A soluble epoxide hydrolase inhibitor—8-HUDE increases pulmonary vasoconstriction through inhibition of KATP channels

Pulmonary Pharmacology & Therapeutics 25 (2012) 69e76

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A soluble epoxide hydrolase inhibitord8-HUDE increases pulmonary vasoconstriction through inhibition of KATP channels Yun Liu a, b,1, Jing Zhang c,1, Lei Yu a, b, Fangyuan Cao d, Jingjing Rao a, b, Jing Li a, b, Chun Jiang e, J.R. Falck f, Elizabeth R. Jacobs g, Daling Zhu a, b, * a

Department of Biopharmaceutical Key Laboratory of Heilongjiang Province, College of Pharmacy, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin, Heilongjiang 150081, PR China Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical University-Daqing, Daqing 163319, China c The First Affiliated Hospital of Harbin Medical University, 23 Youzheng Street, Nangang District, Harbin, Heilongjiang 150001, PR China d Department of Pharmacology, Daqing Campus of Harbin Medical University, Daqing 163319, China e Department of Biology, Georgia State University, Atlanta, GA, 30303, USA f University of Texas Southwestern Medical Center, Dallas, TX 75390, USA g Department of Medicine, Clement J. Zablocki VA and Cardiovascular Center, Medical College of Wisconsin, Milwaukee, WI 53295, USA b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 October 2011 Received in revised form 7 November 2011 Accepted 26 November 2011

Epoxyeicosatrienoic acids (EETs), cytochrome P450-derived metabolites of arachidonic acid, are endogenously produced epoxides that act as substrates for the soluble epoxide hydrolase (sEH). Recent studies indicate that EETs increase the tension of rat pulmonary arteries (PAs), and inhibition of sEH augments hypoxic pulmonary vasoconstriction. However, the mechanisms underlying the proconstrictive effects of sEH inhibitors in pulmonary artery smooth muscle cells (PASMCs) are unclear. In the present study, we used a sEH inhibitor, 12-(3-hexylureido) dodec-8-enoic acid (8-HUDE), to examine the ionic mechanisms underlying the constriction of PAs. 8-HUDE increased the tension of rat PAs to 145% baseline in a manner which was effectively eliminated by 10 mmol/L glibenclamide, an inhibitor of ATP-sensitive Kþ (KATP) channels. Whole cell currents of HEK cells transfected with Kir6.1 or SUR2B were activated by KATP channel opener pinacidil, inhibited by KATP channel inhibitor glibenclamide or inhibited by 8-HUDE in a concentration-dependent manner with an IC50 value of 40 uM. In addition, 8-HUDE inhibited the expression of Kir6.1 and SUR2B at both mRNA and protein level in rat PASMCs. These observations suggest that 8-HUDE exerts acute effects on KATP channel activity as well as subacute effects through decreased channel expression, and these effects are, at least in part, via the Kir6.1/SUR2B channel. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Epoxyeicosatrienoic acids (EETs) 12-(3-hexylureido)dodec-8-enoic acid 8-HUDE Pulmonary arteries ATP-sensitive Kþ (KATP) channels

1. Introduction Cytochrome P450 epoxygenases metabolizes arachidonic acid to 4 regioisomeric epoxyeicosatrienoic acids (EETs): 5, 6-, 8, 9-, 11, 12-, and 14, 15-EET, which products regulate a multitude of cellular and physiological functions, including gene expression, vasoreactivity, inflammation, cellular proliferation, hemostasis, and ischemic

Abbreviations: EETs, epoxyeicosatrienoic acids; Glib, glibenclamide; 8-HUDE, 12-(3-hexylureido)dodec-8-enoic acid; PAs, pulmonary arteries; PASMCs, pulmonary artery smooth muscle cells; PE, phenylephrine; Pin, pinacidil; RT-PCR, reverse transcription polymerase chain reaction; sEH, soluble epoxide hydrolase. * Corresponding author. College of Pharmacy, Harbin Medical University (Daqing), Xinyang Road, Daqing, Heilongjiang 163319, PR China. Tel.: þ86 459 8153555; fax: þ86 459 8153556. E-mail address: [email protected] (D. Zhu). 1 These authors contributed equally to this work. 1094-5539/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.pupt.2011.11.005

