Heavy metals in cigarette smoke strongly inhibit pancreatic ductal function and promote development of chronic pancreatitis.

BACKGROUND AND AIMS: Smoking is recognised as an independent risk factor in the development of chronic pancreatitis (CP). Cystic fibrosis transmembrane conductance regulator (CFTR) function and ductal fluid and bicarbonate secretion are also known to be impaired in CP, so it is crucial to understand the relationships between smoking, pancreatic ductal function and the development of CP. METHODS: We measured sweat chloride (Cl-) concentrations in patients with and without CP, both smokers and non-smokers, to assess CFTR activity. Serum heavy metal levels and tissue cadmium concentrations were determined by mass spectrometry in smoking and non-smoking patients. Guinea pigs were exposed to cigarette smoke, and cigarette smoke extract (CSE) was prepared to characterise its effects on pancreatic HCO3 - and fluid secretion and CFTR function. We administered cerulein to both the smoking and non-smoking groups of mice to induce pancreatitis. RESULTS: Sweat samples from smokers, both with and without CP, exhibited elevated Cl- concentrations compared to those from non-smokers, indicating a decrease in CFTR activity due to smoking. Pancreatic tissues from smokers, regardless of CP status, displayed lower CFTR expression than those from non-smokers. Serum levels of cadmium and mercury, as well as pancreatic tissue cadmium, were increased in smokers. Smoking, CSE, cadmium, mercury and nicotine all hindered fluid and HCO3 - secretion and CFTR activity in pancreatic ductal cells. These effects were mediated by sustained increases in intracellular calcium ([Ca2+]i), depletion of intracellular ATP (ATPi) and mitochondrial membrane depolarisation. CONCLUSION: Smoking impairs pancreatic ductal function and contributes to the development of CP. Heavy metals, notably cadmium, play a significant role in the harmful effects of smoking. KEY POINTS: Smoking and cigarette smoke extract diminish pancreatic ductal fluid and HCO3 - secretion as well as the expression and function of CFTR Cd and Hg concentrations are significantly higher in the serum samples of smokers Cd accumulates in the pancreatic tissue of smokers.

Reduction of intracellular oxidative stress with a copper-incorporated layered double hydroxide.

Biocompatible Cu(II)-doped layered double hydroxide (CMA) nanoparticles were developed to combat reactive oxygen species. The 2-dimensional nanozymes showed both superoxide dismutase- and catalase-like activities in chemical assays, while proving as efficient antioxidants in the reduction of intracellular oxidative stress. The results indicate the great promise of CMA in antioxidant therapies.

Plasminogen Activator Inhibitor 1 Is a Novel Faecal Biomarker for Monitoring Disease Activity and Therapeutic Response in Inflammatory Bowel Diseases.

BACKGROUND AND AIMS: Crohn's disease [CD] and ulcerative colitis [UC] require lifelong treatment and patient monitoring. Current biomarkers have several limitations; therefore, there is an unmet need to identify novel biomarkers in inflammatory bowel disease [IBD]. Previously, the role of plasminogen activator inhibitor 1 [PAI-1] was established in the pathogenesis of IBD and suggested as a potential biomarker. Therefore, we aimed to comprehensively analyse the selectivity of PAI-1 in IBD, its correlation with disease activity, and its potential to predict therapeutic response. METHODS: Blood, colon biopsy, organoid cultures [OC], and faecal samples were used from active and inactive IBD patients and control subjects. Serpin E1 gene expressions and PAI-1 protein levels and localisation in serum, biopsy, and faecal samples were evaluated by qRT-PCR, ELISA, and immunostaining, respectively. RESULTS: The study population comprised 132 IBD patients [56 CD and 76 UC] and 40 non-IBD patients. We demonstrated that the serum, mucosal, and faecal PAI-1 concentrations are elevated in IBD patients, showing clinical and endoscopic activity. In responders [decrease of eMayo ≥3 in UC; or SES-CD  50% in CD], the initial PAI-1 level decreased significantly upon successful therapy. OCs derived from active IBD patients produced higher concentrations of PAI-1 than the controls, suggesting that epithelial cells could be a source of PAI-1. Moreover, faecal PAI-1 selectively increases in active IBD but not in other organic gastrointestinal diseases. CONCLUSIONS: The serum, mucosal, and faecal PAI-1 concentration correlates with disease activity and therapeutic response in IBD, suggesting that PAI-1 could be used as a novel, non-invasive, disease-specific, faecal biomarker in patient follow-up.

