Biological and Pharmaceutical Bulletin
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Up-Regulation of the Voltage-Gated KV2.1 K+ Channel in the Renal Arterial Myocytes of Dahl Salt-Sensitive Hypertensive Rats
Kazunobu OgiwaraSusumu OhyaYoshiaki SuzukiHisao YamamuraYuji Imaizumi
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2017 年 40 巻 9 号 p. 1468-1474

詳細
Abstract

Salt-sensitive hypertension induces renal injury via decreased blood flow in the renal artery (RA), and ion channel dysfunction in RA myocytes (RAMs) may be involved in the higher renal vascular resistance. We examined the effects of several voltage-gated K+ (KV) channel blockers on the resting tension in endothelium-denuded RA strips and delayed-rectifier K+ currents in RAMs of Dahl salt-sensitive hypertensive rats (Dahl-S) fed with low- (Dahl-LS) and high-salt diets (Dahl-HS). The tetraethylammonium (TEA)-induced contraction in RA strips were significantly larger in Dahl-HS than Dahl-LS. Correspondingly, TEA-sensitive KV currents were significantly larger in the RAMs of Dahl-HS than Dahl-LS. Among the TEA-sensitive KV channel subtypes, the expression levels of KV2.1 transcript and protein were significantly higher in the RA of Dahl-HS than Dahl-LS, while those of KV1.5, KV7.1, and KV7.4 transcripts was comparable in two groups. KV2.1 currents detected as the guangxitoxin-1E-sensitive component were larger in the RAMs of Dahl-HS than Dahl-LS. These suggest that the up-regulation of the KV2.1 channel in RAMs may be involved in the compensatory mechanisms against decreased renal blood flow in salt-sensitive hypertension.

K+ channels play key roles in the regulation of the membrane excitability of vascular smooth muscle (VSM).1,2) A number of studies support there being important roles for voltage-gated (KV), delayed-rectifier K+ channels in the regulation of the resting membrane potential in VSM.24) The molecular diversity of delayed-rectifier KV channel subtypes is fundamental to an understanding of the differences in membrane excitability and the regulation of vascular tone. The molecular components of the delayed-rectifier KV channels have been identified in VSMs: KV1.x and KV2.x.57) KV1.x and KV2.x superfamily members have been implicated in the inhibition of myogenic tension in resistant arteries.711) The expression of the KV1.5 transcripts has been shown to be decreased in the pulmonary artery in pulmonary hypertension.12) The loss of KV1.5 and KV2.1 expression stimulates cell proliferation and inhibits apoptosis in pulmonary arterial myocytes in pulmonary hypertension model rats.13) Moreover, the KV channel encoded by KV2.1 was reduced in cerebral arterial myocytes from angiotensin II-induced hypertensive rats.9) These show that the loss of KV channel function is responsible for the development of hypertension. On the other hand, an increase in K+ channel function represents a compensatory reaction in an effort to limit the development of hypertension. In the small mesenteric artery of spontaneously hypertensive rat (SHR), an animal model of primary hypertension, the KV channel function encoded by KV1.2, KV1.5, and KV2.1 was significantly enhanced compared with normotensive rats.14,15) Thus, an understanding of the modification of KV channel function could provide important insight into the mechanisms of increased vascular tone during chronic hypertension and reveal novel targets for the pharmacological treatment and perhaps even prevention of vascular dysfunction.

Excessive salt intake is an important determinant of essential hypertension in humans. Wellman et al. reported significant decreases in both the 4-aminopyridine (4-AP)-sensitive and -insensitive KV current components in the cerebral arteries of Dahl salt-sensitive hypertensive rats, and showed that the resting membrane potential was depolarized.16) However, they did not molecularly characterize the decreased KV channel subtypes or the changes in KV channel function and expression in the other VSMs of Dahl salt-sensitive hypertensive rats. Salt-sensitive hypertension induces renal injury via the decrease in the blood flow through the renal artery myocytes (RAMs), and this is associated with an abnormality in vascular reactivity.17) Therefore, the alterations in KV channel function in the RAMs by salt-sensitive hypertension may be due to the increase in renal vascular resistance.