preconditioning [1e3]. In particular, considerable attention has been given to the role of EETs in regulating vasodilation through activation of Kþ channels, acting as endothelium-derived hyperpolarizing factors [4e7]. However, there are reports that EETs constrict renal arteries and pulmonary arteries (PAs) via cyclooxygenase (COX)-dependent mechanisms [8,9]. The signaling mechanisms through which EETs exert their cellular effects in pulmonary vascular smooth muscle cells (PASMCs) remain unclear. We recently reported that both 8,9-EET and 12-(3-hexylureido) dodec-8-enoic acid (8-HUDE) are involved in the pulmonary vasoconstriction through transient receptor potential channels (TRPCs) [10]. EETs are metabolized by COX and the b-oxidation pathway, but most are hydrolyzed and inactivated by soluble epoxide hydrolase (sEH) enzymes [11]. sEH is highly expressed in a number of organs, such as liver, kidney, heart, vasculature and lung, with the liver and kidney showing the highest levels of enzyme activity. Pharmacological inhibition of sEH has been

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reported to elevate plasma EET concentrations [11]. Inhibition of sEH enhances pulmonary vasoconstriction [12]. Vascular KATP channels are members of the inward rectifier Kþ channel family. Recent studies indicate that KATP channels play a key role in EETs induced modulation of mesenteric vascular tone [13,14]. These channels consist of 4 pore-forming Kir6.x subunits and 4 sulfonylurea receptor (SUR) subunits. Kir6.1/SUR2B is the major isoform in vascular smooth muscles [15e17]. As the major vascular isoform, the Kir6.1/SUR2B channel plays an important role in systemic vascular tone regulation [18,19]. However, there is little information regarding the effect of sEH inhibition on KATP channels in PASMCs. Having reported that the sEH inhibitor 8-HUDE increases PAs tension (10) we asked the question what effects this sEHi would have on KATP channel activation and expression in PASMCs. In present study, we show that 8-HUDE appears to exhibit proconstrictive effects on PAs through inhibited KATP channel via both mechanisms. Namely, 8-HUDE acutely depresses KATP channel activity and subacutely decreases expression of these channels. 2. Material and method 2.1. Materials 8-HUDE and 8,9-EET were synthesized in the laboratory of Dr. J.R. Falck (University of Texas Southwestern); b-Actin, Kir6.1 and SUR2B antibody were purchased from Santa Cruz Biotechnology Inc. (California, USA). Pinacidil and glibenclamide were obtained from SigmaeAldrich (St Louis, MO, USA). Enhanced chemiluminesence (ECL) reagents were provided from Amersham International (Amersham, UK). RT-PCR kit was purchased from Invitrogen (Carlsbad, California, USA). All other chemicals were obtained from common commercial sources. 2.2. Culture preparation and experimental protocol Adult female/male Wistar rats with a mean weight of 200g were from the Experimental Animal Center of Harbin Medical University (Grade II), China. The animals were conditioned at a controlled ambient temperature of 22  2  C with 50  10% relative humidity and at a 12 h lightedark cycle (lights on at 8: 00 AM). Standard rat chow and water ad libitum were provided to all rats. All protocols were approved by the Institutional Animal Care and Use Committee at Harbin Medical University. Primary culture of PASMCs was prepared as we have published [20]. The cells were cultured with Dulbecco’s modified eagle’s medium (DMEM) which contained 20% fetal bovine serum (FBS), 1% penicillin and streptomycin, and in a water jacketed incubator chamber at 37  C under 5% CO2. The purity of primary cultures was confirmed by specific monoclonal antibodies raised against smooth muscle a-actin (Boehringer Mannheim, Germany). Second- through fourth -passage cells were used for further experimentation. Rat Kir6.1 (GenBank accession no. D42145) and mouse SUR2B (GenBank accession no. D86038) were cloned in a eukaryotic expression vector, pcNDA3.1, and used for mammalian cell expression. Human embryonic kidney cells (HEK-293, CRL-1573, batch no. 2187595; American Type Culture Collection, Manassas, VA) were chosen to express the KATP channels. HEK-293 cells were cultured as monolayers in the DMEM-F12 medium with 10% fetal bovine serum and penicillin/streptomycin.