Human pancreatic ductal organoids with controlled polarity provide a novel ex vivo tool to study epithelial cell physiology.

Epithelial ion and fluid secretion determine the physiological functions of a broad range of organs, such as the lung, liver, or pancreas. The molecular mechanism of pancreatic ion secretion is challenging to investigate due to the limited access to functional human ductal epithelia. Patient-derived organoids may overcome these limitations, however direct accessibility of the apical membrane is not solved. In addition, due to the vectorial transport of ions and fluid the intraluminal pressure in the organoids is elevated, which may hinder the study of physiological processes. To overcome these, we developed an advanced culturing method for human pancreatic organoids based on the removal of the extracellular matrix that induced an apical-to-basal polarity switch also leading to reversed localization of proteins with polarized expression. The cells in the apical-out organoids had a cuboidal shape, whereas their resting intracellular Ca2+ concentration was more consistent compared to the cells in the apical-in organoids. Using this advanced model, we demonstrated the expression and function of two novel ion channels, the Ca2+ activated Cl- channel Anoctamin 1 (ANO1) and the epithelial Na+ channel (ENaC), which were not considered in ductal cells yet. Finally, we showed that the available functional assays, such as forskolin-induced swelling, or intracellular Cl- measurement have improved dynamic range when performed with apical-out organoids. Taken together our data suggest that polarity-switched human pancreatic ductal organoids are suitable models to expand our toolset in basic and translational research.

Effects of bowel cleansing on the composition of the gut microbiota in inflammatory bowel disease patients and healthy controls.

Background: In patients with inflammatory bowel disease (IBD), Crohn's disease (CD), and ulcerative colitis (UC), numerous cases of exacerbations could be observed after colonoscopy, raising the possible pathogenetic effect of colonic microbiota alterations in IBD flare. Objectives: We aimed to investigate the changes in the fecal microbiota composition in IBD patients influenced by the bowel preparation with sodium picosulfate. Design: We enrolled patients with IBD undergoing bowel preparation for colonoscopy in the prospective cohort study. The control group (Con) comprised non-IBD patients who underwent colonoscopy. Clinical data, blood, and stool samples were collected before colonoscopy (timepoint A), 3 days later (timepoint B), and 4 weeks later (timepoint C). Methods: Disease activity and gut microbiota changes were assessed at each timepoint. Fecal microbiota structure - at family level - was determined by sequencing the V4 region of the 16S rRNA gene. Statistical analysis included differential abundance analysis and Mann-Whitney tests. Results: Forty-one patients (9 CD, 13 UC, and 19 Con) were included. After bowel preparation, alpha diversity was lower in the CD group than in the UC (p = 0.01) and Con (p = 0.02) groups at timepoint B. Alpha diversity was significantly higher in the UC group than in the CD and Con (p = 0.03) groups at timepoint C. Beta diversity difference differed between the IBD and Con (p = 0.001) groups. Based on the differential abundance analysis, the Clostridiales family was increased, whereas the Bifidobacteriaceae family was decreased in CD patients compared to the Con at timepoint B. Conclusions: Bowel preparation may change the fecal microbial composition in IBD patients, which may have a potential role in disease exacerbation after bowel cleansing.

Orai1 calcium channel inhibition prevents progression of chronic pancreatitis.

Patients with recurrent acute pancreatitis (RAP) are at significant risk of developing early chronic pancreatitis (CP), which progresses into irreversible, end-stage CP with severe symptoms. There is no specific therapy in RAP or in early CP that may hinder disease progression. The pathogenesis of CP is complex and involves interactions among multiple cell types, including pancreatic acinar, ductal, and stellate cells (PSC). Therefore, it is pivotal to identify common pathogenic pathways in these cells that could be targeted pharmacologically. The Orai1-mediated store-operated Ca2+ entry (SOCE) is a ubiquitous signaling mechanism that may become overactivated in pathological states resulting in intracellular Ca2+ overload. In this study, we used ex vivo and in vivo preclinical disease models to demonstrate that Orai1 inhibition prevents progression of RAP and early CP. The selective Orai1 inhibitor CM5480 restored the expression of SOCE-associated regulatory factor in acinar cells, prevented uncontrolled Ca2+ elevation, protected acinar and ductal functions, mitigated immune cell infiltration, and diminished PSC activation, proliferation, and migration. We suggest that the overactivation of Orai1 is a crucial pathogenetic event in the progression of early CP and that inhibition of Orai1 could prevent the development of end-stage CP.

