Studies on isolated smooth muscle cells: The contractile apparatus

1977 ◽  
Vol 24 (1) ◽  
pp. 327-349
Author(s):  
J.V. Small
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Maximum Velocity ◽  
Muscle Cells ◽  
Myosin Filament ◽  
Contractile Apparatus ◽  
Myosin Filaments ◽  
Intact Muscle ◽  
10 Nm Filaments ◽  
Triton X

Smooth muscle cells may be isolated from the taenia coli muscle of the guinea pig which, when made permeable by treatment with Triton X-100 (0-05%) show a sensitivity to Ca for contraction with MgATP. The rate of contraction, about 10 micron s-1, corresponds closely to the maximum velocity of shortening of the intact muscle. Electron microscopy of such partially demembranated muscle cells shows that myosin filaments of about 16-nm diameter are present in both the rigor and the relaxes states. In addition, the actin and myosin filaments are commonly seen to be associated in groups corresponding approximately in size to the fibrils recognizable in cells in rigor in the light microscope. The dense bodies and the 10-nm filaments are found located between the actin-myosin filament groups. The thick myosin filaments may be isolated by fragmentation of the cells under relaxing conditions. These native filaments range up to about 8 micron in length and show the same structural organization as filaments aseembled from purified smooth muscle myosin: there is no central bare zone and bare edges, about 0-2 micrin long, occur at the filament ends. The lack of bipolarity of the native smooth muscle muosin filaments and the absence, in the contractile apparatus, of actin-associated structures equivalent to Z-lines suggests that the amount of shearing that can occur between the actin and myosin filaments is considerably greater than in skeletal muscle.

scholarly journals Smitin, a novel smooth muscle titin–like protein, interacts with myosin filaments in vivo and in vitro

2002 ◽  
Vol 156 (1) ◽  
pp. 101-112 ◽  
Author(s):  
Kyoungtae Kim ◽  
Thomas C.S. Keller
Keyword(s):  
Smooth Muscle ◽  
Ionic Strength ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Smooth Muscle Myosin ◽  
Globular Domain ◽  
Contractile Apparatus ◽  
Myosin Filaments ◽  
Muscle Myosin

Smooth muscle cells use an actin–myosin II-based contractile apparatus to produce force for a variety of physiological functions, including blood pressure regulation and gut peristalsis. The organization of the smooth muscle contractile apparatus resembles that of striated skeletal and cardiac muscle, but remains much more poorly understood. We have found that avian vascular and visceral smooth muscles contain a novel, megadalton protein, smitin, that is similar to striated muscle titin in molecular morphology, localization in a contractile apparatus, and ability to interact with myosin filaments. Smitin, like titin, is a long fibrous molecule with a globular domain on one end. Specific reactivities of an anti-smitin polyclonal antibody and an anti-titin monoclonal antibody suggest that smitin and titin are distinct proteins rather than differentially spliced isoforms encoded by the same gene. Smitin immunofluorescently colocalizes with myosin in chicken gizzard smooth muscle, and interacts with two configurations of smooth muscle myosin filaments in vitro. In physiological ionic strength conditions, smitin and smooth muscle myosin coassemble into irregular aggregates containing large sidepolar myosin filaments. In low ionic strength conditions, smitin and smooth muscle myosin form highly ordered structures containing linear and polygonal end-to-end and side-by-side arrays of small bipolar myosin filaments. We have used immunogold localization and sucrose density gradient cosedimentation analyses to confirm association of smitin with both the sidepolar and bipolar smooth muscle myosin filaments. These findings suggest that the titin-like protein smitin may play a central role in organizing myosin filaments in the contractile apparatus and perhaps in other structures in smooth muscle cells.

scholarly journals Myosin filaments in smooth muscle cells do not have a constant length

2013 ◽  
Vol 591 (23) ◽  
pp. 5867-5878 ◽  
Author(s):  
Jeffrey C.-Y. Liu ◽  
Jörg Rottler ◽  
Lu Wang ◽  
Jenny Zhang ◽  
Chris D. Pascoe ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Constant Length ◽  
Myosin Filaments

beta-Adrenergic catecholamine-dependent properties of rat myometrium primary cultures

