Cigarette toxin 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces experimental pancreatitis through α7 nicotinic acetylcholine receptors (nAChRs) in mice.

Clinical studies have shown that cigarette smoking is a dose-dependent and independent risk factor for acute pancreatitis. Cigarette smoke contains nicotine which can be converted to the potent receptor ligand and toxin, NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone]. Previously, we have shown that NNK induces premature activation of pancreatic zymogens in rats, an initiating event in pancreatitis, and this activation is prevented by pharmacologic inhibition of nicotinic acetylcholine receptors (nAChR). In this study, we determined whether NNK mediates pancreatitis through the α7 isoform of nAChR using α7nAChR knockout mice. PCR analysis confirmed expression of non-neuronal α7nAChR in C57BL/6 (WT) mouse and human acinar cells. NNK treatment stimulated trypsinogen activation in acini from WT but not α7nAChR-/- mice. NNK also stimulated trypsinogen activation in human acini. To further confirm these findings, WT and α7nAChR-/- mice were treated with NNK in vivo and markers of pancreatitis were measured. As observed in acini NNK treatment induced trypsinogen activation in WT but not α7nAChR-/- mice. NNK also induced other markers of pancreatitis including pancreatic edema, vacuolization and pyknotic nuclei in WT but not α7nAChR-/- animals. NNK treatment led to increased neutrophil infiltration, a marker of inflammation, in WT mice and to a significantly lesser extent in α7nAChR-/- mice. We also examined downstream targets of α7nAChR activation and found that calcium and PKC activation are involved down stream of NNK stimulation of α7nAChR. In this study we used genetic deletion of the α7nAChR to confirm our previous inhibitor studies that demonstrated NNK stimulates pancreatitis by activating this receptor. Lastly, we demonstrate that NNK can also stimulate zymogen activation in human acinar cells and thus may play a role in human disease.

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 ß-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.

Effects of increased intracellular cAMP on carbachol-stimulated zymogen activation, secretion, and injury in the pancreatic acinar cell.

A characteristic of acute pancreatitis is the premature activation and retention of enzymes within the pancreatic acinar cell. Because ligands linked to cAMP production may prevent some forms of pancreatitis, we evaluated the effects of increased intracellular cAMP in the rat pancreatic acinar cell. Specifically, this study examined the effects of the cholinergic agonist carbachol and agents that increase cAMP [secretin and 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP)] on zymogen activation (trypsin and chymotrypsin), enzyme secretion, and cellular injury in isolated pancreatic acini. Although cAMP agonists affected the responses to physiological concentrations of carbachol (1 microM), their most prominent effects were observed with supraphysiological concentrations (1 mM). When secretin was added to 1 mM carbachol, there was a slight increase in zymogen activation, but no change in the secretion of amylase or chymotrypsin. Furthermore, coaddition of secretin increased parameters of cell injury (trypan blue exclusion, lactic dehydrogenase release, and morphological markers) compared with carbachol (1 mM) alone. Although directly increasing cellular cAMP by 8-Br-cAMP caused much greater zymogen activation than carbachol (1 mM) alone or with secretin, 8-Br-cAMP cotreatment reduced all parameters of injury to the level of unstimulated acini. Furthermore, 8-Br-cAMP dramatically enhanced the secretion of amylase and chymotrypsin from the acinar cell. This study demonstrates that increasing acinar cell cAMP can overcome the inhibition of enzyme secretion caused by high concentrations of carbachol and eliminate acinar cell injury.

Synapsin I is expressed in epithelial cells: localization to a unique trans-Golgi compartment.

