QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 11 April 2006
doi: 10.1242/jcs.02805

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Woodsome, T. P. Articles by Kitazawa, T. Search for Related Content PubMed PubMed Citation Articles by Woodsome, T. P. Articles by Kitazawa, T. Social Bookmarking                         What's this?

Agonist- and depolarization-induced signals for myosin light chain phosphorylation and force generation of cultured vascular smooth muscle cells

¹ Boston Biomedical Research Institute, 64 Grove St., Watertown, MA 02472, USA
² Center for Cell Signaling, University of Virginia School of Medicine, Charlottesville, VA 22908, USA

View larger version (34K): [in a new window]   Fig. 1. Expression of contractile and/or regulatory proteins in primary cultured rat aorta VSMCs. The VSMCs from rat aorta were cultured up to passage ten. (A) Representative western blot images of various proteins in the cell lysates at passages 5-8. (B-F) Protein extracts were run on SDS gels and immunoblotted. For -actin, caldesmon (CaD) and calponin (CaP) 2 µg of protein extract was used, all other protein extracts were used at 20 µg. Protein expression levels of (B) total actin, (C) smooth muscle -actin isoform, (D) h-calponin, (E) CPI-17 and (F) MYPT1 were measured by immunoblotting and compared with the extract from intact rat aorta, which was normalized to one. n=3-10.

View larger version (144K): [in a new window]   Fig. 2. Representative images of fluorescence of the Fluo-3 Ca2+-indicator in cultured rat aortic smooth muscle cells. Images were captured every 15 seconds to avoid photo-bleaching. Baseline fluorescence was recorded before treatment with ET-1 (-10 seconds). ET-1 was added at 0 seconds and the first image was captured at 5 seconds. Although the cell boundary was not clear, a transient increase in fluorescence intensity was seen in almost all cells.

View larger version (17K): [in a new window]   Fig. 3. Representative traces of fluorescence of the Ca2+-indicator Fluo-3 in cultured VSMCs after various treatments. (A-F) Cells were serum-starved, loaded with Fluo-3 (see Materials and Methods for details), and then subjected to various agonists (1 µM ATII, 0.1 µM ET-1, or 100 µM phenylephrine (PE). (B,C) Average time courses of changes in [Ca2+]i in cells stimulated with (B) ATII and (C) ET-1 (n=3). Dotted lines in A-C show the equivalent levels of [Ca2+]i before stimulation. (D) PE-induced decrease in [Ca2+]i, although ET-1 still increased [Ca2+]i in the presence of PE. (E,F) Cells treated with (E) Ca2+-free solution or (F) of the Ins(1,4,5)P3-receptor antagonist 2-APB m (90 µM) for 10 minutes. Traces are representative of three to four independent experiments for each condition.

View larger version (13K): [in a new window]   Fig. 4. Representative traces of contractile responses to various agonists in (A,B) reconstituted rat aorta VSMC fibers and (C,D) fresh tissues. Reconstituted VSMC fibers were cultured in collagen 3D-matrix in FBS-supplemented medium for 2 weeks and in the serum-free culture medium for an additional day (see Materials and Methods). (A) Contractions were induced with high-K+ solution (124 mM), ATP (200 µM), serotonin (5-HT, 10 µM), ATII (1 µM), PDBu (1 µM) or ET-1 (1 µM). (B) Phenylephrine (PE, 100 µM) and noradrenaline (NA, 10 µM) both induced relaxation, which was blocked by 30 µM propranolol. (C) Contractions of the fresh rat aorta tissues were induced by high-K+ solution, ATP, 5-HT, ATII, or ET-1 at the concentrations given for A. (D) A small contraction induced by 100 µM PE was enhanced by the presence of 30 µM propranolol. Force traces are representative of at least four (in A-C) and three (in D) similar experiments.

View larger version (30K): [in a new window]   Fig. 5. Agonist-induced changes in phosphorylation of MLC, CPI-17 and MYPT1 in cultured VSMCs. (A) MLC phosphorylation is expressed as percent of total MLC (see Materials and Methods). Either ATII (1.0 µM) or ET-1 (0.1 µM) was added at 0 minutes. Inset shows representative 2D gel patterns of 20 kDa MLC of cultured VSMCs at rest (control), stimulated with ATII (+ATII) and ET-1 for 2.5 minutes (+ET-1). (B-D) Western blot results. Of each protein extract 20 µg were loaded onto two identical polyacrylamide gels and each was transferred onto nitrocellulose membrane. The total amount of (nonphosphorylated and phosphorylated) MYPT1 or CPI-17 was then determined with respective antibodies on one membrane (see Materials and Methods). We then compared the ratios of phosphorylated CPI-17 or MYPT1 to the total CPI-17or MYPT1 in the paired set of western blots and expressed relative phosphorylation levels as the percentage of control phosphorylation. (B1,B2) Western blots of total (CPI-17) and phosphorylated CPI-17 (pCPI-17) in cultured VSMCs at rest or VSMCs stimulated for 1, 2.5 and 5 minutes with ET-1 and for 5 minutes with the activator of PKC PDBu (B1). For average CPI-17 phosphorylation, the response to the 5-minute incubation with PDBu was normalized to 1 (B2). (C,D) Phosphorylation of MYPT1 at (C) Thr696 and (D) Thr853 after 2.5-minute incubation with ET-1 (0.1 µM) or ATII (1.0 µM). Responses in the presence of ET-1 were normalized to 1; *, significantly different to control (CT); n=3-5; P0.05.

