Calciumsensitization of smooth musclemediated by a Rho-associated protein kinase in hypertension. Xanya Sofra Weiss

Masayoshi Uehata, Toshimasa Ishizaki, Hiroyuki Satoh, Takashi Ono, Toshio Kawahara, Tamami Morishita, Hiroki Tamakawa, Keiji Yamagami, Jun Inui, Midori Maekawa & Shuh Narumiya Discovery Research (Tokyo), Yoshitomi Pharmaceutical Industries Ltd, Iruma, Saitama 358, Japan Department of Pharmacology, Faculty of Medicine, Kyoto University, Yoshida, Kyoto 606-01, Japan
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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 myofilaments1: the former activates myosin light-chain kinase and the latter is achieved partly by inhibition of myosin phosphatase1–3. The small GTPase Rho and its target, Rho-associated kinase, participate in this latter mechanism in vitro4–6, 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, p160ROCK7. Y- 27632 consistently suppresses Rho-induced, p160ROCKmediated 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. We have developed a series of compounds that are potent relaxants of vascular and bronchial smooth muscles: Y-27632 is a representative compound (Fig. 1a) which potently inhibits phenylephrine- induced contraction of rabbit aortic strips, with a halfmaximal inhibitory concentration (IC50) of 0.7mM (Fig. 1b); however, it had little effect on contraction induced by potassium chloride (IC50 . 30mM). Y-27632 also inhibited the contraction of pig coronary artery strips and guinea-pig trachea induced by various agonists such as phenylephrine, histamine, acetylcholine, serotonin, endothelin and a thromboxane agonist, U-46619 (IC50 values of 0.3–1mM; data not shown). These results indicate that Y- 27632 is a specific and potent inhibitor of agonist-induced smoothmuscle contraction. The effects of this compound were next examined on permeabilized strips of rabbit mesenteric artery. In these preparations, Ca2+- dependent contraction can be induced by an increase in the extracellular Ca2+ concentrations, whereas addition of GTPgS can evoke contraction through a Ca2+-sensitization mechanism8. Y- 27632 inhibited GTPgS-induced contraction in a concentrationdependent way, like the inhibition of phenylephrine-induced contraction in intact strips, but it had no effect on Ca2+-induced contraction (Fig. 1c). Contraction in this system can also be evoked by inhibition of myosin phosphatase with a phosphatase inhibitor, calyculin A9. Y-27632 had no effect on this contraction up to 100mM, whereas a myosin light-chain kinase inhibitor, ML-9 (ref. 10), abolished this contraction (Fig. 1c, bottom trace). These results indicate that Y-27632 inhibits smooth-muscle contraction selectively by inhibiting the Ca2+-sensitization mechanism. To identify the molecular target of Y-27632, we did a radioligand binding assay using [3H]Y-30141, a 3H-labelled analogue of Y- 27632, and found specific binding in the membrane as well as in the cytosol (Fig. 2a). Scatchard analysis in the membrane yielded a single class of binding site (Kd of 8 nM, Bmax of 1.0 pmol mg-1 protein). This binding was efficiently displaced by a series of Y- 27632 analogues as well as by various protein kinase inhibitors.
Displacement potency correlated well with ability to inhibit phenylephrine- induced smooth-muscle contraction: a strong positive correlation was seen, with a slope close to unity (Fig. 2b). This binding protein is likely to be the target for Y-27632-induced inhibition of smooth-muscle contraction. To isolate this binding protein, we used 125I-labelled Y-35526, another Y-27632 analogue, as a photoaffinity ligand. Photoaffinity labelling with this compound gave several bands corresponding to relative molecular masses (Mr) of 150K–160K on SDS–polyacrylamide gel electrophoresis. This labelling was dependent on irradiation time and was inhibited by excess Y-30141 (Fig. 2c). A major 150K protein was purified and subjected to cyanogen bromide fragmentation. Partial amino-acid sequences of the 6.5K and 18K fragments were identified as XRKDFKIDRK (where X is N, D, A or G) and XLDIDKLFHV(XisAorV), respectively. These are identical to amino acids 1,011–1,020 and 1,167–1,176, respectively, of p160ROCK,
a Rho-associated coiled-coil forming kinase7. Consistently, this 150K Nature © Macmillan Publishers Ltd 1997 letters to nature NATURE |VOL 389 | 30 OCTOBER 1997 991 protein reacted with the anti-p160ROCK antibody on western blot analysis (data not shown). To confirm this result, we tested the effects of Y-27632 and related compounds on the kinase activity of p160ROCK purified from human platelets7. All these compoundsinhibited the kinase activity in a dose-dependent manner (Fig. 2d) and their inhibitory potency was well correlated with their ability to inhibit [3H]Y-30141 binding and agonist-induced contraction (data not shown). p160ROCK has an isoenzyme called either ROKa11, Rho kinase12 or ROCK-II13, which is ,90% identical to p160ROCK in its kinase domain. We therefore expressed these kinases in COS cells and examined the inhibition by Y-27632 and related compounds on their activity. Identical profiles and sensitivities were found for the two kinases towards Y-27632, Y-30141, Y-30964 and
HA1077. The Ki values of Y-27632 and related compounds for several kinases are compared in Table 1. Of the five compounds tested, Y-27632 showed the best selectivity: its affinity for p160ROCK was ,200 times or over 2,000 times higher than that for conventional protein kinase C (PKC) from rat brain or cAMPdependent protein kinase, or for myosin light-chain kinase, respectively. The affinity for PKCe, a protein kinase C isoenzyme, was a little higher but still 10–50-fold lower than that for p160ROCK (our unpublished observations). Y-27632 did not appreciably inhibit at
100 mMconcentration PAK14, a protein kinase activated by another Rho-family GTPase, Cdc42. Rho works as a molecular switch for the induction of focal adhesions and stress fibres in cultured cells15, and p160ROCK16 or
ROKa/Rho-kinase/ROCK-II17,18 work as Rho effectors in this process. We therefore tested whether Y-27632 could inhibit Rhoinduced formation of these structures in cultured cells. Treatment of cells with Y-27632 abolished focal adhesions and stress fibres induced not only by overexpression of p160ROCK but also by activated Rho in 30 min after the addition (Fig. 3a, b). It also inhibited induction of stress fibres by lysophosphatidic acid in cultured fibroblasts (data not shown). Y-30141 inhibited induction of this phenotype with a 10-fold higher potency. In addition to stress fibre formation, Rho exerts a variety of actions in the cell, including transcriptional regulation by serum-response factor19; Y- 27632, however, does not inhibit this transcriptional process (E. Sahai and R. Treisman, personal communication). Y-27632 did not inhibit Rac-induced membrane ruffling20 in cultured cells (Fig. 3c).
These findings show that Y-27632 can work as a specific inhibitor of p160ROCK in intact cells. Our results indicate that Y-27632 acts on p160ROCK in intact smooth muscle to inhibit agonist-induced contraction. We next analysed the importance of this mechanism and its pathophysiological role in the regulation of smooth muscle tone in vivo. Y-27632 was administered per os to various hypertensive rat models. As shown in Fig. 4a, this compound significantly decreased the blood.

Xanya Sofra Weiss

Xanya Sofra Weiss