et al., 2009). Endothelial dysfunction resulted primarily from the impaired nitric oxide and endothelium-derived hyperpolarizing factor relaxation pathways (Luksha et al., 2009). Under normal physiological conditions, blood vessels dilate to at least 80% of the agonist-induced contraction. In the present study, due to technical dissection or wire pass-through, some of the endothelium may be damaged before myograph experiments. Thus, the ACh-induced relaxation actually did not reach 60% of the pre-contraction in the fresh group. Thereafter, the endothelium-dependent relaxation was significantly decreased by exposure to 12 week SHS or 8 week SHS + 4 week DMSO. However, endothelium-dependent relaxation was not or was minimally affected by U0126. Damaged endothelium was not improved by MEK1/2 inhibitor treatment. It was suggested that the damaged endothelium may be an important mechanism for the increased vessel constriction that is observed in contractile function. Numerous studies have demonstrated the close relationship between MEK/ERK1/2 activation and receptor upregulation. Previous studies demonstrated that MEK/ERK1/2 pathway inhibition may alleviate the cerebrovascular receptor upregulation, improve cerebral blood flow levels, and reduce infarct volume and neurology score (Ansar et al., 2011; Maddahi and Edvinsson, 2010). Furthermore, increased phosphorylated ERK1/2 immunoactivity and protein levels in arteries were seen in SHS exposure model (Cao et al., 2012a, 2012b, 2013). In the present study, with increased time during treatment, we determined that the MEK1/2 inhibitor U0126 could reduce the SHS exposureinduced ETA receptor upregulation in terms of decreasing the elevated vasoconstriction, increased receptor mRNA and protein expression. The inhibitory effect was influenced by the treatment length and inhibitor dose. There was no difference in the receptor-mediated contraction response in 8 week SHS + 2 week U0126 administration (5 mg/kg) compared with untreated rats. However, the high U0126 dose (15 mg/kg) significantly decreased the ETA receptor contractile response in cerebral arteries. When the treatment period was prolonged to 4 weeks, both doses reduced the contraction responses and protein level of ETA receptor. Interestingly, the ETA receptor remained overexpressed to the level that was reached after 8 week SHS exposure. Additionally, the increased contractile property of ETA receptor did not return to normal after 8 week SHS + 2 week DMSO administration. Similarly, ETA receptor expression was still upregulated following 8 week SHS + 4 week DMSO. However, this alteration was not observed in contractile characteristics in wire myography, which may be because receptor protein expression changes come first in cells, but this alteration has not yet been observed in receptor contractile function.

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5. Conclusions MEK/ERK1/2 pathway inhibition by U0126 offsets SHS exposure-induced ETA receptor upregulation in rat cerebral arteries. These data may advance our knowledge regarding the involvement of the MEK/ERK1/2 signaling pathway in SHS exposure-induced cerebrovascular diseases and provide a new possible target for the treatment. Conflict of interest The authors declare that there is no conflict of interest. Transparency document The Transparency document associated with this article can be found, in online version. Acknowledgements This study was supported by grants from the Swedish Research Council (Grant 5958), the Swedish Heart-Lund Foundation (grant 20070273) and the National Natural Science Foundation of China (grant 81502840). The authors would like to thank Dr. Xue Xiao, Tao Sun., Yinjing Zhu in