RACK1 plays a critical role in mast cell secretion and Ca2+ mobilization by modulating F-actin dynamics.

Although RACK1 is known to act as a signaling hub in immune cells, its presence and role in mast cells (MCs) is undetermined. MC activation via antigen stimulation results in mediator release and is preceded by cytoskeleton reorganization and Ca2+ mobilization. In this study, we found that RACK1 was distributed throughout the MC cytoplasm both in vivo and in vitro. After RACK1 knockdown (KD), MCs were rounded, and the cortical F-actin was fragmented. Following antigen stimulation, in RACK1 KD MCs, there was a reduction in cortical F-actin, an increase in monomeric G-actin and a failure to organize F-actin. RACK1 KD also increased and accelerated degranulation. CD63+ secretory granules were localized in F-actin-free cortical regions in non-stimulated RACK1 KD MCs. Additionally, RACK1 KD increased antigen-stimulated Ca2+ mobilization, but attenuated antigen-stimulated depletion of ER Ca2+ stores and thapsigargin-induced Ca2+ entry. Following MC activation there was also an increase in interaction of RACK1 with Orai1 Ca2+-channels, ß-actin and the actin-binding proteins vinculin and MyoVa. These results show that RACK1 is a critical regulator of actin dynamics, affecting mediator secretion and Ca2+ signaling in MCs. This article has an associated First Person interview with the first author of the paper.

Association of G protein-coupled receptor 78 with salivary dysfunction in male Sjögren's patients.

OBJECTIVE: Sjögren's disease (SjD) has a strong sex bias, suggesting an association with sex hormones. Male SjD represents a distinct subset of the disease, but the pathogenic mechanisms of male SjD is poorly characterized. The aim of this study is to identify initiating events related to the development of gland hypofunction and autoimmunity in male SjD patients. MATERIALS AND METHODS: Human minor salivary glands were transcriptomically analyzed with microarrays to detect differentially expressed genes in male SjD patients. Identified genes were tested on their involvement in the disease using conditional transgenic mice and gene-overexpressing cells. RESULTS: GPR78, an orphan G protein-coupled receptor, was overexpressed in the salivary glands of male SjD patients compared with male healthy controls and female SjD patients. Male GPR78 transgenic mice developed salivary gland hypofunction with increased epithelial apoptosis, which was not seen in control or female transgenic mice. In cell culture, GPR78 overexpression decreased lysosomal integrity, leading to caspase-dependent apoptotic cell death. GPR78-induced cell death in vitro was inhibited by treatment with estradiol. CONCLUSION: GPR78 overexpression can induce apoptosis and salivary gland hypofunction in male mice through lysosomal dysfunction and increased caspase-dependent apoptosis in salivary gland epithelium, which may drive disease in humans.

Lysosome-associated membrane protein 3 misexpression in salivary glands induces a Sjögren's syndrome-like phenotype in mice.

OBJECTIVES: Sjögren's syndrome (SS) is an autoimmune sialadenitis with unknown aetiology. Although extensive research implicated an abnormal immune response associated with lymphocytes, an initiating event mediated by salivary gland epithelial cell (SGEC) abnormalities causing activation is poorly characterised. Transcriptome studies have suggested alternations in lysosomal function are associated with SS, but a cause and effect linkage has not been established. In this study, we demonstrated that altered lysosome activity in SGECs by expression of lysosome-associated membrane protein 3 (LAMP3) can initiate an autoimmune response with autoantibody production and salivary dysfunction similar to SS. METHODS: Retroductal cannulation of the submandibular salivary glands with an adeno-associated virus serotype 2 vector encoding LAMP3 was used to establish a model system. Pilocarpine-stimulated salivary flow and the presence of autoantibodies were assessed at several time points post-cannulation. Salivary glands from the mice were evaluated using RNAseq and histologically. RESULTS: Following LAMP3 expression, saliva flow was significantly decreased and serum anti-Ro/SSA and La/SSB antibodies could be detected in the treated mice. Mechanistically, LAMP3 expression increased apoptosis in SGECs and decreased protein expression related to saliva secretion. Analysis of RNAseq data suggested altered lysosomal function in the transduced SGECs, and that the cellular changes can chemoattract immune cells into the salivary glands. Immune cells were activated via toll-like receptors by damage-associated molecular patterns released from LAMP3-expressing SGECs. CONCLUSIONS: These results show a critical role for lysosomal trafficking in the development of SS and establish a causal relationship between LAMP3 misexpression and the development of SS.

