6 X 10610 ml in selleck screening library culture media containing Hams F 12 nutri ent medium with 2 mM L glutamine, 1% anti biotic, 1. 5% sodium bicarbonate, and 10% fetal bovine serum albumin at 37 C in a 5% CO295% air Inhibitors,Modulators,Libraries at mosphere. On day one, the medium was changed to a serum free nutrient medium. Treatment of cells with nicotine The cells were treated with 10 uM to 500 uM of nicotine in 6. 0 ml of serum free medium for periods of 30 seconds to 10 minutes. Cells were pretreated with inhibitors Inhibitors,Modulators,Libraries for 30 minutes before adding nicotine. Amylase secretion from primary cells after nicotine treatment Cell function studies were performed with or without CCK at its maximal, previ ously determined stimulating dose. These studies were conducted with primary cells that were washed free of media with Hepes Ringer buffer, pH 7.

Inhibitors,Modulators,Libraries 4, incubated in the same buffer with or without nicotine for periods of 0 to 10 minutes and then washed twice with HR buffer to make the cells nico tine free. The cells were then dispersed in fresh HR buffer, incubated Inhibitors,Modulators,Libraries with or without CCK for 30 minutes at 37 C. The selection of this dose of CCK for was based on our initial study, which showed a maximal stimulated response of amylase release in a CCK dose response curve. Following the incubation period, the media was removed by centrifugation and analyzed for amylase activity by the method of with Procion yellow starch as substrate. Cell pellets were washed with ice cold PBS, lysed with water by sonication, and centrifuged. The cell lysate was analyzed for both amylase and protein content. Pro tein concentration was measured by the method of.

The amylase release was expressed as the fractional Inhibitors,Modulators,Libraries amount released from the total. Effect of mecamylamine or conotoxin on primary cell function, with or without nicotine Primary cells were washed and incubated at 37 C with or without 500 uM mecamylamine for 30 minutes, followed by an additional incubation with nico tine for 6 minutes. Cells were washed and then incu bated at 37 C with or without CCK 8 for 30 minutes. Amylase released into the incubation medium was measured with Procion yellow starch as substrate. The data are presented as % initial content and repre sented as the meanSEM of four experiments. For conotoxin experiment, primary cells were washed and incubated at 37 C without or with 80 mM conotoxin for 30 minutes, followed by additional 6 minutes incubation with nicotine.

The rest of the pro cedure was similar as described above. Effect of H 7 on primary toward cell function, with or without nicotine Primary cells were washed and incubated at 37 C with or without 10 uM H 7 for 30 minutes, fol lowed by an additional incubation with nicotine for 6 minutes. Cells were washed and then incubated at 37 C with or without CCK 8 for 30 minutes. Amyl ase released into the incubation medium was measured with Procion yellow starch as substrate.