Department of Pharmacology, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center for tissue collection and technical assistance during the experiment. References Ahnstedt, H., Stenman, E., Cao, L., Henriksson, M., Edvinsson, L., 2012. Cytokines and growth factors modify the upregulation of contractile endothelin ET(A) and ET(B) receptors in rat cerebral arteries after organ culture. Acta Physiol (Oxford) 205, 266–278. Allen, C.L., Bayraktutan, U., 2008. Risk factors for ischaemic stroke. Int. J. Stroke 3, 105–116. Ambrose, J.A., Barua, R.S., 2004. The pathophysiology of cigarette smoking and cardiovascular disease: an update. J. Am. Coll. Cardiol. 43, 1731–1737. Ansar, S., Maddahi, A., Edvinsson, L., 2011. Inhibition of cerebrovascular raf activation attenuates cerebral blood flow and prevents upregulation of contractile receptors after subarachnoid hemorrhage. BMC Neurosci. 12, 107. Barone, F.C., Globus, M.Y., Price, W.J., White, R.F., Storer, B.L., Feuerstein, G.Z., Busto, R., Ohlstein, E.H., 1994. Endothelin levels increase in rat focal and global ischemia. J. Cereb. Blood Flow Metab. 14, 337–342. Bonita, R., Duncan, J., Truelsen, T., Jackson, R.T., Beaglehole, R., 1999. Passive smoking as well as active smoking increases the risk of acute stroke. Tob. Control. 8, 156–160. Cao, L., Xu, C.B., Zhang, Y., Cao, Y.X., Edvinsson, L., 2011. Secondhand smoke exposure induces Raf/ERK/MAPK-mediated upregulation of cerebrovascular endothelin ETA receptors. BMC Neurosci. 12, 109. Cao, L., Zhang, Y., Cao, Y.X., Edvinsson, L., Xu, C.B., 2012a. Cigarette smoke upregulates rat coronary artery endothelin receptors in vivo. PLoS One 7, e33008. Cao, L., Zhang, Y., Cao, Y.X., Edvinsson, L., Xu, C.B., 2012b. Secondhand smoke exposure causes bronchial hyperreactivity via transcriptionally upregulated endothelin and 5-hydroxytryptamine 2A receptors. PLoS One 7, e44170. Cao, L., Xu, C.B., Zhang, Y., Cao, Y.X., Edvinsson, L., 2013. Secondhand cigarette smoke exposure causes upregulation of cerebrovascular 5-HT(1) (B) receptors via the Raf/ERK/ MAPK pathway in rats. Acta Physiol (Oxford) 207, 183–193. Ceppa, F., El Jahiri, Y., Mayaudon, H., Dupuy, O., Burnat, P., 2000. High-performance liquid chromatographic determination of cotinine in urine in isocratic mode. J. Chromatogr. B Biomed. Sci. Appl. 746, 115–122. Czekaj, P., Wiaderkiewicz, A., Florek, E., Wiaderkiewicz, R., 2005. Tobacco smoke-dependent changes in cytochrome P450 1A1, 1A2, and 2E1 protein expressions in fetuses, newborns, pregnant rats, and human placenta. Arch. Toxicol. 79, 13–24. Edvinsson, L.I., Povlsen, G.K., 2011. Vascular plasticity in cerebrovascular disorders. J. Cereb. Blood Flow Metab. 31, 1554–1571. Florek, E., Piekoszewski, W., Wrzosek, J., 2003. Relationship between the level and time of exposure to tobacco smoke and urine nicotine and cotinine concentration. Pol. J. Pharmacol. 55, 97–102. Hansen-Schwartz, J., Edvinsson, L., 2000. Increased sensitivity to ET-1 in rat cerebral arteries following organ culture. Neuroreport 11, 649–652. Hansen-Schwartz, J., Hoel, N.L., Zhou, M., Xu, C.B., Svendgaard, N.A., Edvinsson, L., 2003. Subarachnoid hemorrhage enhances endothelin receptor expression and function in rat cerebral arteries. Neurosurgery 52 (1188–1194) (1194-1185). Huang, L.H., He, J.Y., Yuan, B.X., Cao, Y.X., 2010. Lipid soluble smoke particles upregulate endothelin receptors in rat basilar artery. Toxicol. Lett. 197, 243–255. Kuo, H.W., Yang, J.S., Chiu, M.C., 2002. Determination of urinary and salivary cotinine using gas and liquid chromatography and enzyme-linked immunosorbent assay. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 768, 297–303.

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