NanoLuc Bioluminescence-Driven Photodynamic Activation of Cholecystokinin 1 Receptor with Genetically-Encoded Protein Photosensitizer MiniSOG.

In contrast to reversible activation by agonist, cholecystokinin 1 receptor (CCK1R) is permanently activated by singlet oxygen generated in photodynamic action, with sulphonated aluminium phthalocyanine or genetically encoded mini singlet oxygen generator (miniSOG) as photosensitizer. In these works, a halogen light source was used to power photodynamic action. For possible in vivo application of photodynamic CCK1R physiology, bearing a cumbersome light-delivery device connected to an external light source by experimental animals might interfere with their behavior. Therefore, in the present work, the possibility of bioluminescence-driven miniSOG photodynamic CCK1R activation was examined, as monitored by Fura-2 calcium imaging. In parallel experiments, it was found that, after plasma membrane (PM)-localized expression of miniSOGPM in AR4-2J cells, light irradiation with blue light-emitting diode (LED) (450 nm, 85 mW·cm-2, 1.5 min) induced persistent calcium oscillations that were blocked by CCK1R antagonist devazepide 2 nM. NanoLuc was expressed bicistronically with miniSOGPM via an internal ribosome entry site (IRES) sequence (pminiSOGPM-IRES-NanoLuc). The resultant miniSOGPM-IRES-NanoLuc-AR4-2J cells were found to generate strong bioluminescence upon addition of NanoLuc substrate coelenterazine. Strikingly, coelenterazine 5 microM was found to trigger long-lasting calcium oscillations (a hallmark for permanent CCK1R activation) in perifused miniSOGPM-IRES-NanoLuc-AR4-2J cells. These data indicate that NanoLuc bioluminescence can drive miniSOGPM photodynamic CCK1R activation, laying the foundation for its future in vivo applications.

Permanent Photodynamic Cholecystokinin 1 Receptor Activation: Dimer-to-Monomer Conversion.

The G protein-coupled cholecystokinin 1 receptor (CCK1R) is activated permanently by type II photodynamic action (i.e., by singlet oxygen) in the freshly isolated rat pancreatic acini, in contrast to reversible activation by CCK. But how CCK1R is photodynamically activated is not known. Therefore, in the present work, we subjected membrane proteins extracted from isolated rat pancreatic acini to photodynamic action with photosensitiser sulphonated aluminium phthalocyanine (SALPC), and used reducing gel electrophoresis and Western blot to detect possible changes in CCK1R oligomerization status. Photodynamic action (SALPC 1 µM, light 36.7 mW cm- 2 × 10 min) was found to convert dimeric CCK1R nearly quantitatively to monomers. Such conversion was dependent on both irradiance (8.51-36.7 mW cm- 2) and irradiation time (1-20 min). Minimum effective irradiance was found to be 11.1 mW cm- 2 (× 10 min, with SALPC 1 µM), and brief photodynamic action (SALPC 1 µM, 36.7 mW cm- 2 × 1 min) was effective. Whilst CCK stimulation of purified membrane proteins alone had no effect on CCK1R dimer/monomer balance, sub-threshold photodynamic action (SALPC 100 nM, 36.7 mW cm- 2 × 10 min) plus CCK revealed a bell-shaped CCK dose response curve for CCK1R monomerization, which was remarkably similar to the dose response curve for CCK-stimulated amylase secretion in isolated rat pancreatic acini. These two lines of evidence together suggest that during photodynamic CCK1R activation, CCK1R is permanently monomerized, thus providing a unique approach for permanent G protein-coupled receptor (GPCR) activation which has not been achieved before.

[Photodynamic modulation of cellular functions].

Photodynamic action, due to the rather limited lifetime (1 µs) and effective reactive distance of singlet oxygen (< 10 nm), could subcellular-specifically regulate different cellular functions. Photodynamic action could activate permanently cholecystokinin (CCK) 1 receptors, and sensitize or desensitize other G protein-coupled receptors. The emergence in recent years of genetically- encoded protein photosensitisers has enabled more precisely-targeted photodynamic modulation of subcellular organelles and functional proteins. Protein photosensitisers (such as KillerRed, miniSOG or SOPP) expressed on the plasma membrane, mitochondria, lysosomes or endoplasmic reticulum can modulate photodynamically subcellular functions and fine-tune protein activity by targeted photooxidation. With the newly emerged active illumination technique, simultaneous photodynamic action localized at multiple sites is now possible, and the contribution of subcellular regions to the whole cell or individual cells to a cell cluster could be quantitated. Photodynamic action with protein photosensitiser will be a powerful tool for nano-manipulation in cell physiology research.

