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Previously, we reported that post-translational modifications (PTMs) of MAGI1, including S741 phosphorylation and K931 de-SUMOylation, both of which are regulated by p90RSK activation, lead to endothelial cell (EC) activation. However, roles for p90RSK and MAGI1-PTMs in regulating EC permeability remain unclear despite MAGI1 being a junctional molecule. Here, we show that thrombin (Thb)-induced EC permeability, detected by the electric cell-substrate impedance sensing (ECIS) based system, was decreased by overexpression of dominant negative p90RSK or a MAGI1-S741A phosphorylation mutant, but was accelerated by overexpression of p90RSK, siRNA-mediated knockdown of magi1, or the MAGI1-K931R SUMOylation mutant. MAGI1 depletion also increased the mRNA and protein expression of the large tumor suppressor kinases 1 and 2 (LATS1/2), which inhibited YAP/TAZ activity and increased EC permeability. Because the endothelial barrier is a critical mediator of tumor hypoxia, we also evaluated the role of p90RSK activation in tumor vessel leakiness by using a relatively low dose of the p90RSK specific inhibitor, FMK-MEA. FMK-MEA significantly inhibited tumor vessel leakiness at a dose that does not affect morphology and growth of tumor vessels in vivo. These results provide novel insights into crucial roles for p90RSK-mediated MAGI1 PTMs and the Hippo pathway in EC permeability, as well as p90RSK activation in tumor vessel leakiness.

We construct a model of calcium waves in a three-dimensional anatomically accurate parotid acinar cell, constructed from experimental data. Gradients of inositol trisphosphate receptor (IPR) density are imposed, with the IPR density being greater closer to the lumen, which has a branched structure, and inositol trisphosphate (IP3) is produced only at the basal membrane. We show (1) that IP3 equilibrates so quickly across the cell that it can be assumed to be spatially homogeneous; (2) spatial separation of the sites of IP3 action and IP3 production does not preclude the formation of stable oscillatory Ca2+ waves. However, these waves are not waves in the mathematical sense of a traveling wave with fixed profile. They result instead from a time delay between the Ca2+ rise in the apical and basal regions; (3) the ryanodine receptors serve to reinforce the Ca2+ wave, but are not necessary for the wave to exist; (4) a spatially-independent model is not sufficient to study saliva secretion, although a one-dimensional model might be sufficient. Our results here form the first stages of the construction of a multiscale and multicellular model of saliva secretion in an entire acinus.

The possible association between the membrane-associated guanylate kinase with inverted domain structure-1 (MAGI1) and inflammation has been suggested, but the molecular mechanisms underlying this link, especially during atherogenesis, remain unclear. In endothelial cells (ECs) exposed to disturbed flow (d-flow), p90 ribosomal S6 kinase (p90RSK) bound to MAGI1, causing MAGI1-S741 phosphorylation and sentrin/SUMO-specific protease 2 T368 phosphorylation-mediated MAGI1-K931 deSUMOylation. MAGI1-S741 phosphorylation upregulated EC activation via activating Rap1. MAGI1-K931 deSUMOylation induced both nuclear translocation of p90RSK-MAGI1 and ATF-6-MAGI1 complexes, which accelerated EC activation and apoptosis, respectively. Microarray screening revealed key roles for MAGI1 in the endoplasmic reticulum (ER) stress response. In this context, MAGI1 associated with activating transcription factor 6 (ATF-6). MAGI1 expression was upregulated in ECs and macrophages found in atherosclerotic-prone regions of mouse aortas as well as in the colonic epithelia and ECs of patients with inflammatory bowel disease. Further, reduced MAGI1 expression in Magi1(–/+) mice inhibited d-flow–induced atherogenesis. In sum, EC activation and ER stress–mediated apoptosis are regulated in concert by two different types of MAGI1 posttranslational modifications, elucidating attractive drug targets for chronic inflammatory disease, particularly atherosclerosis.

