The NADPH oxidase activity of phagocytes and its generation of reactive oxygen species (ROS) is c... more The NADPH oxidase activity of phagocytes and its generation of reactive oxygen species (ROS) is critical for host-defense, but ROS overproduction can also lead to inflammation and tissue injury. Here we report that TRPM2, a non-selective and redox-sensitive cation channel, inhibits ROS production in phagocytic cells and prevents endotoxin-induced lung inflammation in mice. TRPM2-deficient mice challenged with endotoxin (lipopolysaccharide) showed an increased inflammatory signature and decreased survival compared to controls. TRPM2 functions by dampening NADPH oxidase-mediated ROS production through depolarization of the plasma membrane in phagocytes. Since ROS also activates TRPM2, our findings establish a negative feedback mechanism inactivating ROS production through inhibition of the membrane potentialsensitive NADPH oxidase.
Mammalian TRP channels are grouped into six subfamilies (TRPC, TRPM, TRPV, TRPA, TRPP, and TRPML)... more Mammalian TRP channels are grouped into six subfamilies (TRPC, TRPM, TRPV, TRPA, TRPP, and TRPML) based on the homology of the amino acid sequence. They are nonselective cation-permeable channels, most of which are permeable for Ca 2+ . Growing evidence demonstrates important roles of TRP channel in controlling vascular function including endothelial permeability, responses to oxidative stress, myogenic tone, cellular proliferative activity, and thermoregulation. TRP channels are activated by a variety of stimuli, including calcium store depletion, mechanical perturbations, receptor activation, and changes in temperature and osmolarity. This diversity of activating mechanisms could be consistent with the potential multiple functions of the TRP superfamily. This review summarizes the burgeoning understanding of these cation channels in the control of vascular function.
Secretion of lysosomes and related organelles is important for immune system function. High-resol... more Secretion of lysosomes and related organelles is important for immune system function. High-resolution membrane capacitance techniques were used to track changes in membrane area in single phagocytes during opsonized polystyrene bead uptake and release. Secretagogue stimulation of cells preloaded with beads resulted in immediate vesicle discharge, visualized as step increases in capacitance. The size of the increases were consistent with phagosome size. This hypothesis was confirmed by direct observation of dye release from bead-containing phagosomes after secretagogue stimulation. Capacitance recordings of exocytosis were correlated with quantal free radical release, as determined by amperometry. Thus, phagosomes undergo regulated secretion in macrophages, one function of which may be to deliver sequestered free radicals to the extracellular space.
Strategies for inhibiting phagolysosome fusion are essential for the intracellular survival and r... more Strategies for inhibiting phagolysosome fusion are essential for the intracellular survival and replication of many pathogens. We found that the lysosomal synaptotagmin Syt VII is required for a mechanism that promotes phagolysosomal fusion and limits the intracellular growth of pathogenic bacteria. Syt VII was required for a form of Ca2+-dependent phagolysosome fusion that is analogous to Ca2+-regulated exocytosis of lysosomes, which can be triggered by membrane injury. Bacterial type III secretion systems, which permeabilize membranes and cause Ca2+ influx in mammalian cells, promote lysosomal exocytosis and inhibit intracellular survival in Syt VII +/+ but not -/- cells. Thus, the lysosomal repair response can also protect cells against pathogens that trigger membrane permeabilization.