An essential role of NAD(P)H oxidase 2 in UVA-induced calcium oscillations in mast cells.

Solar UVA radiation (320-400 nm) is known to have immunomodulatory effects, but the detailed mechanisms involved are not fully elucidated. UVA irradiation has been shown to induce calcium oscillations in rat peritoneal mast cells due to NAD(P)H oxidase (NOX) activation, but the specific NOX isoforms have not been identified. In the present work effects of UVA irradiation were investigated in isolated rat peritoneal mast cells, in cultured rat mast cell line RBL-2H3, and in mouse bone marrow-derived mast cells (BMMC). It was found that UVA irradiation by alternate 340/380 nm (3.2-5.6 µW cm(-2)) or by LED (380 nm, 80 µW cm(-2)) induced calcium oscillations in isolated rat peritoneal mast cells, in RBL-2H3, and in BMMC. Such UVA-induced calcium oscillations resembled closely those induced by surface IgE receptor (FcεRI) activation. It was found that RBL-2H3 expressed high levels of gp91(phox) (NOX2), p22(phox), p67(phox), p47(phox), p40(phox), Rac1, Rac2, moderate levels of DUOX2, but did not express NOX1, NOX3, NOX4, or DUOX1. The specific cellular localizations of gp91(phox) (NOX2), p22(phox), p47(phox), p67(phox), p40(phox) and Rac1/2 were confirmed by immunocytochemistry. UVA-induced reactive oxygen species (ROS) production in RBL-2H3 was completely suppressed by the NOX inhibitor diphenyleneiodonium chloride (DPI) or by the antioxidant N-acetyl-l-cysteine (NAC). siRNA suppression of gp91(phox) (NOX2), p22(phox) and p47(phox) expression inhibited markedly UVA-induced calcium oscillations, ROS and IL-6/LTC4 production in RBL-2H3. Taken together these data indicate that NOX2 plays an essential role in UVA irradiation-induced calcium oscillations, ROS and mediator production in mast cells.

Lasting inhibition of receptor-mediated calcium oscillations in pancreatic acini by neutrophil respiratory burst--a novel mechanism for secretory blockade in acute pancreatitis?

Although overwhelming evidence indicates that neutrophil infiltration is an early event in acute pancreatitis, the effect of neutrophil respiratory burst on pancreatic acini has not been investigated. In the present work, effect of fMLP-induced neutrophil respiratory burst on pancreatic acini was examined. It was found that neutrophil respiratory burst blocked calcium oscillations induced by cholecystokinin or by acetylcholine. Such lasting inhibition was dependent on the density of bursting neutrophils and could be overcome by increased agonist concentration. Inhibition of cholecystokinin stimulation was also observed in AR4-2J cells. In sharp contrast, neutrophil respiratory burst had no effect on calcium oscillations induced by phenylephrine (PE), vasopressin, or by ATP in rat hepatocytes. These data together suggest that inhibition of receptor-mediated calcium oscillations in pancreatic acini by neutrophil respiratory burst would lead to secretory blockade, which is a hallmark of acute pancreatitis. The present work has important implications for clinical treatment and management of acute pancreatitis.

Photodynamic Activation of Cholecystokinin 1 Receptor Is Conserved in Mammalian and Avian Pancreatic Acini.

Cholecystokinin 1 receptor (CCK1R) is the only G protein coupled receptor that is activated in type II photodynamic action, but whether this is a property common to both mammalian and avian species is not known. In this work, pancreatic acini were isolated from the rat, mouse, and Peking duck, and photodynamic CCK1R activation was examined. Isolated pancreatic acini were exposed to photosensitizer sulphonated aluminum phthalocyanine (SALPC) and photodynamic action elicited by a brief light-emitting diode (LED 675 nm) pulse (1.5 min); photodynamic CCK1R activation was assessed by Fura-2 fluorescent calcium imaging. Photodynamic action was found to induce persistent calcium oscillations in rat, mouse, and Peking duck pancreatic acini, with the sensitivity order of mouse > rat > Peking duck. Photodynamically-activated CCK1R could be inhibited reversibly by CCK1R antagonist devazepide (1 µM); photodynamic CCK1R activation was blocked by pre-incubation with 1O2 quencher Trolox C (300 µM). The sensitivity of photodynamic CCK1R activation was correlated with the increasing size of the disordered region in intracellular loop 3. These data suggest that photodynamic CCK1R activation is conserved in both mammalian and avian species, as evidenced by the presence of the photodynamic activation motif "YFM" in transmembrane domain 3.

