Introduction: Dimenhydrinate and scopolamine are frequently used drugs, but they cause drowsiness and performance decrement. Therefore, it is crucial to find peripheral targets and develop new drugs without central side effects. This study aimed to investigate the anti-motion sickness action and inner ear-related mechanisms of atrial natriuretic peptide (ANP). Methods: Endolymph volume in the inner ear was measured with magnetic resonance imaging and expression of AQP2 and p-AQP2 was detected with Western blot analysis and immunofluorescence method. Results: Both rotational stimulus and intraperitoneal arginine vasopressin (AVP) injection induced conditioned taste aversion (CTA) to 0.15% sodium saccharin solution and an increase in the endolymph volume of the inner ear. However, intraperitoneal injection of ANP effectively alleviated the CTA behaviour and reduced the increase in the endolymph volume after rotational stimulus. Intratympanic injection of ANP also inhibited rotational stimulus-induced CTA behaviour, but anantin peptide, an inhibitor of ANP receptor A (NPR-A), blocked this inhibitory effect of ANP. Both rotational stimulus and intraperitoneal AVP injection increased the expression of AQP2 and p-AQP2 in the inner ear of rats, but these increases were blunted by ANP injection. In in vitro experiments, ANP addition decreased AVP-induced increases in the expression and phosphorylation of AQP2 in cultured endolymphatic sac epithelial cells. Conclusion: Therefore, the present study suggests that ANP could alleviate motion sickness through regulating endolymph volume of the inner ear increased by AVP, and this action of ANP is potentially mediated by activating NPR-A and antagonising the increasing effect of AVP on AQP2 expression and phosphorylation.

Motion sickness is a temporary pathophysiological reaction during various modes of transportation or in virtual environments. The main manifestations include autonomic responses, vestibular paraesthesia, and central responses [1, 2]. Motion sickness affects many people travelling or working in special environments. Most people can have their symptoms resolved only through corrective measures or leaving these vehicles [1, 3]. The pathogenesis of motion sickness is still a controversial topic, and medications for the treatment of motion sickness are only partially effective and may have unwanted side effects [3, 4]. Currently, anticholinergic and antihistamine drugs are commonly used, but their side effects, such as central inhibition, are obvious [3, 4]. Therefore, further research is necessary to understand the pathogenesis of motion sickness, identify more suitable targets, and develop new anti-motion sickness drugs without central side effects.

An increasing amount of evidence suggests that motion sickness is associated with a significant increase in plasma arginine vasopressin (AVP) levels [5‒7]. Both intravenous and intracerebroventricular injections of AVP have been shown to induce motion sickness-like symptoms in humans and animals [5‒7]. The increase in plasma AVP levels is thought to be the triggering factor for motion sickness [6, 7]. Activation of the AVP-vasopressin receptor 2 (V2R)-cAMP signalling pathway in the inner ear by rotational stimulation and intraperitoneal injection of AVP may lead to increased expression of AQP2 and p-AQP2 [5]. This in turn can lead to increased production of inner ear endolymph [8] and decreased reabsorption, and an endolymphatic imbalance will induce motion sickness-like symptoms, as in Ménière’s disease [9, 10].

Atrial natriuretic peptide (ANP) is primarily synthesised by atrial myocytes. It increases the glomerular filtration rate in the kidney, inhibits renal tubular reabsorption, and enhances renal drainage and sodium excretion. Natriuretic peptide receptors are categorised into type A (NPR-A), type B (NPR-B), and type C (NPR-C). Among these, ANP has a high affinity for NPR-A [11]. In the kidney, AVP has an antidiuretic effect, while ANP has a natriuretic and diuretic effect. Consequently, AVP and ANP have somewhat opposing effects in the kidney [12, 13], i.e., ANP may inhibit the impact of AVP on water and ion transport in the kidney. To date, many studies have suggested that NPR-A receptors are present in the inner ear [14, 15]. Therefore, ANP may also have some opposite effects on endolymph homoeostasis of the inner ear. Given the background of AVP promoting motion sickness through its action on the inner ear [5], we supposed that ANP might have an anti-motion sickness action. We thus proposed a hypothesis that ANP would regulate the water balance of the endolymph through the NPR-A pathway in the inner ear, especially through effects on the expression and phosphorylation of the downstream protein AQP2, and thus inhibit the development of motion sickness by counteracting the action of AVP on the endolymph homoeostasis in the inner ear.

Animals and Chemicals

Sprague‒Dawley rats (body weight 200–220 g) and guinea pigs (body weight 300–350 g) of both sexes and rat pups at day 10 after birth were obtained from the Experimental Animal Center of Nantong University, Nantong, China. Adult animals were fed under 12-h light/12-h dark cycles (light, 08:00–20:00 h; darkness, 20:00–08:00 h) at room temperature (22–24°C) with free access to standard food and tap water. All procedures used in this study were in accordance with our institutional guidelines, which comply with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and approved by the Institutional Animal Care and Use Committee, Nantong University, approval number (S20220222-025) for guinea pigs and (S20220222-027) for rats.