Synapsin I is abundant in neural tissues. Its phosphorylation is thought to regulate synaptic vesicle exocytosis in the pre-synaptic terminal by mediating vesicle tethering to the cytoskeleton. Using anti-synapsin antibodies, we detected an 85 kDa protein in liver cells and identified it as synapsin I. Like brain synapsin I, non-neuronal synapsin I is phosphorylated in vitro by protein kinase A and yields identical (32)P-peptide maps after limited proteolysis. We also detected synapsin I mRNA in liver by northern blot analysis. These results indicate that the expression of synapsin I is more widespread than previously thought. Immunofluorescence analysis of several non-neuronal cell lines localizes synapsin I to a vesicular compartment adjacent to trans-elements of the Golgi complex, which is also labeled with antibodies against myosin II; no sub-plasma membrane synapsin I is evident. We conclude that synapsin I is present in epithelial cells and is associated with a trans-Golgi network-derived compartment; this localization suggests that it plays a role in modulating post-TGN trafficking pathways.

Exiting the endoplasmic reticulum.

The movement of nascent proteins from sites of synthesis to final cellular or extracellular destinations involves their transport through a distinct series of vesicular compartments. Vesicle biogenesis is regulated by specific proteins and co-factors that control distinct steps including budding, transport, docking, and fusion with target membranes. Budding requires assembly of a coat protein complex on the membrane, membrane deformation and the subsequent cleavage of the nascent vesicle from donor membrane. Coat proteins may also mediate vesicle interactions with the cytoskeleton or insulate the vesicles from fusion with unwanted compartments. Three classes of cytoplasmic coats have been identified. (1) Clathrin, interacting with different adaptor proteins, participates in endocytosis, lysosome biogenesis and as yet unidentified vesicular transport processes that arise in the trans-Golgi region of cells [reviewed in (Kreis, T.E., Lowe, M., Pepperkok, R., 1995. COPs regulating membrane traffic. Ann. Rev. Cell. Dev. Biol. 11, 677--706.)]. (2) The COPI coatomer is involved in retrograde traffic within the Golgi and from the cis-Golgi region to the endoplasmic reticulum (ER). It may also participate in anterograde transport from the ER [reviewed in (Aridor, M., Balch, W.E., 1999. Integration of endoplasmic reticulum signaling in health and disease. Nature 5, 745--751.)]. (3) COPII coats mediate anterograde transport of cargo out of the ER [Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Hamamoto, S., Salama, N., Rexach, M.F., Ravazazola, M., Amherdt, M., Schekman, R., 1994. COPII: a membrane coat formed by sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell 77, 895--907; Scales, S.J., Gomez, M., Kreis, T.E., 2000. Coat proteins regulating membrane traffic. Int. Rev. Cytol. 195, 67--144.]. The COPII coat is required for budding from the ER and ER to Golgi trafficking. Further, COPII proteins also participate in cargo selection and concentrate some nascent proteins in the budding vesicle. Recent studies have shown that human disease may result from mutations that affect proteins in COPII vesicles.

Early trypsinogen activation in acute pancreatitis.

This article discusses zymogen activation within the acinar cell. The authors review advances in respect to the cellular mechanism involved in a premature intrapancreatic protease activation. Critical factors that determine the onset of premature protease activation appear to be the molecular structure of trypsinogen, the presence or absence of functionally intact lysosomal hydrolases, the pH in intracellular compartments, and the calcium signaling cascade in the pancreatic acinar cell.

Etiopathogenesis of acute pancreatitis.

Acute pancreatitis is a disease that has many causes. Each cause seems to affect the acinar cell in some way that results in the premature activation and retention of potent proteolytic enzymes. These activated enzymes then injure the acinar cell and cause the immediate release of cytokines and activate the complement system. Together, these molecules attract and sequester inflammatory cells, in particular neutrophils, which causes further secretion of cytokines, free radicals, and other vasoactive molecules, such as nitric oxide. We propose that the released inflammatory molecules induce local effects, such as pancreatic edema and necrosis, and systemic complications, such as hypotension, tachycardia, fever, capillary leak syndrome, and hypoxia. The cytokines released in the pancreas also stimulate apoptosis, further enhancing the cell death response in pancreatitis. Much of the current research is aimed at understanding the links between these series of events and finding agents that can modulate the cascade of events involved in pancreatitis. What is promising in this endeavor is that the response produced with pancreatitis is nearly identical with all etiologies, suggesting that therapy may not have to be specific to a particular cause. The mechanistic models of AP presented herein are supported by preliminary clinical studies that suggest that protease and cytokine inhibitors may improve the course of AP in specific clinical settings.