Modulating protein activity and cellular function by methionine residue oxidation.

The sulfur-containing amino acid residue methionine (Met) in a peptide/protein is readily oxidized to methionine sulfoxide [Met(O)] by reactive oxygen species both in vitro and in vivo. Methionine residue oxidation by oxidants is found in an accumulating number of important proteins. Met sulfoxidation activates calcium/calmodulin-dependent protein kinase II and the large conductance calcium-activated potassium channels, delays inactivation of the Shaker potassium channel ShC/B and L-type voltage-dependent calcium channels. Sulfoxidation at critical Met residues inhibits fibrillation of atherosclerosis-related apolipoproteins and multiple neurodegenerative disease-related proteins, such as amyloid beta, α-synuclein, prion, and others. Methionine residue oxidation is also correlated with marked changes in cellular activities. Controlled key methionine residue oxidation may be used as an oxi-genetics tool to dissect specific protein function in situ.

Photodynamic Activation of the Cholecystokinin 1 Receptor with Tagged Genetically Encoded Protein Photosensitizers: Optimizing the Tagging Patterns.

Cholecystokinin 1 receptor (CCK1R) is activated photodynamically. For this to happen in situ, genetically encoded protein photosensitizers (GEPP) may be tagged to natively expressed CCK1R, but how to best tag GEPP has not been examined. Therefore, GEPP (miniSOG or KillerRed) was tagged to CCK1R and light-driven photodynamic CCK1R activation was monitored by Fura-2 fluorescent calcium imaging, to screen for optimized tagging patterns. Blue light-emitting diode irradiation of CHO-K1 cells expressing miniSOG fused to N- or C-terminus of CCK1R was found to both trigger persistent calcium oscillations-a hallmark of permanent photodynamic CCK1R activation. Photodynamic CCK1R activation was accomplished also with miniSOG fused to N-terminus of CCK1R via linker (GlySerGly)4 or 8 , but not linker (GSG)12 or an internal ribosomal entry site insert. KillerRed fused to N- or C-terminus of CCK1R after white light irradiation resulted in similar activation of in-frame CCK1R. Photodynamic CCK1R activation in miniSOG-CCK1R-CHO-K1 cells was blocked by singlet oxygen (1 O2 ) quencher uric acid or Trolox C, corroborating the role of 1 O2 as the reactive intermediate. It is concluded that photodynamic CCK1R activation can be achieved either with direct GEPP fusion to CCK1R or fusion via a short linker, fusion via long linkers might serve as the internal control.

Transmembrane Domain 3 Is a Transplantable Pharmacophore in the Photodynamic Activation of Cholecystokinin 1 Receptor.

Cholecystokinin 1 receptor (CCK1R) is activated in photodynamic action by singlet oxygen, but detailed molecular mechanisms are not elucidated. To identify the pharmacophore(s) in photodynamic CCK1R activation, we examined photodynamic activation of point mutants CCK1RM121/3.32A, CCK1RM121/3.32Q, and a chimeric receptor with CCK1R transmembrane domain 3 (TM3) transplanted to muscarinic ACh receptor 3 (M3R) which is unaffected by photodynamic action. These engineered receptors were tagged at the N-terminus with genetically encoded protein photosensitizer miniSOG, and their light-driven photodynamic activation was compared to wild type CCK1R and M3R, as monitored by Fura-2 fluorescent calcium imaging. Photodynamic activations of miniSOG-CCK1RM121/3.32A and miniSOG-CCK1RM121/3.32Q were found to be 55% and 73%, respectively, when compared to miniSOG-CCK1R (100%), whereas miniSOG-M3R was not affected (0% activation). Notably, the chimeric receptor miniSOG-M3R-TM3CCK1R was effectively activated photodynamically (65%). These data suggest that TM3 is an important pharmacophore in photodynamic CCK1R activation, readily transplantable to nonsusceptible M3R for photodynamic activation.

The anti-botulism triterpenoid toosendanin elicits calcium increase and exocytosis in rat sensory neurons.

