Male SHR and WKY rats (~250 g; 20-week-old; n=18 per group) obtained from Charles River Laboratories, Inc. (Wilmington, MA, USA) were used in the present study. Laboratory conditions were maintained at constant humidity (60±5%), temperature (24±1°C) and light cycle (6.00 a.m-6.00 p.m.) and fed a standard rat pellet diet ad libitum. All protocols were approved by the Institutional Animal Care and Use Committee at Shihezi University Medical College (Shihezi, China) and were consistent with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MA, USA). The blood pressure of the rats was measured using a BP-6 non-invasive blood pressure monitor system.

The rats were anesthetized and then sacrificed by exsanguination. The anesthesia was performed by intramuscular injection of a mixture (1 ml/kg) of ketamine (500 mg), xylazine (20 mg) and acepromazine (10 mg) in 8.5 ml H2O. The mesenteric arterioles were rapidly removed and pinned in a dissecting dish filled with physiological saline solution (PSS) containing NaCl (138.0 mM), KCl (5.0 mM), CaCl₂ (1.6 mM), MgCl2 (1.2 mM), Na-HEPES (5.0 mM), HEPES (6.0 mM) and glucose (7.5 mM). The arteries were digested in PSS containing collagenase A (1.5 mg/ml, Roche, Mannheim, Germany) for 15 min at 37°C, and then returned to the PSS. Vascular connective tissue was further removed under a microscope and the vascular segment was transferred to an inverted phase contrast microscope (Olympus Corporation, Tokyo, Japan) for whole-cell patch clamp recording using an Axon 700B amplifier (Molecular Devices, LLC, Sunnyvale, CA, USA) at room temperature. The recording pipettes were pulled with a P-97 pipette puller (Sutter Instrument Co., Novato, CA, USA) and had a resistance of 3–5 MΩ. The pipette was filled with an internal solution containing K-gluconate (130.0 mM), NaCl (10.0 mM), CaCl₂ (2.0 mM), MgCl2 (1.2 mM), HEPES (10.0 mM), EGTA (5.0 mM) and glucose (7.5 mM), and contacted with cells with negative pressure. The seal resistance typically reached 1–20 GΩ before rupture of the membrane. Membrane rupture was achieved by a high-frequency buzz current and/or suction pressure from the pipette. The transient current over the membrane input capacitance (C_input) was routinely uncompensated to monitor and calculate the access resistance and the membrane parameters on-line or off-line. The drug perfusion device was applied to the whole-cell patch clamp recording, as described previously (25).

Mesenteric arterioles with a diameter <300 µm from the SHRs and WKY rats were used in the present study. The vascular segment was pinned in a dissecting dish filled with solution containing NaCl (118.9 mM), KCl (4.69 mM), MgSO4·7H2O (1.17 mM), KH2PO4 (1.18 mM), CaCl₂ (2.5 mM), NaHCO3 (25.0 mM), EDTA (0.26 mM) and glucose (5.5 mM). The initial transmural pressure was set at 20 mmHg at the import end and 5 mmHg at the export end using a pressure myograph system, and allowed to equilibrate for 3 min to wash away residual blood. The segment was pressurized with a 10 mmHg stepwise increase in transmural pressure to 60 mmHg, with 5 min of equilibration at each pressure. The vessel was stabilized for 1 h once the intraluminal pressure reached 60 mmHg and the bath solution was replaced every 20 min. Phenylephrine (PE; 1×10-5 M) was then added to the arteriole to stimulate contraction. On reaching maximal contraction, acetylcholine (1×10-3 M) was added to relax the vessel. If the dilatation rate was >70%, indicating the integrity of the endothelium, the vessel was used for experiments, as described previously (26,27). To perform the experiment, 1×10-5 M PE was added to pre-contract the vessel, following which NFA (10⁻⁵, 3×10⁻⁵, 5×10⁻⁵, 10⁻⁴, 3×10⁻⁴ M) was successively added. The vasomotor responses to PE and NFA were recorded. In order to investigate the effect of the large conductance calcium-activated potassium channels (BK_Ca) on vasodilation, following pre-contraction of the blood vessel with PE, the vessel was incubated with tetraethylammonium (TEA; 1×10-3 M). NFA (3×10-5 M) was then added to observe the vasodilator response.

