Aquaporins (AQPs) are channel proteins that regulate the movement of water through the plasma membrane of secretory and absorptive cells in response to osmotic gradients. In the salivary gland, AQP5 is the major aquaporin expressed on the apical membrane of acinar cells. Previous studies have shown that the volume of saliva secreted by AQP5-deficient mice is decreased, indicating a role for AQP5 in saliva secretion; however, the mechanism by which AQP5 regulates water transport in salivary acinar cells remains to be determined. Here we show that the decreased salivary flow rate and increased tonicity of the saliva secreted by Aqp5 ؊/؊ mice in response to pilocarpine stimulation are not caused by changes in whole body fluid homeostasis, indicated by similar blood gas and electrolyte concentrations in urine and blood in wild-type and AQP5-deficient mice. In contrast, the water permeability in parotid and sublingual acinar cells isolated from Aqp5 ؊/؊ mice is decreased significantly. Water permeability decreased by 65% in parotid and 77% in sublingual acinar cells from Aqp5 ؊/؊ mice in response to hypertonicity-induced cell shrinkage and hypotonicity-induced cell swelling. These data show that AQP5 is the major pathway for regulating the water permeability in acinar cells, a critical property of the plasma membrane which determines the flow rate and ionic composition of secreted saliva.The precise regulation of water and electrolyte transport in the acinar cells of the salivary gland is crucial for proper production of saliva. The fluid component of salivary secretions hydrates the oral cavity, aiding in the mastication and swallowing of food, in the neutralization of acids, and in protection against the invasion of potential pathogens. Clinically, salivary gland hypofunction commonly presents as xerostomia, a symptomatic complaint of dry mouth prevalent in the geriatric population (for review, see Ref. 1) which may result from either systemic or extrinsic causes (for review, see Refs. 1-3).Saliva formation is a two-stage process (4, 5). First, the acinar cells secrete an isotonic plasma-like fluid, and second, ductal cells modify the acinar secretions primarily through the reabsorption of Na ϩ and Cl Ϫ so that the final saliva is hypotonic. This fluid secretion model predicts that saliva formation is primarily caused by transepithelial Cl Ϫ transport and that Cl Ϫ uptake is dependent on an inwardly directed Na ϩ chemical gradient across the basolateral plasma membrane. An increase in intracellular Ca 2ϩ , usually associated with muscarinic receptor stimulation, triggers fluid secretion by simultaneously activating apical Cl Ϫ channels and basolateral K ϩ channels. The efflux of Cl Ϫ and K ϩ across the apical and basolateral membranes, respectively, produces a transepithelial potential difference that is neutralized by paracellular Na ϩ transport across tight junctions. The resulting transepithelial osmotic gradient drives the movement of water, creating a plasma-like primary secretion.In salivary gland acinar cell...

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ClC-2 is localized to the apical membranes of secretory epithelia where it has been hypothesized to play a role in fluid secretion. Although ClC-2 is clearly the inwardly rectifying anion channel in several tissues, the molecular identity of the hyperpolarization-activated Cl ؊ current in other organs, including the salivary gland, is currently unknown. To determine the nature of the hyperpolarization-activated Cl ؊ current and to examine the role of ClC-2 in salivary gland function, a mouse line containing a targeted disruption of the Clcn2 gene was generated. The resulting homozygous Clcn2 ؊/؊ mice lacked detectable hyperpolarization-activated chloride currents in parotid acinar cells and, as described previously, displayed postnatal degeneration of the retina and testis. The magnitude and biophysical characteristics of the volume-and calcium-activated chloride currents in these cells were unaffected by the absence of ClC-2. Although ClC-2 appears to contribute to fluid secretion in some cell types, both the initial and sustained salivary flow rates were normal in Clcn2 ؊/؊ mice following in vivo stimulation with pilocarpine, a cholinergic agonist. In addition, the electrolytes and protein contents of the mature secretions were normal. Because ClC-2 has been postulated to contribute to cell volume control, we also examined regulatory volume decrease following cell swelling. However, parotid acinar cells from Clcn2 ؊/؊ mice recovered volume with similar efficiency to wild-type littermates. These data demonstrate that ClC-2 is the hyperpolarization-activated Cl ؊ channel in salivary acinar cells but is not essential for maximum chloride flux during stimulated secretion of saliva or acinar cell volume regulation.

Multiple Na؉ /H ؉ exchangers (NHEs) are expressed in salivary gland cells; however, their functions in the secretion of saliva by acinar cells and the subsequent modification of the ionic composition of this fluid by the ducts are unclear. Mice with targeted disruptions of the Nhe1, Nhe2, and Nhe3 genes were used to study the in vivo functions of these exchangers in parotid glands. Immunohistochemistry indicated that NHE1 was localized to the basolateral and NHE2 to apical membranes of both acinar and duct cells, whereas NHE3 was restricted to the apical region of duct cells. Na ؉ /H ؉ exchange was reduced more than 95% in acinar cells and greater than 80% in duct cells of NHE1-deficient mice (Nhe1 ؊/؊ ). Salivation in response to pilocarpine stimulation was reduced significantly in both Nhe1 ؊/؊ and Nhe2 ؊/؊ mice, particularly during prolonged stimulation, whereas the loss of NHE3 had no effect on secretion. Expression of Na ؉ /K ؉ /2Cl ؊ cotransporter mRNA increased dramatically in Nhe1 ؊/؊ parotid glands but not in those of Nhe2 ؊/؊ or Nhe3 ؊/؊ mice, suggesting that compensation occurs for the loss of NHE1. The sodium content, chloride activity and osmolality of saliva in Nhe2 ؊/؊ or Nhe3 ؊/؊ mice were comparable with those of wild-type mice. In contrast, Nhe1 ؊/؊ mice displayed impaired NaCl absorption. These results suggest that in parotid duct cells apical NHE2 and NHE3 do not play a major role in Na ؉ absorption. These results also demonstrate that basolateral NHE1 and apical NHE2 modulate saliva secretion in vivo, especially during sustained stimulation when secretion depends less on Na ؉ /K ؉ /2Cl ؊ cotransporter activity.

