Nkx2.3 transcription factor is a key regulator of mucous cell identity in salivary glands.

Saliva is vital to oral health, fulfilling multiple functions in the oral cavity. Three pairs of major salivary glands and hundreds of minor salivary glands contribute to saliva production. The secretory acinar cells within these glands include two distinct populations. Serous acinar cells secrete a watery saliva containing enzymes, while mucous acinar cells secrete a more viscous fluid containing highly glycosylated mucins. Despite their shared developmental origins, the parotid gland (PG) is comprised of only serous acinar cells, while the sublingual gland (SLG) contains predominantly mucous acinar cells. The instructive signals that govern the identity of serous versus mucous acinar cell phenotypes are not yet known. The homeobox transcription factor Nkx2.3 is uniquely expressed in the SLG. Disruption of the Nkx2.3 gene was reported to delay the maturation of SLG mucous acinar cells. To examine whether Nkx2.3 plays a role in directing the mucous cell phenotype, we analyzed SLG from Nkx2.3-/- mice using RNAseq, immunostaining and proteomic analysis of saliva. Our results indicate that Nkx2.3, most likely in concert with other transcription factors uniquely expressed in the SLG, is a key regulator of the molecular program that specifies the identity of mucous acinar cells.

Allosteric modulation of ß-cell M3 muscarinic acetylcholine receptors greatly improves glucose homeostasis in lean and obese mice.

Given the global epidemic in type 2 diabetes, novel antidiabetic drugs with increased efficacy and reduced side effects are urgently needed. Previous work has shown that M3 muscarinic acetylcholine (ACh) receptors (M3Rs) expressed by pancreatic ß cells play key roles in stimulating insulin secretion and maintaining physiological blood glucose levels. In the present study, we tested the hypothesis that a positive allosteric modulator (PAM) of M3R function can improve glucose homeostasis in mice by promoting insulin release. One major advantage of this approach is that allosteric agents respect the ACh-dependent spatiotemporal control of M3R activity. In this study, we first demonstrated that VU0119498, a drug known to act as a PAM at M3Rs, significantly augmented ACh-induced insulin release from cultured ß cells and mouse and human pancreatic islets. This stimulatory effect was absent in islets prepared from mice lacking M3Rs, indicative of the involvement of M3Rs. VU0119498 treatment of wild-type mice caused a significant increase in plasma insulin levels, accompanied by a striking improvement in glucose tolerance. These effects were mediated by ß-cell M3Rs, since they were absent in mutant mice selectively lacking M3Rs in ß cells. Moreover, acute VU0119498 treatment of obese, glucose-intolerant mice triggered enhanced insulin release and restored normal glucose tolerance. Interestingly, doses of VU0119498 that led to pronounced improvements in glucose homeostasis did not cause any significant side effects due to activation of M3Rs expressed by other peripheral cell types. Taken together, the data from this proof-of-concept study strongly suggest that M3R PAMs may become clinically useful as novel antidiabetic agents.

Ano6 disruption impairs acinar cell regulatory volume decrease and protein secretion in murine submandibular salivary glands.

The widely expressed Anoctamin 6 (Ano6) supports different Ca2+ -dependent functions, but little is known about its role in salivary glands. Mouse submandibular gland (SMG) acinar cells exhibited a robust regulatory volume decrease (RVD) following cell swelling that was reduced approximately 70% in Ano6-/- mice. Ca2+ -free conditions nearly eliminated the RVD response suggesting that Ano6 is an obligatory component of the cell volume-activated, Ca2+ -dependent RVD pathway in salivary gland acinar cells. Ex vivo agonist-stimulated secretion of water and ions was unaffected by Ano6 disruption under both isotonic and hypotonic conditions suggesting that Ano6 does not play a major role in fluid and electrolyte secretion. In contrast, the total amount of ß-adrenergic-dependent protein secretion by the SMG was significantly reduced in Ano6-/- mice. Closer inspection of these latter results revealed that protein secretion was affected only in the female SMG by Ano6 disruption. These results indicate that Ano6 modulates the RVD response and protein secretion by salivary gland acinar cells.

Knockout of the LRRC26 subunit reveals a primary role of LRRC26-containing BK channels in secretory epithelial cells.