Confinement of Triple-Enzyme-Involved Antioxidant Cascade in Two-Dimensional Nanostructure.

Application of antioxidant enzymes in medical or industrial processes is limited due to their high sensitivity to environmental conditions. Incorporation of such enzymes in nanostructures provides a promising route to obtain highly efficient and robust biocatalytic system to scavenge reactive oxygen species (ROS). Here, this question was addressed by confinement of superoxide dismutase (SOD), horseradish peroxidase (HRP), and catalase (CAT) enzymes into nanostructures containing polyelectrolyte building blocks (alginate (Alg) and trimethyl chitosan (TMC)) and delaminated layered double hydroxide (dLDH) nanoparticle support. The nanocomposite possessed excellent structural and colloidal stability, while antioxidant tests revealed that the enzymes remained active upon immobilization and the developed composite greatly reduced intracellular oxidative stress in two-dimensional cell cultures. Moreover, it effectively prevented hydrogen peroxide-induced double stranded DNA breaks, which is a common consequence of oxidative stress. The results provide important tools to design complex nanostructures with multienzymatic antioxidant activities for ROS scavenging.

Thiopurines impair the apical plasma membrane expression of CFTR in pancreatic ductal cells via RAC1 inhibition.

BACKGROUND AND AIMS: Thiopurine-induced acute pancreatitis (TIP) is one of the most common adverse events among inflammatory bowel disease patients treated with azathioprine (AZA), representing a significant clinical burden. Previous studies focused on immune-mediated processes, however, the exact pathomechanism of TIP is essentially unclear. METHODS: To model TIP in vivo, we triggered cerulein-induced experimental pancreatitis in mice receiving a daily oral dose of 1.5 mg/kg AZA. Also, freshly isolated mouse pancreatic cells were exposed to AZA ex vivo, and acinar cell viability, ductal and acinar Ca2+ signaling, ductal Cl- and HCO3- secretion, as well as cystic fibrosis transmembrane conductance regulator (CFTR) expression were assessed using microscopy techniques. Ras-related C3 botulinum toxin substrate (RAC1) activity was measured with a G-LISA assay. Super-resolution microscopy was used to determine protein colocalization. RESULTS: We demonstrated that AZA treatment increases tissue damage in the early phase of cerulein-induced pancreatitis in vivo. Also, both per os and ex vivo AZA exposure impaired pancreatic fluid and ductal HCO3- and Cl- secretion, but did not affect acinar cells. Furthermore, ex vivo AZA exposure also inhibited RAC1 activity in ductal cells leading to decreased co-localization of CFTR and the anchor protein ezrin, resulting in impaired plasma membrane localization of CFTR. CONCLUSIONS: AZA impaired the ductal HCO3- and Cl- secretion through the inhibition of RAC1 activity leading to diminished ezrin-CFTR interaction and disturbed apical plasma membrane expression of CFTR. We report a novel direct toxic effect of AZA on pancreatic ductal cells and suggest that the restoration of ductal function might help to prevent TIP in the future.

OGG1 Inhibition Reduces Acinar Cell Injury in a Mouse Model of Acute Pancreatitis.

