Defective NaCl Reabsorption in Salivary Glands of Eda-Null X-LHED Mice.

Mutations in the ectodysplasin A gene ( EDA) cause X-LHED (X-linked hypohidrotic ectodermal dysplasia), the most common human form of ectodermal dysplasia. Defective EDA signaling is linked to hypoplastic development of epithelial tissues, resulting in hypotrichosis, hypodontia, hypohidrosis, and xerostomia. The primary objective of the present study was to better understand the salivary gland dysfunction associated with ectodermal dysplasia using the analogous murine disorder. The salivary flow rate and ion composition of the 3 major salivary glands were determined in adult Eda-deficient Tabby hemizygous male (Ta/Y) and heterozygous female (Ta/X) mice. Submandibular and sublingual glands of Eda-mutant mice were smaller than wild-type littermates, while parotid gland weight was not significantly altered. Fluid secretion by the 3 major salivary glands was essentially unchanged, but the decrease in submandibular gland size was associated with a dramatic loss of ducts in Ta/Y and Ta/X mice. Reabsorption of Na+ and Cl-, previously linked in salivary glands to Scnn1 Na+ channels and Cftr Cl- channels, respectively, was markedly reduced at high flow rates in the ex vivo submandibular glands of Ta/Y mice (~60%) and, to a lesser extent, Ta/X mice (Na+ by 14%). Consistent with decreased Na+ reabsorption in Ta/Y mice, quantitative polymerase chain reaction analysis detected decreased mRNA expression for Scnn1b and Scnn1g, genes encoding the ß and γ subunits, respectively. Moreover, the Na+ channel blocker amiloride significantly inhibited Na+ and Cl- reabsorption by wild-type male submandibular glands to levels comparable to those observed in Ta/Y mice. In summary, fluid secretion was intact in the salivary glands of Eda-deficient mice but displayed marked Na+ and Cl- reabsorption defects that correlated with the loss of duct cells and decreased Scnn1 Na+ channel expression. These results provide a likely mechanism for the elevated NaCl concentration observed in the saliva of affected male and female patients with X-LHED.

Late responses to adenoviral-mediated transfer of the aquaporin-1 gene for radiation-induced salivary hypofunction.

We evaluated late effects of AdhAQP1 administration in five subjects in a clinical trial for radiation-induced salivary hypofunction (http://www.clinicaltrials.gov/ct/show/NCT00372320?order=). All were identified as initially responding to human aquaporin-1 (hAQP1) gene transfer. They were followed for 3-4 years after AdhAQP1 delivery to one parotid gland. At intervals we examined salivary flow, xerostomic symptoms, saliva composition, vector presence and efficacy in the targeted gland, clinical laboratory data and adverse events. All displayed marked increases (71-500% above baseline) in parotid flow 3-4.7 years after treatment, with improved symptoms for ~2-3 years. There were some changes in [Na+] and [Cl-] consistent with elevated salivary flow, but no uniform changes in secretion of key parotid proteins. There were no clinically significant adverse events, nor consistent negative changes in laboratory parameters. One subject underwent a core needle biopsy of the targeted parotid gland 3.1 years post treatment and displayed evidence of hAQP1 protein in acinar, but not duct, cell membranes. All subjects responding to hAQP1 gene transfer initially had benefits for much longer times. First-generation adenoviral vectors typically yield transit effects, but these data show beneficial effects can continue years after parotid gland delivery.

The calcium-activated potassium channel KCa3.1 plays a central role in the chemotactic response of mammalian neutrophils.

AIM: Neutrophils are the first cells to arrive at sites of injury. Nevertheless, many inflammatory diseases are characterized by an uncontrolled infiltration and action of these cells. Cell migration depends on volume changes that are governed by ion channel activity, but potassium channels in neutrophil have not been clearly identified. We aim to test whether KCa3.1 participates in neutrophil migration and other relevant functions of the cell. METHODS: Cytometer and confocal measurements to determine changes in cell volume were used. Cells isolated from human, mouse and horse were tested for KCa3.1-dependent chemotaxis. Chemokinetics, calcium handling and release of reactive oxygen species were measured to determine the role of KCa3.1 in those processes. A mouse model was used to test for neutrophil recruitment after acute lung injury in vivo. RESULTS: We show for the first time that KCa3.1 is expressed in mammalian neutrophils. When the channel is inhibited by a pharmacological blocker or by genetic silencing, it profoundly affects cell volume regulation, and chemotactic and chemokinetic properties of the cells. We also demonstrated that pharmacological inhibition of KCa3.1 did not affect calcium entry or reactive oxygen species production in neutrophils. Using a mouse model of acute lung injury, we observed that Kca3.1(-/-) mice are significantly less effective at recruiting neutrophils into the site of inflammation. CONCLUSIONS: These results demonstrate that KCa3.1 channels are key actors in the migration capacity of neutrophils, and its inhibition did not affect other relevant cellular functions.