Common inorganic salts were purchased in China. Culture medium was purchased from Invitrogen (Carlsbad, USA). Anantin peptide was purchased from Biorbyt (Cambridge, UK). The following primary antibodies were purchased: monoclonal anti-β-actin from Sigma-Aldrich (Saint Louis, USA), and anti-p-AQP2 Ser256 from Abcam Trading (Shanghai) Company Ltd. (China), anti-AQP2 from Santa Cruz Biotechnology, Inc. (Dallas, USA). AVP, desmopressin acetate (ddAVP), mozavaptan hydrochloride, ANP, poly-D-lysine, cytosine β-D-arabinofuranoside, sodium dodecyl sulfate, and other chemicals except those indicated elsewhere were purchased from Sigma-Aldrich.

Motion Sickness-Provoking Rotational Stimulus

Conditioned taste aversion (CTA) was used as a behavioural index for motion sickness in rats and guinea pigs. A rotational stimulator for animal use was manufactured according to the report by Crampton and Lucot [16]. For the induction of CTA, all animals were rotated for a total time of 120 min in the stimulator in alternating acceleration and deceleration modes as shown in our previous study [17]. Before the rotational stimulus, in addition to tap water, the animals were also supplied with 0.15% sodium saccharin solution (SSS) as a novel fluid for 48 h of drinking. When the rotational stimulus was completed, a supply of SSS was maintained, and the intake volume of SSS per 24 h was measured.

Drug Tests in Rats for Motion Sickness

ANP (400 μg/kg) and scopolamine (80 μg/kg) were delivered via intraperitoneal injection 1 h before the 2-h rotational stimulus, and the control rats received an equal injection volume of solvent (2 mL/kg normal saline). To observe the local effect of ANP on the inner ear, ANP was administered via intratympanic injection under ether anaesthesia, and half of the ANP (200 μg/kg) was injected into each side of the middle ear 1 h before the rotational stimulus. The rotation group received an equal volume of saline (50 μL) as a control. To identify whether the anti-motion sickness effect of ANP is mediated via its receptor NPR-A, anantin peptide, an inhibitor of NPR-A was injected intraperitoneally at a dose of 1.2 mg/kg before the use of ANP. The control and rotation groups received an equal volume of solvent for anantin peptide and ANP. Anantin peptide was dissolved in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide was 0.1%. ANP was dissolved in normal saline.

CTA Induction via Intraperitoneal Injection of AVP and Inhibition by ANP and V2R Antagonist

To induce CTA in rats, AVP was intraperitoneally injected at doses of 100, 200, and 400 μg/kg. In addition to drinking water, 0.15% SSS was supplied for the rats to drink for 2 days before and 2 days after AVP injection. The mean intake volume of 0.15% SSS before and after AVP injection was measured. A dose of AVP 200 μg/kg was selected for the following experiment according to the CTA behaviours of the rats. Additionally, ANP was injected intraperitoneally at doses of 200 and 400 μg/kg. Control rats were injected intraperitoneally with an equal volume of solvent (2 mL/kg normal saline).

CTA induction in guinea pigs was the same as in rats described above, but AVP was replaced with ddAVP (1.6 mg/kg, i.p.), an agonist of AVP-V2R and mozavaptan (11.1 mg/kg, i.p.), an antagonist of AVP-V2R, was used 1 h before ddAVP injection.

Swimming Test for Evaluating Vestibular Function

The vestibular function of guinea pigs was assessed using curvature of swim trajectory in a Morris water maze device. The swimming test was performed at a fixed day time. The device is a circular black pool with a height of 60 cm and a diameter of 180 cm, a hidden platform, and a video/computer system. The maze was filled with water at a temperature of 21–25°C to a depth of 40 cm before every trial. Guinea pigs were habituated to the behaviour test room for at least 30 min before starting the test. Five training trials were performed per day for five consecutive days. For each training trial, the hidden platform was removed and each animal was put into the pool at the central point with its head towards the wall of the pool. For each of the 5 training trials, 5 tracings of the head of each animal were recorded using the ANY maze software (Stoelting Company, USA). After each trial, the animals were dried. If an animal will not swim in a straight line within 5 trials on the fifth day, it will be abandoned for the next experiment. All animals showing normal swim trajectory were divided into three groups, namely, control, rotation, and rotation plus ANP, or control, ddAVP, and ddAVP plus mozavaptan. The treatment trials were performed on the sixth day. The open-source Fiji plugin named kappa for measuring curvature was used to calculate the trajectories of swim.

Measurement of Endolymph Volume

Magnetic resonance imaging (MRI) scans on the longitudinal sagittal plane of the head were performed under general anaesthesia. Gadopentetate dimeglumine solution (280 mg/kg, Consum Pharmacy, Guangzhou, China) was intravenously injected 90 min before anaesthesia. All guinea pigs were imaged on a 3.0 Tesla MR scanner (Magnetom Verio, Siemens Healthcare, Erlangen, Germany) with a 4-channel animal head coil. We chose the T2WI-DRIVE-HR sequence (repetition time/echo time, 1,550 ms/251.8 ms; matrix, 124 × 149; flip angle, 90°; slice thickness, 0.2 mm with a total of 200 slices; average scanning time, 21 min 31 s; and field of view, 50 mm × 50 mm) to evaluate the anatomy of whole fluid-filled spaces. The highlighted part of the inner ear is perilymph, and the shaded part is endolymph due to the difficulty of the gadolinium contrast agent entering the endolymph. Data were processed using ImageJ Fiji. A region of interest around the inner ear was cropped via a square bounding box on the full head MRI images. Using ImageJ Fiji, area measurement of the needed target area was performed, and the proportion of endolymph volume to the total lymph volume in the cochlear part was finally calculated. ANP was injected 1 h before rotational stimulus, and MRI scans were started 1 h after rotational stimulus. Additionally, ddAVP was used, MRI scans were performed 3 h after ddAVP use, and mozavaptan was injected 1 h before ddAVP use.

