Molecular and Functional Expression of the Best2 Ca2±activated Cl- Channel in Mouse Submandibular Salivary Gland

[Ca2+]i and the Cl- current were measured in isolated submandibular gland acinar and duct cells to characterize and localize the purinergic receptors expressed in these cells. In both cell types 2'-3'-benzoylbenzoyl (Bz)-ATP and ATP increased [Ca2+]i mainly by activation of Ca2+ influx. UTP had only minimal effect on [Ca2+]i at concentrations between 0.1 and 1 mM. However, a whole cell current recording showed that all nucleotides effectively activated Cl- currents. Inhibition of signal transduction through G proteins by guanyl-5'-beta-thiophosphate revealed that the effect of ATP on Cl- current was mediated in part by activation of a G protein-coupled and in part by a G protein-independent receptor. BzATP activated exclusively the G protein-independent portion, whereas UTP activated only the G protein-dependent portion of the Cl- current. Measurement of [Ca2+]i in the microperfused duct showed that ATP stimulated a [Ca2+]i increase when applied to the luminal or the basolateral sides. BzATP increased [Ca2+]i only when applied to the luminal side, whereas UTP at 100 microM increased -Ca2+-i only when applied to the basolateral side. The combined results suggest that duct and possibly acinar cells express P2z receptors in the luminal and P2u receptors in the basolateral membrane.

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 Ca(2+) dramatically suppressed the initial ATP-induced fluid secretion ( approximately 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 P2X(7) gene were used to evaluate the role of the P2X(7) receptor in nucleotide-evoked fluid secretion. P2X(7) 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 P2X(7)-null mice. Consistent with these observations, the ATP-induced increases in [Ca(2+)](i) were nearly abolished in P2X(7)(-/-) submandibular acinar and duct cells. ATP appeared to also act through the P2X(7) receptor to inhibit muscarinic-induced fluid secretion. These results demonstrate that the ATP-sensitive P2X(7) receptor regulates fluid secretion in the mouse submandibular gland.