This study defines the specific spatial and temporal characteristics of intracellular Ca2+ signaling events, together with their downstream consequences in pancreatic stellate cells during transformation from the quiescent to the activated state, both in culture and in situ, in a disease model of chronic pancreatitis and pancreatic cancer., The specific characteristics of intracellular Ca2+ signaling and the downstream consequences of these events were investigated in mouse pancreatic stellate cells (PSC) in culture and in situ using multiphoton microscopy in pancreatic lobules. PSC undergo a phenotypic transformation from a quiescent state to a myofibroblast-like phenotype in culture. This is believed to parallel the induction of an activated state observed in pancreatic disease such as chronic pancreatitis and pancreatic cancer. By day 7 in culture, the complement of cell surface receptors coupled to intracellular Ca2+ signaling was shown to be markedly altered. Specifically, protease-activated receptors (PAR) 1 and 2, responsive to thrombin and trypsin, respectively, and platelet-derived growth factor (PDGF) receptors were expressed only in activated PSC (aPSC). PAR-1, ATP, and PDGF receptor activation resulted in prominent nuclear Ca2+ signals. Nuclear Ca2+ signals and aPSC proliferation were abolished by expression of parvalbumin targeted to the nucleus. In pancreatic lobules, PSC responded to agonists consistent with the presence of only quiescent PSC. aPSC were observed following induction of experimental pancreatitis. In contrast, in a mouse model of pancreatic disease harboring elevated K-Ras activity in acinar cells, aPSC were present under control conditions and their number greatly increased following induction of pancreatitis. These data are consistent with nuclear Ca2+ signaling generated by agents such as trypsin and thrombin, likely present in the pancreas in disease states, resulting in proliferation of “primed” aPSC to contribute to the severity of pancreatic disease.

Human Adenovirus (HAdv) vectors expressing Mycobacterium tuberculosis (Mtb) derived antigens, have been tested as candidate boosters for BCG vaccine in animal models and Phase I human studies but failed to provide significant additional protection. Presumably, pre-existing immunity to HAdv among humans has been suggested to limit their efficacy as vectors. We earlier reported that novel bovine adenovirus (BAdv)-vectors induced robust T cell mediated immunity in mice and carried less risk of interference by human innate immunity. Thus, we evaluated BAdv vectors expressing Mtb derived antigen 85B and a novel TLR-2 activating peptide as booster vaccines for BCG in vitro and in mouse model for tuberculosis. Both mouse and human APCs (macrophages and dendritic cells) infected with BAdv-85B showed robust presentation of p25 epitope to T cells within 18 hrs of infection. BAdv-Ag85B-TLR2 was better than BAdv-85B in inducing antigen presentation. Antigen presentation by both BAdv-Ag85B and BAdv-Ag85B-TLR2 peptide were significantly decreased when DCs were derived from atg7 knockout mice or when 3-methyladenine was used for blockade, indicating that autophagy played a role during adenovirus-induced antigen presentation. Consistent with in vitro data, BAdv-Ag85B-TLR-2 and BAdv-Ag85B vaccinated mice exhibited reduced lung CFUs of Mtb by 1.5 log and 1.7 log respectively whereas BCG vaccinated mice could reduce CFUs by 0.95 log as compared to unvaccinated mice. In addition, the BAdv vaccines also protected spleens better than BCG suggesting improved protection against disseminated tuberculosis. Since BAdv can be given through mucosal route, we propose that they offer a novel mean to enhance lung and systemic immunity against tuberculosis.

In previous studies, we found that Annexin I (Anx I) was co‐secreted with insulin in response to glucose, and that extracellular Anx I stimulated the release of insulin via the Anx I binding site i...

Inositol 1,4,5-trisphosphate receptors (InsP3R) are a family of ubiquitously expressed intracellular Ca2+ channels. Isoform-specific properties of the three family members may play a prominent role in defining the rich diversity of the spatial and temporal characteristics of intracellular Ca2+ signals. Studying the properties of the particular family members is complicated because individual receptor isoforms are typically never expressed in isolation. In this article, we discuss strategies for studying Ca2+ release through individual InsP3R family members with particular reference to methods applicable following expression of recombinant InsP3R and mutant constructs in the DT40-3KO cell line, an unambiguously null InsP3R expression system.