1983 ◽  
Vol 245 (1) ◽  
pp. C84-C90 ◽  
Author(s):  
M. Fortier ◽  
D. Chase ◽  
S. G. Korenman ◽  
J. F. Krall
Keyword(s):  
Smooth Muscle ◽  
Adenylate Cyclase ◽  
Smooth Muscle Cells ◽  
Primary Cultures ◽  
Morphological Characteristics ◽  
Muscle Cells ◽  
Beta Adrenergic ◽  
Intact Muscle ◽  
Old Rats ◽  
Beta Adrenergic Agonist

We have used cultured trypsin-collagenase-dispersed cells from uteri of 21-day-old rats to investigate the mechanism of control of uterine motility by the beta-adrenergic receptor. After 5 to 7 days in RPMI 1640 the cells started to assume some of the morphological characteristics of smooth muscle cells. When cultures were incubated with 45Ca2+ for 3 h then washed free of isotope and incubated in medium with unlabeled Ca2+, efflux from the prelabeled intracellular pools was linear for up to 60 min. The potent beta-adrenergic agonist isoproterenol had a rapid effect on the rate of efflux and increased it almost sevenfold. Isoproterenol's effect was blocked by propranolol and could be duplicated by the addition of 8-bromo-adenosine 3',5'-cyclic monophosphate or cholera toxin. The cultured myometrial cells had adenylate cyclase properties similar to those of intact muscle strips when these were determined by the conversion of radioactive substrate (alpha-32P-ATP) to 32P-cAMP using a broken-cell preparation. Adenylate cyclase was sensitive to stimulation by GTP and by isoproterenol in the presence but not in the absence of GTP. Adenylate cyclase was also sensitive to stimulation by Ca2+ in the absence of GTP. We conclude that the primary cultures had the properties expected of smooth muscle cells including beta-adrenergic receptors that were coupled to a physiologically important function, Ca2+ flux. The beta-adrenergic receptor's effect on Ca2+ flux was cAMP mediated, and the divalent cation may also regulate its rate of flux by an effect on Ca2+-sensitive cAMP production.

Use of MQAE for measurement of intracellular [Cl-] in cultured aortic smooth muscle cells

1994 ◽  
Vol 267 (6) ◽  
pp. H2114-H2123 ◽  
Author(s):  
C. Koncz ◽  
J. T. Daugirdas
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Primary Cultures ◽  
Chloride Concentration ◽  
Maximum Velocity ◽  
Muscle Cells ◽  
Disulfonic Acid ◽  
Influx Rate ◽  
Aortic Smooth Muscle Cells ◽  
Aortic Smooth Muscle

A novel fluorescent indicator, N-[ethoxycarbonylmethyl]-6-methoxy-quinolinium bromide (MQAE), was used to measure intracellular chloride concentration ([Cl-]i) in primary cultures of rat aortic smooth muscle cells (VSMC). The hydrolytic and fluorescent properties of the dye were characterized. The intracellular Stern-Volmer constant was calculated to be 25 M-1. Cl- efflux curves were characteristic of saturation-type kinetics, with an apparent Michaelis-Menten constant value of 11 +/- 4.8 (SD) mM, a maximum velocity of 0.038 +/- 0.021 mM/s, and a half time (t1/2) of 9.0 +/- 3.7 min. The average efflux rate in the first 10 min (0.023 +/- 0.004 mM/s) was reduced in the presence of either 130 microM 4,4'-diisothiocyanato-dihydrostilbene-2,2'-disulfonic acid (H2DIDS) (0.014 +/- 0.006, P = 0.02) or 40 microM furosemide (0.017 +/- 0.004, P = 0.04). Restoration of physiological extracellular chloride concentration ([Cl-]o) after zero Cl- resulted in net Cl- influx with a t1/2 of 3.6 +/- 1.0 min. The initial Cl- influx rate was reduced after exposure to furosemide, from 0.069 +/- 0.006 to 0.046 +/- 0.008 mM/s, P < 0.002, and was reduced after exposure to H2DIDS from 0.102 +/- 0.013 to 0.033 +/- 0.003 mM/s, P < 0.001. Furosemide reduced the steady-state [Cl-]i from 31.6 +/- 3.2 to 26.1 +/- 2.4 mM, P < 0.01, whereas H2DIDS had little effect on [Cl-]i. Our results demonstrate that MQAE can be used to measure [Cl-]i in primary cultures of VSMC.

scholarly journals Regulation of contraction and thick filament assembly-disassembly in glycerinated vertebrate smooth muscle cells.