Mammalian salivary glands are mainly composed of two epithelial cell types, the acinar cells that secrete the salivary fluid as well as most of the salivary proteins, and the ductal cells that secrete some protein and modify the ionic composition of the saliva as they convey it to the mouth. For a variety of reasons, including their physiological interest, their relative abundance and ease of preparation, and their robust stimulation-secretion responses, salivary acinar cells been intensively studied and many of their properties are well, albeit certainly not completely, understood. Although salivary ducts have also received considerable experimental attention, particularly over the past 10 years, less is known about their behavior. Because of this and because of the central role of the acinar cells in salivary fluid and protein secretion we will concentrate almost exclusively on them in this article. For more information on salivary ducts the interested reader is referred to several recent reviews and the references therein (7; 62; 6). It is also worth mentioning at this point that most of the information presented below has been obtained from the salivary glands of experimental animals (mainly rats and rabbits). Except for morphologic studies and several non-invasive procedures, such as the collection of saliva after the application of various stimuli, experiments using human salivary tissues are rare. However, in those cases where the functional properties of human salivary acini have been investigated they appear to conform well to the results and conclusions derived from animal studies. The easiest way to understand the proposed mechanisms of salivary fluid secretion is to consider a specific model. Figure 1 shows a schematic representation of a salivary acinar cell. The cell contains four ion transporters, the Na+/K+ adenosine triphosphatase (ATPase), a Na+–K+–2Cl− cotransporter and a Ca2+-activated K+ channel, all located in the basolateral membrane, and a Ca2+ activated Cl− channel located in the apical membrane. Fluid secretion is thought to arise from the concerted actions of these four transporters as follows. The Na+/K+ ATPase maintains intracellular Na+ concentration low and intracellular K+ concentration high relative to the interstitium by exchanging 3Na+ for 2K+ at the expense of cellular adenosine triphosphate (ATP). The Na+–K+–2 Cl− cotransporter (also known as NKCC1) is a secondary active transport system that transports 1Na+, 1K+ and 2Cl− into the cell in a tightly coupled fashion. Because of the extracellular to intracellular Na+ gradient maintained by the Na+/K+ ATPase, Cl− is likewise concentrated in the acinar cytoplasm above electrochemical equilibrium by NKCC1. In the resting (unstimulated) state intracellular Ca2+ concentration is low and the Ca2+ activated K+ and Cl− channels are therefore closed. But when the cell is stimulated by secretagogues (in situ, typically the muscarinic agonist acetylcholine) intracellular Ca2+ concentration rises and the K+ and Cl− channels open. These Ca2+ associated changes in K+ and Cl− conductance allow KCl to flow out of the cell resulting in the accumulation of Cl− ions and their associated negative electrical charge in the acinar lumen. Na+ is then thought to follow Cl− by leaking from the interstitium through the tight junctions between the cells to preserve electroneutrality, and the resulting osmotic gradient for NaCl causes a transepithelial movement of water from interstitium to lumen. In the continued presence of the secretagogue a net transepithelial Cl− flux and a concomitant secretion of fluid is sustained as a result of Cl− entry via NKCC1 and exit via the apical Cl− channel. When the stimulus is removed the intracellular Ca2+ concentration falls to resting levels, the K+ and Cl− channels close, and the cell returns to its resting state. Model for salivary fluid secretion based on active transepithelial Cl– transport and the osmotic coupling of salt and water fluxes (see text for details) As discussed in more detail below, considerable experimental evidence indicates that the mechanism presented in Figure 1 can account for most of the salivary secretion from rat, rabbit and presumably human, major salivary glands. However, there is also evidence that two alternate mechanisms based on the same osmotic coupling principle outlined in Figure 1 (viz., transepithelial anion transport driving the secretion of salt followed by osmotically obliged water) can also make significant contributions to salivary fluid secretion. The first of these differs in the Cl− entry step. Here, NKCC1 is replaced by a Cl−/HCO3− exchanger and a Na+/H+ exchanger (Figure 2a). In this model the decrease in intracellular Cl− concentration resulting from secretagogue-induced KCl loss leads to increased Cl− entry via the Cl−/HCO3− exchanger. The HCO3− is then replaced by the diffusion of CO2 into the cell and its conversion into HCO3− plus H+ by carbonic anhydrase (CA). Finally the H+ is pumped out of the cell by the Na+/H+ exchanger using the extracellular to intracellular Na+ gradient generated by Na+/K+ ATPase. The net result is the movement of NaCl into the cell in exchange for CO2 that simply recycles across the basolateral membrane. The other alternate mechanism (Figure 2b) involves the secretion of HCO3− rather than Cl−. Here CO2 enters the acinar cell across the basolateral membrane and is converted to HCO3− plus H+ by intracellular CA. HCO3− is secreted across the apical membrane via an anion channel, possibly the same channel involved in Cl− secretion, and the H+ is extruded by the basolateral Na+/H+ exchanger. Recent data suggest that a basolateral Na+–HCO3− cotransporter may also be responsible for some of the HCO3− entry (38) although this is still rather speculative. Two additional models for salivary fluid secretion based on active transepithelial anion transport (see text for details) Space does not permit us to review all of the experimental data related to the mechanism of salivary fluid secretion. Instead we will briefly discuss some of the early evidence that supports the involvement of the three mechanisms of fluid secretion introduced above and then mention some studies involving more recent experimental approaches. For more comprehensive treatments we suggest several review articles (29; 52; 7; 62) as well as more recent original reports referenced below. Briefly stated, the existing data from whole animal studies, perfused glands, isolated acini, and acinar plasma membranes demonstrate that all of the membrane transport systems illustrated in Figures 1 and 2 are present in salivary acinar cells and function in a way consistent with the proposed mechanisms for fluid secretion. In addition they provide convincing evidence that these mechanisms can account for the fluid secretion observed. As already alluded to above, salivary fluid secretion appears to be a two-stage process as first proposed by 51); that is, saliva is initially formed as a near isotonic plasma-like primary secretion in the acinar lumen (the first stage), then the salivary ducts modify this primary fluid by removing sodium and chloride and adding potassium and bicarbonate to produce a final hypotonic fluid that enters the mouth. The plasma-like ionic composition of the primary saliva has been confirmed by analysing the electrolyte content of fluid collected from the acinar lumen using micropuncture techniques (62; 27). This property of the primary saliva is in general agreement with the operation of some combination of the secretory mechanisms discussed above and with the prevailing view that osmotically driven fluid transport is typically near isotonic owing to the relatively high water permeability of fluid transporting epithelia (43). The impermeability of the salivary ducts to water and their ability to modify the ionic composition of the primary fluid as required have also been confirmed (27; 62). A series of experiments with perfused rat and rabbit submandibular glands carried out in the 1980s were particularly important in focusing experimental attention on the mechanisms discussed above (see 52 for a more detailed review and specific references). These studies showed that acetylcholine-induced salivary secretion is markedly reduced (˜70%) when Cl− is replaced in a Cl−/HCO3−-replete perfusate by a physiologically inert anion, or when inhibitors of NKCC1 were present in the perfusate. The effect of NKCC1 inhibitors was even more dramatic in HCO3−-free media where the inhibition of fluid secretion was >95%. These results, taken together with other supporting data, strongly suggested that the mechanism illustrated in Figure 1 was responsible for most of the fluid secretion. In addition, the residual secretion observed in Cl− free but HCO3− replete media was HCO3− rich and blocked by inhibitors of CA or the Na+/H+ exchanger. This component of fluid secretion was therefore consistent with the presence of the mechanism illustrated in 2Figure 2b. Other less direct observations indicated the operation of the mechanism illustrated in 2Figure 2a as well. In the perfused rat submandibular gland 33) estimated that the mechanisms illustrated in Figures 1, 22a and b contributed to anion secretion and thus fluid secretion in the ratio 16:3:2. Although there is convincing evidence that all three of the mechanisms discussed above make significant contributions to the fluid secreted by the rabbit submandibular, rat submandibular and rat parotid glands, this is not the case for all salivary glands. Thus, for example, the mechanisms shown in 2Figure 2a and b appear to play little if any role in fluid secretion from human labial glands (32), while only the mechanism shown in 2Figure 2b is operative in the bovine parotid (24). This latter observation is consistent with the observation that ruminant saliva contains very high levels of bicarbonate (≈100 mM), which is required to buffer the acid produced by microbial fermentation in the rumen. The reason for the presence of three fluid secretory mechanisms in the same gland is still not yet clear. Moreover, somewhat surprisingly the available evidence indicates that when multiple mechanisms are present they coexist in the same acinar cells (53; 23). It is possible that the cell is able to modulate the contributions of the various mechanisms according to certain physiological circumstances, for example brief versus sustained periods of salivary flow. But at present there is no convincing evidence that anything like this occurs. In a recent paper 13) have studied salivary secretion in mice in which the gene for NKCC1 has been disrupted and no expression of this protein is detectable. Stimulated salivary flow rates in these NKCC1 knockout mice are only 40% of those observed in normal littermates. Interestingly, these authors also found that the activity of the Cl−/HCO3− exchanger was increased in parotid acinar cells from NKCC1 knockout mice suggesting that fluid secretion via the mechanism shown in 2Figure 2a is increased in these animals to compensate for the loss of NKCC1. In addition to reduced salivary flow, NKCC1 knockout mice also exhibited a number of other abnormalities, the most dramatic of which were profound deafness caused by a collapse of the membranous labyrinth of the inner ear (10; 16) and male infertility because of defective spermatogenesis (30). Both of these problems are thought to result from fluid secretory defects in the respective tissues. The mechanisms illustrated in Figures 1 and 2 do not address the question of whether the water that follows salt secretion flows between the cells (via the tight junctions) or through the cell body (via the cytoplasm). In fact this issue has been a source of considerable interest and experimentation in both secretory and absorptive epithelia for many years (43). In salivary acini this question is made more complicated by the pyramidal shape of the acinar cells that results in a rather small area for both the luminal membrane and the tight junctional complex. Thus whichever route the water takes would necessarily require a relatively high water permeability. A major milestone in the resolution of these problems was the discovery and cloning of the aquaporins, a family of plasma membrane water channel proteins (3). It had been known for some time that the membranes of many cells had very high water permeabilities while those of others were relatively water impermeant (e.g. the luminal membranes of salivary ducts, see above) and still others could regulate their water permeability according to need (3). It is now clear that cell membranes have very low intrinsic water permeabilities and that the large water permeabilities observed in the membranes of many tissues are because of the presence of aquaporins. Surprisingly, early studies failed to demonstrate the presence of any aquaporins in salivary acinar cell membranes, however, it was subsequently found that a new aquaporin isoform, AQP5, was localized to the apical membranes of many secretory epithelia, including salivary acinar cells (37). More recent experiments have shown that stimulated salivary flows are reduced >60% in AQP5 knockout mice relative to normal controls (26). Thus it would appear that most, but probably not all of the secreted water flows through the acinar cells. Consistent with its central role in salivary fluid secretion, the activity of NKCC1 has been shown to be dramatically up-regulated by secretagogues. 14) have demonstrated a 20-fold increase in the NKCC1 transport activity of rat parotid acinar cells following the application of muscarinic and other Ca2+ mobilizing stimuli. This effect appears to be mediated by a metabolite of arachadonic acid (14) but the exact mechanism is still uncertain. This observation is consistent with the idea that NKCC1 activity is down-regulated while the cell is at rest to prevent futile cycling of the transporter, then up-regulated during stimulation when its activity is required. Interestingly, in the rat parotid NKCC1 is also activated, although to a lesser degree, by β-adrenergic stimulation (31) which increases intracellular cAMP levels without affecting intracellular Ca2+ concentration. This is because of a phosphorylation of NKCC1 that involves, but does not appear to be directly caused by, cAMP-dependent protein kinase A (PKA) (50; 22). At first glance, it seems odd that NKCC1 would be up-regulated by such a stimulus because, as discussed in detail below, increased intracellular cAMP concentration results in robust acinar protein secretion but typically produces little salivary fluid. However, there is good evidence that sympathetic (adrenergic) stimulation, arising for example from mastication, when superimposed on parasympathetic (muscarinic) stimulation has a synergistic effect on salivary flow (20). This synergetic effect may arise from the cAMP-dependent up regulation of NKCC1. It is also interesting to speculate that the unexplained symptoms of dry mouth (xerostomia) and dry eyes that accompany the use of many commonly prescribed medications (44) may be related to interference with the secretagogue-induced up regulation of NKCC1. Indeed, among these drugs are a number of β-blockers (44). Finally, we should mention that, although the osmotic coupling hypothesis (that water follows salt secretion osmotically) underlying the models in Figures 1 and 2 is widely accepted among physiologists, there are some dissenting views (57). One of the central problems here is that it has not been possible, to date, to experimentally demonstrate the existence of the osmotic gradient between the interstitium and lumen that is required for the functioning of the models. In terms of the osmotic coupling model, the explanation for this problem is that, because of the high water permeability of the epithelium, only a very small gradient is required to account for the water fluxes observed (43). The problem for the dissenting views, a major one in our opinion, is to account for the large body of existing evidence consistent with the osmotic gradient hypothesis not only in salivary glands but also in numerous other secretory and absorptive epithelia. Saliva contains a wide variety of secreted proteins, including: α-amylase, an enzyme involved in the digestion of starch; lysozyme, peroxidase, immunoglobulins (IgA) and many additional proteins that have antibacterial and/or antiviral properties; and mucins, which are multifunctional glycoproteins involved in mechanical protection and prevention of dehydration of the oral epithelia, as well as in lubrication for solid food and trapping of microorganisms (62; Amerongen & Veerman in press). As already indicated, most of the proteins in saliva are secreted by the acinar cells. Salivary proteins exhibit vectorial transport from the rough endoplasmic reticulum, where they are synthesized, through a succession of membrane-bounded compartments including the Golgi complex, condensing vacuoles, and secretory granules (54). The secretory granules migrate to particular locations within the cell close to the apical membrane prior to the release of their contents into the acinar lumen. Exocytosis is the process by which cells release the contents of their secretory granules. This involves the fusion of the granule membrane with the luminal plasma membrane of the secretory cell followed by the rupture of the fused membranes. This process is continuous in most cells (`constitutive' exocytosis), but it can be greatly accelerated following an appropriate cellular signal such as neural stimulation (`regulatory' exocytosis). In the three major salivary glands, parotid, submandibular and sublingual, exocytotic protein secretion is primarily controlled by the autonomic nervous system; sympathetic stimulation elicits protein release from parotid and submandibular gland acini, and parasympathetic stimulation elicits protein release from sublingual gland acini as well as some release from parotid acini (35; 42). We will focus here on amylase secretion from rat parotid acinar cells as recent studies on this system are promoting a better understanding of the cellular events involved in salivary gland exocytosis. Salivary protein secretion, like fluid secretion, is evoked when neurotransmitters bind to specific receptors on the basolateral membrane of the secretory cells and generate intracellular second messengers that, in turn, activate the cellular mechanisms responsible for secretion. cAMP is the primary second messenger for amylase secretion from rat parotid acinar cells (4). Noradrenaline, released from sympathetic nerves, binds to and activates β-adrenergic receptors leading to increased intracellular cAMP levels. cAMP is thought to mediate most of its effects through the activation of a cAMP-dependent protein kinase, also known as PKA. In parotid acinar cells, PKA activation is essential for cAMP-dependent exocytotic secretion (34; 49). However, the target proteins phosphorylated by PKA have not yet been identified. In contrast to rat parotid acini, in many other secretory cells Ca2+ has been found to be the primary intracellular second messenger for exocytosis. But it has been shown that cAMP mediates parotid amylase secretion without the elevation of cytosolic Ca2+ (48). In addition, in permeabilized acinar cells significantly enhanced amylase release was observed at all concentrations of free Ca2+ tested (1). Stimulation of muscarinic, substance P peptidergic or α-adrenergic receptors also elicits significant amylase release from the rat parotid, albeit at levels that are significantly lower than those observed from a β-adrenergic receptor-mediated response. These receptors are activated by acetylcholine and substance P released from parasympathetic nerves, and by noradrenaline released from sympathetic nerves, respectively. The stimulation of these receptors activates phosphatidylinositide metabolism and induces an increase in intracellular Ca2+ concentration without affecting intracellular cAMP levels (4; 46). Yoshimura et al (61; 59, 60) have developed a perfusion system for isolated rat parotid acinar cells that allows one to obtain a detailed time course for amylase release. They have demonstrated that the activation of the cAMP by β-adrenergic results in a increase in the of amylase secretion. the other the activation of Ca2+ mobilizing receptors changes in amylase release of an large but followed by a lower sustained In the of extracellular only the is These time of amylase release the in intracellular Ca2+ that Ca2+ can play a significant role in the regulation of amylase secretion. the stimulation of both cAMP and Ca2+ mobilizing results in that are than the of the by stimulus More detailed discussed below suggest that this synergistic effect is caused by a of the and of the process by It has been shown that the of neurotransmitters can be into three of to the plasma membrane a by and by the elevation of intracellular Ca2+ concentrations that Ca2+ the final fusion process for parotid amylase release and that cAMP the of secretory granules thus in the effect of Ca2+ as the for fusion (Figure confirmed that the experimental data on amylase release from the rat parotid could be for by such a model. More amylase release as two first the first and the second fusion of secretory granules. of the experimental data of with this model indicated that the number of granules is small in cells. Thus considerable amylase release could be by a stimulus even at resting intracellular Ca2+ levels, for the observation that amylase release appears to be mainly It is also worth that in stimulation is to noradrenaline which also results in the stimulation of α-adrenergic that acinar cells a in intracellular Ca2+ concentration with that of A model for the mechanism of and Ca2+ amylase secretion in rat parotid acinar cells. cAMP the of secretory granules and the effect of Ca2+ as the for (see text for details) In cells, proposed the that to a target membrane through the of and target membrane proteins referred to as receptors is a and 1 and are target membrane an protein localized to the secretory granule membrane of rat parotid acinar cells as by that it with an and was by a specific for In rat parotid acinar cells permeabilized with these authors also showed that amylase but not Ca2+ suggesting that is involved in amylase as a In parotid acinar cells, such as 1 and which are found in cells, were not In cells, and are for Both of these proteins were in rat parotid acinar cells, but they were not with suggesting that their with may be studied the of using a rabbit a to of This of the involved in the of This was able to from rat parotid secretory granule membranes from cells in the presence of cAMP and acinar but not in their and this was by an of PKA. These observations suggest that the on to which the was is in resting cells and that PKA is able to this via the phosphorylation of some cytosolic (Figure Two and proteins found to be with are for these proteins, but additional studies are required to this as well as their A possible model for the mechanism of between and by the activation of the cAMP-dependent may be by an as yet protein is removed from as a result of the phosphorylation of a cytosolic protein by cAMP-dependent PKA. of from then allows it to with Two of proteins are known to be involved in signal the proteins, which have well in coupling and the or proteins, which we discuss The small proteins have in the In contrast to the proteins, they of a and have specific proteins that their intrinsic The small proteins are into and These are involved in cell secretion, membrane between the endoplasmic and the Golgi of and protein transport into the respectively. it has been suggested that small proteins are involved in exocytotic in secretory cells, including parotid acinar cells proteins are of the proteins are into two and has been in the secretory granule membrane, plasma membrane and of rat parotid acinar cells and has been observed to to the following β-adrenergic activation proteins have been shown to be involved in the and fusion of transport with membranes in various tissues and have been has been in the secretory granule membrane, plasma membrane and of rat parotid acinar cells like β-adrenergic activation has been shown to the of from the to the membrane has been shown to be localized to secretory granules in rat parotid acinar cells and of from acinar cells following β-adrenergic stimulation was as a for of have been and as multifunctional involved in a number of events such as membrane and have investigated a possible involvement of in from rat parotid acinar cells. They have demonstrated that is mainly in the cytosolic of these cells and is to the secretory granule membrane in a a from the of amylase secretion when it was to permeabilized rat parotid cells The above observations strongly suggest the involvement of small proteins in the of amylase from rat parotid acinar cells. for these proteins based on observations from other tissues are indicated proteins between a cytosolic state and a state with the of many cycling between membranes and can be generated through In cells, proteins are thought to be of is an important in exocytosis. have proposed a role for the in in salivary glands. In human the of protein with has been suggested to be involved in via fusion may be by the of membrane acid may be a In cells, associated with secretory granules to the plasma membrane cell stimulation, resulting in the activation of and its of generated at exocytotic appear to be involved in exocytosis. is also thought to be a of exocytosis. In cells, has been shown to to the activation of following the receptors by the of via an as yet mechanism proteins could have in addition to as yet to those above in protein secretion from salivary acinar cells. The of proteins such as and small proteins in from salivary gland cells to be A number of new and techniques are available to these and other which bind to cytosolic and the of specific proteins, and expression systems for and of specific proteins, and specific should us to understand the of various proteins in salivary protein and as a us to their involvement in mechanisms of oral We for and This was in by for the of and of