connective tissue removed [21]. After tungsten wires were inserted into the freshly prepared isolated PA rings, the rings were mounted in the tension-detecting device, and submerged in Krebs solution (in mmol/L: NaCl 116, KCl 4.2, CaCl2 2.5, NaH2PO4 1.6, MgSO4 1.2, NaHCO3 22, and D-glucose 11) bubbled with 95% oxygen and 5% CO2. PA rings were equilibrated with 0.3 g passive tension for 1e2 h 8-HUDE was added to the bath at concentrations ranging from 108 to 105 mol/L at 10 min intervals. In some experiments as indicated, rings were pretreated for 30 min with 10 mmol/L glibenclamide before addition of 8-HUDE. All rings were contracted with 1.0 mmol/L phenylephrine (PE) to ensure tissue vitality at the end of the experiment. Rings which did not exhibit a doubling of tension to PE were excluded from analysis. Data acquisition was facilitated by CODAS software (Shanghai Alcott Biotech Co, LTD), which allows waveform playback and analysis. 2.4. Electrophysiology Patch clamp experiments were performed at room temperature as described previously [22]. In brief, fire-polished patch pipettes with resistance of 4e6 MU were made with 1.2 mm borosilicate glass capillaries. Whole cell recordings were performed in the single-cell voltage-clamp mode. Current records were low-pass filtered (2 kHz, Bessel 4-pole filter, 3 dB), digitized (20 kHz, 16bit resolution), and stored on a computer hard drive for later analysis using the Clampfit 9 software (Axon Instruments). The bath solution contained (in mmol/L): 10 KCl, 135 potassium gluconate, 5 EGTA, 5 glucose, and 10 HEPES (pH ¼ 7.4). The pipette solution contained (in mmol/L): 10 KCl, 133 potassium gluconate, 5 EGTA, 5 glucose, 1 K2ATP, 0.5 NaADP, and 10 HEPES (pH ¼ 7.4), in which the free Mg2þ concentration was adjusted to 1 mmol/L using MgCl2. HEK cells were co-transfected with Kir6.1 and SUR2B. Inward currents were studied 2 days after transfection using symmetric concentrations of Kþ (145 mmol/L) applied to the bath and pipette solutions. HEK cells show small currents at baseline (BL) under these conditions. In experiments as indicated, pinacidil was added to activate KATP channels to test the capacity of 8-HUDE to effect open channel block. 2.5. Reverse transcriptase-polymerase chain reaction (RT-PCR) Total RNA was extracted by using Trizol reagent from the cultured PASMCs according to the manufacturer’s instructions and detected by ultraviolet spectrophotometry. Extracted total RNAs were reverse-transcribed with the Superscript first-strand cDNA synthesis kit (Invitrogen). The cDNA samples were amplified in a DNA thermal cycler (Thermo). Gene-specific primers were designed from their own coding regions as follows: Kir6.1 (GenBank accession No. AB043636.1: forward:50 -AGGGAGAATGATGACTGGG-30 , reverse: 50 TCCTGCTGGTGAATAGGC-30 , fragment size: 309bp). SUR2B (GenBank accession No. AB-051213: forward: 50 - GATGAGATTGGCGAGGAC-30 , reverse: 50 -AGGATGGCAAGGAGGAGA-30 , fragment size: 320 bp). bActin (GenBank accession No. NM_031144.2: forward: 50 ACTATCGGCAATGAGCG-30 , reverse: 50 -GAGCCAGGGCAGTAATCT-30 , fragment size: 220 bp). Reaction products were separated by electrophoresis on 1% agarose gel stained with ethidium bromide. Images were obtained and band intensities analyzed using gel imaging analysis system (Alpha Innotech, San Leandro, CA). 2.6. Protein preparation/western blotting

2.3. Tension studies of pulmonary arterial (PA) rings PAs were carefully dissected from adult rats, then cut into small pieces about 1 mm diameter and 2e3 mm in length, with

PASMCs in 6-well culture clusters were growth-arrested for 24 h, and then treated with vehicle, 8-HUDE (10 mmol/L) or 8,9-EET (10 mmol/L) in DMEM which contained 5% FBS. Vehicle, native 8,9-