The goal of this study was 1) to examine the role of KV channels in the vascular dysfunction which takes place in the RAMs of Dahl salt-sensitive hypertensive rats (Dahl-S) by the determination of the KV channel blocker-induced tonic contractions and whole-cell KV currents and 2) to determine the salt-sensitive hypertension-induced changes in the expression of the KV channel subtypes in RA strips. The results indicate that there is a larger contribution of KV2.1 to the resting tension maintenance of RA in the Dahl-S rats fed with a high-salt diets (Dahl-HS) than a low-salt diet (Dahl-LS), representing a potent component of the compensatory mechanisms underlying the decrease in the blood flow through RA induced by salt-sensitive hypertension.

MATERIALS AND METHODS

Animals

Male Dahl salt-sensitive rats (Dahl-S) of 6 weeks of age were obtained from Japan SLC, Inc. (Hamamatsu, Japan) and were fed with an 8% (high salt, HS) or a 0.3% (low salt, LS) NaCl diet for 4 weeks, respectively (‘Dahl-HS’ or ‘Dahl-LS’). Systolic arterial pressure (SAP) was measured in awake rats by the tail-cuff method (Softron BP-98 tail-cuff system, Tokyo, Japan). SAP was measured in the morning in a quiet environment, and an average of five successive readings was recorded. All experiments were carried out in accordance with the guiding principles for the care and use of laboratory animals in Nagoya City University, and also with the approval of the President of Nagoya City University.

Measurement of Contractile Response

The RAs were removed and placed in Krebs’ solution containing (in mM): NaCl, 112; KCl, 4.7; CaCl2, 2.2; MgCl2, 1.2; NaHCO3, 25; KH2PO4, 1.2; and glucose, 14 (pH 7.4). Under microscopy, vessels were trimmed of fat and adherent tissue, denuded of endothelium and cut into segments 3 mm long. An arterial strip was attached to a fixed, stainless steel rod at one end and an isometric force transducer at the other. Baths were filled with Krebs’-N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) solution containing (mM): HEPES, 20; NaCl, 120; KCl, 4.8; CaCl2, 1.2; MgCl2, 1.3; NaHCO3, 12.6; KH2PO4, 1.2; and glucose, 5.8 (pH 7.4), warmed to 37°C, and aerated with 95% oxygen and 5% carbon dioxide. Each tissue was placed under resting tension (500 mg) and allowed to equilibrate for 30 min with frequent buffer changes. Strips were then challenged with 80 mM KCl to initiate a maximal contraction and washed repeatedly until the tone returned to baseline. The contractile response was elicited in the presence of 1 µM phentolamine (an α-adrenoceptor antagonist) and 1 µM atropine (a muscarinic receptor antagonist).

Whole-Cell Patch-Clamp Recording

Single myocytes were isolated from the RA of the male Dahl-LS and Dahl-HS as previously reported.18) A whole-cell voltage clamp was applied to individual myocytes with patch pipettes using a CEZ-2400 amplifier (Nihon Kohden, Tokyo, Japan). To isolate K+ currents, normal external solution having the following composition were used: (mM): NaCl 137, KCl 5.9, MgCl2 1.2, CaCl2 2.2, HEPES 10, glucose 14, and CdCl2 0.5 (pH 7.4). The pipette solution contained (mM): KCl 140, MgCl2 3, CaCl2 4.5, ATP-2Na 2, ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetra acetic acid (EGTA) 5 and HEPES 10. The pCa and pH of the pipette solution were adjusted to 6.0 and 7.2, respectively. All of the experiments were performed at room temperature (23±1°C). Data acquisition and analysis were carried out using the analog-digital converter (DIGIDATA 1320 A; Axon Instruments, Foster City, CA, U.S.A.) or pCLAMP software (version 8.2; Axon Instruments).

