Modulation of Ciliary Motility by Na+

The mucociliary system is responsible for maintaining the airways clean of in­haled particles and pathogens. This important task is performed by the beating of cilia and the consequent movement of mucus from the lungs to the upper airways (1,2). Ciliary activity is strongly regulated by hormones and neurotrans­mitters, which provide fine control over the efficiency of mucus transport (1). In various mucociliary systems, including human airways, extracellular ATP po­tently stimulates ciliary activity (3-7). Cell-surface receptors for purines and py­rimidines are classified into two major types (8): G-protein coupled receptors (P2Y receptors) and ligand-gated ion channels (P2X receptors) (9-11). Most P2Y receptors are coupled to phospholipase C, leading to the formation of inositol 1, 4, 5-trisphosphate (InsP3) and, thus, to the mobilization of Ca2+ from internal stores. The P2Y receptor in rabbit airway ciliated cells was recently shown to have a similar sensitivity to extracellular ATP and extracellular UTP and to acti­vate the same signal transduction pathway when activated by either extracellular ATP or extracellular UTP (12). P2X receptors are ligand-gated cation channels that are permeable to Ca2+ (9-11). It is evident, therefore, that the activation of either P2X or P2Y receptors would elevate the cytoplasmic concentration Ca2+ ([Ca2+]j). THE ROLE OF Ca2+ IN EXTRACELLULAR ATP-INDUCED MUCOCILIARY ACTIVATION

It is well established that Ca2+ is an important mediator in mucociliary activation. In many mucociliary systems, including human airway, stimulation of ciliary activity by various agonists is correlated with a rise in [Ca2+]j (4,6,13-17). The dynamic effects of extracellular ATP on ciliary beating frequency (CBF) and [Ca2+]i were simultaneously measured in rabbit airway epithelium (4). Extracellu­lar ATP induces a rapid rise in [Ca2+]i and in CBF. In the continuous presence of extracellular ATP, [Ca2+]j declined over several minutes to a lower elevated [Ca2+]j plateau, while CBF remained at a high level during the ten minutes of exposure to extracellular ATP. In contrast, in low extracellular Ca2+, extracellular ATP induced only a transient rise in both [Ca2+]j and CBF. A similar time course of [Ca2+]i in response to extracellular ATP, and a similar dependence on extracel­lular Ca2+, was observed also in human airway epithelium (18). Taken together, these findings show that in extracellular solutions containing 140 mM Na+, the initial rapid rise in both [Ca2+]j and in CBF induced by extracellular ATP results

primarily from the mobilization of Ca2+ from internal stores, while the sustained elevated ciliary activity requires Ca2+ influx. There is accumulating evidence that a pathway for Ca2+ influx that has not yet been identified is most likely activated by the P2Y pathway. In addition, as outlined below, extracellular ATP can di­rectly activate a Ca2+-permeable ion channel (P2X receptor). EXTRACELLULAR ATP AND Ca2+ INFLUX IN RABBIT AIRWAY CILIATED CELLS

In an attempt to identify the pathway for Ca2+ influx activated by extracellular ATP, we developed a procedure to dissociate rabbit airway epithelium to obtain single viable ciliated cells (19). The patch clamp technique (20) was then used to control the composition of the intracellular environment while simultaneously recording membrane currents activated by various nucleotides. In these whole­cell experiments, the intracellular (pipette) solution contained only a physiologi­cal concentration of salt, Mg-ATP, and GTP. Rabbit airway ciliated cells were found to express an ATP-gated cation-selective channel (P2XciUa channel) that is strongly attenuated by extracellular divalent cations (19). Extracellular ATP (but not extracellular UTP or ADP) activated the P2Xciiia channels, which then remained activated for several minutes in the presence of extracellular ATP. The conductance was permeable to monovalent and divalent cations with approximate relative per­meability for PBa: PCs • P tea of 4:1:0.1. Permeability to Cl was negligible. The permeability of P2Xcilia channels to Ca2+ was estimated to be ninefold greater than the permeability to Cs+, indicating that Ca2+ is more permeant than Ba2+ (21). As Ca2+ plays a pivotal role in ciliary function, and P2XciUa channels remain activated for at least 30 minutes with extracellular ATP, P2XciUa channels might be involved in maintaining ciliary activation during prolonged exposures to extracellular ATP. Indeed, the following section shows that activation of P2XcUia channels contributes to sustained enhancement of ciliary motility by extracellular ATP. EXTRACELLULAR Na+ AND CILIARY MOTILITY

