In the digestive tract, the transit of ingested food induces a local contraction-relaxation reflex of which the smooth muscle cell (SMC) represents the functional unit. Although freshly isolated SMCs have been extensively used for in vitro studies, in specific cases cultured cells appear necessary. Because conventionally cultured SMCs lose their contractile properties, we have developed: (1) differentiated, contractile rabbit gastric SMCs (D-stim cells), cultured in a medium supplemented with insulin, and (2) proliferative, dedifferentiated rabbit gastric SMCs (P-stim cells), cultured in a medium supplemented with insulin, fetal serum, EGF and b-FGF. The proliferative index was 5 +/- 4% and 82 +/- 10%, respectively, for D-stim and P-stim cells. Expression of SM-myosin heavy chain was observed in 90% of D-stim cells, whereas it was progressively lost in P-stim cells. Carbachol (1-100 nM), glicentin (2 nM) and gastrin-17 (100 nM) induced contraction of D-stim cells cultured for 3 or 6 days, whereas they did not induce the contraction of P-stim cells; in contrast, gastrin-17 (10 nM) was able to stimulate DNA synthesis (1.86 +/- 0.09-fold increase) in P-stim cells. The coupling of muscarinic receptors to intracellular transduction pathways was evaluated in D-stim cells: at day 3, carbachol (100 nM) induced a twofold increase in the production of inositol tri-tetra-phosphates; in parallel, a phosphorylation of ERK MAP kinases occurred within 1 min of carbachol stimulation. In conclusion, cultured functional myocytes derived from mature tissue may be used for long-term studies concerning the events coupled either to proliferation or to motility regulation of differentiated SMCs due to the activation of G-protein-coupled receptors.
Copyright 2004 Springer-Verlag