Vascular Smooth Muscle Cell Kinetics: a New Assay for Studying Patterns of Cellular Proliferation in Vivo

Goldberg, Itzhak D.; Stemerman, Michael B.; Schnipper, Lowell E.; Ransil, Bernard J.; Crooks, Gary W.; Fuhro, Robert

doi:10.1126/science.472713

Cited by 51 publications

(34 citation statements)

References 11 publications

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“…: 617-636-0619; Fax: 617-636-1444; E-mail: Hsurks@tufts-nemc.org. 1 The abbreviations used are: MBS, myosin binding subunit; M-RIP, myosin phosphatase-RhoA interacting protein; GST, glutathione S-transferase; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphatebuffered saline; Ni-NTA, nickel-nitrilotriacetic acid; GTP␥S, guanosine 5Ј-3-O-(thio)triphosphate; GDP␤S, guanosine 5Ј-O-2-(thio)diphosphate; cGK, cGMP-dependent protein kinase; CC, coiled-coil; RBD, Rho-binding domain.…”

mentioning

confidence: 99%

“…Myosin phosphatase is a heterotrimer consisting of a PP1 catalytic subunit, a 130-kDa myosin binding subunit (MBS) 1 and a 20-kDa subunit of unknown function (9, 16 -18). The MBS is a regulatory subunit that targets PP1 to its substrate, myosin light chain (9), and has multiple protein interaction domains, including ankyrin repeats at its amino terminus, and a leucine zipper domain at its carboxyl terminus.…”

mentioning

confidence: 99%

“…Diseases characterized by abnormal vascular smooth muscle cell contraction include hypertension, blood vessel spasm, and atherosclerosis (1)(2)(3)(4)(5). Smooth muscle contraction is tightly coupled to myosin light chain phosphorylation (6), which in turn is regulated by the relative activities of myosin light chain kinase and myosin phosphatase.…”

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Myosin Phosphatase-Rho Interacting Protein

Surks

Richards

Mendelsohn

2003

Journal of Biological Chemistry

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Regulation of vascular smooth muscle cell contractile state is critical for the maintenance of blood vessel tone. Abnormal vascular smooth muscle cell contractility plays an important role in the pathogenesis of hypertension, blood vessel spasm, and atherosclerosis. Myosin phosphatase, the key enzyme controlling myosin light chain dephosphorylation, regulates smooth muscle cell contraction. Vasoconstrictor and vasodilator pathways inhibit and activate myosin phosphatase, respectively. G-protein-coupled receptor agonists can inhibit myosin phosphatase and cause smooth muscle cell contraction by activating RhoA/Rho kinase, whereas NO/cGMP can activate myosin phosphatase and cause smooth muscle cell relaxation by activation of cGMP-dependent protein kinase. We have used yeast two-hybrid screening to identify a 116-kDa human protein that interacts with both myosin phosphatase and RhoA. This myosin phosphatase-RhoA interacting protein, or M-RIP, is highly homologous to murine p116 RIP3 , is expressed in vascular smooth muscle, and is localized to actin myofilaments. Blood vessel tone is regulated by the contractile state of vascular smooth muscle cells in the blood vessel wall. Diseases characterized by abnormal vascular smooth muscle cell contraction include hypertension, blood vessel spasm, and atherosclerosis (1-5). Smooth muscle contraction is tightly coupled to myosin light chain phosphorylation (6), which in turn is regulated by the relative activities of myosin light chain kinase and myosin phosphatase. Myosin light chain kinase is activated by intracellular calcium and phosphorylates myosin light chains, leading to cell contraction (7,8). Myosin phosphatase dephosphorylates myosin light chains, leading to smooth muscle cell relaxation (9). Myosin phosphatase activity, once thought to be constitutive, is now known to be highly regulated. Both vasoconstrictor signaling pathways, which lead to inhibition of the phosphatase and cell contraction (reviewed in Ref. 10), and vasodilator signaling pathways, which lead to cell relaxation via activation of myosin phosphatase have been recently defined (11-15).Myosin phosphatase is a heterotrimer consisting of a PP1 catalytic subunit, a 130-kDa myosin binding subunit (MBS) 1 and a 20-kDa subunit of unknown function (9, 16 -18). The MBS is a regulatory subunit that targets PP1 to its substrate, myosin light chain (9), and has multiple protein interaction domains, including ankyrin repeats at its amino terminus, and a leucine zipper domain at its carboxyl terminus. MBS binds PP1 and myosin light chain at its amino terminus and the M20 subunit and cGMP-dependent protein kinase 1␣ (cGK) at its carboxyl terminus (Ref. 11, reviewed in Ref. 19). The MBS-cGK interaction is necessary for NO/cGMP-mediated activation of myosin phosphatase (11).In vascular smooth muscle, G-protein-coupled receptor agonists cause contraction in part by inhibition of myosin phosphatase activity (20). Several downstream signaling pathways that inhibit myosin phosphatase activity have been discovered re...

