All animal care and experimental procedures complied with the policies of Ethics Committee of ZSSOM on Laboratory Animal Care (document No. 2016-080) and conformed to the “Guide for the Care and Use of Laboratory Animals” of the National Institute of Health in China. Sprague-Dawley rats were purchased from the Experimental Animal Center of Sun Yat-sen University. The gut-corrected Cftr knockout mouse strain STOCK Cftr${}^{t\mathit{}m\mathit{}\mathrm{1}\mathit{}U\mathit{}n\mathit{}c}$ Tg(FABPCFTR)1Jaw/J was purchased from the Jackson Laboratory (ME, USA). Females that are heterozygous for the Cftr knockout allele and homozygous for the FABP-hCFTR transgene were bred with males that are homozygous for both Cftr knockout allele and FABP-hCFTR transgene and thus wild-type (Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}}$), heterozygote (Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{-}}$) and Cftr knockout (Cftr${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$) littermates were obtained. Animals were housed in standard plastic rodent cages and maintained at a regulated environment (25 ${}^{\circ }$C, 12 h light, 12 h dark cycle with lights on at 7:00 and off at 19:00) with ad libitum access to a normal chow diet with 2.45 g NaCl/1000 g (D12450B, 10 kcal % Fat, Research Diets). The number used followed the principle of minimal requirement and showed in Figure legends.

Ang II induced hypertension models were established using Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}}$, Cftr${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice (5–8 weeks old) or Sprague-Dawley rats (150–200 g). Ang II was administered via subcutaneously implanted osmotic minipumps (Alzet, Cupertino, CA, Model 1002 for mice and 2002 for rats) for 2 weeks to establish Ang II infusion induced hypertension models. The administration rate was 1000 ng/kg/min [19] for mice and 80 ng/min [20] for rats as previously reported and manufacture’s instruction. BP was measured in conscious mice at 6:00 PM by tail cuff plethysmography (BP-98A, Softron, Japan) as we previously reported [21].

Antibody to CFTR was from Alomone Labs (Jerusalem, Israel) and antibody to MLCK was from Sigma Aldrich Corp. (St. Louis, MO, USA). Antibodies to total myosin light chain (t-MLC), phosphorylated myosin light chain (p-MLC), MYPT1, phosphorylated MYPT1 (p-MYPT1), $\beta$-actin and all secondary antibodies were from Cell Signaling Technology (Boston, MA, USA). CFTR(inh)-172, Y27632, ML-7, Fura-2AM, RIPA Lysis Buffer (10X), protease inhibitor cocktail, L-nitro-arginine methyl ester (L-NAME), TritonX-100, EGTA, MnCl${}_2$, pentobarbital sodium, Ang II, Hanks’ Balanced Salt Solution (HBSS) and Krebs-Henseleit Modified Buffer were from were from Sigma Aldrich Corp. (St. Louis, MO, USA). Polyvinylidene difluoride (PVDF) membrane was from Millipore Corp. (Billerica, MA, USA). Pierce ECL substrate and sequence-specific primers were from Thermo Fisher Scientific (Waltham, MA, USA). Trizol reagent was from Invitrogen (Carlsbad, CA, USA), Transcriptor First Strand cDNA Synthesis Kit (Roche, Mannheim, Germany) and Fast start universal SYBR Green Master (ROX) were from Roche Molecular Biochemicals (Mannheim, BW, Germany). RhoA Activation Assay Kit was from Abcam (Cambridge, UK). Dulbecco’s modified Essential medium/F-12 (DMEM/F12) medium, fetal calf serum were from Gibco (Carlsbad, CA, USA).

Basilar artery smooth muscle cells (BASMCs) were isolated from rat and mice basilar arteries as we previously described [22]. Briefly, 4 weeks old male Sprague–Dawley rats, 5–8 weeks old Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}}$ and Cftr${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice were anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneally). The basilar arteries were collected immediately and immersed in Krebs solution containing 10${}^5$ U/L penicillin and 100 mg/L streptomycin. After the connective tissue was cleaned, the basilar arteries were cut into small pieces about 0.5 mm long and the vessel segments were placed on the surface of the dish. The dish was then incubated in DMEM/F12 supplemented with 20% fetal calf serum at 37 ${}^{\circ }$C and 5% CO${}_2$. Passages 4–6 of the cultured BASMCs were used for experiments.

