Pulmonary neuroendocrine (PNE) cells were harvested and cultivated from peripheral lung tissue of 15 day old fetal hamsters under selective growth conditions, including a 10% CO2 atmosphere. Following 24 h of serum starvation under 10% CO2, PNE cells placed in a 5% CO2 atmosphere failed to proliferate over the next 24 h, while cells kept at 10% CO2 showed a 2.7-fold increase in cell number. Addition of nicotine to the cell culture medium resulted in an additional concentration-dependent increase in cell number under a 10% CO2 atmosphere, while nicotine did not stimulate the proliferation of cells maintained at 5% CO2. The nicotinic receptor antagonist hexamethonium completely blocked the stimulatory effect of nicotine on cell growth. As a first step towards determining the molecular mechanisms regulating the mitogenic stimulation of PNE cells by alterations in CO2/O-2 and nicotine, cells that had been serum starved under a 10% CO2 atmosphere for 24 h were removed from the incubator and either left untreated or treated with 1 mu M nicotine under ambient air. The cells were then returned to either a 5% or 10% CO2 atmosphere. Removal of the cells from a 10% CO2 environment and subsequent reintroduction to a high CO2 concentration resulted in a 3- to 4-fold increase in c-Sos RNA expression within 15-30 min upon return to the cell culture incubator. c-fos RNA levels their decreased back to control values by 1 h. Reintroduction into a 5% CO2 atmosphere, which failed to stimulate cell growth in the proliferation assay, caused only a 2-fold increase in the levels of c-fos transcripts. The CO2-mediated increases in c-Sos transcripts were observed both in the presence and absence of nicotine, suggesting that the effects of nicotine were mediated through a fos-independent pathway. Neither the alterations in CO2/O-2 concentration or treatment with nicotine altered the levels of c-myc or c-jun gene transcripts. Nicotine thus acts indirectly but synergistically with high CO2 concentrations to stimulate PNE cell proliferation.

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