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Vocal fold surface liquid homeostasis contributes to optimal vocal physiology. In this paper we review emerging evidence that vocal fold surface liquid is maintained in part by salt and water fluxes across the epithelium. Based on recent immunolocalization and electrophysiological findings, we describe a transcellular pathway as one mechanism for regulating superficial vocal fold hydration. We propose that the pathway includes the sodium-potassium pump, sodium-potassium-chloride cotransporter, epithelial sodium channels, cystic fibrosis transmembrane regulator chloride channels, and aquaporin water channels. By integrating knowledge of the regulating mechanisms underlying ion and fluid transport with observations from hydration challenges and treatments using in vitro and in vivo studies, we provide a theoretical basis for understanding how environmental and behavioral challenges and clinical interventions may modify vocal fold surface liquid composition. We present converging evidence that clinical protocols directed at facilitating vocal fold epithelial ion and fluid transport may benefit healthy speakers, those with voice disorders, and those at risk for voice disorders.
Vocal folds are covered by a thin layer of liquid.1 This liquid serves as a physical and biochemical barrier that protects the underlying tissue from damage from inhaled particulates and pathogens.2 Presence of surface liquid is also posited to maintain optimal biomechanical characteristics of vocal fold mucosa, increase efficiency of vocal fold oscillation, and promote normal voice quality.1,3-14 This is consistent with the well-accepted clinical practice of recognizing the importance of vocal fold hydration in maintaining optimal vocal physiology. However, the source of surface liquid and mechanisms for maintaining liquid homeostasis are not fully understood.
In this paper we will present an overview of the current understanding of cellular mechanisms that participate in maintaining the composition and depth of the layer of liquid covering the vocal fold surface. This liquid layer constitutes a portion of airway surface liquid that lines the proximal and distal respiratory tract. The depth of airway surface liquid is maintained primarily by sodium ion (Na+) absorption and chloride ion (Cl−) secretion by epithelia of the lungs, bronchi, trachea, and nose.15 Here we will present emerging evidence that vocal fold surface liquid is similarly maintained in part by ion and water fluxes across vocal fold epithelia.
In 2001, Fisher and colleagues16 established a role for vocal fold epithelium in regulating vocal fold surface liquid. Epithelium was shown to be polarized and to demonstrate bidirectional transcellular water fluxes driven by active ion transport. The presence of transcellular water fluxes demonstrates that vocal epithelial cells, in addition to laryngeal gland secretions and mucociliary clearance, determine the depth and composition of surface liquid. Given that water fluxes can be manipulated pharmacologically, epithelial cells provide an important target for therapeutic interventions to regulate vocal fold surface liquid homeostasis. We will describe pathways for Na+, Cl−, and water fluxes across vocal fold epithelial cells that includes the sodium-potassium (Na+K+ATPase) pump, sodium-potassium-chloride (Na+K+2Cl−) cotransporter, epithelial sodium channels (ENaC), cystic fibrosis transmembrane regulator (CFTR) chloride channels, and aquaporin (AQP) water channels. We will outline the role of these transport proteins in maintaining homeostasis of vocal fold surface liquid by regulating transepithelial Na+, Cl−, and water fluxes. Based on recent investigations of transepithelial ion and water fluxes, a preliminary composite model of pathways for ion and water fluxes across epithelial cells will be proposed (Figure 1). An attempt will be made to integrate knowledge of cellular mechanisms underlying salt and water transport with observations of the effects of hydration challenges and treatments on vocal fold function in ex vivo and in vivo studies. The effectiveness of clinical hydration interventions in maintaining phonatory function in healthy speakers exposed to environmental challenges, and restoring voice function in individuals at risk for voice disorders and in speakers with vocal pathologies, will be reviewed.
