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

The ryanodine receptor mediates early zymogen activation in pancreatitis

Acute pancreatitis is characterized by the pathologic activation of zymogens within pancreatic acinar cells. The process requires a rise in cytosolic Ca(2+) from undefined intracellular stores. We hypothesized that zymogen activation is mediated by ryanodine receptor (RYR)-regulated Ca(2+) release, because early zymogen activation takes place in a supranuclear compartment that overlaps in distribution with the RYR. Ca(2+) signals in the basolateral, but not apical, region of acinar cells observed during supraphysiologic agonist stimulation were dependent on RYR Ca(2+) release. Inhibition of RYR or depletion of RYR-sensitive Ca(2+) pools each reduced pathologic zymogen activation in isolated acinar cells, but neither treatment affected amylase secretion. Inhibition of RYR also inhibited zymogen activation in vivo. We propose that Ca(2+) release from the RYR mediates zymogen activation but not enzyme secretion. The findings imply a role for the RYR in acute pancreatitis

NNK stimulation of trypsinogen activation is receptor-dependent in vivo.

<p><b>(A)</b> NNK induces zymogen activation in vivo within WT mice, <b>(B)</b> but not in <i>α</i>7nAChR^-/- mice. Mice were injected with NNK (100 mg/kg), CER (40 <b>μ</b>g/kg), or a combination of both. <b>(A)</b> NNK (100 mg/kg) alone induced trypsin activity, and NNK+CER further enhanced trypsin activity compared to those induced by CER alone. <b>(B)</b> NNK effect on <i>α</i>7nAChR^-/- mice is abrogated. Values are means ± SE; n = 6. *P < 0.05 vs. CTL. #P < 0.05 vs. CER. ns = not significant.</p

Depletion of intracellular calcium or inhibition of PKC blocks NNK induced trypsinogen activation.

<p><b>(A)</b> Trypsinogen activation by NNK (100 nM) was not inhibited in calcium free media. But, preincubation with the membrane permeable calcium chelator BAPTA-AM (10 <b>μ</b>M, 30 min) followed by switching to a calcium free media significantly inhibited trypsinogen activation. <b>(B)</b> NNK induced trypsinogen activation was inhibited by preincubation with the broad-spectrum PCK inhibitor GF-109203X (10 <b>μ</b>M) for 120 min. Values are means ± SE; n = 3. *P < 0.05 vs. control, #P < 0.05 vs. NNK alone.</p

NNK-mediated histological changes in vivo are receptor-dependent.

<p><b>(A & B):</b> no histological changes in PBS-treated pancreatic tissues. (<b>C & D)</b>: NNK caused histological changes in WT, but not in <i>α</i>7nAChR^-/- mice. <b>(E—H)</b>: Histological changes are observed in tissues treated with CER alone or in combination with NNK. Arrows = pyknotic nuclei; Arrowheads = Vacuoles; Sharp arrows = edema. <b>(I)</b>: total histological score of all parameters. <b>(J, K, and L)</b>: categorized scores for pyknotic nuclei, edema, and vacuoles, respectively. *P < 0.05 vs. WT-PBS. #P < 0.05 vs. KO-PBS. **P < 0.05 vs. KO-NNK.</p