Functional differences in the acinar cells of the murine major salivary glands.

In humans, approximately 90% of saliva is secreted by the 3 major salivary glands: the parotid (PG), the submandibular (SMG), and the sublingual glands (SLG). Even though it is known that all 3 major salivary glands secrete saliva by a Cl(-)-dependent mechanism, salivary secretion rates differ greatly among these glands. The goal of this study was to gain insight into the properties of the ion-transporting pathways in acinar cells that might account for the differences among the major salivary glands. Pilocarpine-induced saliva was simultaneously collected in vivo from the 3 major salivary glands of mice. When normalized by gland weight, the amount of saliva secreted by the PG was more than 2-fold larger than that obtained from the SMG and SLG. At the cellular level, carbachol induced an increase in the intracellular [Ca(2+)] that was more than 2-fold larger in PG and SMG than in SLG acinar cells. Carbachol-stimulated Cl(-) efflux and the protein levels of the Ca(2+)-activated Cl(-) channel TMEM16A, the major apical Cl(-) efflux pathway in salivary acinar cells, were significantly greater in PG compared with SMG and SLG. In addition, we evaluated the transporter activity of the Na(+)-K(+)-2Cl(-) cotransporters (NKCC1) and anion exchangers (AE), the 2 primary basolateral Cl(-) uptake mechanisms in acinar cells. The SMG NKCC1 activity was about twice that of the PG and more than 12-fold greater than that of the SLG. AE activity was similar in PG and SLG, and both PG and SLG AE activity was about 2-fold larger than that of SMG. In summary, the salivation kinetics of the 3 major glands are distinct, and these differences can be explained by the unique functional properties of each gland related to Cl(-) movement, including the transporter activities of the Cl(-) uptake and efflux pathways, and intracellular Ca(2+) mobilization.

Scientific frontiers: emerging technologies for salivary diagnostics.

Saliva, a biofluid historically well-studied biochemically and physiologically, has entered the post-genomic 'omics' era, where its proteomic, genomic, and microbiome constituents have been comprehensively deciphered. The translational path of these salivary constituents has begun toward a variety of personalized individual medical applications, including early detection of cancer. Salivary diagnostics is a late-comer, but it is catching up where dedicated resources, like the Salivaomics Knowledge Base (SKB), now have taken center stage in the dissemination of the diagnostic potentials of salivary biomarkers and other translational and clinical utilities.

Acute inhibition of brain-specific Na(+)/H(+) exchanger isoform 5 by protein kinases A and C and cell shrinkage.

Little is known of the functional properties of the mammalian, brain-specific Na(+)/H(+) exchanger isoform 5 (NHE5). Rat NHE5 was stably expressed in NHE-deficient PS120 cells, and its activity was characterized using the fluorescent pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. NHE5 was insensitive to ethylisopropyl amiloride. The transport kinetics displayed a simple Michaelis-Menten relationship for extracellular Na(+) (apparent K(Na) = 27 +/- 5 mM) and a Hill coefficient near 3 for the intracellular proton concentration with a half-maximal activity at an intracellular pH of 6.93 +/- 0.03. NHE5 activity was inhibited by acute exposure to 8-bromo-cAMP or forskolin (which increases intracellular cAMP by activating adenylate cyclase). The kinase inhibitor H-89 reversed this inhibition, suggesting that regulation by cAMP involves a protein kinase A (PKA)-dependent process. In contrast, 8-bromo-cGMP did not have a significant effect on activity. The protein kinase C (PKC) activator phorbol 12-myristrate 13-acetate inhibited NHE5, and the PKC antagonist chelerythrine chloride blunted this effect. Activity was also inhibited by hyperosmotic-induced cell shrinkage but was unaffected by a hyposmotic challenge. These results demonstrate that rat brain NHE5 is downregulated by activation of PKA and PKC and by cell shrinkage, important regulators of neuronal cell function.

Protein kinase A activation phosphorylates the rat ClC-2 Cl- channel but does not change activity.

