Unique geometry of actin-membrane anchorage sites in avian gizzard smooth muscle cells

1989 ◽  
Vol 94 (4) ◽  
pp. 703-711
Author(s):  
A. Draeger ◽  
E.H. Stelzer ◽  
M. Herzog ◽  
J.V. Small
Keyword(s):  
Smooth Muscle ◽  
Cell Surface ◽  
Smooth Muscle Cells ◽  
Laser Scanning ◽  
Smooth Muscles ◽  
Muscle Cells ◽  
Laser Scanning Microscopy ◽  
Surface Pattern ◽  
Confocal Laser ◽  
Dense Bodies

Adherens junctions in isolated avian gizzard smooth muscle cells appear as short longitudinal streaks or chevrons that are arranged in periodic, mainly transverse bands along the cell surface. This barrel-like geometry, revealed by antibodies to either vinculin or talin, was seen also in teased gizzard strips by confocal laser-scanning microscopy and contrasted with the rib-like surface pattern observed here and previously in other avian and mammalian smooth muscles. There were on average 67 transverse bands per gizzard cell and an estimated total of around 800 vinculin/talin sites. The longitudinal spacing between the transverse bands of vinculin streaks in the gizzard cells changed from 4–5 microns in extended cells to around 1 micron in shortened cells and the bands remained essentially transverse at all cell lengths, inconsistent with a screw-like mode of cell shortening as has been invoked for smooth muscle cells by others. The absence of rotation on shortening was confirmed by observations on isolated and bead-decorated skinned cells that were induced to contract with ATP. Counterlabelling of cells with alpha-actinin antibodies produced more or less exclusive staining of the cytoplasmic dense bodies, and little surface label: the total number of dense bodies per cell, estimated from confocal microscope through focal series was in the range of 3000. The data are consistent with a periodic anchorage of actin filaments to the cell surface and, in turn, with the existence of regularly spaced contractile assemblies.

Altered Cytoskeleton in Smooth Muscle of Aganglionic Bowel

2002 ◽  
Vol 126 (6) ◽  
pp. 692-696
Author(s):  
Laszlo Nemeth ◽  
Udo Rolle ◽  
Prem Puri
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Laser Scanning ◽  
Hirschsprung Disease ◽  
Muscle Cells ◽  
Cytoskeletal Proteins ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Normal Bowel ◽  
Pull Through

Abstract Context.—Intestinal motility is under the control of smooth muscle cells, enteric plexus, and hormonal factors. In Hirschsprung disease (HD), the aganglionic colon remains spastic or tonically enhanced and unable to relax. The smooth muscle cell's cytoskeleton consists of proteins or structures whose primary function is to link or connect protein filaments to each other or to the anchoring sites. Dystrophin is a subsarcolemmal protein with a double adhesion property, one between the membrane elements and the contractile filaments of the cytoskeleton and the other between the cytoskeletal proteins and the extracellular matrix. Desmin and vinculin are functionally related proteins that are present in the membrane-associated dense bodies in the sarcolemma of the smooth muscle cells. Objective.—To examine the distribution of the cytoskeletal proteins in the smooth muscle of the aganglionic bowel. Design.—Bowel specimens from ganglionic and aganglionic sections of the colon were collected at the time of pull-through surgery from 8 patients with HD. Colon specimens collected from 4 patients at the time of bladder augmentation acted as controls. Anti-dystrophin, anti-desmin, and anti-vinculin antibodies were used for fluorescein immunostaining using confocal laser scanning microscopy. Results.—Moderate to strong dystrophin immunoreactivity was observed at the periphery of smooth muscle fibers in normal bowel and ganglionic bowel from patients with HD, whereas dystrophin immunoreactivity was either absent or weak in the smooth muscle of aganglionic colon. Moderate to strong cytoplasmic immunostaining for vinculin and desmin was seen in the smooth muscle of normal bowel and ganglionic bowel from patients with HD, whereas vinculin and desmin staining in the aganglionic colon was absent or weak. Conclusion.—This study demonstrates that the cytoskeletal proteins are abundant in the smooth muscle of normal bowel, but are absent or markedly reduced in the aganglionic bowel of HD. As cytoskeletal proteins are required for the coordinated contraction of muscle cells, their absence may be responsible for the motility dysfunction in the aganglionic segment.

scholarly journals OP0141 HIGH DIMENSIONAL ANALYSIS REVEAL A NETWORK OF CERTAIN TRANSCRIPTION FACTORS THAT LINK VASCULOPATHY AND ORGAN FIBROSIS IN SYSTEMIC SCLEROSIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 90.2-91
Author(s):  
C. G. Anchang ◽  
B. Matalobos Lawaree ◽  
S. Weber ◽  
S. Rauber ◽  
T. Wohlfahrt ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Laser Scanning ◽  
Muscle Cells ◽  
Laser Scanning Microscopy ◽  
Tube Length ◽  
Confocal Laser ◽  
Research Support ◽  
Organ Fibrosis

Background:Since vascular manifestations such as Raynaud’s phenomenon often precede the onset of other clinical manifestations of systemic sclerosis (SSc), the identification of pathways linking vasculopathy to organ fibrosis might thus provide important insights into early disease mechanisms and allow early targeted intervention for both fibrotic and vascular events.Objectives:In this study we performed high dimensional (HD) analyses to identify mediators that link vasculopathy to organ fibrosis.Methods:HD techniques including RNA-seq, ChIP-seq, ATAC-seq and FISH-seq have been performed to identify mediators in vessels and fibrotic lesions of human skin samples of SSc patients and healthy volunteers. In addition, murine skin and lung tissue samples were analyzed by multi-channel immunofluorescence (IF) and confocal laser scanning microscopy. Microvascular endothelial cells, smooth muscle cells and fibroblasts have been further processed to address their functional attributes with regard to their proliferative, migratory and chemotactic capacity. In vivo models and ex vivomouse fetal metatarsal assays were performed to study fibrotic and angiogenic processes.Results:Bioinformatic HD analyses revealed the ETS transcription factor PU.1 as molecular checkpoint of a network of factors that drive matrix production and fibrotic imprinting in SSc. Within this network ATF3 was significantly upregulated in fibroblasts of skin biopsies of SSc patients and of various organs of fibrosis models. ATF3 deficiency ameliorated fibrosis in various mouse models. Notably, ATF3 was significantly upregulated in vascular cells of fibrotic tissues of SSc patients. Multi-channel IF and confocal laser scanning microscopy of skin and lung biopsies of SSc patients revealed an increased expression of ATF3 especially in microvascular endothelial cells and smooth muscle cells. ATF3 overexpression in smooth muscle cells led to an extensively enhanced proliferation and increased migratory capacity whereas endothelial cells showed a SSc-like phenotype with reduced proliferation and migration. After ATF3 overexpression, tube formation capacity was completely altered as assessed by cumulative tube length, tube numbers and capillary sprouting. To investigate vessel outgrowth from a different perspective, we used theex vivofetal mouse metatarsal assay. ATF3 knockout mice showed a completely altered angiogenic response as assessed by tube length, number of branches and number junctions compared to wildtype controls.Conclusion:We identified PU.1 and ATF3 as key factors in disturbed vasculature and endogenous activated fibroblasts suggesting this axis as a potential therapeutic target intervening both fibrotic and vascular manifestations.Disclosure of Interests:Charles Gwellem Anchang: None declared, Bettina Matalobos Lawaree: None declared, Stefanie Weber: None declared, Simon Rauber: None declared, Thomas Wohlfahrt: None declared, Markus Luber: None declared, Alexander Kreuter: None declared, