Although often overlooked in our daily lives, saliva performs a host of necessary physiological functions, including lubricating and protecting the oral cavity, facilitating taste sensation and digestion and maintaining tooth enamel. Therefore, salivary gland dysfunction and hyposalivation, often resulting from pathogenesis of the autoimmune disease Sjögren’s syndrome or from radiotherapy of the head and neck region during cancer treatment, severely reduce the quality of life of afflicted patients and can lead to dental caries, periodontitis, digestive disorders, loss of taste and difficulty speaking. Since their initial discovery in the 1970s, P2 purinergic receptors for extracellular nucleotides, including ATP-gated ion channel P2X and G protein-coupled P2Y receptors, have been shown to mediate physiological processes in numerous tissues, including the salivary glands where P2 receptors represent a link between canonical and non-canonical saliva secretion. Additionally, extracellular nucleotides released during periods of cellular stress and inflammation act as a tissue alarmin to coordinate immunological and tissue repair responses through P2 receptor activation. Accordingly, P2 receptors have gained widespread clinical interest with agonists and antagonists either currently undergoing clinical trials or already approved for human use. Here, we review the contributions of P2 receptors to salivary gland function and describe their role in salivary gland dysfunction. We further consider their potential as therapeutic targets to promote physiological saliva flow, prevent salivary gland inflammation and enhance tissue regeneration.
Salivary gland dysfunction and the associated hyposalivation are serious clinical problems that impact millions of people (Atkinson et al., 2005; Qin et al., 2015; Siddiqui and Movsas, 2017). Saliva plays a crucial role in maintaining oral homeostasis by aiding in taste perception and digestion, protecting and lubricating oral tissues, maintaining the integrity of tooth enamel and sustaining the oral microbiome (Dawes et al., 2015). In addition to its physiological roles, saliva contains a plethora of biomarkers and is easy to access allowing clinicians to utilize saliva as a non-invasive diagnostic material to monitor patient health (Chojnowska et al., 2018). Human saliva is increasingly being used to perform screening and risk assessment for systemic diseases, such as HIV, cancer, infections and cardiovascular disorders, demonstrating saliva’s extensive clinical potential (Nunes et al., 2015). Adequate saliva production is essential for maintaining quality of life and salivary gland dysfunction leads to dry mouth, oral bacterial and yeast infections, dental caries and speech problems (Chambers et al., 2004; Meijer et al., 2009).
Hyposalivation and xerostomia (i.e., dry mouth) can present in an iatrogenic manner as side effects of over 400 medications, including antidepressants, antipsychotics, opioids, antihistamines, and others (Furness et al., 2011). Although often transient and reversible, iatrogenic xerostomia contributes to patient non-adherence to medication regimens leaving underlying pathologies untreated. Two common pathophysiological causes of salivary gland dysfunction in humans are Sjögren’s syndrome (SS), an autoimmune disease characterized by xerostomia, autoantibody production and chronic lymphocytic infiltration of the salivary glands (i.e., sialadenitis), and radiotherapy-induced dysfunction where salivary glands sustain collateral damage following γ-radiation to treat head and neck tumors (Pinna et al., 2015; Mariette and Criswell, 2018). In both cases, damage to the salivary parenchyma and the failure to repair saliva-producing salivary acinar epithelium contribute to glandular dysfunction. Current therapies for salivary gland dysfunction are primarily focused on symptom management using muscarinic receptor agonists (i.e., pilocarpine or cevimeline) to stimulate saliva flow from residual salivary epithelium or through the topical use of artificial saliva (Ramos-Casals et al., 2010). While these treatments can provide some relief to patients, they are relatively ineffective because of their transient nature and failure to address the underlying inflammatory and degenerative processes that initiate and sustain glandular tissue damage. Therefore, a better understanding of the pathophysiology of salivary gland dysfunction is crucial to developing novel therapeutic approaches for this serious medical problem.
Purinergic receptors for extracellular nucleosides (i.e., adenosine) or nucleotides (i.e., ATP, ADP, UTP, UDP, and UDP-glucose) mediate numerous physiological processes, including platelet aggregation, neurotransmission, bone remodeling, and inflammatory, and immune responses (Dorsam and Kunapuli, 2004; Orriss et al., 2010; Idzko et al., 2014; Mutafova-Yambolieva and Durnin, 2014; Verkhratsky and Burnstock, 2014). In exocrine tissues, such as salivary gland, lacrimal gland and pancreas, purinergic receptor-mediated ion fluxes and cross-talk with muscarinic receptor signaling have been suggested to modulate secretory function (Novak et al., 2010; Burnstock and Novak, 2012; Hodges and Dartt, 2016). Whereas intracellular nucleotides are well-known for their role in metabolism and enzyme function, it wasn’t until the 1970s that plasma membrane receptors were postulated to respond to extracellular nucleotides, including ATP and ADP, and were suggested to be responsible for non-cholinergic, non-adrenergic neurotransmission (Burnstock et al., 1972; Burnstock, 1976). Under normal conditions, extracellular nucleotides are present at minute concentrations due to the presence of ectonucleotidases (Robson et al., 2006; Zimmermann et al., 2012). However, under pathological conditions nucleotides can accumulate in the extracellular space at abnormally high concentrations, whereupon they activate local purinergic receptors in an autocrine or paracrine manner (Deaglio and Robson, 2011). The purinergic receptor family is subclassified into P1 adenosine receptors (i.e., A1, A2A, A2B, and A3) (Piirainen et al., 2011) or P2 nucleotide receptors. The P2 receptor family is further classified into metabotropic P2Y receptors (i.e., P2Y1,2,4,6,11–14) and ionotropic P2X receptors (i.e., P2X1-7) (Abbracchio et al., 2006; Habermacher et al., 2016).
Pharmacological agonists and antagonists targeting purinergic receptors have gained widespread clinical interest and undergone clinical trials (Burnstock, 2017). P2X7 receptor (P2X7R) antagonists have been previously investigated in phase 2 clinical trials for treatment of inflammatory and autoimmune diseases, including chronic obstructive pulmonary disorder, rheumatoid arthritis and Crohn’s disease (Arulkumaran et al., 2011; Keystone et al., 2012). Recent advances in the development of neuro-permeable P2X7R antagonists have stimulated interest in the use of these compounds to treat neuroinflammatory and neuropsychiatric disorders (Chrovian et al., 2014; Burnstock and Knight, 2018; Bhattacharya and Ceusters, 2019). The P2X3 receptor (P2X3R) contributes to hypersensitivity of lung afferent sensory fibers that mediate cough initiation and phase 2 clinical trials have demonstrated that the P2X3R antagonist gefapixant (AF-219) reduces refractory chronic cough in afflicted patients by 75% (Weigand et al., 2012; Abdulqawi et al., 2015). Follow-up phase 3 clinical trials are currently underway to validate the use of gefapixant for treatment of refractory chronic cough (Muccino and Green, 2019).
Due to its ability to stimulate water transport across epithelial cell membranes following activation of calcium-dependent chloride channels, the P2Y2 receptor (P2Y2R) agonist diquafosol has undergone human clinical trials for the treatment of dry eye disease (DED) and is currently approved for human use in Japan and South Korea under the trade name Diquas (Tauber et al., 2004; Takamura et al., 2012; Koh, 2015). A similar P2Y2R agonist, denufosol, improved lung function relative to placebo in cystic fibrosis patients during phase 2 clinical trials, but failed to achieve its primary endpoints during phase 3 follow-up trials (Accurso et al., 2011). Notably, the FDA-approved anti-coagulant Plavix (clopidogrel), a P2Y12 receptor (P2Y12R) antagonist, was the 2nd most prescribed drug in the world in 2010 and is currently on the World Health Organization’s List of Essential Medicines (Topol and Schork, 2011; Kishore et al., 2018). However, the therapeutic potential of targeting purinergic receptors has not been well-investigated in the context of human salivary dysfunction. In the salivary glands, several purinergic receptors are expressed and upregulated under pathological conditions, including SS (Schrader et al., 2005; Baldini et al., 2013), where their activation mediates inflammatory and immune responses (Baker et al., 2008; Khalafalla M.G. et al., 2017), as well as cell repair mechanisms (El-Sayed et al., 2014). In this review, we summarize the role of purinergic receptors in salivary gland function and highlight their potential as novel therapeutic targets to treat salivary gland dysfunction.
The importance of saliva, as noted above, is clearly exemplified in individuals suffering from salivary gland hypofunction (Chambers et al., 2004; Atkinson et al., 2005; Meijer et al., 2009). In humans, whole unstimulated saliva is formed from the combined secretions of three pairs of major salivary glands, the submandibular (∼65%), parotid (∼20%) and sublingual (∼7%), along with numerous minor glands spread throughout the oral cavity that produce the remainder of saliva (<10%) (Humphrey and Williamson, 2001; de Almeida Pdel et al., 2008; Proctor, 2016). Upon stimulation, the parotid glands contribute the majority of total salivary secretions (Humphrey and Williamson, 2001; de Almeida Pdel et al., 2008; Proctor, 2016). Three basic cell types comprise the salivary glands: acinar epithelial cells that secrete the majority of the water and electrolytes in saliva, ductal cells that modify the electrolyte concentrations in the primary fluid and myoepithelial cells that provide contractile support for acinar cells (Martinez, 1987; Melvin et al., 2005; de Almeida Pdel et al., 2008; Proctor, 2016). Salivary acinar cells are either serous or mucous, whereas ductal cells are classified as intercalated, striated or excretory and the distribution of these cell types is dependent on species and type of gland (Melvin et al., 2005; de Almeida Pdel et al., 2008; Proctor, 2016). Along with the formation and modification of saliva, acinar and ductal cells also secrete important proteins, e.g., amylase and mucins from acinar cells (Boehlke et al., 2015; Frenkel and Ribbeck, 2015), kallikrein from ductal cells (Wong et al., 1983) and growth factors from both cell types (Masahiko et al., 2008), that are integral in maintaining the health of the oral cavity (Proctor, 2016). As shown in Figure 1, saliva formation is initiated in acinar cells by agonist-induced increases in intracellular Ca2+ levels, [Ca2+]i, that induce the opening of apical Ca2+-dependent Cl– channels and basolateral Ca2+-dependent potassium channels, allowing Cl– efflux into the luminal compartment and K+ efflux into the basolateral compartment to maintain membrane potential. The negative electrochemical gradient generated by increased luminal Cl– levels is compensated by the influx of Na+ ions across tight junctions into the lumen leading to Na+Cl– accumulation followed by water movement through water channels, predominately aquaporin-5 (Ma et al., 1999), thus forming saliva in its primary isotonic form. As saliva flows through the salivary gland ducts, electrolyte modification occurs, where Na+ and Cl– ions are exchanged for K+ and HCO3- ions by ductal cells, creating saliva in its final hypotonic form (Martinez, 1987; Melvin et al., 2005; Lee et al., 2012; Ambudkar, 2014; Proctor, 2016). Several types of Ca2+ mobilizing receptors are expressed on acinar cells (i.e., muscarinic, α-adrenergic, substance P), however, stimulation of the Gq protein-coupled M3 muscarinic receptor (M3R) subtype by acetylcholine is accepted as the main receptor signaling pathway that promotes the increases in [Ca2+]i necessary to enhance fluid secretion. Protein secretion from acinar and ductal cells is predominately mediated by activation of the β-adrenergic receptor (β-AR) and subsequent increases in cAMP (Melvin et al., 2005; Proctor, 2016). In addition to the canonical M3R and β-AR pathways, a mechanism of non-cholinergic, non-adrenergic-mediated salivary flow exists (Ekström et al., 1988; Ekström, 1999; Melvin et al., 2005). Because purinergic receptors are expressed in salivary glands and contribute to Ca2+ mobilization, they represent a link between canonical and non-canonical saliva secretion.
