The special case of far-red vision of mesopelagic dragonfish Malacosteus niger facilitated by the presence in the rod outer segment of photosensitizer chlorin e6 from diet has drawn considerable attention both in vision research and in photodynamic action. Rhodopsin binding of Ce6 from either the extracellular or intracellular loops may exert different effects. Theoretical works predict that the extracellularly bound Ce6 upon absorption of red light produces a singlet oxygen, which could via an oxygen tunnel reach the Lys-tethered 11-cis-retinal, by way of peroxy-dioxetane intermediates, to enhance 11-cis- to all-trans-retinal isomerization, therefore triggering the ultrafast phototransduction process. Recent works on the permanent photodynamic activation of some A-class G protein-coupled receptors suggest that the singlet oxygen generated by Ce6 photodynamic action might also oxidize the scotopsin moiety of rhodopsin, leading to direct oxidative rhodopsin activation. More attention needs to be paid to the latter respects of the far-red vision process of the deep-sea dragonfish, with potential translational values.

Different from reversible agonist-stimulated receptor activation, singlet oxygen oxidation activates permanently G protein-coupled receptor (GPCR) cholecystokinin 1 (CCK1R) in type II photodynamic action, with soluble photosensitizer dyes (sulphonated aluminum phthalocyanine, λmax 675 nm) or genetically encoded protein photosensitizers (KillerRed λmax 585 nm; mini singlet oxygen generator λmax 450 nm), together with a pulse of light (37 mW/cm2, 1-2 minutes). Three lines of evidence shed light on the mechanism of GPCR activated by singlet oxygen (GPCR-ABSO): (1) CCK1R is quantitatively converted from dimer to monomer; (2) Transmembrane domain 3, a pharmacophore for permanent photodynamic CCK1R activation, can be transplanted to non-susceptible M3 acetylcholine receptor; and (3) Larger size of disordered region in intracellular loop 3 correlates with higher sensitivity to photodynamic CCK1R activation. GPCR-ABSO will add to the arsenal of engineered designer GPCR such as receptors activated solely by synthetic ligands and designer receptors exclusively activated by designer drugs, but show some clear advantages: Enhanced selectivity (double selectivity of localized photosensitizer and light illumination), long-lasting activation with no need for repeated drug administration, antagonist-binding site remains intact when needed, ease to apply to multiple GPCR. This type of permanent photodynamic activation may be applied to functional proteins other than GPCR.

Reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidases (NOX) are a major cellular source of reactive oxygen species, regulating vital physiological functions, whose dys-regulation leads to a plethora of major diseases. Much effort has been made to develop varied types of NOX inhibitors, but biotechnologies for spatially and temporally controlled NOX activation, however, are not readily available. We previously found that ultraviolet A (UVA) irradiation activates NOX2 in rodent mast cells, to elicit persistent calcium spikes. NOX2 is composed of multiple subunits, making studies of its activation rather complicated. Here we show that the single-subunit nonrodent-expressing NOX5, when expressed ectopically in CHO-K1 cells, is activated by UVA irradiation (380 nm, 0.1-12 mW/cm2, 1.5 min) inducing repetitive calcium spikes, as monitored by Fura-2 fluorescent calcium imaging. UVA-elicited calcium oscillations are inhibited by NOX inhibitor diphenyleneiodonium chloride (DPI) and blocked by singlet oxygen (1O2) quencher Trolox-C (300 μM). A brief pulse of photodynamic action (1.5 min) with photosensitizer sulfonated aluminum phthalocyanine (SALPC 2 μM, 675 nm, 85 mW/cm2) in NOX5-CHO-K1 cells, or with genetically encoded protein photosensitizer miniSOG fused to N-terminus of NOX5 (450 nm, 85 mW/cm2) in miniSOG-NOX5-CHO-K1 cells, induces persistent calcium oscillations, which are blocked by DPI. In the presence of Trolox-C, miniSOG photodynamic action no longer induces any calcium increases in miniSOG-NOX5-CHO-K1 cells. DUOX2 in human thyroid follicular cells SW579 and in DUOX2-CHO-K1 cells is similarly activated by UVA irradiation and SALPC photodynamic action. These data together suggest that NOX   is activated with a brief pulse of photodynamic action.