Aquaporin gene therapy corrects Sjögren's syndrome phenotype in mice.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease that is estimated to affect 35 million people worldwide. Currently, no effective treatments exist for Sjögren's syndrome, and there is a limited understanding of the physiological mechanisms associated with xerostomia and hyposalivation. The present work revealed that aquaporin 5 expression, a water channel critical for salivary gland fluid secretion, is regulated by bone morphogenetic protein 6. Increased expression of this cytokine is strongly associated with the most common symptom of primary Sjögren's syndrome, the loss of salivary gland function. This finding led us to develop a therapy in the treatment of Sjögren's syndrome by increasing the water permeability of the gland to restore saliva flow. Our study demonstrates that the targeted increase of gland permeability not only resulted in the restoration of secretory gland function but also resolved the hallmark salivary gland inflammation and systemic inflammation associated with disease. Secretory function also increased in the lacrimal gland, suggesting this local therapy could treat the systemic symptoms associated with primary Sjögren's syndrome.

Association of bone morphogenetic protein 6 with exocrine gland dysfunction in patients with Sjögren's syndrome and in mice.

OBJECTIVE: Primary Sjögren's syndrome (SS) is characterized by autoimmune activation and loss of function in secretory epithelia. The present study was undertaken to investigate and characterize changes in the epithelia associated with the loss of gland function in primary SS. METHODS: To identify changes in epithelial gene expression, custom microarrays were probed with complementary RNA (cRNA) isolated from minor salivary glands (MSGs) of female patients with primary SS who had low focus scores and low salivary flow rates, and the results were compared with those obtained using cRNA from the MSGs of sex-matched healthy volunteers. The effect of bone morphogenetic protein 6 (BMP-6) on salivary gland function was tested using adeno-associated virus-mediated gene transfer to the salivary glands of C57BL/6 mice. RESULTS: A significant increase in expression of BMP-6 was observed in RNA isolated from SS patients compared with healthy volunteers. Overexpression of BMP-6 locally in the salivary or lacrimal glands of mice resulted in the loss of fluid secretion as well as changes in the connective tissue of the salivary gland. Assessment of the fluid movement in either isolated acinar cells from mice overexpressing BMP-6 or a human salivary gland cell line cultured with BMP-6 revealed a loss in volume regulation in these cells. Lymphocytic infiltration in the submandibular gland of BMP-6 vector-treated mice was increased. No significant changes in the production of proinflammatory cytokines or autoantibodies associated with SS (anti-Ro/SSA and anti-La/SSB) were found after BMP-6 overexpression. CONCLUSION: In addition to identifying BMP-6 expression in association with xerostomia and xerophthalmia in primary SS, the present results suggest that BMP-6-induced salivary and lacrimal gland dysfunction in primary SS is independent of the autoantibodies and immune activation associated with the disease.

Extracellular Ca(2+) sensing in salivary ductal cells.

Ca(2+) is secreted from the salivary acinar cells as an ionic constituent of primary saliva. Ions such as Na(+) and Cl(-) get reabsorbed whereas primary saliva flows through the salivary ductal system. Although earlier studies have shown that salivary [Ca(2+)] decreases as it flows down the ductal tree into the oral cavity, ductal reabsorption of Ca(2+) remains enigmatic. Here we report a potential role for the G protein-coupled receptor, calcium-sensing receptor (CSR), in the regulation of Ca(2+) reabsorption by salivary gland ducts. Our data show that CSR is present in the apical region of ductal cells where it is co-localized with transient receptor potential canonical 3 (TRPC3). CSR is activated in isolated salivary gland ducts as well as a ductal cell line (SMIE) by altering extracellular [Ca(2+)] or by aromatic amino acid, L-phenylalanine (L-Phe, endogenous component of saliva), as well as neomycin. CSR activation leads to Ca(2+) influx that, in polarized cells grown on a filter support, is initiated in the luminal region. We show that TRPC3 contributes to Ca(2+) entry triggered by CSR activation. Further, stimulation of CSR in SMIE cells enhances the CSR-TRPC3 association as well as surface expression of TRPC3. Together our findings suggest that CSR could serve as a Ca(2+) sensor in the luminal membrane of salivary gland ducts and regulate reabsorption of [Ca(2+)] from the saliva via TRPC3, thus contributing to maintenance of salivary [Ca(2+)]. CSR could therefore be a potentially important protective mechanism against formation of salivary gland stones (sialolithiasis) and infection (sialoadenitis).