1983 ◽  
Vol 97 (4) ◽  
pp. 1062-1071 ◽  
Author(s):  
W Z Cande ◽  
P J Tooth ◽  
J Kendrick-Jones
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Spatial Information ◽  
Muscle Cells ◽  
Thick Filament ◽  
Length Change ◽  
Intact Cells ◽  
Myosin Filaments ◽  
Skeletal Myosin

Isolated smooth muscle cells and cell fragments prepared by glycerination and subsequent homogenization will contract to one-third their normal length, provided Ca++ and ATP are present. Ca++-independent contraction was obtained by preincubation in Ca++ and ATP gamma S, or by addition of trypsin-treated myosin light chain kinase (MLCK) that no longer requires Ca++ for activation. In the absence of Ca++, myosin was rapidly lost from the cells upon addition of ATP. Glycerol-urea-PAGE gels showed that none of this myosin is phosphorylated. The extent of myosin loss was ATP- and pH-dependent and occurred under conditions similar to those previously reported for the in vitro disassembly of gizzard myosin filaments. Ca++-dependent contraction was restored to extracted cells by addition of gizzard myosin under rigor conditions (i.e., no ATP), followed by addition of MLCK, calmodulin, Ca++, and ATP. Function could also be restored by adding all these proteins in relaxing conditions (i.e., in EGTA and ATP) and then initiating contraction by Ca++ addition. Incubation with skeletal myosin will restore contraction, but this was not Ca++-dependent unless the cells were first incubated in troponin and tropomyosin. These results strengthen the idea that contraction in glycerinated cells and presumably also in intact cells is primarily thick filament regulated via MLCK, that the myosin filaments are unstable in relaxing conditions, and that the spatial information required for cell length change is present in the thin filament-intermediate filament organization.

scholarly journals Selective phosphorylation of the IP3R-I in vivo by cGMP-dependent protein kinase in smooth muscle

2003 ◽  
Vol 284 (2) ◽  
pp. G221-G230 ◽  
Author(s):  
Karnam S. Murthy ◽  
Huiping Zhou
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Specific Site ◽  
Kinase Substrate ◽  
Gastric Smooth Muscle ◽  
Intact Muscle ◽  
Dependent Protein

This study examined the expression of inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) types and PKG isoforms in isolated gastric smooth muscle cells and determined the ability of PKG and PKA to phosphorylate IP3Rs and inhibit IP3-dependent Ca2+ release, which mediates the initial phase of agonist-induced contraction. PKG-Iα and PKG-Iβ were expressed in gastric smooth muscle cells, together with IP3-R-associated cG-kinase substrate, a protein that couples PKG-Iβ to IP3R-I. IP3R-I and IP3R-III were also expressed, but only IP3R-I was phosphorylated by PKA and PKG in vitro and exclusively by PKG in vivo. Sequential phosphorylation by PKA and by PKG-Iα in vitro showed that PKA phosphorylated the same site as PKG (presumably S1755) and an additional PKA-specific site (S1589). In intact muscle cells, agents that activated PKG or both PKG and PKA induced IP3R-I phosphorylation that was reversed by the PKG inhibitor (8R,9S,11s)-(−)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,1H,-2,7b,11a-trizadizo-benzo9(a,g)cycloocta(c,d,e)-trinden-1-one. Agents that activated PKA induced IP3R-I phosphorylation in permeabilized but not intact muscle cells, implying that PKA does not gain access to IP3R-I in intact muscle cells. The pattern of IP3R-I phosphorylation in vivo and in vitro was more consistent with phosphorylation by PKG-Iα. Phosphorylation of IP3R-I in microsomes by PKG, PKA, or a combination of PKG and PKA inhibited IP3-induced Ca2+ release to the same extent, implying that inhibition was mediated by phosphorylation of the PKG-specific site. We conclude that IP3R-I is selectively phosphorylated by PKG-I in intact smooth muscle resulting in inhibition of IP3-dependent Ca2+ release.