Leucine-rich-repeat-containing protein 26 (LRRC26) is the regulatory γ1 subunit of Ca2+- and voltage-dependent BK-type K+ channels. BK channels that contain LRRC26 subunits are active near normal resting potentials even without Ca2+, suggesting they play unique physiological roles, likely limited to very specific cell types and cellular functions. By using Lrrc26 KO mice with a ß-gal reporter, Lrrc26 promoter activity is found in secretory epithelial cells, especially acinar epithelial cells in lacrimal and salivary glands, and also goblet and Paneth cells in intestine and colon, although absent from neurons. We establish the presence of LRRC26 protein in eight secretory tissues or tissues with significant secretory epithelium and show that LRRC26 protein coassembles with the pore-forming BK α-subunit in at least three tissues: lacrimal gland, parotid gland, and colon. In lacrimal, parotid, and submandibular gland acinar cells, LRRC26 KO shifts BK gating to be like α-subunit-only BK channels. Finally, LRRC26 KO mimics the effect of SLO1/BK KO in reducing [K+] in saliva. LRRC26-containing BK channels are competent to contribute to resting K+ efflux at normal cell membrane potentials with resting cytosolic Ca2+ concentrations and likely play a critical physiological role in supporting normal secretory function in all secretory epithelial cells.

Slc26a6 is an apical membrane anion exchanger that drives HCO3--dependent fluid secretion in murine pancreatic acinar cells.

The nonselective anion exchanger Slc26a6, also known as putative anion transporter 1 and chloride/formate exchanger, is thought to play a major role in HCO3- transport in exocrine glands. In this study, Slc26a6 null mice were used to explore the function of Slc26a6 in the exocrine pancreas. Slc26a6 primarily localized to the apical membrane of pancreatic exocrine acinar cells. The volume of stimulated juice secretion by the ex vivo pancreas was significantly reduced ~35% in Slc26a6-/- mice, but no changes occurred in the gross structure or gland weights of Slc26a6 null mice. The secretion of pancreatic juice by Slc26a6+/+ mice was dependent on HCO3- while, in contrast, fluid secretion by Slc26a6-/- mice was independent of HCO3-, suggesting that Slc26a6 mediates the HCO3--dependent component of fluid secretion. Consistent with these observations, disruption of Slc26a6 also significantly reduced HCO3- secretion by the pancreas ~35%. Taken together, these results demonstrate that the apical Slc26a6 anion exchanger in acinar cells is involved in HCO3--dependent fluid secretion but that another major HCO3--independent pathway is the primary driver of the fluid secretion process in the mouse pancreas.

Physiological cAMP-elevating secretagogues differentially regulate fluid and protein secretions in mouse submandibular and sublingual glands.

The mechanisms underlying the functional differences in sympathetic and parasympathetic regulation of the major salivary glands have received little attention. The acute effects of parasympathetic muscarinic (carbachol)-dependent and combined parasympathetic-dependent plus cAMP-dependent pathways on fluid secretion rates, ion composition, and protein content were assessed using a newly developed ex vivo preparation that allows the simultaneous perfusion of the mouse submandibular (SMGs) and sublingual glands (SLGs). Our results confirm that the muscarinic-dependent pathway accounts for the bulk of salivation in SMGs and SLGs, whereas costimulation with a cAMP-increasing agent (forskolin, isoproterenol, or vasoactive intestinal peptide) did not increase the flow rate. Costimulation with carbachol plus the ß-adrenergic agonist isoproterenol decreased the concentration of NaCl and produced a substantial increase in the protein and Ca2+ content of SMG but not SLG saliva, consistent with a sparse sympathetic innervation of the SLGs. On the other hand, forskolin, which bypasses receptors to increase intracellular cAMP by directly activating the enzyme adenylate cyclase, enhanced the secretion of protein and Ca2+ by both the SMGs and SLGs. In contrast, isoproterenol and vasoactive intestinal peptide specifically stimulated protein secretion in SMG and SLG salivas, respectively. In summary, cAMP-dependent signaling does not play a major role in the stimulation of fluid secretion in SMGs and SLGs, whereas each cAMP-increasing agonist behaves differently in a gland-specific manner suggesting differential expression of G protein-coupled receptors in the epithelial cells of SMGs and SLGs.

The apical anion exchanger Slc26a6 promotes oxalate secretion by murine submandibular gland acinar cells.