Membrane-type MMPs are indispensable for placental labyrinth formation and development.

The membrane-type matrix metalloproteinases (MT-MMPs) are essential for pericellular matrix remodeling in late stages of development, as well as in growth and tissue homeostasis in postnatal life. Although early morphogenesis is perceived to involve substantial tissue remodeling, the roles of MT-MMPs in these processes are only partially characterized. Here we explore the functions of 2 prominently expressed MT-MMPs, MT1-MMP and MT2-MMP, and describe their roles in the process of placental morphogenesis. The fetal portion of the placenta, in particular the labyrinth (LA), displays strong overlapping expression of MT1-MMP and MT2-MMP, which is critical for syncytiotrophoblast formation and in turn for fetal vessels. Disruption of trophoblast syncytium formation consequently leads to developmental arrest with only a few poorly branched fetal vessels entering the LA causing embryonic death at embryonic day 11.5. Through knockdown of MMP expression, we demonstrate that either MT1-MMP or MT2-MMP is crucial specifically during development of the LA. In contrast, knockdown of MT-MMP activity after LA formation is compatible with development to term and postnatal life. Taken together these data identify essential but interchangeable roles for MT1-MMP or MT2-MMP in placental vasculogenesis and provide the first example of selective temporal and spatial MMP activity required for development of the mouse embryo.

Increased T-bet+ cytotoxic effectors and type I interferon-mediated processes in chronic graft-versus-host disease of the oral mucosa.

Although chronic graft-versus-host disease (cGVHD) is a major long-term complication of allogeneic hematopoietic stem cell transplantation, little is known of its pathogenesis. We have systematically examined oral mucosa among cGVHD patients and determined that the clinical severity of oral cGVHD was correlated with apoptotic epithelial cells, often found adjacent to infiltrating effector-memory T cells expressing markers of cytotoxicity and type I cytokine polarization. Accumulation of T-bet(+) T-cell effectors was associated with both increased proliferation and the expression of the type I chemokine receptor CXCR3. Concurrently, in both infiltrating cells and keratinocytes, we observed increased expression of the CXCR3 ligand MIG (CXCL9) and interleukin-15 (IL-15), type I interferon (IFN)-inducible factors that support the migration, type I differentiation, and expansion of alloreactive effectors. In severely affected mucosa, we observed high levels of MxA, a protein specifically induced by type I IFN, and signal transducer and activator of transcription 1 (STAT1) phosphorylation, a critical step in the IFN-signaling pathway, along with increased numbers of plasmacytoid dendritic cells. These data challenge the current paradigm of cGVHD as a type II cytokine-driven disorder and support the model that oral cGVHD results from type I IFN-driven immigration, proliferation, and differentiation of T-bet(+) type I T effectors. The clinical trials are registered at http://www.clinicaltrials.gov as NCT00331968.

Conditional overexpression of TGF-beta1 disrupts mouse salivary gland development and function.

Transforming growth factor-beta (TGF-beta) signaling is known to affect salivary gland physiology by influencing branching morphogenesis, regulating ECM deposition, and controlling immune homeostasis. To study the role of TGF-beta1 in the salivary gland, we created a transgenic mouse (beta1(glo)) that conditionally overexpresses active TGF-beta1 upon genomic recombination by Cre recombinase. beta1(glo) mice were bred with an MMTV (mouse mammary tumor virus)-Cre (MC) transgenic line that expresses the Cre recombinase predominantly in the secretory cells of both the mammary and salivary glands. Although most of the double positive (beta1(glo)/MC) pups die either in utero or just after birth, clear defects in salivary gland morphogenesis such as reduced branching and increased mesenchyme could be seen. Those beta1(glo)/MC mice that survived into adulthood, however, had hyposalivation due to salivary gland fibrosis and acinar atrophy. Increased TGF-beta signaling was observed in the salivary gland with elevated phosphorylation of Smad2 and concomitant increase in ECM deposition. In particular, aberrant TGF-beta1 overexpression caused salivary gland hypofunction in this mouse model because of the replacement of normal glandular parenchyma with interstitial fibrous tissue. These results further implicate TGF-beta in pathological cases of salivary gland inflammation and fibrosis that occur with chronic infections in the glands or with the autoimmune disease, Sjögren's syndrome, or with radiation therapy given to head-and-neck cancer patients.