The present study was aimed at localization of plasma membrane (PMCA) and intracellular (SERCA) Ca2+ pumps and characterizing their role in initiation and propagation of Ca2+ waves. Specific and polarized expression of Ca2+ pumps was observed in all epithelial cells examined. Immunolocalization revealed expression of PMCA in both the basolateral and luminal membranes of all cell types. SERCA2a appeared to be expressed in the luminal pole, whereas SERCA2b was expressed in the basal pole and the nuclear envelope of pancreatic acini. Interestingly, SERCA2b was found in the luminal pole of submandibular salivary gland acinar and duct cells. These cells expressed SERCA3 in the basal pole. To examine the significance of the polarized expression of SERCA and perhaps PMCA pumps in secretory cells, we compared the effect of inhibition of SERCA pumps with thapsigargine and partial Ca2+ release with ionomycin on Ca2+ release evoked by agonists and Ca2+ uptake induced by antagonists. Despite their polarized expression, Ca2+ uptake by SERCA pumps and Ca2+ efflux by PMCA resulted in uniform reduction in [Ca2+]i. Surprisingly, inhibition of the SERCA pumps, but not Ca2+ release by ionomycin, eliminated the distinct initiation sites and propagated Ca2+ waves, leading to a uniform increase in [Ca2+]i. In addition, inhibition of SERCA pumps reduced the rate of Ca2+ release from internal stores. The implication of these findings to rates of Ca2+ diffusion in the cytosol, compartmentalization of Ca2+ signaling complexes, and mechanism of Ca2+ wave propagation are discussed.

