Hypoxia is a phenomenon where tissues are deprived of sufficient supply of oxygen, which can be recurrently interspersed with episodes of normoxia (intermittent hypoxia [IH]) [1]. Chronic hypoxia can have a wide range of effects, including endothelial dysfunction, oxidative stress, vascular tone, and inflammation [2,3]. Chronic IH (CIH) causes vascular dysfunction, which can boost cardiac output and peripheral vasoconstriction, resulting in higher blood pressure [4]. In addition, CIH-induced endothelial dysfunction is accompanied by downregulated nitric oxide (NO) and upregulated endothelin-1 (ET-1) [5]. Moreover, CIH-caused cardiovascular disease is fueled by aortic vascular remodeling which involves aortic smooth muscle cells (ASMCs) [6]. However, pathologies underlying CIH-induced vascular dysfunction remain poorly clarified.

Large conductance calcium-activated potassium (BK) channels have specific single-conductance selectivity for K (+), combined activation mechanisms of membrane depolarization and increased intracellular calcium ion (Ca²⁺), and diverse expression patterns, making them common mediators of cell excitability [7]. BK channels consist of the pore-forming α (BKα) subunit and regulatory β and γ subunits [8]. Each subunit exhibits different gating characteristics to BK channels. For instance, BKβ1 activates the opening of BK channels in SMCs in response to Ca²⁺ sparks by sensitizing BK channels to Ca²⁺ and voltage, which is a critical mechanism for vasodilation and regulation of vessel diameter [9]. In smooth muscle, the α subunit and β1 subunit are co-expressed, thereby resulting in an enhancement in the apparent sensitivity to Ca²⁺ and slowing down the channel-gating kinetics. The BK α/β1 channels lower the risk of vascular tone-related pathologies through their functions of membrane hyperpolarization and vasodilatation [10]. Importantly, the opening of BK channels causes hyperpolarization by allowing K⁺ efflux across the plasma membrane. In contrast, the closure of the channels induces depolarization, which underlines the pivotal role of BK channel activity in determining the membrane potential of vascular SMCs and, therefore, vascular tone [11]. Additionally, BK channels are the key to the mechanism of oxygen sensing, whose activity is correlated with hypoxic responses [12]. It is worth noting that a previous study found that the activity of BK channels decreased in CIH-induced rats [13]. Another study elucidated that chronic hypoxia diminished the activity of BK channels in uterine arteries [14].

These findings support the hypothesis that BK channels may be involved in the mechanism of CIH-induced vascular dysfunction. In our study, we analyzed whether BK channels modulated CIH-induced vascular dysfunction by using models of rats and rat ASMCs (RASMCs).

CIH rat models

Sprague-Dawley (SD) male rats (n = 30, 5 weeks old; Hunan SJA Laboratory Animal Co., Ltd.) were housed in the plastic cages of the Animal Care Center in Jiangxi Provincial People’s Hospital, with ad libitum access to water and food, a constant temperature (21°C), and a 12 h/12 h light/dark cycle. All animal experiments were conducted by conforming to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, and the protocol for this study was ratified by the Animal Care and Use Committee of Jiangxi Provincial People’s Hospital (JXSRM-24-0513). NS1619 (an agonist of the BK channel) was purchased from Merck (S0462) and Iberiotoxin (an inhibitor of the BK channel) was purchased from Selleck (E0015).

Rats were randomized into five groups (N = 6 rats/group): normoxia (normoxia treatment), CIH (IH treatment), dimethyl sulfoxide (DMSO; injection of an equal volume of 0.001% DMSO before CIH modeling), NS1619 (intraperitoneal injection of 20 μM NS1619 before CIH modeling), and Iberiotoxin (intraperitoneal injection of 1 nM Iberiotoxin before CIH modeling) groups.