Mechanisms of intracellular zymogen activation.

The pancreatic acinar cell is potentially the initial site of injury that begins the series of events leading to acute pancreatitis. Pathological intrapancreatic zymogen activation occurs in experimental pancreatitis in animals and in human pancreatitis. Intracellular activation has been clearly linked to aberrant zymogen processing in one form of hereditary pancreatitis; in this genetic disease a mutation in cationic trypsinogen may eliminate the degradation of any trypsin activated in the acinar cell. Recent studies have also provided the first direct evidence that trypsinogen activation takes place early in the course of caerulein-induced pancreatitis; parallel studies have used isolated pancreatic acini and conditions that simulate those that cause pancreatitis in vivo to demonstrate that zymogens can be pathologically activated in isolated cells. A unique acinar cell pathway regulates the intracellular proteinase processing of zymogens to their active forms. Stimulating the acinar cell with supramaximal concentrations of cholecystokinin (CCK) or carbamylcholine can activate this pathway. The activation depends on a low pH compartment within the acinar cell and activation of an intracellular serine protease. A marker of trypsinogen processing, the trypsinogen activation peptide (TAP), is generated in acinar cell compartments that do not overlap with secretory granules. This compartment overlaps with a marker of recycling endosomes and lysosomes. Thus, zymogen processing within the acinar cell proceeds in a distinct subcellular compartment and is dependent on a low pH environment and activation of serine proteases.

Codistribution of TAP and the granule membrane protein GRAMP-92 in rat caerulein-induced pancreatitis.

The pathological activation of zymogens within the pancreatic acinar cell plays a role in acute pancreatitis. To identify the processing site where activation occurs, antibodies to the trypsinogen activation peptide (TAP) were used in immunofluorescence studies using frozen sections from rat pancreas. Saline controls or animals receiving caerulein in amounts producing physiological levels of pancreatic stimulation demonstrated little or no TAP immunoreactivity. However, after caerulein hyperstimulation (5 micrograms. kg-1. h-1) for 30 min and the induction of pancreatitis, TAP immunoreactivity appeared in a vesicular, supranuclear compartment that demonstrated no overlap with zymogen granules. The number of vesicles and their size increased with time. After 60 min of hyperstimulation with caerulein, most of the TAP reactivity was localized within vacuoles >/=1 micrometer that demonstrated immunoreactivity for the granule membrane protein GRAMP-92, a marker for lysosomes and recycling endosomes. Pretreatment with the protease inhibitor FUT-175 blocked the appearance of TAP after hyperstimulation. These studies provide evidence that caerulein hyperstimulation stimulates trypsinogen processing to trypsin in distinct acinar cell compartments in a time-dependent manner.

Zymogen proteolysis within the pancreatic acinar cell is associated with cellular injury.

The pathological activation of digestive zymogens within the pancreatic acinar cell probably plays a central role in initiating many forms of pancreatitis. To examine the relationship between zymogen activation and acinar cell injury, we investigated the effects of secretagogue treatment on isolated pancreatic acini. Immunofluorescence studies using antibodies to the trypsinogen-activation peptide demonstrated that both CCK (10(-7) M) hyperstimulation and bombesin (10(-5) M) stimulation of isolated acini resulted in trypsinogen processing to trypsin. These treatments also induced the proteolytic processing of procarboxypeptidase A1 to carboxypeptidase A1 (CA1). After CCK hyperstimulation, most CA1 remained in the acinar cell. In contrast, the CA1 generated by bombesin was released from the acinar cell. CCK hyperstimulation of acini was associated with cellular injury, whereas bombesin treatment did not induce injury. These studies suggest that 1) proteolytic zymogen processing occurs within the pancreatic acinar cell and 2) both zymogen activation and the retention of enzymes within the acinar cell may be required to induce injury.