In the context of an increasingly escalating antibiotics crisis, phototherapy has emerged as a promising therapeutic approach due to its inherent advantages, including high selectivity, noninvasiveness, and low drug resistance. Photothermal therapy (PTT) and photodynamic therapy (PDT) are two complementary and promising phototherapies albeit with inherent limitations, noted as the challenges in achieving precise heat confinement and the associated risk of off-target damage for PTT, while the constraints due to the hypoxic microenvironment are prevalent in biofilms faced by PDT. Herein, we have designed a supramolecular nanoformulation that leverages the complexation-induced quenching of guanidinium-modified calix[5]arene grafted with fluorocarbon chains (GC5AF5), the efficient recognition of adenosine triphosphate (ATP), and the oxygen-carrying capacity of the fluorocarbon chain. This intelligent nanoformulation enables the adaptive enhancement of both photothermal therapy (PTT) and photodynamic therapy (PDT), allowing for on-demand switching between the two modalities. Our nanoformulation utilizes ATP released by dead bacteria to accelerate the elimination of biofilms, rendering bacteria unable to resist while minimizing harm to healthy tissues. This research highlights the particular recognition and assembly capabilities of macrocycles, offering a promising strategy for creating potent, combined antibiofilm therapies.

Little is known about the blood-feeding physiology of arbovirus vector Aedes aegypti although this type of mosquito is known to transmit infectious diseases dengue, Zika, yellow fever, and chikungunya. Blood feeding in the female A. aegypti mosquito is essential for egg maturation and for transmission of disease agents between human subjects. Here, we identify the A. aegypti sulfakinin receptor gene SKR from the A. aegypti genome and show that SKR is expressed at different developmental stages and in varied anatomical localizations in the adult mosquito (at three days after eclosion), with particularly high expression in the CNS. Knockingdown sulfakinin and sulfakinin receptor gene expression in the female A. aegypti results in increased blood meal intake, but microinjection in the thorax of the sulfakinin peptide 1 and 2 both inhibits dose dependently blood meal intake (and delays the time course of blood intake), which is reversible with receptor antagonist. Sulfakinin receptor expressed ectopically in mammalian cells CHO-K1 responds to sulfakinin stimulation with persistent calcium spikes, blockable with receptor antagonist. These data together suggest that activation of the Gq protein-coupled (i.e., calcium-mobilizing) sulfakinin receptor inhibits blood meal intake in female A. aegypti mosquitoes and could serve as a strategic node for the future control of A. aegypti mosquito reproduction/population and disease transmission.

Phototherapies, including photothermal therapy and photodynamic therapy, have gained booming development over the past several decades for their attractive non-invasiveness nature, negligible adverse effects, minimal systemic toxicity, and high spatial selectivity. Phototherapy usually requires three components: light irradiation, photosensitizers, and molecular oxygen. Photosensitizers can convert light energy into heat or reactive oxygen species, which can be used in the tumor-killing process. The direct application of photosensitizers in tumor therapy is restricted by their poor water solubility, fast clearance, severe toxicity, and low cellular uptake. The encapsulation of photosensitizers into nanostructures is an attractive strategy to overcome these critical limitations. Poly(glutamic acid) (PGA) is a kind of poly(amino acid)s containing the repeating units of glutamic acid. PGA has superiority for cancer treatment because of its good biocompatibility, low immunogenicity, and modulated pH responsiveness. The hydrophilicity nature of PGA allows the physical entrapment of photosensitizers and anticancer drugs via the construction of amphiphilic polymers. Moreover, the pendent carboxyl groups of PGA enable chemical conjugation with therapeutic agents. In this mini-review, we highlight the stateof- the-art design and fabrication of PGA-based nanoplatforms for phototherapy. We also discuss the potential challenges and future perspectives of phototherapy, and clinical translation of PGA-based nanomedicines.

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.

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.

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.

Chronic pancreatitis (CP) is a complex inflammatory disorder with numerous associated genetic and environmental risk factors. The most distressing characteristic of CP is recalcitrant pain, often requiring surgical resection including total pancreatectomy with islet autotransplantation (TPIAT). We studied five consented subjects undergoing pancreatic resection and processed isolated cells for single-cell RNA sequencing (scRNA-Seq). Using high-dimensional transcriptomic cluster analysis, we identified 11 unique cell clusters in the pancreas tissue. These cell clusters include a cluster of undifferentiated/dedifferentiated cells and two unique clusters of acinar cells, one of which appears to be in a transitional stage. To determine the cellular response to protease inhibitor and stimulation, we treated aliquots of cells from one subject with a protease inhibitor cocktail with and without bethanechol (a muscarinic receptor agonist) at 100 and 400 µM and compared gene expression profiles. The protease inhibitors appeared to reduce cell stress. Pancreatic digestive enzymes and islet hormones were upregulated in both doses of bethanechol-treated cells compared with naïve cells. High-dose bethanechol appeared to be toxic and consistent with hyperstimulation. These studies demonstrate the feasibility of investigating human acinar cell physiology at the single-cell level and initial evidence that these cells retain responsiveness to agonist stimulation with predicted second messenger and transcriptomic responses.NEW & NOTEWORTHY We conducted single cell RNA sequencing on pancreas tissue from five individuals. We identified eleven unique cell clusters including a large population of dedifferentiated cells as well as two unique clusters of acinar cells, one of which appears to exist in a transitional state. We also examined the cellular response of pancreas tissue to stimulation and identified affected genes and pathways, including pancreatic digestive enzymes.