Measurement of [Cl-]i and the Cl- current in the rat salivary submandibular gland (SMG) acinar and duct cells was used to evaluate the role of Cl- channels in the regulation of [Cl-]i during purinergic stimulation. Under resting conditions [Cl-]i averaged 56 +/- 8 and 26 +/- 7 mM in acinar and duct cells, respectively. In both cells, stimulation with 1 mM ATP resulted in Cl- efflux and subsequent influx. Inhibition of NaKCl2 cotransport had no effect on [Cl-]i changes in duct cells and inhibited only about 50% of Cl- uptake in acinar cells. Accordingly, low levels of expression of NaKCl2 cotransporter protein were found in duct cells. Acinar cells expressed high levels of the cotransporter. Measurement of Cl- current under selective conditions revealed that acinar and duct cells express at least five distinct Cl- channels; a ClCO-like, volume-sensitive, inward rectifying, Ca2+-activated and CFTR-like Cl- currents. ATP acting on both cell types activated at least two channels, the Ca2+-activated Cl- channel and a Ca2+-independent glibenclamide-sensitive Cl--current, possibly cystic fibrosis transmembrane regulator (CFTR). Of the many nucleotides tested only 2'-3'-benzoylbenzoyl (Bz)-ATP and UTP activated Cl- channels in SMG cells. Despite their relative potency in increasing [Ca2+]i, BzATP in both SMG cell types largely activated the Ca2+-independent, glibenclamide-sensitive Cl- current, whereas UTP activated only the Ca2+-dependent Cl- current. We interpret this to suggest that BzATP and UTP largely activate Cl- channels residing in the membrane expressing the receptor for the active nucleotide. The present studies reveal a potentially new mechanism for transcellular Cl- transport in a CFTR-expressing tissue, the SMG. Coordinated action of the P2z (luminal) and P2u (basolateral) receptors can mediate part of the transcellular Cl- transport by acinar and duct cells to determine the final electrolyte composition of salivary fluid.

Salivary glands secrete K(+) and HCO(-)(3) and reabsorb Na(+) and Cl(-), but the identity of transporters involved in HCO(-)(3) transport remains unclear. We investigated localization of Cl(-)/HCO(-)(3) exchanger isoform AE2 and of Na(+)-HCO(-)(3) cotransporter (NBC) in rat parotid gland (PAR) and submandibular gland (SMG) by immunoblot and immunocytochemical techniques. Immunoblotting of PAR and SMG plasma membranes with specific antibodies against mouse kidney AE2 and rat kidney NBC revealed protein bands at approximately 160 and 180 kDa for AE2 and approximately 130 kDa for NBC, as expected for the AE2 full-length protein and consistent with the apparent molecular mass of NBC in several tissues other than kidney. Immunostaining of fixed PAR and SMG tissue sections revealed specific basolateral staining of PAR acinar cells for AE2 and NBC, but in SMG acinar cells only basolateral AE2 labeling was observed. No AE2 expression was detected in any ducts. Striated, intralobular, and main duct cells of both glands showed NBC expression predominantly at basolateral membranes, with some cells being apically stained. In SMG duct cells, NBC staining exhibited a gradient of distribution from basolateral localization in more proximal parts of the ductal tree to apical localization toward distal parts of the ductal tree. Both immunoblotting signals and immunostaining were abolished in preabsorption experiments with the respective antigens. Thus the mechanisms of fluid and anion secretion in salivary acinar cells may be different between PAR and SMG, and, because NBC was detected in acinar and duct cells, it may play a more important role in transport of HCO(-)(3) by rat salivary duct cells than previously believed.