Toosendanin, a triterpenoid from Melia toosendan Sieb et Zucc, has been found before to be an effective anti-botulism agent, with a bi-phasic effect at both motor nerve endings and central synapse: an initial facilitation followed by prolonged depression. Initial facilitation may be due to activation of voltage-dependent calcium channels plus inhibition of potassium channels, but the depression is not fully understood. Toosendanin has no effect on intracellular calcium or secretion in the non-excitable pancreatic acinar cells, ruling out general toosendanin inhibition of exocytosis. In this study, toosendanin effects on sensory neurons isolated from rat nodose ganglia were investigated. It was found that toosendanin stimulated increases in cytosolic calcium and neuronal exocytosis dose dependently. Experiments with membrane potential indicator bis-(1,3-dibutylbarbituric acid)trimethine oxonol found that toosendanin hyperpolarized capsaicin-insensitive but depolarized capsaicin-sensitive neurons; high potassium-induced calcium increase was much smaller in hyperpolarizing neurons than in depolarizing neurons, whereas no difference was found for potassium-induced depolarization in these two types of neurons. In neurons showing spontaneous calcium oscillations, toosendanin increased the oscillatory amplitude but not frequency. Toosendanin-induced calcium increase was decreased in calcium-free buffer, by nifedipine, and by transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine. Simultaneous measurements of cytosolic and endoplasmic reticulum (ER) calcium showed an increase in cytosolic but a decrease in ER calcium, indicating that toosendanin triggered ER calcium release. These data together indicate that toosendanin modulates sensory neurons, but had opposite effects on membrane potential depending on the presence or absence of capsaicin receptor/TRPV 1 channel.

Substance P conjugated to CdTe quantum dots triggers cytosolic calcium concentration oscillations and induces quantum dots internalization in the pancreatic carcinoma cell line AR4-2J.

Highly fluorescent CdTe quantum dots (QDs) stabilized by 3-mercaptopropionic acid were prepared by an aqueous solution approach and used as a fluorescent label to link substance P (SP) in studying the interaction of SP with NK-1 receptor, which was expressed on the AR4-2J cell line. Nonspecific adsorptions of CdTe QDs on the AR4-2J cell membrane were observed, whereas the QD-SP conjugates successfully crossed the cell membrane and entered the cytosol. SP is a neurotransmitter, and neurotransmitter-induced calcium concentration oscillation is a common phenomenon in diverse cells especially of secretory type. Cytosolic calcium concentration responses were studied in the AR4-2J cell line during stimulation with SP and QD-SP conjugates. The oscillations triggered by SP and QD-SP conjugates were dose-dependent and very similar. Such QD-SP conjugates readily internalized into the cytosol as would be expected of an active NK-1 ligand. Therefore QD-SP conjugates could be used successfully to study ligand and NK-1 receptor interactions in live cells. Our research may provide a meaningful reference for congener research.

Ketone Body 3-Hydroxybutyrate Ameliorates Atherosclerosis via Receptor Gpr109a-Mediated Calcium Influx.

Atherosclerosis is a chronic inflammatory disease that can cause acute cardiovascular events. Activation of the NOD-like receptor family, pyrin domain containing protein 3 (NLRP3) inflammasome enhances atherogenesis, which links lipid metabolism to sterile inflammation. This study examines the impact of an endogenous metabolite, namely ketone body 3-hydroxybutyrate (3-HB), on a mouse model of atherosclerosis. It is found that daily oral administration of 3-HB can significantly ameliorate atherosclerosis. Mechanistically, 3-HB is found to reduce the M1 macrophage proportion and promote cholesterol efflux by acting on macrophages through its receptor G-protein-coupled receptor 109a (Gpr109a). 3-HB-Gpr109a signaling promotes extracellular calcium (Ca2+) influx. The elevation of intracellular Ca2+ level reduces the release of Ca2+ from the endothelium reticulum (ER) to mitochondria, thus inhibits ER stress triggered by ER Ca2+ store depletion. As NLRP3 inflammasome can be activated by ER stress, 3-HB can inhibit the activation of NLRP3 inflammasome, which triggers the increase of M1 macrophage proportion and the inhibition of cholesterol efflux. It is concluded that daily nutritional supplementation of 3-HB attenuates atherosclerosis in mice.

Thioredoxin 1 downregulates MCP-1 secretion and expression in human endothelial cells by suppressing nuclear translocation of activator protein 1 and redox factor-1.