CIH modeling was performed by controlling the oxygen concentration in the cages with an automated and computer-controlled gas exchange system. Specifically, rats were exposed to hypoxia for 6 h/day at 60 times/h (cages were introduced with 20% O₂ for 40 sec and 5% O₂ for 20 sec per min) during the light phase (10 a.m. to 4 p.m.). Rats in the normoxia group breathed normoxia gas and were placed in an identical system for 28 days. NS1619 and Iberiotoxin were dissolved in 0.001% DMSO. SD rats were pre-injected intraperitoneally with DMSO, NS1619, or Iberiotoxin (1 mg/kg), followed by CIH modeling. Afterward, subsequent experiments were performed.

Cell culture and grouping

RASMCs (CP-R076; Procell) were cultured with the complete medium (CM-R076; Procell) in a humidified incubator under the conditions of 37°C and 5% CO₂.

The RASMC CIH model was developed by immersing RASMCs in 1% O₂ for 5 min and then in 21% O₂ for 5 min (6 cycles/h) [15]. Cells were randomly categorized into five groups: control (normoxia treatment), CIH (hypoxia [1% O₂/21% O₂] treatment to establish CIH cell model), DMSO (DMSO treatment before CIH treatment), NS1619 (20 μM NS1619 treatment before CIH treatment), and Iberiotoxin (1 nM Iberiotoxin treatment before CIH treatment) [16].

Measurement of intracellular Ca²⁺ concentration

After cell treatment, Fluo-3/AM (S1056; Beyotime) was dissolved in DMSO and stored at –20°C in the dark to analyze changes in Ca²⁺ in RASMCs. Specifically, the treated cells were added to a 96-well plate (FCP966-10pcs; Beyotime) and cultured with 5 μM Fura-3/AM for 45 min under the conditions of 37°C and 5% CO₂, followed by protein blocking with 0.2% bovine serum albumin and three rinses with Ca²⁺-free Hank’s balanced salt solution. The fluorescence intensity was measured using Varioskan Flash (Thermo Fisher Scientific) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Fluorescence was calculated based on the following formula: fluorescence percentage = F treatment/F control × 100%. In addition, Ca²⁺ fluorescence images of cells loaded with Fluo-3/AM were captured in the ultraviolet region under an inverted microscope [17].

Immunofluorescence staining

RASMCs were fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde for 15 min and subjected to blocking with 10% goat serum in PBS containing 0.2% Triton X-100 and overnight incubation with antibodies against BKα (1:100, bs-4775R; Bioss Antibodies) and BKβ (1:500, DF9301; Affinity) at 4°C. RASMCs were then incubated for 2 h with the secondary antibodies (1:100, Affinity), goat anti-rabbit immunoglobulin G (IgG; H + L), Fluor594 (S0006) and Fluor488 (S0018). Fluorescence images were obtained with a confocal laser scanning microscope (IX53; Olympus).

Hemodynamic measurement

Rats were anesthetized with an intraperitoneal injection of 3% phenobarbital (3 ml/kg), after which the skin was incised along the centerline of the neck. Then the carotid artery was carefully separated. After the distal end of the carotid artery was ligated, a slight break was made at the proximal end of the carotid artery. A Millar pressure catheter was inserted into the carotid artery, followed by recording systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean blood pressure (MBP) with a biological function detection system.

