Tetsuji Nakamoto

A Role for AQP5 in Activation of TRPV4 by Hypotonicity CONCERTED INVOLVEMENT OF AQP5 AND TRPV4 IN REGULATION OF CELL VOLUME RECOVERY

Regulation of cell volume in response to changes in osmolarity is critical for cell function and survival. However, the molecular basis of osmosensation and regulation of cell volume are not clearly understood. We have examined the mechanism of regulatory volume decrease (RVD) in salivary gland cells and report a novel association between osmosensing TRPV4 (transient receptor potential vanalloid 4) and AQP5 (aquaporin 5), which is required for regulating water permeability and cell volume. Exposure of salivary gland cells and acini to hypotonicity elicited an increase in cell volume and activation of RVD. Hypotonicity also activated Ca2+ entry, which was required for subsequent RVD. Ca2+ entry was associated with a distinct nonselective cation current that was activated by 4αPDD and inhibited by ruthenium red, suggesting involvement of TRPV4. Consistent with this, endogenous TRPV4 was detected in cells and in the apical region of acini along AQP5. Importantly, acinar cells from mice lacking either TRPV4 or AQP5 displayed greatly reduced Ca2+ entry and loss of RVD in response to hypotonicity, although the extent of cell swelling was similar. Expression of N terminus-deleted AQP5 suppressed TRPV4 activation and RVD but not cell swelling. Furthermore, hypotonicity increased the association and surface expression of AQP5 and TRPV4. Both these effects and RVD were reduced by actin depolymerization. These data demonstrate that (i) activation of TRPV4 by hypotonicity depends on AQP5, not on cell swelling per se, and (ii) TRPV4 and AQP5 concertedly control regulatory volume decrease. These data suggest a potentially important role for TRPV4 in salivary gland function.

Purinergic P2X7 Receptors Mediate ATP-induced Saliva Secretion by the Mouse Submandibular Gland

Salivary glands express multiple isoforms of P2X and P2Y nucleotide receptors, but their in vivo physiological roles are unclear. P2 receptor agonists induced salivation in an ex vivo submandibular gland preparation. The nucleotide selectivity sequence of the secretion response was BzATP ≫ ATP > ADP ≫ UTP, and removal of external Ca2+ dramatically suppressed the initial ATP-induced fluid secretion (∼85%). Together, these results suggested that P2X receptors are the major purinergic receptor subfamily involved in the fluid secretion process. Mice with targeted disruption of the P2X7 gene were used to evaluate the role of the P2X7 receptor in nucleotide-evoked fluid secretion. P2X7 receptor protein and BzATP-activated inward cation currents were absent, and importantly, purinergic receptor agonist-stimulated salivation was suppressed by more than 70% in submandibular glands from P2X7-null mice. Consistent with these observations, the ATP-induced increases in [Ca2+]i were nearly abolished in P2X7–/– submandibular acinar and duct cells. ATP appeared to also act through the P2X7 receptor to inhibit muscarinic-induced fluid secretion. These results demonstrate that the ATP-sensitive P2X7 receptor regulates fluid secretion in the mouse submandibular gland.

Regulation of membrane potential and fluid secretion by Ca2+-activated K+ channels in mouse submandibular glands.

We have recently shown that the IK1 and maxi‐K channels in parotid salivary gland acinar cells are encoded by the KCa3.1 and KCa1.1 genes, respectively, and in vivo stimulated parotid secretion is severely reduced in double‐null mice. The current study tested whether submandibular acinar cell function also relies on these channels. We found that the K+ currents in submandibular acinar cells have the biophysical and pharmacological footprints of IK1 and maxi‐K channels and their molecular identities were confirmed by the loss of these currents in KCa3.1‐ and KCa1.1‐null mice. Unexpectedly, the pilocarpine‐stimulated in vivo fluid secretion from submandibular glands was essentially normal in double‐null mice. This result and the possibility of side‐effects of pilocarpine on the nervous system, led us to develop an ex vivo fluid secretion assay. Fluid secretion from the ex vivo assay was substantially (about 75%) reduced in animals with both K+ channel genes ablated – strongly suggesting systemic complications with the in vivo assay. Additional experiments focusing on the membrane potential in isolated submandibular acinar cells revealed mechanistic details underlying fluid secretion in K+ channel‐deficient mice. The membrane potential of submandibular acinar cells from wild‐type mice remained strongly hyperpolarized (−55 ± 2 mV) relative to the Cl− equilibrium potential (−24 mV) during muscarinic stimulation. Similar hyperpolarizations were observed in KCa3.1‐ and KCa1.1‐null mice (−51 ± 3 and −48 ± 3 mV, respectively), consistent with the normal fluid secretion produced ex vivo. In contrast, acinar cells from double KCa3.1/KCa1.1‐null mice were only slightly hyperpolarized (−35 ± 2 mV) also consistent with the ex vivo (but not in vivo) results. Finally, we found that the modest hyperpolarization of cells from the double‐null mice was maintained by the electrogenic Na+,K+‐ATPase.

Apical maxi-K (KCa1.1) channels mediate K secretion by the mouse submandibular exocrine gland

The exocrine salivary glands of mammals secrete K+ by an unknown pathway that has been associated with HCO3(-) efflux. However, the present studies found that K+ secretion in the mouse submandibular gland did not require HCO3(-), demonstrating that neither K+/HCO3(-) cotransport nor K+/H+ exchange mechanisms were involved. Because HCO3(-) did not appear to participate in this process, we tested whether a K channel is required. Indeed, K+ secretion was inhibited >75% in mice with a null mutation in the maxi-K, Ca2+-activated K channel (KCa1.1) but was unchanged in mice lacking the intermediate-conductance IKCa1 channel (KCa3.1). Moreover, paxilline, a specific maxi-K channel blocker, dramatically reduced the K+ concentration in submandibular saliva. The K+ concentration of saliva is well known to be flow rate dependent, the K+ concentration increasing as the flow decreases. The flow rate dependence of K+ secretion was nearly eliminated in KCa1.1 null mice, suggesting an important role for KCa1.1 channels in this process as well. Importantly, a maxi-K-like current had not been previously detected in duct cells, the theoretical site of K+ secretion, but we found that KCa1.1 channels localized to the apical membranes of both striated and excretory duct cells, but not granular duct cells, using immunohistochemistry. Consistent with this latter observation, maxi-K currents were not detected in granular duct cells. Taken together, these results demonstrate that the secretion of K+ requires and is likely mediated by KCa1.1 potassium channels localized to the apical membranes of striated and excretory duct cells in the mouse submandibular exocrine gland.

Molecular identification and physiological roles of parotid acinar cell maxi-K channels.

The physiological success of fluid-secreting tissues relies on a regulated interplay between Ca2+-activated Cl– and K+ channels. Parotid acinar cells express two types of Ca2+-activated K+ channels: intermediate conductance IK1 channels and maxi-K channels. The IK1 channel is encoded by the KCa3.1 gene, and the KCa1.1 gene is a likely candidate for the maxi-K channel. To confirm the genetic identity of the maxi-K channel and to probe its specific roles, we studied parotid glands in mice with the KCa1.1 gene ablated. Parotid acinar cells from these animals lacked maxi-K channels, confirming their genetic identity. The stimulated parotid gland fluid secretion rate was normal, but the sodium and potassium content of the secreted fluid was altered. In addition, we found that the regulatory volume decrease in acinar cells was substantially impaired in KCa1.1-null animals. We examined fluid secretion from animals with both K+ channel genes deleted. The secretion rate was severely reduced, and the ion content of the secreted fluid was significantly changed. We measured the membrane potentials of acinar cells from wild-type mice and from animals with either or both K+ channel genes ablated. They revealed that the observed functional effects on fluid secretion reflected alterations in cell membrane voltage. Our findings show that the maxi-K channels are critical for the regulatory volume decrease in these cells and that they play an important role in the sodium uptake and potassium secretion process in the ducts of these fluid-secreting salivary glands.