Activation of an apical Ca(2+)-dependent Cl(-) channel (CaCC) is the rate-limiting step for fluid secretion in many exocrine tissues. Here, we compared the properties of native CaCC in mouse submandibular salivary gland acinar cells to the Ca(2+)-gated Cl(-) currents generated by Tmem16A and Best2, members from two distinct families of Ca(2+)-activated Cl(-) channels found in salivary glands. Heterologous expression of Tmem16A and Best2 transcripts in HEK293 cells produced Ca(2+)-activated Cl(-) currents with time and voltage dependence and inhibitor sensitivity that resembled the Ca(2+)-activated Cl(-) current found in native salivary acinar cells. Best2(-/-) and Tmem16A(-/-) mice were used to further characterize the role of these channels in the exocrine salivary gland. The amplitude and the biophysical footprint of the Ca(2+)-activated Cl(-) current in submandibular gland acinar cells from Best2-deficient mice were the same as in wild type cells. Consistent with this observation, the fluid secretion rate in Best2 null mice was comparable with that in wild type mice. In contrast, submandibular gland acinar cells from Tmem16A(-/-) mice lacked a Ca(2+)-activated Cl(-) current and a Ca(2+)-mobilizing agonist failed to stimulate Cl(-) efflux, requirements for fluid secretion. Furthermore, saliva secretion was abolished by the CaCC inhibitor niflumic acid in wild type and Best2(-/-) mice. Our results demonstrate that both Tmem16A and Best2 generate Ca(2+)-activated Cl(-) current in vitro with similar properties to those expressed in native cells, yet only Tmem16A appears to be a critical component of the acinar Ca(2+)-activated Cl(-) channel complex that is essential for saliva production by the submandibular gland.

Cystic fibrosis results from defective Cl- channel activity mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) gene product. In the gastrointestinal tract this is manifested in abnormal salivary secretion and pancreatic insufficiency. This is generally attributed to defective Cl- transport by the ductal system of the glands. We provide the first immunocytochemical and functional evidence for expression of CFTR protein and Cl- current in rat and mouse submandibular gland (SMG) and pancreatic acinar cells, a site proximal to the ductal system of these secretory glands. Monoclonal and polyclonal antibodies recognizing COOH-terminal epitopes of CFTR show that duct and acinar cells from the two glands express CFTR in the luminal membrane. Specificity of the polyclonal antibody was verified by absence of staining in duct and acinar cells of the SMG of cf-/cf- and delta F/delta F mice. Identification of CFTR in acinar cells was aided by demonstrating coexpression of CFTR and type 3 inositol 1,4,5-trisphosphate receptors in the luminal pole of acini and absence of type 3 inositol 1,4,5-trisphosphate receptors in ducts. Electrophysiological characterization in single SMG duct and acinar cells shows the presence of a protein kinase A-activated, voltage- and time-independent, ohmic Cl- current and absence of repolarization-dependent tail currents, all of which are kinetic properties of the CFTR-dependent Cl- channel. In addition, the channel was activated by the nonhydrolyzable ATP analog 5'-adenylylimidodiphosphate and the benzimidazalone NS-004. Channels activated by all activators were inhibited by glibenclamide and a known inhibitory antiserum [anti-CFTR-(505-511)]. Combined immunologic, functional, and pharmacological evidence allows us to conclude that acinar cells of the SMG and pancreas express functional CFTR-dependent Cl- channels. Because this site is proximal to the duct, modification of activity of this channel in acinar cells is likely to contribute to abnormal salivary secretion and pancreatic insufficiency typical of cystic fibrosis.

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.

