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.

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 α7-nicotinic acetylcholine receptor (α7-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.

Protein kinase C (PKC) has been shown to have multiple roles in the regulation of both physiology and pathophysiology of the pancreas. We propose that PKC participates in premature zymogen activation within the pancreatic acinar cell, a key event in the initiation of acute pancreatitis. To examine this hypothesis we used both whole-cell and reconstituted systems and a series of isoform-specific PKC inhibitors and antibodies. Isolated pancreatic acinar cells were stimulated with supraphysiologic concentrations of the cholecystokinin orthologue cerulein (10-7 M) in the presence of PKC δ, ζ and ζ inhibitors. Reduction of cerulein-induced trypsinogen and chymotrypsinogen activation was observed following inhibition of PKC δ and ζ but not ζ. Stimulated amylase secretion was unaffected by PKC δ and ζ inhibition, but increased following PKC ζ inhibition. Using a cell-free reconstitution system (Thrower, AJP 2006) we have shown that zymogen granule-enriched and microsomal fractions from the pancreas can support cytosol-dependent zymogen activation. In the presence of inhibitors for PKC δ and ζ, a reduction in trypsin and chymotrypsin activity was seen. Similar results were observed when isoform-specific PKC antibodies were used in place of peptide inhibitors. Translocation of PKC isoforms following cerulein stimulation was investigated by cellular fractionation and immunoblot. PKC δ and ζ isoforms translocate to a microsomal fraction whereas ζ does not. These results collectively indicate that PKC δ and ζ isoforms participate in premature zymogen activation by translocating to their target site. PKC ζ, however, does not affect zymogen activation, but may modulate secretion. These findings imply that distinct isoforms of PKC can mediate zymogen activation in a distinct acinar cell compartment.

Pathological activation of digestive zymogens within the pancreatic acinar cell initiates acute pancreatitis. Cytosolic events regulate this activation within intracellular compartments of unclear identity. In an in vivo model of acute pancreatitis, zymogen activation was detected in both zymogen granule-enriched and microsomal cellular fractions. To examine the mechanism of this activation in vitro, a reconstituted system was developed using pancreatic cytosol, a zymogen granule-enriched fraction, and a microsomal fraction. Addition of cytosol to either particulate fraction resulted in a prominent increase in both trypsin and chymotrypsin activities. The percentage of the pool of trypsinogen and chymotrypsinogen activated was about twofold and sixfold greater, respectively, in the microsomal than in the zymogen granule-enriched fraction. Activation of chymotrypsinogen but not trypsinogen was significantly enhanced by ATP (5 mM) but not by the inactive ATP analog AMP-PNP. The processing of procarboxypeptidase B to its mature form also demonstrated a requirement for ATP and cytosol. E64d, an inhibitor of cathepsin B, a thiol protease that can activate trypsin, completely inhibited trypsin activity but did not affect chymotrypsin activity or carboxypeptidase B generation. These studies demonstrate that both zymogen granule-enriched and microsomal fractions from the pancreas can support cytosol-dependent zymogen activation. A component of the activation of some zymogens, such as chymotrypsinogen and procarboxypeptidase, may depend on ATP but not on trypsin or cathepsin B.

Chromogranins A and B (CGA and CGB) are high capacity, low affinity calcium (Ca2+) storage proteins found in many cell types most often associated with secretory granules of secretory cells but also with the endoplasmic reticulum (ER) lumen of these cells. Both CGA and CGB associate with inositol 1,4,5-trisphosphate receptor (InsP3R) in a pH-dependent manner. At an intraluminal pH of 5.5, as found in secretory vesicles, both CGA and CGB bind to the InsP3R. When the intraluminal pH is 7.5, as found in the ER, CGA totally dissociates from InsP3R, whereas CGB only partially dissociates. To investigate the functional consequences of the interaction between the InsP3R and CGB monomers or CGA/CGB heteromers, purified mouse InsP3R type I were fused to planar lipid bilayers and activated by 2 μm InsP3. In the presence of luminal CGB monomers or CGA/CGB heteromers the InsP3R/Ca2+ channel open probability and mean open time increased significantly. The channel activity remained elevated when the pH was changed to 7.5, a reflection of CGB binding to the InsP3R even at pH 7.5. These results suggest that CGB may play an important modulatory role in the control of Ca2+ release from the ER. Furthermore, the difference in the ability of CGA and CGB to regulate the InsP3R/Ca2+ channel and the variability of CGA/CGB ratios could influence the pattern of InsP3-mediated Ca2+ release.

