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ISSN : 1226-7155(Print)
ISSN : 2287-6618(Online)
International Journal of Oral Biology Vol.50 No.1 pp.31-39
DOI : https://doi.org/10.11620/IJOB.2025.50.1.31

Adenosine receptors activate cyclic adenosine monophosphate signaling in salivary gland cells

Ji-Ah Kang1, Yoo-Bin Kim1, Sunghan Lee2, Hee-Kyung Park2,3*, Se-Young Choi1*
1Department of Physiology, Dental Research Institute, Seoul National University School of Dentistry, Seoul 03080, Republic of Korea
2Department of Oral Medicine and Oral Diagnosis, Dental Research Institute, Seoul National University School of Dentistry, Seoul
03080, Republic of Korea
3Department of Oral Medicine, Seoul National University Dental Hospital, Seoul 03080, Republic of Korea

†Ji-Ah Kang, Yoo-Bin Kim, and Sunghan Lee contributed equally to this work.


*Correspondence to: Se-Young Choi, E-mail: sychoi@snu.ac.krhttps://orcid.org/0000-0001-7534-5167
*Correspondence to: Hee-Kyung Park, E-mail: dentopark@snu.ac.krhttps://orcid.org/0000-0003-4388-0338
February 8, 2025 February 19, 2025 February 20, 2025

Abstract


Sympathetic innervation stimulates β-adrenergic receptors, triggering cyclic adenosine monophosphate (cAMP) production and enhancing protein secretion in salivary gland cells. While cAMP signaling, in conjunction with Ca2+ signaling, is essential for salivary gland function, the identified cAMP-producing G-protein-coupled receptors (GPCRs) remains limited. Here, we report the presence of cAMP-producing adenosine receptors in salivary gland cells. By reanalyzing publicly available single-cell transcriptome datasets of human and mouse submandibular glands, we identified mRNA expression of adenosine A1, A2A, A2B, and A3 receptors. Additionally, we confirmed that 5’-N-ethylcarboxamidoadenosine (NECA), an adenosine A2B receptor agonist, increases cAMP levels in human salivary gland cells, suggesting a physiological role for adenosine A2B receptors. Our findings enhance understanding of adenosine’s regulatory function in salivary glands and highlight new avenues for research on cAMPproducing adenosine receptors.


Salivary glands , G-protein-coupled receptor , Adenosine receptors , Cyclic AMP , Single cell transcriptome analysis

초록

    © The Korean Academy of Oral Biology. All rights reserved.

    This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/bync/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Introduction

    Salivary glands are regulated by nerves, specifically through the autonomous nervous system, which includes both parasympathetic and sympathetic innervation [1]. Originating from the medulla, the superior salivatory nucleus (via the submandibular ganglion and facial nerve) controls submandibular and sublingual glands, whereas the inferior salivatory nucleus (via the otic ganglion and glossopharyngeal nerve) controls the parotid gland [2-5]. Autonomic nerves release neurotransmitters and activate G-protein-coupled receptors (GPCRs) in salivary gland cells to transmit signals [1]. GPCR signaling regulates various aspects of salivary gland function, including secretion and cellular processes. The parasympathetic system primarily regulates salivary secretion [6]. Muscarinic receptors are the primary GPCRs in salivary glands, coupled to Gq-proteins and their activation stimulates Ca2+ signaling and salivary secretion [7,8].

    The sympathetic inputs stimulate β-adrenergic receptors activate cyclic adenosine monophosphate (cAMP) signaling pathways, leading to increased cAMP production and protein kinase A (PKA) activation which is crucial for protein secretion and fluid modulation in salivary glands [9,10]. cAMP signaling it interacts with Ca2+ signaling pathways, enhancing IP3R sensitivity and increasing Ca2+ release from the endoplasmic reticulum [11,12]. Recent findings indicate the expression of cystic fibrosis transmembrane-conductance regulator (CFTR), a well-known cAMP-regulated anion channel, in acinar cells of human salivary glands, challenging the role of cAMP in the salivary gland cells [13]. Despite the crucial role of cAMP signaling in salivary gland function, there have been few comprehensive studies on GPCRs known to increase cAMP, with the exception of the β-adrenergic receptor.

    In this study, we examined publicly available single-cell RNA sequencing (scRNA-seq) data and identified the expression of adenosine receptors in salivary gland cells. Consequently, we analyzed cAMP production induced by 5’-N-ethylcarboxamidoadenosine (NECA), an adenosine receptor agonist, in the A253 cell line, a well-established human submandibular gland (SMG) cell line [14].

    Materials and Methods

    The study was approved by the Institutional Animal Care and Use Committee of Seoul National University (SNU-240124-2-1).

    1. Materials

    Carbachol, NECA, forskolin, isoproterenol, ATP, bradykinin were obtained from Sigma-Aldrich. Collagenase P was sourced from Roche Molecular Biochemicals. Fura-2 AM was acquired from Molecular Probes. Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum, and penicillin-streptomycin were supplied by GIBCO. [3H]cAMP was procured from NEN Life Science Products. Lipofectamine 2000 was secured from Invitrogen.

    2. Cell culture

    A253 cells were cultured in DMEM enriched with 10% heatinactivated fetal bovine serum and 1% penicillin-streptomycin. These cells were maintained in a humidified atmosphere comprising 95% air and 5% CO2. The culture medium was refreshed daily, and the cells were subcultured every three days.

    3. Analysis of scRNA-seq data

    Two publicly accessible scRNA-seq datasets from human (Gene Expression Omnibus [GEO]: GSE199209) [15] and mouse (GEO: GSE175649) [16] SMGs were analyzed using the Seurat 4.1.0 R package. For quality control, only cells with a total number of detected molecules (nCount_RNA) below 40,000, a number of detected genes (nFeature_RNA) between 200 and 2,500 (for human) or 5,000 (for mouse), and mitochondrial transcript content below 50% were included. Normalization was performed using Seurat’s LogNormalize method with a scale factor of 10,000. Dimensional reduction utilized t-distributed stochastic neighbor embedding (tSNE) and Uniform Manifold Approximation and Projection (UMAP) on the 2,000 most highly variable genes. Clustering was carried out via a shared nearest neighbor (SNN) modularity optimization- based algorithm, and clusters were annotated based on the expression of established cell type-specific markers. In the human SMG, cell populations were identified by the expression of markers such as TP63, Krt5, Krt14, and ACTA2 for basal and myoepithelial cells; KRT7/19 and SLC5A5 for ductal cells; PIP, LPO, STATH, MUC5B, and TFF3 for acinar cells; PLVAP and IFI27 for endothelial cells; DCN for fibroblasts; CD79A, IGHM, and MZB1 for B cells; CD3G, CD3D, and CD3E for T cells; AIF1, CD68, and ITGAX for monocytes; HBA1 and HBB for red blood cells; GPM6B and S100B for Schwann cells. In mouse SMG, epithelial cells were broadly identified by EpCAM expression, with subpopulations classified based on markers such as CFTR for striated duct cells; NGF and EGF for granular convoluted tubule cells; KRT14 and KRT5 for basal duct cells; GSTT1 for intercalated duct cells; ACTA2 for myoepithelial cells; and aquaporin-5 (AQP5) for acinar cells. Non-epithelial populations were annotated by the expression of COL1A1 for stromal cells; PECAM1 for endothelial cells; CD68 for macrophages; ICOS for T cells; KIT for mast cells; NKG7 and GZMA for natural killer cells; ACTA2+ EpCAM– for smooth muscle cells; and ALAS2+ for erythroid cells.

