Divergent differentiation paths in airway smooth muscle culture: inductionof functionally contractile myocytes

We tested the hypothesis that prolonged serum deprivation would allow a sub set of cultured airway myocytes to reacquire the abundant contractile prote in content, marked shortening capacity, and elongated morphology characteri stic of contractile cells within intact tissue. Passage 1 or 2 canine trach eal smooth muscle (SM) cells were grown to confluence, then serum deprived for up to 19 days. During serum deprivation, two differentiation pathways e merged. One-sixth of the cells developed an elongated morphology and aligne d into bundles. Elongated myocytes contained cables of contractile myofilam ents, dense bodies, gap junctions, and membrane caveoli, ultrastructural fe atures of contractile SM in tissue. These cells immunostained intensely for SM alpha-actin, SM myosin heavy chain (MHC), and SM22 (an SM-specific acti n-binding protein), and Western analysis of culture lysates disclosed 1.8 ( SM alpha-actin)-, 7.7 (SM MHC)-, and 5.8 (SM22)-fold protein increases duri ng serum deprivation. Immunoreactive M-3 muscarinic receptors were present in dense foci distributed throughout elongated, SM MHC-positive myocytes. A Ch (10(-3) M) induced a marked shortening (59.7 +/- 14.4% of original lengt h) in 62% of elongated myocytes made semiadherent by gentle proteolytic dig estion, and membrane bleb formation (a consequence of contraction) occurred in all stimulated cells that remained adherent and so did not shorten. Cul tured airway myocytes that did not elongate during serum deprivation instea d became short and flattened, lost immunoreactivity for contractile protein s, lacked the M-3 muscarinic-receptor expression pattern seen in elongated cells, and exhibited no contractile response to ACh. Thus we demonstrate th at prolonged serum deprivation induces distinct differentiation pathways in confluent cultured tracheal myocytes and that one subpopulation acquires a n unequivocally functional contractile phenotype in which structure and fun ction resemble contractile myocytes from intact tissue.