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Myosin Phosphatase-Rho Interacting Protein

Surks

Richards

Mendelsohn

2003

Journal of Biological Chemistry

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“…This was determined according to the method of Goldberg et al 11 and involved the dissolution of the buffered tissue homogenate by nuclease-free Pronase B (Calibiochem-Behring Corporation, Lucerne, Switzerland), followed by precipitation of DNA in cold trichloracetic acid (TCA). After washing, the precipitate was collected on a nitrocellulose filter (Miilipore Corporation, Bedford, Massachusetts), which was then put into a liquid scintillation counting vial.…”

Section: Radioactivity In Dnamentioning

confidence: 99%

A system for long-term perfusion of rabbit aorta in vitro.

Björnheden¹,

Bylock²,

Hansson³

et al. 1983

Arteriosclerosis

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In an in vitro system for the perfusion of arterial tissue, the volume of the incubation chamber should be small, and the composition of the medium should be easily modified. The tissue should consist of media and intima only, and the interaction with the medium should occur via the intimal side. Consecutive sampling of the medium and the tissue should be possible. This paper describes a system with these characteristics. Scanning and transmission electron microscopy of the perfused tissue indicated that the endothelium was intact during the first day and that it still covered more than 95% of the surface after 3 days. On the 2nd day, the nonthrombogenic properties of the endothelium were maintained. The medial smooth muscle cells of the inner twothirds of the preparation were viable during the perfusion, while the cells in the outer one-third were dead from the start. Still, the metabolic activity of the tissue was stable, at least during the 2nd day as assessed by the study of DNA, protein, and lipid synthesis, as well as by oxygen consumption. We conclude that the perfusion system presented here might be useful in the study of the interaction between cellular and humoral components of the blood and the arterial wall.

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“…16 Following a 1-hour pulse label, the animals were sacrificed with a lethal dose of anesthetic, and the heart was immediately removed.…”

Section: Pulse Labeling With 3 H-thymldlnementioning

confidence: 99%

Smooth muscle cell proliferation in response to endothelial injury in coronary arteries of normal and von Willebrand's disease swine.

Lamb¹,

Manning²,

Reddick³

et al. 1984

Arteriosclerosis

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The proliferative response of medial smooth muscle cells to balloon-induced endothelial denudation was measured in coronary arteries of normal and von Willebrand's disease swine, a strain of swine with a genetic defect affecting platelet-vessel wall interactions. Animals were sacrificed 48 hours following endothelial injury. Incorporation of a 1 -hour pulse label of 3 H-thymidine was determined by measuring DNA specific activity. Autoradiography of coronary artery step segments was also performed. The ratio of DNA specific activity in ballooned vessels to that in nonballooned vessels ranged from 1.2 to 26.

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Vascular Smooth Muscle Cell Kinetics: a New Assay for Studying Patterns of Cellular Proliferation in Vivo

Cited by 51 publications

References 11 publications

Myosin Phosphatase-Rho Interacting Protein

Myosin Phosphatase-Rho Interacting Protein

A system for long-term perfusion of rabbit aorta in vitro.

Smooth muscle cell proliferation in response to endothelial injury in coronary arteries of normal and von Willebrand's disease swine.

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