James E. Melvin

The Proteomes of Human Parotid and Submandibular/Sublingual Gland Salivas Collected as the Ductal Secretions

Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications--914 in parotid and 917 in submandibular/sublingual saliva--were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.

Mouse Down-regulated in Adenoma (DRA) Is an Intestinal Cl−/HCO3 − 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−/HCO3 − 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−/CHO3 − exchange activity. Removal of extracellular Cl− from medium containing HCO3 − caused a rapid intracellular alkalinization, whereas the intracellular pH increase following Cl−removal from HCO3 −-free medium was reduced greater than 7-fold. The intracellular alkalinization in Cl−-free, HCO3 −-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−/HCO3 − 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.

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 byAqp5 − /− 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 fromAqp5 − /− 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.

Proteomic Analysis of Human Parotid Gland Exosomes by Multidimensional Protein Identification Technology (MudPIT)

Human ductal saliva contributes over a thousand unique proteins to whole oral fluids. The mechanism by which most of these proteins are secreted by salivary glands remains to be determined. The present study used a mass spectrometry-based, shotgun proteomics approach to explore the possibility that a subset of the proteins found in saliva are derived from exosomes, membrane-bound vesicles of endosomal origin within multivesicular endosomes. Using MudPIT (multidimensional protein identification technology) mass spectrometry, we catalogued 491 proteins in the exosome fraction of human parotid saliva. Many of these proteins were previously observed in ductal saliva from parotid glands (265 proteins). Furthermore, 72 of the proteins in parotid exosomes overlap with those previously identified as urinary exosome proteins, proteins which are also frequently associated with exosomes from other tissues and cell types. Gene Ontology (GO) and KEGG pathway analyses found that cytosolic proteins comprise the largest category of proteins in parotid exosomes (43%), involved in such processes as phosphatidylinositol signaling system, calcium signaling pathway, inositol metabolism, protein export, and signal transduction, among others; whereas the integral plasma membrane proteins and associated/peripheral plasma membrane proteins (26%) were associated with extracellular matrix-receptor interaction, epithelial cell signaling, T-cell and B-cell receptor signaling, cytokine receptor interaction, and antigen processing and presentation, among other biological functions. In addition, these putative saliva exosomal proteins were linked to specific diseases (e.g., neurodegenerative disorders, prion disease, cancers, type I and II diabetes). Consequently, parotid glands secrete exosomes that reflect the metabolic and functional status of the gland and may also carry informative protein markers useful in the diagnosis and treatment of systemic diseases.

A Role for AQP5 in Activation of TRPV4 by Hypotonicity CONCERTED INVOLVEMENT OF AQP5 AND TRPV4 IN REGULATION OF CELL VOLUME RECOVERY

Regulation of cell volume in response to changes in osmolarity is critical for cell function and survival. However, the molecular basis of osmosensation and regulation of cell volume are not clearly understood. We have examined the mechanism of regulatory volume decrease (RVD) in salivary gland cells and report a novel association between osmosensing TRPV4 (transient receptor potential vanalloid 4) and AQP5 (aquaporin 5), which is required for regulating water permeability and cell volume. Exposure of salivary gland cells and acini to hypotonicity elicited an increase in cell volume and activation of RVD. Hypotonicity also activated Ca2+ entry, which was required for subsequent RVD. Ca2+ entry was associated with a distinct nonselective cation current that was activated by 4αPDD and inhibited by ruthenium red, suggesting involvement of TRPV4. Consistent with this, endogenous TRPV4 was detected in cells and in the apical region of acini along AQP5. Importantly, acinar cells from mice lacking either TRPV4 or AQP5 displayed greatly reduced Ca2+ entry and loss of RVD in response to hypotonicity, although the extent of cell swelling was similar. Expression of N terminus-deleted AQP5 suppressed TRPV4 activation and RVD but not cell swelling. Furthermore, hypotonicity increased the association and surface expression of AQP5 and TRPV4. Both these effects and RVD were reduced by actin depolymerization. These data demonstrate that (i) activation of TRPV4 by hypotonicity depends on AQP5, not on cell swelling per se, and (ii) TRPV4 and AQP5 concertedly control regulatory volume decrease. These data suggest a potentially important role for TRPV4 in salivary gland function.