RNA Extraction, cDNA Synthesis and Real-Time PCR

Total RNA extraction and cDNA synthesis from the RA, mesenteric artery (large), and thoracic aorta of Dahl-S rats were performed as previously reported.18,19) The resulting cDNA product was amplified with gene specific primers and designated using Primer Express software (Ver 1.5, Applied Biosystems, Foster City, CA, U.S.A.). Quantitative, real-time PCR was performed with the use of Syber Green chemistry on an ABI 7700 sequence detection system (Applied Biosystems) as previously reported.18,19) Regression analysis of the mean values of three multiplex RT-PCRs for the log10 diluted cDNA was used to generate standard curves. Unknown quantities relative to the standard curve for a particular set of primers were calculated, yielding transcriptional quantitation of the gene products for the voltage-gated K+ channel subtypes relative to the endogenous standard, β-actin (ACTB). The following PCR primers were used for real-time PCR: KV1.1 (GenBank accession number: X12589, 357–485), amplicon=129 bp; KV1.2 (NM_012970, 1285–1385), 101 bp; KV1.3 (NM_019270, 1205–1326), 122; KV1.5 (NM_012972, 1385–1491), 107 bp; KV1.6 (AJ276137, 155–259), 105 bp; KV2.1 (NM_013186, 1058–1168), 111 bp; KV2.2 (M77482, 944–1061), 118 bp; KV3.1 (NM_012856, 2498–2603), 106 bp; KV3.2 (NM_139217, 54–173), 120 bp; KCa1.1 (NM_031828, 408–509), 102 bp; KV7.1 (NM_032073, 1891–1991), 101 bp; KV7.4 (AF249748, 230–330), 101 bp; and ACTB (NM_031144, 338–438), 101 bp.

Western Blot

Protein fractions of plasma membrane were prepared from rat RA as previously reported,18) and proteins (50 µg/lane) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (10%). The blots were incubated with anti-KV2.1 (Alomone Labs, Jerusalem, Israel) polyclonal antibody and then incubated with anti-rabbit horseradish peroxidase-conjugated immunoglobulin G (IgG) (Chemicon International, Inc., Temecula, CA, U.S.A.). An enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, U.S.A.) was used for the detection of the bound antibody. Resulting images were analyzed using a LAS-1000 device (FUJIFILM, Tokyo, Japan).

Statistical Analysis

Statistical significance between two or among multiple groups was evaluated using Student’s t-test after F test or ANOVA. Data are presented as means±standard error of the mean (S.E.M.)

Statistical significance between two and among multiple groups was evaluated using Student’s t-, Welch’s t-tests or Tukey’s test after F test or ANOVA.

RESULTS

Changes in Blood Pressure, Left Ventricle Hypertrophy and Tissue Weight in Dahl Salt Sensitive Hypertensive Rats

We first confirmed the changes in blood pressure and the other parameters induced by a high salt diet given for 4 weeks. SAP was significantly increased in Dahl-HS compared with Dahl-LS: 120±1 (n=43) and 205±1 (n=30) mmHg in Dahl-LS and Dahl-HS, respectively (p<0.01). In addition, the ratios of 1) the heart weight (HW) to body weight (BW) (HW/BW) and 2) kidney weight (KW) to BW (KW/BW), and 3) the weight of the renal artery (RAW) were significantly increased in Dahl-HS compared with in Dahl-LS: HW/BW, 0.34±0.01% (n=43) and 0.47±0.01 (n=30) (p<0.01); KW/BW: 0.79±0.02 (n=39) and 1.02±0.01 (n=26) % (p<0.01); RAW, 0.27±0.01 mg/mm (n=28) and 0.47±0.09 (n=26) (p<0.05) in Dahl-LS and Dahl-HS, respectively.