Culture of the Endolymphatic Sac Epithelial Cells

Epithelial cell culture of the endolymphatic sac was conducted based on other reports [18, 19]. Rat pups at day 10 after birth were used for primary culture. After ether anaesthesia, the temporal bones were removed and placed in a cold (4°C) endolymph-like solution containing: 150 mM KCl, 5 mM NaCl, 2 mM KH2PO4, 1 mM MgCl2, 3 mM glucose, 25 mM HEPES, and 1 mg/mL albumin. The endolymphatic sacs were carefully isolated with the aid of a dissecting microscope, divided into small pieces, transferred to culture dishes, and attached as explants to poly-lysine-coated cover slips with the gentle pressure of a scalpel. The explants were cultured for 5 days at 37°C in a humidified 5% CO2 incubator. The culture medium used was DMEM with the addition of 15% foetal calf serum, penicillin (85 IU/mL), streptomycin (85 μg/mL), and EGF (100 μg/mL). Half of the culture medium was changed every 3 days. On day 6 in vitro, the explants were digested with 0.125% trypsin, dispersed gently with a heat-polished pipette, and then cultured with the above culture medium containing BrdU (0.1 mmol/L). On day 11 in vitro, the cultures were digested once more with 0.125% trypsin containing EDTA∙4Na (190 mg/L, Gibco, Shanghai, China) and centrifuged at 1,500 rpm for 4 min. The supernatants were discarded, and the cells were resuspended with the above culture medium and seeded on poly-lysine-coated cover slips or culture dishes. On day 15–16 in vitro, cultured epithelial cells from the endolymphatic sac were used for the following experiments.

Western Blot Analysis

The inner ears and endolymphatic sacs isolated from the rat temporal bones or the cultured epithelial cells from the endolymphatic sacs were lysed in tissue or cell lysis buffer containing phenylmethanesulfonyl fluoride (Beyotime, Nantong, China), homogenised, and centrifuged at 14,000 rpm for 20 min at 4°C. The protein contents of the supernatants were determined spectrophotometrically using the bicinchoninic acid method. Equal amounts of protein (40 μg per lane) from each sample were loaded on a 10% sodium dodecyl sulfate-polyacrylamide gel, electrophoresed, and transferred onto polyvinylidene difluoride membranes (Merck Millipore, Temecula, USA). These membranes were blocked in 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 for 2 h at room temperature and then incubated overnight at 4°C with primary antibodies against AQP2 (1:100), p-AQP2 (1:300), and β-actin (1:5,000). Finally, the membranes were incubated with a secondary antibody (1:6,000, Bioworld, Nanjing, China) conjugated with horseradish peroxidase for 2 h at room temperature, and the immunoreactive bands were visualised by enhanced chemiluminescent agents (Pierce Biotechnology, Rockford, USA) and captured using a Tanon 5200 multisystem (Tanon, Shanghai, China). The expression level of each protein was quantified using ImageJ analysis software and normalised to the β-actin value in the same lane. The same samples were repeatedly analysed at least two times.

Cellular Immunofluorescence Imaging

The cultured epithelial cells from the endolymphatic sacs were fixed with 4% paraformaldehyde at room temperature for 15 min and then incubated with precooled methanol at −20°C for 8 min. After incubation with 10% bovine serum albumin for 1 h, the cells were incubated with primary antibodies at different dilutions overnight at 4°C. Simultaneously, a negative control was performed without each primary antibody. For immunofluorescence imaging of target molecule expression, the cultured epithelial cells were incubated with donkey anti-rabbit Alexa-488 secondary antibodies (1:1,000, Jackson, West Grove, USA) for 1–2 h at room temperature. The immunostaining images were collected using a laser confocal microscope (TCS SP8, Leica Microsystems, Wetzlar, Germany) at room temperature and processed with LASX 2.0 software.

Statistical Analysis

All data are presented as the mean ± SEM. Student’s t test was used for the comparison of two groups. One-way ANOVA was used for the comparison of data from three or more group design experiments with the least significant difference test for post hoc comparisons. Differences were considered statistically significant at a level of p < 0.05.

Anti-Motion Sickness Action of ANP

Rotational stimulus significantly reduced the amount of SSS consumed by the rats, but intraperitoneal injection of scopolamine (Sco.), a positive control drug, and ANP significantly ameliorated this CTA behaviour (Fig. 1a). Similarly, intratympanic injection of ANP also alleviated CTA behaviour after rotational stimulus (Fig. 1b). However, intraperitoneal injection of anantin peptide, an inhibitor of the ANP receptor NPR-A, blocked this inhibition of intratympanic ANP injection on rotational stimulus-induced CTA behaviour (Fig. 1b). Moreover, AVP also induced CTA, i.e., the consumption of SSS was significantly reduced by AVP at 200 and 400 μg/kg (Fig. 1c). ANP at 400 µg/kg also significantly ameliorated AVP-induced CTA behaviour, whereas the effect of ANP at 200 µg/kg was not significant (p > 0.05, Fig. 1d). The above results suggest that ANP exerts an antagonistic effect on motion sickness, but this effect could be counteracted by an NPR-A inhibitor.