Phosphorylation-dependent events have been shown to modulate the activity of several members of the mammalian CLC Cl- channel gene family, including the inward rectifier ClC-2. In the present study we investigated the regulation of rat ClC-2 expressed in the TSA-201 cell line (a transformed HEK293 cell line that stably expresses the SV40 T-antigen) by protein kinases. Protein kinase A activation phosphorylated ClC-2 in vivo, whereas stimulation of protein kinase C with phorbol 12-myristate 13-acetate did not. In vitro labeling studies confirmed that protein kinase A could directly phosphorylate ClC-2, and that protein kinase C and Ca2+/calmodulin-dependent protein kinase II did not. Nevertheless, protein kinase A-dependent phosphorylation of CLC-2 failed to regulate either the magnitude or the kinetics of the hyperpolarization-activated Cl- currents. Considered together, we demonstrate that protein kinase A activation results in the phosphorylation of rat ClC-2 in vivo, but this event is independent of Cl- channel activity.

Defective fluid secretion and NaCl absorption in the parotid glands of Na+/H+ exchanger-deficient mice.

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.

Salivary acinar cells from aquaporin 5-deficient mice have decreased membrane water permeability and altered cell volume regulation.

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.

Targeted disruption of the Nhe1 gene fails to inhibit beta(1)-adrenergic receptor-induced parotid gland hypertrophy.

Chronic beta(1)-adrenergic receptor activation results in hypertrophy and hyperplasia of rodent salivary gland acinar cells. Na(+)/H(+) exchanger isoform 1 (NHE1) regulates cell volume and the induction of cell proliferation in many tissues. To investigate the relationship between NHE1 and the response of parotid glands to beta(1)-adrenergic agonists, we examined by Northern blot analysis NHE1 expression in saline-treated mice and mice 30 min and 2, 6, and 24 h after isoproterenol injection. NHE1 transcripts increased approximately 50% by 2 h, and a more than twofold increase was noted at 24 h. Isoproterenol did not acutely increase Na(+)/H(+) exchanger activity; however, exchanger activity was significantly elevated by 24 h. To test whether NHE1 activity is essential for inducing salivary gland hypertrophy in vivo, mice with targeted disruption of Nhe1 were treated with isoproterenol. Na(+)/H(+) exchanger activity was absent in acinar cells from Nhe1(-/-) mice, nevertheless, the lack of NHE1 failed to inhibit isoproterenol-induced hypertrophy. These data directly demonstrate that acinar cell hypertrophy induced by chronic beta(1)-adrenergic receptor stimulation occurs independently of NHE1 activity.

Into ion channel and transporter function. Caenorhabditis elegans ClC-type chloride channels: novel variants and functional expression.

Six ClC-type chloride channel genes have been identified in Caenorhabditis elegans, termed clh-1 through clh-6. cDNA sequences from these genes suggest that clh-2, clh-3, and clh-4 may code for multiple channel variants, bringing the total to at least nine channel types in this nematode. Promoter-driven green fluorescent protein (GFP) expression in transgenic animals indicates that the protein CLH-5 is expressed ubiquitously, CLH-6 is expressed mainly in nonneuronal cells, and the remaining isoforms vary from those restricted to a single cell to those expressed in over a dozen cells of the nematode. In an Sf9 cell expression system, recombinant CLH-2b, CLH-4b, and CLH-5 did not form functional plasma membrane channels. In contrast, both CLH-1 and CLH-3b produced strong, inward-rectifying chloride currents similar to those arising from mammalian ClC2, but which operate over different voltage ranges. Our demonstration of multiple CLH protein variants and comparison of expression patterns among the clh gene family provides a framework, in combination with the electrical properties of the recombinant channels, to further examine the physiology and cell-specific role each isoform plays in this simple model system.

Severe impairment of salivation in Na+/K+/2Cl- cotransporter (NKCC1)-deficient mice.