Tetraethylammonium (TEA)-Induced Contractile Responses in the RA Strips of Dahl Salt-Sensitive Hypertensive Rats

To determine the contribution of TEA (Wako Pure Chemical Industries, Ltd., Osaka, Japan)-sensitive KV channels to the resting tension in the RA strips, we examined the effects of TEA in both groups. Contractile responses were measured in the presence of 1 µM phentolamine and 1 µM atropine. When TEA was applied, the amplitude of tonic contraction increased in a concentration-dependent manner in the RA strips of Dahl-LS (Fig. 1Aa) and Dahl-HS (Fig. 1Ab). The TEA-induced contractile responses were expressed as the relative contraction (%) of the 80 mM KCl-induced level. A significant difference in the TEA-induced contractile response was found in the groups [1 mM TEA: 9.6±2.0 (n=10) and 35.1±6.0% (n=10), p<0.01; 10 mM TEA: 53.3±6.0 (n=10) and 76.8±4.0% (n=10), p<0.01] (Fig. 1B). The difference in the 80 mM KCl-induced contraction was not significant between the groups [29.8±1.2 (n=28) and 26.6±1.0 mN (n=30), p>0.05].

Fig. 1. Tetraethylammonium (TEA)-Induced Contractile Responses in Endothelium-Denuded RA Strips of Dahl-S Rats

A: Traces showing the tonic contractions elicited by the application of 1 or 10 mM TEA in RA strips of Dahl-LS (Aa) and Dahl-HS (Ab). B: Summarized data from ‘A.’ TEA-induced contractile responses were expressed as a % of the 80 mM KCl-induced ones. Columns and bars indicate means±S.E.M. (n=10 for each, **: p<0.01 vs. Dahl-LS).

Large-Conductance Ca2+-Activated K+ Channels in the RAMs of Dahl Salt-Sensitive Hypertensive Rats

In addition to KV channels, large-conductance Ca2+-activated K+ (BKCa) channels are an important contributor to the regulation of the resting membrane potential in VSMs.2) Our previous report demonstrated increases in BKCa channel activity and BKCa protein expression in the aortic myocytes of SHR.18) We examined the contribution of BKCa channel to resting tension in the RA strips of both groups by the measurement of paxilline (PAX: a selective BKCa channel inhibitor) (Sigma, St. Louis, MO, U.S.A.)-induced tonic contraction (not shown). When 1 µM PAX was applied, PAX-induced contractile responses were much smaller than 1 mM TEA-induced ones (less than 0.2 mN, n=3 for each) in the RA strips of both groups and no significant differences were observed in the groups. Subsequently, whole-cell patch clamp recordings of the BKCa currents (IBK) were performed in the RAMs of both groups. IBK was determined to be a PAX (1 µM)-sensitive outward current in this study.

Figure 2A shows the effects of PAX on outward currents elicited by a 150 ms depolarizing voltage step to +40 mV from the holding potential of −60 mV in the RAMs of Dahl-LS (Fig. 2Aa) and Dahl-HS (Fig. 2Ab). The PAX-sensitive IBK currents were 2.6±0.5 (n=15) and 2.4±0.3 (n=15) pA/pF, respectively (Fig. 2B), and comprised approximately 10% of the total outward currents in both groups: 10.7±1.9 and 13.1±1.2%, respectively (p>0.05) (Fig. 2C). Another BKCa channel inhibitor (100 nM iberiotoxin) exhibited similar results (not shown). In addition, the expression of KCa1.1 transcripts encoding BKCa channel α subunit did not exhibit any significant difference in the groups (p>0.05, n=4 for each) (Fig. 2D). These results indicate that the BKCa channel in RA makes a small contribution to the control of the resting tension and membrane potential, and that the BKCa channel function in RA is unchanged by salt-sensitive hypertension.