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EET or 8-HUDE were added every 6 h. After 24 or 48 h, cells were washed with cold PBS three times, then lysed with a lysis buffer (Tris 50 mmol/L, pH 7.4, NaCl 150 mmol/L, TritonX-100 1%, EDTA 1 mmol/L, and PMSF 2 mmol/L) on ice. The lysates were sonicated for 1min and then centrifuged at 15,000 rpm for 15 min at 4  C. Protein concentrations were determined by the Bradford assay using bovine serum albumin (BSA) as standard. Samples containing 20 mg total protein were heated with SDS-PAGE sample buffer for 5 min at 95  C and separated by 8% or 10% SDS-PAGE gels. After separation, proteins were transferred to nitrocellulose membrane. After 1 h incubation at 22e24  C in a blocking buffer (Tris 20 mmol/ L, pH 7.6, NaCl 150 mmol/L, and Tween 20 0.1%) containing 5% nonfat dry milk powder, the membranes were incubated overnight at 4  C in TBS-T containing BSA and appropriate antibody, such as anti-Kir6.1 (1:200 dilution) and anti-SUR2B (1:200 dilution), antib-actin (1:5000 dilution). Imaging and semi-quantitation of bands of interest were analyzed according to our previously published methods [23]. 2.7. Statistical analysis The experimental data are expressed as means  SEM. Statistical analysis was performed with one-way ANOVA followed by Dunnett’s test where appropriate. P < 0.05 was considered statistically significant. 3. Results 3.1. Chemical structure of 8-HUDE The chemical structures of 8-HUDE and 8,9-EET appear in Fig. 1. The compound of 8-HUDE has sEH inhibitor activity [24] as measured using recombinant human EH. 8-HUDE also inhibits the expression of sEH in PASMCs [10]. 3.2. KATP channels underlie 8-HUDE induced PA vasoconstriction As our previous report, we found that the tension of PA rings was enhanced by 8-HUDE [10]. At a concentration of 105 mol/L 8HUDE increased PA tension to w144% baseline. Pretreatment of PA rings with the inward rectifier Kþ channels inhibitor glibenclamide, effectively blocked 8-HUDE-evoked increases in tension (Fig. 2).

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Fig. 1. The structure of compounds 8-HUDE and the native isoform 8,9-EET.

low as 10 mmol/L 100 mmol/L 8-HUDE decreased pinacidilstimulated currents to w25% that of vehicle control (Fig. 3D). 3.4. Time-dependent changes in Kir6.1 and SUR2B expression in PASMCs treated with 8-HUDE To determine the effect of 8-HUDE on the expression of KATP channels, we performed RT-PCR and Western blot analyses on total RNA and protein extracted from PASMCs at 6, 12, 24 and 48 h after the addition of vehicle or compound. Our results show that mRNA levels of Kir6.1 and SUR2B decreased after 24 h of exposure to 8HUDE (Fig. 4A and C n ¼ 3 separated experiments in three groups). Immunospecific protein levels of Kir6.1 and SUR2B were also reduced after 24 h exposure to 8-HUDE (Fig. 4B, D n ¼ 3 separated experiments in three groups). PASMCs treated with vehicle showed no change in either mRNA or protein levels of Kir6.1 and SUR2B (Fig. 5A,B,C,D n ¼ 3 separated experiments in three groups). 3.5. Like 8-HUDE, 8,8-EETs modulates Kir6.1 and SUR2B mRNA and protein expression Next we compared the effects of 8-HUDE to that of native 8,9EETs on Kir6.1 and SUR2B expression. Like 8-HUDE, 8,9-EET decreased Kir6.1 and SUR2B at the mRNA level (Fig. 6A, C) and protein level (Fig. 6B, D). 4. Discussion In our previous work, we showed that the tension of PA rings was enhanced by both native 8,9-EET and 8-HUDE [10]. Our results from the present study indicate that the PASMC KATP channel may be one of the important targets of 8-HUDE. After blocking KATP

3.3. Inhibition of Kir6.1/SUR2B channels by 8-HUDE in HEK-293 cells Kir6.1/SUR2B channels transiently expressed in the HEK-293 cells were identified by a GFP tag. Whole cell voltage-clamp studies were performed on GFP-positive cells. Small amplitude currents typically reached steady state within a few minutes (Fig. 3A). These currents were activated by 10 mmol/L pinacidil (KATP channel opener) and were densely inhibited by 10 mmol/L glibenclamide, consistent with KATP currents. Addition of 100 mmol/L 8HUDE to the bath blocked pinacidil activated currents. (Fig. 3B). The effect of 8-HUDE was quantified as the fraction of current inhibited after maximum channel activation by 10 mmol/L pinacidil. Inhibition of the Kir6.1/SUR2B currents was seen with 10 mmol/L, and the maximum inhibition was reached with 100 mmol/L 8HUDE. The concentration-current relationship was described using the Hill equation with an IC50 40 uM, where the y axis is normalized Kir6.1/SUR2B currents, and the x axis is 8-HUDE concentration (Fig. 3C). Inhibition of the Kir6.1/SUR2B channel by 8-HUDE showed clear concentration-dependence. Decreases in the current amplitude were observed with 8-HUDE concentrations as

Fig. 2. Effects of glibenclamide (Glib), inhibitors of ATP-sensitive Kþ (KATP) channels on the tension of PAs rings. 8-HUDE (10 mmol/L) increases the tension of PA rings, and pretreatment with 10 mmol/L GLIB effectively blocks this increase in tension (n ¼ 6 each group; *p < 0.05 Glib pretreatment vs. vehicle control pretreatment, each value represents the mean  SEM).