Background & Aims: Diarrhea is one of the major complications of inflammatory bowel disease. The ... more Background & Aims: Diarrhea is one of the major complications of inflammatory bowel disease. The role of oxidants in promoting net intestinal secretion is important, but the cellular mechanisms underlying their effects are unclear. We examined the effects and defined the cellular actions of the oxidant monochloramine (NH 2 Cl) on anion secretion in human colonic T84 cells. Methods: Effects of NH 2 Cl on basal and agonist-stimulated short-circuit current (Isc) of T84 monolayers were determined. Apical Cl ؊ and basolateral K ؉ conductances were measured by efflux of 125 I ؊ and 86 Rb ؉ , respectively. Results: NH 2 Cl alone had little effect on Isc and 125 I ؊ efflux. However, pretreatment with NH 2 Cl led to a concentration-dependent potentiation of the Ca 2؉mediated Isc and of submaximal cAMP-mediated responses. These effects were associated with increased basolateral K ؉ channel conductance and were blocked by increasing cellular Ca 2؉ buffering capacity with Quin-2. Whole-cell voltage clamp experiments showed that NH 2 Cl potentiated Ca 2؉ activation of basolateral K ؉ channel conductance. Conclusions: Oxidants potentiate both Ca 2؉ -and cAMP-stimulated Cl ؊ secretion by a direct effect on calcium-activated basolateral K ؉ channel conductance, lowering its Ca 2؉ activation threshold. This effect may play an important role in amplifying and prolonging the secretory response of inflamed intestinal mucosa and enhancing the severity of diarrhea.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride chann... more The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized primarily at the apical or luminal surfaces of epithelial cells that line the airway, gut, and exocrine glands; it is well established that CFTR plays a pivotal role in cholera toxin (CTX)-induced secretory diarrhea. Lysophosphatidic acid (LPA), a naturally occurring phospholipid present in blood and foods, has been reported to play a vital role in a variety of conditions involving gastrointestinal wound repair, apoptosis, inflammatory bowel disease, and diarrhea. Here we show, for the first time, that type 2 LPA receptors (LPA 2 ) are expressed at the apical surface of intestinal epithelial cells, where they form a macromolecular complex with Na ؉ /H ؉ exchanger regulatory factor-2 and CFTR through a PSD95/Dlg/ZO-1based interaction. LPA inhibited CFTR-dependent iodide efflux through LPA 2 -mediated G i pathway, and LPA inhibited CFTR-mediated short-circuit currents in a compartmentalized fashion. CFTR-dependent intestinal fluid secretion induced by CTX in mice was reduced substantially by LPA administration; disruption of this complex using a cell-permeant LPA 2 -specific peptide reversed LPA 2 -mediated inhibition. Thus, LPA-rich foods may represent an alternative method of treating certain forms of diarrhea.
Acidification of phagosomes has been proposed to have a key role in the microbicidal function of ... more Acidification of phagosomes has been proposed to have a key role in the microbicidal function of phagocytes. Here, we show that in alveolar macrophages the cystic fibrosis transmembrane conductance regulator Clchannel (CFTR) participates in phagosomal pH control and has bacterial killing capacity. Alveolar macrophages from Cftr -/mice retained the ability to phagocytose and generate an oxidative burst, but exhibited defective killing of internalized bacteria. Lysosomes from CFTR-null macrophages failed to acidify, although they retained normal fusogenic capacity with nascent phagosomes. We hypothesize that CFTR contributes to lysosomal acidification and that in its absence phagolysosomes acidify poorly, thus providing an environment conducive to bacterial replication.
Biochimica Et Biophysica Acta-molecular Cell Research, 2007
The cystic fibrosis transmembrane conductance regulator (CFTR) undergoes rapid turnover at the pl... more The cystic fibrosis transmembrane conductance regulator (CFTR) undergoes rapid turnover at the plasma membrane in various cell types. The ubiquitously expressed N-WASP promotes actin polymerization and regulates endocytic trafficking of other proteins in response to signaling molecules such as Rho-GTPases. In the present study we investigated the effects of wiskostatin, an N-WASP inhibitor, on the surface expression and activity of CFTR. We demonstrate, using surface biotinylation methods, that the steady-state surface CFTR pool in stably transfected BHK cells was dramatically decreased following wiskostatin treatment with a corresponding increase in the amount of intracellular CFTR. Similar effects were observed for latrunculin B, a specific actin-disrupting reagent. Both reagents strongly inhibited macroscopic CFTR-mediated Cl − currents in two cell types including HT29-Cl19A colonic epithelial cells. As previously reported, CFTR internalization from the cell surface was strongly inhibited by a cyclic-AMP cocktail. This effect of cyclic-AMP was only partially blunted in the presence of wiskostatin, which raises the possibility that these two factors modulate different steps in CFTR traffic. In kinetic studies wiskostatin appeared to accelerate the initial rate of CFTR endocytosis as well as inhibit its recycling back to the cell surface over longer time periods. Our studies implicate a role for N-WASP-mediated actin polymerization in regulating CFTR surface expression and channel activity.
Biochemical and Biophysical Research Communications, 2007
Blood can provide a valuable source for the generation of stem cells. Herein we identified a nove... more Blood can provide a valuable source for the generation of stem cells. Herein we identified a novel cell population from adult human blood, designated peripheral blood insulin-producing cells (PB-IPC). Phenotypic analysis demonstrated that PB-IPC displayed the embryonic stem (ES) cell-associated transcription factors including Oct-4 and Nanog, along with the hematopoietic markers CD9, CD45, and CD117; but lacked expression of the hematopoietic stem cell marker CD34 as well as lymphocyte and monocyte/macrophage markers. Notably, in vitro and in vivo characterization revealed that PB-IPC demonstrated characteristics of islet b cell progenitors including the expression of b cell-specific insulin gene transcription factors and prohormone convertases, production of insulin, formation of insulin granules, and the ability to reduce hyperglycemia and migrate into pancreatic islets after transplantation into the diabetic mice. These findings may open up new avenues for autologous blood stem cell-based therapies for diabetes.