Fig. 2. Effects of a BKCa Channel Inhibitor, Paxilline (PAX) on Outward KV Currents in RAMs of Dahl-S Rats

A. Currents were elicited by 150 ms depolarizing voltage step to +40 mV from the holding potential (−60 mV), and 1 µM PAX was applied in the RAMs of Dahl-LS (Aa) and Dahl-HS (Ab). B. Summarized data of the PAX-sensitive current density. Currents were normalized by cell capacitance and are expressed as the current density (pA/pF) (n=5 for each). C. PAX-sensitive current density was expressed as a % of the inhibition of the outward currents (n=5 for each). D. Transcriptional expression of KCa1.1 in the RA of Dahl-LS and Dahl-HS was examined by real-time PCR analysis (n=4 for each). The values of KCa1.1 expressions in the RA of Dahl-HS are expressed as the ratio to those in the RA of Dahl-LS. In ‘B’–‘D,’ the results are expressed as means±S.E.M.

TEA-Sensitive, Delayed-Rectifier KV Currents in the RAMs of Dahl Salt-Sensitive Hypertensive Rats

We next determined the effects of TEA on outward KV currents in the presence of 1 µM PAX (PAX-insensitive KV currents) in the RAMs of Dahl-S rats. The PAX-insensitive KV currents were 22.7±2.2 (n=15) and 17.1±2.2 (n=15) pA/pF in the RAMs of Dahl-LS and Dahl-HS (p>0.05). Figure 3A shows that the TEA (1, 10, and 30 mM)-induced inhibition of KV current elicited by a 150 ms depolarizing voltage step to +40 mV from holding potential of −60 mV in the RAMs of Dahl-LS (Fig. 3Aa) and Dahl-HS (Fig. 3Ab). TEA inhibited the PAX-insensitive KV currents in a concentration-dependent manner in the RAMs of both groups. When the TEA-sensitive KV current components were expressed as the % of the amplitude of the PAX-insensitive KV currents, they were significantly larger in the RAMs of Dahl-HS than Dahl-LS in the range of 1–30 mM (Fig. 3B). The 30 mM TEA-sensitive currents at +40 mV were 7.0±1.4 (n=5) and 12.7±2.0 (n=5) pA/pF (32.7±3.3 and 66.5±5.3% of the PAX-insensitive KV currents) in the RAMs of Dahl-LS and Dahl-HS, respectively (p<0.01).

Fig. 3. Effects of TEA on the PAX-Insensitive KV Currents in the RAMs of Dahl-S Rats

A. Currents were elicited by 150 ms depolarizing voltage step to +40 mV from (a) the holding potential of −60 mV in the RAMs of Dahl-LS (Aa) and Dahl-HS (Ab). Application of TEA (1, 10, and 30 mM) inhibited the PAX-insensitive KV currents. B. Summarized data of the TEA-sensitive KV currents. The TEA-sensitive KV currents (were) are expressed as a % of the amplitude of the PAX-insensitive KV currents. Results are expressed as means±S.E.M. (n=5 for each). **: p<0.01 vs. Dahl-LS.

Effects of a KV2.1-Selective Blocker, Guangxitoxin-1E (GxTX), on TEA-Sensitive KV Currents

In contrast to TEA as a non-selective KV channel blocker, guangxitoxin-1E (GxTX-1E) (Peptide Institute, Osaka, Japan) is a novel peptidyl inhibitor of KV2.x, and 50 nM GxTX-1E almost completely suppresses cloned human KV2.1 currents (IC50=approximately 1 nM).20) We determined the effects of GxTX-1E on PAX-insensitive KV currents in the RAMs of both groups. Figure 4A shows that 50 nM GxTX-1E induced an inhibition of the PAX-insensitive KV currents elicited by a 150 ms depolarizing voltage step to +40 mV from the holding potential of −60 mV in the RAMs of both groups. When the GxTX-1E-sensitive KV currents were expressed as a % of the amplitude of PAX-insensitive KV currents, they were significantly larger in the RAMs of Dahl-HS than Dahl-LS. The GxTX-1E-sensitive currents at +40 mV were 3.9±0.3 (n=5) and 7.5±1.2 (n=5) pA/pF (Fig. 4B) and 18.0±1.8 (n=5) and 43.7±4.6% (n=5) of PAX-insensitive KV currents (Fig. 4C) in the RAMs of Dahl-LS and Dahl-HS, respectively (p<0.01).

