Understanding salivary fluid and protein secretion (2002)

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 to that the of and that the of many may be related to with the secretagogue-induced up regulation of NKCC1. these are a number of we mention although the osmotic coupling water follows salt secretion the models in Figures 1 and 2 is there are some of the central problems is that it has not been to to demonstrate the of the osmotic gradient between the interstitium and lumen that is required for the of the In of the osmotic coupling the for this is because of the high water permeability of the only a very small gradient is required to account for the water fluxes observed (43). The for the a major in is to account for the large body of existing evidence consistent with the osmotic gradient not only in salivary glands but also in other secretory and absorptive contains a variety of secreted proteins, an involved in the of and many additional proteins that have and which are involved in and of of the epithelia, as well as in for and of (62; in As already most of the proteins in saliva are secreted by the acinar cells. proteins transport from the where they are through a of including the and secretory The secretory to the cell to the apical membrane to the of their into the acinar lumen. is the process by which cells the of their secretory This involves the of the membrane with the luminal plasma membrane of the secretory cell followed by the of the This process is in most cells but it can be following an cellular such as stimulation In the three major salivary glands, submandibular and protein secretion is by the sympathetic stimulation protein from parotid and submandibular gland acini, and parasympathetic stimulation protein from gland acini as well as some from parotid acini will on secretion from rat parotid acinar cells as recent studies on this system are a of the cellular involved in salivary gland protein secretion, like fluid secretion, is when to specific on the basolateral membrane of the secretory cells and intracellular in the cellular mechanisms responsible for secretion. cAMP is the primary for secretion from rat parotid acinar cells from sympathetic to and β-adrenergic to increased intracellular cAMP cAMP is thought to most of its through the of a cAMP-dependent protein also known as In parotid acinar is for cAMP-dependent secretion However, the proteins by have not yet been In to rat parotid acini, in many other secretory cells Ca2+ has been found to be the primary intracellular for But it has been shown that cAMP parotid secretion without the of Ca2+ In addition, in acinar cells was observed at all of free Ca2+ of or also significant from the rat albeit at levels that are than those observed from a β-adrenergic These are activated by and from parasympathetic and by from sympathetic The stimulation of these and an increase in intracellular Ca2+ concentration without affecting intracellular cAMP levels have a system for isolated rat parotid acinar cells that to a detailed time for have demonstrated that the of the cAMP by β-adrenergic results in a increase in the of secretion. the other the of Ca2+ mobilizing changes in of an large but followed by a sustained In the of extracellular only the is These time of the in intracellular Ca2+ that Ca2+ can play a significant role in the regulation of 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 can be into three of to the plasma membrane a by and by the of intracellular Ca2+ that Ca2+ the final process for parotid and that cAMP the of secretory thus in the effect of Ca2+ as the for (Figure confirmed that the experimental data on from the rat parotid could be for by such a model. More as two first the first and the of secretory of the experimental data of with this model indicated that the number of is small in cells. Thus considerable could be by a stimulus even at resting intracellular Ca2+ levels, for the observation that appears to be mainly It is also worth that in stimulation is to which also results in the stimulation of that acinar cells a in intracellular Ca2+ concentration with that of A model for the mechanism of and Ca2+ secretion in rat parotid acinar cells. cAMP the of secretory and the effect of Ca2+ as the for (see text for details) In proposed the that to a membrane through the of and membrane proteins referred to as is a and 1 and are membrane an protein localized to the secretory membrane of rat parotid acinar cells as by that it with an and was by a specific for In rat parotid acinar cells with these authors also showed that but not Ca2+ suggesting that is involved in as a In parotid acinar such as 1 and which are found in were not In and are for Both of these proteins were in rat parotid acinar 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 membranes from cells in the presence of cAMP and acinar but not in their and this was by an of These observations suggest that the on to which the was is in resting cells and that is able to this via the phosphorylation of some (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 of the cAMP-dependent may be by an as yet protein is removed from as a result of the phosphorylation of a protein by cAMP-dependent of from then it to with Two of proteins are known to be involved in the proteins, which have well in coupling and the or proteins, which we discuss The small proteins have in the In 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 and the of and protein transport into the it has been suggested that small proteins are involved in in secretory including parotid acinar cells proteins are of the proteins are into two and has been in the secretory membrane, plasma membrane and of rat parotid acinar cells and has been observed to to the following β-adrenergic proteins have been shown to be involved in the and of transport with membranes in various tissues and have been has been in the secretory membrane, plasma membrane and of rat parotid acinar cells like β-adrenergic has been shown to the of from the to the membrane has been shown to be localized to secretory in rat parotid acinar cells and of from acinar cells following β-adrenergic stimulation was as a for of have been and as involved in a number of such as membrane and have investigated a possible involvement of in from rat parotid acinar cells. have demonstrated that is mainly in the of these cells and is to the secretory membrane in a a from the of secretion when it was to rat parotid cells The above observations strongly suggest the involvement of small proteins in the of from rat parotid acinar cells. for these proteins based on observations from other tissues are indicated proteins between a state and a state with the of many cycling between membranes and can be generated through In proteins are thought to be of is an important in have proposed a role for the in in salivary glands. In human the of protein with has been suggested to be involved in via may be by the of membrane acid may be a In associated with secretory to the plasma membrane cell stimulation, resulting in the of and its of generated at appear to be involved in is also thought to be a of In has been shown to to the of following the 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 to and the of specific proteins, and expression systems for and of specific proteins, and specific us to understand the of various proteins in salivary protein and as a us to their involvement in mechanisms of for and This was in by for the of and of