Molecular analysis of ATP-sensitive K+ channel subunits expressed in mouse portal vein

VPH-06224; No of Pages 11 Vascular Pharmacology xxx (2015) xxx–xxx

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Molecular analysis of ATP-sensitive K+ channel subunits expressed in mouse portal vein Tadashi Yamamoto a,b, Kohei Takahara a, Tetsuichiro Inai c, Koichi Node b, Noriyoshi Teramoto a,d,⁎ a

Department of Pharmacology Faculty of Medicine, Saga University, Saga 849-8501, Japan Department of Cardiovascular Medicine, Faculty of Medicine, Saga University, Saga 849-8501, Japan Department of Morphological Biology, Division of Biomedical Sciences, Fukuoka Dental College, Fukuoka 814-0193, Japan d Laboratory of Biomedical Engineering, Graduate School of Biomedical Engineering, Tohoku University, Sendai 980-8575, Japan b c

a r t i c l e

i n f o

Article history: Received 24 March 2015 Received in revised form 16 June 2015 Accepted 29 June 2015 Available online xxxx Keywords: ATP-sensitive K+ channels KIR6.1 SUR2B Vascular smooth muscle cells

a b s t r a c t Background: Several combinations of inwardly rectifying K+ channel 6.x family pore-forming (KIR6.x) subunits associated with sulphonylurea receptor (SUR.x) subunits have been detected among ATP-sensitive K+ (KATP) channels. It remains to be established which of these is expressed in native vascular smooth muscle. Methods: Pharmacological and electrophysiological properties of KATP channels in mouse portal vein were investigated using tension measurements and patch-clamp techniques. Molecular biological analyses were also performed to investigate the structural properties of these channels. Results: Spontaneous contractions in mouse portal vein were reversibly reduced by pinacidil and MCC-134, and the pinacidil-induced relaxation was antagonized by glibenclamide and U-37883A. In cell-attached mode, pinacidil activated glibenclamide-sensitive K+ channels with a conductance (35 pS) similar to that of KIR6.1. RT-PCR analysis revealed the expression of KIR6.1, KIR6.2 and SUR2B transcripts. Using real-time PCR methods, the quantitative expression of KIR6.1 was much greater than that of KIR6.2. Immunohistochemical studies indicated the presence of KIR6.1 and SUR2B proteins in the smooth muscle layers of mouse portal vein and in single smooth muscle cells dispersed from mouse portal vein. Conclusions: The results indicate that native KATP channels in mouse portal vein are likely to be composed of a heterocomplex of KIR6.1 and SUR2B subunits. © 2015 Elsevier Inc. All rights reserved.

1. Introduction ATP-sensitive K+ channels (KATP channels) are broadly expressed in the vasculature and play critical roles in the regulation of blood vessel tone and blood pressure [24]. Indeed, it has been reported that modulation of vascular smooth muscle-type KATP channels allows the vasocontractility of smooth muscle to be very finely regulated, suggesting that vascular smooth muscle-type KATP channels are involved in the fine tuning of vascular tone [24]. Vascular smooth muscle-type KATP channels couple intracellular metabolic events (ATP/ADP ratio, pH, etc.) to plasma membrane electrical activity and respond to several endogenous vasodilators (adenosine, VIP, CGRP, etc.) as well as vasoconstrictors (noradrenaline, angiotensin II, histamine, etc.) [5]. It is well known that vascular smooth muscle-type KATP channel activity can also be regulated by synthetic compounds such as anti-diabetic agents, KATP channel openers, and so on [19]. Molecular studies have revealed that KATP channels are tetrameric ion channel complexes assembled from two different protein subunits, ⁎ Corresponding author at: Department of Pharmacology Faculty of Medicine, Saga University, Saga 849-8501, Japan. E-mail address: [email protected] (N. Teramoto).

namely an inwardly rectifying K+ channel 6.x family pore-forming subunit (KIR6.x), and a modulatory SUR.x subunit of the ATP-binding cassette (ABC) protein superfamily [10]. In general, experiments using recombinant expression of KATP channels have provided good evidence that SUR1, associated with KIR6.2 (i.e. KIR6.2/SUR1 channels), represents the predominant isoform present in pancreatic ß-cells [1,19]. It has been demonstrated that KIR6.2/SUR2A channels and KIR6.2/SUR2B channels are present in cardiac myocytes and smooth muscle cells, respectively [5,11]. A wide variety of KATP channel openers (including cromakalim and aprikalim) induce a significant glibenclamide-sensitive relaxation in both rat [9] and human [18] portal vein, suggesting that KATP channels are present and may be functionally activated in portal vein. However, these experiments were performed in the presence of high concentrations of agonists (noradrenaline, antazoline, etc.) in order to enhance the basal vascular tone, which may not only interfere with the roles of KATP channel openers in relaxing intact vascular preparations but also modify several intracellular signalling pathways. Thus, under such extreme experimental conditions, it is somewhat difficult to investigate the effects of KATP channel openers on vascular tone in isolation and to compare their potencies. Furthermore, although several types of KATP channels have been identified in native freshly dispersed smooth muscle cells using single-channel patch-clamp recordings, the molecular

http://dx.doi.org/10.1016/j.vph.2015.06.018 1537-1891/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: T. Yamamoto, et al., Molecular analysis of ATP-sensitive K+ channel subunits expressed in mouse portal vein, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.06.018

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T. Yamamoto et al. / Vascular Pharmacology xxx (2015) xxx–xxx

subunits of the KATP channels have solely been identified by the use of reverse transcription (RT)-PCR analytical methods [24]. Recently, it has been reported that it is still necessary to elucidate unequivocally the full details of the molecular subunit composition of KATP channels using investigational techniques such as their pharmacological properties, the sizes of the unitary conductances measured in symmetrical 140 mM K+ conditions, transcript, and protein expression analysis [12]. In the present experiments, the effects of KATP channel openers on spontaneous myogenic activity in mouse portal vein were investigated in the absence of agonists. Moreover, we obtained electrophysiological, molecular and biochemical evidence for the subunit composition of KATP channels in mouse portal vein. The electrophysiological and pharmacological properties of unitary KATP currents were investigated using single-channel recordings in order to measure the single-channel conductance of KATP channels in freshly dispersed cells from vascular smooth muscle. Furthermore, RT-PCR, real-time PCR and immunohistochemical analyses were carried out to determine the transcript and protein expressions of KATP channel subunits.

2. Materials and methods 2.1. Animal studies and animal rights All experiments were approved by the Saga University Animal Care and Use Committee (Saga, Japan; 22-034-4). Male BALB/c mice (8–10 weeks old; Charles River Laboratories Japan, INC., Yokohama, Japan) were killed by cervical dislocation. Portal vein smooth muscle was isolated and immediately placed in modified physiological salt solution (PSS, vide infra). The endothelial layer was mechanically removed from mouse portal vein.