Fig. 1.

Anti-motion sickness action of ANP. a Influence of intraperitoneal injection (i.p.) of ANP on the intake of 0.15% SSS by rats after rotational stimulus (n = 12). b Influence of intratympanic injection of ANP on the intake of 0.15% SSS by rats after rotational stimulus and blockage of this action by pretreatment with anantin peptide (i.p.), an NPR-A inhibitor (n = 10–12). c Changes in the intake of 0.15% SSS by rats that received different doses of AVP (i.p., n = 12). d Influence of ANP pretreatment (i.p.) on the intake of 0.15% SSS by rats after systemic treatment (i.p.) of AVP (n = 12).

Fig. 1.

Anti-motion sickness action of ANP. a Influence of intraperitoneal injection (i.p.) of ANP on the intake of 0.15% SSS by rats after rotational stimulus (n = 12). b Influence of intratympanic injection of ANP on the intake of 0.15% SSS by rats after rotational stimulus and blockage of this action by pretreatment with anantin peptide (i.p.), an NPR-A inhibitor (n = 10–12). c Changes in the intake of 0.15% SSS by rats that received different doses of AVP (i.p., n = 12). d Influence of ANP pretreatment (i.p.) on the intake of 0.15% SSS by rats after systemic treatment (i.p.) of AVP (n = 12).

Close modal

ANP Blockage of Rotation-Induced Increase in Endolymph Volume

MRI enhanced with gadolinium showed that the endolymph volume of the inner ear in guinea pigs was increased significantly after rotation stimulation, and ANP treatment blocked this increase (Fig. 2a, b). Moreover, ddAVP, an agonist of AVP-V2R, also induced an increase in the endolymph volume, and mozavaptan, an antagonist of V2R, blocked this increase (Fig. 2f–g). Due to the technical limits, we could not measure the endolymph volume of the inner ear in rats. To make up for this deficiency, we also observed the anti-motion sickness effect of ANP in guinea pigs. We found that ANP at 400 µg/kg significantly ameliorated rotation-induced CTA behaviour and increase in the curvature of swim trajectory (Fig. 2c–e). This anti-motion sickness effect of ANP in guinea pigs is consistent with that in rats. In addition, mozavaptan also reduced ddAVP-induced CTA behaviour and increase in the curvature of swim trajectory (Fig. 2h–j).

Fig. 2.

ANP inhibition of the rotation-induced increase in the endolymph volume of guinea pig inner ears. a Examples of MRI enhanced with gadolinium before or after rotational stimulus and after ANP pretreatment. b Mean values of endolymph volume (n = 5). c Influence of ANP injection (i.p.) on the intake of 0.15% SSS by guinea pigs after rotational stimulus (n = 12). d Examples of swim trajectory in a Morris water maze in animals of each group. e Influence of ANP injection (i.p.) on the curvature of swim trajectory after rotational stimulus (n = 6). f Examples of MRI enhanced with gadolinium before or after ddAVP use and after mozavaptan pretreatment. g Mean values of endolymph volume (n = 5). h Influence of mozavaptan (Moz.) injection (i.p.) on the intake of 0.15% SSS by guinea pigs after ddAVP injection (i.p., n = 12). i Examples of swim trajectory in a Morris water maze in animals of each group. j Influence of mozavaptan injection (i.p.) on the curvature of swim trajectory after ddAVP injection (i.p., n = 6). Upper row images in (a) and (f) reveal examples of the cochlea and vestibule of each group, and the lower row images were amplification of upper cochlea images. The white arrow in the lower left image of (a, f) indicates the endolymph space (the shaded area in black), and the arrowhead indicates the perilymph space (the enhanced area in white). Scale bar, 1 mm.

Fig. 2.

ANP inhibition of the rotation-induced increase in the endolymph volume of guinea pig inner ears. a Examples of MRI enhanced with gadolinium before or after rotational stimulus and after ANP pretreatment. b Mean values of endolymph volume (n = 5). c Influence of ANP injection (i.p.) on the intake of 0.15% SSS by guinea pigs after rotational stimulus (n = 12). d Examples of swim trajectory in a Morris water maze in animals of each group. e Influence of ANP injection (i.p.) on the curvature of swim trajectory after rotational stimulus (n = 6). f Examples of MRI enhanced with gadolinium before or after ddAVP use and after mozavaptan pretreatment. g Mean values of endolymph volume (n = 5). h Influence of mozavaptan (Moz.) injection (i.p.) on the intake of 0.15% SSS by guinea pigs after ddAVP injection (i.p., n = 12). i Examples of swim trajectory in a Morris water maze in animals of each group. j Influence of mozavaptan injection (i.p.) on the curvature of swim trajectory after ddAVP injection (i.p., n = 6). Upper row images in (a) and (f) reveal examples of the cochlea and vestibule of each group, and the lower row images were amplification of upper cochlea images. The white arrow in the lower left image of (a, f) indicates the endolymph space (the shaded area in black), and the arrowhead indicates the perilymph space (the enhanced area in white). Scale bar, 1 mm.