Fig. 4. Effects of a KV2.1-Selective Blocker, Guangxitoxin-1E (GxTX-1E), on PAX-Insensitive KV Currents in RAMs of Dahl-S Rats

A. Currents were elicited by a 150 ms depolarizing voltage step to +40 mV from (a) the holding potential of −60 mV in the RAMs of Dahl-LS (Aa) and Dahl-HS (Ab). The application of 50 nM GxTX-1E inhibited the PAX-insensitive KV currents. B. Summarized data of the GxTX-1E-sensitive KV currents from ‘A.’ The currents were normalized by cell capacitance and are expressed as current density (pA/pF). C. GxTX-1E-sensitive KV currents were expressed as a % of the amplitude of the PAX-insensitive KV currents. Results are expressed as means±S.E.M. (n=5 for each). **: p<0.05, 0.01 vs. Dahl-LS.

Expression of KV Channel Subtypes in the RA of Dahl Salt-Sensitive Hypertensive Rats

We examined the expression patterns of delayed-rectifier-type KV channel subtypes (KV1.1, 1.2, 1.3, 1.5, 1.6, 2.1, 2.2, 3.1, 3.2) in the RAs of both groups using quantitative, real-time RT-PCR. The expression levels of KV1.2, KV1.5, and KV2.1 transcripts were relatively high, and those of the other KV subtype transcripts were undetectable (not shown). No significant differences in the threshold cycle (Ct) values for β-actin (ACTB) expression were found between the groups (not shown). The expression of the KV2.1 transcripts was approximately 4-fold higher in Dahl-HS compared with Dahl-LS rats (n=4 for each, p<0.01) (Fig. 5A). We examined the expression of the KV2.1 transcripts in the mesenteric artery (large) and thoracic aorta of Dahl-HS and Dahl-LS, and there were no significant difference in the KV2.1 transcript expression levels between the groups (Fig. 5C). As shown in Fig. 5A, there are no significant differences in the expression of KV1.2 and KV1.5 transcripts between the RAs of the two groups (p>0.05, n=4 for each). We further determined the protein expressions of KV2.1 by Western blot analysis. As shown in Fig. 5B, Western blot analysis identified the band specific for anti-KV2.1 antibody with a molecular weight of approximately 100 kDa in the RA plasma membrane fractions in both groups. The band signals for the anti-KV2.1 antibody were significantly increased in Dahl-HS compared with in Dahl-LS (Fig. 5Ba). Densitometric analysis revealed that the KV2.1 protein levels in Dahl-HS were approximately 3-fold those in Dahl-LS (n=4) (Fig. 5Bc). These signals disappeared upon pre-incubation with excess antigen (+antigen) (Fig. 5Bb).

Fig. 5. Expression of the KV Channel Subtypes in the RA of Dahl-S Rats

A. Transcriptional expression of KV1.2, KV1.5, and KV2.1 in the RA of Dahl-LS and Dahl-HS was examined by real-time PCR analysis (n=4 for each). The values of KV subtype expressions in the RA of Dahl-HS are expressed as the ratio to those in the RA of Dahl-LS. **: p<0.01 vs. Dahl-LS. B. Protein expression of KV2.1 in the RA of Dahl-LS and Dahl-HS was examined by Western blotting. The membrane proteins extracted from RAs were immunoblotted with the anti-KV2.1 antibody (a). When the anti-KV2.1 antibody was pre-incubated with the peptide against which the antibody was generated, the immunostaining was faint (+antigen) (b). Summarized data of relative optical density from ‘b’ (c). The values of optical band density in the RA of Dahl-HS are expressed as the ratio to those in the RA of Dahl-LS. C. Transcriptional expression levels of KV2.1 in mesenteric artery and thoracic aorta of Dahl-LS and Dahl-HS (n=4 for each). The results are expressed as means±S.E.M. (n=4). **: p<0.01 vs. Dahl-LS.

