Abnormal smooth-muscle contractility may be a major cause of disease states such as hypertension, and a smooth-muscle relaxant that modulates this process would be useful therapeutically. Smooth-muscle contraction is regulated by the cytosolic Ca2+ concentration and by the Ca2+ sensitivity of myofilaments: the former activates myosin light-chain kinase and the latter is achieved partly by inhibition of myosin phosphatase. The small GTPase Rho and its target, Rho-associated kinase, participate in this latter mechanism in vitro, but their participation has not been demonstrated in intact muscles. Here we show that a pyridine derivative, Y-27632, selectively inhibits smooth-muscle contraction by inhibiting Ca2+ sensitization. We identified the Y-27632 target as a Rho-associated protein kinase, p160ROCK. Y-27632 consistently suppresses Rho-induced, p160ROCK-mediated formation of stress fibres in cultured cells and dramatically corrects hypertension in several hypertensive rat models. Our findings indicate that p160ROCK-mediated Ca2+ sensitization is involved in the pathophysiology of hypertension and suggest that compounds that inhibit this process might be useful therapeutically.

Genomic typing of class I HLA alleles adds substantially to the success of transplantation of hematopoietic stem cells from unrelated donors, even if the donors are serologically identical to their recipients with respect to HLA-A, B, and DR antigens.

A p21Cip1/Waf1/Sdi1 is known to act as a negative cellcycle regulator by inhibiting kinase activity of a variety of cyclin-dependent kinases. In addition to binding of the cyclin-dependent kinase to the N-terminal region of p21, p21 is also bound at its C-terminal region by proliferating cell nuclear antigen (PCNA), SET/TAF1, and calmodulin, indicating the versatile function of p21. In this study, we cloned cDNA encoding a novel protein named TOK-1 as a p21 C-terminal-binding protein by a twohybrid system. Two splicing isoforms of TOK-1, TOK-1␣ and TOK-1␤, comprising 322 and 314 amino acids, respectively, were co-localized with p21 in nuclei and showed a similar expression profile to that of p21 in human tissues. TOK-1␣, but not TOK-1␤, directly bound to the C-terminal proximal region of p21, and both were expressed at the G 1 /S boundary of the cell cycle. TOK-1␣ also preferentially bound to an active form of cyclin-dependent kinase 2 (CDK2) via p21, and these made a ternary complex in human cells. Furthermore, the results of three different types of experiments showed that TOK-1␣ enhanced the inhibitory activity of p21 toward histone H1 kinase activity of CDK2. TOK-1␣ is thus thought to be a new type of CDK2 modulator.Cell-cycle movement is known to be coordinately regulated by several combinations of cyclin-cyclin-dependent kinase (CDK) 1 complex and their inhibitors. INK4 family proteins, including p15, p16, p18, and p19, inhibit CDK4/CDK6, and Cip/Kip family proteins, including p21, p27, and p57, inhibit all of the CDKs (see recent reviews, Refs. 1-3 and references therein). A cDNA of p21Cip1/Waf1/Sdi1 was cloned independently by different three procedures: Cip1 (4), a cyclin-dependent kinase 2 (CDK2)-binding protein; Waf1, p53-inducible protein (5); and Sdi1, a senescent-inducible protein (6). The p21 gene is induced dependently or independently by p53 in cells after various stresses. p53-dependent p21 induction occurs by x-ray or UV irradiation, which causes DNA damage, and by heat shock or osmotic shock to cells (5). Nutrition starvation, contact inhibition, terminal differentiation, or aging of cells triggers p53-independent p21 induction that is brought about by transcription factors, including STAT family protein (7), C/EBP␣ (8), MyoD (9, 10), and vitamin D3 receptor (11). In any case, p21 induces cell cycle arrest, thus inhibiting CDK activity necessary for Rb inactivation.In addition to binding of CDK-cyclin to the N-terminal region of p21 (12-16), a variety of proteins were found to bind to the C-proximal region of p21. DNA replication activity of DNA polymerase ␦ is inhibited by p21 by binding to a proliferating cell nuclear antigen (PCNA), indicating that p21 also has the function of stopping the cell cycle during the S phase (12,13,15,(17)(18)(19)(20). Furthermore, SET/TAF1 (21), human papilloma virus E7 (22, 23), c-Myc (24), and calmodulin (25) are reported to bind to the C-terminal region of p21. The molecular mechanisms explaining the versatile functions of p21, however, remain to be determin...

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