Close modal

Inhibitory Effects of ANP on the Expression and Phosphorylation of AQP2 in the Inner Ear

Both rotational stimulus and AVP significantly increased the expression of AQP2 and p-AQP2 proteins (Fig. 3a–d). However, ANP injection (i.p.) reduced both rotational stimulus- and AVP-induced increases in the expression of AQP2 and p-AQP2 (Fig. 3a–d) while ANP itself also inhibited the expression of AQP2 and p-AQP2 (Fig. 3e–f).

Fig. 3.

Inhibitory effects of ANP on the expression and phosphorylation levels of AQP2 in the inner ear of rats after rotational stimulus and AVP injection (i.p.). a Western blot analysis for AQP2 and p-AQP2 after rotational stimulus and ANP use. b Mean values of the expression of AQP2 and p-AQP2 after rotational stimulus and ANP use (n = 6). c Western blot analysis for AQP2 and p-AQP2 after AVP injection and ANP use (i.p.). d Mean values of the expression of AQP2 and p-AQP2 after AVP injection and ANP use (i.p., n = 6). e Western blot analysis for AQP2 and p-AQP2 after ANP injection (i.p.). f Mean values of the expression of AQP2 and p-AQP2 after ANP injection (i.p., n = 6).

Fig. 3.

Inhibitory effects of ANP on the expression and phosphorylation levels of AQP2 in the inner ear of rats after rotational stimulus and AVP injection (i.p.). a Western blot analysis for AQP2 and p-AQP2 after rotational stimulus and ANP use. b Mean values of the expression of AQP2 and p-AQP2 after rotational stimulus and ANP use (n = 6). c Western blot analysis for AQP2 and p-AQP2 after AVP injection and ANP use (i.p.). d Mean values of the expression of AQP2 and p-AQP2 after AVP injection and ANP use (i.p., n = 6). e Western blot analysis for AQP2 and p-AQP2 after ANP injection (i.p.). f Mean values of the expression of AQP2 and p-AQP2 after ANP injection (i.p., n = 6).

Close modal

Inhibitory Effects of ANP on the Expression and Phosphorylation of AQP2 in Cultured Endolymphatic Sac Epithelial Cells

Time-dependent and dose-dependent influences of ANP treatment on the expression and phosphorylation of AQP2 were investigated in cultured endolymphatic sac epithelial cells. The expression of AQP2 and p-AQP2 was significantly decreased after 1 h of ANP treatment at 100 and 1,000 nm, respectively (Fig. 4a–d). Similar results of ANP treatment on the expression of AQP2 were obtained using cellular immunofluorescence imaging (Fig. 4e–f). Moreover, after ANP treatment (100 nm) for different durations, a significant decrease was found after 30 min and 60 min of ANP treatment (Fig. 5a–d). Similar effects of ANP treatment on the expression of AQP2 were also found using cellular immunofluorescence imaging (Fig. 5e–f).

Fig. 4.

Dose-dependent effect of 1-h ANP treatment on the expression and phosphorylation levels of AQP2 in cultured endolymphatic sac epithelial cells. a Western blot analysis for AQP2. b Mean values of AQP2 expression (n = 4). c Western blot analysis for p-AQP2. d Mean values of p-AQP2 expression (n = 4). e Immunofluorescence images of AQP2 expression. f Mean fluorescence intensity for AQP2 (n = 4).

Fig. 4.

Dose-dependent effect of 1-h ANP treatment on the expression and phosphorylation levels of AQP2 in cultured endolymphatic sac epithelial cells. a Western blot analysis for AQP2. b Mean values of AQP2 expression (n = 4). c Western blot analysis for p-AQP2. d Mean values of p-AQP2 expression (n = 4). e Immunofluorescence images of AQP2 expression. f Mean fluorescence intensity for AQP2 (n = 4).

Close modal
Fig. 5.

Time-dependent effect of ANP treatment (100 nM) on the expression and phosphorylation levels of AQP2 in cultured endolymphatic sac epithelial cells. a Western blot analysis for AQP2. b Mean values of AQP2 expression (n = 4). c Western blot analysis for p-AQP2. d Mean values of p-AQP2 expression (n = 4). e Immunofluorescence images of AQP2 expression. f Mean fluorescence intensity for AQP2 (n = 4).

Fig. 5.

Time-dependent effect of ANP treatment (100 nM) on the expression and phosphorylation levels of AQP2 in cultured endolymphatic sac epithelial cells. a Western blot analysis for AQP2. b Mean values of AQP2 expression (n = 4). c Western blot analysis for p-AQP2. d Mean values of p-AQP2 expression (n = 4). e Immunofluorescence images of AQP2 expression. f Mean fluorescence intensity for AQP2 (n = 4).