Transduction of Salivary Gland Acinar Cells with a Novel AAV Vector 44.9.

The loss of salivary gland function caused by radiation therapy of the head and neck or autoimmune disease such as Sjögren's syndrome is a serious condition that affects a patient's quality of life. Due to the combined exocrine and endocrine functions of the salivary gland, gene transfer to the salivary glands holds the potential for developing therapies for disorders of the salivary gland and the expression of therapeutic proteins via the exocrine pathway to the mouth, upper gastrointestinal tract, or endocrine pathway, systemically, into the blood. Recent clinical success with viral vector-mediated gene transfer for the treatment of irradiation-induced damage to the salivary glands has highlighted the need for the development of novel vectors with acinar cell tropism able to result in stable long-term transduction. Previous studies with adeno-associated virus (AAV) focused on the submandibular gland and reported mostly ductal cell transduction. In this study, we have screened AAV vectors for acinar cell tropism in the parotid gland utilizing membrane-tomato floxed membrane-GFP transgenic mice to screen CRE recombinase encoding AAV vectors of different clades to rapidly identify capsid isolates able to transduce salivary gland acinar cells. We determined that AAVRh10 and a novel isolate found as a contaminant of a laboratory stock of simian adenovirus SV15, AAV44.9, are both able to transduce parotid and sublingual acinar cells. Persistence and localization of transduction of these AAVs were tested using vectors encoding firefly luciferase, which was detected 6 months after vector administration. Most luciferase expression was localized to the salivary gland compared to that of distal organs. Transduction resulted in robust secretion of recombinant protein in both blood and saliva. Transduction was species specific, with AAVRh10 having stronger transduction activity in rats compared with AAV44.9 or AAV2 but weaker in human primary salivary gland cells. This work demonstrates efficient transduction of parotid acinar cells by AAV that resulted in secretion of recombinant protein in both serum and saliva.

Inhibition of bone morphogenetic protein 6 receptors ameliorates Sjögren's syndrome in mice.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease, with only palliative treatments available. Recent work has suggested that increased bone morphogenetic protein 6 (BMP6) expression could alter cell signaling in the salivary gland (SG) and result in the associated salivary hypofunction. We examined the prevalence of elevated BMP6 expression in a large cohort of pSS patients and tested the therapeutic efficacy of BMP signaling inhibitors in two pSS animal models. Increased BMP6 expression was found in the SGs of 54% of pSS patients, and this increased expression was correlated with low unstimulated whole saliva flow rate. In mouse models of SS, inhibition of BMP6 signaling reduced phosphorylation of SMAD1/5/8 in the mouse submandibular glands, and led to a recovery of SG function and a decrease in inflammatory markers in the mice. The recovery of SG function after inhibition of BMP6 signaling suggests cellular plasticity within the salivary gland and a possibility for therapeutic intervention that can reverse the loss of function in pSS.

Lead line in rodents: an old sign of lead intoxication turned into a new method for environmental surveillance.

The "lead line" was described by Henry Burton in 1840. Rodents are used as sentinels to monitor environmental pollution, but their teeth have not been used to determine lead. To determine whether lead deposits can be observed in the teeth of lead-exposed animals, since the gingival deposits known as "lead line" would likely have a correlate in the calcified tissue to which the gums are opposed during life. Male Wistar rats were exposed to lead in the drinking water (30 mg/L) since birth until 60 days-old. Molars and the incisors of each hemimandible were analyzed by scanning electron microscopy (SEM) on regular and backscattered electrons (BSE) mode. Elements were determined using electron dispersive spectroscopy (EDS). Clean cervical margins were observed on control teeth, as opposed to the findings of extensive deposits on lead-exposed animals, even in hemimandibles that had been exhumed after being buried for 90 days. BSE/EDS indicated that those deposits were an exogenous material compatible with lead sulfite. Presence of calcium, phosphorus, magnesium, carbon, lead, and oxygen is presented. Lead-exposed animals presented marked root resorption. The lead deposits characterized here for the first time show that the "lead line" seen in gums has a calcified tissue counterpart, that is detectable post-mortem even in animals exposed to a low dose of lead. This is likely a good method to detect undue lead exposure and will likely have wide application for pollution surveillance using sentinels.

