Motility disorders are frequently observed in intestinal inflammation. We previously reported that in vitro treatment of intestinal smooth muscle tissue with IL-1beta decreases the expression of CPI-17, an endogenous inhibitory protein of smooth muscle serine/threonine protein phosphatase, thereby inhibiting contraction. The present study was performed to examine the pathophysiological importance of CPI-17 expression in the motility disorders by using an in vivo model of intestinal inflammation and to define the regulatory mechanism of CPI-17 expression by proinflammatory cytokines. After the induction of acute ileitis with 2,4,6,-trinitrobenzensulfonic acid, CPI-17 expression declined in a time-dependent manner. This decrease in CPI-17 expression was parallel with the reduction of cholinergic agonist-induced contraction of smooth muscle strips and sensitivity of permeabilized smooth muscle fibers to Ca(2+). Among the various proinflammatory cytokines tested, TNF-alpha and IL-1beta were observed to directly inhibit CPI-17 expression and contraction in cultured rat intestinal tissue. Moreover, both TNF-alpha and IL-1beta inhibited CPI-17 expression and contraction of smooth muscle tissue isolated from wild-type and IL-1alpha/beta double-knockout mice. However, IL-1beta treatment failed to inhibit CPI-17 expression and contraction in TNF-alpha knockout mice. In beta-escin-permeabilized ileal tissues, pretreatment with anti-phosphorylated CPI-17 antibody inhibited the carbachol-induced Ca(2+) sensitization in the presence of GTP. These findings suggest that CPI-17 was downregulated during intestinal inflammation and that TNF-alpha plays a central role in this process. Downregulation of CPI-17 may play a role in motility impairments in inflammation.

In addition to its kinase activity, the myosin light chain kinase (MLCK) of smooth muscle has an actin binding activity through which it can regulate the actinmyosin interaction of smooth muscle (Kohama, K., Okagaki, T., Hayakawa, K., Lin, Y., Ishikawa, R., Shimmen In addition to this kinase activity, MLCK can act as an actin-binding protein; MLCK is present in association with the sarcomeric I-band (2), and it binds to actin filaments with a high affinity (3-5). We have shown that MLCK can inhibit the ATP-dependent interaction between actin and myosin by binding to actin filaments. This inhibition can be relieved by Ca 2ϩ /CaM, as was demonstrated by avoiding the complication derived from its kinase activity (6 -8), although the demonstration was limited to in vitro only.Such an inhibition, however, is not peculiar to MLCK. Caldesmon (see Ref. 9 for a review) and calponin (10) The relationship between the structure of MLCK and the function of the kinase activity in phosphorylating the myosin light chain and how Ca 2ϩ /CaM modulates the activity has been well established (see Ref. 13 for a review). However, there has been little study of the structure-function relationship of the actin binding activity except for the purification of an actinbinding fragment from MLCK (14).In this study, we have investigated which sequence of MLCK is responsible for the actin binding activity, which sequence inhibits the actin-myosin interaction, and which sequence binds CaM to relieve the inhibition. Our approach has been to cleave MLCK to prepare native fragments containing the actin binding activity (14), to design MLCK cDNA to express the recombinant fragments of actin binding activity in Escherichia coli, and then to analyze them biochemically. A few peptides have been synthesized to confirm these analyses. We have shown that: (i) MLCK has two actin-binding sites on the Nterminal side away from the central kinase domain, (ii) the binding site responsible for the inhibitory effect is at the Met MATERIALS AND METHODSPreparation of Proteins-All procedures were carried out at 0 -4°C. The purity of proteins was routinely monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (see below) so * This work was supported in part by grants from the Fujisawa Foundation and the Mitsubishi Foundation and by grants-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.ʈ To whom correspondence should be addressed. Tel.: 81-27-220-7960; Fax: 81-27-235-1401 or 81-27-220-7962. 1 The abbreviations used are: MLCK, myosin light chain kinase; CaM, calmodulin; PAGE, polyacrylamide gel electrophoresis; NTCB fragment, chicken gizzard MLCK fragment produced by the cleavage with 2-nitro-5-thiocyanatobenzoic acid; NN fragment, expressed fragment (amino acids 1-337 ...