Nuclear calcium (Ca 2+ ) regulates a number of important cellular processes, including gene transcription, growth, and apoptosis. However, it is unclear whether Ca 2+ signaling is regulated differently in the nucleus and cytosol. To investigate this possibility, we examined subcellular mechanisms of Ca 2+ release in the HepG2 liver cell line. The type II isoform of the inositol 1,4,5-trisphosphate (InsP 3 ) receptor (InsP 3 R) was expressed to a similar extent in the endoplasmic reticulum and nucleus, whereas the type III InsP 3 R was concentrated in the endoplasmic reticulum, and the type I isoform was not expressed. Ca 2+ signals induced by low InsP 3 concentrations started earlier or were larger in the nucleus than in the cytosol, indicating higher sensitivity of nuclear Ca 2+ stores for InsP 3 . Nuclear InsP 3 R channels were active at lower InsP 3 concentrations than InsP 3 R from cytosol. Enriched expression of type II InsP 3 R in the nucleus results in greater sensitivity of the nucleus to InsP 3 , thus providing a mechanism for independent regulation of Ca 2+ -dependent processes in this cellular compartment.

We report here the first three‐dimensional structure of the type 1 inositol 1,4,5‐trisphosphate receptor (IP3R). From cryo‐electron microscopic images of purified receptors embedded in vitreous ice, a three‐dimensional structure was determined by use of standard single particle reconstruction techniques. The structure is strikingly different from that of the ryanodine receptor at similar resolution despite molecular similarities between these two calcium release channels. The 24 Å resolution structure of the IP3R takes the shape of an uneven dumbbell, and is ∼170 Å tall. Its larger end is bulky, with four arms protruding laterally by ∼50 Å and, in comparison with the receptor topology, probably corresponds to the cytoplasmic domain of the receptor. The lateral dimension at the height of the protruding arms is ∼155 Å. The smaller end, whose lateral dimension is ∼100 Å, has structural features indicative of the membrane‐spanning domain. A central opening in this domain, which is occluded on the cytoplasmic half, outlines a pathway for calcium flow in the open state of the channel.

Secretory granules of neuroendocrine cells are inositol 1,4,5-trisphosphate (InsP(3))-sensitive Ca(2+) stores in which the Ca(2+) storage protein, chromogranin A (CGA), couples with InsP(3)-gated Ca(2+) channels (InsP(3)R) located in the granule membrane. The functional aspect of this coupling has been investigated via release studies and planar lipid bilayer experiments in the presence and absence of CGA. CGA drastically increased the release activity of the InsP(3)R by increasing the channel open probability by 9-fold and the mean open time by 12-fold. Our results show that CGA-coupled InsP(3)Rs are more sensitive to activation than uncoupled receptors. This modulation of InsP(3)R channel activity by CGA appears to be an essential component in the control of intracellular Ca(2+) concentration by secretory granules and may regulate the rate of vesicle fusion and exocytosis.

Three isoforms of the inositol (1,4,5)-trisphosphate [Ins(1,4,5)P-3] receptor have been identified. Each receptor isoform has been functionally characterized using many different techniques. Although these receptor isoforms possess high homology, interesting differences in their Ca2+ dependence, Ins(1,4,5)P-3 sensitivity and subcellular distribution exist, implying distinct cellular roles. Indeed, interplay among the isoforms might be necessary for a cell to control spatial and temporal aspects of cytosolic Ca2+ signals, which are important for many cellular processes. In this review isoform-specific functions, primarily at the single-channel level, will be highlighted and these properties will be correlated with Ca2+ signals in intact cells.

Ca(2+) within intracellular stores (luminal Ca(2+)) is believed to play a role in regulating Ca(2+) release into the cytosol via the inositol (1,4,5)-trisphosphate (Ins(1,4,5)P(3))-gated Ca(2+) channel (or Ins(1,4,5)P(3) receptor). To investigate this, we incorporated purified Type 1 Ins(1,4,5)P(3) receptor from rat cerebellum into planar lipid bilayers and monitored effects at altered luminal [Ca(2+)] using K(+) as the current carrier. At a high luminal [Ca(2+)] and in the presence of optimal [Ins(1,4,5)P(3)] and cytosolic [Ca(2+)], a short burst of Ins(1,4,5)P(3) receptor channel activity was followed by complete inactivation. Lowering the luminal [Ca(2+)] caused the channel to reactivate indefinitely. At luminal [Ca(2+)], reflecting a partially empty store, channel activity did not inactivate. The addition of cytosolic ATP to a channel inactivated by high luminal [Ca(2+)] caused reactivation. We provide evidence that luminal Ca(2+) is exerting its effects via a direct interaction with the luminal face of the receptor. Activation of the receptor by ATP may act as a device by which cytosolic Ca(2+) overload is prevented when the energy state of the cell is compromised.