Strategies for inhibiting phagolysosome fusion are essential for the intracellular survival and r... more Strategies for inhibiting phagolysosome fusion are essential for the intracellular survival and replication of many pathogens. We found that the lysosomal synaptotagmin Syt VII is required for a mechanism that promotes phagolysosomal fusion and limits the intracellular growth of pathogenic bacteria. Syt VII was required for a form of Ca2+-dependent phagolysosome fusion that is analogous to Ca2+-regulated exocytosis of lysosomes, which can be triggered by membrane injury. Bacterial type III secretion systems, which permeabilize membranes and cause Ca2+ influx in mammalian cells, promote lysosomal exocytosis and inhibit intracellular survival in Syt VII +/+ but not -/- cells. Thus, the lysosomal repair response can also protect cells against pathogens that trigger membrane permeabilization.
Biochemical and Biophysical Research Communications, 2005
AMP-activated protein kinase (AMPK) is an important signaling effector that couples cellular meta... more AMP-activated protein kinase (AMPK) is an important signaling effector that couples cellular metabolism and function. The effects of AMPK activation on pancreatic b-cell function remain unresolved. We used 5-amino-imidazole carboxamide riboside (AICAR), an activator of AMPK, to define the signaling mechanisms linking the activation of AMPK with insulin secretion. Application of 300 lM AICAR to mouse islets incubated in 5-14 mM glucose significantly increased AMPK activity and potentiated insulin secretion. AICAR inhibited ATP-sensitive K + (K ATP ) channels and increased the frequency of glucose-induced calcium oscillations in islets incubated in 8-14 mM glucose. At lower glucose concentration (5 mM) AICAR did not affect K ATP activity or intracellular ([Ca 2+ ] i ). AICAR also did not inhibit 86 Rb + efflux from islets isolated from Sur1 À/À mice that lack K ATP channels yet significantly potentiated glucose stimulated insulin secretion. Our data suggest that AICAR stimulates insulin secretion by both K ATP channel-dependent and -independent pathways.
The NADPH oxidase activity of phagocytes and its generation of reactive oxygen species (ROS) is c... more The NADPH oxidase activity of phagocytes and its generation of reactive oxygen species (ROS) is critical for host-defense, but ROS overproduction can also lead to inflammation and tissue injury. Here we report that TRPM2, a non-selective and redox-sensitive cation channel, inhibits ROS production in phagocytic cells and prevents endotoxin-induced lung inflammation in mice. TRPM2-deficient mice challenged with endotoxin (lipopolysaccharide) showed an increased inflammatory signature and decreased survival compared to controls. TRPM2 functions by dampening NADPH oxidase-mediated ROS production through depolarization of the plasma membrane in phagocytes. Since ROS also activates TRPM2, our findings establish a negative feedback mechanism inactivating ROS production through inhibition of the membrane potentialsensitive NADPH oxidase.
Mammalian TRP channels are grouped into six subfamilies (TRPC, TRPM, TRPV, TRPA, TRPP, and TRPML)... more Mammalian TRP channels are grouped into six subfamilies (TRPC, TRPM, TRPV, TRPA, TRPP, and TRPML) based on the homology of the amino acid sequence. They are nonselective cation-permeable channels, most of which are permeable for Ca 2+ . Growing evidence demonstrates important roles of TRP channel in controlling vascular function including endothelial permeability, responses to oxidative stress, myogenic tone, cellular proliferative activity, and thermoregulation. TRP channels are activated by a variety of stimuli, including calcium store depletion, mechanical perturbations, receptor activation, and changes in temperature and osmolarity. This diversity of activating mechanisms could be consistent with the potential multiple functions of the TRP superfamily. This review summarizes the burgeoning understanding of these cation channels in the control of vascular function.
Secretion of lysosomes and related organelles is important for immune system function. High-resol... more Secretion of lysosomes and related organelles is important for immune system function. High-resolution membrane capacitance techniques were used to track changes in membrane area in single phagocytes during opsonized polystyrene bead uptake and release. Secretagogue stimulation of cells preloaded with beads resulted in immediate vesicle discharge, visualized as step increases in capacitance. The size of the increases were consistent with phagosome size. This hypothesis was confirmed by direct observation of dye release from bead-containing phagosomes after secretagogue stimulation. Capacitance recordings of exocytosis were correlated with quantal free radical release, as determined by amperometry. Thus, phagosomes undergo regulated secretion in macrophages, one function of which may be to deliver sequestered free radicals to the extracellular space.
