Biomedical subjects

John A Williams

Publications and source records attributed to John A Williams.

At least 19 recordsLinked to original sources

PubMed2006

Induction of early response genes in trypsin inhibitor-induced pancreatic growth.

Endogenous CCK release induced by a synthetic trypsin inhibitor, camostat, stimulates pancreatic growth; however, the mechanisms mediating this growth are not well established. Early response genes often couple short-term signals with long-term responses. To study their participation in the pancreatic growth response, mice were fasted for 18 h and refed chow containing 0.1% camostat for 1-24 h. Expression of 18 early response genes were evaluated by quantitative PCR; mRNA for 17 of the 18 increased at 1, 2, 4, or 8 h. Protein expression for c-jun, c-fos, ATF-3, Egr-1, and JunB peaked at 2 h. Nuclear localization was confirmed by immunohistochemistry of c-fos, c-jun, and Egr-1. Refeeding regular chow induced only a small increase of c-jun and none in c-fos expression. JNKs and ERKs were activated 1 h after camostat feeding as was the phosphorylation of c-jun and ATF-2. AP-1 DNA binding evaluated by EMSA showed a significant increase 1-2 h after camostat feeding with participation of c-jun, c-fos, ATF-2, ATF-3, and JunB shown by supershift. The CCK antagonist IQM-95,333 blocked camostat feeding-induced c-jun and c-fos expression by 67 and 84%, respectively, and AP-1 DNA binding was also inhibited. In CCK-deficient mice, the maximal response of c-jun induction and AP-1 DNA binding were reduced by 64 and 70%, respectively. These results indicate that camostat feeding induces a spectrum of early response gene expression and AP-1 DNA binding and that these effects are mainly CCK dependent.

Activating Transcription Factor 2↗

PubMed2006

Activation of the mTOR signalling pathway is required for pancreatic growth in protease-inhibitor-fed mice.

Cholecystokinin (CCK)-induced pancreatic growth in mice involves parallel increases in DNA and protein. The mammalian target of rapamycin (mTOR) signalling pathway regulates mRNA translation and its activation is implicated in growth of various tissues. The aim of this study was to elucidate whether mTOR activation is required for pancreatic growth in a mouse model of increased endogenous CCK release. In mice fed chow containing the synthetic protease inhibitor camostat, protein synthetic rates and phosphorylation of two downstream targets of mTOR, eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and the ribosomal protein S6 (S6), increased in comparison with fasted controls. The camostat-induced increases in protein synthesis and 4E-BP1 and S6 phosphorylation were almost totally abolished by administration of the mTOR inhibitor rapamycin 1 h prior to camostat feeding. In contrast, the phosphorylation of ERK1/2 and JNK and the expression of the early response genes c-jun, c-fos, ATF3 and egr-1 induced by camostat feeding were not affected by rapamycin. In mice fed camostat for 7 days, the ratio of pancreatic to body weight increased by 143%, but when rapamycin was administered daily this was reduced to a 22% increase. Changes in pancreatic mass were paralleled by protein and DNA content following camostat feeding and rapamycin administration. Moreover, while BrdU incorporation, an indicator of DNA synthesis, was increased to 448% of control values after 2 days of camostat feeding, rapamycin administration completely inhibited this increase. We conclude that the mTOR signalling pathway is required for CCK-induced cell division and pancreatic growth.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Early activation of endoplasmic reticulum stress is associated with arginine-induced acute pancreatitis.