    4. Determination of cAMP concentration

    The cellular concentration of cAMP was measured using a [3H]cAMP competition assay for binding to the cAMP-binding protein [17]. For cAMP production determination, cells were harvested and washed with Locke’s solution (154 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl2, 2.2 mM CaCl2, 5 mM HEPES, 10 mM glucose, pH 7.4). Subsequently, cells were preincubated in Locke’s solution containing 1 mM isobutylmethylxanthine (IBMX) to inhibit phosphodiesterase activity. IBMX was also added to the stimulating buffer with agonists. Following 15 minutes of stimulation at 37℃, the reaction was terminated by adding twice the volume of ice-cold absolute ethanol. Cells were incubated for 2 hours at –20℃ to facilitate cAMP extraction. The ethanol-suspended cells were centrifuged at 10,000 × g for 10 minutes at 4℃. The supernatant was evaporated and the remaining residues were dissolved in TE buffer (0.2 mM Tris-HCl, 4 mM EDTA, pH 7.5). A 50 μL sample solution was used for the cAMP assay. This assay involves competition between [3H]cAMP and unlabeled cAMP from the sample for a crude cAMP-binding protein sourced from bovine adrenal glands. Free [3H]cAMP is adsorbed by charcoal and removed by centrifugation, while bound [3H]cAMP in the supernatant is quantified via liquid scintillation counting. Each unknown sample was incubated with 50 μL [3H]cAMP (5 μCi) and 100 μL binding protein for 2 hours at 4℃. The separation of proteinbound cAMP and unbound cAMP was achieved by adsorption of free cAMP onto charcoal (100 μL), followed by centrifugation at 12,000 × g at 4℃. The radioactivity was measured by adding 200 μL supernatant to an Eppendorf tube containing 1.2 mL scintillation cocktail. cAMP concentration in the sample was determined from a standard curve and expressed as pmol per cell number.

    5. Statistical analysis

    Statistical analysis was performed using IBM SPSS Statistics version 23 (IBM Corporation). Origin 8.0 (OriginLab Corporation) software was used for calculation of EC50. Quantitative data are expressed as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was employed to assess statistical significance, followed by Bonferroni’s post hoc test for multiple comparisons between groups. Differences were considered significant at p ＜ 0.05.

    Results

    1. Investigating adenosine receptor expression in the salivary gland through reanalysis of scRNA-seq datasets

    We reanalyzed publicly available single-cell transcriptome datasets of human SMG tissue [15]. Dimensional reduction and unsupervised clustering using affinity propagation, based on the expression of high-variance genes, identified 12 transcriptionally distinct cell clusters (Fig. 1A and 1B), as shown through UMAP. The expression levels of adenosine receptors A1, A2A, A2B, and A3 were examined (Fig. 1C–1F). Notably, the A1 receptor was relatively well expressed in SMG acinar cells, aligning with previous reports of its presence in salivary glands [18]. To further validate these findings, we also analyzed publicly available scRNA-seq datasets of mouse SMG tissue [16]. Dimensional reduction and unsupervised clustering using affinity propagation, based on the expression of high-variance genes, identified 16 transcriptionally distinct cell clusters (Fig. 2A and 2B), as shown through UMAP. The expression levels of adenosine receptors A1, A2A, A2B, and A3 were examined (Fig. 2C–2F). However, in contrast to human SMG, A2A, and A2B receptors showed higher expression levels in mouse SMG tissue, suggesting potential species-specific differences in adenosine receptor signaling.