2.2. Contraction studies For tension measurements, a modified Krebs solution was used (mM): Na+ 137, K+ 5.9, Mg2 + 1.2, Ca2 + 2.5, Cl− 133.7, HCO− 3 15.4, H2PO− 4 1.2 and glucose 11.5, which was bubbled with 97% O2 and 3% CO2 to pH 7.40. An initial tension equivalent to 0.3 g weight was applied to tissues which were then allowed to equilibrate for 3 h at 37 °C in a multi-chamber organ bath system (PL3508B5/C-V, Panlab SLU, Barcelona, Spain). Isometric contraction of the longitudinal muscle layer was recorded and digitized (100 Hz) using LabChart 7 software (ADInstruments, Bella Vista, New South Wales, Australia) and a personal computer (Inspiron 15, Dell Japan Inc., Kawasaki, Kanagawa, Japan). Tension is expressed as mN mg− 1 of tissue and the area under the trace was measured (in mN mg− 1 per min) and compared to the control.

2.3. Cell dispersion Mouse portal vein myocytes were freshly isolated by the gentle tapping method [25,27]. Briefly, thin strips of smooth muscle (5–6 mm × 2–3 mm) were placed in a salt solution (mM): Na+ 140, K+ 5.0, Mg2+ 0.5, Ca2+ 0.5, Cl− 147, glucose 10, HEPES 10/Tris, titrated to pH 7.35–7.40, containing 0.2–0.3 mg mL−1 papain (Sigma-Aldrich Japan K.K., Tokyo, Japan) at 4 °C for approximately 20 min. The digested strips were washed in a Ca2+-free salt solution, and pre-incubated in this solution at 37 °C for 4–5 min. The strips were then incubated in Ca2+-free salt solution containing 0.3–0.4 mg mL−1 Type IA collagenase (Sigma-Aldrich Japan K.K., Tokyo, Japan) at 37 °C for 10–15 min. Relaxed spindle-shaped cells were isolated and used for patch-clamp, PCR and immunohistochemical analyses within 3–4 h following isolation (after storage at 4 °C).

2.4. Electrophysiological recordings Single-channel recordings were carried out at room temperature (21–23 °C) in symmetrical 140 mM K+ conditions, as previously described [27]. Glass pipettes (resistances 3–5 MΩ) were fabricated using a micropipette puller (P-97, Sutter Instrument, Novato, CA, USA). Junction potentials between the bath and pipette solutions were measured with a 3 M KCl reference electrode and were b 1 mV; therefore, a correction for these small potentials was not made. The capacitance noise was kept to a minimum by minimizing the level of the test solution within the recording electrode. 2.5. Data analysis The data recording system used was similar to that previously described [27]. For single-channel recordings, the stored data were low-pass filtered at 2 kHz (−3 dB) and sampled at a digitisation interval of 100 μs using ‘PAT’ software (kindly provided by Dr J. Dempster, University of Strathclyde, UK); events briefer than 100 μs were not included in the analysis. The continuous traces displayed in the figures were obtained from records filtered at 1 kHz for presentation purposes (current–voltage relationships, digital sampling interval, 100 μs; long duration traces, digital sampling interval, 5 ms). Values for the channel open state probability (Popen) were measured at several holding potentials during 1 min recordings. Channel open probability was determined according to the equation: 0 1 N X NPo ¼ @ t j  J A=T j¼1

where tj is the time spent at each current level corresponding to j = 0, 1, 2, …N, T is the duration of the recording, and N is the number of channels detected in the patch. Data points were fitted using a leastsquares method. 2.6. Solutions and drugs Modified PSS contained (mM): Na+ 140, K+ 5.0, Mg2+ 1.2, Ca2+ 2.0, Cl− 151.4, glucose 10, HEPES 10, titrated to pH 7.35–7.40 with Tris base. For single-channel recordings, symmetrical 140 mM K+ conditions were generated; the pipette and bath solutions contained (in mM): K+ 140, Ca2+ 1.0, Mg2+ 5.5, Cl− 153, glucose 5.5, HEPES 10 (pH 7.35–7.40 with Tris), and K+ 140, Mg2+ 4.6, Cl− 149.2, EGTA 1.0, glucose 10, HEPES 10 (pH 7.35–7.40 with Tris), respectively. Cells were allowed to settle in the small experimental chamber (approximately 80 μL in volume) before perfusion with bath solution was initiated. The gravity driven bath solution superfused the experimental chamber at a rate of 2 mL·min−1. Pinacidil, glibenclamide, MCC-134 (N-methyl-1-[4(1H-imidazol-1-yl) benzoyl]-N-methylcyclobutane carbothioamide) and U-37883A (4-morpholinecarboximidine-N-1-adamantyl-N′-cyclohexylhydrochloride) were prepared daily as 100 mM stock solutions in dimethyl sulfoxide (DMSO). The final concentration of DMSO was less than 0.3% and did not affect K+ channel activity [25,27]. U-37883A, a selective KIR6.1 blocker [14,24], was purchased from Biomol Research Labs Inc. (Plymouth Meeting, PA, USA). MCC-134, a SUR modulator [20], was kindly provided by Mitsubishi Tanabe Pharma Co. (Osaka, Japan). All other chemicals were purchased from SigmaAldrich (Sigma-Aldrich Japan K.K., Tokyo, Japan). 2.7. RNA preparation, RT-PCR and quantitative real-time PCR analyses Total RNA was extracted from mouse ventricle and portal vein using TRIzol reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer's instructions. The RNA was dissolved in nuclease-free water and quantitated using a NanoDrop 1000

Please cite this article as: T. Yamamoto, et al., Molecular analysis of ATP-sensitive K+ channel subunits expressed in mouse portal vein, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.06.018

T. Yamamoto et al. / Vascular Pharmacology xxx (2015) xxx–xxx Table 1 Nucleotide sequences for the custom-designed primers used to detect the KATP channel subunits (KIR6.x and SUR.x) with RT-PCR analysis. Encoding protein name

Gene name

Reference sequence ID

Primer sequence (5′-to 3′)

KIR6.1

Kcnj8

NM_008428

KIR6.2

Kcnj11

NM_010602

SUR1

Abcc8

NM_011510

SUR2A

Abcc9 variant2

NM_021041

SUR2B

Abcc9 variant1

NM_011511

F-TGCTCTTCGCTATCATGT R-GTTTTCTTGACCACCTGGAT F-TCTGCCTTCCTTTTCTCCAT R-TGCATGTGGATGGTGGCGCT F-CCCTCTACCAGCACACCAAT R-CAGTCTGCATGAGGCAGGTA F-ATGAAGCCACTGCTTCCATC R-ATCCGTCAAAGTTGGCAAAG F-ATGAAGCCACTGCTTCCATC R-ATCCGTCAAAGTTGGCAAAG