Involvement of KV7 K+ Channel in TEA-Sensitive KV Currents in the RAMs

Over 60% of the PAX-insensitive KV currents were inhibited by 30 mM TEA in the RAMs of Dahl-HS (Fig. 3), however a selective KV2.1 blocker, GxTX-1E (50 nM) (IC50=1–4 nM for KV2.1) inhibited approximately 45% of them (Fig. 4). Several reports have shown that delayed-rectifier KV7 K+ channels are responsible for TEA-sensitive KV currents in arterial myocytes.2123) We therefore examined the contribution of KV7 to the TEA-sensitive KV currents in the RAMs using a KV7 channel blocker, XE-991 (Tocris Bioscience, Bristol, U.K.). Three micromolar XE-991 inhibited the KV currents elicited by the 150 ms depolarizing voltage step to +40 mV from the holding potential (−60 mV) in the RAMs of both groups (Fig. 6A). When the XE-991-sensitive components were expressed as a percentage of the amplitude of PAX-insensitive outward currents, no significant differences of the XE-991-sensitive KV currents were found between the RAMs of Dahl-LS and Dahl-HS (approximately 10% of PAX-insensitive KV currents) (p>0.05) (Fig. 6B). Quantitative, real-time PCR analysis showed that among five KV7 subtypes (KV7.1–7.5), the KV7.1 and KV7.4 transcripts were predominantly expressed in the RAs, and no significant differences were found between the groups (p>0.05, n=5 for each) (Fig. 6C).

Fig. 6. Effects of a KV7-Selective Blocker, XE-991 on PAX-Insensitive KV Currents in RAMs of Dahl-S Rats and Expression Levels of KV7.1 and KV7.4 Transcripts in the RA of Dahl-S Rats

A. Currents were elicited by a 150 ms depolarizing voltage step to +40 mV from (a) the holding potential of −60 mV in the RAMs of Dahl-LS (Aa) and Dahl-HS (Ab). The application of XE-991 (3 and 10 µM) inhibited the PAX-insensitive KV currents. B. Summarized data of the XE-991-sensitive KV currents from ‘A.’ XE-991-sensitive KV currents were expressed as a % of the amplitude of the PAX-insensitive KV currents. Results are expressed as means±S.E.M. (n=5 for each). C. Transcriptional expression of KV7.1 and KV7.4 in the RA of Dahl-LS and Dahl-HS was examined by real-time PCR analysis (n=4 for each). The values of KV7.1 and KV7.4 expressions in the RA of Dahl-HS are expressed as the ratio to those in the RA of Dahl-LS.

DISCUSSION

Cox et al. have showed that voltage-gated, delayed-rectifier KV currents are larger in isolated arterial myocytes from the mesenteric and tail arteries of SHR than Wistar Kyoto rat (WKY), and significant increases in the KV1.2 and/or KV1.5 genes are detected in the arteries of SHR, and that both KV2.1 gene expression and functional activity are larger in the mesenteric artery of SHR than WKY.14,24) The present study showed that 1) the increase in the contribution of TEA-sensitive KV channels to the resting tension in the RAs of Dahl-HS (Fig. 1) and 2) the gene and protein expression levels of KV2.1 in the RAs were up-regulated in Dahl-HS (Fig. 5), which is consistent with the increase in the TEA- and GxTX-sensitive KV current components (Figs. 3, 4). Indeed, the IC50 of TEA for PAX-insensitive KV currents in the RAMs of Dahl-HS (approximately 2.5 mM) was similar to that for the cloned KV2.1 currents (4.5 mM).25) However, no up-regulation of KV1.2 and KV1.5 genes was observed in the RAs of Dahl-HS (Fig. 5A). We demonstrated 4-AP exerts an effect on the PAX-insensitive currents, with the result that there was no significant current inhibition in the range of 0.1–10 mM between the groups, and the IC50 of 4-AP was approximately 50 µM (unpublished observation by Ogiwara). 4-AP inhibits the cloned KV1.2 and KV1.5 currents with IC50 values of approximately 600 and 50 µM,26) respectively, in agreement with the predominant expression of the KV1.5 transcripts but not KV1.2 in the RAMs of both groups (Fig. 5A). Taken together, up-regulation of KV2.1 in the RA of Dahl-HS may be a compensatory increase in the contribution of KV2.1 activity to the normal resting membrane potential. Indeed, 50 nM GxTX-1E induced an approximate 5 mV depolarization in the RAMs of Dahl-HS, but no GxTX-1E-induced contraction was observed (not shown).