    2. Adenosine receptor agonist NECA evokes cAMP production in human salivary gland cells

    To confirm the expression and physiological function of adenosine receptors in salivary gland cells, we investigated the adenosine-induced cAMP signaling in human salivary gland cell lines, A253 cells. We measured cAMP formation following treatment with NECA, an adenosine A2B receptor-specific agonist. As previously reported [19], forskolin, an adenylyl cyclase activator, and isoproterenol, a β-adrenergic receptor agonist, significantly increased cAMP levels in A253 cells (Fig. 3A). Additionally, bradykinin and carbachol, known to elevate cytosolic Ca2+ concentration ([Ca2+]i) in A253 cells [20], did not induce cAMP increases, indicating that cAMP formation is independent of Ca2+ signaling (Fig. 3A). Notably, NECA and ATP successfully increased cytosolic cAMP concentration under these conditions (Fig. 3A), suggesting the presence of a cAMP-producing adenosine receptor in A253 cells. To further elucidate the response via adenosine receptor, we measured NECA-mediated cAMP production across various concentrations. The NECA-mediated cAMP production displayed a concentration-dependent manner with an EC50 value of 199 ± 4 nM (Fig. 3B). These findings clearly demonstrate that NECAmediated cAMP production is facilitated via the adenosine A2B receptor.

    Discussion

    cAMP plays a critical role as an intracellular signaling molecule in the sympathetic modulation of salivary gland function. However, cAMP’s contribution to salivary secretion is relatively minor compared to Ca2+ signaling. Intracellular calcium signaling, which induces the translocation of AQP5 and directly facilitates water movement, is more pivotal in salivary secretion [7]. Instead, cAMP signaling influences the development of salivary glands [21] and regulates the synthesis of proteins such as amylase and AQP5 [10,22-24]. Adrenergic stimulation regulates salivary secretion indirectly by interacting with Ca2+ signaling. β-adrenergic receptors facilitate the movement of ions and water into the acinar lumen, promoting the secretion of protein-rich serous saliva through interaction with Ca2+ signaling [12,25]. This contrasts with cAMP signaling in the kidney, another exocrine organ. In the kidney, cAMP signaling is crucial for the movement of aquaporin in the distal convoluted tubule and collecting duct, playing a significant role in maintaining body water homeostasis. Arginine vasopressin (also known as antidiuretic hormone) binds to the vasopressin V2 receptor, inducing cAMP synthesis and activating PKA to phosphorylate serine 256 of aquaporin-2 (AQP2) [26,27]. Phosphorylated AQP2 moves from intracellular vesicles to the cell membrane, increasing water permeability [28]. However, AQP5, the aquaporin in salivary glands, is mainly regulated by Ca2+, not cAMP [29,30]. Consequently, research on GPCRs that increase cAMP has received relatively less attention.

    However, recent advances in transcriptomics study have revealed the presence of various GPCR mRNAs in salivary glands, including previously unpublished cAMP-producing GPCRs. In this study, we analyzed the scRNA-seq database of salivary gland transcriptomes and discovered that adenosine receptors are expressed in salivary gland cells. Empirical experiments further confirmed that NECA, an adenosine receptor agonist, increases cAMP levels in these cells.

    Adenosine receptors play essential roles in various organs, influencing physiological and pathological processes through four subtypes: A1, A2A, A2B, and A3 receptors [31]. A1 receptor is a Gi/o-coupled receptor that inhibits cAMP production, whereas A2A and A2B receptors are positively coupled to adenylyl cyclase, stimulating cAMP production. A3 receptors have dual coupling―negatively regulating adenylyl cyclase through Gi-proteins and activating phospholipase C via Gβγ subunits. NECA used in our experiments showed slightly stronger affinity for A2B receptor than for A2A receptor. We think that adenosine-induced cAMP accumulation in salivary gland cells may depend on A2B receptor.

