Hiroko Kishi

Ablation and Mutation of Nonmuscle Myosin Heavy Chain II-B Results in a Defect in Cardiac Myocyte Cytokinesis

Abstract— We have identified a novel form of cardiac myocyte enlargement in nonmuscle myosin heavy chain II-B (NMHC II-B) ablated mice, based on a partial failure in cytokinesis. In contrast to most cells, cardiac myocytes lack NMHC II-A, and ablation of NMHC II-B results in a heart with 70% fewer myocytes at embryonic day 14.5 (E14.5) than control mice (B+/B− and B+/B+). In addition, B−/B− cardiac myocytes show a marked increase in binucleation at E12.5, reflecting the occurrence of karyokinesis in the absence of cytokinesis. An increase in binucleation and cell size is also found in hypomorphic, homozygous mice harboring a single amino acid mutation (R709C) in the gene encoding NMHC II-B. The nonmyocytes in B−/B− hearts and homozygous mutant hearts, all of which contain NMHC II-A, do not show either of these abnormalities. B−/B− cardiac myocytes at E14.5 show a decreased bromodeoxyuridine (BrdU) labeling index compared with controls, consistent with the decrease in myocyte proliferation. This decreased BrdU labeling is not seen in nonmyocyte cells in the heart. In addition to these changes, both B−/B− mice as well as homozygous mutated mice show an increase in cyclin D2 and D3 reflecting an abnormality in earlier steps in the cell cycle. Whereas cardiac myocytes completely ablated for NMHC II-B show enlargement and binucleation, mice expressing as little as 6% of the normal amount of wild-type NMHC II-B in the heart do not show these abnormalities.

Identification of the active metabolite of ticlopidine from rat in vitro metabolites

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. An active moiety (UR‐4501) was isolated by high‐performance liquid chromatography after large‐scale preparation of metabolites. 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. UR‐4501 produced a concentration‐dependent inhibition (3–100 μM) of ADP (10 μM)‐induced human platelet aggregation, whereas 2‐oxo‐ticlopidine (3–100 μM) did not elicit inhibitory responses. UR‐4501 (10–100 μM) strongly inhibited ADP‐ and collagen‐induced aggregation and slightly inhibited thrombin‐induced aggregation. The inhibition of rat washed platelet aggregation by UR‐4501 (100 μM) persisted, even after the platelets had been washed twice. These results suggest that UR‐4501 is the molecule responsible for the in vivo activities of ticlopidine.

Myosin light chain kinase as a multifunctional regulatory protein of smooth muscle contraction

Myosin light chain kinase (MLCK) is a regulatory protein for smooth muscle contraction, which acts by phosphorylating 20‐kDa myosin light chain (MLC20) to activate the myosin ATPase activity. Although this mode of action is well‐established, there are numerous reports of smooth muscle contraction that is not associated with MLC20 phosphorylation. The kinase activity for the phosphorylation is localized at the central part of MLCK, which is also furnished with actin‐binding activity at its N terminal and myosin‐binding activity at its C terminal. This article overviews as to how such multifunctional properties of MLCK modify the actin‐myosin interaction and presents our observations that the phosphorylation is not obligatory in induction of smooth muscle contraction.

Cholesterol Primes Vascular Smooth Muscle to Induce Ca2 Sensitization Mediated by a Sphingosylphosphorylcholine–Rho-Kinase Pathway Possible Role for Membrane Raft

Hypercholesterolemia is a major risk factor involved in abnormal cardiovascular events. Rho-kinase–mediated Ca2+ 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 Ca2+ sensitization. Here we report the strong linkage between cholesterol and the Ca2+ 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 &bgr;-cyclodextrin resulted in a loss of membrane caveolin-1, a marker of cholesterol-enriched lipid raft, and inhibited the SPC-induced Ca2+ 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 Ca2+ 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.