Endoplasmic reticulum (ER) stress mechanisms have been found to play critical roles in a number of diseases states, such as diabetes mellitus and Alzheimer disease, but whether they are involved in acute pancreatitis is unknown. Here we show for the first time that all major ER stress sensing and signaling mechanisms are present in exocrine acini and are activated early in the arginine model of experimental acute pancreatitis. Pancreatitis was induced in rats by intraperitoneal injection of 4.0 g/kg body wt arginine. Pancreatitis severity was assessed by analysis of serum amylase, pancreatic trypsin activity, water content, and histology. ER stress-related molecules PERK, eIF2alpha, ATF6, XBP-1, BiP, CHOP, and caspase-12 were analyzed. Arginine treatment induced rapid and severe pancreatitis, as indicated by increased serum amylase, pancreatic tissue edema, and acinar cell damage within 4 h. Arginine treatment also caused an early activation of ER stress, as indicated by phosphorylation of PERK and its downstream target eIF2alpha, ATF6 translocation into the nucleus (within 1 h), and upregulation of BiP (within 4 h). XBP-1 splicing and CHOP expression were observed within 8 h. After 24 h, increased activation of the ER stress-related proapoptotic molecule caspase-12 was observed along with an increase in caspase-3 activity and TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labeling (TUNEL) staining in exocrine acini. These results indicate that ER stress is an important early acinar cell event that likely contributes to the development of acute pancreatitis in the arginine model.

Animals↗

PubMed2006

Leucine activates pancreatic translational machinery in rats and mice through mTOR independently of CCK and insulin.

Feeding stimulates pancreatic digestive enzyme synthesis at the translational level, and this is thought to be mediated by hormones and neurotransmitters. However, BCAAs, particularly leucine, stimulate protein synthesis in several tissues. We investigated whether BCAA stimulated the translational machinery in murine pancreas and whether their effects were independent of hormones. Rats and mice were administered (i.g. gavage) individual BCAA at 1.35 mg/g (body weight) and rat isolated pancreatic acini were incubated with BCAA under different conditions. Activation of translation initiation factors and total protein synthesis were analyzed. BCAA gavage stimulated the phosphorylation of the initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) and the ribosomal protein S6 kinase (S6K), with leucine being the most effective. Leucine also increased the association of the initiation factors eIF4E and eIF4G, but did not affect the activity of the guanine nucleotide exchange factor eIF2B, nor total protein synthesis. BCAA acted independently of insulin signaling on isolated pancreatic acini from diabetic rats. The ability of leucine to promote phosphorylation of 4E-BP1 and S6K as well as enhance the assembly of the eIF4F complex was unimpaired in CCK-deficient mice. Finally, rapamycin (0.75 mg/kg) administered to rats 2 h before leucine gavage inhibited the phosphorylation of S6 and 4E-BP1 induced by leucine. We conclude that leucine may participate, as a signal as well as a substrate, in activating the translational machinery in pancreatic acinar cells independently of hormonal effects and that this action is through the mTOR pathway.

Amino Acids, Branched-Chain↗

PubMed2006

Regulation of pancreatic acinar cell function.

PURPOSE OF REVIEW: Recent investigations into the regulation of pancreatic acinar cell function have led to a more detailed understanding of the mechanisms regulating digestive enzyme synthesis and secretion. This review identifies and puts into context those articles which further our understanding in this area. RECENT FINDINGS: The secretagogue receptors present on acinar cells, especially muscarinic and cholecystokinin, have been better identified and characterized. The complex control of intracellular Ca by intracellular messengers such as inositol trisphosphate, cellular ion pumps and membrane channels has become more clearly understood, including the identification of organelles sequestering intracellular Ca. In the area of Ca driven exocytosis, progress has been made in understanding the proteins present on the zymogen granules, especially Rabs and SNARE proteins, and the dynamic changes in actin filaments. Secretagogues have also been shown to enhance the translation of new protein by activation of the mammalian target of rapamycin pathway. Finally, considerable progress has been made in understanding the mechanisms regulating pancreatic growth in response to nutrients and following pancreatectomy or pancreatitis. SUMMARY: Understanding the mechanisms that regulate pancreatic acinar cell function is contributing to our knowledge of normal pancreatic function and alterations in diseases such as pancreatitis and pancreatic cancer.

Animals↗

PubMed2006

Calcineurin-dependent and calcineurin-independent signal transduction pathways activated as part of pancreatic growth.