The solute carrier family 26 (SLC26) gene family encodes at least 10 different anion exchangers. SLC26 member 6 (SLC26A6 or CFEX/PAT-1) and the cystic fibrosis transmembrane conductance regulator (CFTR) co-localize to the apical membrane of pancreatic duct cells, where they act in concert to drive HCO3- and fluid secretion. In contrast, in the small intestine, SLC26A6 serves as the major pathway for oxalate secretion. However, little is known about the function of Slc26a6 in murine salivary glands. Here, RNA sequencing-based transcriptional profiling and Western blots revealed that Slc26a6 is highly expressed in mouse submandibular and sublingual salivary glands. Slc26a6 localized to the apical membrane of salivary gland acinar cells with no detectable immunostaining in the ducts. CHO-K1 cells transfected with mouse Slc26a6 exchanged Cl- for oxalate and HCO3-, whereas two other anion exchangers known to be expressed in salivary gland acinar cells, Slc4a4 and Slc4a9, mediated little, if any, Cl-/oxalate exchange. Of note, both Cl-/oxalate exchange and Cl-/HCO3- exchange were significantly reduced in acinar cells isolated from the submandibular glands of Slc26a6-/- mice. Oxalate secretion in submandibular saliva also decreased significantly in Slc26a6-/- mice, but HCO3- secretion was unaffected. Taken together, our findings indicate that Slc26a6 is located at the apical membrane of salivary gland acinar cells, where it mediates Cl-/oxalate exchange and plays a critical role in the secretion of oxalate into saliva.

The apical Na+ -HCO3 - cotransporter Slc4a7 (NBCn1) does not contribute to bicarbonate transport by mouse salivary gland ducts.

The HCO3 - secretion mechanism in salivary glands is unclear but is thought to rely on the co-ordinated activity of multiple ion transport proteins including members of the Slc4 family of bicarbonate transporters. Slc4a7 was immunolocalized to the apical membrane of mouse submandibular duct cells. In contrast, Slc4a7 was not detected in acinar cells, and correspondingly, Slc4a7 disruption did not affect fluid secretion in response to cholinergic or ß-adrenergic stimulation in the submandibular gland (SMG). Much of the Na + -dependent intracellular pH (pH i ) regulation in SMG duct cells was insensitive to 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, S0859, and to the removal of extracellular HCO 3 - . Consistent with these latter observations, the Slc4a7 null mutation had no impact on HCO 3 - secretion nor on pH i regulation in duct cells. Taken together, our results revealed that Slc4a7 targets to the apical membrane of mouse SMG duct cells where it contributes little if any to pH i regulation or stimulated HCO 3 - secretion.

Transcriptional profiling reveals gland-specific differential expression in the three major salivary glands of the adult mouse.

RNA-Seq was used to better understand the molecular nature of the biological differences among the three major exocrine salivary glands in mammals. Transcriptional profiling found that the adult murine parotid, submandibular, and sublingual salivary glands express greater than 14,300 protein-coding genes, and nearly 2,000 of these genes were differentially expressed. Principle component analysis of the differentially expressed genes revealed three distinct clusters according to gland type. The three salivary gland transcriptomes were dominated by a relatively few number of highly expressed genes (6.3%) that accounted for more than 90% of transcriptional output. Of the 912 transcription factors expressed in the major salivary glands, greater than 90% of them were detected in all three glands, while expression for ~2% of them was enriched in an individual gland. Expression of these unique transcription factors correlated with sublingual and parotid specific subsets of both highly expressed and differentially expressed genes. Gene ontology analyses revealed that the highly expressed genes common to all glands were associated with global functions, while many of the genes expressed in a single gland play a major role in the function of that gland. In summary, transcriptional profiling of the three murine major salivary glands identified a limited number of highly expressed genes, differentially expressed genes, and unique transcription factors that represent the transcriptional signatures underlying gland-specific biological properties.

A Mathematical Model Supports a Key Role for Ae4 (Slc4a9) in Salivary Gland Secretion.

