To investigate the molecular mechanism that regulates the mechanical properties of smooth muscle, we determined the effect of forced expression of MLC 17a and MLC 17b on the rate of force activation during agonist-stimulated contractions of single cultured chicken embryonic aortic and gizzard smooth muscle cells. Forced expression of MLC 17a in aortic smooth muscle cells increased (p < 0.05) the rate of force activation, forced expression of MLC 17b in gizzard smooth muscle cells decreased (p < 0.05) the rate of force activation, while forced expression of the endogenous MLC 17 isoform had no effect on the rate of force activation. These results demonstrate that MLC 17 is a molecular determinant of the contractile properties of smooth muscle. MLC 17 could affect the contractile properties of smooth muscle by either changing the stiffness of the myosin lever arm or modulating the rate of a load-dependent step and/or transition in the actomyosin ATPase cycle.The mechanical properties of smooth muscle are broadly classified as tonic and phasic (3-5). Tonic smooth muscle has slow rates of force activation, force relaxation, maximum velocity of muscle shortening (V max ), and actomyosin ATPase, while phasic smooth muscle has rapid rates of force activation, force relaxation, V max , and actomyosin ATPase (3-5). The molecular basis for tonic and phasic contractile properties is unknown, but has been suggested to be regulated by a variety of factors including splice variant isoforms of the myosin heavy chain (MHC) 1 at the 25/50-kDa junction (1) and/or splice variant isoforms of the essential myosin light chain (2) producing an acidic (MLC 17a ) and basic (MLC 17b ) isoform. The rate of force activation (6, 7), V max (6, 7), the velocity of actin movement in the in vitro motility assay (1,8), and ATPase activity (1, 9) have been correlated with the expression of splice variant isoforms both of the MHC and MLC 17 . In this study, we tested the hypothesis that the mechanical properties of smooth muscle are regulated by splice variant expression of MLC 17 . We determined the effect of forced expression of MLC 17a and MLC 17b on the rate of force activation for agonist stimulated contractions of single cultured chicken embryonic aortic and gizzard smooth muscle cells (SMC).
EXPERIMENTAL PROCEDURESAorta and gizzard cells were isolated from primary cultures of ED 13-15 chicken embryos, as described previously (10). Briefly, after dissecting these tissues and removing the surrounding connective and epithelial tissues, gizzard or aorta were minced into fine pieces and resuspended in growth media (Dulbecco's modified Eagle's medium/ Ham's F-12, 1:1 mixture supplemented with 10% fetal calf serum and 50 units/ml penicillin, 50 g/ml streptomycin, Life Technologies, Inc.). The fine pieces in suspension medium were pipetted into culture dishes, and sedimented larger fragments were re-minced. This procedure was repeated for 2-4 cycles and all tissue fragments were incubated at 37°C with 5% CO 2. The culture media was changed the ne...