OBJECTIVE: We have recently reported that pancreatic growth driven by cholecystokinin released endogenously by feeding the synthetic trypsin inhibitor camostat requires the Ca-activated phosphatase calcineurin. In the present study, we evaluated a number of signal transduction pathways for their activation as part of the growth response and whether their activation was dependent on calcineurin. METHODS: Male ICR mice were fed with either chow or chow plus 1 mg/g of camostat. FK506 was administered at 3 mg/kg. After various times from 12 hours to 10 days, pancreatic samples were prepared and assayed for activity of various signal transduction pathway components. RESULTS: Camostat feeding increased the activation of extracellular signal-regulated kinases, c-Jun NH2-terminal kinases, and phosphorylation of the translation factor eukaryotic initiation factor 4E and activated the mammalian target of rapamycin pathway that leads to phosphorylation of the ribosomal protein S6 and of the eukaryotic initiation factor 4E binding protein but with different time courses. Treatment of mice with the calcineurin inhibitor FK506 totally blocked c-Jun NH2-terminal kinase activation, partially blocked the mammalian target of rapamycin pathway, and had no effect on extracellular signal-regulated kinase activation or the phosphorylation of eukaryotic initiation factor 4E. CONCLUSIONS: The pancreatic growth response is accompanied by activation of a number of signaling pathways regulating transcription and translation, some of which are dependent on and some independent of calcineurin.

Animals↗

PubMed2005

Organellar proteomics: analysis of pancreatic zymogen granule membranes.

The zymogen granule (ZG) is the specialized organelle in pancreatic acinar cells for digestive enzyme storage and regulated secretion and has been a model for studying secretory granule functions. In an initial effort to comprehensively understand the functions of this organelle, we conducted a proteomic study to identify proteins from highly purified ZG membranes. By combining two-dimensional gel electrophoresis and two-dimensional LC with tandem mass spectrometry, 101 proteins were identified from purified ZG membranes including 28 known ZG proteins and 73 previously unknown proteins, including SNAP29, Rab27B, Rab11A, Rab6, Rap1, and myosin Vc. Moreover several hypothetical proteins were identified that represent potential novel proteins. The ZG localization of nine of these proteins was further confirmed by immunocytochemistry. To distinguish intrinsic membrane proteins from soluble and peripheral membrane proteins, a quantitative proteomic strategy was used to measure the enrichment of intrinsic membrane proteins through the purification process. The iTRAQ ratios correlated well with known or Transmembrane Hidden Markov Model-predicted soluble or membrane proteins. By combining subcellular fractionation with high resolution separation and comprehensive identification of proteins, we have begun to elucidate zymogen granule functions through proteomic and subsequent functional analysis of its membrane components.

Animals↗

PubMed2005

Rho and Rac promote acinar morphological changes, actin reorganization, and amylase secretion.

Supramaximal stimulation of isolated pancreatic acini with specific agonists such as CCK induces the formation of large basolateral blebs, redistributes filamentous actin, and inhibits secretion. Rho family small G proteins are well documented for their function in actin reorganization that determines cell shape and have been suggested to play a role in secretion. Here, we determined whether Rho and Rac are involved in the morphological changes, actin redistribution, and inhibition of amylase secretion induced by high concentrations of CCK. Introduction of constitutively active RhoV14 and RacV12 but not Cdc42V12 in mouse pancreatic acini by adenoviral vectors stimulated acinar morphological changes including basolateral protrusions, increased the total amount of F-actin, and reorganized the actin cytoskeleton. Dominant-negative RhoN19, Clostridium botulinum C3 exotoxin, which inhibits Rho, and dominant-negative RacN17 all partially blocked CCK-induced acinar morphological changes and actin redistribution. To study the correlation between actin polymerization and acinar shape changes, two marine toxins were employed. Jasplakinolide, a reagent that facilitates actin polymerization and stabilizes F-actin, stimulated acinar basolateral protrusions, whereas latrunculin, which sequesters actin monomers, blocked CCK-induced acinar blebbing. Unexpectedly, RhoV14, RacV12, and jasplakinolide all increased amylase secretion by CCK from 30 pM to 10 nM. The data suggest that Rho and Rac are involved in CCK-evoked changes in acinar morphology, actin redistribution, and secretion and that inhibition of secretion by high concentrations of CCK is not directly coupled to the changes in acinar morphology.

