We show that an extract of a new green tea cultivar Cha Chuukanbohon Nou6 (Nou6), which rich in anthocyanin, has an inhibitory effect on myosin regulatory light chain (MRLC) phosphorylation in rat aortic smooth muscle cell line A7r5. The inhibitory effect of Nou6 extract was abolished when the extract was treated with polyvinylpyrrolidone which traps polyphenols. However, none of the major tea constituents tested had an inhibitory effect. In addition, we found that Nou6 extract had a strong effect on reducing the phosphorylation of myosin phosphatase targeting subunit 1 at Thr696. These results suggest that Nou6 extract inhibits MRLC phosphorylation induced by thrombin through activating myosin light chain phosphatase, and polyphenols in the tea extract may contribute to this effect.Keywords: anthocyanin, smooth muscle cell, myosin regulatory light chain, polyphenol, myosin light chain phosphatase, myosin phosphatase targeting subunit 1 *To whom correspondence should be addressed. E-mail: tatibana@agr.kyushu-u.ac.jp IntroductionContractile state of vascular smooth muscle cells is the primary determinant of blood vessel tone (Ogita et al., 2003). Contractile state has been shown to be directly related to the phosphorylation of myosin regulatory light chain (MRLC) at Ser-19 (Murthy et al., 2006). MRLC phosphorylation controls the activity of myosin II, a major motor protein in animal cells, which is involved in a wide range of processes, including muscle contraction, cell locomotion, cell division, and receptor capping (Maciver, 1996). Phosphorylation of MRLC is regulated by two classes of enzymes: myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) (Somlyo, Somlyo, 2003). MLCP is composed with three subunits: a 37-kDa catalytic subunit, a 20-kDa subunit of unknown function, and a myosin phosphatase targeting subunit 1 (MYPT1). The activity of myosin phosphatase is known to be regulated by phosphorylation of MYPT1, and two major sites, Thr696 and Thr853, have been extensively investigated and identified as inhibitory sites (Hartshorne et al., 2004).Tea (Camellia sinensis L.) has been consumed worldwide for a long time. Among the green tea polyphenols, (-)-epigallocatechin-3-O-gallate (EGCG) is the most abundant and most active polyphenol in inhibiting experimental carcinogenesis and related reactions (Yang et al., 2009). Recently, we have identified 67-kDa laminin receptor (67LR) as a cell surface EGCG receptor that mediates the anticancer action of EGCG (Tachibana et al., 2004). We also reported that EGCG induced reduction of the phosphorylation of MRLC at Thr18/ Ser19 through 67LR in HeLa cells (Umeda et al., 2005), human basophilic KU812 cells (Fujimura et al., 2006), mouse melanoma B16 cells , and human colon adenocarcinoma Caco-2 cells . We found that MYPT1 phosphorylation at Thr696, which inhibits myosin phosphatase and promotes MRLC phosphorylation, was reduced in response to EGCG .New green tea cultivars (Cha Chuukanbohon Nou6 and Sunrouge) are characterized as having a large anthocy...

Recently we found a small hepatocyte-specific protein, annexin A3 (AnxA3), in fractionated adult rat hepatocytes. Here we describe the results of an in vivo demonstration of AnxA3-expressing cellular phenotypes in the liver with 2-acetylaminofluoren (2-AAF)/carbon tetrachloride (CCl(4))-injury. In association with an elevation of alanine amino transferase (ALT) and aspartic acid amino transferase (AST) activities, hepatic AnxA3 mRNA increased markedly. AnxA3-positive cells were detected in clustered cells present in or emerging from the pericentral region. These albumin-expressed cells were histologically similar to cells expressing CD34, a hematopoietic cell marker protein. The number of clusters decreased in the days following CCl(4) treatment, and annexin-negative, but albumin-positive, oval cells appeared. We concluded that the agent-induced liver defect initially recruits bone marrow-derived cells, and that it promotes differentiation of these cells into AnxA3-positive cells, followed by emergence of the oval cells, which might have a role in the restitution of the damaged liver.