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Fig. 3. Inhibition of Kir6.1/SUR2B channels by 8-HUDE in HEK-293 cells. Whole cell currents were recorded from a cell transfected with Kir6.1/SUR2B. The bath solutions contained 145 mmol/L Kþ so that the reversal potential of Kþ currents is close to 0 mV. (A) Small inward currents were seen upon the formation of the whole cell configuration. The currents were increased by 10 mmol/L pinacidil (KATP channel opener; see the line in the figure indicating when pinacidil was added). The maximal activation was reached in 2min, whereas 10 mmol/L glibenclamide (Glib, KATP channel inhibitor) reduced currents to a level even below the baseline. (B) The currents activated by 10 mmol/L pinacidil were strongly inhibited with exposure to 100 mmol/L 8-HUDE (C) The concentration-response relationship can be described by using the Hill equation with IC50 40 uM. (D) The inhibition of the Kir6.1/ SUR2B channel by 8-HUDE showed clear concentration-dependence. Evident decrease in the current amplitude was observed with 8-HUDE concentrations as low as 10 mmol/L (n ¼ 9, *p < 0.005, **p < 0.001). “Glib” stands for glibenclamide.

channels with 10 mmol/L glibenclamide, the capacity of 8-HUDE to increase PAs tension was effectively abolished. 8-HUDE strongly, though incompletely inhibits currents through Kir6.1/SUR2B channels expressed in the HEK cell line. In addition to acute effects on KATP currents, subacute exposure of PASMCs to 8-HUDE diminished expression of kir6.1 and SUR2B.

EETs, cytochrome P450 epoxygenase metabolites of arachidonic acid, have potent vasoactive effects in a number of vascular beds and are considered biologically important regulators of vascular tone [25]. In most vascular beds studied, EETs have vasodilator actions [1,25]. However, EETs have been reported to contract rat kidney [8,26] and PAs [9]. Our results show that 8-HUDE constricts

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Fig. 4. (A, B, C, D) Time course of the expression of Kir6.1 and SUR2B mRNA and protein in cultured rat PASMCs exposed to 8-HUDE. Kir6.1 (A, B) and SUR2B (C, D) mRNA and protein expression were analyzed separately in PASMCs cultured in the presence of 8-HUDE or vehicle for different times as indicated. Bar graphs show means  SEM data for Kir6.1 (A, B) and SUR2B (C, D) expression normalized to beactin mRNA and protein. All values are denoted as mean  SEM from at least three separate experiments (*p < 0. 05).

rat PAs rings and inhibits KATP current in dose-dependent manner. Our data cannot determine whether these effects are attributable to direct actions of 8-HUDE on ion channels or through enhanced local concentrations of endogenous EETs due to sEH properties of this agent. As a vasoconstrictor, 8-HUDE appears to modulate contractions of vascular smooth muscle cells (VAMCs) through effects on multiple ion channels. Our previous work showed that the compound evoked 1) Ca2þ release from intracellular Ca2þ stores via IP3 receptor and ryanodine receptors and 2) calcium influx via

SOCCs, specifically TRPC1 and TRPC6 in isolated PASMCs. In addition, 8-HUDE decreased expression of sEH in PASMCs, an effect which would be anticipated to result in sustained increases in tension in PAs over hours to days [10]. Our present studies support an action of 8-HUDE on KATP as well as TRP channels. As an important player in vascular tone regulation, the KATP channels are subject to multiple-levels of controls. KATP channels regulate resting membrane potentials, control voltage-gate Ca2þ channels, cytosolic Ca2þ levels, and the excitation-contraction coupling in smooth muscles. At the post-translational level, KATP channels are targeted

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Fig. 5. (A, B, C, D) Time course of the expression of Kir6.1 and SUR2B mRNA and protein in cultured rat PASMCs exposed to vehicle. Kir6.1 (A, B) and SUR2B (C, D) mRNA and protein expression were analyzed separately in PASMCs cultured in the presence of vehicle for different times as indicated. No significant change in the expression was found within 6, 12, 24, 48 h of exposure to vehicle. All values are denoted as mean  SEM from at least three separate experiments (*p < 0. 05).