Vascular tone assessment

Rats were euthanized with an overdose of pentobarbital sodium (100 mg/kg), and the vessel was separated from the connective tissues before being removed and immediately immersed in an oxygenated Kreb’s solution (pH 7.4) containing 133.1 mM NaCl, 4.7 mM KCl, 0.61 mM MgSO₄, 1.3 mM NaH₂PO₄, 16.7 mM NaHCO₃, 2.5 mM CaCl₂, and 7.6 mM glucose at 4°C. The aorta was separated and divided into 3-mm rings. The endothelium was then mechanically removed from the vessel by inserting watchmaker’s forceps into the lumen of the vessel, and the vessels were repeatedly rotated on saline-soaked filter paper. Endothelium-intact (E+) and endothelium-denuded (E-) rings were suspended horizontally between two stainless steel wires in organ chambers containing 7 ml of Kreb's solution and in the control solution maintained at 37°C and aerated with 95% O₂ and 5% CO₂. Prior to the experiment, the tissues were equilibrated in Kreb’s solution for 60 min, which was changed every 15 min. During this period, the aortic rings were stretched to a passive tension of 1.5 g. A Power-Lab/8sp recording and analysis system (ML785; AD Instruments) was utilized for the measurement of isometric tension. To validate viability, phenylephrine (PE) (10^-6 M) was contracted, followed by the examination of vasorelaxant responses to the endothelium-dependent vasodilator acetylcholine (ACh, 10^-6 M) and the endothelium-independent vasodilator sodium nitroprusside (SNP, 10^-6 M) [5,18].

NO level detection

Using a NO assay kit, total nitrate and nitrite concentrations were tested to determine the amount of NO present in rat aortic tissues and serum (S0021S; Beyotime). Based on the enzymatic conversion of nitrate to nitrite by nitrate reductase, NO levels were investigated in this assay. After the reaction, nitrite as a product of azo dyes in the Griess reaction was detected with colorimetry. The absorbance of the compounds at 550 nm was examined with a microplate reader.

Vascular endothelin detection

After anesthesia of rats with ether, rats’ blood was collected and left at room temperature for 30 min before being centrifuged at 1,500 g for 15 min to separate the serum. The supernatant was stored at –80°C. Next, 100 μl of diluted samples were added to the ET-1 antibody-coated reaction wells. Subsequently the concentration of ET-1 protein in serum was measured as instructed in the manuals of the enzyme-linked immunosorbent assay (ELISA) kit (D731152-0048; Sangon).

Oxidative stress evaluation

The levels of reactive oxygen species (ROS) and malondialdehyde (MDA) were determined with corresponding commercial ELISA kits (S0033S and S0131S; Beyotime). Briefly, aortic tissue homogenates were added to 96-well plates along with standards for 90 min of culture at room temperature, followed by 90-min culture with coupling reagents. The samples were then colored for 15 min with tetramethylbenzidine solution before being treated with the termination solution. The absorbance at 450 nm was detected with a fully automatic microplate reader (WD-2102B; Beijing Liuyi Biotechnology Co., Ltd.).

Measurement of inflammatory factor levels

Aortic tissue homogenates were acquired for the examination of expression levels of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) as directed in the manuals of corresponding ELISA kits (PI328 and PT516; Beyotime).

Hematoxylin-eosin (H&E) staining

Aortic tissues attained from rats were fixed in 4% paraformaldehyde for 48 h and subjected to gradient alcohol dehydration, paraffin embedding, and sectioning. Then, 5 μm tissue sections were prepared and stained with H&E for histologic analysis. Every section was observed using a light microscope (Olympus) under 10 × 40 light microscope fields.

Immunohistochemistry

Rat aortic tissues were fixed in 4% paraformaldehyde (P0099; Beyotime) for 24 h, paraffin-embedded, and sliced into 5 μm. The sections were then stained with antibodies (1:100, Affinity) against BKα (DF8570) and BKβ (DF9301) by immunohistochemistry. Subsequent to three PBS washes (5 min each time), the sections were incubated with biotin-labeled secondary antibody (A0277; Beyotime) for 1 h at room temperature, labeled with diaminobenzidine, and finally analyzed under a light microscope (BX-50 ; Olympus).