We develop a mathematical model of a salivary gland acinar cell with the objective of investigating the role of two [Formula: see text] exchangers from the solute carrier family 4 (Slc4), Ae2 (Slc4a2) and Ae4 (Slc4a9), in fluid secretion. Water transport in this type of cell is predominantly driven by [Formula: see text] movement. Here, a basolateral [Formula: see text] adenosine triphosphatase pump (NaK-ATPase) and a [Formula: see text]-[Formula: see text]-[Formula: see text] cotransporter (Nkcc1) are primarily responsible for concentrating the intracellular space with [Formula: see text] well above its equilibrium potential. Gustatory and olfactory stimuli induce the release of [Formula: see text] ions from the internal stores of acinar cells, which triggers saliva secretion. [Formula: see text]-dependent [Formula: see text] and [Formula: see text] channels promote ion secretion into the luminal space, thus creating an osmotic gradient that promotes water movement in the secretory direction. The current model for saliva secretion proposes that [Formula: see text] anion exchangers (Ae), coupled with a basolateral [Formula: see text] ([Formula: see text]) (Nhe1) antiporter, regulate intracellular pH and act as a secondary [Formula: see text] uptake mechanism (Nauntofte in Am J Physiol Gastrointest Liver Physiol 263(6):G823-G837, 1992; Melvin et al. in Annu Rev Physiol 67:445-469, 2005. https://doi.org/10.1146/annurev.physiol.67.041703.084745 ). Recent studies demonstrated that Ae4 deficient mice exhibit an approximate [Formula: see text] decrease in gland salivation (Peña-Münzenmayer et al. in J Biol Chem 290(17):10677-10688, 2015). Surprisingly, the same study revealed that absence of Ae2 does not impair salivation, as previously suggested. These results seem to indicate that the Ae4 may be responsible for the majority of the secondary [Formula: see text] uptake and thus a key mechanism for saliva secretion. Here, by using 'in-silico' Ae2 and Ae4 knockout simulations, we produced mathematical support for such controversial findings. Our results suggest that the exchanger's cotransport of monovalent cations is likely to be important in establishing the osmotic gradient necessary for optimal transepithelial fluid movement.

A fluid secretion pathway unmasked by acinar-specific Tmem16A gene ablation in the adult mouse salivary gland.

Activation of an apical Ca(2+)-activated Cl(-) channel (CaCC) triggers the secretion of saliva. It was previously demonstrated that CaCC-mediated Cl(-) current and Cl(-) efflux are absent in the acinar cells of systemic Tmem16A (Tmem16A Cl(-) channel) null mice, but salivation was not assessed in fully developed glands because Tmem16A null mice die within a few days after birth. To test the role of Tmem16A in adult salivary glands, we generated conditional knockout mice lacking Tmem16A in acinar cells (Tmem16A(-/-)). Ca(2+)-dependent salivation was abolished in Tmem16A(-/-) mice, demonstrating that Tmem16A is obligatory for Ca(2+)-mediated fluid secretion. However, the amount of saliva secreted by Tmem16A(-/-) mice in response to the ß-adrenergic receptor agonist isoproterenol (IPR) was comparable to that seen in controls, indicating that Tmem16A does not significantly contribute to cAMP-induced secretion. Furthermore, IPR-stimulated secretion was unaffected in mice lacking Cftr (Cftr(∆F508/∆F508)) or ClC-2 (Clcn2(-/-)) Cl(-) channels. The time course for activation of IPR-stimulated fluid secretion closely correlated with that of the IPR-induced cell volume increase, suggesting that acinar swelling may activate a volume-sensitive Cl(-) channel. Indeed, Cl(-) channel blockers abolished fluid secretion, indicating that Cl(-) channel activity is critical for IPR-stimulated secretion. These data suggest that ß-adrenergic-induced, cAMP-dependent fluid secretion involves a volume-regulated anion channel. In summary, our results using acinar-specific Tmem16A(-/-) mice identify Tmem16A as the Cl(-) channel essential for muscarinic, Ca(2+)-dependent fluid secretion in adult mouse salivary glands.

Ae4 (Slc4a9) Anion Exchanger Drives Cl- Uptake-dependent Fluid Secretion by Mouse Submandibular Gland Acinar Cells.