KV channels play important roles in the regulation of cell proliferation and apoptosis.13,27,28) In pulmonary hypertension, the membrane depolarization induced by dysfunction of the arterial KV channels (KV1.5 and KV2.1) increases Ca2+ entry through voltage-gated, L-type Ca2+ channel, and thereby leads to not only vasoconstriction but also cell proliferation, with the phenotypic modulation in atherosclerosis and restenosis.29) These indicate that the loss of KV2.1 activity causes the arterial hypertrophy by stimulating cell proliferation and inhibiting apoptosis in arterial myocytes.29) Similar to the previous study reported by Hampton et al.,30) the present study showed the RA hypertrophy (the increase in RAW) in Dahl-HS, however, KV2.1 activity was rather increased in RAMs of Dahl-HS (Figs. 1, 2, 4, 5). These suggest that up-regulation of KV2.1 in the RAMs of Dahl-HS RA may be also involved in the suppressive regulation of the RA hypertrophy induced by salt-sensitive hypertension.

In angiotensin (ANG II)-induced hypertensive rats, ANG II decreases ASM KV currents by the down-regulation of KV2.1 expression via the activation of the nuclear factor of activated T-cells c3, NFATc3.31) In Dahl-HS, a high salt diet causes a reduction in the plasma ANG II levels in Dahl-HS.32,33) We therefore investigated a possible contribution of the down-regulation of NFATc3 to the up-regulation of KV2.1 transcripts in RAMs of Dahl-HS. However, the NFAT3c transcripts were expressed at very low levels in the RAs of Dahl-HS, and no significant differences were observed between Dahl-LS and Dahl-HS groups (not shown). The mechanisms underlying the up-regulation of KV2.1 transcripts by salt-sensitive hypertension remains to be determined.

Sung et al. have shown that 4-AP-sensitive KV channels are the crucial regulator of the resting tone in rat mesenteric artery, and serotonin elicits the contraction by inhibiting KV channels via 5-hydroxytryptamine (5-HT)2A receptor and Src tyrosine kinase pathaway.34) This study showed that KV2.1 inhibition-induced contractile responses were enhanced in the RAs of Dahl-HS. On the other hand, Song et al. have shown that KV2.1 activity is enhanced by Src-mediated phosphorylation in neurons.35) These suggest that vasoconstrictor-induced responses may be altered in the RA of patients with salt-sensitive hypertension by up-regulation of KV2.1. Recently, Hong da et al. have shown that up-regulation of KV1 and KV2 in the mesenteric artery during the early phase of diabetes using type 2 diabetic model rats.35) The increased KV channel-related contractile responses were reversed during the chronic phase. These suggest that up-regulation of KV2.1 in the RA of Dahl-HS may be altered during chronic phase of salt-sensitive hypertension. Further studies will be needed to clarify the clinical significance of up-regulation of RA KV2.1 during salt-sensitive hypertension.

In conclusion, this study demonstrated the up-regulation of the voltage-gated, delayed-rectifier K+ channel subtype KV2.1 in RAMs by salt-sensitive hypertension. The results indicate that KV2.1 is a potent contributor to the compensatory mechanisms underlying the decrease in blood flow through the RA induced by salt-sensitive hypertension.

Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (20056027) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a Grant-in-Aid for Scientific Research (B) (20390027) from the Japan Society for the Promotion of Science (JSPS) to Y.I.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES
 
© 2017 The Pharmaceutical Society of Japan
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