The salivary fluid secretory mechanism is thought to require Na(+)/K(+)/2Cl(-) cotransporter-mediated Cl(-) uptake. To directly test this possibility we studied the in vivo and in vitro functioning of acinar cells from the parotid glands of mice with targeted disruption of Na(+)/K(+)/2Cl(-) cotransporter isoform 1 (Nkcc1), the gene encoding the salivary Na(+)/K(+)/2Cl(-) cotransporter. In wild-type mice NKCC1 was localized to the basolateral membranes of parotid acinar cells, whereas expression was not detected in duct cells. The lack of functional NKCC1 resulted in a dramatic reduction (>60%) in the volume of saliva secreted in response to a muscarinic agonist, the primary in situ salivation signal. Consistent with defective Cl(-) uptake, a loss of bumetanide-sensitive Cl(-) influx was observed in parotid acinar cells from mice lacking NKCC1. Cl(-)/ HCO(3)(-) exchanger activity was increased in parotid acinar cells isolated from knockout mice suggesting that the residual saliva secreted by mice lacking NKCC1 is associated with anion exchanger-dependent Cl(-) uptake. Indeed, expression of the Cl(-)/ HCO(3)(-) exchanger AE2 was enhanced suggesting that this transporter compensates for the loss of functional Na(+)/K(+)/2Cl(-) cotransporter. Furthermore, the ability of the parotid gland to conserve NaCl was abolished in NKCC1-deficient mice. This deficit was not associated with changes in the morphology of the ducts, but transcript levels for the alpha-, beta-, and gamma-subunits of the epithelial Na(+) channel were reduced. These data directly demonstrate that NKCC1 is the major Cl(-) uptake mechanism across the basolateral membrane of acinar cells and is critical for driving saliva secretion in vivo.

Muscarinic receptor-induced acidification in sublingual mucous acinar cells: loss of pH recovery in Na+-H+ exchanger-1 deficient mice.

1. Intracellular pH (pHi) 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 pHi during muscarinic stimulation in mouse sublingual mucous acinar cells. 2. In the presence of HCO3-, muscarinic stimulation caused a rapid decrease in pHi (0.24 +/- 0.02 pH units) followed by a slow recovery rate (0.042 +/- 0.002 pH units min-1) to the initial resting pHi 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 approximately 4-fold faster (0.181 +/- 0.005 pH units min-1). 3. The agonist-induced intracellular acidification was inhibited by the anion channel blocker niflumate, and was prevented in the absence of HCO3- by treatment with the carbonic anhydrase inhibitor methazolamide. These results indicate that the muscarinic-induced acidification is due to HCO3- 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 pHi 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 pHi 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 pHi recovery from an acid load. These results demonstrate that NHE1 is critical for regulating pHi 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 load in the presence of bicarbonate. These results confirm that the major regulatory mechanism involved in pHi recovery from an acid load is not Na+-HCO3- cotransport, but amiloride-sensitive Na+-H+ exchange via isoform 1.

What can transgenic and gene-targeted mouse models teach us about salivary gland physiology?

Thousands of genetically modified mice have been developed since the first reports of stable expression of recombinant DNA in this species nearly 20 years ago. This mammalian model system has revolutionized the study of whole-animal, organ, and cell physiology. Transgenic and gene-targeted mice have been widely used to characterize salivary-gland-specific expression and to identify genes associated with tumorigenesis. Moreover, several of these mouse lines have proved to be useful models of salivary gland disease related to impaired immunology, i.e., Sjögren's syndrome, and disease states associated with pathogens. Despite the availability of genetically modified mice, few investigators have taken advantage of this resource to better their understanding of salivary gland function as it relates to the production of saliva. In this article, we describe the methods used to generate transgenic and gene-targeted mice and provide an overview of the advantages of and potential difficulties with these models. Finally, using these mouse models, we discuss the advances made in our understanding of the salivary gland secretion process.

Targeted disruption of the Nhe1 gene prevents muscarinic agonist-induced up-regulation of Na(+)/H(+) exchange in mouse parotid acinar cells.

The onset of salivary gland fluid secretion in response to muscarinic stimulation is accompanied by up-regulation of Na(+)/H(+) exchanger (NHE) activity. Although multiple NHE isoforms (NHE1, NHE2, and NHE3) have been identified in salivary glands, little is known about their specific function(s) in resting and secreting acinar cells. Mice with targeted disruptions of the Nhe1, Nhe2, and Nhe3 genes were used to investigate the contribution of these proteins to the stimulation-induced up-regulation of NHE activity in mouse parotid acinar cells. The lack of NHE1, but not NHE2 or NHE3, prevented intracellular pH recovery from an acid load in resting acinar cells, in acini stimulated to secrete with the muscarinic agonist carbachol, and in acini shrunken by hypertonic addition of sucrose. In HCO(3)(-)-containing solution, the rate of intracellular pH recovery from a muscarinic agonist-stimulated acid load was significantly inhibited in acinar cells from mice lacking NHE1, but not in cells from NHE2- or NHE3-deficient mice. These data demonstrate that NHE1 is the major regulator of intracellular pH in both resting and muscarinic agonist-stimulated acinar cells and suggest that up-regulation of NHE1 activity has an important role in modulating saliva production in vivo.

