Nicotine-induced proliferation of isolated rat pancreatic acinar cells: effect on cell signalling and function

Abstract

Objectives: The aim of the current study was to investigate whether nicotine treatment would induce the proliferation of isolated rat primary pancreatic acinar cells in culture by activating mitogen-activated protein kinase (MAPK) signalling and exocrine secretion.

Materials and methods: A nicotine dose- and time-response curve was initially developed to determine the optimal dose and time used for all subsequent studies. Proliferation studies were conducted by cell counting and confirmed further by bromodeoxyuridine (BrdU) incorporation and flow cytometry assays. MAPK signalling studies were conducted by Western blot analysis. Localization of ERK1/2 signals, with or without nicotine and the MAPK inhibitor, was visualized by immunofluorescence.

Results: Nicotine treatment caused dose-dependent activation of extracellular signal-regulated kinases (ERK1/2), the maxima occurring at 100 micro m and at 3 min after treatment; the response was suppressed by the ERK1/2 inhibitor. Maximal nicotine-induced cell proliferation occurred at 24 h, and UO126-treatment significantly reduced this response. Exposure of cells to 100 microm nicotine for 6 min significantly enhanced both baseline and cholecystokinin-stimulated cell function, and these effects were not affected by treatment with the inhibitor of ERK1/2 but were suppressed by mecamylamine, a nicotinic receptor antagonist.

Conclusions: Our results suggest that nicotine treatment induced cell proliferation of isolated pancreatic acinar cells and that this is coupled with the activation of MAPK signalling with no effect on its function. Hence, in primary cells, the mechanism of induction and regulation of these two processes, cell proliferation and cell function, by nicotine treatment are independent of each other.

Figure 1

Dose‐ and time‐dependent induction of ERK1/2 in isolated pancreatic acinar cells. Cell lysates were loaded on SDS gel, separated by electrophoresis, blocked by casein and probed with antibodies to total and phospho ERK1/2. Horseradish peroxidase‐coupled anti‐IgG was used as the secondary antibody. Bands were visualized with an enhanced chemiluminescence kit and were quantified with a storm imager. Panels a and b: representative ERK1/2 bands on Western blots. Panels c and d: increased induction (band intensity in arbitrary units) of ERK1/2 as the mean ± SEM of five separate studies.

Figure 2

Effect of MEK1/2 inhibitor and mecamylamine on ERK1/2 induction in primary cells. Primary cells were incubated with MEK1/2 inhibitor, UO126 (10 µm) or nicotine receptor antagonist, mecamylamine (500 µm) for 30 min, after which the cells were incubated with 100 µm nicotine for 3 min. Cell extracts were prepared and used for Western blotting of total and phospho ERK1/2, as described in the legends of Figure 1. Panels a and b: representative ERK1/2 bands on Western blots. Panels c and d: induction (band intensity in arbitrary units) of phospho ERK1/2 as the mean ± SEM of five separate studies. *P < 0.05 between control and nicotine‐added samples; **P < 0.05 between nicotine‐added and UO126 + nicotine‐added samples.

Figure 3

Effect of nicotine on the proliferation of primary cells. Primary cells were seeded in 96‐well microplates, allowed to attach by overnight incubation, and were transferred to 0.05% serum prior to the study. Nicotine, at a concentration of 100 µm, was added, and cell proliferation was measured for 18–40 h, using the cell counting kit‐8 from Dojindo Molecular Technologies (panel a) or BrdU incorporation method (panel b) according to the manufacturer's instructions. Data points represent mean ± SEM of five separate studies for control (circles and solid line) and nicotine added (triangles and dashed line) samples. *P < 0.05.

Figure 4

Cell cycle study after nicotine treatment of primary cells. Primary pancreatic cells were treated with nicotine for 24 h and were sorted for their DNA contents using Guava EasyCyte mini flowcytometer and ‘Guava Cell cycle kit’. Panel a represents control cells. Panel b represents cells treated with 10 µm of ERK1/2 inhibitor, UO126. Panel c represents cells treated with 100 µm nicotine alone. Panel d represents cells treated with UO126 and nicotine. In each panel, region 1 (purple) represents cells in G₀ + G₁ phase, region 2 (green) represents cells in S phase and region 3 (blue) represents cells in G₂ + M phase. Cells on the left of region 1 (dark green) represent debris and sub‐G₀ cells, whereas cells on the right of region 3 represent aggregates. Percentages in different phases of the cell cycle were calculated, excluding debris and sub‐G₀ cells as well as aggregates.

Figure 5

Immunolocalization of phospho‐ERK1/2 in primary cells after nicotine treatment, as indicated by immunohistochemistry. Primary cells were treated with 100 µm of nicotine for 3 min. Cells were fixed with paraformaldehyde and were covered with antibody to phospho ERK1/2 for 1 h. After washing, they were treated with secondary antibody and labelled with FITC. Slides were observed under a fluorescence microscope. Panel a: primary cells untreated control. Panel b: primary cells following treatment with nicotine for 3 min. Panel c: primary cells following ERK1/2 inhibitor and nicotine treatment. The increase in phospho ERK1/2 is clearly seen following treatment with nicotine for 3 min. This is absent in cells treated with the ERK1/2 inhibitor, UO126. No corresponding increase in fluorescence was seen after nicotine treatment when antibody to total ERK1/2 was used.

Figure 6

Effect of nicotine treatment on primary cell function. Primary cells were washed, incubated at 37 °C with 100 µm nicotine for 0–10 min (panel a), or with various concentrations of nicotine (panel b), and then were incubated with CCK‐8 (10⁻⁹ M, stimulated, triangles with dashed line) or without CCK‐8 (un‐stimulated, circles with solid line) for 30 min. Amylase released into the medium was measured with Procion‐yellow starch as substrate. Data are presented as percentage initial content and represented as the mean ± SEM of four separate experiments. *P < 0.05.

Figure 7

Effect of ERK1/2 inhibitor and mecamylamine on primary cell function, with or without nicotine. Panel a: primary cells (4–6 × 10⁶) were washed and incubated at 37 °C with or without 10 µm UO126 for 30 min, followed by an additional incubation with nicotine for 6 min. The cells were washed and then incubated at 37 °C with or without CCK‐8 (10⁻⁹ M) for 30 min. Amylase released into the incubation medium was measured with Procion‐yellow starch as substrate. The data are presented as percentage initial content and represent the mean ± SEM of four experiments. *P < 0.05 between control and nicotine‐added samples. Panel b: primary cells (4–6 × 10⁶) were washed and incubated at 37 °C without or with 500 µm mecamylamine for 30 min, followed by additional 6 min incubation with nicotine. The rest of the procedure is similar to panel a. The data are presented as percentage initial content and represent the mean ± SEM of four experiments. *P < 0.05 between control and nicotine‐added samples; **P < 0.05 between nicotine‐added and mecamylamine + nicotine‐added samples.