Spectrophotometer (Thermo Fisher Scientific, Inc.). First-strand cDNA was synthesized from 200 ng or 1 μg of total RNA using a HighCapacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific Inc.). The PCR reaction was performed using 3 μL of cDNA in 50 μL of KOD-Plus-Ver.2 (Toyobo Co., Ltd., Osaka, Japan) containing 0.3 μM of each primer. Generic specific primers for KIR6.1, KIR6.2, SUR1 and SUR2A/B were the same sequences that we have previously described (Table 1) [12]. The cycling condition for KIR6.1 was 94 °C for 2 min, followed by 40 cycles at 94 °C for 15 s, 55 °C for 30 s and 68 °C for 30 s. The cycling condition for KIR6.2 was 94 °C for 2 min, followed by 40 cycles at 94 °C for 15 s, 62 °C for 30 s and 68 °C for 30 s. The cycling conditions for SUR.x (SUR1 and SUR2A/B) were 94 °C for 2 min, followed by 40 cycles at 94 °C for 15 s, 66 °C for 30 s and 68 °C for 30 s. An aliquot of the RT-PCR product (25 μL) was loaded onto 1.5% agarose gels and was run in parallel as a molecular size marker. Gels were stained with ethidium bromide to reveal DNA bands under UV illumination. Subsequently, each cDNA sample of KATP channel subunits (i.e. KIR6.x and SUR.x) was analysed for gene expression by quantitative real-time PCR using SYBR Premix Ex Taq II (Takara Bio Inc., Otsu, Shiga, Japan) and a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Inc.). Amplification data were analysed with StepOne Software Version 2.3 (Thermo Fisher Scientific Inc.) using the relative standard curve method. The conditions of real-time PCR were as follows: an initial denaturation at 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. Oligonucleotide primers were designed using published sequence data from GenBank with the aid of primer design software. The primer sequences used are shown in Table 2. The mRNA expression was normalized to the levels of Gapdh mRNA as the housekeeping gene. 2.8. Immunofluorescence studies Mouse portal vein was fixed in cold 1% (w/v) paraformaldehyde (PFA) in phosphate buffered saline (PBS) (i.e., 1% PFA-PBS) for 1 h, washed in PBS, incubated in solutions of sucrose in PBS (10, 20 and 30% w/v), and stored for 1–2 h at 4 °C. Fixed tissue was embedded in Table 2 Primers used to detect KATP channel subunits (KIR6.x and SUR.x) in quantitative real-time PCR studies. Encoding protein name

Gene name

Reference sequence ID

Primer sequence (5′-to 3′)

KIR6.1

Kcnj8

NM_008428

KIR6.2

Kcnj11

NM_010602

SUR1

Abcc8

NM_011510

SUR2

Abcc9 variant1 variant2 Gapdh

NM_011511 NM_021041

F-AGGTCATTCACGTCTGCGTTT R-GCATTCCTCAGTCATCATTCTCC F-AAGCCCAAGTTTAGCATCTCTCC R-CACCCCACCACTCTACATACCA F-GCTTCTGGTGATCCTCTACGG R-CACTTCCCTTGGCGTCTTG F-TTCCACATCCTCGTCACACC R-CTCACCAATCTCATCGCTCAA

Gapdh

NM_008084

F-TGTGTCCGTCGTGGATCTGA R-TTGCTGTTGAAGTCGCAGGAG

3

optimal cutting temperature compound (Tissues-Tek, Sakura Finetek Japan Co., Ltd., Tokyo, Japan). Tissue in the embedding medium was immediately frozen on dry ice. Frozen sections (5 μm thick) were cut using a cryostat (Microm Cryo-Star HM 560 Cryostat, GMI, Ramsey, MN, USA) and mounted on pre-coated glass slides (MAS-coated glass slide S9441, Matsunami Glass Ind., Ltd., Kishiwada, Osaka, Japan), and allowed to dry in air at room temperature for 1 h. After the sections were washed with PBS, transverse sections of mouse portal vein were permeabilized in 0.2% polyoxyethylene-p-isooctylphenol (Triton X-100) in PBS (i.e., 0.2% Triton X-PBS) for 15 min at room temperature. The sections were then washed with PBS and blocked with 1% (w/v) bovine serum albumin (BSA) in PBS (i.e., 1% BSA–PBS) for 30 min at room temperature. In order to detect the localization of KIR6.1 and KIR6.2 proteins, the sections were next incubated with a primary purified rabbit antiKIR6.1 primary antibody (sc-20808, Santa Cruz Biotechnology; diluted 1:100) and a goat anti-KIR6.2 primary antibody (sc-11228, Santa Cruz Biotechnology; diluted 1:100) in the blocking solution at 4 °C for 40 min in a humidified chamber. Following washing with PBS (three times for 5 min), sections were incubated with Alexa Fluor 594 donkey anti-rabbit IgG and Alexa Fluor 488 donkey anti-goat IgG (Invitrogen, Carlsbad, CA, USA) both diluted to 1:200 in blocking solution for 20 min in the dark at room temperature. In order to detect the histological co-localization of KIR6.1 and SUR2B proteins, the sections of mouse portal vein were incubated with the primary purified rabbit anti-KIR6.1 primary antibody (sc-20808, Santa Cruz Biotechnology; diluted 1:100) and a goat anti-SUR2B primary antibody (sc-5793, Santa Cruz Biotechnology; diluted 1:100) in blocking solution at 4 °C for 40 min in a humidified chamber. Following washing with PBS (3 times for 5 min), sections were incubated with Alexa Fluor 594 donkey anti-rabbit IgG and Alexa Fluor 488 donkey anti-goat IgG (Invitrogen) both diluted to 1:200 in blocking solution for 20 min at room temperature in the dark. Sections were subsequently washed with PBS (3 times for 5 min) and cover-slipped with a mounting reagent containing DAPI (4′,6diamidino-2-phenylindole; Vectashield H-1200, Vector Laboratories Ltd., Peterborough, UK). Fluorescence images of stained portal vein were obtained using a confocal microscope (Zeiss LSM710, Carl Zeiss Microscopy GmbH, Jena, Germany). Non-specific staining of the secondary antibodies was tested by negative controls in which the primary antibodies were omitted (data not shown). Single, dissociated myocytes were plated onto glass slides (Matsunami Glass Ind., Ltd.) and incubated at 37 °C for overnight to allow them to adhere to the slides before being fixed. Portal vein myocytes were fixed in 1% PFA–PBS for 10–15 min at room temperature, and then washed thoroughly in PBS (twice for 2 min). The cells were permeabilised in 0.2% Triton X-PBS for 10–15 min at room temperature. The isolated cells were then washed with PBS (twice for 2 min), and 1% BSA–PBS was applied as a blocking solution for 15 min at room temperature. For KIR6.x staining, dispersed cells were incubated, for approximately 1 h at room temperature, with a rabbit anti-KIR6.1 primary antibody (sc-20808, Santa Cruz Biotechnology; diluted 1:100) and a goat anti-KIR6.2 primary antibody (sc-11228, Santa Cruz Biotechnology; diluted 1:100). Following wash with PBS (three times for 2 min), myocytes were incubated with Alexa Fluor 594 donkey anti-rabbit IgG and Alexa Fluor 488 donkey anti-goat IgG (all 1:100 dilution in blocking solution; Invitrogen) for approximately 30 min at room temperature in the dark. The dispersed smooth muscle cells were then washed with PBS (three times for 2 min) and mounted in Vectashield (Vector Laboratories Ltd.) mounting medium with DAPI. In order to detect the histological co-localization of KIR6.1 and SUR2B proteins, dispersed cells were incubated, for approximately 1 h at room temperature, with rabbit anti-KIR6.1 primary antibody (sc-20808, Santa Cruz Biotechnology; diluted 1:100) and goat anti-SUR2B primary antibody (sc-5793, Santa Cruz Biotechnology; diluted 1:100). Following wash with PBS (three times for 2 min), the myocytes were incubated with Alexa Fluor 594