Telenzepine-sensitive muscarinic receptors on rat pancreatic acinar cells.

To identify the muscarinic subtype present on the rat pancreatic acinar cell, we examined the effects of different muscarinic receptor antagonists on amylase secretion and proteolytic zymogen processing in isolated rat pancreatic acini. Maximal zymogen processing required a concentration of carbachol 10- to 100-fold greater (10(-3) M) than that required for maximal amylase secretion (10(-5) M). Although both secretion and conversion were inhibited by the M3 antagonist 4-diphenylacetoxy-N-methyl-piperidine (4-DAMP) (50% inhibition approximately 6 x 10(-7) M and 1 x 10(-8) M, respectively), the most potent inhibitor was the M1 antagonist telenzepine (50% inhibition approximately 5 x 10(-10) M and 1 x 10(-11) M, respectively). Pirenzepine, another M1 antagonist, and the M2 antagonist methoctramine did not reduce amylase secretion or zymogen processing in concentrations up to 1 x 10(-5) M. Analysis of acinar cell muscarinic receptor by PCR revealed expression of both m1 and m3 subtypes. The pancreatic acinar cell has a distinct pattern of muscarinic antagonist sensitivity (telenzepine >> 4-DAMP > pirenzepine) with respect to both amylase secretion and zymogen conversion.

Cellular localization of calmodulin-dependent protein kinases I and II to A-cells and D-cells of the endocrine pancreas.

Ca2+/calmodulin-dependent protein kinases I and II, initially identified in brain on the basis of their ability to phosphorylate synapsin I, have been implicated in the regulation of Ca2+-dependent synaptic neurosecretion. Specific recombinant and synthetic peptide antibodies were used to examine the distribution of CaM kinases I and II in the rat pancreas and other tissues. The CaM kinase I antibodies detected a doublet of cytosolic proteins of approximately 38 and approximately 42 kD by immunoblot. CaM kinase I was observed in glucagon-containing A-cells at the periphery of the islet of Langerhans but had little or no overlap with pancreatic polypeptide or somatostatin cells. In contrast, CaM kinase II was localized to somatostatin-containing D-cells. CaM kinase I co-localized with glucagon secretory granules. CaM kinase II was not associated with the somatostatin granule but rather was enriched in areas of the cells that contained relatively little somatostatin. Because glucagon secretion is Ca2+-dependent, it is attractive to speculate that CaM kinase I may play a regulatory role in glucagon secretion. Glucagon and somatostatin cells both utilize intracellular Ca2+ for signaling. Therefore, specific CaM kinases may act as effectors of Ca2+ in these different cell types.

Serologic assay for secretory component distinguishes mechanical from hepatocellular cholestasis in humans.

In rats, serum secretory component (SC) is elevated in mechanical but not hepatocellular cholestasis. To determine if serum SC might distinguish cholestatic syndromes in humans, serum samples were obtained from control subjects and patients with mechanical and hepatocellular cholestasis. Equal volumes of serum were assayed for SC by immunoblotting with an antibody specific for human SC. Quantitative densitometry of these immunoblots showed that in mechanically obstructed patients serum SC was reversibly elevated to a level approximately 10-fold higher than that of patients with hepatocellular cholestasis (P < 0.001). When comparing the two cholestatic groups, levels of serum alkaline phosphatase, but not bilirubin and alanine aminotransferase, were significantly higher in the group with mechanical cholestasis (P < 0.01). When comparing individual patients, serum SC was more reliable than alkaline phosphatase in distinguishing the two cholestatic syndromes (P < 0.05). Thus, serum SC may distinguish mechanical from hepatocellular cholestasis in humans.

Distribution of a synthetic protease inhibitor in rat pancreatic acini after supramaximal secretagogue stimulation.