Acute pancreatitis (AP) is a potentially life-threatening gastrointestinal disease with a complex pathology including oxidative stress. Oxidative stress triggers oxidative DNA lesions such as formation of 7,8-dihydro-8-oxo-2'-oxoguanine (8-oxoG) and also causes DNA strand breaks. DNA breaks can activate the nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) which contributes to AP pathology. 8-oxoG is recognized by 8-oxoG glycosylase 1 (OGG1) resulting in the removal of 8-oxoG from DNA as an initial step of base excision repair. Since OGG1 also possesses a DNA nicking activity, OGG1 activation may also trigger PARP1 activation. In the present study we investigated the role played by OGG1 in AP. We found that the OGG1 inhibitor compound TH5487 reduced edema formation, inflammatory cell migration and necrosis in a cerulein-induced AP model in mice. Moreover, TH5487 caused 8-oxoG accumulation and reduced tissue poly(ADP-ribose) levels. Consistent with the indirect PARP inhibitory effect, TH5487 shifted necrotic cell death (LDH release and Sytox green uptake) towards apoptosis (caspase activity) in isolated pancreatic acinar cells. In the in vivo AP model, TH5487 treatment suppressed the expression of various cytokine and chemokine mRNAs such as those of TNF, IL-1ß, IL1ra, IL6, IL16, IL23, CSF, CCL2, CCL4, CCL12, IL10 and TREM as measured with a cytokine array and verified by RT-qPCR. As a potential mechanism underlying the transcriptional inhibitory effect of the OGG1 inhibitor we showed that while 8-oxoG accumulation in the DNA facilitates NF-κB binding to its consensus sequence, when OGG1 is inhibited, target site occupancy of NF-κB is impaired. In summary, OGG1 inhibition provides protection from tissue injury in AP and these effects are likely due to interference with the PARP1 and NF-κB activation pathways.

Tricetin Reduces Inflammation and Acinar Cell Injury in Cerulein-Induced Acute Pancreatitis: The Role of Oxidative Stress-Induced DNA Damage Signaling.

Acute pancreatitis (AP) poses a worldwide challenge due to the growing incidence and its potentially life-threatening course and complications. Specific targeted therapies are not available, prompting the identification of new pathways and novel therapeutic approaches. Flavonoids comprise several groups of biologically active compounds with wide-ranging effects. The flavone compound, tricetin (TCT), has not yet been investigated in detail but sporadic reports indicate diverse biological activities. In the current study, we evaluated the potential protective effects of TCT in AP. TCT (30 µM) protected isolated primary murine acinar cells from the cytotoxic effects of cerulein, a cholecystokinin analog peptide. The protective effects of TCT were observed in a general viability assay (calcein ester hydrolysis), in an apoptosis assay (caspase activity), and in necrosis assays (propidium iodide uptake and lactate dehydrogenase release). The effects of TCT were not related to its potential antioxidant effects, as TCT did not protect against H2O2-induced acinar cell death despite possessing radical scavenging activity. Cerulein-induced expression of IL1ß, IL6, and matrix metalloproteinase 2 and activation of nuclear factor-κB (NFκB) were reduced by 30 µM TCT. In vivo experiments confirmed the protective effect of TCT in a mouse model of cerulein-induced AP. TCT suppressed edema formation and apoptosis in the pancreas and reduced lipase and amylase levels in the serum. Moreover, TCT inhibited interleukin-1ß (IL1ß), interleukin-6 (IL6), and tumor necrosis factor-α (TNFα) expression in the pancreas and reduced the activation of the oxidative DNA damage sensor enzyme poly(ADP-ribose) polymerase-1 (PARP-1). Our data indicate that TCT can be a potential treatment option for AP.

Impaired regulation of PMCA activity by defective CFTR expression promotes epithelial cell damage in alcoholic pancreatitis and hepatitis.

Alcoholic pancreatitis and hepatitis are frequent, potentially lethal diseases with limited treatment options. Our previous study reported that the expression of CFTR Cl- channel is impaired by ethanol in pancreatic ductal cells leading to more severe alcohol-induced pancreatitis. In addition to determining epithelial ion secretion, CFTR has multiple interactions with other proteins, which may influence intracellular Ca2+ signaling. Thus, we aimed to investigate the impact of ethanol-mediated CFTR damage on intracellular Ca2+ homeostasis in pancreatic ductal epithelial cells and cholangiocytes. Human and mouse pancreas and liver samples and organoids were used to study ion secretion, intracellular signaling, protein expression and interaction. The effect of PMCA4 inhibition was analyzed in a mouse model of alcohol-induced pancreatitis. The decreased CFTR expression impaired PMCA function and resulted in sustained intracellular Ca2+ elevation in ethanol-treated and mouse and human pancreatic organoids. Liver samples derived from alcoholic hepatitis patients and ethanol-treated mouse liver organoids showed decreased CFTR expression and function, and impaired PMCA4 activity. PMCA4 co-localizes and physically interacts with CFTR on the apical membrane of polarized epithelial cells, where CFTR-dependent calmodulin recruitment determines PMCA4 activity. The sustained intracellular Ca2+ elevation in the absence of CFTR inhibited mitochondrial function and was accompanied with increased apoptosis in pancreatic epithelial cells and PMCA4 inhibition increased the severity of alcohol-induced AP in mice. Our results suggest that improving Ca2+ extrusion in epithelial cells may be a potential novel therapeutic approach to protect the exocrine pancreatic function in alcoholic pancreatitis and prevent the development of cholestasis in alcoholic hepatitis.