RNA was isolated from mesenteric arterioles using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) from WKY and SHR rats. To remove genomic DNA, extracted RNA was digested with DNase I (Promega Corporation, Madison, WI, USA), followed by first-strand cDNA synthesis using 500 g total RNA, oligo(dT)16 primers and M-MLV reverse transcriptase according to the manufacturer's protocol (Promega Corporation). To analyze gene expression, the Cx43 cDNA was quantified and normalized using GAPDH as a reference gene. All semi-quantitative PCR amplification were performed in a total volume of 25 µl under the following PCR conditions in a thermal cycler (TP650; Takara Biotechnology Co., Ltd., Dalian, China): 94°C for 5 min, followed by 94°C for 40 sec, 58°C for 40 sec, and finally 72°C for 10 min for 26 (GAPDH) or 30 (CX42) cycles, respectively. The rat Cx43 genome sequence was obtained from GenBank (GenBank accession no. NM_012567.2). The following gene-specific primers were used: Cx43, forward 5′-aag tta gac gaa gtc cac gta gag-3′, reverse 5′-aac cac aga gag cga aac ttg tag-3′; GAPDH, forward 5′-caa ggt cat cca tga caa ctttg-3′, reverse 5′-gtc cac cac cct gtt gct gtag-3′. PCR products were separated by agarose gel (1.0%) electrophoresis and observed by ethidium bromide staining. Semi-quantitative analysis was performed using Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA, USA) following scanning of agarose gel electrophoretic images.

The mesenteric arterioles from the SHRs were rapidly dissected and transferred into a petri dish with Krebs solution containing NaCl (138.0 mM), KCl (5.0 mM), CaCl₂ (1.6 mM), MgCl2 (1.2 mM), Na HEPES (5.0 mM), HEPES (6.0 mM) and glucose (7.5 mM) at pH 7.4. The perivascular adipose tissue and residual blood were cleaned out using a fine forceps under a microscope and the shredded vessel was digested using Krebs solution with 0.1% collagenase. Following centrifugation at 168 × g for 5 min at 4°C, the precipitated cells (5×10⁵ cells/ml) were plated in dishes with culture medium containing 20% fetal bovine serum. As a marker of VSMCs, α-smooth muscle actin (α-SMA) was determined using immunohistochemistry.

The mesenteric arterioles were homogenized in radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, Inc.) (ratio of 10 mg tissue to 100 µl RIPA buffer) with freshly added protease inhibitor, phenylmethylsulfonyl fluoride (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany). The homogenates were incubated at 4°C for 30 min and centrifuged at 12,000 × g for 15 min at 4°C. The supernatant was collected, and the protein concentration in the supernatant was determined using a bicinchoninic acid assay. Protein aliquots (40 µg/lane) were separated by Tris-glycine denaturing gradient gel electrophoresis on a 10% SDS-PAGE gel. The proteins were then transferred to a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk in TBS-Tween buffer (pH 8.0, 10 mmol/l Tris-HCl, 150 mmol/l NaCl and 0.2% Tween 20) for 1 h at room temperature, and then probed the following primary antibodies: Anti-Cx43 polyclonal antibody (1:1,000; cat. no. 3512; Cell Signaling Technology, Inc., Danvers, MA, USA), and anti-β-actin monoclonal antibody (1:10,000; cat. no. A5316; Sigma-Aldrich; Merck Millipore) overnight at 4°C. Following incubation of primary antibodies, the blots were washed three times with TBS-Tween for 5 min and incubated with horseradish peroxidase-conjugated goat anti-rabbit (cat. no. A0545) or goat anti-mouse (cat. no. A9044) secondary antibodies (1:20,000; Sigma-Aldrich; Merck Millipore) for 2 h at room temperature. The blots were washed five times with TBS-Tween, 5 min each time, and visualized on X-ray film using the ECL chemiluminescence reagents (Thermo Fisher Scientific, Inc.). The optical density of each target protein band was assessed with Quantity One software (version 4.6.2; Bio-Rad Laboratories, Inc.) and normalized to the corresponding β-actin bands in the same sample.