Upon continuous stimulation, the pore of the monovalent cation-selective P2X7 receptor (P2X7R) expands to accommodate large molecules such as N-methyl-D-glucamine (NMDG+). How the change in P2X7R permeability is regulated is not known. Here we report that extracellular Cl- (Cl-(o)) regulates the outward current, whereas extracellular Na+ (Na+(o)) regulates the inward current of large molecules by P2X7Rs. The P2X7R-mediated current was measured in parotid acinar and duct cells of wild type and P2X7R-/- mice and in HEK293 cells expressing the human or mouse P2X7R isoforms. In symmetrical NaCl, triethylammonium chloride, and NMDG+ chloride solutions, the P2X7R current followed a linear current/voltage relationship. In symmetrical NaCl, removal of Cl-(o) reduced the inward Na+ current by approximately 35% and the outward Na+ current by only 10%. By contrast, in the absence of Na+(i) and the presence of Na+(o) or NMDG+(o), the removal of Cl-(o) reduced the inward Na+ or NMDG+ currents by 35% but the outward NMDG+ current by >95%. The effect of Cl-(o) was half-maximal at approximately 60 mm. Reducing Cl-(i) from 150 to 10 mm did not reproduce the effects of Cl-(o). All currents were eliminated in P2X7R-/- cells and reproduced by expressing the P2X7Rs in HEK cells. These findings suggest that Cl-(o) primarily regulates the outward P2X7R current of large molecules. When cells dialyzed with NMDG+ were stimulated in the presence of Na+(o), subsequent removal of Na+(o) resulted in a strongly outward rectifying NMDG+ current, indicating maintained high selectivity for Na+ over NMDG+. During continuous incubation in Na+-free medium, the permeability of the P2X7Rs to NMDG+ gradually increased. On the other hand, when the cells were incubated in symmetrical NMDG+ and only then stimulated with ATP, the NMDG+ current by P2X7Rs followed a linear current/voltage relationship and did not change with time. These findings suggest that the P2X7R has a "Na+(o) memory" and that Na+(o) regulates the inward permeability of P2X7Rs to large molecules. The novel regulation of P2X7R outward and inward permeability to large molecules by Cl-(o) and Na+(o), respectively, may have an important protective function, particularly in secretory epithelial cells.

Currently, all salivary ducts (intercalated, striated and collecting) are assumed to function broadly in a similar manner, reclaiming ions that were secreted by the secretory acinar cells while preserving fluid volume and delivering saliva to the oral cavity. Nevertheless, there has been minimal investigation into the structural and functional differences between distinct types of salivary duct cells. Therefore, in this study, the expression profile of proteins involved in stimulus-secretion coupling, as well as the function of the intercalated duct (ID) and striated duct cells, was examined. Particular focus was placed on defining differences between distinct duct cell populations. To accomplish this, immunohistochemistry and in situ hybridization were utilized to examine the localization and expression of proteins involved in reabsorption and secretion of ions and fluid. Further, in vivo calcium imaging was employed to investigate cellular function. Based on the protein expression profile and functional data, marked differences between the IDs and striated ducts were observed. Specifically, the ID cells express proteins native to the secretory acinar cells while lacking proteins specifically expressed in the striated ducts. Further, the ID and striated duct cells display different calcium signalling characteristics, with the IDs responding to a neural stimulus in a manner similar to the acinar cells. Overall, our data suggest that the IDs have a distinct role in the secretory process, separate from the reabsorptive striated ducts. Instead, based on our evidence, the IDs express proteins found in secretory cells, generate calcium signals in a manner similar to acinar cells, and, therefore, are likely secretory cells. KEY POINTS: Current studies examining salivary intercalated duct cells are limited, with minimal documentation of the ion transport machinery and the overall role of the cells in fluid generation. Salivary intercalated duct cells are presumed to function in the same manner as other duct cells, reclaiming ions, maintaining fluid volume and delivering the final saliva to the oral cavity. Here we systematically examine the structure and function of the salivary intercalated duct cells using immunohistochemistry, in situ hybridization and by monitoring in vivo Ca2+ dynamics. Structural data revealed that the intercalated duct cells lack proteins vital for reabsorption and express proteins necessary for secretion. Ca2+ dynamics in the intercalated duct cells were consistent with those observed in secretory cells and resulted from GPCR-mediated IP3 production.