To know whether thioredoxin 1 (Trx1) works for an antioxidant defense mechanism in atherosclerosis, the effect of Trx1 on the release of monocyte chemoattractant protein-1 (MCP-1), a potent chemoattractant for recruitment and accumulation of monocytes/macrophages in the intima of artery vessel, was investigated in human endothelial-like EA.hy 926 cells. It was found that overexpression of Trx1 suppressed, whereas knockdown of endogenous Trx1 enhanced, oxidized low-density lipoprotein (oxLDL)-stimulated MCP-1 release and expression in the cells. It was also observed that overexpression of Trx1 suppressed, whereas depletion of endogenous Trx1 greatly promoted, nuclear translocation of c-Jun and the redox factor-1 (Ref-1). Electrophoretic mobility shift assay showed significantly reduced DNA-binding activity of activator protein-1 (AP-1) in Trx1-overexpressing cells but apparently enhanced DNA binding activity of AP-1 in Trx1-knockdown cells, indicating that nuclear Ref-1 rather than Trx1 itself finally dominates the regulation of AP-1 activity, although Trx1 is considered to upregulate AP-1 activity. It was also observed that Trx1 depressed intracellular generation of reactive oxygen species (ROS). Diphenyleneiodonium (DPI), the inhibitor of NADPH oxidase, suppressed MCP-1 secretion, whereas transient expression of Nox1 enhanced transcription of MCP-1 in endothelial cells. Assays with AP-1 and MCP-1 luciferase reporters further demonstrated that transient expression of Trx1 significantly depressed the transcriptional activity of c-Jun/c-Fos and consequent MCP-1 transcription. This study suggests that Trx1 inherently suppresses MCP-1 expression in vascular endothelium and may prevent atherosclerosis by depressing MCP-1 release. Besides the suppression of intracellular ROS generation, the inhibition of nuclear translocation of AP-1 and Ref-1 are mainly responsible for the downregulation of MCP-1 by Trx1.

Photodynamic Activation of Cholecystokinin 1 Receptor with Different Genetically Encoded Protein Photosensitizers and from Varied Subcellular Sites.

Cholecystokinin 1 receptor (CCK1R) is activated by singlet oxygen (1O2) generated in photodynamic action with sulphonated aluminum phthalocyanine (SALPC) or genetically encoded protein photosensitizer (GEPP) KillerRed or mini singlet oxygen generator (miniSOG). A large number of GEPP with varied 1O2 quantum yields have appeared recently; therefore, in the present work, the efficacy of different GEPP to photodynamically activate CCK1R was examined, as monitored by Fura-2 calcium imaging. KillerRed, miniSOG, miniSOG2, singlet oxygen protein photosensitizer (SOPP), flavin-binding fluorescent protein from Methylobacterium radiotolerans with point mutation C71G (Mr4511C71G), and flavin-binding fluorescent protein from Dinoroseobacter shibae (DsFbFP) were expressed at the plasma membrane (PM) in AR4-2J cells, which express endogenous CCK1R. Light irradiation (KillerRed: white light 85.3 mW‧cm-2, 4' and all others: LED 450 nm, 85 mW·cm-2, 1.5') of GEPPPM-expressing AR4-2J was found to all trigger persistent calcium oscillations, a hallmark of permanent photodynamic CCK1R activation; DsFbFP was the least effective, due to poor expression. miniSOG was targeted to PM, mitochondria (MT) or lysosomes (LS) in AR4-2J in parallel experiments; LED light irradiation was found to all induce persistent calcium oscillations. In miniSOGPM-AR4-2J cells, light emitting diode (LED) light irradiation-induced calcium oscillations were readily inhibited by CCK1R antagonist devazepide 2 nM; miniSOGMT-AR4-2J cells were less susceptible, but miniSOGLS-AR4-2J cells were not inhibited. In conclusion, different GEPPPM could all photodynamically activate CCK1R. Intracellular GEPP photodynamic action may prove particularly suited to study intracellular GPCR.

Permanent Photodynamic Activation of the Cholecystokinin 2 Receptor.