Protease inhibitors may have a beneficial effect in acute pancreatitis. The effects of E3123, a new low molecular weight protease inhibitor, on the ultrastructure of isolated pancreatic acini were examined using transmission electron microscopy. Acini supramaximally stimulated with cerulein (10(-8) M) formed large cytoplasmic vacuoles similar to those generated in the cerulein-induced in vivo model of pancreatitis. Pretreatment of isolated acini with E3123 significantly reduced the size and number of vacuoles associated with cerulein treatment. The distribution of 3H-E3123 in acinar cells was examined using a pulse-chase protocol and electron microscopic autoradiography. Cellular levels of 3H-E3123 increased about 30-fold in acinar cells treated with cerulein (10(-8) M) compared to unstimulated controls. In cerulein-treated acini examined after a 5-min chase, 47.4% of the autoradiographic grains were associated with the rough endoplasmic reticulum and 13.2% were associated with zymogen granules. After 30 min of incubation, the grains associated with the endoplasmic reticulum decreased to 18.5% but increased to 26.3% over zymogen granules. Thus, E3123 is taken up by the acinar cell and follows a cellular itinerary similar to that of newly synthesized secretory proteins. One potential conclusion from these studies is that the ability of E3123 to reduce the formation of vacuoles in supra-maximally stimulated acini may be due to its inhibition of proteases within the secretory pathway.

Co-distribution of calmodulin-dependent protein kinase II and inositol trisphosphate receptors in an apical domain of gastrointestinal mucosal cells.

The Type 3 inositol 1,4,5-trisphosphate (InsP3) receptor is expressed at high levels in gastrointestinal tissues. This receptor has 16 potential phosphorylation sites for calcium/calmodulin-dependent protein kinase II (CaM kinase II). To determine if the Type 3 InsP3 receptor is likely to be a physiologic substrate for CaM kinase II, localizations of the Type 3 InsP3 receptor and CaM kinase II were compared in tissues of the gastrointestinal tract. Cellular and subcellular localizations were determined by immunofluorescence microscopy in rat intestine, pancreas, and stomach, and in isolated rabbit gastric glands. Both proteins were found in the apical region of intestinal enterocytes, pancreatic acinar cells, and gastric parietal, chief, and surface mucous cells. CaM kinase II was found throughout the entire intracellular canalicular F-actin domain of parietal cells, whereas the type 3 InsP3 receptor was restricted to the neck region. Thus, in several gastrointestinal tissues the Type 3 InsP3 receptor is specifically localized to a portion of the apical cytoskeletal domain in which resides the calcium-responsive effector CaM kinase II.

Ca2+/calmodulin-dependent protein kinase II activation and regulation of adrenal glomerulosa Ca2+ signaling.

We recently reported that elevations in the intracellular Ca2+ concentration ([Ca2+]i) enhance low-voltage-activated, T-type, Ca2+ channel activity via Ca2+/calmodulin-dependent protein kinase II (CaMKII). Here, we document CaMKII activity in bovine adrenal glomerulosa (AG) cells and assess the importance of CaMKII in depolarization-induced Ca2+ signaling. AG cell extracts exhibited kinase activity toward a CaMKII-selective peptide substrate that was dependent on both Ca2+ [half-maximal concentration for Ca2+ activation (K0.5) = 1.5 microM] and calmodulin (K0.5 = 46 nM) and was sensitive to a calmodulin antagonist and a CaMKII peptide inhibitor. On cell treatment with elevated extracellular potassium (10-60 mM) or angiotensin II, Ca(2+)-independent CaMKII activity increased to 133-205% of basal activity. Ca(2+)-independent kinase activity in agonist-stimulated extracts was inhibited by the CaMKII inhibitor peptide, 1(-)[N,O-bis(1,5- isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a cell-permeable inhibitor of CaMKII, reduced the agonist-induced stimulation of Ca(2+)-independent CaMKII activity. KN-62 also diminished depolarization-induced increases in [Ca2+]i without affecting the membrane potential. These observations suggest that CaMKII is activated in situ by aldosterone secretagogues and augments Ca2+ signaling through voltage-gated Ca2+ channels.