Transcellular Cl(-) movement across acinar cells is the rate-limiting step for salivary gland fluid secretion. Basolateral Nkcc1 Na(+)-K(+)-2Cl(-) cotransporters play a critical role in fluid secretion by promoting the intracellular accumulation of Cl(-) above its equilibrium potential. However, salivation is only partially abolished in the absence of Nkcc1 cotransporter activity, suggesting that another Cl(-) uptake pathway concentrates Cl(-) ions in acinar cells. To identify alternative molecular mechanisms, we studied mice lacking Ae2 and Ae4 Cl(-)/HCO3 (-) exchangers. We found that salivation stimulated by muscarinic and ß-adrenergic receptor agonists was normal in the submandibular glands of Ae2(-/-) mice. In contrast, saliva secretion was reduced by 35% in Ae4(-/-) mice. The decrease in salivation was not related to loss of Na(+)-K(+)-2Cl(-) cotransporter or Na(+)/H(+) exchanger activity in Ae4(-/-) mice but correlated with reduced Cl(-) uptake during ß-adrenergic receptor activation of cAMP signaling. Direct measurements of Cl(-)/HCO3 (-) exchanger activity revealed that HCO3 (-)-dependent Cl(-) uptake was reduced in the acinar cells of Ae2(-/-) and Ae4(-/-) mice. Moreover, Cl(-)/HCO3 (-) exchanger activity was nearly abolished in double Ae4/Ae2 knock-out mice, suggesting that most of the Cl(-)/HCO3 (-) exchanger activity in submandibular acinar cells depends on Ae2 and Ae4 expression. In conclusion, both Ae2 and Ae4 anion exchangers are functionally expressed in submandibular acinar cells; however, only Ae4 expression appears to be important for cAMP-dependent regulation of fluid secretion.

Severe defects in absorptive ion transport in distal colons of mice that lack ClC-2 channels.

BACKGROUND & AIMS: The fluid secretion model predicts that intestinal obstruction disorders can be alleviated by promoting epithelial Cl(-) secretion. The adenosine 3',5'-cyclic monophosphate (cAMP)-activated anion channel CFTR mediates Cl(-)-dependent fluid secretion in the intestine. Although the role of the ClC-2 channel has not been determined in the intestine, this voltage-gated Cl(-) channel might compensate for the secretory defects observed in patients with cystic fibrosis and other chronic constipation disorders. We investigated whether mice that lack ClC-2 channels (Clcn2(-/-)) have defects in intestinal ion transport. METHODS: Immunolocalization and immunoblot analyses were used to determine the cellular localization and the amount of ClC-2 expressed in mouse early distal colon (EDC) and late distal colon (LDC). Colon sheets from wild-type and Clcn2(-/-) littermates were mounted in Ussing chambers to determine transepithelial bioelectrical parameters and Na(+), K(+), and Cl(-) fluxes. RESULTS: Expression of ClC-2 was higher in the basolateral membrane of surface cells in the EDC compared with the LDC, with little expression in crypts. Neither cAMP nor Ca(2+)-induced secretion of Cl(-) was affected in the EDC or LDC of Clcn2(-/-) mice, whereas the amiloride-sensitive short-circuit current was increased approximately 3-fold in Clcn2(-/-) EDC compared with control littermates. Conversely, electroneutral Na(+), K(+), and Cl(-) absorption was dramatically reduced in colons of Clcn2(-/-) mice. CONCLUSIONS: Basolateral ClC-2 channels are required for colonic electroneutral absorption of NaCl and KCl. The increase in the amiloride-sensitive short-circuit current in Clcn2(-/-) mice revealed a compensatory mechanism that is activated in the colons of mice that lack the ClC-2 channel.

Quantitative proteomics of parotid saliva in primary Sjögren's syndrome.

The diagnosis of primary Sjögren's syndrome (pSS) is difficult due to the lack of specific laboratory and clinical tests. As an initial step for the global discovery of changes in the abundance of parotid salivary proteins in pSS, a pooled sample was compared to that from healthy control subjects by multidimensional protein identification technology (MudPIT). A total of 1246 proteins were identified by MudPIT. The abundance of 477 of these proteins did not change, 529 were only detected in either the pSS or HC sample, while 206 of these proteins were significantly upregulated ≥ twofold and 34 were downregulated ≤ 0.5. Ingenuity Pathway Analyses of differentially expressed proteins identified by MudPIT resulted in the identification of 100 significant pathways. The same samples were quantified in parallel using RP MS. Fifty-eight of 71 proteins identified by RP overlapped with MudPIT results. Five proteins were further analyzed by targeted label-free quantification to confirm the similar relative differential expression observed by RP and MudPIT approaches. The present study supports the use of MS for global discovery and validation of marker proteins for improved and early diagnosis of pSS.