Intercellular calcium signaling and nitric oxide feedback during constriction of rabbit renal afferent arterioles

2007 ◽  
Vol 292 (4) ◽  
pp. F1124-F1131 ◽  
Author(s):  
T. R. Uhrenholt ◽  
J. Schjerning ◽  
P. M. Vanhoutte ◽  
B. L. Jensen ◽  
O. Skøtt
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Smooth Muscle ◽  
Endothelial Cell ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Calcium Sensitivity ◽  
No Production ◽  
Contractile Apparatus ◽  
Afferent Arterioles

Vasoconstriction and increase in the intracellular calcium concentration ([Ca2+]i) of vascular smooth muscle cells may cause an increase of endothelial cell [Ca2+]i, which, in turn, augments nitric oxide (NO) production and inhibits smooth muscle cell contraction. This hypothesis was tested in microperfused rabbit renal afferent arterioles, using fluorescence imaging microscopy with the calcium-sensitive dye fura-2 and the NO-sensitive dye 4-amino-5-methylamino-2′,7′-difluorescein. Both dyes were loaded into smooth muscle and endothelium. Depolarization with 100 mmol/l KCl led to a transient vasoconstriction which was converted into a sustained response by N-nitro-l-arginine methyl ester (l-NAME). Depolarization increased smooth muscle cell [Ca2+]ifrom 162 ± 15 nmol/l to a peak of 555 ± 70 nmol/l ( n = 7), and this response was inhibited by 80% by the l-type calcium channel blocker calciseptine. After a delay of 10 s, [Ca2+]iincreased in endothelial cells immediately adjacent to reactive smooth muscle cells, and this calcium wave spread in a nonregenerative fashion laterally into the endothelial cell layer with a velocity of 1.2 μm/s. Depolarization with 100 mmol/l KCl led to a significant increase in NO production ([NO]i) which was inhibited by l-NAME ( n = 5). Acetylcholine caused a rapid increase in endothelial [Ca2+]i, which did not transfer to the smooth muscle cells. l-NAME treatment did not affect changes in smooth muscle [Ca2+]iafter depolarization, but it did increase the calcium sensitivity of the contractile apparatus. We conclude that depolarization increases smooth muscle [Ca2+]iwhich is transferred to the endothelial cells and stimulates NO production which curtails vasoconstriction by reducing the calcium sensitivity of the contractile apparatus.

scholarly journals Myosin filament structure in vertebrate smooth muscle.

1996 ◽  
Vol 134 (1) ◽  
pp. 53-66 ◽  
Author(s):  
J Q Xu ◽  
B A Harder ◽  
P Uman ◽  
R Craig
Keyword(s):  
Smooth Muscle ◽  
Striated Muscle ◽  
Smooth Muscles ◽  
Helical Structure ◽  
Structure Characteristic ◽  
Myosin Filament ◽  
Filament Structure ◽  
Myosin Filaments ◽  
Intact Muscle

The in vivo structure of the myosin filaments in vertebrate smooth muscle is unknown. Evidence from purified smooth muscle myosin and from some studies of intact smooth muscle suggests that they may have a nonhelical, side-polar arrangement of crossbridges. However, the bipolar, helical structure characteristic of myosin filaments in striated muscle has not been disproved for smooth muscle. We have used EM to investigate this question in a functionally diverse group of smooth muscles (from the vascular, gastrointestinal, reproductive, and visual systems) from mammalian, amphibian, and avian species. Intact muscle under physiological conditions, rapidly frozen and then freeze substituted, shows many myosin filaments with a square backbone in transverse profile. Transverse sections of fixed, chemically skinned muscles also show square backbones and, in addition, reveal projections (crossbridges) on only two opposite sides of the square. Filaments gently isolated from skinned smooth muscles and observed by negative staining show crossbridges with a 14.5-nm repeat projecting in opposite directions on opposite sides of the filament. Such filaments subjected to low ionic strength conditions show bare filament ends and an antiparallel arrangement of myosin tails along the length of the filament. All of these observations are consistent with a side-polar structure and argue against a bipolar, helical crossbridge arrangement. We conclude that myosin filaments in all smooth muscles, regardless of function, are likely to be side-polar. Such a structure could be an important factor in the ability of smooth muscles to contract by large amounts.

Sign in / Sign up

Export Citation Format

Share Document