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did not affect the frequency of occurrence of spontaneous contractions (n = 33). Cumulative application of pinacidil (30–500 nM; 8 min duration) produced a concentration-dependent inhibition of the spontaneous contractions. The integrated area of spontaneous contractions was normalized as one, just before the application of pinacidil (i.e. the control for 2 min duration) and a relative value for the spontaneous contractions was determined for each concentration of pinacidil tested (Fig. 1C, Ki = 130 nM, n = 6). Similarly, when MCC-134 (0.1–10 μM, 8 min duration), a sulphonylurea modulator, was cumulatively applied, MCC-134 produced a concentration-dependent inhibition of spontaneous contractions (Fig. 1B, n = 7). Fig. 1C summarizes the concentrationdependent inhibitory effects of MCC-134 on the relative value of the spontaneous contractions (Fig. 1C, Ki = 1.6 μM, n = 7), where the integrated area of the spontaneous contractions is normalized as one, just before the application of each concentration of MCC-134 (2 min duration). The pinacidil-induced relaxation (500 nM) was reversibly inhibited by glibenclamide (300 nM, n = 5; Fig. 2A, C), an KATP channel inhibitor, and by U-37883A (300 nM, n = 5; Fig. 2B, D), a selective KIR6.1 blocker. Although MCC-134 (10 μM)-induced relaxations were partially inhibited by the additional application of 300 nM glibenclamide (n = 5; Fig. 3A, C) and by the subsequent application of 300 nM U-37883A

donkey anti-rabbit IgG and Alexa Fluor 488 donkey anti-goat IgG (both 1:100 dilution in blocking solution; Invitrogen) for approximately 30 min at room temperature in the dark. The dispersed smooth muscle cells were then washed with PBS (three times for 2 min) and mounted in Vectashield (Vector Laboratories Ltd.) mounting medium with DAPI. Fluorescence images of the stained portal vein myocytes were obtained using a confocal microscope (Zeiss LSM710, Carl Zeiss Microscopy GmbH). 2.9. Statistical analysis Statistical analyses were performed using ANOVA tests (two-factor with replication). Changes were considered significant at P b 0.01 (*). Data are expressed as the mean ± S.D. 3. Results 3.1. Effects of pinacidil on spontaneous contractions of mouse portal vein Muscle strips of mouse portal vein exhibited spontaneous contractions at a range of amplitudes and frequencies (Fig. 1A). Pinacidil (≥ 30 nM) gradually inhibited the amplitude of the contractions but

A 0.03

Pinacidil (µM) 0.3 0.1

0.5

1 mN 10 min

B

MCC-134 (µM) 0.3

0.1

1

3

5

10

1 mN 10 min

C Relative contraction

1.2 1 0.8

Pinacidil MCC-134

0.6 0.4 0.2 0 0.001 0.01 0.1 1 10 100 Concentrations of KATP channel openers (µM)

Fig. 1. Effects of KATP channel openers (pinacidil and MCC-134) on spontaneous contractions of mouse portal vein. (A) The effects of cumulative application of pinacidil (8 min duration). The dashed line indicates the mean resting vascular tone of portal vein. (B) The effects of cumulative application of MCC-134 (8 min duration). The dashed line indicates the mean resting vascular tone of portal vein. (C) Relationships between the relative inhibitory value of the KATP channel opener (pinacidil and MCC-134)-induced relaxation and the concentration of KATP channel opener. The integrated area of the spontaneous contractions (2 min duration) just before the application of a KATP channel opener was normalized as one. The curves were drawn by the application of the following equation, using the least-squares method:   Relative amplitude ¼ 1 = 1 þ ðKi = D ÞnH where Ki, D and nH are the inhibitory dissociation constant, the concentration of each inhibitor and Hill's coefficient respectively. The following values were used for the curve fitting: pinacidil, Ki = 130 nM, nH = 1.4 (open circle); MCC-134, Ki = 1.6 μM, nH = 1.4 (open square). Each symbol indicates the mean with ±S.D. shown by the vertical line. Some of the S.D. bars are smaller than the symbols.

Please cite this article as: T. Yamamoto, et al., Molecular analysis of ATP-sensitive K+ channel subunits expressed in mouse portal vein, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.06.018

T. Yamamoto et al. / Vascular Pharmacology xxx (2015) xxx–xxx

A

5

Glibenclamide 300 nM Pinacidil 500 nM 1 mN 10 min

B U-37883A 300 nM Pinacidil 500 nM

1 mN 10 min * 1.4 1.2 1 0.8 0.6 0.4 0.2 0

D Relative contraction

Relative contraction

C

Control

Pinacidil

Pinacidil + Glibenclamide

1.4 1.2 1 0.8 0.6 0.4 0.2 0

*

Control

Pinacidil

Pinacidil + U-37883A

Fig. 2. Effects of KATP channel inhibitors (glibenclamide and U-37883A) on the 500 nM pinacidil-induced relaxation in mouse portal vein. (A) The effects of 300 nM glibenclamide on the 500 nM pinacidil-induced relaxation in mouse portal vein. The dashed line indicates the mean resting vascular tone of portal vein. (B) The effects of 300 nM U-37883A on the 500 nM pinacidil-induced relaxation in mouse portal vein. The dashed line indicates the mean resting vascular tone of portal vein. (C) Relative inhibitory value of KATP channel inhibitors on the pinacidil-induced relaxation under the indicated conditions. The integrated area of the spontaneous contractions (2 min duration) just before the application of 500 nM pinacidil was normalized as one (control, open column). The solid column indicates the relative value of integrated area of the spontaneous contractions in the presence of 500 nM pinacidil. The grey column represents the relative value of integrated area of the spontaneous contractions in the presence of both 500 nM pinacidil and 300 nM glibenclamide. Each column represents the mean + S.D. shown by the vertical line. (D) Relative inhibitory value of KATP channel inhibitors on the pinacidil-induced relaxation under the indicated conditions. The integrated area of the spontaneous contractions (2 min duration) just before the application of KATP channel inhibitor was normalized as one (control, open column). The solid column indicates the relative value of integrated area of the spontaneous contractions in the presence of 500 nM pinacidil. The grey column represents the relative value of integrated area of the spontaneous contractions in the presence of both 500 nM pinacidil and 300 nM U-37883A. Each column indicates the mean + S.D. shown by the vertical line.

(n = 5; Fig. 3B, D), spontaneous contractions of mouse portal vein did not recover to the control levels.

3.2. KATP channel unitary current in single-channel recordings The cell-attached patch configuration was utilised to determine the conductances of pinacidil-induced channel openings. When myocytes were exposed to 100 μM pinacidil at a holding potential of − 60 mV, an increase of approximately 2.1 pA in the K+ channel-gating current was observed. KATP channel activity of this amplitude was observed in more than 95% of the patches tested. To document fully the current– voltage relationship of the unitary currents, the holding membrane potential was changed from −120 mV to 60 mV, in increments of 10 mV, when pinacidil was present in the bath solution (Fig. 4A; n = 5). The conductance, obtained from the peak amplitude of the unitary K+ channel was 35 ± 1 pS (Fig. 4B, n = 5). The unitary current–voltage relationship demonstrated a significant departure from linearity at positive potentials, and exhibited a weak but significant inward rectification positive to the reversal potential for current flow through the channel pore (i.e. 0 mV, Fig. 4B). When 100 μM pinacidil was present in the bath solution, KATP channels were activated at a holding potential of − 60 mV. Application of 10 μM glibenclamide inhibited the activity of KATP channels and, about 30 s later, caused complete inhibition. Upon removal of glibenclamide, channel reappeared and activity fully recovered to control levels (Fig. 4C). Similarly, U-37883A (100 μM) reversibly suppressed the activity of the pinacidil-induced KATP channels at −60 mV (Fig. 4D).