Modifying the NH2 and COOH termini of aquaporin-5: effects on localization in polarized epithelial cells.

To reengineer polarized epithelial cell functions directly in situ, or ex vivo in the fabrication of an artificial organ, it is necessary to understand mechanisms that account for polarized membrane sorting. We have used the aquaporins (AQPs), a family of homotetrameric water channel proteins, as model membrane proteins for this purpose. AQP monomers contain six transmembrane-spanning domains linked by five interconnecting loops, with the NH2 and COOH termini residing in the cytosol. AQP5 is localized in the apical membranes of several different epithelia in vivo, and in stably transfected MDCK-II cells grown as a polarized monolayer. We wished to identify a structural region(s) within rat AQP5 (rAQP5) important for apical localization, and to study the MDCK-II cell localization of rAQP5s modified in either their NH2 or COOH terminus. We show that the NH2- terminal region does not play a major role in apical localization as deletion of the NH2 terminus produced a modified rAQP5 construct (AQP5-NT(del)) that was stably expressed and localized primarily to the apical membranes of MDCK-II cells. Attachment of a FLAG epitope to the NH2 terminus of AQP5 (AQP5(flag) construct) also did not perturb apical localization. In addition, we found that the exchange of NH2-terminal regions between rAQP5 and human AQP1 (hAQP1; a nonpolarized AQP isoform) produced a modified rAQP5 construct (AQP5-1NT) and a modified hAQP1 construct (AQP1-5NT), each of which localized as the parental AQP (apically, and to both apical and basolateral membranes, respectively). In contrast, we found that deletion of the COOH terminus resulted in a modified rAQP5 construct (AQP5-CT(del)) that was unstably expressed and localized to intracellular site(s) in MDCK-II cells. Substitution of the COOH terminus of AQP1 with the COOH terminus of AQP5 also produced a construct (AQP1-5CT) transiently expressed in intracellular compartment(s). However, substitution of the COOH terminus of rAQP5 with the COOH terminus of hAQP1 produced a modified rAQP5 construct (AQP5-1CT) that was stably expressed and localized to basolateral membranes, suggesting the loss of an apical targeting/retention signal from rAQP5, the gain of a basolateral targeting/retention signal from hAQP1, or a combination of these two possibilities.

Matriptase deletion initiates a Sjögren's syndrome-like disease in mice.

OBJECTIVE: The objective of this study was to determine the effect of epithelial barrier disruption, caused by deficiency of the membrane-anchored serine protease, matriptase, on salivary gland function and the induction of autoimmunity in an animal model. METHODS: Embryonic and acute ablation of matriptase expression in the salivary glands of mice was induced, leading to decreased epithelial barrier function. Mice were characterized for secretory epithelial function and the induction of autoimmunity including salivary and lacrimal gland dysfunction, lymphocytic infiltration, serum anti-Ro/SSA, anti-La/SSB and antinuclear antibodies. Salivary glands immune activation/regulation, barrier function as well as tight junction proteins expression also were determined. Expression of matriptase in minor salivary gland biopsies was compared among pSS patients and healthy volunteers. RESULTS: Embryonic ablation of matriptase expression in mice resulted in the loss of secretory epithelial cell function and the induction of autoimmunity similar to that observed in primary Sjögren's syndrome. Phenotypic changes included exocrine gland dysfunction, lymphocytic infiltrates, production of Sjögren's syndrome-specific autoantibodies, and overall activation of the immune system. Acute ablation of matriptase expression resulted in significant salivary gland dysfunction in the absence of overt immune activation. Analysis of the salivary glands indicates a loss of electrical potential across the epithelial layer as well as altered distribution of a tight junction protein. Moreover, a significant decrease in matriptase gene expression was detected in the minor salivary glands of pSS patients compared with healthy volunteers. CONCLUSIONS: Our findings demonstrate that local impairment of epithelial barrier function can lead to loss of exocrine gland function [corrected] in the absence of inflammation while systemic deletion can induce a primary Sjögren's syndrome like phenotype with autoimmunity and loss of gland function.