Close modal

ANP Blockage of AVP-Induced Increases in the Expression and Phosphorylation of AQP2

Furthermore, we investigated the expression and phosphorylation of AQP2 in cultured endolymphatic sac epithelial cells after cotreatment with ANP and AVP. Western blot analysis showed that the expression of AQP2 and p-AQP2 was significantly increased after 1 h of AVP treatment at 10 nm, whereas ANP at 100 nm blocked this increase induced by AVP at 10 nm (Fig. 6a–d). Similar results of ANP treatment on the expression of AQP2 were obtained using cellular immunofluorescence imaging (Fig. 6e–f).

Fig. 6.

ANP blocked the AVP-induced increase in the expression and phosphorylation levels of AQP2 in cultured endolymphatic sac epithelial cells. a Western blot analysis for AQP2. b Mean values of AQP2 expression (n = 4). c Western blot analysis for p-AQP2. d Mean values of p-AQP2 expression (n = 4). e Immunofluorescence images of AQP2 expression. f Mean fluorescence intensity for AQP2 (n = 4).

Fig. 6.

ANP blocked the AVP-induced increase in the expression and phosphorylation levels of AQP2 in cultured endolymphatic sac epithelial cells. a Western blot analysis for AQP2. b Mean values of AQP2 expression (n = 4). c Western blot analysis for p-AQP2. d Mean values of p-AQP2 expression (n = 4). e Immunofluorescence images of AQP2 expression. f Mean fluorescence intensity for AQP2 (n = 4).

Close modal

In the present study, motion sickness in rats was induced by rotational stimulus and intraperitoneal injection of AVP. CTA to sodium saccharin solution was selected as a marker of this sickness. After rotational stimulus and intraperitoneal injection of AVP, the consumption of sodium saccharin solution in rats was significantly reduced. The present motion sickness-inductive action of AVP further supports the viewpoint that AVP is potentially a predisposing factor for motion sickness [6, 7]. This CTA behaviour induced after rotational stimulus or AVP injection was significantly alleviated by pretreatment with scopolamine, a positive control drug for motion sickness, and intraperitoneal and transtympanic injections of ANP, and this effect of ANP was blunted by the NPR-A antagonist. These results suggest that ANP could exert an effective anti-motion sickness action, potentially through NPR-A in the inner ear. Many studies have shown that ANP inhibits sodium and urine absorption, promotes urine production and sodium excretion, and antagonises the effect of AVP on water and ion transport in the kidney [12, 13]. In the present study, we found an antagonistic effect on AVP-induced motion sickness behaviour by intraperitoneal injection of ANP, suggesting that ANP may play an anti-motion sickness role through an effect opposite to AVP in the inner ear.

To further verify the underlying mechanism of the anti-motion sickness action of ANP, we investigated changes in inner ear-related variables after rotational stimulus or AVP injection and influences of ANP injection. We found that rotational stimulus induced an increase in the endolymph volume of the inner ear in guinea pigs, but intraperitoneal injection of ANP inhibited this increase. Moreover, intraperitoneal injection of ddAVP, an AVP-V2R agonist, also induced an increase in the endolymph volume, and mozavaptan, an AVP-V2R antagonist, reduced this action of AVP. However, there is a gap between the results from rats and guinea pigs because we did not measure the endolymph volume in the rats due to our technical limits. To make up for this deficiency, we also observed the anti-motion sickness effect of ANP in guinea pigs and found that ANP reduced both rotational stimulus-induced CTA behaviour and changes in the swim trajectory. It is consistent with the anti-motion sickness effect of ANP in the rats. In addition, injection of ddAVP also induced CTA behaviour and increase in the curvature of swim trajectory, and mozavaptan reduced ddAVP-induced CTA behaviour and changes in the swim trajectory, being consistent with the above results of the changes in the endolymph volume in the guinea pigs.

Furthermore, we observed the expression of AQP2 and phosphorylation level at the S256 site of AQP2 in the inner ear of rats after rotational stimulus and AVP injection because increasing evidence suggests that the expression and phosphorylation of AQP2 and modulation by AVP are involved in modulation of endolymph homoeostasis in the inner ear [20, 21]. AVP has been reported to increase the expression and phosphorylation at the S256 site of AQP2, as well as translocation to the apical membrane and via the AVP-V2R-cAMP-PKA pathway in the inner ear [5, 20, 21]. The present results revealed that rotational stimulus and AVP increased the expression and phosphorylation level of AQP2 in the inner ear, but ANP inhibited the expression of AQP2 and p-AQP2 and reduced rotation- or AVP-induced increases in the expression of AQP2 and p-AQP2. Moreover, we examined the influence of ANP on the expression and phosphorylation level of AQP2 in cultured endolymphatic sac epithelial cells because the endolymphatic sac, where AQP2 and V2R are expressed [22], is an important structure for the endolymph homoeostasis of the inner ear and is regulated by AVP [23, 24]. ANP reduced the expression and phosphorylation level of AQP2 in cultured endolymphatic sac epithelial cells in a dose- and time-dependent manner. In addition, ANP inhibited the AVP-induced increase in the expression and phosphorylation level of AQP2 in cultured endolymphatic sac epithelial cells, consistent with the result in cultured inner medullary collecting duct cells of rat kidney [13]. These results suggest that ANP could reduce the endolymph volume of the inner ear after rotational stimulus potentially by inhibiting the expression and phosphorylation level of AQP2 and especially the AVP-induced increase and may thus contribute to its anti-motion sickness action.