Blood-brain barrier dysfunction in L-ornithine induced acute pancreatitis in rats and the direct effect of L-ornithine on cultured brain endothelial cells.

BACKGROUND: In severe acute pancreatitis (AP) the CNS is affected manifesting in neurological symptoms. Earlier research from our laboratory showed blood-brain barrier (BBB) permeability elevation in a taurocholate-induced AP model. Here we aimed to further explore BBB changes in AP using a different, non-invasive in vivo model induced by L-ornithine. Our goal was also to identify whether L-ornithine, a cationic amino acid, has a direct effect on brain endothelial cells in vitro contributing to the observed BBB changes. METHODS: AP was induced in rats by the intraperitoneal administration of L-ornithine-HCl. Vessel permeability and the gene expression of the primary transporter of L-ornithine, cationic amino acid transporter-1 (Cat-1) in the brain cortex, pancreas, liver and lung were determined. Ultrastructural changes were followed by transmission electron microscopy. The direct effect of L-ornithine was tested on primary rat brain endothelial cells and a triple co-culture model of the BBB. Viability and barrier integrity, including permeability and TEER, nitrogen monoxide (NO) and reactive oxygen species (ROS) production and NF-κB translocation were measured. Fluorescent staining for claudin-5, occludin, ZO-1, ß-catenin, cell adhesion molecules Icam-1 and Vcam-1 and mitochondria was performed. Cell surface charge was measured by laser Doppler velocimetry. RESULTS: In the L-ornithine-induced AP model vessel permeability for fluorescein and Cat-1 expression levels were elevated in the brain cortex and pancreas. On the ultrastructural level surface glycocalyx and mitochondrial damage, tight junction and basal membrane alterations, and glial edema were observed. L-ornithine decreased cell impedance and elevated the BBB model permeability in vitro. Discontinuity in the surface glycocalyx labeling and immunostaining of junctional proteins, cytoplasmic redistribution of ZO-1 and ß-catenin, and elevation of Vcam-1 expression were measured. ROS production was increased and mitochondrial network was damaged without NF-κB, NO production or mitochondrial membrane potential alterations. Similar ultrastructural changes were seen in L-ornithine treated brain endothelial cells as in vivo. The basal negative zeta potential of brain endothelial cells became more positive after L-ornithine treatment. CONCLUSION: We demonstrated BBB damage in the L-ornithine-induced rat AP model suggesting a general, AP model independent effect. L-ornithine induced oxidative stress, decreased barrier integrity and altered BBB morphology in a culture BBB model. These data suggest a direct effect of the cationic L-ornithine on brain endothelium. Endothelial surface glycocalyx injury was revealed both in vivo and in vitro, as an additional novel component of the BBB-related pathological changes in AP.

Fentanyl but Not Morphine or Buprenorphine Improves the Severity of Necrotizing Acute Pancreatitis in Rats.

Opioids are widely used for the pain management of acute pancreatitis (AP), but their impact on disease progression is unclear. Therefore, our aim was to study the effects of clinically relevant opioids on the severity of experimental AP. Various doses of fentanyl, morphine, or buprenorphine were administered as pre- and/or post-treatments in rats. Necrotizing AP was induced by the intraperitoneal injection of L-ornithine-HCl or intra-ductal injection of Na-taurocholate, while intraperitoneal caerulein administration caused edematous AP. Disease severity was determined by laboratory and histological measurements. Mu opioid receptor (MOR) expression and function was assessed in control and AP animals. MOR was expressed in both the pancreas and brain. The pancreatic expression and function of MOR were reduced in AP. Fentanyl post-treatment reduced necrotizing AP severity, whereas pre-treatment exacerbated it. Fentanyl did not affect the outcome of edematous AP. Morphine decreased vacuolization in edematous AP, while buprenorphine pre-treatment increased pancreatic edema during AP. The overall effects of morphine on disease severity were negligible. In conclusion, the type, dosing, administration route, and timing of opioid treatment can influence the effects of opioids on AP severity. Fentanyl post-treatment proved to be beneficial in AP. Clinical studies are needed to determine which opioids are best in AP.