Ad-Cftr and Ad-Cftr-shRNA were designed and produced by GENECHEM (Shanghai, CN). The cDNA of Cftr plasmid was a kind gift from Dr. Jim Hu (University of Toronto, ON, CA). The sequence of Cftr (NM_031506) shRNA was 5${}^{\prime }$-CCGGGCTGAAAGCAGGTGGGATTCTCAAGAGAAATCCCACCTGCTTTCAGCTTTTTTG-3${}^{\prime }$. Adenovirus was transfected using the protocol previously described [21]. Briefly, cultured BASMCs at 50% confluence were infected with adenovirus encoding Cftr or Cftr shRNA for 6 h in serum-free medium, and then cells were washed and incubated in fresh complete medium for another 48 h before harvested.

Western blot was performed as previously described [22]. Briefly, cells or tissues were lysed with RIPA lysis buffer containing protease inhibitor cocktail. Protein was separated with 8% or 10% SDS-PAGE and transformed to a PVDF membrane. After blocked with 5% milk for 1 h at room temperature, the membrane was incubated with primary antibody at 4 ${}^{\circ }$C overnight and then with secondary antibody for 1 h at room temperature. For analysis of CFTR expression in arteries or in VSMCs, proteins were extracted in lysis buffer and immunoblotted with anti-CFTR primary antibody (1:500, Alomone Labs) [23], and detected with HRP-linked secondary antibodies. Immunogen of CFTR antibody: Peptide (C) KEETEEEVQDTRL, corresponding to amino acid residues 1468–1480 of human CFTR (Accession P13569). Final band detection was performed with a Pierce ECL substrate by using a BIO-RAD molecular imager ChemiDoc XRS+ (Bio-Red, Hercules, CA). The densities of the target bands were determined by ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Quantitative real time PCR (qRT-PCR) was performed as previously described [24]. Briefly, the total RNA was extracted from basilar arteries using Trizol reagent according to the manufacturer’s instructions. Purity and concentration of isolated total RNA were measured using the TECAN infinite M100 PRO Biotek microplate reader (Tecan Group, Ltd., Männedorf, Switzerland). First-strand cDNA was generated from 1 $\mu$g of total RNA using Transcriptor First Strand cDNA Synthesis Kit and qRT-PCR was performed using the Fast Start Universal SYBR Green Master (ROX) according to the manufacturer’s instructions. The total reaction volume was 20 $\mu$L, including 10 $\mu$L of 2 $\times$ SYBR Green Master, 0.6 $\mu$L of PCR Forward Primer (10 $\mu$M), 0.6 $\mu$L of PCR Reverse Primer (10 $\mu$M), 2 $\mu$L of cDNA (20 ng) and 7 $\mu$L of double-distilled water. The qRT-PCR was set at an initial step of 10 min at 95 ${}^{\circ }$C, followed by 40 cycles at 95 ${}^{\circ }$C for 15 seconds and then 60 ${}^{\circ }$C for 60 seconds. qRT-PCR was executed using iQ5 real-time PCR detection system (Bio-Rad Laboratories, Inc, CA, USA). All experiments were done in triplicate, and all samples were normalized to GAPDH. The expression levels were calculated using 2${}^{-\mathrm{\Delta }\mathrm{\Delta }Ct}$ methods. Melting curve analysis was performed in the range of 60 ${}^{\circ }$C to 95 ${}^{\circ }$C, 0.5 ${}^{\circ }$C per 5 seconds increments. The sequence-specific primers were used as follows: CFTR, 5${}^{\prime }$-GGATGCTGAGGAAGCAACTC-3${}^{\prime }$ (forward) and 5${}^{\prime }$-CCAGCCTGGAACTCTCTTTG-3${}^{\prime }$ (reverse); GAPDH, 5${}^{\prime }$-AGGTCGGTGTGAACGGATTTG-3${}^{\prime }$ (forward) and 5${}^{\prime }$-TGTAGACCATG TAGTTGAGGTCA-3${}^{\prime }$ (reverse).

Vascular rings of thoracic aorta and basilar artery were prepared and isometric tension measurement experiments were performed as previously described [25]. Briefly, mice were euthanized and then thoracic aortas or basilar arteries were quickly removed and placed into ice-cold Krebs-Henseleit Modified Buffer containing 2.5 mM Ca${}^{2+}$ (Krebs buffer), and detached from fatty and connective tissues. Aortic ring segments (3–5 mm) and basilar artery ring segments (2 mm) were fixed on the Wire Myograph System (Danish Myo Technology, ​Denmark) and immersed in Krebs buffer aerated with 95% O${}_2$/5% CO${}_2$ and maintained at 37 ${}^{\circ }$C. The rings were stretched to an optimal resting tension (3 mN) and allowed to stabilize for 1 h. 60 mM of KCl was used to stimulate vessel contraction for 3 times, the difference of isometric force should be less than 5% between each time. At the end of each stimulation, the original equilibrium tension of vessels were recovered after washed by Krebs buffer for 3 times. The vessels were then balanced for 30 min, followed by subsequent experiments. During the measurement process, endothelium of the arteries was chemically denudated by L-NAME (10 $\mu$M). Isometric force were recorded using PowerLab data acquisition system (AD Instruments Inc, US).

[Ca${}^{2+}$]${}_i$ was measured as we previously described [26]. Briefly, VSMCs were digested and incubated with DMEM/F12 medium containing 2 $\mu$M of Fura-2/AM for 45 min at 37 ${}^{\circ }$C, then the cells were washed with HBSS buffer containing 2 mM Ca${}^{2+}$ 3 times and resuspended. The number of cells was adjusted to 10${}^6$ cells per mL. Fluorescence emission was monitored at 500 nm using RF-5301 fluorescence Spectrophotometer (Shimadzu, Tokyo, Japan) with an excitation at 340 and 380 nm. [Ca${}^{2+}$]${}_i$ was determined from the formula: [Ca${}^{2+}$]${}_i$ = Kd $\times$ Sf380/b380(R–Rmin)/(Rmax–R), where Kd is 224 nm at 37 ${}^{\circ }$C; Sf380/b380 is the ratio of the intensities of the free and bound dye forms at 380 nm; R is the fluorescence ratio (340 nm/380 nm) of the intracellular Fura-2/AM; Rmax and Rmin are the maximal and minimal fluorescence ratios obtained by addition of Triton X-100 (0.09%) and EGTA (3 mM) respectively. Ang II (100 nM) was added to the cell suspension when the fluorescence intensity was stable.

As we described previously [27], VSMCs were digested and incubated with 2 $\mu$M of Fura-2/AM for 45 min at 37 ${}^{\circ }$C, then the cells were washed with HBSS buffer containing 1.3 mM Ca${}^{2+}$ 3 times and resuspended with Ca${}^{2+}$-free HBSS buffer. The number of cells was adjusted to 10${}^6$ cells per ml. After baseline fluorescence got to steady state, Mn${}^{2+}$ was injected into the cuvette with the final concentration of 500 $\mu$M. Fluorescence intensity was detected by RF-5301 Fluorescence Spectrophotometer (Shimadzu, Tokyo, Japan) with magnetic stirring at 37 ${}^{\circ }$C. The excitation wavelength was 360 nm and the absorption wavelength was 510 nm. After the fluorescence value was relatively stable, Ang II were added to stimulate the flow of divalent cations.