Calcium release through inositol (1,4,5)-trisphosphate receptors (InsP(3)R) is the primary signal driving digestive enzyme and fluid secretion from pancreatic acinar cells. The type 2 (InsP(3)R2) and type 3 (InsP(3)R3) InsP(3)R are the predominant isoforms expressed in acinar cells and are required for proper exocrine gland function. Both InsP(3)R2 and InsP(3)R3 are positively regulated by cytosolic ATP, but InsP(3)R2 is 10-fold more sensitive than InsP(3)R3 to this form of modulation. In this study, we examined the role of InsP(3)R2 in setting the sensitivity of InsP(3)-induced Ca(2+) release (IICR) to ATP in pancreatic acinar cells. IICR was measured in permeabilized acinar cells from wild-type (WT) and InsP(3)R2 knock-out (KO) mice. ATP augmented IICR from WT pancreatic cells with an EC(50) of 38 microm. However, the EC(50) was 10-fold higher in acinar cells isolated from InsP(3)R2-KO mice, indicating a role for InsP(3)R2 in setting the sensitivity of IICR to ATP. Consistent with this idea, heterologous expression of InsP(3)R2 in RinM5F cells, which natively express predominately InsP(3)R3, increased the sensitivity of IICR to ATP. Depletion of ATP attenuated agonist-induced Ca(2+) signaling in WT pancreatic acinar cells. This effect was more profound in acinar cells prepared from InsP(3)R2-KO mice. These data suggest that the sensitivity of IICR to ATP depletion is regulated by the particular complement of InsP(3)R expressed in an individual cell. The effects of metabolic stress on intracellular Ca(2+) signals can therefore be determined by the relative amount of InsP(3)R2 expressed in cells.

Intracellular calcium concentration ([Ca2+]i) signals are central to the mechanisms underlying fluid and protein secretion in pancreatic and parotid acinar cells. Calcium release was studied in natively buffered cells following focal laser photolysis of caged molecules. Focal photolysis of caged-inositol 1,4,5 trisphosphate (InsP3) in the apical region resulted in Ca2+release from the apical trigger zone and, after a latent period, the initiation of an apical-to-basal Ca2+wave. The latency was longer and the wave speed significantly slower in pancreatic compared with parotid cells. Focal photolysis in basal regions evoked only limited Ca2+release at the photolysis site and never resulted in a propagating wave. Instead, an apical-to-basal wave was initiated following a latent period. Again, the latent period was significantly longer under all conditions in pancreas than parotid. Although slower in pancreas than parotid, once initiated, the apical-to-basal wave speed was constant in a particular cell type. Photo release of caged-Ca2+failed to evoke a propagating Ca2+wave in either cell type. However, the kinetics of the Ca2+signal evoked following photolysis of caged-InsP3were significantly dampened by ryanodine in parotid but not pancreas, indicating a more prominent functional role for ryanodine receptor (RyR) following InsP3receptor (InsP3R) activation. These data suggest that differing expression levels of InsP3R, RyR, and possibly cellular buffering capacity may contribute to the fast kinetics of Ca2+signals in parotid compared with pancreas. These properties may represent a specialization of the cell type to effectively stimulate Ca2+-dependent effectors important for the differing primary physiological role of each gland.

This study investigated the effect of extracellular annexin I on regulating insulin secretion in MIN6N8a (an insulin secreting cell line) cells. The properties of annexin I receptor in MIN6N8a cells were also determined. Annexin I stimulated insulin release in MIN6N8a cells, regardless of the presence or absence of extracellular Ca2+. Confocal microscopy revealed that annexin I bound to the surface of MIN6N8a cells. In addition, FACs analysis showed that annexin I bound to the surface of MIN6N8a cells in a dose-dependent manner. However, the annexin I-stimulated insulin secretion and the annexin I binding were abolished in MIN6N8a cells treated with proteases. Annexin I receptors were regenerated time-dependently. Furthermore, annexin I-stimulated insulin secretion was inhibited by cycloheximide but not by actinomycin D. These results showed that annexin I binds to the surface receptor in order to regulate the stimulation of insulin release in MIN6N8a cells.

