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.

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.

Large volumes of saliva are generated by transepithelial Cl(-) movement during parasympathetic muscarinic receptor stimulation. To gain further insight into a major Cl(-) uptake mechanism involved in this process, we have characterized the anion exchanger (AE) activity in mouse serous parotid and mucous sublingual salivary gland acinar cells. The AE activity in acinar cells was Na(+) independent, electroneutral, and sensitive to the anion exchange inhibitor DIDS, properties consistent with the AE members of the SLC4A gene family. Localization studies using a specific antibody to the ubiquitously expressed AE2 isoform labeled acini in both parotid and sublingual glands. Western blot analysis detected an approximately 170-kDa protein that was more highly expressed in the plasma membranes of sublingual than in parotid glands. Correspondingly, the DIDS-sensitive Cl(-)/HCO(3)(-) exchanger activity was significantly greater in sublingual acinar cells. The carbonic anhydrase antagonist acetazolamide markedly inhibited, whereas muscarinic receptor stimulation enhanced, the Cl(-)/HCO(3)(-) exchanger activity in acinar cells from both glands. Intracellular Ca(2+) chelation prevented muscarinic receptor-induced upregulation of the AE, whereas raising the intracellular Ca(2+) concentration with the Ca(2+)-ATPase inhibitor thapsigargin mimicked the effects of muscarinic receptor stimulation. In summary, carbonic anhydrase activity was essential for regulating Cl(-)/HCO(3)(-) exchange in salivary gland acinar cells. Moreover, muscarinic receptor stimulation enhanced AE activity through a Ca(2+)-dependent mechanism. Such forms of regulation may play important roles in modulating fluid and electrolyte secretion by salivary gland acinar cells.

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The regulation of intracellular pH (pHé) in epithelial cells is critical for maintaining normal enzyme activity as well as for modulating fluid and electrolyte absorption and secretion (Aronson, 1985). There are several ion transport pathways involved in epithelial pHé regulation including Na¤-H¤ exchangers, Cl¦-HCOצ exchangers and Na¤-HCOצ cotransporters (Geibel et al. 1990;Kopito, 1990;Steward et al. 1996). In salivary acinar cells, Na¤-H¤ exchange plays a significant role in regulating Cl¦-and HCOצ-dependent fluid secretion during muscarinic stimulation via at least two mechanisms. Upregulation of Na¤-H¤ exchanger activity maintains a neutral intracellular pH, thereby enhancing the production of HCOצ (Turner, 1993) and the activity of the intracellular pH-sensitive anion channel (Arreola et al. 1995). Moreover, Na¤-H¤ and Cl¦-HCOצ exchangers act in concert to drive NaCl uptake in exchange Journal of Physiology (2000) 1. Intracellular pH (pHé) plays an important role in regulating fluid and electrolyte secretion by salivary gland acinar cells. The pH-sensitive, fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxylfluorescein (BCECF) was used to characterize the mechanisms involved in regulating pHé during muscarinic stimulation in mouse sublingual mucous acinar cells. 2. In the presence of HCOצ, muscarinic stimulation caused a rapid decrease in pHé (0·24 ± 0·02 pH units) followed by a slow recovery rate (0·042 ± 0·002 pH units min¢) to the initial resting pHé in sublingual acinar cells. The muscarinic receptor-induced acidification in parotid acinar cells was of a similar magnitude (0·25 ± 0·02 pH units), but in contrast, the recovery rate was •4-fold faster (0·181 ± 0·005 pH units min¢). 3. The agonist-induced intracellular acidification was inhibited by the anion channel blocker niflumate, and was prevented in the absence of HCOצ by treatment with the carbonic anhydrase inhibitor methazolamide. These results indicate that the muscarinic-induced acidification is due to HCOצ loss, probably mediated by an anion conductive pathway. 4. The Na¤-H¤ exchange inhibitor 5-(N-ethyl-N-isopropyl)amiloride (EIPA) amplified the magnitude of the agonist-induced acidification and completely blocked the Na¤-dependent pHé recovery. 5. To examine the molecular nature of the Na¤-H¤ exchange mechanism in sublingual acinar cells, pH regulation was investigated in mice lacking Na¤-H¤ exchanger isoforms 1 and 2 (NHE1 and NHE2, respectively). The magnitude and the rate of pHé recovery in response to an acid load in acinar cells isolated from mice lacking NHE2 were comparable to that observed in cells from wild-type animals. In contrast, targeted disruption of the Nhe1 gene completely abolished pHé recovery from an acid load. These results demonstrate that NHE1 is critical for regulating pHé during a muscarinic agonist-stimulated acid challenge and probably plays an important role in regulating fluid secretion in the sublingual exocrine gland. 6. In NHE1-deficient mice, sublingual acinar cells failed to recover from an acid lo...