Mouse down-regulated in adenoma (DRA) is an intestinal Cl(-)/HCO(3)(-) exchanger and is up-regulated in colon of mice lacking the NHE3 Na(+)/H(+) exchanger.

Mutations in human DRA cause congenital chloride diarrhea, thereby raising the possibility that it functions as a Cl(-)/HCO(3)(-) exchanger. To test this hypothesis we cloned a cDNA encoding mouse DRA (mDRA) and analyzed its activity in cultured mammalian cells. When expressed in HEK 293 cells, mDRA conferred Na(+)-independent, electroneutral Cl(-)/CHO(3)(-) exchange activity. Removal of extracellular Cl(-) from medium containing HCO(3)(-) caused a rapid intracellular alkalinization, whereas the intracellular pH increase following Cl(-) removal from HCO(3)(-)-free medium was reduced greater than 7-fold. The intracellular alkalinization in Cl(-)-free, HCO(3)(-)-containing medium was unaffected by removal of extracellular Na(+) or by depolarization of the membrane by addition of 75 mM K(+) to the medium. Like human DRA mRNA, mDRA transcripts were expressed at high levels in cecum and colon and at lower levels in small intestine. The expression of mDRA mRNA was modestly up-regulated in the colon of mice lacking the NHE3 Na(+)/H(+) exchanger. These results show that DRA is a Cl(-)/HCO(3)(-) exchanger and suggest that it normally acts in concert with NHE3 to absorb NaCl and that in NHE3-deficient mice its activity is coupled with those of the sharply up-regulated colonic H(+),K(+)-ATPase and epithelial Na(+) channel to mediate electrolyte and fluid absorption.

Molecular cloning and functional expression of a rat Na+/H+ exchanger (NHE5) highly expressed in brain.

We report here the cloning, primary structure, heterologous expression, tissue distribution, and localization of a cDNA encoding rat NHE5, a fifth member of the mammalian plasma membrane Na+/H+ exchanger (NHE) gene family. The full-length open reading frame as well as 34 nucleotides of 5'-untranslated and 1443 nucleotides of 3'-untranslated sequences were obtained using a polymerase chain reaction strategy involving reverse transcription-polymerase chain reaction and 5'/3'-rapid amplification of cDNA ends. The NHE5 cDNA encodes a protein of 898 amino acids with a calculated Mr of 99,044 and is predicted to contain 11-13 transmembrane domains. An amino acid comparison of the coding region of rat NHE5 reveals 95% identity with human NHE5. Northern hybridization analysis showed that high level expression of NHE5 mRNA is restricted to brain. Transfection of the coding region of rat NHE5 into NHE-deficient PS120 cells resulted in Na+/H+ exchange activity that was relatively insensitive to the amiloride analogue, 5-(N-ethyl-N-isopropyl) amiloride, with a half-maximal inhibitory concentration (IC50) of 1. 53 +/- 0.25 microM. In situ hybridization of rat brain sections revealed significant NHE5 mRNA levels in the dentate gyrus with lower levels observed in the hippocampus and cerebral cortex. These results suggest a specialized role for this fifth NHE isoform in neuronal tissues.

Expression of multiple Na+/H+ exchanger isoforms in rat parotid acinar and ductal cells.

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 microM), 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 microM) than cell lines expressing NHE1 or NHE2 (IC50 = 0.076 +/- 0.013 or 0.055 +/- 0.015 microM, 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 microM). 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.

Chloride channels and salivary gland function.

Fluid and electrolyte transport is driven by transepithelial Cl- movement. The opening of Cl- channels in the apical membrane of salivary gland acinar cells initiates the fluid secretion process, whereas the activation of Cl- channels in both the apical and the basolateral membranes of ductal cells is thought to be necessary for NaCl re-absorption. Saliva formation can be evoked by sympathetic and parasympathetic stimulation. The composition and flow rate vary greatly, depending on the type of stimulation. As many as five classes of Cl- channels with distinct gating mechanisms have been identified in salivary cells. One of these Cl- channels is activated by intracellular Ca2+, while another is gated by cAMP. An increase in the intracellular free Ca2+ concentration is the dominant mechanism triggering fluid secretion from acinar cells, while cAMP may be required for efficient NaCl re-absorption in many ductal cells. In addition to cAMP- and Ca(2+)-gated Cl- channels, agonist-induced changes in membrane potential and cell volume activate different Cl- channels that likely play a role in modulating fluid and electrolyte movement. In this review, the properties of the different types of Cl- currents expressed in salivary gland cells are described, and functions are proposed based on the unique properties of these channels.