RhoA-GTP and total RhoA were detected using a RhoA Activation Assay Kit according to manufacturer’s instruction. Briefly, the anti-active RhoA (RhoA-GTP) mouse monoclonal antibody was incubated with cell lysates. The bound RhoA-GTP was then pulled down by protein A/G agarose beads at 4 ${}^{\circ }$C for 1 h. The beads were centrifuged and washed, and then boiled in SDS-PAGE sample buffer. Samples were resolved on 10% SDS-PAGE gels and transferred to PVDF membranes and incubated with anti-RhoA Rabbit polyclonal antibody. Total RhoA was detected by western blot of the cell lysates using anti-RhoA rabbit polyclonal antibody provided in the kit.

All data are expressed as mean $\pm$ SEM with n representing the number of independent experiments on different batches of cells or different mice. All statistical analyses were performed using Prism 9 (GraphPad software, San Diego, CA). Student’s t test (2-tailed) was used to detect significant differences between two groups while one-way ANOVA followed by Bonferroni multiple comparison test was used to compare differences between three groups or more. Values were considered statistically significant when p 0.05.

Ang II of various infusion rates (125, 250, 500 and 1000 ng/kg/min) was administrated to Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}}$ mice using osmotic pumps for 14 days. In comparison to sham group, mice administrated with 500 and 1000 ng/kg/min Ang II exhibited significant higher systolic blood pressure (SBP), which were 139.33 $\pm$ 2.58 and 159.67 $\pm$ 4.57 mmHg (Fig. 1A). Meanwhile, Cftr mRNA level in basilar arteries was significantly reduced in these two groups (Fig. 1B) and negative correlation between Cftr mRNA and SBP was observed (Fig. 1C). Moreover, CFTR protein expression was decreased in basilar arteries from Ang II induced hypertensive rats (Fig. 1D). These in vivo data suggested that arterial CFTR was downregulated only if SBP was elevated.

Fig. 1.

Expression of CFTR is downregulated in hypertensive arteries and Cftr knockout enhanced Ang II induced blood pressure elevation. (A) Ang II of 0, 125, 250, 500 and 1000 ng/kg/min was administrated for 14 days to obtain Ang II induced hypertensive mice model. Systolic blood pressure (SBP) was significantly elevated in 500 and 1000 ng/kg/min groups. (B) Cftr mRNA level in basilar arteries was also significantly reduced in 500 and 1000 ng/kg/min groups (n = 6, *p 0.05 and ***p 0.001 vs. 0 ng/kg/min group). (C) Correlation analysis of basilar artery Cftr mRNA expression and SBP in Ang II induced hypertensive mice. (D) CFTR protein expression in basilar arteries was reduced in Ang II induced hypertensive rats (n = 6, *p 0.05 vs. Sham group). (E,F) No significant difference was observed in baseline SBP and DBP levels between Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}}$, Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{-}}$ and Cftr${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice (n = 6). (G,H) Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}}$ and Cftr${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice were used to establish Ang II induced hypertensive model, SBP and DBP were measured 0, 3, 6, 9, 12, 14 days after 1000 ng/kg/min of Ang II infusion operation. In comparison to Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}}$ mice, SBP and DBP measured in Cftr${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice were increased (n = 7–8, *p 0.05 and **p 0.01 vs. Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}}$ group).

In previous study, average SBP and DBP of 24-hour, daytime and night time of Cftr${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice were all significantly higher than those of Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}}$ mice, however, Cftr knockout did not alter SBP or DBP recorded from 4:00 PM to 8:00 PM [12]. To investigate whether CFTR play a part in Ang II induced hypertension, Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}}$ and Cftr${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice were used to establish Ang II infusion induced hypertension model, SBP and DBP was measured at 6:00 PM 0, 3, 6, 9, 12 and 14 days after the pumps were implanted. We found that SBP of Cftr${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice was significantly higher than that of Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}}$ mice since day 6 (Fig. 1G). DBP was also additionally increased in Cftr${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice on day 3, 12, and 14 (Fig. 1H). Baseline SBP and DBP of Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}}$, Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{-}}$ and Cftr${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice were also measured at 6:00 PM, as shown in Supplementary Fig. 1, Cftr knockout did not affect BP in physiological state. These in vivo data indicated that Cftr knockout aggravated Ang II induced hypertension.