3.3. Molecular expression of KATP channel subunits in mouse portal vein In order to determine the identity of subunits that could potentially contribute to the formation of KATP channel pores, samples of RNA were isolated from mouse ventricular and portal vein myocytes, and used in RT-PCR experiments with primers specific for KIR6.x subunits. Specific primers were designed for the amplification of both KIR6.1 and KIR6.2 mRNAs, to produce cDNA fragments for KIR6.1 and KIR6.2, respectively (see Table 1). Amplicons were generated from mouse heart RNA samples that were consistent with the products generated using mRNAs encoding KIR6.1 and KIR6.2 (Fig. 5A). Using the same primers, both KIR6.1 and KIR6.2, transcripts were detected in portal vein myocytes. To identify the subtypes of the modulatory subunits in KATP channels, samples of RNA were obtained from mouse ventricular and portal vein myocytes. Specific primers were designed for the amplification of SUR.x (SUR1, SUR2A and SUR2B) subunits, to produce cDNA fragments for the genes of these SUR.x isoforms (see Table 1). Positive amplicons for SUR1, SUR2A and SUR2B were detected in cardiac myocytes, whilst only SUR2B was detected in portal vein myocytes (Fig. 5B).

3.4. Quantitative real-time PCR analysis for KIR6.x subunits and SUR subunits Using mouse cardiac cDNA, melting curve analysis of the real-time PCR program showed single product-specific melting temperature peaks when the amplicons generated from all the primers were analysed. Similar specific band patterns were observed on agarose gels

Please cite this article as: T. Yamamoto, et al., Molecular analysis of ATP-sensitive K+ channel subunits expressed in mouse portal vein, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.06.018

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T. Yamamoto et al. / Vascular Pharmacology xxx (2015) xxx–xxx

A

Glibenclamide 300 nM MCC-134 10 µM

1 mN 10 min

B

U-37883A 300 nM MCC-134 10 µM

1 mN 10 min

D

* 1.4 1.2 1 0.8 0.6 0.4 0.2 0

*

Control

MCC-134

Relative contraction

Relative contraction

C

30 min 10 min MCC-134 + Glibenclamide

* 1.4 1.2 1 0.8 0.6 0.4 0.2 0

*

Control

MCC-134

30 min 10 min MCC-134 + U-37883A

Fig. 3. Effects of KATP channel inhibitors (glibenclamide and U-37883A) on the 10 μM MCC-134-induced relaxation in mouse portal vein. (A) The effects of 300 nM glibenclamide on the 10 μM MCC-134-induced relaxation in mouse portal vein. The dashed line indicates the mean resting vascular tone of portal vein. (B) The effects of 300 nM U-37883A on the 10 μM MCC-134-induced relaxation in mouse portal vein. The dashed line indicates the mean resting vascular tone of portal vein. (C) Relative inhibitory value of KATP channel inhibitors on the MCC-134-induced relaxation under the indicated conditions. The integrated area of the spontaneous contractions (2 min duration) just before the application of 10 μM MCC-134 was normalized as one (control, open column). The solid column indicates the relative value of integrated area of the spontaneous contractions in the presence of 10 μM MCC-134. The grey column represents the relative value of integrated area of the spontaneous contractions in the presence of both 10 μM MCC-134 and 300 nM glibenclamide (after 10 min and 30 min, respectively). Each column represents the mean + S.D. shown by the vertical line. (D) Relative inhibitory value of KATP channel inhibitors on the MCC-134-induced relaxation under the indicated conditions. The integrated area of the spontaneous contractions (2 min duration) just before the application of KATP channel inhibitor was normalized as one (control, open column). The solid column indicates the relative value of integrated area of the spontaneous contractions in the presence of 10 μM MCC-134. The grey column represents the relative value of integrated area of the spontaneous contractions in the presence of both 10 μM MCC-134 and 300 nM U-37883A (after 10 min and 30 min, respectively). Each column indicates the mean + S.D. shown by the vertical line.

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Fig. 4. Relationship between the holding membrane potential and the unitary amplitude of the single-channel current activated by 100 μM pinacidil. (A) The current traces show channel activities recorded from the same patch at the indicated membrane potentials (−120 to 60 mV, in increments of 20 mV). The dashed line indicates the current baseline, when the channel was not open. (B) Current–voltage relationship obtained using a cell-attached patch. The unitary amplitudes of the K+ channel currents were taken from the all-points amplitude histograms for 30 s. The line was fitted by the least-squares method at negative potentials (the channel conductance, 35 pS) and demonstrated the property of inward rectification at positive potentials. Note BKCa channel activity at positive membrane potentials (≥20 mV). (C) When 100 μM pinacidil was present in the bath solution, KATP channel was activated at −60 mV. Application of 10 μM glibenclamide reversibly inhibited the activity of KATP channels (D) U-37883A (100 μM) reversibly suppressed the activity of the pinacidil-induced KATP channels at −60 mV.

Please cite this article as: T. Yamamoto, et al., Molecular analysis of ATP-sensitive K+ channel subunits expressed in mouse portal vein, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.06.018

T. Yamamoto et al. / Vascular Pharmacology xxx (2015) xxx–xxx

B

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Ventricle

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Fig. 5. Molecular identification of the KATP channel subunits by RT-PCR analysis. RT-PCR was performed as described in Section 2, and a ladder was used to indicate the size of the amplified fragments. (A) Specific primers for the KIR6.x gene isoforms (KIR6.1 and KIR6.2) were used, and mRNA was extracted from freshly dissected mouse ventricular myocytes and portal vein myocytes. Amplicons of sizes consistent with those of KIR6.1 (445 bp) and KIR6.2 (299 bp) were evident for the cardiac myocytes and the portal vein myocytes. (B) Specific primers for the SUR.x gene isoforms (SUR1, SUR2A and SUR2B) were used, and mRNA was extracted from freshly dissected mouse ventricular myocytes and portal vein myocytes. Amplicons of sizes consistent with those of SUR1 (169 bp), SUR2A (495 bp) and SUR2B (319 bp) were observed in the ventricular myocytes, but only SUR2B was evident in the portal vein myocytes.

when the PCR reactions were tested after quantitative real-time PCR studies, to verify the target/subunit-specific primers. No PCR bands were detected when template controls and water blank samples were analysed (data not shown). In addition, only one PCR product was detected when the post-PCR reactions were analysed by agarose gel electrophoresis and the products amplified by each primer pair were detected at the expected sequence size. Both KIR6.1 and KIR6.2 transcripts were detected at similar levels in mouse cardiac myocytes (Fig. 6A, n = 5). Based on quantitative real-time PCR studies in mouse portal vein myocytes, KIR6.1 was found to be the predominant mRNA transcript and KIR6.2 was barely detectable (Fig. 6A, n = 5). Furthermore, both SUR1 and SUR2 transcripts were detected in mouse cardiac myocytes (Fig. 6B, n = 5). In mouse portal vein myocytes, SUR2 was found to be the predominant mRNA transcript and SUR1 was close to the detectable limit (Fig. 6B, n = 5). 3.5. Immunohistochemical localisation of KATP channel subunits in mouse portal vein In order to identify and localize molecular markers for the KATP channel subunits KIR6.x and SUR.x, immunohistochemical studies were performed. KIR6.1 immunoreactivity was clearly visible in mouse portal vein sections (Fig. 7A), whilst no specific immunoreactive signal was detected for KIR6.2 (Fig. 7B). Since only the SUR2B amplicon in the portal vein was detected in mRNA level studies, immunohistochemical methods were employed to confirm the presence of an