Phototherapy such as photothermal therapy and photodynamic therapy in cancer treatment has been developed quickly over the past few years for its noninvasive nature and high efficiency. However, there are still many drawbacks in phototherapy that prevent it from clinical applications. Thus, scientists have designed different systems to overcome the issues associated with phototherapy, including enhancing the targeting ability of phototherapy, low-temperature photothermal therapy, replacing near-infrared light with other excitation sources, and so on. This article discusses the problems and shortcomings encountered in the development of phototherapy and highlights possible solutions to address them so that phototherapy may become a useful cancer treatment approach in clinical practice. This article aims to give a brief summary about current research advancements in phototherapy research and provides a quick guideline toward future developments in the field.

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.

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.

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.

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.

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.

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.

Singlet oxygen generated in a type II photodynamic action, due to its limited lifetime (1 μs) and reactive distance (<10 nm), could regulate live cell function nanoscopically. The genetically-encoded protein photosensitizers (engineered fluorescent proteins such as KillerRed, TagRFP, and flavin-binding proteins such as miniSOG, Pp2FbFPL30M) could be expressed in a cell type- and/or subcellular organelle-specific manner for targeted protein photo-oxidative activation/desensitization. The newly emerged active illumination technique provides an additional level of specificity. Typical examples of photodynamic activation include permanent activation of G protein-coupled receptor CCK1 and photodynamic activation of ionic channel TRPA1. Protein photosensitizers have been used to photodynamically modulate major cellular functions (such as neurotransmitter release and gene transcription) and animal behavior. Protein photosensitizers are increasingly used in photon-driven nanomanipulation in cell physiology research.

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.

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.

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.

UVA is a major bio-active component in solar irradiation, and is shown to have immunomodulatory and anti-inflammatory effects. The detailed molecular mechanism of UVA action in regard to calcium signaling in mast cells, however, is not fully understood. In this study, it was found that UVA induced ROS formation and cytosolic calcium oscillations in individual rat mast cells. Exogenously added H2O2 and hypoxanthine/xanthine oxidase (HX/XOD) mimicked UVA effects on cytosolic calcium increases. Regular calcium oscillation induced by UVA irradiation was inhibited completely by the phosphatidylinositol-specific phospholipase C inhibitor U73122, but U73343 was without effect. Tetrandrine, a calcium entry blocker, or calcium-free buffer abolished UVA-induced calcium oscillations. L-type calcium channel blocker nifedipine and stores-operated calcium channel blocker SK&F96365 had no such inhibitory effect. ROS induction by UVA was abolished after pre-incubation with anti-oxidant NAC or with NAD(P)H oxidase inhibitor DPI; such treatment also made UVA-induced calcium oscillation to disappear. UVA irradiation did not increase mast cell diameter, but it made mast cell structure more granular. Spectral confocal imaging revealed that the emission spectrum of the endogenous fluorophore in single mast cell contained a sizable peak which corresponded to that of NAD(P)H. Taken together, these data suggest that UVA in rat mast cells could activate NAD(P)H oxidase, to produce ROS, which in turn activates phospholipase C signaling, to trigger regular cytosolic calcium oscillation.

The field of cholecystokinin (CCK) stimulation of exocrine pancreatic secretion has experienced major changes in the recent past. This review attempts to summarize the present status of the field. CCK production in the intestinal I cells, the molecular forms of CCK produced and subsequently circulated in the blood, the presence or absence of CCK receptors on the isolated pancreatic acinar cells and the associated signaling for acinar cell secretion, and the actual circuits and sites of action for CCK regulation of exocrine pancreatic secretion in vivo are reviewed in different animal species with an emphasis on birds, rodents, and humans. Clear differences in the relative importance of neural and direct modes of CCK action on pancreatic acinar cells were identified. Rodents seem to be endowed with both modes of action, whereas in humans the neural mode may predominate. In birds, such as duck, the direct mode needs further assistance from pituitary adenylate cyclase-activating peptide/VIP receptors. However, much further work needs to be directed to the neural mode to map out all sites of CCK action and details of the full circuits, and we foresee a major revival for this field of research in the near future.