Several members of the Na+/H+exchanger gene family (NHE1, NHE2, NHE3, and NHE4) with unique functional properties have been cloned from rat epithelial tissues. The present study examined the molecular and pharmacological properties of Na+/H+exchange in rat parotid salivary gland cells. In acinar cells superfused with a physiological salt solution (145 mM Na+), Na+/H+exchanger activity was inhibited by low concentrations of the amiloride derivative ethylisopropyl amiloride (EIPA; IC50 = 0.014 ± 0.005 μM), suggesting the expression of amiloride-sensitive isoforms NHE1 and/or NHE2. Semiquantitative RT-PCR confirmed that NHE1 transcripts are most abundant in this cell type. In contrast, the intermediate sensitivity of ductal cells to EIPA indicated that inhibitor-sensitive and -resistant Na+/H+exchanger isoforms are coexpressed. Ductal cells were about one order of magnitude more resistant to EIPA (IC50 = 0.754 ± 0.104 μM) than cell lines expressing NHE1 or NHE2 (IC50 = 0.076 ± 0.013 or 0.055 ± 0.015 μM, respectively). Conversely, ductal cells were nearly one order of magnitude more sensitive to EIPA than a cell line expressing the NHE3 isoform (IC50= 6.25 ± 1.89 μM). Semiquantitative RT-PCR demonstrated that both NHE1 and NHE3 transcripts are expressed in ducts. NHE1 was immunolocalized to the basolateral membranes of acinar and ductal cells, whereas NHE3 was exclusively seen in the apical membrane of ductal cells. Immunoblotting, immunolocalization, and semiquantitative RT-PCR experiments failed to detect NHE2 expression in either cell type. Taken together, our results demonstrate that NHE1 is the dominant functional Na+/H+exchanger in the plasma membrane of rat parotid acinar cells, whereas NHE1 and NHE3 act in concert to regulate the intracellular pH of ductal cells.

Salivary gland acinar cells shrink when Cl− currents are activated following cell swelling induced by exposure to a hypotonic solution or in response to calcium‐mobilizing agonists. The molecular identity of the Cl− channel(s) in salivary cells involved in these processes is unknown, although ClC‐3 has been implicated in several tissues as a cell‐volume‐sensitive Cl− channel. We found that cells isolated from mice with targeted disruption of the Clcn3 gene undergo regulatory volume decrease in a fashion similar to cells from wild‐type littermates. Consistent with a normal regulatory volume decrease response, the magnitude and the kinetics of the swell‐activated Cl− currents in cells from ClC‐3‐deficient mice were equivalent to those from wild‐type mice. It has also been suggested that ClC‐3 is activated by Ca2+‐calmodulin‐dependent protein kinase II; however, the magnitude of the Ca2+‐dependent Cl− current was unchanged in the Clcn3−/‐ animals. In addition, we observed that ClC‐3 appeared to be highly expressed in the smooth muscle cells of glandular blood vessels, suggesting a potential role for this channel in saliva production by regulating blood flow, yet the volume and ionic compositions of in vivo stimulated saliva from wild‐type and null mutant animals were comparable. Finally, in some cells ClC‐3 is an intracellular channel that is thought to be involved in vesicular acidification and secretion. Nevertheless, the protein content of saliva was unchanged in Clcn3−/‐ mice. Our results demonstrate that the ClC‐3 Cl− channel is not a major regulator of acinar cell volume, nor is it essential for determining the secretion rate and composition of saliva.

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We investigated the role of M1 and M3 receptors in regulating exocrine secretion from acini isolated from rat sublingual glands. In secretion experiments, we derived affinity values (KB) from Schild regression analysis for the antagonists pirenzepine (61.0 nM) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP; 1.06 nM). The KB for 4-DAMP is similar to its affinity value [equilibrium dissociation constant from competition studies (Ki); 1.81 nM] determined from radioligand competition experiments. In contrast, the KB for pirenzepine is between its high-affinity (17.6 nM) and low-affinity (404 nM) Ki values. In separate secretion experiments, we found that the M1 receptor antagonist, M1-toxin, induces a rightward shift in the concentration-response curve to muscarinic agonist and inhibits maximal secretion by 40%. The inhibitory effect of M1-toxin appears specific for M1 receptor blockade, since the toxin abolishes acinar high-affinity pirenzepine-binding sites and does not inhibit secretion induced by nonmuscarinic agents. Additional pharmacological studies indicate muscarinic receptors do not function through putative neural elements within isolated acini. Our combined results are consistent with both M1 and M3 receptors directly regulating mucous acinar exocrine secretion and indicate M3 receptors alone are insufficient to induce a maximal muscarinic response.