A

immunoreactive signal for SUR2B. Immunohistochemical studies to detect the likely co-localization of KIR6.1 and SUR2B proteins in mouse portal vein were also carried out on thin transverse sections. Both KIR6.1 and SUR2B immunoreactivities were clearly visible in smooth muscle layers of mouse portal vein (Fig. 8). Note that the autofluorescence originating from elastin was detected in both the tunica intima and the tunica adventitia (Figs. 7 and 8). 3.6. Immunohistochemical localisation of KATP channel subunits in mouse portal vein myocytes In order to identify and localize molecular markers for KATP channel subunits (KIR6.x and SUR.x), immunohistochemical studies were performed using staining methods for single smooth muscle cells. KIR6.1 immunoreactivity was clearly visible in portal vein myocytes (Fig. 9A), whilst no specific immunoreactive signal was seen for KIR6.2 (Fig. 9A). Immunohistochemical methods were also employed to confirm the presence of an immunoreactive signal for both KIR6.1 and SUR2B. Both KIR6.1 and SUR2B immunoreactivities were clearly visible in single smooth muscle cells dispersed from mouse portal vein (Fig. 9B). 4. Discussion We have demonstrated that the main molecular composition of KATP channels in the vascular smooth muscle of mouse portal vein is likely to be KIR6.1/SUR2B channels.

B Ventricular myocytes

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Fig. 6. Relative expression levels of the genes encoding KATP channel subunits (KIR6.x and SUR.x) in cardiac myocytes (open column) and portal vein myocytes (solid column). Quantitative real-time PCR was performed to analyse the expression of KIR6.x (A) and SUR.x (B). All values were normalized to Gapdh expression with the +S.D. shown by the vertical lines.

Please cite this article as: T. Yamamoto, et al., Molecular analysis of ATP-sensitive K+ channel subunits expressed in mouse portal vein, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.06.018

8

T. Yamamoto et al. / Vascular Pharmacology xxx (2015) xxx–xxx

A

Anti-KIR6.1

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Fig. 7. Images of transverse sections of portal vein showing fluorescent labelling of immunoreactivity for KIR6.1 and KIR6.2. (A) Immunoreactivity detected with anti-KIR6.1 antibody. (B) Immunoreactivity of the anti-KIR6.2 antibody. (C) DAPI nucleic acid stain. (D) An overlay of panels A, B and C. (E) Structure revealed with Nomarski differential interference contrast imaging. Bar (black line) in (E) represents 20 μm.

4.1. KATP channel opener-induced vascular relaxation of mouse portal vein spontaneous contractions To date, the comparative potencies of KATP channel openers have been examined by studying their relaxant effects on either excess [K+]o (20 mM KCl, 80 mM KCl, etc.) or agonist-induced contractions (adrenaline, noradrenaline, phenylephrine) in intact vascular smooth

muscle. However, it is well known that excess [K+]o activates voltagedependent mechanisms (voltage-dependent Ca2 + channels, voltagegated Na+ channels), Ca2 +-activated mechanisms following Ca2 + entry into myocytes (Ca2 +-induced Ca2+ release, Ca2 +-activated K+ channels, Ca2+-activated Cl− channels) and that agonists also activate stimulatory mechanisms (such as TRPC channels, muscarinic receptormodulated pathways and IP3-induced Ca2+ release mechanisms) [15].

A

Anti-KIR6.1

B

Anti-SUR2B

D

Merged image

E

Transmission image

C

DAPI

Fig. 8. Images of transverse sections of portal vein showing fluorescent labelling of immunoreactivity for KIR6.1 and SUR2B. (A) Immunoreactivity detected with anti-KIR6.1 antibody. (B) Immunoreactivity of the anti-SUR2B antibody. (C) DAPI nucleic acid stain. (D) An overlay of panels A, B and C. (E) Structure revealed with Nomarski differential interference contrast imaging. Bar (black line) in (E) represents 20 μm.

Please cite this article as: T. Yamamoto, et al., Molecular analysis of ATP-sensitive K+ channel subunits expressed in mouse portal vein, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.06.018

T. Yamamoto et al. / Vascular Pharmacology xxx (2015) xxx–xxx

A

9

B

Anti-KIR6.1

Anti-KIR6.2

Anti-KIR6.1

Anti-SUR2B

DAPI

Merged image

DAPI

Merged image

Transmission image

Transmission image

Fig. 9. Immunohistochemical localization of KATP channel subunits in myocytes isolated from the mouse portal vein. (A) Immunohistochemical localization of KIR6.1 and KIR6.2 subunits. Immunoreactivity of the anti-KIR6.1 antibody. Immunoreactivity of the anti-KIR6.2 antibody. DAPI nucleic acid stain, an overlay of panels. Transmission image of a mouse portal vein myocyte. Black bar represents 10 μm. (B) Immunohistochemical localization of KIR6.1 and SUR2B subunits. Immunoreactivity of the anti-KIR6.1 antibody. Immunoreactivity of the antiSUR2B antibody. DAPI nucleic acid stain. An overlay of panels. Transmission image of the mouse portal vein myocyte. Black bar represents 10 μm.

Therefore, it is rather difficult to estimate the effects of KATP channel openers on vascular contraction mechanisms alone and compare the potency of KATP channel openers in the presence of either excess [K+]o or agonists. In tension measurements, spontaneous contractions of mouse portal vein were recorded in the absence of any agonists (i.e. non-stimulated conditions). Similarly, spontaneous contractions, with a range of amplitudes and frequencies, were also recorded in guineapig portal vein [26,31]. Furthermore, since KATP channel openerinduced smooth muscle relaxation was measureable with reasonable accuracy in mouse portal vein, the potency of various KATP channel openers could be comparably estimated with reasonable certainty. 4.2. Molecular properties of SUR.x subunits in mouse portal vein KATP channels The molecular properties of SUR.x in mouse portal vein KATP channels were determined as follows: (i) MCC-134-induced relaxation: It has been reported that MCC-134 is a useful pharmacological tool to identify the SUR.x subtype [12,20]. It has been reported that MCC-134 acts as an inverse agonist at SUR1, as a partial agonist at SUR2A, and as a full agonist at SUR2B; hence its effects depend on the type of SUR.x present in the KATP channel [20]. In the present experiments, MCC-134 (10 μM) produced a relaxation of mouse portal vein similar to that induced by pinacidil (500 nM). The MCC-134-induced relaxation was inhibited by the subsequent application of glibenclamide and U-37883A, gradually recovering to the control level. The Ki value of the MCC-134-induced relaxation in mouse portal vein was 1.6 μM and the Ki value of the pinacidil-induced relaxation was 130 nM. Similarly, in recombinant expression studies of SUR2B associated with KIR6.2 (i.e. KIR6.2/SUR2B channels) in HEK293 cells, the Ki value of the MCC134-induced currents was 5.2 μM and the Ki value of the pinacidilinduced currents was 1.4 μM [20,21]. These findings show that the potency of MCC-134 was weaker than that of pinacidil. However, in KIR6.2/SUR2A channels, the Ki value of the MCC-134-induced currents was 8.5 μM and the Ki value of the pinacidil-induced currents was 9.8 μM, showing a similar potency between the KATP channel openers

