CAUSALES DEL RECURSO Y SUS FUNDAMENTOS.

Uterine, similar to other smooth muscles, are composed of two major contractile proteins; thick and thin filaments, with thin filaments composed of actin and tropomyosin along with the regulatory proteins caldesmon and calponin (Hodgkinson 2000). Smooth muscle generate force with cross-bridge cycling as found in striated muscle, and contains generally the same contractile apparatus, with a few obvious exceptions; troponin is not expressed in smooth muscle, and the addition of the regulatory protein caldesmon, calponin and calmodulin. The structural organisation is very different, while striated muscles are composed of highly structured sarcomeres, smooth muscle is less organised, and maintained by dense bodies (Ashton et al. 1975; Ali et al. 2005)(See figure 1.5.2 for schematic diagram of contractile proteins and organisation).

1.5.2.1 Myosin (thick filaments)

Myosin comprises a large family of ATPases, who in conjunction with actin are responsible for the movement during cross-bridge cycling. Myosin II is responsible for the contraction of muscle, and there are multiple homologues, having the same basic structure; hexamers composed of two myosin heavy chains (230KDa) and two pairs of myosin light chains (MLC) (17 and 20KDa). The two heavy chains form an α-helical coiled-coiled structure that includes the sites of myosin molecule association for filament formation, and extends through the hinge region (converter domain), responsible for the movement of the globular head, into the globular head, which is the site of actin binding and ATP hydrolysis (Eddinger et al. 2007). The MLCs are associated with the globular head region of the heavy chain, were MLC20 is thought to inhibit the actin-activated ATPase, upon phosphorylation, at either

Ser19 or Thr18, there is a conformational change and this inhibition is removed (Ikebe et al. 1983; Ikebe et al. 1988).

1.5.2.2 Thin filaments

While myosin phosphorylation is the determining factor in smooth muscle contractility, it is the smooth muscle thin filaments that are thought to provide the fine tuning regulation required for effective contractility. Smooth muscle thin filaments are composed of actin, tropomyosin, caldesmon and calmodulin in ratios of 14:2:1:1. All four components are needed for correct cross-bridge cycling and its regulation.

The principal component of thin filaments is actin. In smooth muscle, actin is made up of smooth muscle α-actin (ACTA2 gene) and γ-actin (ACTAG2 gene), with proportions

dependent upon the smooth muscle (Marston et al. 2008). Actin is a highly conserved protein with a molecular mass of 42 KDa. With all actin monomers (globular G-actin) able to polymerise and form fibrillar F-actin macromolecules, forming a right handed helix composed of two strands that cross every 36 nm, containing 13 G-actin (Hodgkinson et al. 1997a; Hodgkinson et al. 1997b; Hodgkinson 2000). Each G-actin has a high-affinity myosin head binding site.

Tropomyosin is the second most abundant thin filament protein. There are two smooth muscle isoforms α- and β- tropomyocin, produced by alternative splicing. They are approximately 284 amino acids in length, existing as heterodimers in a predominantly α- helical coiled-coiled structure, wrapped around the α-helical actin strands (Jancso et al. 1991; Hodgkinson 2000).

Caldesmon is the third most abundant thin filament protein, after actin and tropomyocin. It is an elongated molecule of approximately 87 KDa, composed of three domains. The C- terminal region is considered to be the most important domain with primary binding sites for; actin, calmodulin and other phosphorylation sites (Wang 2001). While the N-terminal is also able to interact with actin and calmodulin, it can only do this weakly, it also contains a myosin binding domain (Wang 2001). The two regions are interspaced by a middle spanning region, composed of a highly charge repeating sequence with no clear function. It is this spanning region that gives the shape to caldesmon, appearing as a stretch dumbbell (Wang 2001).

Caldesmon acts to inhibit myosin ATPase activity (Ngai et al. 1984) acting as a break to myometrial contractility. In conjunction with its role as an inhibitor to myometrial contractility, expression is significantly increased in pregnancy (Word et al. 1993).It is

thought to act as a myometrial contractile inhibitor by tethering actin and myosin together and blocking their direct interaction. The N-terminal region of caldesmon binds myosin, while its C-terminal binds actin, in a low [Ca2+]i /unstipulated state this configuration blocks

the binding of unphosphorylated myosin heads to actin. Upon stimulation, Ca2+-calmodulin interacts with caldesmon resulting in the movement of caldesmon away from actin/myosin interaction sites (Wang 2001). In addition to Ca2+-calmodulin inhibition of caldesmon, it is also able to be phosphorylated in a non-Ca2+ dependent manner by PKC and protein kinase II (Gorenne et al. 2004).

Calponin is another regulatory protein associated with the thin filaments, and found in all smooth muscles. It is a monomeric protein of 34 KDa (Winder et al. 1993), with similar binding characteristic to caldesmon, able to interact with actin (Childs et al. 1992) myosin (Szymanski et al. 1997) and Ca2+-calmodulin (Winder et al. 1993). Unlike caldesmon, it appears that the only physiological relevant interaction partner is actin (Winder et al. 1993). Calponin inhibits the myosin ATPase activity rather than reducing Ca2+-dependent myosin phosphorylation (Winder et al. 1990) and is thought to involve inhibition of a catalytic step ATPase cycle, most probably the rate limiting step of ADP or inorganic phosphate (Pi) release.

Calponin itself is regulated by phosphorylation, resulting in the abolishment of calponin inhibition on the actin-activated ATPase (Winder et al. 1993). Both PKC and calmodulin kinase II have been shown to phosphorylate calponin (Winder et al. 1990) while dephosphorylated by a type 2A protein serine/threonine phosphatase (Winder et al. 1992).