Vocal fold surface hydration is subjected to persistent behavioral and environmental challenges.8,9,12,17 These challenges may compromise vocal fold defense and physiology. If optimal vocal fold function is to be maintained, it is necessary that there be an intrinsic mechanism for ensuring homeostasis of surface liquid in the face of these daily challenges. Based on observations of epithelial cell function in other airway epithelia,18 Fisher and colleagues16 hypothesized that the depth and ionic composition of vocal fold surface liquid is determined in part by active ion transport in vocal fold epithelial cells. Specifically, it was proposed that epithelial cells provide a pathway for Na+- and Cl−-coupled fluid fluxes.16,19,20 Recent studies have sought to identify the pathways and cellular mechanisms for maintaining local vocal fold surface hydration using three approaches: immunohistochemistry, electrophysiology, and measurement of water fluxes. Using these approaches, Na+, Cl−, and water transport proteins have been localized to vocal fold epithelial cells and net ion and water fluxes supported by these proteins have been quantified.
Integral membrane transport proteins believed to support transepithelial ion and water fluxes have been localized to vocal fold epithelial cells (Table 1). Immunolocalization assays have revealed that the α-subunit of the sodium-potassium (Na+K+ATPase) pump protein is localized to the plasma membrane of the most luminal and most basal vocal fold epithelial cells.16 This Na+K+ATPase pump supports active ion transport across cells. By transporting three Na+ ions out of the cell and two potassium ions (K+) into the cell, the Na+K+ATPase pump creates an asymmetric distribution of ions across the cell wall, resulting in the build up of transepithelial electrochemical gradients21 and driving ion transport. Other proteins that play an important role in ion transport have also been localized to vocal fold epithelia. The carboxy (C)-terminus of the sodium-potassium-chloride cotransporter (Na+K+2Cl−) has been localized to the plasma membranes of vocal fold epithelial cells where it may provide a point of entry for sodium (Na+), potassium (K+), and chloride ions (Cl−) into epithelial cells.20 Two primary ion channels, the epithelial sodium channels (ENaC)19 and the cystic fibrosis transmembrane regulator (CFTR) chloride channels20 have also been localized to vocal fold epithelial cells. ENaC and CFTR provide a pathway for transmembrane Na+ and Cl− fluxes, respectively.19,20 ENaC is a heterotetramer composed of two α, one β, and one γ homologous subunits.22 The α- and β- subunits of ENaC have been localized to the plasma membrane of epithelial cells, with greatest density noted in the luminal cell layer. 19 Luminal sodium absorption occurs predominantly through these ENaC, however sodium ions may also enter the cell coupled with potassium and chloride ions via the Na+K+2Cl− cotransporter.20 A pathway for vocal fold epithelial Cl− secretion similar to that observed in other airway epithelia15 has also been proposed.19 Chloride ion entry into vocal fold epithelial cells is believed to occur via the Na+K+2Cl− cotransporter described above. Cl− secretion is thought to occur via the CFTR chloride channels. The carboxy (C)-terminus and regulatory (R)-domain of CFTR have been localized to the plasma membranes of the two most superficial vocal fold epithelial cell layers.20 Based on electrophysiological findings described below, a second Cl− specific channel, the calcium-activated chloride channel (CaCC) may also provide a pathway for Cl− secretion from the cell.20 Localization of CaCC to vocal fold epithelial cells is awaited.
Recent electrophysiological studies have revealed that the transport proteins localized to vocal fold epithelial cells support transepithelial ion fluxes. Short-circuit current (Isc) provides a measure of the net flow of ions across epithelium. To assess the extent to which each protein contributes to measures of the Isc, viable excised ovine and canine mucosae have been treated with pharmacological agents to selectively inhibit or stimulate protein function (Table 2). Changes in Isc capture the effects of stimulation or inhibition of transport protein activity on ion fluxes. For example, inhibition of Na+K+ATPase with acetylstrophanthidin reduced transepithelial Isc.16 This finding is consistent with speculation that functional Na+K+ATPase participates in active ion transport across epithelial cells. Inhibition of ENaC with amiloride, a known epithelial sodium channel (ENaC) inhibitor, reduced Isc consistent with decreased Na+ absorption.19 Inhibition of CFTR with diphenylamine-2-carboxylate (DPC), a broad-spectrum Cl− transport inhibitor, decreased Isc consistent with a reduction in transepithelial Cl− movement. Conversely, stimulation of CFTR with secretagogues, isobutymethylxanthine (IBMX) and uridine triphosphate (UTP), induced a Cl−-dependent increase in Isc consistent with an increase in Cl− movement across the epithelium.20 Closer examination of the kinetics of the Isc response to UTP revealed a biphasic response consistent with the presence of CaCC in vocal fold epithelial cells.
Bidirectional water fluxes across excised vocal fold epithelium have been quantified using a Transepithelial Water and Ion Measurement System (TWIMS; Bio-Tech Plex, San Marco, CA).16,19,23 Transepithelial ion movements provide the driving force for bidirectional water fluxes across vocal fold epithelium. The effects of ion transport inhibitors on the magnitude of water fluxes have been examined. Inhibition of Na+K+ATPase by acetylstrophantidin resulted in a reduction in both secretory and absorptive water fluxes.16,19 Similarly, a reduction in absorption of ENaC mediated ion fluxes across vocal fold mucosae following treatment with amiloride resulted in decreased absorptive water consistent with decreased Na+ absorption.19
The interaction between ENaC and CFTR may play an important role in dictating the net driving forces for water secretion and absorption across vocal fold epithelium. Airway epithelium can be both absorptive and secretory. The transport of Na+ and Cl− ions across the epithelium creates a local osmotic gradient that serves as a driving force for transepithelial water fluxes. These water fluxes may occur via specialized water transport proteins (aquaporin, AQP). Two members of the AQP family, AQP1 and AQP2, have been immunolocalized to the luminal plasma membrane and cytoplasmic structures of vocal folds.24 The interaction between ENaC and CFTR also determines whether the epithelial tissue is absorptive or secretory. At rest, airway tissue is absorptive.25 When stimulated, a net secretion of Cl− towards the surface occurs. Activation of CFTR provides a pathway for Cl− secretion towards the surface while reducing Na+ absorption through inhibition of ENaC activity.26 When CFTR are absent or mutated (as in airway diseases such as cystic fibrosis) Cl− flux towards the surface is reduced and the inhibitory effect on ENaC is absent.27 Consequently, Na+ absorption remains unchecked and epithelial dehydration ensues.28 The mechanism underlying CFTR-mediated inhibition of ENaC is not known.26 Future studies are warranted to identify the mechanisms underlying CFTR-mediated inhibition of ENaC and the impact of the interaction of CFTR and ENaC on relative movements of Na+ and Cl− and, therefore on vocal fold surface hydration.
A review of electrophysiological and immunohistochemical data suggests that vocal fold epithelium may participate in regulating and maintaining vocal fold surface liquid homeostasis via ion transport and bi-directional water fluxes. Based on these data, we propose a functional pathway for transcellular ion and water fluxes (Figure 1). This model provides a theoretical basis for understanding how epithelial cells may alter the depth and composition of surface liquid in response to behavioral and environmental challenges, clinical interventions, and pharmacological treatment. Since ion-coupled water fluxes can be manipulated through luminal application of drugs to the vocal fold surface suggests that bidirectional water fluxes that contribute to vocal fold surface hydration and function can be controlled pharmacologically.
An understanding of the mechanisms by which vocal fold epithelial cells regulate local hydration offers a theoretical framework for appreciating the effects of behavioral and environmental challenges on surface hydration and provides the knowledge base necessary for the development of effective clinical interventions to maintain superficial and systemic hydration.
Drying of the vocal fold surface can occur due to environmental and behavioral challenges associated with mouth breathing, exercising, and inhaling poorly conditioned air (Table 3). 8,9,13,17 Vocal fold dehydration can also occur secondary to reduced systemic hydration,17,29–31 emotional factors,32 and the normal aging process.33,34 The relationship between dehydration and vocal fold physiology has been examined empirically in in vitro and in vivo studies.
Bench models have allowed study of the effects of hydration on the biomechanical and, consequently, phonatory characteristics of vocal folds (Table 4). Evaporative water loss from the airway surface due to dry air exposure can increase the stiffness and viscosity of ovine vocal fold mucosa.35 Adherent, viscous mucus on the vocal fold surface can also reduce vocal fold separation and increase vocal fold contact excised larynges,36 affecting vocal quality. Optimal viscoelastic properties of vocal folds are necessary to maintain ease of phonation,37,38 and the detrimental effects of surface dehydration on vocal fold viscoelasticity are consistent with the clinical adage to avoid dry environments that could adversely affect voice production.39 In excised larynges, dehydration of vocal folds raised phonation threshold pressure (PTP), the minimum subglottal pressure required to initiate and sustain vocal fold oscillation,5,40 and increased tissue stiffness.3
The negative effects of dehydration on efficiency of vocal fold function in bench models are consistent with those observed in clinical trials. Challenges to systemic and superficial vocal fold dehydration compromise vocal quality and phonatory efficiency in vocally healthy and disordered participants (Table 3). Decreased systemic hydration increased PTP14,29 and compromised voice quality.41 A presumed reduction in systemic hydration following ingestion of a diuretic, Lasix, increased phonatory effort at high pitches in healthy participants.31 Increased superficial and systemic hydration through ingestion of water and a mucolytic expectorant resulted in an improvement in phonatory efficiency in vocally healthy participants13,14 and participants with vocal nodules or polyps.42 Fisher and colleagues29 demonstrated that phonatory effort increased temporarily in patients following dialysis. Measures of phonatory effort returned to baseline values in these patients following rehydration. Improved phonatory efficiency following interventions purported to increase systemic hydration have also been reported.41,42 For example, ingestion of water and mucolytic agents decreased PTP and perceived phonatory effort (PPE) in participants presenting with voice disorders.42 Drinking water in combination with vocal rest between demanding vocal tasks improved voice quality in healthy amateur singers.41 Behavioral, environmental, and medical treatments to increase superficial and systemic hydration appear to improve vocal function. Notwithstanding differences in the nature of challenges, attributes of participants, and measures of vocal fold function including efficiency of vocal fold oscillation, vocal quality, and perception, these clinical studies provide a rationale for inclusion of interventions to increase systemic and superficial hydration in vocal hygiene treatment. A meta-analysis of this growing body of literature is underway to assess the clinical effectiveness of treatment on vocal fold function.
Challenges to superficial vocal fold hydration result in decreased efficiency of vocal fold vibration and compromised voice quality. Inhaling desiccated air increased jitter and shimmer in vocally healthy individuals.4 Superficial vocal fold dehydration induced by short-term oral breathing increased PTP in healthy female speakers8 and individuals reporting symptoms of vocal fatigue. The detrimental phonatory effects of challenges to systemic and superficial hydration in combination with vocal loading have been documented in healthy adults.10,11,39 While the adverse phonatory effects of behavioral and environmental challenges that perturb vocal fold hydration are well-recognized, the mechanisms by which these challenges alter the state of vocal fold hydration is not completely understood. Understanding of the cellular mechanisms underlying transepithelial ion and water flux may shed light on the manner in which behavioral and environmental challenges affect vocal fold function.
Investigations of the cellular mechanisms governing salt and water transport across vocal fold epithelium outlined above suggest potential ion transport related mechanisms by which vocal folds may respond to dehydration challenges to the luminal surface. It has been demonstrated in other airway epithelia that drying of the respiratory surface increases the salt concentration and decreases the depth of airway surface liquid (ASL).44–48 These changes to the depth and volume of ASL are transient as epithelia lining the nose, trachea, and lungs detect the increased ionic and osmotic concentration49–50 and generate water fluxes to replenish surface hydration. The secretory water fluxes observed in response to threats to airway surface liquid homeostasis are predominantly associated with ion and water transport processes.51
Based on observed increases in ion-coupled water fluxes in other airway epithelia in response to hyperosmotic and ionic challenges in in vitro studies, Sivasankar and Fisher posited that vocal fold epithelium would respond to perturbations in the composition of vocal fold surface liquid in vivo. 23,52 While the beneficial effects of osmotic agents on superficial hydration have not been universally supported in clinical trials,11 Roy and colleagues demonstrated that a nebulized osmotic agent transiently lowered PTP in vocally healthy volunteers.7 This decrease in PTP is consistent with increased vocal fold surface hydration resulting from compensatory secretory ion-coupled water fluxes in response to a threat to local surface hydration. The manner by which epithelium detects changes in surface fluid composition and depth awaits further study. It has been posited that the peripheral nervous system plays an important role in detecting changes in the composition and depth of airway surface liquid in vivo.53–55 However, the mechanisms for detecting ionic and osmotic perturbations in vocal fold surface liquid in excised, de-innervated vocal folds has yet to be determined.
Improved superficial and systemic vocal fold hydration promote efficient voice production.1,3-14 An understanding of the relationship between superficial and systemic vocal fold hydration is emerging; however, the distinct roles of superficial and systemic hydration remain unknown. It has been traditionally suggested that superficial vocal fold surface liquid is maintained by glandular secretions and that internal vocal fold liquid is provided by local vasculature.56 However, based on the presence of transepithelial ion-coupled water fluxes, we suggest that superficial and internal vocal fold hydration are interdependent. We further posit that ion-coupled water fluxes towards the vocal fold surface may influence internal ion and water composition, potentially altering the biomechanical properties of the vocal folds. It has been demonstrated that the ionic and osmotic composition of airway surface liquid overlying the trachea impacts the ionic environment of underlying tissue.57 These effects are greater in the presence of epithelial cell damage (for example, in cystic fibrosis) in which airway epithelial cells are unable to regulate transepithelial ion and water fluxes.
Here we propose a model of cellular mechanisms by which vocal fold epithelium may contribute to maintaining vocal fold surface liquid homeostasis. The preliminary model outlines pathways for transcellular ion and water fluxes that may regulate the composition and depth of surface liquid in the face of challenges. For example, vocal fold epithelial cells may respond to dehydration through activation of transport proteins. Ionic and osmotic challenges to surface liquid as a result of vocal fold drying may induce increased transepithelial secretory ion and fluid fluxes to restore surface liquid homeostasis. The proposed model also provides a theoretical basis for understanding the changes in vocal fold surface liquid associated with clinical interventions. We posit that clinical protocols directed at facilitating vocal fold epithelial ion and water transport may benefit individuals who experience systemic and superficial dehydration. While the presence of functional ion and water transport proteins in vocal fold epithelial cells suggests a role for epithelial cells in regulating vocal fold hydration, other possible sources of hydration are recognized. Vocal fold hydration may also be regulated through paracellular ion-coupled water fluxes, laryngeal glandular secretions29 and mucociliary clearance.1 The relative contribution and mechanisms underlying these sources of vocal fold surface liquid await further study.
This work was supported by a grant from the National Institute of Deafness and Other Communication Disorders (K08-DC0068) awarded to the last author.
This work was presented in part at the 34th Annual Symposium of the Voice Foundation: Care of the Professional Voice, Philadelphia, PA, June2, 2005
Ciara Leydon, Department of Speech Communication Arts and Sciences, Brooklyn College of The City University of New York.
Mahalakshmi Sivasankar, Department of Speech, Language, and Hearing Sciences, Purdue University.
Danielle Lodewyck Falciglia, Department of Otolaryngology, New York University School of Medicine.
Christopher Atkins, Department of Communication Sciences and Disorders, Northwestern University.
Kimberly V. Fisher, Department of Communication Sciences and Disorders, Northwestern University.