PAR2 exerts local protection against acute pancreatitis via modulation of MAP kinase and MAP kinase phosphatase signaling.

During acute pancreatitis, protease-activated receptor 2 (PAR2) can be activated by interstitially released trypsin. In the mild form of pancreatitis, PAR2 activation exerts local protection against intrapancreatic damage, whereas, in the severe form of pancreatitis, PAR2 activation mediates some systemic complications. This study aimed to identify the molecular mechanisms of PAR2-mediated protective effects against intrapancreatic damage. A mild form of acute pancreatitis was induced by an intraperitoneal injection of caerulein (40 microg/kg) in rats. Effects of PAR2 activation on intrapancreatic damage and on mitogen-activated protein (MAP) kinase signaling were assessed. Caerulein treatment activated extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK) within 15 min and maintained phosphorylation of ERK and JNK for 2 h in the rat pancreas. Although PAR2 activation by the pretreatment with PAR2-activating peptide (AP) itself increased ERK phosphorylation in rat pancreas, the same treatment remarkably decreased caerulein-induced activation of ERK and JNK principally by accelerating their dephosphorylation. Inhibition of ERK and JNK phosphorylation by the pretreatment with MAP/ERK kinase (MEK) or JNK inhibitors decreased caerulein-induced pancreatic damage that was similar to the effect induced by PAR2-AP. Notably, in caerulein-treated rats, PAR2-AP cotreatment highly increased the expression of a group of MAP kinase phosphatases (MKPs) that deactivate ERK and JNK. The above results imply that downregulation of MAP kinase signaling by MKP induction is a key mechanism involved in the protective effects of PAR2 activation on caerulein-induced intrapancreatic damageope

NHERF4 PDZ 연결단백에 의한 SLC26A3 이온수송 기능 조절

Dept. of Medical Science/박사[한글] [영문]The solute linked carrier (SLC) 26A3, also known as the downregulated in adenomas (DRA), functions as a Cl-/HCO3- exchanger and is expressed at the apical membrane of secretory epithelial cells in the intestines, pancreas and salivary glands. In the exploratory yeast two-hybrid assay, the sodium/proton exchanger regulatory factor (NHERF) 4, a PDZ-containing scaffold protein also known as the intestinal and kidney-enriched PDZ protein (IKEPP), was found to interact with the SLC26A3. In this study, the functional role of interaction between NHERF4 and SLC26A3 was investigated using an integrated molecular physiological approach. Immunoprecipitation with the C-terminus-deleted SLC26A3 mutant revealed that the C-terminal PDZ binding motif of SLC26A3 was required for the SLC26A3-NHERF4 interaction. In addition, a direct protein-protein interaction between the C-terminus of SLC26A3 and the third PDZ domain of NHERF4 (NHERF4-PDZ3) was observed in the GST-based pull-down assay. Of note, co-expression of NHERF4 decreased the surface expression of SLC26A3 by accelerating endocytosis and consequently reduced the SLC26A3-mediated Cl-/HCO3- exchange activities. In contrast, knockdown of the NHERF4 expression by treatment with small interfering RNAs increased the DIDS-insensitive Cl-/HCO3- exchange activities in the HT-29 human colonic epithelial cells. Interestingly, modulation of phosphorylation at the NHERF4-PDZ3 altered the intensity of interaction between NHERF4 and SLC26A3. These results imply that NHERF4 is a physiological regulator of SLC26A3 by affecting its surface expression and that phosphorylation of NHERF4-PDZ3 may be one of the important regulatory factors to control the SLC26A3-NHERF4 interaction.ope

한국의 정보보호 활동과 시사점(Information security activities in Korea and implications)

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Effects of KR-33028, a novel Na+/H+ exchanger-1 inhibitor, on glutamate-induced neuronal cell death and ischemia-induced cerebral infarct

We investigated the effects of a novel Na(+)/H(+) exchanger-1 (NHE-1) inhibitor KR-33028 on glutamate excitotoxicity in cultured neuron cells in vitro and cerebral infarct in vivo by comparing its potency with that of zoniporide, a well-known, highly potent NHE-1 inhibitor. KR-33028 inhibited NHE-1 activation in a concentration-dependent manner (IC(50)=2.2 nM), with 18-fold greater potency than that of zoniporide (IC(50)=40.7 nM). KR-33028 significantly attenuated glutamate-induced LDH release with approximately 100 times lower EC(25) than that of zoniporide in cortical neurons in vitro (EC(25) of 0.007 and 0.81 microM, respectively), suggesting its 100-fold greater potency than zoniporide in producing anti-necrotic effect. in addition, the EC(50) of KR-33028 for anti-apoptotic effect was 100 times lower than that of zoniporide shown by TUNEL positivity (0.005 and 0.62 microM, respectively) and caspase-3 activity (0.01 and 2.64 microM, respectively). Furthermore, the EC(50) value of KR-33028 against glutamate-induced intracellular Ca(2+) overload was also 100 times lower than that of zoniporide (EC(50) of 0.004 and 0.65 microM, respectively). in the in vivo cerebral infarct model (60 min middle cerebral artery occlusion followed by 24 h reperfusion), KR-33028 reduced infarct size in a dose-dependent manner. Its ED(25) value, however, was quite similar to that of zoniporide (ED(25) of 0.072 and 0.097 mg/kg, respectively). Hence these results suggest that the novel NHE-1 inhibitor, KR-33028, could be an efficient therapeutic tool to protect neuronal cells against ischemic injury.ope

Heterogeneity in the processing defect of SLC26A4 mutants.

BACKGROUND: Mutations in the SLC26A4 gene are responsible for Pendred syndrome and non-syndromic hearing loss (DFNB4). This study analysed non-synonymous SLC26A4 mutations newly identified in East Asians, as well as three common mutations in Caucasians, to characterise their molecular pathogenic mechanisms and to explore the possibility of rescuing their processing defects. METHODS: A total of 11 non-synonymous disease associated mutations were generated and their effects on protein processing and on ion transporting activities were examined. RESULTS: Most of the mutations caused retention of the SLC26A4 gene product (pendrin) in the intracellular region, while wild-type pendrin reached the plasma membrane. Accordingly, these mutations abolished complex glycosylation and Cl(-)/HCO(3)(-) exchange activities of pendrin. However, significant heterogeneity in the processing of mutant pendrin molecules was observed. Each mutant protein exhibited a different cellular localisation, a different degree of N-glycosylation, and a different degree of sensitivity to the treatments that rescue processing defects. For example, H723R-pendrin, the most common mutation in East Asians, was mostly expressed in endoplasmic reticulum (ER), and its defects in protein processing and ion transporting activities were restored considerably by low temperature incubation. On the other hand, L236P-pendrin, the most common mutation in Caucasians, was mainly in the centrosomal region and was temperature insensitive. CONCLUSION: These results indicate that the processing of pendrin mutant protein is determined by mutant specific mechanisms, and that a mutant specific method would be required to rescue the conformational defects of each folding mutantope

Dynamic regulation of CFTR bicarbonate permeability by [Cl-]i and its role in pancreatic bicarbonate secretion.

BACKGROUND & AIMS: Pancreatic bicarbonate (HCO3-) secretion is important for a healthy pancreas as well as digestive physiology. However, how human pancreatic duct cells secrete copious amounts of HCO3- has long been a puzzle. Here, we report that a dynamic increase in the cystic fibrosis transmembrane conductance regulator (CFTR) HCO3- permeability by intracellular Cl- concentration ([Cl-]i)-sensitive mechanisms plays a pivotal role in pancreatic HCO3- secretion. METHODS: The role of [Cl-]i-sensitive kinases in CFTR-mediated HCO3- transport was examined in heterologous expression systems, PANC1 human pancreatic duct cells, and human and guinea pig pancreatic tissues using an integrated molecular and physiologic approach. RESULTS: In human pancreatic tissues, CFTR-positive duct cells abundantly expressed with-no-lysine (WNK1) kinase, oxidative stress-responsive kinase 1 (OSR1), and sterile 20/SPS1-related proline/alanine-rich kinase (SPAK), which are known to be activated by low [Cl-]i. Interestingly, CFTR activation rapidly decreased [Cl-]i in response to luminal Cl- depletion in polarized PANC1 human pancreatic duct cells. Notably, the WNK1-mediated OSR1 and SPAK activation by low [Cl-]i strongly increased CFTR HCO3- permeability in CFTR-transfected HEK 293T, PANC1, and guinea pig pancreatic duct cells, making CFTR primarily an HCO3- channel, which is essential for the secretion of pancreatic juice containing HCO3- at a concentration greater than 140 mmol/L. In contrast, OSR1 and SPAK activation inhibited CFTR-dependent Cl-/HCO3- exchange activity that may reabsorb HCO3- from the high HCO3--containing pancreatic juice. CONCLUSIONS: These results indicate that the [Cl-]i-sensitive activation of the WNK1-OSR1/SPAK pathway is the molecular switch to generate HCO3--rich fluid in the human pancreatic duct.ope