As many reports have suggested, water homoeostasis of the inner ear is regulated in part via the AVP-AQP2 system in the same fashion as in the kidney [20, 21]. Regulation of AVP on the expression and phosphorylation of AQP2 through V2R-AC-PKA signalling pathway is a special case, where PKA not only phosphorylates the AQP2 but also promotes the expression of AQP2 through phosphorylation of CREB and resultant transcriptional activation [20]. Present results are consistent with previous reports. AVP promotes both the expression and phosphorylation of AQP2, and the relative proportion of pAQP2/AQP2 may not change. The role AQP2 plays depends on the content of pAQP2 membrane distribution, and in the case of AVP regulation on the effect of AQP2, we thus considered the content of pAQP2 and the total expression of AQP2, but not the relative proportion of pAQP2/AQP2, and increase in the content of AQP2 will afford a basic condition for the increase of pAQP2. Therefore, present study did not calculate the relative proportion of pAQP2/AQP2.

The distributions of ANP and its receptors have been found in the inner ear [14, 15, 25]; however, the functions of ANP in the inner ear have not been investigated extensively [26, 27]. Luo et al. [28] reported that ANP reduces mRNA expression of the α-subunit of the epithelial sodium channel in the mouse stria vascularis, suggesting a potential effect of ANP on the ion and water balance and endolymph homoeostasis in the inner ear. The present result is a direct suggestion of ANP function in the modulation of inner ear endolymph homoeostasis. Moreover, the present study suggests that future drug development for anti-motion sickness could consider V2R antagonists targeting the receptors of AVP [5] or NPR-A agonists targeting the receptors of ANP in the inner ear to avoid the central side effects of present drugs.

In conclusion, the present results suggest that ANP could play an anti-motion sickness role by regulating the expression of AQP2 and its phosphorylation in the inner ear through the NPR-A signalling pathway, which in turn regulates the endolymph homoeostasis. Therefore, the present study preliminarily provides a theoretical basis for the development of anti-motion sickness drugs that target the receptors of AVP (V2R antagonist) and ANP (NPR-A agonist) in the inner ear.

All procedures of animal experiments in this study were in accordance with the ARRIVE guidelines and were approved by the Animal Care and Use Committee of Nantong University, Nantong, China.

The authors declare no conflict of interest.

This work was supported by grants from the National Natural Science Foundation of China (No. 81671859 and 82171869).

L.-H.X., J.-G.G., X.Z., X.L., and Z.L.J. designed research; L.-H.X., J.-G.G., S.-F.X., W.J., Y.-Q.M., Q.-C.L., J.-Y.S., X.-Y.Z., and M.-L.C. performed research; L.-H.X., J.-G.G., S.-F.X., X.Z., and Z.-L.J. analysed data; and L.-H.X., X.Z., and Z.-L.J. wrote the manuscript.

Additional Information

Li-Hua Xu, Jian-Gang Ge, and Shui-Feng Xiao contributed equally to this work.

All data generated or analysed during this study are included in this article. Further enquiries can be directed to the corresponding author.

