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.

Prostanoids are the cyclooxygenase metabolites of arachidonic acid and include prostaglandin (PG) D(2), PGE(2), PGF(2alpha), PGI(2), and thromboxne A(2). They are synthesized and released upon cell stimulation and act on cells in the vicinity of their synthesis to exert their actions. Receptors mediating the actions of prostanoids were recently identified and cloned. They are G protein-coupled receptors with seven transmembrane domains. There are eight types and subtypes of prostanoid receptors that are encoded by different genes but as a whole constitute a subfamily in the superfamily of the rhodopsin-type receptors. Each of the receptors was expressed in cultured cells, and its ligand-binding properties and signal transduction pathways were characterized. Moreover, domains and amino acid residues conferring the specificities of ligand binding and signal transduction are being clarified. Information also is accumulating as to the distribution of these receptors in the body. It is also becoming clear for some types of receptors how expression of their genes is regulated. Furthermore, the gene for each of the eight types of prostanoid receptor has been disrupted, and mice deficient in each type of receptor are being examined to identify and assess the roles played by each receptor under various physiological and pathophysiological conditions. In this article, we summarize these findings and attempt to give an overview of the current status of research on the prostanoid receptors.

The actin cytoskeleton undergoes extensive remodeling during cell morphogenesis and motility. The small guanosine triphosphatase Rho regulates such remodeling, but the underlying mechanisms of this regulation remain unclear. Cofilin exhibits actin-depolymerizing activity that is inhibited as a result of its phosphorylation by LIM-kinase. Cofilin was phosphorylated in N1E-115 neuroblastoma cells during lysophosphatidic acid-induced, Rho-mediated neurite retraction. This phosphorylation was sensitive to Y-27632, a specific inhibitor of the Rho-associated kinase ROCK. ROCK, which is a downstream effector of Rho, did not phosphorylate cofilin directly but phosphorylated LIM-kinase, which in turn was activated to phosphorylate cofilin. Overexpression of LIM-kinase in HeLa cells induced the formation of actin stress fibers in a Y-27632-sensitive manner. These results indicate that phosphorylation of LIM-kinase by ROCK and consequently increased phosphorylation of cofilin by LIM-kinase contribute to Rho-induced reorganization of the actin cytoskeleton.

We have identified a novel gene containing CAG repeats and mapped it to chromosome 14q32.1, the genetic locus for Machado-Joseph disease (MJD). In normal individuals the gene contains between 13 and 36 CAG repeats, whereas most of the clinically diagnosed patients and all of the affected members of a family with the clinical and pathological diagnosis of MJD show expansion of the repeat-number (from 68-79). Southern blot analyses and genomic cloning demonstrates the existence of related genes. These results raise the possibility that similar abnormalities in related genes may give rise to diseases similar to MJD.

Prostaglandin (PG) E 2 exerts its actions by acting on a group of G-protein-coupled receptors (GPCRs). There are four GPCRs responding to PGE 2 designated subtypes EP1, EP2, EP3, and EP4 and multiple splicing isoforms of the subtype EP3. The EP subtypes exhibit differences in signal transduction, tissue localization, and regulation of expression. This molecular and biochemical heterogeneity of PGE receptors leads to PGE 2 being the most versatile prostanoid. Studies on knock-out mice deficient in each EP subtype have defined PGE 2 actions mediated by each subtype and identified the role each EP subtype plays in various physiological and pathophysiological responses. Here we review recent advances in PGE receptor research. Prostanoids including various prostaglandins (PGs)2 and thromboxanes (TXs) are cyclooxygenase (COX) metabolites of C20-unsaturated fatty acids such as arachidonic acid. These substances are synthesized in response to various stimuli in a variety of cells, released immediately after synthesis, and act in the vicinity of their synthesis to maintain local homeostasis (1). Among prostanoids, the E type PGs, particularly PGE 2 derived from arachidonic acid, is most widely produced in the body, most widely found in animal species, and exhibits the most versatile actions. Receptors mediating prostanoid actions were characterized first by pharmacological analysis, which indicated the presence of one receptor each, named DP, FP, IP, and TP, for PGs of the D, F, and I types and TXA, respectively, and four different receptors designated EP1, EP2, EP3, and EP4 for the E type PGs (reviewed in Refs. 2 and 3). Molecular identification of these receptors was achieved by their cDNA cloning, which revealed that the prostanoid receptors are G-proteincoupled receptors (GPCRs) and that there is indeed a family of eight GPCRs that correspond to the pharmacologically defined receptors. In addition, a recent study revealed the presence of the ninth prostanoid receptor that belongs not to the prostanoid receptor family described above but to the chemoattractant receptor family (4). This receptor called CRTH2 or DP2 is expressed in Th2 cells and eosinophils and mediates some of the PGD 2 actions on these cells such as chemotaxis. cDNA cloning also revealed the presence of several splicing variants for EP3. Thus, there are four GPCRs designated subtypes EP1, EP2, EP3, and EP4 and EP3 variants mediating PGE 2 actions. Subsequent analysis has revealed distinct biochemical properties and tissue and cellular localization of each EP subtype. The cloned EP subtypes have also been used in the development of compounds specific to each subtype. Biochemical Properties of PGE Receptor Subtypes and IsoformsMolecular Structures- Fig. 1 shows an alignment of the primary amino acid sequences of the mouse EP1, EP2, and EP4 and three isoforms of mouse EP3 receptors. The mouse EP1, EP2, EP3 (EP3␣), and EP4 receptors consist of 405, 362, 366, and 513 amino acids, respectively. EP4 has the longest intracellular C terminus and a relatively l...