The Structure and Function of the Actin-binding Domain of Myosin Light Chain Kinase of Smooth Muscle

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 actin-myosin interaction of smooth muscle (Kohama, K., Okagaki, T., Hayakawa, K., Lin, Y., Ishikawa, R., Shimmen, T., and Inoue, A. (1992) Biochem. Biophys. Res. Commun. 184, 1204–1211). In this study, we have analyzed the actin binding activity of MLCK and related it to its amino acid sequence by producing native and recombinant fragments of MLCK. Parent MLCK exhibited both calcium ion (Ca2+) and calmodulin (Ca2+/CaM)-sensitive and Ca2+/CaM-insensitive binding to actin filaments. The native fragment, which consists of the Met1–Lys114 sequence (Kanoh, S., Ito, M., Niwa, E., Kawano, Y., and Hartshorne, D. J. (1993)Biochemistry 32, 8902–8907), and the recombinant NN fragment, which contains this 1–114 sequence, showed only Ca2+/CaM-sensitive binding. An inhibitory effect of the NN fragment on the actin-myosin interaction was observed by assayingin vitro motility and by measuring the actin-activated ATPase activity of myosin. The recombinant NN/41 fragment, which is constructed without the Met1–Pro41 sequence of the NN fragment, lost both the actin binding activity and the inhibitory effect. We confirmed the importance of the 1–41 sequence by using a few synthetic peptides to compete against the NN fragment in binding to actin filaments. The experiments using recombinant fragments and synthetic peptides also revealed that the site for CaM-binding is the Pro26–Pro41 sequence. The site for the Ca2+/CaM-insensitive binding, which is shown to be localized between the Ca2+/CaM-sensitive site and the central kinase domain of MLCK, exerted no regulatory effects on the actin-myosin interaction.

Stable Transfectants of Smooth Muscle Cell Line Lacking the Expression of Myosin Light Chain Kinase and Their Characterization with Respect to the Actomyosin System

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).

Sphingomyelinase causes endothelium-dependent vasorelaxation through endothelial nitric oxide production without cytosolic Ca2+ elevation

Neutral sphingomyelinase (N‐SMase) elevated nitric oxide (NO) production without affecting intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells in situ on aortic valves, and induced prominent endothelium‐dependent relaxation of coronary arteries, which was blocked by N ω ‐monomethyl‐L‐arginine, a NO synthase (NOS) inhibitor. N‐SMase induced translocation of endothelial NOS (eNOS) from plasma membrane caveolae to intracellular region, eNOS phosphorylation on serine 1179, and an increase of ceramide level in endothelial cells. Membrane‐permeable ceramide (C8‐ceramide) mimicked the responses to N‐SMase. We propose the involvement of N‐SMase and ceramide in Ca2+‐independent eNOS activation and NO production in endothelial cells in situ, linking to endothelium‐dependent vasorelaxation.

Elevated concentrations of sphingosylphosphorylcholine in cerebrospinal fluid after subarachnoid hemorrhage: A possible role as a spasmogen

This study investigates the role of sphingosylphosphorylcholine (SPC) in the mechanisms underlying cerebral vasospasm after subarachnoid hemorrhage (SAH). The levels of SPC were measured in cerebrospinal fluid (CSF) of patients with SAH and also in an experimental canine model. CSF samples were collected from 11 patients with SAH, and from dogs that had received an injection of SPC into the cisterna magna to examine SPC kinetics in the CSF. SPC was assayed using solid-phase extraction and triple quadrupole mass spectrometry. The SPC concentrations in SAH patients on days 3, 8, and 14 after the onset of SAH were significantly higher than those in normal CSF. In the canine model, rapid dilution of SPC in CSF was observed. In combination with data from previous studies, these results suggest that SPC is involved in the development of cerebral vasospasm. Rapid dilution of SPC in CSF suggests that SPC is released into CSF at higher concentrations than those measured in the present study.

Synchronous phosphorylation of CPI-17 and MYPT1 is essential for inducing Ca2+ sensitization in intestinal smooth muscle

Background  Myosin phosphatase activity is regulated by mechanisms involving the phosphorylation of CPI‐17 and MYPT1, primarily based on studies with tonic‐type vascular smooth muscles. This study examined how these mechanisms contribute to the regulation of contraction of a phasic‐type intestinal smooth muscle.

Involvement of Fyn tyrosine kinase in actin stress fiber formation in fibroblasts

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.