Tuberculosis due to Mycobacterium tuberculosis (Mtb) kills around 1.5 million people each year. Although only 10% of humans develop active disease after aerosol infection, the basis for differential susceptibility of humans remains unclear. To determine if human macrophages are heterogeneous in their ability to kill Mtb, cord blood derived macrophages were treated with either IFN-γ or IL-4 to prepare M1 and M2 phenotype in vitro. Human THP-1 macrophages were also similarly prepared. Macrophages were infected with a 1:1 ratio of Mtb, incubated and on days 3 and 5 post infection, macrophage lysates were plated for CFU counts. In addition, intracellular autophagy was measured in gfpMtb infected macrophages using cytoID stains to detect autophagosomes and LC3 lipidation through western blot of macrophage lysates. Finally, Mtb infected macrophages were overlaid using CD4 T cells specific for the Antigen-85B epitope to measure antigen presentation. M1 macrophages showed a bacteriostatic ability and restricted the growth of Mtb while IL-4 treated M2 macrophages showed nearly 1 log increase in the growth of Mtb. Thus M2 macrophages were permissive for growth of Mtb. Consistent with this observation, gfpMtb co-localized with autophagosomes more frequently in M1 macrophages suggesting a role for autophagy during growth inhibition. LC3 lipidation was better in M1 compared to M2 macrophages. Finally, M1 macrophages presented Ag85B to CD4 T cells better than M2 macrophages. These observations suggest that macrophage heterogeneity into M1 and M2 is associated with an innate ability to kill intracellular tuberculosis and trigger immune activation.

Ca(2+) activation of Cl and K channels is a key event underlying stimulated fluid secretion from parotid salivary glands. Cl channels are exclusively present on the apical plasma membrane (PM), whereas the localization of K channels has not been established. Mathematical models have suggested that localization of some K channels to the apical PM is optimum for fluid secretion. A combination of whole cell electrophysiology and temporally resolved digital imaging with local manipulation of intracellular [Ca(2+)] was used to investigate if Ca(2+)-activated K channels are present in the apical PM of parotid acinar cells. Initial experiments established Ca(2+)-buffering conditions that produced brief, localized increases in [Ca(2+)] after focal laser photolysis of caged Ca(2+). Conditions were used to isolate K(+) and Cl(-) conductances. Photolysis at the apical PM resulted in a robust increase in K(+) and Cl(-) currents. A localized reduction in [Ca(2+)] at the apical PM after photolysis of Diazo-2, a caged Ca(2+) chelator, resulted in a decrease in both K(+) and Cl(-) currents. The K(+) currents evoked by apical photolysis were partially blocked by both paxilline and TRAM-34, specific blockers of large-conductance "maxi-K" (BK) and intermediate K (IK), respectively, and almost abolished by incubation with both antagonists. Apical TRAM-34-sensitive K(+) currents were also observed in BK-null parotid acini. In contrast, when the [Ca(2+)] was increased at the basal or lateral PM, no increase in either K(+) or Cl(-) currents was evoked. These data provide strong evidence that K and Cl channels are similarly distributed in the apical PM. Furthermore, both IK and BK channels are present in this domain, and the density of these channels appears higher in the apical versus basolateral PM. Collectively, this study provides support for a model in which fluid secretion is optimized after expression of K channels specifically in the apical PM.

Coordination of Ca(2+) signaling among cells contributes to synchronization of salivary gland cell function. However, mechanisms that underlie this signaling remain elusive. Here, intercellular Ca(2+) waves (ICW) in submandibular gland cells were investigated using Fura-2 fluorescence imaging. Mechanical stimulation of single cells induced ICW propagation from the stimulated cells through approximately 7 layers of cells or approximately 120microm. Our findings indicate that an extracellular ATP-dependent pathway is involved because the purinergic receptor antagonist suramin and the ATP hydrolyzing enzyme apyrase blocked ICW propagation. However, the gap junction uncoupler oleamide had no effect. ATP is released from mechanically stimulated cells possibly through opening of mechanosensitive maxi-anion channels, and does not appear to be directly linked to cytosolic Ca(2+). The ICW is propagated by diffusing ATP, which activates purinergic receptors in neighboring cells. This purinergic signaling induces a Ca(2+) transient that is dependent on Ca(2+) release via IP(3) receptors in the ER and store operated Ca(2+) entry (SOCE). Finally, inhibition of mitochondrial Ca(2+) uptake modified ICW indicating an important role of these organelles in this phenomenon. These studies increase our understanding of purinergic receptor signaling in salivary gland cells, and its role as a coordination mechanism of Ca(2+) signals induced by mechanical stimulation.