The results are expressed as the mean ± standard error of the mean. Statistical analysis was performed using the SPSS 17.0 statistical package (SPSS, Inc., Chicago, IL, USA). The statistical tests used were one-way analysis of variance (ANOVA), Student's t-test and q-test. When the overall F test of the ANOVA was significant, Tukey's multiple-comparison test was applied. Student's t-test was used to compare between two means. P<0.05 was considered to indicate a statistically significant difference.

The voltage step induced whole-cell current tracings prior to the application of NFA (Fig. 1A). The electrophysiological characteristics of the VSMCs were similar to a single VSMC following the addition of NFA (Fig. 1B). The charging and discharging of VSMCs in a single exponential equation were applied to a current curve prior to (Fig. 1C) and during (Fig. 1D) application of NFA, which indicated that simulation of a single exponential equation was improved following the application of NFA and cell C_input charge-discharge time decreased significantly from 9.73 to 0.48 ms. NFA at 1×10⁻⁴ M decreased VSMC membrane C_input from 87±16 to 11.2±0.64 pF (P<0.05; n=6; Fig. 1E) and decreased membrane conductance (G_input) from 3.31±1.15 to 0.39±0.11 nS (P<0.05; n=6; Fig. 1F).

Following pre-contraction of the blood vessels with 1×10-5 M PE, various concentrations (30, 50 and 100 µM) of NFA were added to compare the relaxation of mesenteric arterioles of WKY rats (Fig. 2A) with SHRs (Fig. 2B). The relaxation response of the mesenteric arterioles of the WKY rats was significantly higher, compared with that in the SHRs. The half maximal effective concentration (EC50) of NFA was also higher in the WKY rats (EC50=192 µM), compared with that in the SHRs (EC50=366 µM; P<0.05; n=6), as shown in Fig. 2C. To assess the contribution of BK_Ca to the vasodilator reactivity of the mesenteric arteriole during application of NFA, 1×10-3 M TEA was applied to inhibit BK_Ca channels following pre-contraction of the blood vessel with PE. No significant change in the diameter of the blood vessels was observed following the application of TEA (142.3±22.7 µm diameter). However, the vasodilator reactivity of the mesenteric arterioles was significant (210.5±18.6 µm diameter; P<0.05; n=6) following the addition of 3×10-5 M NFA (data not shown).

The mRNA (Fig. 3A) and protein (Fig. 3B) levels of Cx43 in the third-level branches of the mesenteric arterioles were significantly higher, compared with those in the primary branch of the mesenteric arterioles from the SHRs and WKY rats (P<0.05; n=6; Fig. 3C and D). When comparing the same branch of mesenteric arterioles from the SHRs with those of the WKY rats, the mRNA (Fig. 3A) and protein (Fig. 3B) levels of Cx43 in the SHRs were significantly higher, compared with those in the WKY rats (P<0.05; n=6; Fig. 3C and D).

Following treatment with 30, 50 and 100 µM NFA, the mRNA levels of Cx43 (Fig. 4A) were significantly decreased in the VSMCs of the SHRs in a concentration-dependent manner (P<0.05; n=6; Fig. 4B).

Similar to mRNA, the protein levels of Cx43 (Fig. 5A) were significantly reduced in the VSMCs of the SHRs in a concentration-dependent manner (P<0.05; n=6; Fig. 5B). The expression of Cx43 was almost completely inhibited in the VSMCs when the NFA concentration reached 100 µM.