by numerous hormones and neurotransmitters which effect PKA and PKC phosphorylations [27]. There are numerous examples of a single eicosanoid having distinct effects on several channel types in the VAMCs. For example, 20-HETE has independent actions to increase the open state probability of L-type calcium channels and inhibit calcium activated K channels in cerebral VAMCs [28]. The patch clamp data in this report demonstrate that the vascular isoform of KATP channels Kir6.1/SUR2B is indeed targeted by 8-HUDE. The effect of 8-HUDE is not limited to the heterologous expression system. We found that the VAMCs -endogenous KATP current is inhibited by 8-HUDE to roughly the same degree as the Kir6.1/SUR2B channels expressed in HEK-293 cells. If the effect of a KATP channel blocker depends on the channel open state, it may exert limited effects when the channels are closed. When KATP channels in PASMCs are opened by pinacidil, we observed that pinacidil activated currents were strongly, though incompletely inhibited by 8-HUDE (Fig. 3B). Although open channel block by 8-HUDE suggests a direct effect on KATP channels, there may well be indirect effects that may contribute to regulation of channel activity in intact PASMCs, including actions of enhanced local concentrations of endogenous EETs. Indeed because 8-HUDE inhibition of pinacidil activated currents was less complete than

that of glibenclamide, effects of 8-HUDE on other channel types is a reasonable speculation. Studies of the effects of 8-HUDE on KATP, L-type calcium, and TRP channels in excised membrane patches of PASMCs represent reasonable next steps in exploring direct and indirect effects in distinct ion channel types. Another finding from the present study is that 8-HUDE inhibits Kir6.1/SUR2B expression in PASMCs. Previous studies using RT-PCR and Northern blot have demonstrated that Kir6.1 and SUR2B form KATP channels in blood vessels [29,30]. Yamada et al. [31] and Satoh et al. [32] demonstrated that recombinant KATP consisting of Kir6.1 and SUR2B proteins share biophysical and pharmacological properties with potassium selective, nucleotide diphosphate activated (KNDP) channels [33e36]. KATP currents have been identified in a variety of vascular myocytes, and Kþ channel-opening drugs induce a voltage-independent Kþ current that is suppressed by glibenclamide or reductions in intracellular MgATP [37]. ATP-sensitive Kþ channels (KATP), acting in concert with at least 3 other Kþ channels, including delayed rectifier, large-conductance Ca2þ-activated, and inward rectifier Kþ channels, play an important role in the control of membrane potential and tone in VAMCs [38]. Several vasoconstrictors affect Kþ channel activity in vascular myocytes. For example, voltage-clamp studies provide evidence for the contribution of

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Fig. 6. Effect of 8-HUDE or native 8,9-EET on KATP mRNA and protein expression. A and C: PCR amplified products were displayed in agarose gels stained with ethidium bromide for Kir6.1 and SUR2B and b-actin. Both native 8,9-EET and 8-HUDE decreased expression of Kir6.1 and SUR2B mRNA in PASMCs (n ¼ 6 separated experiments in three groups, *p < 0.05). B and D: Kir6.1 and SUR2B protein expression in PASMCs treated for 48 h with 8-HUDE or 8,9-EET. All values are denoted as mean  SEM from three or more separate experiments (*p < 0.05 compared with control group). Both 8,9-EET and 8-HUDE decreased expression of Kir6.1 and SUR2B protein (B,D, n ¼ 6 separated experiments in three groups, *p < 0.05).

delayed rectifier, Ca2þ-activated and KATP currents to membrane potential in these cells. Since 8-HUDE inhibits the Kir6.1/SUR2B KATP channels our data suggest that inhibition of the Kir6.1/SUR2B channel could play a role in 8-HUDE induced vasoconstriction. In conclusion, our results indicate that 8-HUDE-evoked increases in the contractility of PASMCs are attributable in part to the inhibited activity of KATP channels. The acute effects of 8-HUDE

appear to be amplified by decreased expression of Kir6.1 and SUR2B under conditions of sustained exposure. Therapeutic applications of 8-HUDE or other eicosatrienoic analogs depend upon identification of its target molecules and critical intracellular signal pathways. In this regard, our studies constitute a significant step toward the understanding of regulation by 8-HUDE of vascular ion channels.

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