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

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-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

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

Pancreatic neutrophil infiltration during acute pancreatitis is receptor dependent.

<p>NNK caused neutrophil infiltration in WT but not <i>α</i>7nAChR^-/- mice. Also, knocking out <i>α</i>7nAChR decreases neutrophils migration into CER-treated pancreatic tissues, and this effect was more intense when NNK was given in combination with CER. Studies in wild type and <i>α</i>7nAChR^-/- (KO) mice. Values are means ± SE; n ≥ 4. *P < 0.05 vs. PBS WT. #P < 0.05 vs. analogous WT.</p