1 Ticlopidine is a well-known anti-platelet agent, but is not active by itself in vitro. We identified a metabolite with anti-platelet activity, which was generated after incubation of 2-oxo-ticlopidine with phenobarbital-induced rat liver homogenate in vitro. 2 An active moiety (UR-4501) was isolated by high-performance liquid chromatography after largescale preparation of metabolites. 3 The chemical structure of UR-4501 was determined by a combination of liquid chromatography mass/mass spectrometry (LC/MS/MS) and nuclear magnetic resonance (NMR) analysis. 4 UR-4501 produced a concentration-dependent inhibition (3-100 mM) of ADP (10 mM)-induced human platelet aggregation, whereas 2-oxo-ticlopidine (3-100 mM) did not elicit inhibitory responses. 5 UR-4501 (10-100 mM) strongly inhibited ADP-and collagen-induced aggregation and slightly inhibited thrombin-induced aggregation. 6 The inhibition of rat washed platelet aggregation by UR-4501 (100 mM) persisted, even after the platelets had been washed twice. 7 These results suggest that UR-4501 is the molecule responsible for the in vivo activities of ticlopidine.

We constructed a plasmid vector having a 1.4-kilobase pair insert of myosin light chain kinase (MLCK) cDNA in an antisense direction to express antisense mRNA. The construct was then transfected to SM3, a cell line from vascular smooth muscle cells, producing a few stable transfectants. The down-regulation of MLCK expression in the transfectants was confirmed by both Northern and Western blots. The control SM3 showed chemotaxic motility to platelet-derived growth factor-BB, which was supported by lamellipodia. However, the transfectants showed neither chemotaxic motility nor developed lamellipodia, indicating the essential role of MLCK in the motility. The specificity for the targeting was assessed by a few tests including the rescue experiment. Despite this importance of MLCK, platelet-derived growth factor-BB failed to induce MLC20 phosphorylation in not only the transfectants but also in SM3. The mode in which MLCK was involved in the development of membrane ruffling is discussed with special reference to the novel property of MLCK that stimulates the ATPase activity of smooth muscle myosin without phosphorylating its light chain (Ye, L.-H., Kishi, H., Nakamura, A., Okagaki, T., Tanaka, T., Oiwa, K., and Kohama, K. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6666 -6671).

Abstract-Hypercholesterolemia is a major risk factor involved in abnormal cardiovascular events. Rho-kinase-mediated Ca 2ϩ sensitization of vascular smooth muscle (VSM) plays a critical role in vasospasm and hypertension. We recently identified sphingosylphosphorylcholine (SPC) and Src family tyrosine kinase (Src-TK) as upstream mediators for the Rho-kinase-mediated Ca 2ϩ sensitization. Here we report the strong linkage between cholesterol and the Ca 2ϩ sensitization of VSM mediated by a novel SPC/Src-TK/Rho-kinase pathway in both humans and rabbits. The extent of the sensitization correlated well with the total cholesterol or low-density lipoprotein cholesterol levels in serum.However, an inverse correlation with the serum level of high-density lipoprotein cholesterol was observed, and a correlation with other cardiovascular risk factors was nil. When cholesterol-lowering therapy was given to patients and rabbits with hypercholesterolemia, the SPC-induced contractions diminished. Depletion of VSM cholesterol by ␤-cyclodextrin resulted in a loss of membrane caveolin-1, a marker of cholesterol-enriched lipid raft, and inhibited the SPC-induced Ca 2ϩ sensitization and translocation of Rho-kinase from cytosol to the cell membrane. Vasocontractions induced by membrane depolarization and by an adrenergic agonist were cholesterol-independent. Our data support the previously unreported concept that cholesterol potentiates the Ca 2ϩ sensitization of VSM mediated by a SPC/Src-TK/ Rho-kinase pathway, and are also compatible with a role for cholesterol-enriched membrane microdomain, a lipid raft. This process may play an important role in the development of abnormal vascular contractions in patients with hypercholesterolemia. ( Key Words: Ca 2ϩ sensitization Ⅲ contraction Ⅲ membrane lipid raft Ⅲ Rho-kinase Ⅲ sphingosylphosphorylcholine Ⅲ vascular smooth muscle A bnormal vascular contraction as a result of Ca 2ϩ sensitization of vascular smooth muscles (VSMs) has attracted attention as a cause of hypertension and vasospasm. 1 Kureishi et al showed that constitutively active Rho-kinase applied to the cytosol of permeabilized VSM induced Ca 2ϩ sensitization and increased myosin light chain phosphorylation through a mechanism that is independent of a Ca 2ϩ -dependent myosin light chain kinase pathway. 2 In addition, the Rho-kinase blocker Y27632 cures hypertension in rats without affecting normal blood pressure, 3 and it also inhibits vasospasm of coronary arteries in a porcine model. 4 However, the mechanism by which Rho-kinase-mediated Ca 2ϩ sensitization of VSM is triggered in cardiovascular diseases has not been evident.Hyperlipidemia is a major risk factor for abnormal cardiovascular events. Evidence for this is provided by the J-shaped curve showing a strong relationship between serum cholesterol levels and the relative risk for coronary disease 5-7 (see also Figure I in the online data supplement, available at http://circres.ahajournals.org). Atheromatous plaques may be involved in cardiovascular events, 8,9 but the ext...