In both sham and Ang II induced hypertensive mice, Ang II induced tension of thoracic aorta was significantly higher in Cftr${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice vs. Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}}$ mice (Fig. 2A,B). Furthermore, preincubation of CFTR specific inhibitor CFTR(inh)-172 (5 $\mu$M) for 5 min significantly increased Ang II induced vasoconstriction in thoracic aorta and basilar artery, which were isolated from Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}}$ mice and SD rats, respectively (Fig. 2C,D). Additionally, KCl induced arterial tension was also enhanced by CFTR(inh)-172 (Fig. 2E), while no significant effect could be observed when thromboxane analogue U46619, 5-HT, or phenylephrine were used as stimulators (Fig. 2F–H). These data demonstrated that short-term pharmacological or genetic inhibition of CFTR increased vasoconstriction in response to Ang II in both normotensive and hypertensive arteries.

Fig. 2.

Effect of Cftr knockout or pharmacological inhibition on vasoconstriction in response to different stimulus. (A–B) Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}}$ and Cftr${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice were used to establish Ang II infusion induced hypertensive model and thoracic aortas were isolated. Endothelium of the vessel was chemically denudated by L-NAME (10 $\mu$M). Representative tracings of isometric tension in response to Ang II (1 $\mu$M) in thoracic aorta were shown. Maximum tension was adopted for analysis (n = 5–6, *p 0.05). (C,D) Thoracic aortas and basilar arteries were isolated from C57BL/6J mice and SD rats, respectively, and conducted endothelial denudation. CFTR was pharmacologically inhibited by 5 min of preincubation with CFTR(inh)-172 (5 $\mu$M), while DMSO was used as solvent control. Arterial rings were incubated with Ang II (1 $\mu$M) for 5 min and representative tracings of isometric tension were shown. Ang II induced tension was expressed in the percentage of KCl (60 mM) induced tension in the absence of CFTR(inh)-172 (n = 6, *p 0.05). (E) Arteries were pretreated with CFTR(inh)-172 (5 $\mu$M) for 5 min and KCl (60 mM) induced tension were measured (n = 8, *p 0.05). (F–H) Concentration-response curves of U46619, 5-HT and phenylephrine (Phe) were drawn in the presence or absence of CFTR(inh)-172, no significant difference in tension could be observed (n = 5).

The reversible phosphorylation of regulatory myosin light chain (MLC) is the core of VSMCs contraction. To investigate the effect of CFTR on MLC phosphorylation in vivo, Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}}$ and Cftr${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice were used to establish Ang II infusion induced hypertension model, and the phosphorylated MLC (p-MLC) and total MLC (t-MLC) were detected. As shown in Fig. 3A, p-MLC in thoracic aortas was increased by Ang II infusion, and significantly higher in Cftr${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice in comparison to Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}}$ ones in the hypertension group. No significant alteration was observed in t-MLC level (Fig. 3B). In vitro, CFTR overexpression adenovirus (Ad-Cftr), CFTR shRNA silencing adenovirus (Ad-Cftr-shRNA) and CFTR specific inhibitor CFTR(inh)-172 were used to alter the expression or activity of CFTR. As shown in Fig. 3C,D, p-MLC in primarily isolated basilar artery smooth muscle cells (BASMCs) was decreased by CFTR overexpression and increased by CFTR silencing or CFTR(inh)-172 incubation for 48 h, while no significant alteration was observed in t-MLC level (Supplementary Fig. 1). These data demonstrated that both CFTR expression and long-term activity inhibition could affect MLC phosphorylation level in resting VSMCs.

Fig. 3.