Bile acid- and ethanol-mediated activation of Orai1 damages pancreatic ductal secretion in acute pancreatitis.

Regardless of its aetiology, sustained intracellular Ca2+ overload is a well-known hallmark of acute pancreatitis (AP). Toxic Ca2+ elevation induces pancreatic ductal cell damage characterized by impaired ion and fluid secretion - essential to wash out the protein-rich fluid secreted by acinar cells while maintaining the alkaline intra-ductal pH under physiological conditions - and mitochondrial dysfunction. While prevention of ductal cell injury decreases the severity of AP, no specific drug target has yet been identified in the ductal cells. Although Orai1, a store-operated Ca2+ influx channel, is known to contribute to sustained Ca2+ overload in acinar cells, details concerning its expression and function in ductal cells are currently lacking. In this study, we demonstrate that functionally active Orai1 channels reside predominantly in the apical plasma membrane of pancreatic ductal cells. Selective CM5480-mediated Orai1 inhibition impairs Stim1-dependent extracellular Ca2+ influx evoked by bile acids or ethanol combined with non-oxidative ethanol metabolites. Furthermore, prevention of sustained extracellular Ca2+ influx protects ductal cell secretory function in vitro and decreases pancreatic ductal cell death. Finally, Orai1 inhibition partially restores and maintains proper exocrine pancreatic secretion in in vivo AP models. In conclusion, our results indicate that Orai1 inhibition prevents AP-related ductal cell function impairment and holds the potential of improving disease outcome. KEY POINTS: Sustained intracellular Ca2+ overload in pancreatic acinar and ductal cells is a hallmark of biliary and alcohol-induced acute pancreatitis, which leads to impaired ductal ion and fluid secretion. Orai1 is a plasma membrane Ca2+ channel that mediates extracellular Ca2+ influx upon endoplasmic reticulum Ca2+ depletion. Results showed that Orai1 is expressed on the luminal plasma membrane of the ductal cells and selective Orai1 inhibition impaired Stim1-dependent extracellular Ca2+ influx evoked by bile acids or ethanol combined with non-oxidative ethanol metabolites. The prevention of sustained extracellular Ca2+ influx protected ductal cell secretory functions in in vitro models and maintained exocrine pancreatic secretion in in vivo acute pancreatitis models. Orai1 inhibition prevents the bile acid- and alcohol-induced damage of the pancreatic ductal secretion and holds the potential of improving the outcome of acute pancreatitis.

Development of polymer-based multifunctional composite particles of protease and peroxidase activities.

A hybrid material (SL-PPN-HEP-HRP) of dual enzyme function was prepared by co-immobilization of papain (PPN) and horseradish peroxidase (HRP) on sulphate latex (SL) microspheres using heparin (HEP) polyelectrolyte as a building block in the sequential adsorption method. The doses of PPN, HEP and HRP were optimized in each step of the preparation process to achieve high functional and colloidal stability. The enzymes and the polyelectrolyte strongly adsorbed on the oppositely charged surfaces via electrostatic forces, and enzyme leakage was not observed from the hybrid material, as confirmed by colorimetric protein tests and microscopy measurements. It was found that the polyelectrolyte acted as a separator between PPN and HRP to prevent hydrolytic attack on the latter enzyme, which otherwise prevents the joint use of these important biocatalysts. Excellent colloidal stability was obtained for the SL-PPN-HEP-HRP composite and the embedded PPN and HRP showed remarkable protease and peroxidase activities, respectively, at least until five days after preparation. The present results offer a promising approach to develop biocatalytic systems of dual function, which are often required in manufacturing processes in the food industry, where the colloidal stability of such multifunctional materials is a key parameter to achieve remarkable efficiency.