Overview of confocal microscopy.

Born out of the need to overcome an imaging problem in the 1950s, confocal microscopes today allow researchers to go beyond simple imaging and ask biochemical questions. This chapter provides background information on the development of modern confocal microscopes, their uses and applications. Sample preparation and observation are also discussed. Information is also provided about more advanced applications such as FRAP, FRET and 2-photon imaging. The requirements for setting up a confocal laboratory and the instrumentation needs are also discussed.

Prolonged analgesic response of cornea to topical resiniferatoxin, a potent TRPV1 agonist.

Analgesics currently available for the treatment of pain following ophthalmic surgery or injury are limited by transient effectiveness and undesirable or adverse side effects. The cornea is primarily innervated by small-diameter C-fiber sensory neurons expressing TRPV1 (transient receptor potential channel, subfamily V, member 1), a sodium/calcium cation channel expressed abundantly by nociceptive neurons and consequently a target for pain control. Resiniferatoxin (RTX), a potent TRPV1 agonist, produces transient analgesia when injected peripherally by inactivating TRPV1-expressing nerve terminals through excessive calcium influx. The aim of the present study was to evaluate topical RTX as a corneal analgesic. In rat cornea, a single application of RTX dose dependently eliminated or reduced the capsaicin eye wipe response for 3-5 days, with normal nociceptive responses returning by 5-7 days. RTX alone produced a brief but intense noxious response, similar to capsaicin, necessitating pretreatment of the cornea with a local anesthetic. Topical lidocaine, applied prior to RTX, blocks acute nociceptive responses to RTX without impairing the subsequent analgesic effect. Importantly, RTX analgesia (a) did not impair epithelial wound healing, (b) left the blink reflex intact and (c) occurred without detectable histological damage to the cornea. Immunohistochemistry showed that loss of CGRP immunoreactivity, a surrogate marker for TRPV1-expressing fibers, extended at least to the corneal-scleral boundary and displayed a progressive return, coincident with the return of capsaicin sensitivity. These data suggest that RTX may be a safe and effective treatment for post-operative or post-injury ophthalmic pain.

Interleukin-12 induces salivary gland dysfunction in transgenic mice, providing a new model of Sjögren's syndrome.

OBJECTIVE: Interleukin-12 (IL-12) is a pleiotropic cytokine that is elevated in the affected organs of patients with Sjögren's syndrome (SS). We have previously reported that overexpression of IL-12 in CBA mice leads to mononuclear infiltration of salivary and lacrimal glands, as well as to expansion of bronchial lymphoid tissue and decreased mucociliary clearance. Because xerostomia is one of the most important clinical features in SS patients, our main objective in the current study was to evaluate salivary gland function in IL-12-transgenic mice. Our secondary objective was to further characterize this animal model and to determine if the changes observed in these mice are representative of those observed in patients with SS overall. METHODS: Pilocarpine-stimulated salivary flow was used to address salivary gland function in a large group of IL-12-transgenic mice bred onto the autoimmune-prone SJL background. Furthermore, salivary glands were removed to assess the formation of infiltrates in the glands and gland morphology. Serum was also collected from these animals to investigate the formation of autoantibodies. RESULTS: Pilocarpine-stimulated salivary flow was significantly lower in IL-12-transgenic mice than in wild-type controls. Salivary glands from transgenic mice exhibited an increase in both the number and the size of lymphocytic foci, versus glands from age-matched controls. Furthermore, the acini in transgenic mice were fewer in number and larger in size compared with acini in controls. An age-dependent increase in anti-SSB/La antibodies was observed in IL-12-transgenic mice and was accompanied by an increase in antinuclear antibodies. CONCLUSION: Our findings indicate that a number of conditions associated with SS are exhibited by IL-12-transgenic SJL mice and that this model might be useful in researching multiple aspects of the disease.

Localization of S100A8 and S100A9 expressing neutrophils to spinal cord during peripheral tissue inflammation.

