THIN FILAMENTS AND VERTEBRATE SMOOTH MUSCLE CONTRACTION

调节蛋白通过赋予

Regulatory proteins control muscular contraction by conferring Ca2+-dependence on actin-myosin interactions with ATP. In vertebrate striated muscle, regulatory proteins (tropomyosin and troponin) have convincingly been shown to be linked to the actin-containing thin filaments. In contrast, vertebrate smooth muscle is, apparently, myosin-regulated whereby Ca2+-dependent phosphorylation of its light-chain subunits activates the actomyosin ATPase and the contractile event. Controversy exists, however, on the possible coexistence of thin filament-linked Ca2+-regulation in vertebrate smooth muscle. To date, several possible thin filament-linked regulatory proteins have been isolated in a number of laboratories, but they have not been demonstrated to be components of intact "native" thin filaments. We will report on the isolation of such native thin filaments from smooth muscle which contain besides actin and tropomyosin, caldesmon, a possible thin filament-linked regulatory protein. Caldesmon is a major calmodulin-binding protein found in smooth muscle and in non-muscle cells. It is known to influence actomyosin ATPase and to cause F-actin aggregation. We aim to assess caldesmon function and its possible modulating role as a component of the native thin filament. In these studies we will test its influence on actin-myosin interaction using established ATPase assay techniques. We will also attempt to determine the influence of caldesmon on tension development and maintenance using skinned fibers. Additionally we will try to reverse caldesmon effects using specific antibodies to caldesmon as inhibitors. We will also localize and determine possible periodicity of caldesmon in smooth muscle by staining native thin filaments with anti-caldesmon anti-bodies and by utilizing immunoelectron microscopic techniques. The dual-regulation of smooth muscle myosin and thin filaments may be involved in the fine-tuning of the contractile response, and disturbances of such modulation may contribute to the development of such disease processes as hypertension and asthma. Dual regulation also may be associated with the "latch" process, whereby smooth muscle tone is maintained at low energy cost.