Kynurenic Acid and Its Analogue SZR-72 Ameliorate the Severity of Experimental Acute Necrotizing Pancreatitis.

The pathophysiology of acute pancreatitis (AP) is not well understood, and the disease does not have specific therapy. Tryptophan metabolite L-kynurenic acid (KYNA) and its synthetic analogue SZR-72 are antagonists of the N-methyl-D-aspartate receptor (NMDAR) and have immune modulatory roles in several inflammatory diseases. Our aims were to investigate the effects of KYNA and SZR-72 on experimental AP and to reveal their possible mode of action. AP was induced by intraperitoneal (i.p.) injection of L-ornithine-HCl (LO) in SPRD rats. Animals were pretreated with 75-300 mg/kg KYNA or SZR-72. Control animals were injected with physiological saline instead of LO, KYNA and/or SZR-72. Laboratory and histological parameters, as well as pancreatic and systemic circulation were measured to evaluate AP severity. Pancreatic heat shock protein-72 and IL-1ß were measured by western blot and ELISA, respectively. Pancreatic expression of NMDAR1 was investigated by RT-PCR and immunohistochemistry. Viability of isolated pancreatic acinar cells in response to LO, KYNA, SZR-72 and/or NMDA administration was assessed by propidium-iodide assay. The effects of LO and/or SZR-72 on neutrophil granulocyte function was also studied. Almost all investigated laboratory and histological parameters of AP were significantly reduced by administration of 300 mg/kg KYNA or SZR-72, whereas the 150 mg/kg or 75 mg/kg doses were less or not effective, respectively. The decreased pancreatic microcirculation was also improved in the AP groups treated with 300 mg/kg KYNA or SZR-72. Interestingly, pancreatic heat shock protein-72 expression was significantly increased by administration of SZR-72, KYNA and/or LO. mRNA and protein expression of NMDAR1 was detected in pancreatic tissue. LO treatment caused acinar cell toxicity which was reversed by 250 µM KYNA or SZR-72. Treatment of acini with NMDA (25, 250, 2000 µM) did not influence the effects of KYNA or SZR-72. Moreover, SZR-72 reduced LO-induced H2O2 production of neutrophil granulocytes. KYNA and SZR-72 have dose-dependent protective effects on LO-induced AP or acinar toxicity which seem to be independent of pancreatic NMDA receptors. Furthermore, SZR-72 treatment suppressed AP-induced activation of neutrophil granulocytes. This study suggests that administration of KYNA and its derivative could be beneficial in AP.

Mislocalization of CFTR expression in acute pancreatitis and the beneficial effect of VX-661 + VX-770 treatment on disease severity.

Cystic fibrosis transmembrane conductance regulator (CFTR) has an essential role in maintaining pancreatic ductal function. Impaired CFTR function can trigger acute pancreatitis (AP) and exacerbate disease severity. We aimed to investigate the localization and expression of CFTR during AP, and determined the effects of a CFTR corrector (VX-661) and potentiator (VX-770) on disease severity. AP was induced in FVB/n mice by 6-10 hourly intraperitoneal injections of 50 µg/kg cerulein. Some mice were pre-treated with five to six daily injections of 2 mg/kg VX-661 + VX-770. Control animals were administered physiological saline instead of cerulein and dimethyl sulfoxide instead of VX compounds. AP severity was determined by measuring laboratory and histological parameters; CFTR and CK19 expression was measured. Activity of ion transporters was followed by intracellular pH or fluid secretion measurement of isolated pancreatic intra-/interlobular ducts. Cerulein-induced AP severity was greatest between 12 and 24 h. CFTR mRNA expression was significantly increased 24 h after AP induction. Immunohistochemistry demonstrated disturbed staining morphology of CFTR and CK19 proteins in AP. Mislocalization of CFTR protein was observed from 6 h, while expression increased at 24 h compared to control. Ductal HCO3- transport activity was significantly increased 6 h after AP induction. AP mice pre-treatment with VX-661 + VX-770 significantly reduced the extent of tissue damage by about 20-30%, but other parameters were unchanged. Interestingly, VX-661 + VX-770 in vitro administration significantly increased the fluid secretion of ducts derived from AP animals. This study described the course of the CFTR expression and mislocalization in cerulein-induced AP. Our results suggest that the beneficial effects of CFTR correctors and potentiators should be further investigated in AP. KEY POINTS: Cystic fibrosis transmembrane conductance regulator (CFTR) is an important ion channel in epithelial cells. Its malfunction has several serious consequences, like developing or aggravating acute pancreatitis (AP). Here, the localization and expression of CFTR during cerulein-induced AP in mice were investigated and the effects of CFTR corrector (VX-661) and a potentiator (VX-770) on disease severity were determined. CFTR mRNA expression was significantly increased and mislocalization of CFTR protein was observed in AP compared to the control group. Interestingly, pre-treatment of AP mice with VX-661 + VX-770 significantly reduced the extent of pancreatic tissue damage by 20-30%. In vitro administration of VX-661 + VX-770 significantly increased the fluid secretion of ducts derived from AP animals. Based on these results, the utilization of CFTR correctors and potentiators should be further investigated in AP.

