Last Month's Most Accessed Feature: Pancreatic poser: Interspecies effects of nicotine on proteome

Nicotine and pancreatic disease

One of the many ways in which cigarette smoke affects our bodies is its tendency to initiate diseases of the pancreas, specifically pancreatitis, pancreatic fibrosis and pancreatic cancer. Although it has not been strongly associated with these diseases, nicotine is strongly suspected of having a role, given its relatively high concentration in tobacco smoke. It has been identified in pancreatic fluid and is known to bind to many cell receptors to influence their behaviour.

A team of scientists in the US has decided to investigate the significance of nicotine by conducting the first cross-species study of its effects on pancreatic cells. Specifically, they examined the proteomes of rat, mouse and human pancreatic stellar cells that had been stimulated with nicotine. Sustained activation of these cells, named after their star shape, has been implicated in the progression of chronic pancreatitis and pancreatic cancer.

The study design and results were described in Proteomics by senior reporter Hanno Steen from Boston Children's Hospital. A mass spectrometric-based procedure was used to identify as many proteins as possible and compare them between the species.

Spectral counting

The cell lines for each species were incubated for 24 hours in the absence or presence of nicotine. The proteins were extracted from the cell homogenates and reduced and alkylated for separation by SDS-PAGE. Each gel lane was divided into six and the proteins present were digested on the gel to produce a complex mixture of peptides.

The individual peptides in each digest were analysed by liquid chromatography-tandem mass spectrometry, using the UniProt database to identify the proteins from rat, mouse and human sections.

The relative abundances of the proteins were estimated by spectral counting, comparing the number of tandem mass spectra for the same protein over the various data sets. In this way, an average of 1000 proteins were identified, with 1247 from murine cells, 921 from rat cells and 1586 from human stellate cells.

Protein pathways

When all of the proteins from the untreated and nicotine-treated cells were considered together, there was a large overlap between those found across more than one species, with about 75% found in two species and approximately 50% found in all three.

Given this commonality, the researchers were surprised to find that only six proteins were identified in the nicotine-treated cells of all three species, and 38 in at least two species. Equally, only two proteins from the untreated cells were found in all species and 47 in at least two of them.

Three of the proteins which were more abundant following nicotine treatment were involved in cytokine signalling, which occurs during the activation of pancreatic stellar cells and the onset of pancreatic fibrosis. They are amyloid beta, toll-interacting protein and integral membrane protein 2B. A fourth protein common to all species was procollagen type VI alpha1, which helps to form the extracellular matrix, one of the initial steps in pancreatic fibrosis.

These exclusive proteins and those which had different abundances in the untreated and nicotine-treated cells played key roles in the primary metabolism and nucleic acid binding in the cells. Other enriched proteins were involved with cytokine, integrin and nicotinic acetylcholine receptor pathways.

These preliminary results show that nicotine affects similar pathways in the pancreatic cells of rats, mice and humans in a similar way but the low number of common proteins indicates that the similarity does not extend to the level of individual proteins, given the low number of common ones identified.

The information will help to gain further insight into the molecular mechanisms that are brought into play when nicotine is involved in pancreatic disease. However, there is much more to be done. Improved fractionation of the peptides and proteins, variation of the nicotine concentration and incubation times, and special experiments that target particular proteins will help to confirm the results obtained so far and provide a clearer picture.