Actins↗

PubMed2005

A role for Rho and Rac in secretagogue-induced amylase release by pancreatic acini.

The actin cytoskeleton has long been implicated in protein secretion. We investigated whether Rho and Rac, known regulators of the cytoskeleton, are involved in amylase secretion by mouse pancreatic acini. Secretagogues, including cholecystokinin (CCK) and the acetylcholine analog carbachol, increased the amount of GTP-bound RhoA and Rac1 and induced translocation from cytosol to a membrane fraction. Immunocytochemistry revealed the translocation of Rho and Rac within the apical region of the cell. Expression by means of adenoviral vectors of dominant-negative Rho (RhoN19), dominant-negative Rac (RacN17), and Clostridium Botulinum C3 exotoxin, which ADP ribosylates and inactivates Rho, significantly inhibited amylase secretion by CCK and carbachol; inhibiting both Rho and Rac resulted in a greater reduction. This inhibitory effect of RhoN19 on CCK-induced amylase secretion was apparent in both the early and late phases of secretion, whereas RacN17 was more potent on the late phase of secretion. None of these three affected the basal Ca2+ or the peak intracellular Ca2+ concentration stimulated by CCK. Latrunculin, a marine toxin that sequesters actin monomers, time-dependently decreased the total amount of filamentous actin (F-actin) and dose-dependently decreased secretion by secretagogues without affecting Ca2+ signaling. These data suggest that Rho and Rac are both involved in CCK-induced amylase release in pancreatic acinar cell possibly through an effect on the actin cytoskeleton.

ADP Ribose Transferases↗

PubMed2004

Rab27b localizes to zymogen granules and regulates pancreatic acinar exocytosis.

To understand the function of pancreatic zymogen granules, we performed a proteomics analysis to identify ZG membrane components. Here we report the identification of Rab27b through this proteomics study and validate its role in granule function. MALDI-MS peptide mass fingerprint was matched to rat Rab27b with 43% sequence coverage, and the identification was also confirmed by tandem mass spectrometry. The localization of Rab27b on ZGs was confirmed by Western blotting and immunocytochemistry. To examine the function of Rab27b in acinar secretion, we overexpressed wild type and mutant Rab27b protein in pancreatic acini using recombinant adenoviruses. Wild type Rab27b had no effect on amylase secretion, while Rab27b Q78L enhanced, and Rab27b N133I inhibited, CCK-induced amylase release by 92+/-13% and 53+/-8%, respectively. This enhancement and inhibition occurred at all points on the CCK dose-response curve and over a 30min time course. These results demonstrate that Rab27b is present on ZGs and plays an important role in regulating acinar exocytosis.

Amino Acid Sequence↗

PubMed2004

Regulation of CCK-induced amylase release by PKC-delta in rat pancreatic acinar cells.

PKC is known to be activated by pancreatic secretagogues such as CCK and carbachol and to participate along with calcium in amylase release. Four PKC isoforms, alpha, delta, epsilon, and zeta, have been identified in acinar cells, but which isoforms participate in amylase release are unknown. To identify the responsible isoforms, we used translocation assays, chemical inhibitors, and overexpression of individual isoforms and their dominant-negative variants by means of adenoviral vectors. CCK stimulation caused translocation of PKC-alpha, -delta, and -epsilon, but not -zeta from soluble to membrane fraction. CCK-induced amylase release was inhibited approximately 30% by GF109203X, a broad spectrum PKC inhibitor, and by rottlerin, a PKC-delta inhibitor, but not by Gö6976, a PKC-alpha inhibitor, at concentrations from 1 to 5 microM. Neither overexpression of wild-type or dominant-negative PKC-alpha affected CCK-induced amylase release. Overexpression of PKC-delta and -epsilon enhanced amylase release, whereas only dominant-negative PKC-delta inhibited amylase release by 25%. PKC-delta overexpression increased amylase release at all concentrations of CCK, but dominant-negative PKC-delta only inhibited the maximal concentration; both similarly affected carbachol and JMV-180-induced amylase release. Overexpression of both PKC-delta and its dominant-negative variant affected the late but not the early phase of amylase release. GF109203X totally blocked the enhancement of amylase release by PKC-delta but had no further effect in the presence of dominant-negative PKC-delta. These results indicate that PKC-delta is the PKC isoform involved with amylase secretion.

Acetophenones↗

PubMed2004

Regulation of translation elongation and phosphorylation of eEF2 in rat pancreatic acini.

While pancreatic protein synthesis and the initiation of translation are regulated by hormones and neurotransmiters, whether the elongation process is also regulated is unknown. Stimulatory doses of cholecystokinin (CCK) (100 pM), bombesin (10 nM), and carbachol (10 microM) increased elongation rates (measured as ribosomal half-transit time) in pancreatic acini in vitro. At the same time these secretagogues reduced elongation factor 2 (eEF2) phosphorylation, the main factor known to regulate elongation, and increased the phosphorylation of the eEF2 kinase. The mTOR inhibitor rapamycin reversed the dephosphorylation of eEF2 induced by CCK, as did treatment with the p38 MAPK inhibitor SB202190, the MEK inhibitor PD98059, and the phosphatase inhibitor calyculin A. Neither rapamycin, SB202190, PD98059 nor calyculin A had an effect on CCK mediated eEF2 kinase phosphorylation. Translation elongation in pancreatic acinar cells is likely regulated by eEF2 through the mTOR, p38, and MEK pathways, and modulated through PP2A.

Animals↗

PubMed2004

Feeding activates protein synthesis in mouse pancreas at the translational level without increase in mRNA.

To determine the mechanism of meal-regulated synthesis of pancreatic digestive enzymes, we studied the effect of fasting and refeeding on pancreatic protein synthesis, relative mRNA levels of digestive enzymes, and activation of the translational machinery. With the use of the flooding dose technique with L-[3H]phenylalanine, morning protein synthesis in the pancreas of Institute for Cancer Research mice fed ad libitum was 7.9 +/- 0.3 nmol phenylalanine.10 min(-1).mg protein(-1). Prior fasting for 18 h reduced total protein synthesis to 70 +/- 1.4% of this value. Refeeding for 2 h, during which the mice consumed 29% of their daily food intake, increased protein synthesis to 117.3 +/- 4.9% of the control level. Pancreatic mRNA levels of amylase, lipases, trypsins, chymotrypsin, elastases, as well as those for several housekeeping genes tested were not significantly changed after refeeding compared with fasted mice. By contrast, the major translational control pathway involving Akt, mTOR, and S6K was strongly regulated by fasting and refeeding. Fasting for 18 h decreased phosphorylation of ribosomal protein S6 to almost undetectable levels, and refeeding highly increased it. The most highly phosphorylated form of the eIF4E binding protein (4E-BP1) made up the 14.6% of total 4E-BP1 in normally fed animals, was only 2.8% after fasting, and was increased to 21.4% after refeeding. This was correlated with an increase in the formation of the eIF4E-eIF4G complex after refeeding. By contrast, feeding did not affect eIF2B activity. Thus food intake stimulates pancreatic protein synthesis and translational effectors without increasing digestive enzyme mRNA levels.

Animals↗

PubMed2004

Calcineurin is required for translational control of protein synthesis in rat pancreatic acini.

CCK increases the rate of net protein synthesis in rat pancreatic acini by activating initiation and elongation factors required for translation. The immunosuppressant FK506 inhibits the Ca(2+)-calmodulin-dependent phosphatase calcineurin in pancreatic acinar cells and blocks pancreatic growth induced by chronic CCK treatment. To test a requirement for calcineurin in the activation of the translational machinery stimulated by CCK, we evaluated the effects of FK506 on protein synthesis and on regulatory initiation and elongation factors in rat pancreatic acini in vitro. CCK acutely increased protein synthesis in acini from normal rats with a maximum increase at 100 pM CCK to 170 +/- 11% of control. The immunosuppressant FK506 dose-dependently inhibited CCK-stimulated protein synthesis over the same concentration range that blocked calcineurin activity, as assessed by dephosphorylation of the calcineurin substrate calcium-regulated heat-stable protein of 24 kDa. Another immunosuppressant, cyclosporin A, inhibited protein synthesis, but its effects appeared more complex. FK506 also inhibited protein synthesis stimulated by bombesin and carbachol. FK506 did not significantly affect the activity of the initiation factor-2B, or the phosphorylation of the initiation factor-2alpha, ribosomal protein protein S6, or the mRNA cap binding protein eukaryotic initiation factor (eIF) 4E. Instead, blockade of calcineurin with FK506 reduced the phosphorylation of the eIF4E binding protein, reduced the formation of the eIF4F complex, and increased the phosphorylation of eukaryotic elongation factor 2. From these results, we conclude that calcineurin activity is required for protein synthesis, and this action may be related to an effect on the formation of the mRNA cap binding complex and the elongation processes.

Animals↗

PubMed2004

Endothelial nitric oxide synthase is protective in the initiation of caerulein-induced acute pancreatitis in mice.

The effect of inhibiting nitric oxide (NO) synthase (NOS) or enhancing NO on the course of acute pancreatitis (AP) is controversial, in part because three NOS isoforms exist: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). We investigated whether inhibition or selective gene deletion of NOS isoforms modified the initiation phase of caerulein-induced AP in mice and explored whether this affected pancreatic microvascular blood flow (PMBF). We investigated the effects of nonspecific NOS inhibition with N(omega)-nitro-l-arginine (l-NNA; 10 mg/kg ip) or targeted deletion of eNOS, nNOS, or iNOS genes on the initiation phase of caerulein-induced AP in mice using in vivo and in vitro models. Western blot analysis was performed to assess eNOS phosphorylation status, an indicator of enzyme activity, and microsphere studies were used to measure PMBF. l-NNA and eNOS deletion, but not nNOS or iNOS deletion, increased pancreatic trypsin activity and serum lipase during the initiation phase of in vivo caerulein-induced AP. l-NNA and eNOS did not affect trypsin activity in caerulein-hyperstimulated isolated acini, suggesting that nonacinar events mediate the effect of NOS blockade in vivo. The initiation phase of AP in wild-type mice was associated with eNOS Thr(495) residue dephosphorylation, which accompanies eNOS activation, and a 178% increase in PMBF; these effects were absent in eNOS-deleted mice. Thus eNOS is the main isoform influencing the initiation of caerulein-induced AP. eNOS-derived NO exerts a protective effect through actions on nonacinar cell types, most likely endothelial cells, to produce greater PMBF.

Acute Disease↗

PubMed2004

Raising high-density lipoprotein in humans through inhibition of cholesteryl ester transfer protein: an initial multidose study of torcetrapib.

OBJECTIVE: The ability of the potent cholesteryl ester transfer protein (CETP) inhibitor torcetrapib (CP-529,414) to raise high-density lipoprotein cholesterol (HDL-C) levels in healthy young subjects was tested in this initial phase 1 multidose study. METHODS AND RESULTS: Five groups of 8 subjects each were randomized to placebo (n=2) or torcetrapib (n=6) at 10, 30, 60, and 120 mg daily and 120 mg twice daily for 14 days. Torcetrapib was well tolerated, with all treated subjects completing the study. The correlation of plasma drug levels with inhibition (EC50=43 nM) was as expected based on in vitro potency (IC50 approximately 50 nM), and increases in CETP mass were consistent with the proposed mechanism of inhibition. CETP inhibition increased with escalating dose, leading to elevations of HDL-C of 16% to 91%. Total plasma cholesterol did not change significantly because of a reduction in nonHDL-C, including a 21% to 42% lowering of low-density lipoprotein cholesterol at the higher doses. Apolipoprotein A-I and E were elevated 27% and 66%, respectively, and apoB was reduced 26% with 120 mg twice daily. Cholesteryl ester content decreased and triglyceride increased in the nonHDL plasma fraction, with contrasting changes occurring in HDL. CONCLUSIONS: These effects of CETP inhibition resemble those observed in partial CETP deficiency. This work serves as a prelude to further studies in subjects with low HDL, or combinations of dyslipidemia, in assessing the role of CETP in atherosclerosis.

Adolescent↗

PubMed2004

Overexpression of heat shock protein Hsp27 protects against cerulein-induced pancreatitis.

BACKGROUND & AIMS: Heat shock protein (Hsp) 27 regulates actin cytoskeletal dynamics, and overexpression of Hsp27 in fibroblasts protects against stress in a phosphorylation-dependent manner. Induction of Hsps occurs in acute pancreatitis, but Hsp27 has not been ascribed a specific role. To examine whether Hsp27 would ameliorate acute pancreatitis, we generated transgenic mice overexpressing human Hsp27 (huHsp27) or Hsp27 with the phosphorylatable residues Ser(15,78,82) mutated to aspartic acid (huHsp27-3D) to mimic phosphorylation or to alanine (huHsp27-3A), which is nonphosphorylatable. METHODS: huHsp27 was expressed at high levels in the exocrine pancreas by use of a cytomegalovirus promoter. Protein expression was analyzed by Western blotting and immunofluorescence. Acute pancreatitis was induced with 6 or 12 hourly cerulein injections (50 microg/kg intraperitoneally) and its severity assessed by measuring serum amylase and lipase levels, pancreatic trypsin activity, edema, and morphologic changes by quantitative scoring of multiple histologic sections and visualization of filamentous actin. Systemic inflammatory effects were monitored by measuring lung myeloperoxidase activity (a marker of neutrophil infiltration). RESULTS: huHsp27 protein was overexpressed in the pancreas and localized to pancreatic acini. Acute pancreatitis was ameliorated by overexpression of huHsp27 and the huHsp27-3D mutant, which were associated with suppression of pancreatic trypsin activity and acinar cell injury and preservation of the actin cytoskeleton. In contrast, these changes were unaffected by overexpression of the nonphosphorylatable huHsp27-3A mutant. CONCLUSIONS: Pancreatic overexpression of huHsp27 protects against cerulein-induced acute pancreatitis in a specific phosphorylation-dependent manner and is associated with preservation of the actin cytoskeleton.

Actins↗

PubMed2004

Receptor biology and signal transduction in pancreatic acinar cells.

PURPOSE OF REVIEW: Secretagogue receptors and their intracellular signaling pathways regulate pancreatic physiology and may be altered in pathophysiology. Therefore, understanding of the continued progress into their nature and function is relevant to both biology and disease. RECENT FINDINGS: The major secretagogue receptors on acinar cells include those binding cholecystokinin and acetylcholine, whereas secretin receptors regulate duct cells. Two physical models of the cholecystokinin receptor and ligand binding have been proposed through extensive structure-activity studies. Receptor oligomerization has been described for both cholecystokinin and secretin receptors. Ca plays a central role in the control of digestive enzyme secretion and is largely mobilized from intracellular stores. Inositol trisphosphate has been joined by two other Ca-releasing messengers, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate, in initiating and coordinating Ca signaling. Progress has also been made in determining the roles of specific organelles in Ca release. Ca triggers secretion, and knowledge of the function and regulation of the proteins involved in exocytosis is accumulating. Continuing advances have also been made in understanding the signaling pathways regulating protein synthesis and growth in adult pancreas. The protein kinase mammalian target of rapamycin and its downstream targets play a central role in protein synthesis, whereas the protein phosphatase calcineurin was recently reported to regulate pancreatic growth. Other signaling molecules include the MAP kinases, PKCs, cytoplasmic tyrosine kinases, and nitric oxide. SUMMARY: The current findings reviewed here are illuminating the structure and function of receptors on pancreatic acinar and duct cells and the multiple intracellular signaling pathways that they initiate. Understanding of these mechanisms is contributing to knowledge of normal pancreatic functions and alterations in disease such as pancreatitis and pancreatic cancer.

Journal Article↗