View larger version (14K): [in a new window]   Fig. 6. Effect on phosphorylation of MYPT1 at (A) Thr696 and (B) Thr853, and of (C) CPI-17 at Thr38 after pretreatment with inhibitors. Cells were pretreated for 30 minutes with Y-27632 (30 µM) before addition of either ATII (1 µM) or ET-1 (0.1 µM) for 2.5 minutes. ET-1-treated samples were normalized to 1 for (A) MYPT1 Thr696, (B) MYPT1 Thr853 and (C) CPI-17 Thr38. *, significantly different to normal agonist treatments (n=3-4).

View larger version (14K): [in a new window]   Fig. 7. Effect of 2-APB, GF-109203X and Y-27632 on agonist-induced contraction in reconstituted rat aortic VSMC fibers. (A) High levels of K+ induced a contraction in the Ca2+-free [EGTA-containing (2 mM)] solution similar to that of the control solution containing 2 mM Ca2+. Y-27632 (10 µM) was added at the peak of contraction in response to the addition of high-K+ solution to the Ca2+-free solution. (B) The first contraction induced by 1 µM ATII was used as the control for the reconstituted VSMC fibers. After a 45-minute wash, the second addition of ATII resulted in a contraction to a level similar to the one after the first addition of ATII. Addition of 2-APB (30 µM) caused a faster relaxation in the presence of ATII than that by simple removal of ATII. (C) ET-1-induced contraction in the reconstituted VSMC fibers was partially inhibited by 2-APB (30 µM) alone and subsequently completely inhibited by the mixture of 2-APB and Y-27632 (10 µM). (D) ET-1-induced contraction was not affected by addition of GF-109203X (3 µM). The figures are representative of 3-5 similar experiments.

View larger version (15K): [in a new window]   Fig. 8. Effect of various inhibitors on high-K+- and agonist-induced contractions in fresh rat aorta tissues. (A) Ca2+-free, EGTA-containing solution inhibited the development of contractions induced by high K+. (B) The first force trace shows a control contraction, transient and practically irreversible, induced by 1 µM ATII after a contraction induced by high K+. The second trace (from another tissue strip) shows that the development of transient ATII-induced contraction is prevented by in presence of 30 µM 2-APB. (C) ET-1 (1 µM) produced a large sustained contraction, which was relaxed to the base line by 3 µM GF-109203X, 30 µM 2-APB and 10 µM Y-27632. (D) PDBu (1 µM) generated a large contraction, which was reduced by 3 µM GF-109203X. All figures are representative of 3-5 similar experiments.

View larger version (30K): [in a new window]   Fig. 9. Schematic diagram of signal transduction pathways towards phosphorylation of myosin and contraction in cultured VSMCs. In smooth muscle tissues, both pathways of signal transduction (black and gray) are significant in development of contraction. However, only signaling pathways indicated in black are active in cultured VSMCs. GPCR, G protein-coupled receptor; PLCß, phospholipase Cß; PLA2, phospholipase A2; Ins(1,4,5)P3, inositol 1,4,5-trisphosphatase; DAG, diacylglycerol; AA, arachidonic acid; GEF, guanine nucleotide exchange factor; PKC, protein kinase C; CPI-17, protein kinase C-potentiated myosin phosphatase inhibitor protein 17 kDa; MYPT1, myosin targeting subunit of myosin phosphatase; PP1C, -isoform of type 1 protein phosphatase catalytic subunit; MLCK, myosin light chain kinase; CaM, calmodulin. All pathways represent signaling pathways leading to an increase in myosin-P and contraction. Pathway1 through voltage-dependent Ca2+ channels, pathway 2 through Ins(1,4,5)P3-induced Ca2+ release from the SR, pathway 3 through phosphorylation of CPI-17 at Thr38 by PKC, pathway 4 through phosphorylation of CPI-17 by Rho-kinase and pathway-5 through phosphorylation of MYPT1 at Thr853 by Rho-kinase.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter