Thomas R. Kolodecik

Reducing Extracellular pH Sensitizes the Acinar Cell to Secretagogue-Induced Pancreatitis Responses in Rats

BACKGROUND & AIMS Protease activation within the pancreatic acinar cell is a key early event in acute pancreatitis and may require low pH intracellular compartments. Clinical studies suggest that acidosis may affect the risk for developing pancreatitis. We hypothesized that exposure to an acid load might sensitize the acinar cell to secretagogue-induced pancreatitis. METHODS Secretagogues (cerulein, carbachol, and bombesin) can induce protease activation in acinar cells at high (100 nmol/L, 1 mmol/L, and 10 micromol/L, respectively) but not at physiologically relevant concentrations. The effects of decreasing extracellular pH (pHe) in early secretagogue-induced pancreatitis (zymogen activation and injury) were examined in rats (1) in vitro with isolated acini and (2) in vivo with an acid challenge. RESULTS In acini, lowering pHe from 7.6 to 6.8 enhanced secretagogue-induced zymogen activation and injury, but did not affect secretion. For cerulein, this sensitization was seen over a range of concentrations (0.01-100.00 nmol/L). However, reduced pHe alone had no effect on zymogen activation, amylase secretion, or cell injury. We have reported that zymogen activation is mediated by the vacuolar ATPase (vATPase), a proton transporter. vATPase inhibition, using concanamycin (100 nmol/L), blocked the low pHe effects on zymogen activation. An acute acid load given in vivo enhanced cerulein-induced (50 microg/kg) trypsinogen activation and pancreatic edema. CONCLUSION These studies suggest that acid challenge sensitizes the pancreatic acinar cell to secretagogue-induced zymogen activation and injury and may increase the risk for the development and severity of acute pancreatitis.

Risk factors for pancreatic cancer: underlying mechanisms and potential targets

Purpose of the review: Pancreatic cancer is extremely aggressive, forming highly chemo-resistant tumors, and has one of the worst prognoses. The evolution of this cancer is multi-factorial. Repeated acute pancreatic injury and inflammation are important contributing factors in the development of pancreatic cancer. This article attempts to understand the common pathways linking pancreatitis to pancreatic cancer. Recent findings: Intracellular activation of both pancreatic enzymes and the transcription factor NF-κB are important mechanisms that induce acute pancreatitis (AP). Recurrent pancreatic injury due to genetic susceptibility, environmental factors such as smoking, alcohol intake, and conditions such as obesity lead to increases in oxidative stress, impaired autophagy and constitutive activation of inflammatory pathways. These processes can stimulate pancreatic stellate cells, thereby increasing fibrosis and encouraging chronic disease development. Activation of oncogenic Kras mutations through inflammation, coupled with altered levels of tumor suppressor proteins (p53 and p16) can ultimately lead to development of pancreatic cancer. Summary: Although our understanding of pancreatitis and pancreatic cancer has tremendously increased over many years, much remains to be elucidated in terms of common pathways linking these conditions.

The novel protein kinase C isoforms -δ and -ε modulate caerulein-induced zymogen activation in pancreatic acinar cells

Isoforms of protein kinase C (PKC) have been shown to modulate some cellular responses such as pathological secretion and generation of inflammatory mediators during acute pancreatitis (AP). We propose that PKC also participates in premature zymogen activation within the pancreatic acinar cell, a key event in the initiation of AP. This hypothesis was examined in in vivo and cellular models of caerulein-induced AP using PKC activators and inhibitors. Phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA, 200 nM), a known activator of PKC, enhanced zymogen activation at both 0.1 nM and 100 nM caerulein, concentrations which mimic physiological and supraphysiological effects of the hormone cholecystokinin, respectively, in preparations of pancreatic acinar cells. Isoform-specific PKC inhibitors for PKC-delta and PKC-epsilon reduced supraphysiological caerulein-induced zymogen activation. Using a cell-free reconstitution system, we showed that inhibition of PKC-delta and -epsilon, reduced zymogen activation in both zymogen granule-enriched and microsomal fractions. In dispersed acinar cells, 100 nM caerulein stimulation caused PKC-delta and -epsilon isoform translocation to microsomal membranes using cell fractionation and immunoblot analysis. PKC translocation was confirmed with in vivo studies and immunofluorescence microscopy in pancreatic tissues from rats treated with or without 100 nM caerulein. PKC-epsilon redistributed from an apical to a supranuclear region following caerulein administration. The signal for PKC-epsilon overlapped with granule membrane protein, GRAMP-92, an endosomal/lysosomal marker, in a supranuclear region where zymogen activation takes place. These results indicate that PKC-delta and -epsilon isoforms translocate to specific acinar cell compartments and modulate zymogen activation.

Protein Kinase C δ-Mediated Processes in Cholecystokinin-8-Stimulated Pancreatic Acini

Objectives: To define the role of protein kinase C &dgr; (PKC &dgr;) in acinar cell responses to the hormone cholecystokinin-8 (CCK) using isoform-specific inhibitors and a previously unreported genetic deletion model. Methods: Pancreatic acinar cells were isolated from (1) rat, and pretreated with a PKC &dgr;-specific inhibitor or (2) PKC &dgr;-deficient and wild type mice. Isolated cells were stimulated with CCK (0.001-100 nmol/L) and cell responses were measured. Results: The PKC &dgr; inhibitor did not affect stimulated amylase secretion from rat pancreatic acinar cells. Cholecystokinin-8 stimulation induced a typical biphasic dose-response curve for amylase secretion in acinar cells isolated from both PKC &dgr;−/− and wild type mice, with maximal stimulation at 10-pmol/L CCK. Cholecystokinin-8 (100 nmol/L) induced zymogen and nuclear factor &kgr;B activation in both PKC &dgr;−/− and wild type mice, although it was up to 50% less in PKC &dgr;−/−. Conclusions: In contrast to previous studies, this study has used specific and complementary approaches to examine PKC &dgr;-mediated acinar cell responses. We could not confirm that it mediates amylase release but corroborated its role in the early stages of acute pancreatitis.

Tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone initiates and enhances pancreatitis responses

Clinical studies indicate that cigarette smoking increases the risk for developing acute pancreatitis. The nicotine metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a major cigarette smoke toxin. We hypothesized that NNK could sensitize to pancreatitis and examined its effects in isolated rat pancreatic acini and in vivo. In acini, 100 nM NNK caused three- and fivefold activation of trypsinogen and chymotrypsinogen, respectively, above control. Furthermore, NNK pretreatment in acini enhanced zymogen activation in a cerulein pancreatitis model. The long-term effects of NNK were examined in vivo after intraperitoneal injection of NNK (100 mg/kg body wt) three times weekly for 2 wk. NNK alone caused zymogen activation (6-fold for trypsinogen and 2-fold for chymotrypsinogen vs. control), vacuolization, pyknotic nuclei, and edema. This NNK pretreatment followed by treatment with cerulein (40 μg/kg) for 1 h to induce early pancreatitis responses enhanced trypsinogen and chymotrypsinogen activation, as well as other parameters of pancreatitis, compared with cerulein alone. Potential targets of NNK include nicotinic acetylcholine receptors and β-adrenergic receptors; mRNA for both receptor types was detected in acinar cell preparations. Studies with pharmacological inhibitors of these receptors indicate that NNK can mediate acinar cell responses through an nonneuronal α(7)-nicotinic acetylcholine receptor (α(7)-nAChR). These studies suggest that prolonged exposure to this tobacco toxin can cause pancreatitis and sensitize to disease. Therapies targeting NNK-mediated pathways may prove useful in treatment of smoking-related pancreatitis.

Activation of Soluble Adenylyl Cyclase Protects against Secretagogue Stimulated Zymogen Activation in Rat Pancreaic Acinar Cells

An early feature of acute pancreatitis is activation of zymogens, such as trypsinogen, within the pancreatic acinar cell. Supraphysiologic concentrations of the hormone cholecystokinin (CCK; 100 nM), or its orthologue cerulein (CER), induce zymogen activation and elevate levels of cAMP in pancreatic acinar cells. The two classes of adenylyl cyclase, trans-membrane (tmAC) and soluble (sAC), are activated by distinct mechanisms, localize to specific subcellular domains, and can produce locally high concentrations of cAMP. We hypothesized that sAC activity might selectively modulate acinar cell zymogen activation. sAC was identified in acinar cells by PCR and immunoblot. It localized to the apical region of the cell under resting conditions and redistributed intracellularly after treatment with supraphysiologic concentrations of cerulein. In cerulein-treated cells, pre-incubation with a trans-membrane adenylyl cyclase inhibitor did not affect zymogen activation or amylase secretion. However, treatment with a sAC inhibitor (KH7), or inhibition of a downstream target of cAMP, protein kinase A (PKA), significantly enhanced secretagogue-stimulated zymogen activation and amylase secretion. Activation of sAC with bicarbonate significantly inhibited secretagogue-stimulated zymogen activation; this response was decreased by inhibition of sAC or PKA. Bicarbonate also enhanced secretagogue-stimulated cAMP accumulation; this effect was inhibited by KH7. Bicarbonate treatment reduced secretagogue-stimulated acinar cell vacuolization, an early marker of pancreatitis. These data suggest that activation of sAC in the pancreatic acinar cell has a protective effect and reduces the pathologic activation of proteases during pancreatitis.

Cerulein hyperstimulation decreases AMP-activated protein kinase levels at the site of maximal zymogen activation

The premature activation of digestive enzyme zymogens in the pancreatic acinar cell is an important initiating event in acute pancreatitis. We have previously demonstrated that vacuolar ATPase (vATPase) activity is required for zymogen activation. Adenosine monophosphate-activated protein kinase (AMPK) regulates vATPase function in kidney and epididymal clear cells. To determine whether AMPK could affect pancreatitis responses, its effects were first examined in a cellular model of pancreatitis, cerulein-hyperstimulated (100 nM) pancreatic acini. This treatment caused a prominent increase in trypsin and chymotrypsin activities. Pretreatment with AICAR or metformin (AMPK activators) or compound C (an AMPK inhibitor) reduced or increased cerulein-induced zymogen activation, respectively. The association of the vATPase E subunit with membranes, a marker of its activation, tended to be inversely related to AMPK activity (assessed by AICAR and compound C treatments). Cerulein treatment did not change AMPK (α and β) levels but did lead to an increase in its activation (phosphorylation of Thr172) and induced the time-dependent translocation of the enzyme to a Triton-insoluble compartment. Basal in vivo studies showed that AMPK was widely distributed between membrane and soluble fractions generated by differential centrifugation. After cerulein hyperstimulation, AMPK levels selectively decreased in fractions containing the highest levels of active zymogens. These studies suggest that AMPK activity has a protective role in the pancreatic acinar cell that inhibits zymogen activation in the basal state, and this AMPK effect is reduced during pancreatitis. Therapies that prevent the selective reduction of AMPK in compartments that support zymogen activation could reduce injury during pancreatitis.

Ascorbic acid-dependent cytoprotection of ovarian cells by leukocyte and nonleukocyte peroxidases.

Luteal cells contain high levels of ascorbic acid that is secreted by stimulation with agents like luteinizing hormone (LH) and prostaglandin F2 alpha (PGF2 alpha). One role for interstitial ascorbic acid, we propose, may be the detoxification of H2O2 by regeneration of catalytically active peroxidase. By serving as a preferred secondary substrate, ascorbic acid regenerates the catalytically active peroxidase that is inhibited irreversibly by H2O2 alone. To test this hypothesis, luteal cells were incubated in the absence and presence of peroxidases and H2O2, and the maximal cyclic AMP and steroidogenic response to LH was examined. In luteal cells, H2O2 is known to severely inhibit LH-sensitive cyclic AMP accumulation and steroidogenesis, and the addition of lactoperoxidase, myeloperoxidase, eosinophil peroxidase, or ascorbic acid (1 mM) alone had no effect on these responses to H2O2. However, co-incubation of ascorbic acid and the peroxidases completely reversed the inhibition of cyclic AMP accumulation and steroidogenesis produced by H2O2. These findings and the results that show direct oxidation of ascorbic acid in the presence of peroxidase and H2O2, but not with H2O2 alone, support the conclusion that ascorbic acid released from cells may detoxify H2O2 by regenerating the catalytically active state of peroxidases.

IP3 Receptor Type 2 Deficiency Is Associated with a Secretory Defect in the Pancreatic Acinar Cell and an Accumulation of Zymogen Granules

Acute pancreatitis is a painful, life-threatening disorder of the pancreas whose etiology is often multi-factorial. It is of great importance to understand the interplay between factors that predispose patients to develop the disease. One such factor is an excessive elevation in pancreatic acinar cell Ca2+. These aberrant Ca2+ elevations are triggered by release of Ca2+ from apical Ca2+ pools that are gated by the inositol 1,4,5-trisphosphate receptor (IP3R) types 2 and 3. In this study, we examined the role of IP3R type 2 (IP3R2) using mice deficient in this Ca2+ release channel (IP3R2−/−). Using live acinar cell Ca2+ imaging we found that loss of IP3R2 reduced the amplitude of the apical Ca2+ signal and caused a delay in its initiation. This was associated with a reduction in carbachol-stimulated amylase release and an accumulation of zymogen granules (ZGs). Specifically, there was a 2-fold increase in the number of ZGs (P<0.05) and an expansion of the ZG pool area within the cell. There was also a 1.6- and 2.6-fold increase in cellular amylase and trypsinogen, respectively. However, the mice did not have evidence of pancreatic injury at baseline, other than an elevated serum amylase level. Further, pancreatitis outcomes using a mild caerulein hyperstimulation model were similar between IP3R2−/− and wild type mice. In summary, IP3R2 modulates apical acinar cell Ca2+ signals and pancreatic enzyme secretion. IP3R-deficient acinar cells accumulate ZGs, but the mice do not succumb to pancreatic damage or worse pancreatitis outcomes.

Low pH enhances connexin32 degradation in the pancreatic acinar cell.

Decreased extracellular pH is observed in a number of clinical conditions and can sensitize to the development and worsen the severity of acute pancreatitis. Because intercellular communication through gap junctions is pH-sensitive and modulates pancreatitis responses, we evaluated the effects of low pH on gap junctions in the rat pancreatic acinar cell. Decreasing extracellular pH from 7.4 to 7.0 significantly inhibited gap junctional intracellular communication. Acidic pH also significantly reduced levels of connexin32, the predominant gap junction protein in acinar cells, and altered its localization. Increased degradation through the proteasomal, lysosomal, and autophagic pathways mediated the decrease in connexin32 under low-pH conditions. These findings provide the first evidence that low extracellular pH can regulate gap junctional intercellular communication by enhancing connexin degradation.