Clcn2 encodes the hyperpolarization-activated chloride channel in the ducts of mouse salivary glands

Transepithelial Cl(-) transport in salivary gland ducts is a major component of the ion reabsorption process, the final stage of saliva production. It was previously demonstrated that a Cl(-) current with the biophysical properties of ClC-2 channels dominates the Cl(-) conductance of unstimulated granular duct cells in the mouse submandibular gland. This inward-rectifying Cl(-) current is activated by hyperpolarization and elevated intracellular Cl(-) concentration. Here we show that ClC-2 immunolocalized to the basolateral region of acinar and duct cells in mouse salivary glands, whereas its expression was most robust in granular and striated duct cells. Consistent with this observation, nearly 10-fold larger ClC-2-like currents were observed in granular duct cells than the acinar cells obtained from submandibular glands. The loss of inward-rectifying Cl(-) current in cells from Clcn2(-/-) mice confirmed the molecular identity of the channel responsible for these currents as ClC-2. Nevertheless, both in vivo and ex vivo fluid secretion assays failed to identify significant changes in the ion composition, osmolality, or salivary flow rate of Clcn2(-/-) mice. Additionally, neither a compensatory increase in Cftr Cl(-) channel protein expression nor in Cftr-like Cl(-) currents were detected in Clcn2 null mice, nor did it appear that ClC-2 was important for blood-organ barrier function. We conclude that ClC-2 is the inward-rectifying Cl(-) channel in duct cells, but its expression is not apparently required for the ion reabsorption or the barrier function of salivary ductal epithelium.

Enhanced Formation of a Transport Metabolon in Exocrine Cells of Nhe1–/– Mice

Cl- influx across the basolateral membrane is a limiting step in fluid production in exocrine cells and often involves functionally linked \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Cl}^{-}{/}\mathrm{HCO}_{3}^{-}\) \end{document} (Ae) and Na+/H+ (Nhe) exchange mechanisms. The dependence of this major Cl- uptake pathway on Na+/H+ exchanger expression was examined in the parotid acinar cells of Nhe1-/- and Nhe2-/- mice, both of which exhibited impaired fluid secretion. No change in \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Cl}^{-}{/}\mathrm{HCO}_{3}^{-}\) \end{document} exchanger activity was detected in Nhe2-deficient mice. Conversely, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Cl}^{-}{/}\mathrm{HCO}_{3}^{-}\) \end{document} exchanger activity increased nearly 4-fold in Nhe1-deficient mice, despite only minimal or any change in mRNA and protein levels of the anion exchanger Ae2. Acetazolamide completely blocked the increase in \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Cl}^{-}{/}\mathrm{HCO}_{3}^{-}\) \end{document} exchanger activity in Nhe1-null mice suggesting that increased anion exchange required carbonic anhydrase activity. Indeed, the parotid glands of Nhe1-/- mice expressed higher levels of carbonic anhydrase 2 (Car2) polypeptide. Moreover, the enhanced \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Cl}^{-}{/}\mathrm{HCO}_{3}^{-}\) \end{document} exchange activity was accompanied by an increased abundance of Car2·Ae2 complexes in the parotid plasma membranes of Nhe1-/- mice. Anion exchanger activity was also significantly reduced in Car2-deficient mice, consistent with an important role of a putative Car2·Ae2 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} transport metabolon in parotid exocrine cell function. Increased abundance of this \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} transport metabolon is likely one of the multiple compensatory changes in the exocrine parotid gland of Nhe1-/- mice that together attenuate the severity of in vivo electrolyte and acid-base balance perturbations.

The Electrolyte and Water Secretion Mechanism

The currently accepted salivary gland secretion model describes the process of fluid secretion as the coordinated action of water and ion channels and transporters. The secretion of electrolytes and water by salivary glands is thought to be activated by an agonist-induced increase in the intracellular free [Ca2+] and to be driven by transepithelial chloride movement. The Cl- transport is supported by upregulation of several ion transporters, K+ and Cl- channels, and the Na+/K+-ATPase. This review will focus on the details of the transport mechanisms as well as recent developments in confirming the molecular identities of the involved transporter and channel proteins.