Calcium oscillations can, by default, encode diverse and specific signals by different modes of modulation. Frequency modulation is illustrated by the activation of calcium/calmodulin-dependent protein kinase II at unit Hz, and of calcineurin at 10 mHz frequencies, respectively. The submandibular gland secretory axis is characterized by both potassium and osmolarity gradients from the luminal side of the secretory cells. Such gradients may play significant physiological roles through the feedback modulation of cholinergic stimulation. High potassium transforms plateau calcium increases induced by cholinergic stimulation of the submandibular acinar cells into oscillatory calcium increases. The ductal cells may have similar mechanisms of feedback modulation both by high potassium and by hypoosmolarity. Such feedback mechanisms could modulate the decision-making process for determining which secretory products are selectively released after nerve stimulation.

Oxidative stress has been involved in several biological and pathological processes. Reactive oxygen species have been shown to play both beneficial and deleterious roles. The present work contributes to the understanding of the very early events of cellular response to oxidative stress. Oxidative stress was produced intracellularly by light activation of zinc phthalocyanine (ZnPc) at a light dose that did not lead to apoptosis or necrosis. Phthalocyanine was photoactivated using the 647-nm laser line of a confocal microscope through the objective lens causing oxidative stress and allowing observation of the evoked phenomena at the single cell level and in real time. Mitochondria membrane potential (DeltaPsi(m)), intracellular pH, calcium concentration, and generation of reactive oxygen species (ROS) were recorded using specific vital fluorescent probes and quantified by image processing and analysis. Subcellular localization of ZnPc was also studied in order to determine the primary and intermediate ROS target.

Concentration-dependent transformation of hormone- and neurotransmitter-induced calcium oscillation is a common phenomenon in diverse types of cells especially of the secretory type. The rodent submandibular acinar cells are an exception to this rule, which show elevated plateau increase in intracellular calcium under all stimulatory concentrations of both norepinephrine and acetylcholine. However, under depolarized state this cell type could also show a variation of periodic calcium changes. This reserved mechanism of calcium oscillation is jump-started by depolarization only with muscarinic cholinergic stimulation, but not with adrenergic stimulation. This latter effect is attributable to alpha receptor activation, not due to simultaneous activation of alpha and beta receptors, with beta receptor activation only serving to enhance the magnitude. These data suggest that this reserved mechanism for inducing calcium oscillation can be selectively used only by specific receptor-signaling pathways, and may therefore partly explain the long-known differences between secretion induced by sympathetic and parasympathetic stimulation in the submandibular gland.

In photodynamic therapy (PDT) a sensitizer, light and oxygen are used to induce death of tumor cells and in the treatment of certain noncancerous conditions. Cell death in PDT may occur by apoptosis or by necrosis, depending on the sensitizer, on the PDT dose and on the cell genotype. Some sensitizers that have been used in PDT are accumulated in the mitochondria, and this may explain their efficiency in inducing apoptotic cell death, both in vitro and in vivo. In this review we will focus on the events that characterize apoptotic death in PDT and on the intracellular signaling events that are set in motion in photosensitized cells. Activation of phospholipases, changes in ceramide metabolism, a rise in the cytosolic free Ca2+ concentration, stimulation of nitric oxide synthase (NOS), changes in protein phosphorylation and alterations in the activity of transcription factors and on gene expression have all been observed in PDT-treated cells. Although many of these metabolic reactions contribute to the demise process, some of them may antagonize cell death. Understanding the signaling mechanisms in PDT may provide means to modulate the PDT effects at the molecular level and potentiate its antitumor effectiveness.

Photodynamic action of a plasma membrane-specific photosensitizer sulphonated aluminium phthalocyanine (SALPC) has been found to regulate cellular signalling pathways. The present study aimed to investigate whether SALPC photodynamic action modulates the structure of plasma membrane proteins, and as control, of model proteins. To check the photodynamic effect, intrinsic fluorescence of model proteins bovine serum albumin (BSA), phospholipase A2 (PLA2), and calmodulin were monitored continuously during photodynamic action (SALPC 1 microM, light 14,000 1x at > 580 nm). Significant decrease in fluorescence intensity was observed in BSA and PLA2, whereas the fluorescence of calmodulin was not affected. Confirming a major change in protein structure, difference IR spectrum revealed a significant downward deflection after photodynamic action in both BSA and in pancreatic acinar cells, whereas SALPC alone or light illumination alone resulted in no major deflection. Quantitative FTIR analysis indicated that in BSA, photodynamic action decreased the content of alpha-helix, increased the content of beta-turn and random structures, whereas beta-sheet remained the same; in freshly isolated rat pancreatic acini, photodynamic action decreased the content of alpha-helix and beta-sheet, increased the content of 1-turn and random structures. Taken together the fact that under the present experimental conditions SALPC mainly localized at the plasma membrane, it is concluded that SALPC photodynamic action directly modulates plasma membrane protein structure.