The cholecystokinin 2 receptor (CCK2R) is expressed in the central nervous system and peripheral tissues, playing an important role in higher nervous and gastrointestinal functions, pain sensation, and cancer growth. CCK2R is reversibly activated by cholecystokinin or gastrin, but whether it can be activated permanently is not known. In this work, we found that CCK2R expressed ectopically in CHO-K1 cells was permanently activated in the dark by sulfonated aluminum phthalocyanine (SALPC / AlPcS4, 10-1,000 nM), as monitored by Fura-2 fluorescent calcium imaging. Permanent CCK2R activation was also observed with AlPcS2, but not PcS4. CCK2R previously exposed to SALPC (3 and 10 nM) was sensitized by subsequent light irradiation (> 580 nm, 31.5 mW·cm-2). After the genetically encoded protein photosensitizer mini singlet oxygen generator (miniSOG) was fused to the N-terminus of CCK2R and expressed in CHO-K1 cells, light irradiation (450 nm, 85 mW·cm-2) activated in-frame CCK2R (miniSOG-CCK2R), permanently triggering persistent calcium oscillations blocked by the CCK2R antagonist YM 022 (30 nM). From these data, it is concluded that SALPC is a long-lasting CCK2R agonist in the dark, and CCK2R is photogenetically activated permanently with miniSOG as photosensitizer. These properties of SALPC and CCK2R could be used to study CCK2R physiology and possibly for pain and cancer therapies.

Duck pancreatic acinar cell as a unique model for independent cholinergic stimulation-secretion coupling.

This paper investigated the role of acetylcholine (ACh) in physiological regulation of amylase secretion in avian exocrine pancreas. In the isolated duck pancreatic acini, ACh dose dependently stimulated amylase secretion, with a maximal effective concentration at 10 muM. The cAMP-mobilizing compounds forskolin, vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase activating peptide (PACAP) receptor (VPAC) agonists PACAP-38 and PACAP-27 had no effect on the dose-response curve. ACh dose dependently induced increases in cytosolic Ca(2+) concentration ([Ca(2+)]( c )), with increasing concentrations transforming oscillations into plateau increases. Forskolin (10 muM), PACAP-38 (1 nM), PACAP-27 (1 nM), or VIP (10 nM) alone did not stimulate [Ca(2+)]( c ) increase; neither did they modulate ACh-induced oscillations, nor made ACh low concentration effective. These data indicate that ACh-stimulated zymogen secretion in duck pancreatic acinar cells is not subject to modulation from the cAMP signaling pathway; whereas it has been widely reported in the rodents that ACh-stimulated exocrine pancreatic secretion is significantly enhanced by cAMP-mobilizing agents. This makes the duck exocrine pancreas unique in that cholinergic stimulus-secretion coupling is not subject to cAMP regulation.

Pancreatic Stellate Cells Serve as a Brake Mechanism on Pancreatic Acinar Cell Calcium Signaling Modulated by Methionine Sulfoxide Reductase Expression.

Although methionine sulfoxide reductase (Msr) is known to modulate the activity of multiple functional proteins, the roles of Msr in pancreatic stellate cell physiology have not been reported. In the present work we investigated expression and function of Msr in freshly isolated and cultured rat pancreatic stellate cells. Msr expression was determined by RT-PCR, Western blot and immunocytochemistry. Msr over-expression was achieved by transfection with adenovirus vectors. Pancreatic stellate cells were co-cultured with pancreatic acinar cells AR4-2J in monolayer culture. Pancreatic stellate and acinar cell function was monitored by Fura-2 calcium imaging. Rat pancreatic stellate cells were found to express MsrA, B1, B2, their expressions diminished in culture. Over-expressions of MsrA, B1 or B2 were found to enhance ATP-stimulated calcium increase but decreased reactive oxygen species generation and lipopolysaccharide-elicited IL-1 production. Pancreatic stellate cell-co-culture with AR4-2J blunted cholecystokinin- and acetylcholine-stimulated calcium increases in AR4-2J, depending on acinar/stellate cell ratio, this inhibition was reversed by MsrA, B1 over-expression in stellate cells or by Met supplementation in the co-culture medium. These data suggest that Msr play important roles in pancreatic stellate cell function and the stellate cells may serve as a brake mechanism on pancreatic acinar cell calcium signaling modulated by stellate cell Msr expression.

TRPV1 mediates cell death in rat synovial fibroblasts through calcium entry-dependent ROS production and mitochondrial depolarization.