Downregulation/inhibition of CFTR increased the phosphorylation level of myosin light chain in vivo and in vitro. (A,B) Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}}$ and Cftr${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice were used to establish Ang II infusion induced hypertensive model. Thoracic aortas were isolated from hypertensive group and sham group, phosphorylated myosin light chain (p-MLC) and total myosin light chain (t-MLC) were detected by western blot (n = 6 mice, *p 0.05). Cftr knockout increased p-MLC in Ang II induced hypertensive arteries. (C,D) p-MLC level in basilar artery smooth muscle cells (BASMCs) were measured with CFTR overexpression, silencing or pharmacological inhibition. Ad-Cftr, Ad-Cftr-shRNA or CFTR(inh)-172 (5 $\mu$M, 48 h) were used, respectively. BASMCs were treated with Ad-Cftr or Ad-Cftr-shRNA for 6 h followed by cultivation with DMEM/F12 medium containing 10% FBS for another 48 h. Ad-lacZ and Ad-Scr were used as negative control, respectively. p-MLC (C,D) and t-MLC (see in Supplementary Fig. 1) in cell lysates were detected by western blot (n = 6, *p 0.05).

Cytosolic Ca${}^{2+}$ concentration elevation is a trigger for vascular contraction. A defect in the regulation of Ca${}^{2+}$ signaling plays a role in hypertension associated vascular dysfunction. For smooth muscle cell contraction to occur, cytosolic Ca${}^{2+}$ binds to calmodulin, and Ca${}^{2+}$/calmodulin complex interacts with MLC, leading to the activation of MLCK and phosphorylation of MLC. To investigate whether CFTR works through Ca${}^{2+}$-dependent pathway, effect of CFTR on Ang II induced increase in [Ca${}^{2+}$]${}_i$ was detected. BASMCs were cultured from Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}}$ or Cftr${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice, and CFTR overexpression in Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}}$ BASMCs was conducted using Ad-Cftr. 100 nM of Ang II was added to stimulate [Ca${}^{2+}$]${}_i$ increase with 2 mM of extracellular Ca${}^{2+}$. As shown in Fig. 4A,C, CFTR did not affect basal [Ca${}^{2+}$]${}_i$ in resting BASMCs, however, BASMCs from Cftr${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice exhibited higher [Ca${}^{2+}$]${}_i$ in response to Ang II compared to that from Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}}$ mice (Fig. 4B), while CFTR overexpression caused a decrease in Ang II induced [Ca${}^{2+}$]${}_i$ rise (Fig. 4D). Then we went further for the Mn${}^{2+}$ quenching rate measurement and found that Cftr knockout also enhanced Ang II induced Mn${}^{2+}$ influx (Fig. 4E). These results for the first time demonstrated that CFTR was involved in Ang II induced Ca${}^{2+}$ influx, and thus initiated MLCK activation and the consequent MLC phosphorylation.

Fig. 4.

Effect of CFTR on Ang II induced Ca${}^{\mathrm{𝟐}\mathbf{+}}$ influx in cultured BASMCs. (A) BASMCs were isolated from Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}}$ and Cftr${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice and cultured in DMEM/F12 medium with 20% FBS. Intracellular calcium concentration ([Ca${}^{2+}$]${}_i$) was monitored and recorded every 10 seconds with 2 mM of extracellular Ca${}^{2+}$. Ang II (100 nM) was added after the fluorescence was stable. (B) Maximum of [Ca${}^{2+}$]${}_i$ in response to Ang II were compared between BASMCs from Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}}$ and Cftr${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice (n = 6, **p 0.01). (C) BASMCs were treated with Ad-lacZ (100 MOI) or Ad-Cftr (100 MOI) for 6 h followed by cultivation with DMEM/F12 medium containing 10% FBS for another 48 h. [Ca${}^{2+}$]${}_i$ was monitored and recorded every 10 seconds with 2 mM of extracellular calcium concentration. Ang II (100 nM) was added after the fluorescence was stable. (D) Maximum of Ang II induced [Ca${}^{2+}$]${}_i$ rise were compared between Ad-lacZ and Ad-Cftr group. (n = 6, **p 0.01). (E) Measurement of Mn${}^{2+}$ quenching rate. BASMCs were cultured from Cftr${}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}}$ and Cftr${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice, digested and incubated with 2 $\mu$M Fura-2AM for 45 min and then washed and resuspended with Ca${}^{2+}$ free HBSS. Fluorescence in response to Ang II was recorded every second. Quenching rate was expressed as percentage change of the maximum rate.

