Phorbol Ester-Stimulated Contractile Responses

Given the fundamental importance of a second regulatory system in controlling smooth muscle contrac- 

The role of LC20 phosphorylation in phorbol ester-stimulated contractile responses has been largely resolved by defining the specific phosphorylated residues. In vitro studies using purified PKC and smooth muscle LC20 as a substrate identified Thr followed by Seri and/or 2 gg the primary PKC phosphorylation sites and phosphorylation of these sites was associated with a decrease in actomyosin ATPase activity (Ikebe et al., 1987). In contrast, MLCK is known to phosphory-late Seri as a primary site and Thri as a secondary site, both of which result in activation of actomyosin ATPase (Ikebe and Hartshorne, 1985). Stimulation of intact 32p-labeled bovine tracheal strips (Kamm et al., 

rabbit aortic rings (Singer etal., 1989), and swine carotid artery strips (Singer, 1990b BSrany et al., 1990) with high concentrations of phorbol ester ( 1 jlM) was found to increase LC20 phosphorylation primarily on Seri 2 with only low amounts of net Thr phosphor- 

The mechanism underlying the selective phosphorylation of Thr s is not clear, but it suggests the activity of an unidentified kinase. Based on these experiments and studies characterizing the mechanical properties of arterial smooth muscle activated by phor-bol esters (Fulginiti et al., 1993), it was concluded that phorbol ester-stimulated contraction in smooth muscle can occur by a mechanism that is independent of LC20 phosphorylation. 

Phosphorylation of LC20 in permeabilized gizzard smooth muscle by direct addition of PKC (Sutton and Haeberle, 1990) or its constitutively active proteolytic fragment, PKM (Parente et al., 1992), results in phosphorylation of LC20 mainly on Seri 2 with a small amount on Thr. The only mechanical response observed in those studies in response to direct addition of PKC or PKM was an attenuation of Ca +-induced contraction in permeabilized gizzard smooth muscle fibers (Parente et al., 1992). Phorbol ester-induced attenuation of contractile responses has been observed in ileal and uterine smooth muscle (Baraban et al., 

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Studies aimed at defining how phorbol ester activators of PKC stimulate contractile responses in smooth muscle have pointed to the existence of an unidentified mechanism for activating cross-bridges that is independent of LC20 phosphorylation of SeH. The... 

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