Western blotting

Utilizing Radio-Immunoprecipitation Assay lysis buffer (ST507; Beyotime) to isolate the total protein in aortic tissues, 30 μg of the protein was combined with 12% sodium dodecyl sulfate-polyacrylamide gel, separated via gel electrophoresis, and subsequently placed onto polyvinylidene fluoride membranes (FFP24; Beyotime). The membranes were then sealed with 5% skimmed milk at room temperature and mixed with primary antibodies (Affinity) against endothelial nitric oxide synthase (eNOS; 1:1,000, AF0096), phosphorylation (p)-eNOS (1:1,000, AF3247), ET-1 (1:500, DF612), and β-actin (1:3,000, AF7018; the internal reference) for overnight incubation at 4°C. At 37°C, the secondary goat anti-rabbit antibody (1:3,000, S0001; Affinity) was also incubated with the membrane. Lastly, the Image J program was used to quantify the band intensities.

Statistical analysis

Statistical analysis was carried out with the GraphPad Prism 8 software (GraphPad Software). Differences were analyzed between two groups with the unpaired two-tailed t-test and among multiple groups with analysis of variance followed by the Tukey’s post-hoc test. In all statistical analyses, p < 0.05 represented a statistically significant difference.

CIH elicited vascular dysfunction and inhibited BK channel activity in rats

To investigate the effects of CIH on vascular function, CIH rat models were created by intermittently inhaling hypoxic gas mixtures in SD rats. The hemodynamic results showed that SBP, DBP, and MBP significantly increased in SD rats after exposure to IH (Fig. 1A–C). Vascular tone results demonstrated that for E+ rings, ACh-induced vasorelaxant responses were markedly lower in the CIH group than in the normoxia group. For E- rings, ACh-induced vasorelaxant responses were insignificantly different between the CIH and normoxia groups. ACh-induced vasorelaxant responses were substantially lower in all E- groups than in all E+ groups. Meanwhile, no marked difference was observed in SNP-induced vasorelaxant responses among all groups (Fig. 1D). The above results illustrated that CIH impaired vascular endothelial function.

Figure 1. Vascular dysfunction occurs, and BK channel activity is repressed in chronic intermittent hypoxia (CIH)-treated rats. (A) Systolic blood pressure (SBP) in the normoxia and CIH groups. (B) Diastolic blood pressure (DBP). (C) Mean blood pressure (MBP). (D) Vascular tone assessment. (E) H&E staining to detect pathological changes in rat aorta. (F) Immunohistochemistry to measure the expression of BK channels in rat aortic tissues. N = 6. The data were expressed as mean ± standard deviation. Data comparison between two groups was performed with the unpaired student’s t-test, and data comparison among multiple groups was conducted with a one-way analysis of variance, with Tukey’s for post-hoc test. IOD, integrated optical density; PE, phenylephrine; ACh, acetylcholine; SNP, sodium nitroprusside. *p < 0.05 compared with the normoxia group.

Following that, the pathological features of the aortic tissues were examined using H&E staining (Fig. 1E), which revealed regularly shaped and arranged vascular endothelial cells in the normoxia group, whereas the vascular endothelium in the CIH group exhibited obvious histopathological alterations, evidenced by cellular edema and partial detachment of endothelial cells. Immunohistochemistry of rat aortic tissues (Fig. 1F) showed that the CIH group had significantly fewer BKα- and BKβ-positive cells than the normoxia group. In conclusion, CIH led to vascular dysfunction and diminished BK channel activity in rat aortic tissues.

Activation of BK channels enhanced eNOS expression to increase NO levels in CIH rats

To determine the effect of BK channels on vascular endothelial dysfunction in CIH-treated rats, NS1619 was injected into rats prior to CIH modeling. Then, NO levels were examined in rat aortic tissues and serum, which displayed that the activation of BK channels elevated NO levels in rat aortic tissues and serum, whereas the inhibition of BK channels considerably lowered NO levels in rat aortic tissues and serum (Fig. 2A, B). Additionally, western blotting clarified that the protein expression and phosphorylation of eNOS were conspicuously augmented in CIH-treated rats after NS1619 injection but appreciably decreased by Iberiotoxin injection (Fig. 2C, D). Overall, activation of BK channels can indirectly stimulate eNOS and NO expression in CIH-treated rats.