[20,21]. It is recognized that mouse ventricular myocytes are useful positive control cells to detect three different subunits of SUR.x at the protein level [16,17,30]. Thus, it has been recently reported that three different types of SUR.x (i.e. SUR1, SUR2A and SUR2B) genes were present in mouse ventricular myocytes using RT-PCR analysis [12]. Using the same primer for each SUR.x gene, the presence of three different types of SUR.x subunit in cardiac myocytes was detected at the mRNA levels, confirming that each primer was able to detect the individual gene of each SUR.x as the positive control. Under these experimental conditions, only transcripts of the SUR2B gene, but not transcripts of SUR1 or SUR2A genes, were detected at the mRNA level in mouse portal vein. Furthermore, in quantitative real-time PCR analysis, the mRNA SUR2 was the predominant transcript, whilst the expression level of mRNA SUR1 was not detectible. Anti-SUR2B immunoreactivity was clearly visible in single cardiac myocytes (mouse, [16]; rat, [17]), human detrusor [2] and mouse vas deferens [12]. Using the same anti-SUR2B primary antibody, anti-SUR2B immunoreactivity was detected in both thin transverse sections of portal vein and single smooth muscle cells dispersed from mouse portal vein. Based on the above observations (namely, the relaxing effects of MCC-134 on spontaneous contractions, RT-PCR analysis and anti-SUR2B immunoreactivity), it is highly likely that the major functional SUR.x modulatory subunit in mouse portal vein is SUR2B. 4.3. Molecular properties of KIR6.x subunits forming KATP channels in mouse portal vein The molecular properties of KIR6.x in mouse portal vein KATP channels were as follows: (i) U-37883A-sensitivity: pinacidil caused a concentration-dependent vascular relaxation, which was suppressed not only by glibenclamide, an KATP channel inhibitor, but also by U-37883A, a selective KIR6.1 blocker [14,23]. The channel activity of KATP channels activated by 100 μM pinacidil was also reversibly inhibited by U-37883A. It is probable that the channel pore subunits of KATP channels are functionally related to the KIR6.1 channel subunit. (ii) Single-channel conductance: the conductance was ~ 35 pS in the cell-attached mode, which is similar to that of KIR6.1. (iii) RT-PCR

Please cite this article as: T. Yamamoto, et al., Molecular analysis of ATP-sensitive K+ channel subunits expressed in mouse portal vein, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.06.018

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T. Yamamoto et al. / Vascular Pharmacology xxx (2015) xxx–xxx

analysis and quantitative real-time PCR analysis: in the present experiments, the expression of transcripts for both KATP channel pore-forming subunits (Kcnj8 for KIR6.1 gene and Kcnj11 for KIR6.2 gene) was detected at the mRNA level in mouse portal vein. Similar observations were made for other vascular (rat basilar and cerebral) arteries [13], human coronary artery [28], and visceral smooth muscles (guinea-pig stomach, [22]; pig urethra, [27]). In the present experiments, we further analysed the mRNA expression levels of KIR6.1 and KIR6.2 using real-time PCR techniques in order to facilitate accurate quantification of each subunit mRNA transcript in mouse portal vein. The mRNA KIR6.1 was the predominant transcript, whilst the expression level of mRNA KIR6.2 was barely detectable and negligibly small in comparison with mRNA KIR6.1. (iv) Anti-KIR6.x immunoreactivity: we performed the crossmatch test of primary antibodies for KIR6.x (KIR6.1 or KIR6.2) in order to show selectivity in the expression system of HEK293 cells [12]. Using the same anti-KIR6.1 and anti-KIR6.2 primary antibodies, KIR6.1 protein but not KIR6.2 protein was detected in thin transverse sections of portal vein. Based on these findings, it is highly likely that the mouse portal vein KATP channel pore is composed of KIR6.1 subunits. Similarly, anti-KIR6.1 immunoreactivity but not anti-KIR6.2 immunoreactivity was detected in single smooth muscle cells isolated from mouse portal vein. Taken together, these results indicate that SUR2B is the major modulatory SUR.x subunit and that KIR6.1 is almost certainly the main subunit of the channel pore protein in mouse portal vein, showing co-localization with KIR6.1 and SUR2B. A KIR6.1/SUR2B complex (i.e. KIR6.1/SUR2B channels), which is similar to the KNDP channel subtype found in vascular smooth muscle [3,24], is the most plausible channel complex structure.

4.4. Multiple types of KIR6.x subunits in smooth muscle-type KATP channels In some visceral smooth muscles, RT-PCR analysis revealed the expression of transcripts for both KIR6.1 and KIR6.2 subunits at the mRNA level. For example, KATP channels have been suggested to be a heteromultimerization of KIR6.1 and KIR6.2 subunits in pig urethral myocytes [27] and to comprise a homotetrameric structure of KIR6.1 subunits in gastric myocytes, neglecting the presence of the KIR6.2 transcript [22]. Interestingly, in rat portal vein, multiple homotetrameric structural pore regions with two different channel conductances (KIR6.1 and KIR6.2) were recorded in the same membrane patches using the cell-attached mode [7,29]. Furthermore, RT-PCR analysis also confirmed the presence of both KIR6.1 and KIR6.2 subunit transcripts in rat portal vein [6]. It was concluded that the tissue-specific expression patterns of KATP channels in rat portal vein are unique [6]. This helps our understanding of the heteromultimeric compositions of native vascular KATP channels and provides clues to the selective modulating mechanisms of native vascular KATP channels. In the present experiments, in mouse portal vein, RT-PCR analysis revealed the expression of transcripts for both KIR6.1 and KIR6.2 subunits at the mRNA level. However, in quantitative real-time PCR studies, the expression level of KIR6.2 in mouse portal vein was substantially lower than that of KIR6.1. Moreover, the present immunohistochemical analysis also revealed that KIR6.1 immunoreactivity but not KIR6.2 immunoreactivity was clearly visible. Furthermore, from electrophysiological, pharmacological and molecular studies, the KIR6.1 subunit seems to be mainly involved in the activity of mouse portal vein KATP channels, given the openings of the glibenclamide-sensitive 35 pS K+ channels. It has been reported that the expression of KIR6.x subunits may be altered in some disease states [4,8]. For example, in hypertension, the KIR6.1 subunit expression in rat aorta was decreased with SUR2B [4] and in aorta from obese rats, the KIR6.1 subunit was downregulated in comparison with normal rat aorta [8]. These results lead us to conclude that heteromultimeric expression of KIR6.x subunits is preserved in some disease states. To more accurately compare the expression levels of two different KIR6.x subunit genes, Northern blotting and other techniques will have to be