Synoviocyte hyperplasia is critical for rheumatoid arthritis, therefore, potentially an important target for therapeutics. It was found in this work that a TRPV1 agonist capsaicin, and acidic solution (pH 5.5) induced increases in cytosolic calcium concentration ([Ca(2+)](c)) and reactive oxygen species (ROS) production in synoviocytes isolated from a rat model of collagen-induced arthritis. The increases in both [Ca(2+)](c) and ROS production were completely abolished in calcium-free buffer or by a TRPV1 antagonist capsazepine. Further experiments revealed that capsaicin and pH 5.5 solution caused mitochondrial membrane depolarization and reduction in cell viability; such effects were inhibited by capsazepine, or the NAD(P)H oxidase inhibitor diphenylene iodonium. Both capsaicin and pH 5.5 buffer induced apoptosis as shown by nuclear condensation and fragmentation. Furthermore, RT-PCR readily detected TRPV1 mRNA expression in the isolated synoviocytes. Taken together, these data indicated that TRPV1 activation triggered synoviocyte death by [Ca(2+)](c) elevation, ROS production, and mitochondrial membrane depolarization.

Extracellular Histones Activate Plasma Membrane Toll-Like Receptor 9 to Trigger Calcium Oscillations in Rat Pancreatic Acinar Tumor Cell AR4-2J.

In acute pancreatitis, histones are released by infiltrating neutrophils, but how histones modulate pancreatic acinar cell function has not been investigated. We have examined histone modulation of rat pancreatic acini and pancreatic acinar tumor cell AR4-2J by calcium imaging. Histones were found to have no effect on calcium in pancreatic acini but blocked calcium oscillations induced by cholecystokinin or acetylcholine. Both mixed (Hx) and individual (H1, H2A, H2B, H3, H4) histones induced calcium oscillations in AR4-2J. RT-PCR and Western blot verified the expression of histone-targeted Toll-like receptor (TLR) 2, 4 and 9. Immunocytochemistry identified TLR2/TLR4 on apical plasma membrane and TLR9 in zymogen granule regions in pancreatic acini. TLR2 was found on neighboring and TLR9 on peripheral plasma membranes, but TLR4 was in the nucleus in AR4-2J clusters. Neither TLR2 agonist zymosan-A nor TLR4 agonist lipopolysaccharide had any effect on calcium, but TLR9 agonist ODN1826 induced calcium oscillations; TLR9 antagonist ODN2088 blocked H4-induced calcium oscillations in AR4-2J, which also disappeared after treatment of AR4-2J with glucocorticoid dexamethasone, with concurrent TLR9 migration from plasma membrane to cell interiors. TLR9 down regulation with siRNA suppressed H4-induced calcium oscillations. These data together suggest that extracellular histones activate plasma membrane TLR9 to trigger calcium oscillations in AR4-2J cells.

Cholecystokinin 1 Receptor - A Unique G Protein-Coupled Receptor Activated by Singlet Oxygen (GPCR-ABSO).

Plasma membrane-delimited generation of singlet oxygen by photodynamic action with photosensitizer sulfonated aluminum phthalocyanine (SALPC) activates cholecystokinin 1 receptor (CCK1R) in pancreatic acini. Whether CCK1R retains such photooxidative singlet oxygen activation properties in other environments is not known. Genetically encoded protein photosensitizers KillerRed or mini singlet oxygen generator (miniSOG) were expressed in pancreatic acinar tumor cell line AR4-2J, CCK1R, KillerRed or miniSOG were expressed in HEK293 or CHO-K1 cells. Cold light irradiation (87 mW⋅cm-2) was applied to photosensitizer-expressing cells to examine photodynamic activation of CCK1R by Fura-2 fluorescent calcium imaging. When CCK1R was transduced into HEK293 cells which lack endogenous CCK1R, photodynamic action with SALPC was found to activate CCK1R in CCK1R-HEK293 cells. When KillerRed or miniSOG were transduced into AR4-2J which expresses endogenous CCK1R, KillerRed or miniSOG photodynamic action at the plasma membrane also activated CCK1R. When fused KillerRed-CCK1R was transduced into CHO-K1 cells, light irradiation activated the fused CCK1R leading to calcium oscillations. Therefore KillerRed either expressed independently, or fused with CCK1R can both activate CCK1R photodynamically. It is concluded that photodynamic singlet oxygen activation is an intrinsic property of CCK1R, independent of photosensitizer used, or CCK1R-expressing cell types. Photodynamic singlet oxygen CCK1R activation after transduction of genetically encoded photosensitizer in situ may provide a convenient way to verify intrinsic physiological functions of CCK1R in multiple CCK1R-expressing cells and tissues, or to actuate CCK1R function in CCK1R-expressing and non-expressing cell types after transduction with fused KillerRed-CCK1R.