Strategies for inhibiting phagolysosome fusion are essential for the intracellular survival and r... more Strategies for inhibiting phagolysosome fusion are essential for the intracellular survival and replication of many pathogens. We found that the lysosomal synaptotagmin Syt VII is required for a mechanism that promotes phagolysosomal fusion and limits the intracellular growth of pathogenic bacteria. Syt VII was required for a form of Ca2+-dependent phagolysosome fusion that is analogous to Ca2+-regulated exocytosis of lysosomes, which can be triggered by membrane injury. Bacterial type III secretion systems, which permeabilize membranes and cause Ca2+ influx in mammalian cells, promote lysosomal exocytosis and inhibit intracellular survival in Syt VII +/+ but not -/- cells. Thus, the lysosomal repair response can also protect cells against pathogens that trigger membrane permeabilization.
Background & Aims: Diarrhea is one of the major complications of inflammatory bowel disease. The ... more Background & Aims: Diarrhea is one of the major complications of inflammatory bowel disease. The role of oxidants in promoting net intestinal secretion is important, but the cellular mechanisms underlying their effects are unclear. We examined the effects and defined the cellular actions of the oxidant monochloramine (NH 2 Cl) on anion secretion in human colonic T84 cells. Methods: Effects of NH 2 Cl on basal and agonist-stimulated short-circuit current (Isc) of T84 monolayers were determined. Apical Cl ؊ and basolateral K ؉ conductances were measured by efflux of 125 I ؊ and 86 Rb ؉ , respectively. Results: NH 2 Cl alone had little effect on Isc and 125 I ؊ efflux. However, pretreatment with NH 2 Cl led to a concentration-dependent potentiation of the Ca 2؉mediated Isc and of submaximal cAMP-mediated responses. These effects were associated with increased basolateral K ؉ channel conductance and were blocked by increasing cellular Ca 2؉ buffering capacity with Quin-2. Whole-cell voltage clamp experiments showed that NH 2 Cl potentiated Ca 2؉ activation of basolateral K ؉ channel conductance. Conclusions: Oxidants potentiate both Ca 2؉ -and cAMP-stimulated Cl ؊ secretion by a direct effect on calcium-activated basolateral K ؉ channel conductance, lowering its Ca 2؉ activation threshold. This effect may play an important role in amplifying and prolonging the secretory response of inflamed intestinal mucosa and enhancing the severity of diarrhea.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride chann... more The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized primarily at the apical or luminal surfaces of epithelial cells that line the airway, gut, and exocrine glands; it is well established that CFTR plays a pivotal role in cholera toxin (CTX)-induced secretory diarrhea. Lysophosphatidic acid (LPA), a naturally occurring phospholipid present in blood and foods, has been reported to play a vital role in a variety of conditions involving gastrointestinal wound repair, apoptosis, inflammatory bowel disease, and diarrhea. Here we show, for the first time, that type 2 LPA receptors (LPA 2 ) are expressed at the apical surface of intestinal epithelial cells, where they form a macromolecular complex with Na ؉ /H ؉ exchanger regulatory factor-2 and CFTR through a PSD95/Dlg/ZO-1based interaction. LPA inhibited CFTR-dependent iodide efflux through LPA 2 -mediated G i pathway, and LPA inhibited CFTR-mediated short-circuit currents in a compartmentalized fashion. CFTR-dependent intestinal fluid secretion induced by CTX in mice was reduced substantially by LPA administration; disruption of this complex using a cell-permeant LPA 2 -specific peptide reversed LPA 2 -mediated inhibition. Thus, LPA-rich foods may represent an alternative method of treating certain forms of diarrhea.
Acidification of phagosomes has been proposed to have a key role in the microbicidal function of ... more Acidification of phagosomes has been proposed to have a key role in the microbicidal function of phagocytes. Here, we show that in alveolar macrophages the cystic fibrosis transmembrane conductance regulator Clchannel (CFTR) participates in phagosomal pH control and has bacterial killing capacity. Alveolar macrophages from Cftr -/mice retained the ability to phagocytose and generate an oxidative burst, but exhibited defective killing of internalized bacteria. Lysosomes from CFTR-null macrophages failed to acidify, although they retained normal fusogenic capacity with nascent phagosomes. We hypothesize that CFTR contributes to lysosomal acidification and that in its absence phagolysosomes acidify poorly, thus providing an environment conducive to bacterial replication.