carried out to quantify the mRNA expression levels of KIR6.x subunit genes. In conclusion, this study provides novel evidence that native KATP channels in mouse portal vein are likely to be a heterocomplex of KIR6.1 channels and SUR2B subunits. Conflict of interest The authors declare no conflict of interest, financial or otherwise. Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (B) from the Japanese Society for the Promotion of Science (Noriyoshi Teramoto, grant number 26282148). References [1] L. Aguilar-Bryan, J. Bryan, Molecular biology of adenosine triphosphate-sensitive potassium channels, Endocr. Rev. 20 (1999) 101–135. [2] M. Aishima, T. Tomoda, T. Yunoki, T. Nakano, N. Seki, Y. Yonemitsu, et al., Actions of ZD0947, a novel ATP-sensitive K+ channel opener, on membrane currents in human detrusor myocytes, Br. J. Pharmacol. 149 (2006) 542–550. [3] D.J. Beech, H. Zhang, K. Nakao, T.B. Bolton, K channel activation by nucleotide diphosphates and its inhibition by glibenclamide in vascular smooth muscle cells, Br. J. Pharmacol. 110 (1993) 573–582. [4] J. Blanco-Rivero, C. Gamallo, R. Aras-López, L. Cobeño, A. Cogolludo, F. PérezVizcaino, et al., Decreased expression of aortic KIR6.1 and SUR2B in hypertension does not correlate with changes in the functional role of KATP channels, Eur. J. Pharmacol. 587 (2008) 204–208. [5] J.E. Brayden, Functional roles of KATP channels in vascular smooth muscle, Clin. Exp. Pharmacol. Physiol. 29 (2002) 312–316. [6] K. Cao, G. Tang, D. Hu, R. Wang, Molecular basis of ATP-sensitive K+ channels in rat vascular smooth muscles, Biochem. Biophys. Res. Commun. 296 (2002) 463–469. [7] W.C. Cole, T. Malcolm, M.P. Walsh, P.E. Light, Inhibition by protein kinase C of the KNDP subtype of vascular smooth muscle ATP-sensitive potassium channel, Circ. Res. 87 (2000) 112–117. [8] L.H. Fan, H.Y. Tian, J. Wang, J.H. Huo, Z. Hu, A.Q. Ma, et al., Downregulation of Kir6.1/ SUR2B channels in the obese rat aorta, Nutrition 25 (2009) 359–363. [9] T. Ibbotson, G. Edwards, A.H. Weston, Antagonism of levcromakalim by imidazolineand guanidine-derivatives in rat portal vein: involvement of the delayed rectifier, Br. J. Pharmacol. 110 (1993) 1556–1564. [10] N. Inagaki, T. Gonoi, J.P. Clement IV, N. Namba, J. Inazawa, G. Gonzalez, et al., Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor, Science 270 (1995) 1166–1170. [11] S. Isomoto, C. Kondo, M. Yamada, S. Matsumoto, O. Higashiguchi, Y. Horio, et al., A novel sulfonylurea receptor forms with BIR (Kir6.2) a smooth muscle type ATPsensitive K+ channel, J. Biol. Chem. 271 (1996) 24321–24324. [12] K. Iwasa, H.L. Zhu, A. Shibata, Y. Maehara, N. Teramoto, Molecular analysis of ATPsensitive K+ channel subunits expressed in mouse vas deferens myocytes, Br. J. Pharmacol. 171 (2014) 145–157. [13] I. Jansen-Olesen, C.H. Mortensen, N. El-Bariaki, K.B. Ploug, Characterization of KATPchannels in rat basilar and middle cerebral arteries: studies of vasomotor responses and mRNA expression, Eur. J. Pharmacol. 523 (2005) 109–118. [14] H. Kovalev, J.M. Quayle, T. Kamishima, D. Lodwick, Molecular analysis of the subtype-selective inhibition of cloned KATP channels by PNU-37883A, Br. J. Pharmacol. 141 (2004) 867–873. [15] H. Kuriyama, K. Kitamura, H. Nabata, Pharmacological and physiological significance of ion channels and factors that modulate them in vascular tissues, Pharmacol. Rev. 47 (1995) 387–573. [16] A. Morrissey, L. Parachuru, M. Leung, G. Lopez, T.Y. Nakamura, X. Tong, et al., Expression of ATP-sensitive K+ channel subunits during perinatal maturation in the mouse heart, Pediatr. Res. 58 (2005) 185–192. [17] A. Morrissey, E. Rosner, J. Lanning, L. Parachuru, P. Dhar Chowdhury, S. Han, et al., Immunolocalization of KATP channel subunits in mouse and rat cardiac myocytes and the coronary vasculature, BMC Physiol. 5 (2005) 1. [18] J. Pataricza, J. Hõhn, A. Petri, A. Balogh, J.G. Papp, Comparison of the vasorelaxing effect of cromakalim and the new inodilator, levosimendan, in human isolated portal vein, J. Pharm. Pharmacol. 52 (2000) 213–217. [19] S. Seino, ATP-sensitive potassium channels: a model of heteromultimeric potassium channel/receptor assemblies, Annu. Rev. Physiol. 61 (1999) 337–362. [20] T. Shindo, Y. Katayama, Y. Horio, Y. Kurachi, MCC-134, a novel vascular relaxing agent, is an inverse agonist for the pancreatic-type ATP-sensitive K+ channel, J. Pharmacol. Exp. Ther. 292 (2000) 131–135. [21] T. Shindo, M. Yamada, S. Isomoto, Y. Horio, Y. Kurachi, SUR2 subtype (A and B)dependent differential activation of the cloned ATP-sensitive K+ channels by pinacidil and nicorandil, Br. J. Pharmacol. 124 (1998) 985–991. [22] J.H. Sim, D.K. Yang, Y.C. Kim, S.J. Park, T.M. Kang, I. So, et al., ATP-sensitive K+ channels composed of Kir6.1 and SUR2B subunits in guinea pig gastric myocytes, Am. J. Physiol. Gastrointest. Liver Physiol. 282 (2002) G137–G144.

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[28] H. Yoshida, J.E. Feig, A. Morrissey, I.A. Ghiu, M. Artman, W.A. Coetzee, KATP channels of primary human coronary artery endothelial cells consist of a heteromultimeric complex of Kir6.1, Kir6.2, and SUR2B subunits, J. Mol. Cell. Cardiol. 37 (2004) 857–869. [29] H.-L. Zhang, T.B. Bolton, Two types of ATP-sensitive potassium channels in rat portal vein smooth muscle cells, Br. J. Pharmacol. 118 (1996) 105–114. [30] M. Zhou, H.J. He, R. Suzuki, K.X. Liu, O. Tanaka, M. Sekiguchi, et al., Localization of sulfonylurea receptor subunits, SUR2A and SUR2B, in rat heart, J. Histochem. Cytochem. 55 (2007) 795–804. [31] H.L. Zhu, T. Tomoda, M. Aishima, Y. Ito, N. Teramoto, The actions of azelnidipine, a dihydropyridine-derivative Ca antagonist, on voltage-dependent Ba2+ currents in guinea-pig vascular smooth muscle, Br. J. Pharmacol. 149 (2006) 786–796.

Please cite this article as: T. Yamamoto, et al., Molecular analysis of ATP-sensitive K+ channel subunits expressed in mouse portal vein, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.06.018