TRPV4 activation in mouse submandibular gland modulates Ca2+ influx and salivation.

Transient receptor potential vanilloid subtype 4 (TRPV4) is a ligand-gated nonselective cation channel that participates in the transduction of mechanical and osmotic stimuli in different tissues. TRPV4 is activated by endogenous arachidonic acid metabolites, 4α-phorbol-12,13 didecanoate, GSK1016790A, moderate heat, and mechanical stress. TRPV4 is expressed in the salivary glands, but its expression pattern and function are poorly understood. The aim of this study was to evaluate the functional role of TRPV4 channels in the mouse submandibular gland. Using RT-PCR and Western blot analysis, we detected expression of TRPV4 message and protein, respectively, in the submandibular gland. Immunolocalization studies showed that TRPV4 targeted to the basolateral membrane of mouse submandibular gland acinar cells. Pharmacological TRPV4 activation using the selective agonist GSK1016790A caused Ca(2+) influx in isolated acinar cells in a basal-to-apical wave. Consistent with these observations, GSK1016790A elicited salivation in the perfused submandibular gland that was dependent on extracellular Ca(2+). In summary, we report that activation of TRPV4 channels induced Ca(2+) influx and salivation and, thus, may contribute a novel nonadrenergic, noncholinergic secretion pathway in the mouse submandibular gland.

A quantitative analysis of electrolyte exchange in the salivary duct.

A healthy salivary gland secretes saliva in two stages. First, acinar cells generate primary saliva, a plasma-like, isotonic fluid high in Na(+) and Cl(-). In the second stage, the ducts exchange Na(+) and Cl(-) for K(+) and HCO(3)(-), producing a hypotonic final saliva with no apparent loss in volume. We have developed a tool that aims to understand how the ducts achieve this electrolyte exchange while maintaining the same volume. This tool is part of a larger multiscale model of the salivary gland and can be used at the duct or gland level to investigate the effects of genetic and chemical alterations. In this study, we construct a radially symmetric mathematical model of the mouse salivary gland duct, representing the lumen, the cell, and the interstitium. For a given flow and primary saliva composition, we predict the potential differences and the luminal and cytosolic concentrations along a duct. Our model accounts well for experimental data obtained in wild-type animals as well as knockouts and chemical inhibitors. Additionally, the luminal membrane potential of the duct cells is predicted to be very depolarized compared with acinar cells. We investigate the effects of an electrogenic vs. electroneutral anion exchanger in the luminal membrane on concentration and the potential difference across the luminal membrane as well as how impairing the cystic fibrosis transmembrane conductance regulator channel affects other ion transporting mechanisms. Our model suggests the electrogenicity of the anion exchanger has little effect in the submandibular duct.

Quantitative salivary proteomic differences in oral chronic graft-versus-host disease.

PURPOSE: Chronic graft-versus-host disease (cGVHD) is a severe immunological complication that occurs after allogeneic hematopoietic stem cell transplantation (HSCT). Although oral cGVHD occurs in >25% of cGVHD patients and leads to decreased quality of life, its etiology is poorly understood. The present retrospective cross-sectional analysis of oral cGVHD patients sought to (1) test the feasibility of liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify protein biomarkers of oral cGVHD and (2) to gain a clearer understanding of salivary proteins impacted by oral cGVHD. METHODS: Using unstimulated whole saliva, we compared pooled saliva from five patients with a diagnosis of moderate or severe oral cGVHD, with a gender-and age- matched pool of five cGVHD patients with no oral mucosal findings. LC-MS/MS was used to identify salivary proteins, followed by Ingenuity Pathway Analysis (IPA). Selected mass spectrometric findings, including lactotransferrin, lactoperoxidase, and albumin, were confirmed by targeted label-free quantification. RESULTS: LC-MS/MS led to confident identification of 180 proteins. Of these proteins, 102 changed in abundance at least 2 fold, including 12 proteins identified only in the No oral cGVHD group. Downregulation of ~0.4 fold was confirmed for both lactotransferrin and lactoperoxidase in Oral cGVHD saliva using targeted label-free quantification. IPA analysis implicated pathways involved in cellular metabolism and immunoregulation. CONCLUSIONS: Reduction of salivary lactoperoxidase, lactotransferrin, and several cysteine proteinase inhibitor family proteins suggests impaired oral antimicrobial host immunity in cGVHD patients. This shotgun proteomic analysis of oral cGVHD saliva using targeted label-free quantification of select proteins supports the use of mass spectrometry for future validation in a large patient population as noninvasive tests for screening, early detection, and monitoring of cGVHD.

