Abstract
Mixing feed fibroblasts with soluble collagen and serum-supplemented culture medium at 37 degrees C results in the entrapment of cells within the polymerizing collagen matrix. This cellular-collagen complex is referred to as a fibroblast-populated collagen lattice (FPCL). In time, this FPCL undergoes a reduction in size called lattice contraction. The proposed mechanism for lattice contraction is cellular force produced by cytoplasmic microfilaments which organize collagen fibrils compacting the matrix. When the regulatory subunits of myosin, myosin light chains, are phosphorylated by myosin light chain kinase (MLCK), myosin ATPase activity is increased and actin-myosin dynamic filament sliding occurs. Elevated levels of myosin ATPase are required for maximal lattice contraction. Cholera toxin inhibits lattice contraction by increasing intracellular levels of cAMP. It is proposed that increased cytoplasmic concentrations of cAMP promote phosphorylation of MLCK, the enzyme important for maximizing myosin ATPase activity. Phosphorylating MLCK in vitro inhibits activity by decreasing its sensitivity to calcium-calmodulin complex. A decrease in MLCK activity would result in lower levels of myosin ATPase activity. MLCK, purified from turkey gizzard, was subjected to limited proteolytic digestion to produce calmodulin-independent-MLCK. The partially digested kinase does not require calcium-calmodulin for activation. Independent-MLCK is not subject to inhibition by phosphorylation. The electroporetic inoculation of independent-MLCK into fibroblasts before FPCL manufacture produced enhanced lattice contraction. Lattice contraction, in the presence of cholera toxin, was restored to normal levels by the prior electroporetic introduction of independent-MLCK. These findings support the hypothesis that increases in cAMP hinder lattice contraction by a mechanism involving inhibition of MLCK and myosin ATPase.
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