PDAC development is associated with the formation of premalignant duct-like lesions called pancreatic intraepithelial neoplasias (PanIN). PanIN are thought to initially form as low-grade hypermucinous PanIN1 lesions then become less mucinous and contain nuclear nuclear atypia. Although these lesions are known to be associated with PDAC, with the exception of PanIN3 lesions, it is unknown whether PanINs directly contribute to PDAC. To model PanIN and PDAC initiation, animal models have been created to express one of the most common PDAC mutations, oncogenic KrasG12D, in pancreatic cells. Expression of KrasG12D specifically in acinar cells results in widespread formation of low-grade PanIN lesions, where as KrasG12D expression in ductal cells results in low numbers of low-grade PanIN. This suggests that both acinar and ductal cells can contribute to low-grade PanIN, but these lesions form more readily from acinar cells in the presence of KrasG12D. The contribution of acinar and ductal cells to PDAC, however, is less clear. Recent studies have shown that loss of Brg1 or biallelic expression of Trp53R172H are required for ductal cell transformation, however loss of Brg1 in acinar cells prevents PanIN formation in the presence of KrasG12D and only one copy of Trp53R172H is required for transformation of KrasG12D-expressing acinar cells. This suggests that the genetic or epigenetic changes needed for ductal and acinar cell transformation are different. However, the underlying impact of cellular origin to tumorigenesis remains unclear. To address this open question, we examined whether acinar or ductal cells expressing oncogenic Kras contribute to PDAC in the absence of Trp53. Using our novel Sox9CreER;KrasG12D or Ptf1aCreER;KrasG12D mouse models combined with a condition knockout allele for Trp53, we created Sox9CreER;KrasG12D;Trp53f/f (Duct:Kras-p53) or Ptf1aCreER;KrasG12D;Trp53f/f (Acinar:Kras-p53) mice. Our unpublished analysis of these mice show that KrasG12D induces lethal PDAC from ductal and acinar cells in the absence of Trp53. Remarkably, the survival of mice with ductal-cell-derived PDAC is significantly shorter than the survival of mice with acinar-cell-derived PDAC. Through analysis of these mice at earlier time points, we demonstrated that this difference in survival appears to be primarily due to the rapid induction of high-grade PanIN lesions and PDAC from ductal cells by as early as four weeks after the induction of recombination. In contrast, tumor development from acinar cells is initiated at a slower pace, requires loss of the mature acinar cell fate, and is associated with the formation of large numbers of low-grade PanIN, as well as a lower number of high-grade lesions. Altogether, these early events appear to delay tumor formation from acinar cells compared to ductal cells. Thus, cellular origin has important affects on tumor initiation and progression. Finally, cellular origin not only appears to impact initiation of tumorigenesis, but the tumors arising from ductal and acinar cells also appear to differ. Our studies revealed that gross distant metastases occurred from ductal-cell-derived PDAC, but was not observed in mice with acinar-cell-derived PDAC. This suggests that the inherent phenotype of the tumors resulting from acinar and ductal cells may also differ. In sum, work by our group and others have shown that both acinar and ductal cells can contribute to PDAC. Importantly, our work shows that cell of origin alone can impact the course of tumor development and affect the metastatic nature of PDAC. Citation Format: Alex Lee, Claire Dubois, David Schaeffer, Maike Sander, Janel Kopp.{Authors}. PDAC initiation and progression occurs more rapidly from KrasG12D-expressing ductal cells than KrasG12D-expressing acinar cells in the absence of Trp53. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A84.

In Vivo Lineage Tracing Defines the Role of Acinar-to-Ductal Transdifferentiation in Inflammatory Ductal Metaplasia

Cystic fibrosis is caused by mutations in CFTR, the cystic fibrosis transmembrane conductance regulator gene. Disruption of CFTR-mediated anion conductance results in defective fluid and electrolyte movement in the epithelial cells of organs such as the pancreas, airways and sweat glands, but the function of CFTR in salivary glands is unclear. Salivary gland acinar cells produce an isotonic, plasma-like fluid, which is subsequently modified by the ducts to produce a hypotonic, NaCl-depleted final saliva. In the present study we investigated whether submandibular salivary glands (SMGs) in F508 mice (Cftr(F/F)) display ion transport defects characteristic of cystic fibrosis in other tissues. Immunolocalization and whole-cell recordings demonstrated that Cftr and the epithelial Na(+) (ENaC) channels are co-expressed in the apical membrane of submandibular duct cells, consistent with the significantly higher saliva [NaCl] observed in vivo in Cftr(F/F) mice. In contrast, Cftr and ENaC channels were not detected in acinar cells, nor was saliva production affected in Cftr(F/F) mice, implying that Cftr contributes little to the fluid secretion process in the mouse SMG. To identify the source of the NaCl absorption defect in Cftr(F/F) mice, saliva was collected from ex vivo perfused SMGs. Cftr(F/F) glands secreted saliva with significantly increased [NaCl]. Moreover, pharmacological inhibition of either Cftr or ENaC in the ex vivo SMGs mimicked the Cftr(F/F) phenotype. In summary, our results demonstrate that NaCl absorption requires and is likely to be mediated by functionally dependent Cftr and ENaC channels localized to the apical membranes of mouse salivary gland duct cells.

Salivary glands consist of multiple phenotypically and functionally unique cell populations, such as the acinar, ductal, and myoepithelial cells that help produce, modify, and secrete saliva (Lombaert et al., 2011). Identification of mechanisms and factors that regulate these populations has been of key interest, as salivary gland-related diseases have detrimental effects on these cell populations. A variety of approaches have been used to understand the roles different signaling mechanisms and transcription factors play in regulating salivary gland development and homeostasis. Differentiation assays have been performed with primary salivary cells in the past (Maimets et al., 2016), however this approach may sometimes be limiting due to tissue availability, labor intensity of processing the tissue samples, and/or inability to long-term passage the cells. Here we describe in detail a 3D differentiation assay to analyze the differentiation potential of a salivary gland cell line, SIMS, which was immortalized from an adult mouse submandibular salivary gland (Laoide et al., 1996). SIMS cells express cytokeratin 7 and 19, which is characteristic for a ductal cell type. Although adult acinar and myoepithelial cells were found in vivo to preserve their own cell population through self-duplication (Aure et al., 2015; Song et al. 2018), in some cases duct cells can differentiate into acinar cells in vivo, such as after radiation injury (Lombaert et al., 2008; Weng et al., 2018). Thus, utilization of SIMS cells allows us to target and analyze the self-renewal and differentiation effects of ductal cells under specific in vitro controlled conditions.