An organotypic slice preparation of the adult mouse parotid salivary gland amenable to a variety of optical assessments of fluid and protein secretion dynamics is described. The semi-intact preparation rendered without the use of enzymatic treatment permitted live-cell imaging and multiphoton analysis of cellular and supracellular signals. Toward this end we demonstrated that the parotid slice is a significant addition to the repertoire of tools available to investigators to probe exocrine structure and function since there is currently no cell culture system that fully recapitulates parotid acinar cell biology. Importantly, we show that a subpopulation of the acinar cells of parotid slices can be maintained in short-term culture and retain their morphology and function for up to 2 days. This in vitro model system is a significant step forward compared with enzymatically dispersed acini that rapidly lose their morphological and functional characteristics over several hours, and it was shown to be long enough for the expression and trafficking of exogenous protein following adenoviral infection. This system is compatible with a variety of genetic and physiological approaches used to study secretory function.

In nonexcitable cells, such as exocrine cells from the pancreas and salivary glands, agonist-stimulated Ca2+signals consist of both Ca2+release and Ca2+influx. We have investigated the contribution of these processes to membrane-localized Ca2+signals in pancreatic and parotid acinar cells using total internal reflection fluorescence (TIRF) microscopy (TIRFM). This technique allows imaging with unsurpassed resolution in a limited zone at the interface of the plasma membrane and the coverslip. In TIRFM mode, physiological agonist stimulation resulted in Ca2+oscillations in both pancreas and parotid with qualitatively similar characteristics to those reported using conventional wide-field microscopy (WFM). Because local Ca2+release in the TIRF zone would be expected to saturate the Ca2+indicator (Fluo-4), these data suggest that Ca2+release is occurring some distance from the area subjected to the measurement. When acini were stimulated with supermaximal concentrations of agonists, an initial peak, largely due to Ca2+release, followed by a substantial, maintained plateau phase indicative of Ca2+entry, was observed. The contribution of Ca2+influx and Ca2+release in isolation to these near-plasma membrane Ca2+signals was investigated by using a Ca2+readmission protocol. In the absence of extracellular Ca2+, the profile and magnitude of the initial Ca2+release following stimulation with maximal concentrations of agonist or after SERCA pump inhibition were similar to those obtained with WFM in both pancreas and parotid acini. In contrast, when Ca2+influx was isolated by subsequent Ca2+readmission, the Ca2+signals evoked were more robust than those measured with WFM. Furthermore, in parotid acinar cells, Ca2+readdition often resulted in the apparent saturation of Fluo-4 but not of the low-affinity dye Fluo-4-FF. Interestingly, Ca2+influx as measured by this protocol in parotid acinar cells was substantially greater than that initiated in pancreatic acinar cells. Indeed, robust Ca2+influx was observed in parotid acinar cells even at low physiological concentrations of agonist. These data indicate that TIRFM is a useful tool to monitor agonist-stimulated near-membrane Ca2+signals mediated by Ca2+influx in exocrine acinar cells. In addition, TIRFM reveals that the extent of Ca2+influx in parotid acinar cells is greater than pancreatic acinar cells when compared using identical methodologies.

A cytosolic phospholipase A2 (PLA2) was purified 640-fold from rat liver by sequential anion-exchange chromatography, Ca2+-precipitation/KCl-solubilization, gel filtration chromatography, and affinity chromatography. A single peak of PLA2 activity was eluted at an apparent molecular mass of 197 kDa from a Superdex 200HR gel filtration column. In the presence of Ca2+, the purified enzyme catalyzed the hydrolysis of 81.8 nmol of phosphatidylethanolamine per hour per mg of protein. The apparent Km was 1.83 nM. The enzyme was inhibited by arachidonyl trifluoromethyl ketone (AACOCF3), an inhibitor of cPLA2. However, it was not inhibited by bromoenol lactone (BEL), an inhibitor of iPLA2, and p-bromophenacyl bromide (p-BPB), an inhibitor of sPLA2. These data suggest that the purified enzyme is a novel Ca2+-dependent cytosolic PLA2.

This study investigates the effect of extracellular annexin I (Anx I) on regulating insulin secretion in isolated rat pancreatic islets. Results show that Anx I stimulates insulin release in pancreatic islets regardless of the presence or absence of extracellular Ca2+. In particular, confocal microscopy shows that Anx I binds to the surface of islet cells in the absence of extracellular Ca2+. However, insulin secretion through Anx I significantly decreases in trypsin-treated islets. Likewise, there is minimal binding of Anx I to the surface of trypsin-treated islets. Anti-Anx I polyclonal antibody also inhibits the stimulating effect of Anx I on insulin secretion. These results indicate that Anx I is capable of binding to the cell surface receptor, in order to regulate the stimulation of insulin release in rat pancreatic islets.