Biochimica Et Biophysica Acta-molecular Cell Research, 2007
The cystic fibrosis transmembrane conductance regulator (CFTR) undergoes rapid turnover at the pl... more The cystic fibrosis transmembrane conductance regulator (CFTR) undergoes rapid turnover at the plasma membrane in various cell types. The ubiquitously expressed N-WASP promotes actin polymerization and regulates endocytic trafficking of other proteins in response to signaling molecules such as Rho-GTPases. In the present study we investigated the effects of wiskostatin, an N-WASP inhibitor, on the surface expression and activity of CFTR. We demonstrate, using surface biotinylation methods, that the steady-state surface CFTR pool in stably transfected BHK cells was dramatically decreased following wiskostatin treatment with a corresponding increase in the amount of intracellular CFTR. Similar effects were observed for latrunculin B, a specific actin-disrupting reagent. Both reagents strongly inhibited macroscopic CFTR-mediated Cl − currents in two cell types including HT29-Cl19A colonic epithelial cells. As previously reported, CFTR internalization from the cell surface was strongly inhibited by a cyclic-AMP cocktail. This effect of cyclic-AMP was only partially blunted in the presence of wiskostatin, which raises the possibility that these two factors modulate different steps in CFTR traffic. In kinetic studies wiskostatin appeared to accelerate the initial rate of CFTR endocytosis as well as inhibit its recycling back to the cell surface over longer time periods. Our studies implicate a role for N-WASP-mediated actin polymerization in regulating CFTR surface expression and channel activity.
Biochemical and Biophysical Research Communications, 2007
Blood can provide a valuable source for the generation of stem cells. Herein we identified a nove... more Blood can provide a valuable source for the generation of stem cells. Herein we identified a novel cell population from adult human blood, designated peripheral blood insulin-producing cells (PB-IPC). Phenotypic analysis demonstrated that PB-IPC displayed the embryonic stem (ES) cell-associated transcription factors including Oct-4 and Nanog, along with the hematopoietic markers CD9, CD45, and CD117; but lacked expression of the hematopoietic stem cell marker CD34 as well as lymphocyte and monocyte/macrophage markers. Notably, in vitro and in vivo characterization revealed that PB-IPC demonstrated characteristics of islet b cell progenitors including the expression of b cell-specific insulin gene transcription factors and prohormone convertases, production of insulin, formation of insulin granules, and the ability to reduce hyperglycemia and migrate into pancreatic islets after transplantation into the diabetic mice. These findings may open up new avenues for autologous blood stem cell-based therapies for diabetes.
Strategies for inhibiting phagolysosome fusion are essential for the intracellular survival and r... more Strategies for inhibiting phagolysosome fusion are essential for the intracellular survival and replication of many pathogens. We found that the lysosomal synaptotagmin Syt VII is required for a mechanism that promotes phagolysosomal fusion and limits the intracellular growth of pathogenic bacteria. Syt VII was required for a form of Ca2+-dependent phagolysosome fusion that is analogous to Ca2+-regulated exocytosis of lysosomes, which can be triggered by membrane injury. Bacterial type III secretion systems, which permeabilize membranes and cause Ca2+ influx in mammalian cells, promote lysosomal exocytosis and inhibit intracellular survival in Syt VII +/+ but not -/- cells. Thus, the lysosomal repair response can also protect cells against pathogens that trigger membrane permeabilization.
Biochemical and Biophysical Research Communications, 2005
AMP-activated protein kinase (AMPK) is an important signaling effector that couples cellular meta... more AMP-activated protein kinase (AMPK) is an important signaling effector that couples cellular metabolism and function. The effects of AMPK activation on pancreatic b-cell function remain unresolved. We used 5-amino-imidazole carboxamide riboside (AICAR), an activator of AMPK, to define the signaling mechanisms linking the activation of AMPK with insulin secretion. Application of 300 lM AICAR to mouse islets incubated in 5-14 mM glucose significantly increased AMPK activity and potentiated insulin secretion. AICAR inhibited ATP-sensitive K + (K ATP ) channels and increased the frequency of glucose-induced calcium oscillations in islets incubated in 8-14 mM glucose. At lower glucose concentration (5 mM) AICAR did not affect K ATP activity or intracellular ([Ca 2+ ] i ). AICAR also did not inhibit 86 Rb + efflux from islets isolated from Sur1 À/À mice that lack K ATP channels yet significantly potentiated glucose stimulated insulin secretion. Our data suggest that AICAR stimulates insulin secretion by both K ATP channel-dependent and -independent pathways.
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