Investigation of hyperalgesia at the spinal transcriptome level indicated that carrageenan-induced inflammation of rat hind paws leads to a rapid but sustained increase in S100A8 and S100A9 expression, two genes implicated in the pathology of numerous inflammatory diseases including rheumatoid arthritis and gout. In situ hybridization revealed that the elevation occurred in neutrophils that migrate to the spinal cord vasculature during peripheral inflammation, not in spinal neurons or glial cells. Immunohistochemical analysis suggests, but does not prove, that these neutrophils abundantly release S100A8 and S100A9. Consistent with this, we detected an increase in ICAM and VCAM, both indicators of endothelial activation, a known trigger for secretion of S100A8 and S100A9. Migration of S100A8- and S100A9-expressing neutrophils to spinal cord is selective, since MCP-1- and CD68-expressing leukocytes do not increase in spinal cord vasculature during hind paw inflammation. Examination of many neutrophil granule mediators in spinal cord indicated that they are not regulated to the same degree as S100A8 and S100A9. Neutrophil migration also occurs in the vasculature of brain and pituitary gland during peripheral inflammation. Together, these findings suggest an interaction between a subpopulation of leukocytes and the CNS during peripheral tissue inflammation, as implied by an apparent release and possible diffusion of S100A8 and S100A9 through the endothelial blood-brain barrier. Although the present findings do not establish the neurophysiological or behavioral relevance of these observations to nociceptive processing, the data raise the possibility that selective populations of leukocytes may communicate the presence of disease or tissue damage from the periphery to cells in the central nervous system.

Relocalization of STIM1 for activation of store-operated Ca(2+) entry is determined by the depletion of subplasma membrane endoplasmic reticulum Ca(2+) store.

STIM1 (stromal interacting molecule 1), an endoplasmic reticulum (ER) protein that controls store-operated Ca(2+) entry (SOCE), redistributes into punctae at the cell periphery after store depletion. This redistribution is suggested to have a causal role in activation of SOCE. However, whether peripheral STIM1 punctae that are involved in regulation of SOCE are determined by depletion of peripheral or more internal ER has not yet been demonstrated. Here we show that Ca(2+) depletion in subplasma membrane ER is sufficient for peripheral redistribution of STIM1 and activation of SOCE. 1 microM thapsigargin (Tg) induced substantial depletion of intracellular Ca(2+) stores and rapidly activated SOCE. In comparison, 1 nM Tg induced slower, about 60-70% less Ca(2+) depletion but similar SOCE. SOCE was confirmed by measuring I(SOC) in addition to Ca(2+), Mn(2+), and Ba(2+) entry. Importantly, 1 nM Tg caused redistribution of STIM1 only in the ER-plasma membrane junction, whereas 1 microM Tg caused a relatively global relocalization of STIM1 in the cell. During the time taken for STIM1 relocalization and SOCE activation, 1 nM Bodipy-fluorescein Tg primarily labeled the subplasma membrane region, whereas 1 microM Tg labeled the entire cell. The localization of Tg in the subplasma membrane region was associated with depletion of ER in this region and activation of SOCE. Together, these data suggest that peripheral STIM1 relocalization that is causal in regulation of SOCE is determined by the status of [Ca(2+)] in the ER in close proximity to the plasma membrane. Thus, the mechanism involved in regulation of SOCE is contained within the ER-plasma membrane junctional region.

Attenuation of store-operated Ca2+ current impairs salivary gland fluid secretion in TRPC1(-/-) mice.

Agonist-induced Ca(2+) entry via store-operated Ca(2+) (SOC) channels is suggested to regulate a wide variety of cellular functions, including salivary gland fluid secretion. However, the molecular components of these channels and their physiological function(s) are largely unknown. Here we report that attenuation of SOC current underlies salivary gland dysfunction in mice lacking transient receptor potential 1 (TRPC1). Neurotransmitter-regulated salivary gland fluid secretion in TRPC1-deficient TRPC1(-/-) mice was severely decreased (by 70%). Further, agonist- and thapsigargin-stimulated SOC channel activity was significantly reduced in salivary gland acinar cells isolated from TRPC1(-/-) mice. Deletion of TRPC1 also eliminated sustained Ca(2+)-dependent potassium channel activity, which depends on Ca(2+) entry and is required for fluid secretion. Expression of key proteins involved in fluid secretion and Ca(2+) signaling, including STIM1 and other TRPC channels, was not altered. Together, these data demonstrate that reduced SOC entry accounts for the severe loss of salivary gland fluid secretion in TRPC1(-/-) mice. Thus, TRPC1 is a critical component of the SOC channel in salivary gland acinar cells and is essential for neurotransmitter-regulation of fluid secretion.