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Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, Chicago, Illinois 60637
We
tested the hypothesis that prolonged serum deprivation would allow a
subset of cultured airway myocytes to reacquire the abundant
contractile protein content, marked shortening capacity, and elongated
morphology characteristic of contractile cells within intact tissue.
Passage 1 or
2 canine tracheal smooth muscle (SM) cells were grown to confluence, then serum deprived for up to 19 days.
During serum deprivation, two differentiation pathways emerged.
One-sixth of the cells developed an elongated morphology and aligned
into bundles. Elongated myocytes contained cables of contractile
myofilaments, dense bodies, gap junctions, and membrane caveoli,
ultrastructural features of contractile SM in tissue. These cells
immunostained intensely for SM 
-actin, SM myosin heavy chain (MHC),
and SM22 (an SM-specific actin-binding protein), and Western analysis
of culture lysates disclosed 1.8 (SM -actin)-, 7.7 (SM MHC)-, and
5.8 (SM22)-fold protein increases during serum deprivation.
Immunoreactive M3 muscarinic
receptors were present in dense foci distributed throughout elongated,
SM MHC-positive myocytes. ACh
(10
3 M) induced a marked
shortening (59.7 ± 14.4% of original length) in 62% of elongated
myocytes made semiadherent by gentle proteolytic digestion, and
membrane bleb formation (a consequence of contraction) occurred in all
stimulated cells that remained adherent and so did not shorten.
Cultured airway myocytes that did not elongate during serum deprivation
instead became short and flattened, lost immunoreactivity for
contractile proteins, lacked the
M3 muscarinic-receptor expression
pattern seen in elongated cells, and exhibited no contractile response
to ACh. Thus we demonstrate that prolonged serum deprivation induces
distinct differentiation pathways in confluent cultured tracheal
myocytes and that one subpopulation acquires an unequivocally functional contractile phenotype in which structure and function resemble contractile myocytes from intact tissue.
phenotypic modulation; serum deprivation; contraction; ultrastructure; heterogeneity; muscarinic receptors; contractile proteins
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