The intermediate-conductance calcium-activated potassium channel KCa3.1 contributes to atherogenesis in mice and humans

Atherosclerosis remains a major cause of death in the developed world despite the success of therapies that lower cholesterol and BP. The intermediate-conductance calcium-activated potassium channel KCa3.1 is expressed in multiple cell types implicated in atherogenesis, and pharmacological blockade of this channel inhibits VSMC and lymphocyte activation in rats and mice. We found that coronary vessels from patients with coronary artery disease expressed elevated levels of KCa3.1. In Apoe(-/-) mice, a genetic model of atherosclerosis, KCa3.1 expression was elevated in the VSMCs, macrophages, and T lymphocytes that infiltrated atherosclerotic lesions. Selective pharmacological blockade and gene silencing of KCa3.1 suppressed proliferation, migration, and oxidative stress of human VSMCs. Furthermore, VSMC proliferation and macrophage activation were reduced in KCa3.1(-/-) mice. In vivo therapy with 2 KCa3.1 blockers, TRAM-34 and clotrimazole, significantly reduced the development of atherosclerosis in aortas of Apoe(-/-) mice by suppressing VSMC proliferation and migration into plaques, decreasing infiltration of plaques by macrophages and T lymphocytes, and reducing oxidative stress. Therapeutic concentrations of TRAM-34 in mice caused no discernible toxicity after repeated dosing and did not compromise the immune response to influenza virus. These data suggest that KCa3.1 blockers represent a promising therapeutic strategy for atherosclerosis.

Tmem16A Encodes the Ca2+-activated Cl− Channel in Mouse Submandibular Salivary Gland Acinar Cells

Activation of an apical Ca2+-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 Ca2+-gated Cl− currents generated by Tmem16A and Best2, members from two distinct families of Ca2+-activated Cl− channels found in salivary glands. Heterologous expression of Tmem16A and Best2 transcripts in HEK293 cells produced Ca2+-activated Cl− currents with time and voltage dependence and inhibitor sensitivity that resembled the Ca2+-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 Ca2+-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 Ca2+-activated Cl− current and a Ca2+-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 Ca2+-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 Ca2+-activated Cl− channel complex that is essential for saliva production by the submandibular gland.

Loss of Hyperpolarization-activated Cl− Current in Salivary Acinar Cells from Clcn2 Knockout Mice

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 theClcn2 gene was generated. The resulting homozygousClcn2 −/− 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 inClcn2 −/− mice following in vivostimulation 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.

Secretion and cell volume regulation by salivary acinar cells from mice lacking expression of the Clcn3 Cl− channel gene

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.

Interaction between transcellular and paracellular water transport pathways through Aquaporin 5 and the tight junction complex

To investigate potential physiological interactions between the transcellular and paracellular pathways of water transport, we asked whether targeted deletion of Aquaporin 5 (AQP5), the major transcellular water transporter in salivary acinar cells, affected paracellular transport of 4-kDa FITC-labeled dextran (FITC-D), which is transported through the paracellular but not the transcellular route. After i.v. injection of FITC-D into either AQP5 wild-type or AQP5−/− mice and saliva collection for fixed time intervals, we show that the relative amount of FITC-D transported in the saliva of AQP5−/− mice is half that in matched AQP5+/+ mice, indicating a 2-fold decrease in permeability of the paracellular barrier in mice lacking AQP5. We also found a significant difference in the proportion of transcellular vs. paracellular transport between male and female mice. Freeze-fracture electron microscopy revealed an increase in the number of tight junction strands of both AQP5+/+ and AQP5−/− male mice after pilocarpine stimulation but no change in strand number in female mice. Average acinar cell volume was increased by ≈1.4-fold in glands from AQP5−/− mice, suggesting an alteration in the volume-sensing machinery of the cell. Western blots revealed that expression of Claudin-7, Claudin-3, and Occludin, critical proteins that regulate the permeability of the tight junction barrier, were significantly decreased in AQP5−/− compared with AQP5+/+ salivary glands. These findings reveal the existence of a gender-influenced molecular mechanism involving AQP5 that allows transcellular and paracellular routes of water transport to act in conjunction.