Endoplasmic Reticulum-Plasma Membrane Contact Sites as an Organizing Principle for Compartmentalized Calcium and cAMP Signaling.

In eukaryotic cells, ultimate specificity in activation and action-for example, by means of second messengers-of the myriad of signaling cascades is primordial. In fact, versatile and ubiquitous second messengers, such as calcium (Ca2+) and cyclic adenosine monophosphate (cAMP), regulate multiple-sometimes opposite-cellular functions in a specific spatiotemporal manner. Cells achieve this through segregation of the initiators and modulators to specific plasma membrane (PM) subdomains, such as lipid rafts and caveolae, as well as by dynamic close contacts between the endoplasmic reticulum (ER) membrane and other intracellular organelles, including the PM. Especially, these membrane contact sites (MCSs) are currently receiving a lot of attention as their large influence on cell signaling regulation and cell physiology is increasingly appreciated. Depletion of ER Ca2+ stores activates ER membrane STIM proteins, which activate PM-residing Orai and TRPC Ca2+ channels at ER-PM contact sites. Within the MCS, Ca2+ fluxes relay to cAMP signaling through highly interconnected networks. However, the precise mechanisms of MCS formation and the influence of their dynamic lipid environment on their functional maintenance are not completely understood. The current review aims to provide an overview of our current understanding and to identify open questions of the field.

Poly(ADP-Ribose) Polymerase 1 Promotes Inflammation and Fibrosis in a Mouse Model of Chronic Pancreatitis.

Chronic pancreatitis (CP) is an inflammatory disease of the pancreas characterized by ductal obstructions, tissue fibrosis, atrophy and exocrine and endocrine pancreatic insufficiency. However, our understanding is very limited concerning the disease's progression from a single acute inflammation, via recurrent acute pancreatitis (AP) and early CP, to the late stage CP. Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor enzyme activated mostly by oxidative DNA damage. As a co-activator of inflammatory transcription factors, PARP1 is a central mediator of the inflammatory response and it has also been implicated in acute pancreatitis. Here, we set out to investigate whether PARP1 contributed to the pathogenesis of CP. We found that the clinically used PARP inhibitor olaparib (OLA) had protective effects in a murine model of CP induced by multiple cerulein injections. OLA reduced pancreas atrophy and expression of the inflammatory mediators TNFα and interleukin-6 (IL-6), both in the pancreas and in the lungs. Moreover, there was significantly less fibrosis (Masson's trichrome staining) in the pancreatic sections of OLA-treated mice compared to the cerulein-only group. mRNA expression of the fibrosis markers TGFß, smooth muscle actin (SMA), and collagen-1 were markedly reduced by OLA. CP was also induced in PARP1 knockout (KO) mice and their wild-type (WT) counterparts. Inflammation and fibrosis markers showed lower expression in the KO compared to the WT mice. Moreover, reduced granulocyte infiltration (tissue myeloperoxidase activity) and a lower elevation of serum amylase and lipase activity could also be detected in the KO mice. Furthermore, primary acinar cells isolated from KO mice were also protected from cerulein-induced toxicity compared to WT cells. In summary, our data suggest that PARP inhibitors may be promising candidates for repurposing to treat not only acute but chronic pancreatitis as well.