1.
Cha
YH
,
Golding
JF
,
Keshavarz
B
,
Furman
J
,
Kim
JS
,
Lopez-Escamez
JA
, et al
.
Motion sickness diagnostic criteria: consensus document of the classification committee of the barany society
.
J Vest Res
.
2021
;
31
(
5
):
327
44
.
2.
Golding
JF
.
Motion sickness
.
Handb Clin Neurol
.
2016
;
137
:
371
90
.
3.
Keshavarz
B
,
Golding
JF
.
Motion sickness: current concepts and management
.
Curr Opin Neurol
.
2022
;
35
(
1
):
107
12
.
4.
Rahimzadeh
G
,
Tay
A
,
Travica
N
,
Lacy
K
,
Mohamed
S
,
Nahavandi
D
, et al
.
Nutritional and behavioral countermeasures as medication approaches to relieve motion sickness: a comprehensive review
.
Nutrients
.
2023
;
15
(
6
):
1320
.
5.
Xu
LH
,
Yang
Y
,
Liu
HX
,
Xiao
SF
,
Qiu
WX
,
Wang
JX
, et al
.
Inner ear arginine vasopressin-vasopressin receptor 2-aquaporin 2 signaling pathway is involved in the induction of motion sickness
.
J Pharmacol Exp Ther
.
2020
;
373
:
248
60
.
6.
Cheung
BS
,
Kohl
RL
,
Money
KE
,
Kinter
LB
.
Etiologic significance of arginine vasopressin in motion sickness
.
J Clin Pharmacol
.
1994
;
34
(
6
):
664
70
.
7.
Kim
MS
,
Chey
WD
,
Owyang
C
,
Hasler
WL
.
Role of plasma vasopressin as a mediator of nausea and gastric slow wave dysrhythmias in motion sickness
.
Am J Physiol
.
1997
;
272
(
4 Pt 1
):
G853
62
.
8.
Degerman
E
,
in’t Zandt
R
,
Pålbrink
AK
,
Magnusson
M
.
Vasopressin induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4 T MRI
.
Hear Res
.
2015
;
330
(
Pt A
):
119
24
.
9.
Frejo
L
,
Lopez-Escamez
JA
.
Recent advances in understanding molecular bases of Ménière’s disease
.
Fac Rev
.
2023
;
12
:
11
.
10.
Chabbert
C
.
Pathophysiological mechanisms at the sources of the endolymphatic hydrops, and possible consequences
.
J Vestib Res
.
2021
;
31
(
4
):
289
95
.
11.
Pandey
KN
.
Guanylyl cyclase/natriuretic peptide receptor-A: identification, molecular characterization, and physiological genomics
.
Front Mol Neurosci
.
2022
;
15
:
1076799
.
12.
Dillingham
MA
,
Anderson
RJ
.
Inhibition of vasopressin action by atrial natriuretic factor
.
Science
.
1986
;
231
(
4745
):
1572
3
.
13.
Klokkers
J
,
Langehanenberg
P
,
Kemper
B
,
Kosmeier
S
,
von Bally
G
,
Riethmüller
C
, et al
.
Atrial natriuretic peptide and nitric oxide signaling antagonizes vasopressin-mediated water permeability in inner medullary collecting duct cells
.
Am J Physiol Ren Physiol
.
2009
;
297
(
3
):
F693
703
.
14.
Lamprecht
J
,
Meyer zum Gottesberge
AM
.
The presence and localization of receptors for atrial natriuretic peptide in the inner ear of the Guinea pig
.
Arch Oto-Rhino-Laryngol
.
1988
;
245
(
5
):
300
1
.
15.
Long
LL
,
Tang
YD
,
Xia
QJ
,
Xia
ZL
,
Liu
J
.
Detection of atrial natriuretic peptide receptor in the labyrinth of the mouse inner ear
.
Neuroendocrinol Lett
.
2008
;
29
(
4
):
577
80
.
16.
Crampton
GH
,
Lucot
JB
.
A stimulator for laboratory studies of motion sickness in cats
.
Aviat Space Environ Med
.
1985
;
56
:
462
5
.
17.
Li
X
,
Jiang
ZL
,
Wang
GH
,
Fan
JW
.
Plasma vasopressin, an etiologic factor of motion sickness in rat and human
.
Neuroendocrinology
.
2005
;
81
(
6
):
351
9
.
18.
Agrup
C
,
Berggren
PO
,
Bagger-Sjoback
D
.
Morphological and functional characteristics of cells cultured from the endolymphatic sac
.
Hear Res
.
2001
;
157
(
1–2
):
43
51
.
19.
Kumagami
H
,
Terakado
M
,
Sainoo
Y
,
Baba
A
,
Fujiyama
D
,
Fukuda
T
, et al
.
Expression of the osmotically responsive cationic channel TRPV4 in the endolymphatic sac
.
Audiol Neurotol
.
2009
;
14
(
3
):
190
7
.
20.
Takeda
T
,
Taguchi
D
.
Aquaporins as potential drug targets for Méniere’s disease and its related diseases
.
Handb Exp Pharmacol
.
2009
;
190
:
171
84
.
21.
Takeda
T
,
Takeda
S
,
Kakigi
A
,
Okada
T
,
Nishioka
R
,
Taguchi
D
, et al
.
Hormonal aspects of Ménière’s disease on the basis of clinical and experimental studies
.
ORL J Otorhinolaryngol Relat Spec
.
2010
;
71
(
Suppl 1
):
1
9
.
22.
Asmar
MH
,
Gaboury
L
,
Saliba
I
.
Ménière’s disease pathophysiology: endolymphatic sac immunohistochemical study of aquaporin-2, V2R vasopressin receptor, NKCC2, and TRPV4
.
Otolaryngol Head Neck Surg
.
2018
;
158
(
4
):
721
8
.
23.
Sawada
S
,
Takeda
T
,
Kitano
H
,
Takeuchi
S
,
Kakigi
A
,
Azuma
H
.
Aquaporin-2 regulation by vasopressin in the rat inner ear
.
Neuroreport
.
2002
;
13
:
1127
9
.
24.
Maekawa
C
,
Kitahara
T
,
Kizawa
K
,
Okazaki
S
,
Kamakura
T
,
Horii
A
, et al
.
Expression and translocation of aquaporin-2 in the endolymphatic sac in patients with Méniere’s disease
.
J Neuroendocrinol
.
2010
;
22
(
11
):
1157
64
.
25.
Yoon
YJ
,
Anniko
M
.
Distribution of alpha-ANP in the cochlea and the vestibular organs
.
ORL J Otorhinolaryngol Relat Spec
.
1994
;
56
(
2
):
73
7
.
26.
Yoon
YJ
,
Lee
EJ
,
Hellstrom
S
,
Kim
JS
.
Atrial natriuretic peptide modulates auditory brainstem response of rat
.
Acta Otolaryngol
.
2015
;
135
(
12
):
1293
7
.
27.
Rachel
JD
,
Dziadziola
JK
,
Quirk
WS
.
Atrial natriuretic peptide participates in the regulation of vestibular blood flow
.
Hear Res
.
1995
;
89
(
1–2
):
181
6
.
28.
Luo
Y
,
Xia
QJ
,
Xia
ZL
,
Tang
YD
.
Atrial natriuretic peptide reduces the α-subunit of the epithelial sodium channel mRNA expression in the mouse stria vascularis
.
Biomed Rep
.
2015
;
3
(
2
):
159
62
.