Muscarinic receptor expression in the primary culture of tracheal smooth muscle cells

Tracheal smooth muscle cells (TSMCs) were isolated from dog trachea in order to analyze the direct effects of growth factors and hormones on cell proliferation and muscarinic receptor (mAchR) expression. Dissection and dissociation of tracheal smooth muscle tissue with a collagenase I, deoxyribonuclease I and elastase IV mixture resulted in high yield and viability of TSMCs. A screen of growth factors, hormones, and serum concentration for the stimulation of cell growth, reveald that insulin-like growth factor, basic fibroblast growth factor, epidermal growth factor, insulin, transferrin, or hydrocortisone alone at the concentration used was not necessary or sufficient to stimulate growth of TSMCs in the primary culture with DMEM/F-12 containing 1% FBS. The regulation of cell surface mAchR expression in response to serum and cell growth in primary culture of TSMCs has been examined. In the presence of 1% serum, TSMCs withdraw from the cell cycle and express high levels of cell surface mAchRs. Exposure of quiescent TSMCs to 10% serum results in a loss of surface mAchRs. In addition, insulin-like growth factor, insulin or transferrin could stimulate the expression of mAchRs on TSMCs cultured in DMEM/F-12 containing 1% FBS. The results demonstrated that low serum concentration culture system may provide a useful model to elucidate the expression of mAchRs in the culture of TSMCs.