Recently we discovered that extracellular Na+ (Na+0) inhibits P2Xcilia channels and thereby attenuates extracellular ATP-induced ciliary motility (22). The major observations can be summarized as follows: (1) Na+0 directly and specifically inhibits P2Xcma channels, (2) inhibition by Na+0 of an extracellular ATP-activated conductance is seen in airway ciliated cells from a variety of mammalian species including rabbit, rat, pig, and human, and (3) the degree of inhibition by Na+0 depends on interplay between the effective concentrations of extracellular ATP and Na+0.In order to investigate the contribution of P2XcUia channels to ciliary func­tion and consequently the possible contribution of Na+0 to mucociliary clearance,

we performed experiments in which the P2Y-mediated pathway was suppressed by depleting the intracellular Ca2+ stores with thapsigargin and by inhibiting phospholipase C with U-73122. Ciliary activity and [Ca2+]j were simultaneously measured from these cells in extracellular solutions containing either low (10 mM) or high (140 mM) Na+. In control cells (with functional P2Y receptors), in high Na+0, extracellular ATP (100 jiM) increased [Ca2+]j and CBF (Fig. 1A). In contrast, in high Na+0, after suppressing the P2Y pathway, extracellular ATP (100 pM) failed to elevate [Ca2+h or to enhance ciliary activity (Fig. IB). This result clearly shows that the P2Y pathway contributes to sustained ciliary activity. In addition, these results showed that the experimental procedure effectively sup­pressed ciliary activation via the P2Y pathway and thus provided a means to investigate the possible contribution of the P2Xciiia channels to ciliary activation by extracellular ATP.Contrary to the inability of extracellular ATP to enhance CBF after sup­pressing the P2Y pathway when Na+0 is high, in low Na+0> extracellular ATP elevated [Ca2+h and enhanced CBF (Fig. 2A). To show that the effects of extra­cellular ATP in low Na+0 are mediated via P2Xcma channels, two additional sets of experiments were performed. First, extracellular Ca2+ was lowered from 0.5 mM to 0.5 pM, demonstrating that the ATP-induced rise in [Ca2+]j and in CBF requires extracellular Ca2+ (Fig. 2B). Second, the effect of extracellular UTP on

[Ca2+]j and CBF in low Na+0 was examined. Since extracellular UTP does not activate P2XcUia channels, we predicted that extracellular UTP would not elevate [Ca2+]j or stimulate the cilia. Indeed, extracellular UTP (100 pM) was without effect (Fig. 2B). Taken together, these results show that Ca2+ influx through P2Xana channels can increase ciliary activity and that Na+ inhibits both Ca2+ influx through P2XciUa channels and the resulting ciliary activity. THE ROLE OF P2Xcilia CHANNELS IN MUCOCILIARY FUNCTION

Little is known on the concentrations of extracellular ATP in the airways, and thus it is presently not possible to estimate the extent to which P2XciUa channels are physiologically activated. Nevertheless, comparing the effects of Na+0 on

ciliary activation induced by 100 pM extracellular ATP to the effects of Na+0 on ciliary activation induced by 6 pM extracellular ATP (Fig. 3) provides insight as to the relative contributions of the P2Y pathway and the P2XciUa pathway to ciliary activation. Assuming that ATP4-is the active agonist (19), then under the experimental conditions used, 100 pM and 6 pM extracellular ATP are equivalent to approximately 95 and 6 nM of ATP4-, respectively.The initial rise in CBF induced by 100 pM extracellular ATP is the same in low and high Na+0 (Fig. 3B), indicating that robust activation of the P2Y pathway can maximally stimulate the cilia. However, when the P2Xcitia pathway is inhibited (i.e., in high Na+0), ciliary activation induced by extracellular ATP is transient. Thus, the P2Xcma pathway contributes to sustaining the cilia at a high level of activation. In contrast to the ability of 100 pM ATP to maximally stimu­late the cilia in both high and low Na+0, the initial rise in ciliary activation induced by 6 pM extracellular ATP is partially inhibited by Na+0 (Fig. 3A). This suggests that the P2Xcilia pathway can also contribute to the initial rise in ciliary activation induced by extracellular ATP when the concentration of extracellular ATP is relatively low. Thus, modulation of P2Xcilia channels by Na+0 might be a physio­logical mechanism for regulating ciliary function. A POSSIBLE ROLE FOR Na+0 IN AIRWAY PHYSIOLOGY

The physiological relevance of the findings described above may depend on the fundamental question of whether P2Xcina channels are exposed to the airway sur­

face fluid. Using the patch clamp technique, we have detected P2XciUa channels in the basolateral membrane of isolated ciliary cells, suggesting that P2Xcina chan­nels are exposed to the interstitial fluid in which the concentration of Na+ is high. However, it is well established that dissociated epithelial cells lose their polarity and membrane proteins redistribute in the membrane. Thus, the patch-clamp data do not undermine the possibility that in intact epithelium the P2Xcilia channels are restricted to the apical membrane of the ciliary cells. Without specific antibod­ies raised against P2XciUa channels, this question cannot easily be addressed since it is presently not possible to record single-channel currents from the membrane of the cilium.Another question of key importance is what are the physiological concen­trations of ATP and Na+ in the airways. In the proximal airways, the Na+ content of the airway surface fluid (ASF) has been reported to be lower than in plasma (23-26) and has been reported to be either unchanged (24-27) or elevated (23,28) in CF. Little information is available, however, on the composition of the ASF in the distal airways (29,30). If Na+ in the distal airways is elevated in disorders such as CF, then one of the early events leading to impaired mucociliary clearance in CF may be a reduced ability of extracellular ATP to regulate ciliary motility. Since human (3-defensin, the peptide antibiotic expressed by the surface epithelia in lung, is also modulated by Na+ (31-33), modulation of protein function by Na+0 may be a fundamental physiological mechanism of airway function.While the above results suggest that P2Xcina channels contribute to the regu­lation of ciliary motility, the physiological role of Na+ as a modulator of mucocil­iary clearance remains to be determined. Preventing the inhibition of P2XcUia channels by Na+ may, nevertheless, provide a novel route for improving mucocil­iary clearance. ACKNOWLEDGMENTS

This work was supported by the Israel Science Foundation, founded by the Israel Academy of Sciences and Humanities-Dorot Science Foundation. REFERENCES

1. A Wanner, M Salathe, TG O’Riordan. Mucociliary clearance in the airways. Am J Respir Crit Care Med 154:1868-1902, 1996.2. H Matsui, SH Randell, SW Peretti, CW Davis, RC Boucher. Coordinated clearance of periciliary liquid and mucus from airway surfaces. J Clin Invest 102:1125-1131, 1998.3. CA Geary, CW Davis, AM Paradiso, RC Boucher. Role of CNP in human airways: cGMP-mediated stimulation of ciliary beat frequency. Am J Physiol 268:L 1021-L1028, 1995.4. A Komgreen, Z Priel. Purinergic stimulation of rabbit ciliated airway epithelia: con­trol by multiple Ca2+ sources. J Physiol (Lond) 497:53-66, 1996.