    Adenosine receptors have diverse functions throughout the body. Adenosine acts as a vasodilator in the cardiovascular system, reducing blood pressure and protecting the heart by controlling heart rate and contractility [32,33]. In the central nervous system, it functions as a neuromodulator, affecting synaptic transmission, neuronal excitability, and sleep-wake cycles [34,35]. The A1 receptor reduces neurotransmitter release, while the A2A receptor, found in the basal ganglia, influences motor control and is linked to neurodegenerative diseases like Parkinson’s [36]. In the immune system, adenosine serves as an anti-inflammatory agent by modulating cytokine production, inhibiting T cell proliferation, and resolving inflammation [37]. In the respiratory system, adenosine receptors regulate airway tone; A1 receptor activation causes bronchoconstriction, and A2B receptors promote inflammation and mucus secretion, relevant in conditions like asthma [38]. What role does adenosine play in salivary glands? In the salivary gland, an adenosine A1 activator, N6-cyclopentyladenosine exerts an increase in amylase release, inositol phosphate accumulation, cAMP production, and nitric oxide synthase activity. [18]. However, the A1 receptor, together with A3, is a typical Gi/o-coupled receptor and inhibits cAMP production. Our result that neither bradykinin nor muscarinic receptor, which are Ca2+-mobilizing GPCRs, induced cAMP production in salivary cells, makes it difficult to conclude that Ca2+ signals had a significant effect on cAMP under our experimental conditions. While it is challenging to entirely rule out other indirect effects, we believe that the adenosine A2B receptor may have induced adenosine-mediated cAMP production in salivary glands. Future research is expected to raise many intriguing questions, including whether interactions between A1 and A2B receptors exist. Additionally, it will be essential to reproduce these findings in primary cultured human salivary gland cells.

    Taken together, we investigate the expression profiles of adenosine receptors in salivary gland cells using scRNA-seq database. Subsequently, we evaluated the functional activity of these receptors by measuring cAMP production in response to NECA, an adenosine receptor agonist, in human SMG A253 cells. Our research advances the current understanding of cAMP signaling in salivary glands and provides new insights into their functions.

    Funding

    This work was supported by the National Research Foundation of Korea (2018R1A5A2024418 to S.Y.C; 2021R1A2C200 5573 to H.K.P).

    Conflicts of Interest

    No potential conflict of interest relevant to this article was reported.

    Figure

    IJOB-50-1-31_F1.gif

    mRNA expression of adenosine receptors in human submandibular glands (SMGs), analyzed by single-cell RNA sequencing database. (A) Uniform Manifold Approximation and Projection (UMAP) plot of human submandibular glands. (B) Dot plot showing expression of known SMG cell-type markers used for annotation. Blue indicates maximum gene expression and gray indicates low or no expression. Violin plots showing expression of adenosine receptors, including (C) ADORA1, (D) ADORA2A, (E) ADORA2B, and (F) ADORA3 genes in human SMGs.

    RBC, red blood cell.

    IJOB-50-1-31_F2.gif

    mRNA expression of adenosine receptors in mouse submandibular glands (SMGs), analyzed by single-cell RNA sequencing database. (A) Uniform Manifold Approximation and Projection (UMAP) plot of adult mouse submandibular glands. (B) Dot plot showing expression of known SMG cell-type markers used for annotation. Blue indicates maximum gene expression and gray indicates low or no expression. Violin plots showing expression of adenosine receptors, including (C) Adora1, (D) Adora2a, (E) Adora2b, and (F) Adora3 genes in mouse SMGs.

    GCT, granular convoluted tubule; NK, natural killer.

    IJOB-50-1-31_F3.gif

    NECA increase the intracellular cAMP concentration in A253 cells. (A) Cells were stimulated with 10 µM forskolin, 100 µM isoproterenol, 300 µM ATP, 3 µM bradykinin, 300 µM carbachol, or 10 µM NECA in the presence of 1 mM IBMX for 15 minutes; cAMP production was then monitored. The relative cAMP productions are depicted as percent of vehicle control-induced cAMP production. (B) Cells were challenged with the indicated NECA concentration, and cAMP production was then monitored. Each point represents mean ± SEM.

    NECA, 5’-N-ethylcarboxamidoadenosine; cAMP, cyclic adenosine monophosphate; ATP, adenosine triphosphate; IBMX, isobutylmethylxanthine; SEM, standard error of the mean.

    **p < 0.01, ***p < 0.001.

    Table

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