Figure 2. BK channel activation upregulates eNOS, raising NO expression in CIH-treated rats. (A) NO levels in rat aortic tissues. (B) NO levels in rat serum. (C) eNOS protein expression in rat aortic tissues examined with Western blotting. (D) p-eNOS expression in rat aortic tissues tested with Western blotting. N = 6. The data were presented as mean ± standard deviation. Data comparison between two groups was performed with the unpaired student’s t-test, and data comparison among multiple groups was conducted with a one-way analysis of variance, with Tukey’s for post-hoc test. NO, nitric oxide; CIH, chronic intermittent hypoxia; eNOS, endothelial nitric oxide synthase; p-eNOS, phosphorylation-eNOS; DMSO, dimethyl sulfoxide. *p < 0.05 compared with the DMSO group.

BK channel activation suppressed ET-1 expression and indirectly influenced vascular endothelial dysfunction in CIH-treated rats

Subsequently, we measured ET-1 levels in the serum of rats. The results showed that in the serum of CIH rats, ET-1 expression was reduced by NS1619 but increased after Iberiotoxin injection (Fig. 3A). Western blotting of the rat aortic tissues also corroborated that NS1619 decreased ET-1 protein expression in tissues (Fig. 3B). Next, vascular tone assessment (Fig. 3C) exhibited that ACh-induced vasorelaxant responses in E+ rings of CIH-treated rats were prominently enhanced following NS1619 injection but remarkably decreased by Iberiotoxin and that ACh-induced vasorelaxant responses in all E- groups were strikingly lower than those in all E+ groups. The SNP-induced vasorelaxant response was not different among all groups. In summary, activation of BK channels effectively reduced ET-1 levels, which facilitated vasorelaxation and indirectly depressed vascular endothelial dysfunction in CIH-treated rats.

Figure 3. BK channel activation downregulates ET-1 and mitigates vascular endothelial dysfunction in CIH-treated rats. (A) Detection of ET-1 levels in rat serum. (B) Western blotting analysis of ET-1 protein expression in rat aortic tissues. (C) Vascular tone assessment. N = 6. The data were presented as mean ± standard deviation. Data between two groups was compared using the unpaired student’s t-test, and data comparison among multiple groups was conducted with a one-way analysis of variance, with Tukey’s for post-hoc test. ET-1, endothelin-1; CIH, chronic intermittent hypoxia; DMSO, dimethyl sulfoxide, PE, phenylephrine; ACh, acetylcholine; SNP, sodium nitroprusside. *p < 0.05 compared to the DMSO group.

BK channel activation effectively repressed vascular inflammation and oxidative stress in CIH-treated rats

Vascular dysfunction has been widely reported to include impaired vascular endothelial cells, as well as increased oxidative stress and inflammation. Therefore, the expression of relevant cytokines was detected in aortic tissues with ELISA after activation and inhibition of BK channels. Iberiotoxin caused significant increases in oxidative stress (MDA and ROS), whereas NS1619 reduced oxidative stress to some extent in CIH-treated rats. Meanwhile, activation of BK channels markedly lowered the levels of related inflammatory factors (IL-6 and TNF-α) in CIH-treated rats (Fig. 4C, D). In addition, H&E staining results (Fig. 4E) demonstrated that NS1619 injection remarkably improved the pathological changes of aortic tissues, as well as reducing cell edema and partial detachment in endothelial cells. On the contrary, the pathological features of aortic tissues were further promoted in Iberiotoxin-treated CIH rats. Altogether, activation of BK channels impeded oxidative stress and inflammation in aortic tissues and effectively attenuated the pathological features of tissues in CIH-treated rats.

Figure 4. BK channel activation curbs vascular inflammation and oxidative stress in CIH-treated rats. (A) ROS levels. (B) MDA levels. (C) IL-6 levels. (D) TNF-α levels. (E) H&E staining results. Scale bar = 50 μm. N = 6. Data were expressed as mean ± standard deviation. Data comparison between two groups was performed with the unpaired student’s t-test, and data comparison among multiple groups was conducted with a one-way analysis of variance, with Tukey’s for post-hoc test. CIH, chronic intermittent hypoxia; ROS, reactive oxygen species; MDA, malondialdehyde; IL-6, interleukin-6; TNF-α, tumor necrosis factor alpha; DMSO, dimethyl sulfoxide. *p < 0.05 compared to the DMSO group.

Activated BK channels via BKα and BKβ subunits indirectly regulate vascular dysfunction

To further discuss the regulatory mechanisms of BK channels in RASMCs, CIH cell models were established in RASMCs after co-culture with NS1619 or Iberiotoxin. Ca²⁺ concentration was augmented in the membrane of RASMCs after CIH modeling, while this trend was counteracted after NS1619 treatment but was further promoted by Iberiotoxin treatment (Fig. 5A). To better understand Bkα and Bkβ expression, BK channel-related subunits were examined in RASMCs using immunofluorescence. The results (Fig. 5B) displayed a reduction in the fluorescence intensity of Bkα and Bkβ subunits in RASMCs after CIH modeling. Conversely, activation of BK channels substantially elevated the fluorescence intensity of Bkα and Bkβ in CIH-modeled RASMCs. To sum up, activating BK channels reduced intracellular Ca²⁺ concentration by upregulating α and β subunits, indirectly reducing vascular dysfunction.

Figure 5. BK channel activation increases Bk α and Bkβ subunits, indirectly inhibiting vascular dysfunction. (A, B) Measurement of intracellular Ca²⁺ concentration. (B) Immunofluorescence observation of fluorescence intensity of Bkα (red) and Bkβ (green) proteins. Scale bar = 50 μm. The data were expressed as mean ± standard deviation. Data comparison between two groups was performed with the unpaired student’s t-test, and data comparison among multiple groups was conducted with a one-way analysis of variance, with Tukey’s for post-hoc test. CIH, chronic intermittent hypoxia; DMSO, dimethyl sulfoxide. *p < 0.05 compared with the control group, ^#p < 0.05 compared with the DMSO + CIH group.

CIH is a risk factor for cardiovascular diseases because it affects cardiac systolic function and the function of coagulation-fibrinolysis system and endothelia in the vasculature [19]. For example, CIH can cause and exacerbate arteriosclerosis by participating in several processes including platelet activation, vascular endothelial injury, oxidative stress, and inflammation [20]. Accordingly, early intervention with CIH is beneficial for reducing vascular injury and treating cardiovascular diseases. However, little is known about the mechanisms underlying CIH. Our study explored the mechanism of CIH from the aspect of vascular dysfunction and our data demonstrated that the activation of BK channels facilitated vasorelaxation and depressed vascular endothelial dysfunction, oxidative stress, and inflammation, thereby indirectly mitigating CIH-induced vascular dysfunction.

CIH has been reported to be associated with increased blood pressure [21]. Our results showed that CIH modeling led to an elevation in SBP, DBP, and MBP of rats. Similarly, a prior study revealed that SBP and DBP were increased after CIH modeling in rats, suggesting the occurrence of hypertension [22]. The endothelium is critically involved in the mediation of vascular tone, and endothelium-dependent dilatations have been observed in multiple arteries of many mammalian species and can be affected by several factors, including increases in blood flow and hypoxia [23]. According to a prior study, CIH treatment reduced ACh-induced vasorelaxation in rats [24]. Consistently, our data showed that ACh-induced vasorelaxant responses of E+ rings were significantly lowered in the CIH group and that ACh-induced vasorelaxant responses were greatly higher in all E+ groups than in all E- groups. Furthermore, our H&E staining results revealed significant histopathological changes in the vascular endothelium of CIH-treated rats, including cellular edema and partial detachment of endothelial cells. These observations illustrated that CIH elicited vascular endothelial dysfunction.

Several studies unveiled that the opening probability of BK channels was diminished by CIH treatment in rats [13,25], indicating the potential involvement of decreased BK channel activity in CIH-induced vascular dysfunction. Our data showed that CIH modeling decreased BKα and BKβ levels in rats and RASMCs. Therefore, we determined the function of BK channels with the use of their agonist, NS1619, or inhibitor, Iberiotoxin, in rats and RASMCs. Endothelial function can be modulated by the enzymatic conversion of eNOS to NO, and oxidative stress can disturb the regulation of eNOS and induce endothelial dysfunction [26]. ET-1 can activate eNOS [27]. Endothelial dysfunction is associated with increased ET-1 but decreased NO and eNOS [28,29]. Interestingly, Wang et al. [30] discovered that IH increased ET-1 expression while decreasing NO and eNOS levels in rat pulmonary arteries, resulting in endothelial dysfunction. IL-6 and TNF-α are classical pro-inflammatory cytokines [31]. Under oxidative stress conditions, ROS is accumulated to induce lipid peroxidation and glycoxidation reactions, thus promoting the endogenous production of reactive aldehydes and their derivatives such as MDA [32]. CIH treatment increases MDA, IL-6, and TNF-α levels in mouse cardiac tissues [33]. Our study found that activating BK channels reduced ET-1, ROS, MDA, IL-6, and TNF-α levels while increasing NO and eNOS levels. BKβ1 mRNA expression is involved in the alleviatory effects of continuous positive airway pressure on endothelial dysfunction [34]. The activation of BK channels was demonstrated to participate in the protective effects of unoprostone against oxidative stress [35] and suppress TNF-α-induced inflammation in pulmonary endothelial cells [36]. BK channels are abundantly expressed on SMCs and modulate vascular tone [37]. In this case, RASMCs were used for CIH modeling in vitro to confirm the role of BK channels. The results showed that activating BK channels reduced intracellular Ca²⁺ concentrations in CIH-treated RASMCs. The result of a previous study demonstrated after the removal of an endothelium, the inhibitory effect of Ca²⁺ induced vasorelaxation by inhibition on BK channels can no longer be observed, indicating the stimulation of eNOS, NO and BK channels on vascular SMCs in the endothelium-dependent fashion [38]. Prior research has shown that BK channel activation contributes to SMC relaxation [39], and that decreased BK channel activity is associated with intracellular free Ca overload [40]. The possible mechanism related to the mediation of BK channels on vasorelaxation may include the activation of potassium channel. The rise in intracellular calcium concentration can lead to contraction of vascular smooth muscle. Actin-myosin interaction and cross-bridge creation subsequently occurred after the calcium concentration increased. Several potassium channels’ physiological functions play a crucial role in controlling the membrane potential and vascular tone. The activation of potassium channels inhibits voltage-gated calcium channels. As a result, vascular SMCs experience hyperpolarization and decreased intracellular calcium influx [41]. Although this study explores the alternations of intracellular Ca²⁺ concentration and BK channels in CIH-induced vascular dysfunction, but limited emphasis was put on determining how BK channels can mediate NO increasing and endothelium-dependent relaxation. As a result, additional studies are encouraged to conduct further research. Finally, our findings shed light on how BK channel activation may reduce CIH-induced vascular dysfunction. Specifically, BK channel activation suppressed vascular endothelial dysfunction by increasing NO and eNOS while decreasing ET-1, as well as reducing oxidative stress and inflammation. Meanwhile, BK channel activation exerts vasorelaxant effects by decreasing intracellular Ca²⁺ concentration via BKα and BKβ subunits. Our findings indicate BK channels as a potential mechanism in CIH-induced vascular dysfunction, thus providing a potential therapeutic target for CIH-induced cardiovascular disease. However, further studies are warranted due to the lack of clinical experiments.

None.

This study was supported by the Interventional Therapy Clinical Medical Research Center of Jiangxi Province (No.20223BCG74005).

The authors declare no conflicts of interest.