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

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 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

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

<p><b>(A)</b> NNK induces zymogen activation in pancreatic acinar cells isolated from WT mice, <b>(B)</b> but not in <i>α</i>7nAChR^-/- isolated acini. Acinar cells were treated with NNK (100 nM), CER (100 nM), and combination of both. NNK (100 nM) alone induced trypsin activity, and NNK+CER further enhanced trypsin activity compared to those induced by CER (100 nM) alone. Values are means ± SE; n = 6. *P < 0.05 vs. CTL. #P < 0.05 vs. CER. ns = not significant.</p