The Dietary Approaches to Stop Hypertension (DASH) diet is recommended by the American Heart Association to lower blood pressure (BP); however, its effects in Japanese participants have not been rigorously studied. We assessed the effects of the DASH-Japan Ube Modified diet Program (DASH-JUMP), a modified DASH diet, on cardiometabolic and inflammatory biomarkers in Japanese participants with untreated high-normal BP or stage 1 hypertension. Fifty-eight participants (30 men and 28 women; mean age 54.1±8.1 years) with untreated high-normal BP or stage 1 hypertension followed the DASH-JUMP (salt 8.0 g per day) for 2 months. After the intervention period, they resumed their usual diets for 4 months. The DASH-JUMP significantly decreased the participants' body mass index values (24.6±3.5 kg m−2 at baseline→23.2±3.3 kg m−2 at 2 months, P=0.000), BP (153±14/91±11 mm Hg at baseline→130±16/80±9 mm Hg at 2 months, P=0.000 and 139±16/85±10 mm Hg at 6 months, P=0.000), fasting serum glucose level (100±26 mg dl−1→94±15 mg dl−1 at 2 months, P=0.003) and fasting insulin level (6.9±5.9 μIU ml−1→4.4±2.7 μIU ml−1 at 2 months, P=0.000). The mean compliance of the participants for the DASH-JUMP diet was 88.5%. The DASH-JUMP diet reduced cardiovascular risk factors and may be an effective nutritional strategy for preventing cardiovascular events.

Fatty acid-binding proteins (FABPs) bind and solubilize long-chain fatty acids, controlling intracellular lipid dynamics. FABP7 is expressed by astrocytes in the developing brain, and suggested to be involved in the control of astrocyte lipid homeostasis. In this study, we sought to examine the role of FABP7 in astrocytes, focusing on plasma membrane lipid raft function, which is important for receptor-mediated signal transduction in response to extracellular stimuli. In FABP7-knockout (KO) astrocytes, the ligand-dependent accumulation of Toll-like receptor 4 (TLR4) and glial cell-line-derived neurotrophic factor receptor alpha 1 into lipid raft was decreased, and the activation of mitogen-activated protein kinases and nuclear factor-κB was impaired after lipopolysaccharide (LPS) stimulation when compared with wild-type astrocytes. In addition, the expression of caveolin-1, not cavin-1, 2, 3, caveolin-2, and flotillin-1, was found to be decreased at the protein and transcriptional levels. FABP7 re-expression in FABP7-KO astrocytes rescued the decreased level of caveolin-1. Furthermore, caveolin-1-transfection into FABP7-KO astrocytes significantly increased TLR4 recruitment into lipid raft and tumor necrosis factor-α production after LPS stimulation. Taken together, these data suggest that FABP7 controls lipid raft function through the regulation of caveolin-1 expression and is involved in the response of astrocytes to the external stimuli. GLIA 2015;63:780-794.

Lysophosphatidic acid (LPA) and sphingosylphosphorylcholine (SPC) activated Fyn tyrosine kinase and induced stress fiber formation, which was blocked by pharmacological inhibition of Fyn, gene silencing of Fyn, or dominant negative Fyn. Overexpressed constitutively active Fyn localized at both ends of F-actin bundles and triggered stress fiber formation, only the latter of which was abolished by Rho-kinase (ROCK) inhibition. SPC, but not LPA, induced filopodia-like protrusion formation, which was not mediated by Fyn and ROCK. Thus, Fyn appears to act downstream of LPA and SPC to specifically stimulate stress fiber formation mediated by ROCK in fibroblasts.