Design and Synthesis of Brain Penetrant Glycopeptide Analogues of PACAP With Neuroprotective Potential for Traumatic Brain Injury and Parkinsonism.

There is an unmet clinical need for curative therapies to treat neurodegenerative disorders. Most mainstay treatments currently on the market only alleviate specific symptoms and do not reverse disease progression. The Pituitary adenylate cyclase-activating polypeptide (PACAP), an endogenous neuropeptide hormone, has been extensively studied as a potential regenerative therapeutic. PACAP is widely distributed in the central nervous system (CNS) and exerts its neuroprotective and neurotrophic effects via the related Class B GPCRs PAC1, VPAC1, and VPAC2, at which the hormone shows roughly equal activity. Vasoactive intestinal peptide (VIP) also activates these receptors, and this close analogue of PACAP has also shown to promote neuronal survival in various animal models of acute and progressive neurodegenerative diseases. However, PACAP's poor pharmacokinetic profile (non-linear PK/PD), and more importantly its limited blood-brain barrier (BBB) permeability has hampered development of this peptide as a therapeutic. We have demonstrated that glycosylation of PACAP and related peptides promotes penetration of the BBB and improves PK properties while retaining efficacy and potency in the low nanomolar range at its target receptors. Furthermore, judicious structure-activity relationship (SAR) studies revealed key motifs that can be modulated to afford compounds with diverse selectivity profiles. Most importantly, we have demonstrated that select PACAP glycopeptide analogues (2LS80Mel and 2LS98Lac) exert potent neuroprotective effects and anti-inflammatory activity in animal models of traumatic brain injury and in a mild-toxin lesion model of Parkinson's disease, highlighting glycosylation as a viable strategy for converting endogenous peptides into robust and efficacious drug candidates.

Characterization of the human submandibular/sublingual saliva glycoproteome using lectin affinity chromatography coupled to multidimensional protein identification technology.

In-depth analysis of the salivary proteome is fundamental to understanding the functions of salivary proteins in the oral cavity and to reveal disease biomarkers involved in different pathophysiological conditions, with the ultimate goal of improving patient diagnosis and prognosis. Submandibular and sublingual glands contribute saliva rich in glycoproteins to the total saliva output, making them valuable sources for glycoproteomic analysis. Lectin-affinity chromatography coupled to mass spectrometry-based shotgun proteomics was used to explore the submandibular/sublingual (SM/SL) saliva glycoproteome. A total of 262 N- and O-linked glycoproteins were identified by multidimensional protein identification technology (MudPIT). Only 38 were previously described in SM and SL salivas from the human salivary N-linked glycoproteome, while 224 were unique. Further comparison analysis with SM/SL saliva of the human saliva proteome, revealed 125 glycoproteins not formerly reported in this secretion. KEGG pathway analyses demonstrated that many of these glycoproteins are involved in processes such as complement and coagulation cascades, cell communication, glycosphingolipid biosynthesis neo-lactoseries, O-glycan biosynthesis, glycan structures-biosynthesis 2, starch and sucrose metabolism, peptidoglycan biosynthesis or others pathways. In summary, lectin-affinity chromatography coupled to MudPIT mass spectrometry identified many novel glycoproteins in SM/SL saliva. These new additions to the salivary proteome may prove to be a critical step for providing reliable biomarkers in the diagnosis of a myriad of oral and systemic diseases.

Tmem16A encodes the Ca2+-activated Cl- channel in mouse submandibular salivary gland acinar cells.

Activation of an apical Ca(2+)-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 Ca(2+)-gated Cl(-) currents generated by Tmem16A and Best2, members from two distinct families of Ca(2+)-activated Cl(-) channels found in salivary glands. Heterologous expression of Tmem16A and Best2 transcripts in HEK293 cells produced Ca(2+)-activated Cl(-) currents with time and voltage dependence and inhibitor sensitivity that resembled the Ca(2+)-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 Ca(2+)-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 Ca(2+)-activated Cl(-) current and a Ca(2+)-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 Ca(2+)-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 Ca(2+)-activated Cl(-) channel complex that is essential for saliva production by the submandibular gland.