As shown in Fig. 3C,D,4A,C, in resting VSMCs, CFTR could regulate the phosphorylation of MLC without affecting resting [Ca${}^{2+}$]${}_i$. In addition, CFTR overexpression did not alter MLCK protein expression level (Fig. 5A), and with MLCK activity inhibited by ML-7, CFTR overexpression could still decrease p-MLC (Fig. 5B), suggesting that CFTR might regulate resting tension of VSMCs through Ca${}^{2+}$-independent pathway.

Fig. 5.

Effect of CFTR on phosphorylation of MYPT1 via adjusting RhoA activity. (A) BASMCs were treated with Ad-Cftr or Ad-lacZ for 6 h and cultured with 10% FBS DMEM/F12 medium for another 48 h. MLCK expression was detected by western blot and no significant difference was observed (n = 6). (B) ML-7 (inhibitor of MLCK, 10 $\mu$M) was applied during the whole process of CFTR overexpression treatment in BASMCs. Cell lysates were collected and p-MLC was detected by western blot (n = 6, *p 0.05). (C–F) BASMCs were treated with Ad-Cftr, Ad-Cftr-shRNA or negative control vector for 6 h and cultured with DMEM/F12 medium with 10% FBS for another 48 h. Cell lysates were collected and MYPT1 phosphorylation at Ser507, Thr696 and Thr855 and total MYPT1 expression were detected using western blot (see in Supplementary Fig. 2), $\beta$-actin was used as loading control. (n = 6, **p 0.01). (G) BASMCs were treated with CFTR(inh)-172 (5 $\mu$M, 48 h) and/or Y-27632 (10 $\mu$M, 48 h) and terminated by liquid nitrogen. Cell lysates were collected and p-MYPT1${}^{Thr855}$ was detected by western blot (n = 6, *p 0.05, ***p 0.001). (H) BASMCs were treated with Ad-Cftr or Ad-lacZ, Ang II (100 nM) was added for 5 min to induce cell contraction and then liquid nitrogen was used to terminate the process immediately. Cell lysates were collected and RhoA-GTP and total RhoA were detected using RhoA Activation Assay Kit (n = 6, *p 0.05, **p 0.01).

While MLCK-mediated constriction is strictly dependent on [Ca${}^{2+}$]${}_i$, MLC phosphorylation and the subsequent constriction can be maintained even after [Ca${}^{2+}$]${}_i$ returns to basal levels via inhibition MLCP. MLCP holoenzyme consists of three subunits: catalytic subunit of protein phosphatase 1 (PP1), myosin phosphatase target subunit 1 (MYPT1) and a subunit of unknown function [28]. The binding of MYPT1 to PP1 unit increases MLCP catalytic activity, while phosphorylated MYPT1 (p-MYPT1) represses the catalytic activity of MLCP and thus enhances MLC phosphorylation and VSMCs contraction. p-MYPT1 is modulated by RhoA/ROCK pathway, which can be activated by various stimuli, including Ang II.

As shown in Fig. 5C–F, CFTR affected p-MYPT1 at site Thr855, but not at Ser507 or Thr696 in BASMCs. Incubation with selective Rock inhibitor Y-27632 decreased p-MYPT1 in resting BASMCs and abolished the enhancement of p-MYPT1 induced by long-term CFTR activity inhibition (48 h) (Fig. 5G), while CFTR overexpression decreased RhoA activation in resting and Ang II stimulated BASMCs (Fig. 5H). These data indicated that RhoA/Rock mediated MYPT1 phosphorylation might be another signaling pathway involved in the regulating effect of CFTR on vasoconstriction and thus hypertension. Our finding is in agreement with a previous report demonstrating that continuous inhibition CFTR-Cl${}^{-}$ conductance for 3–5 days resulted in a threefold increase in RhoA expression [29].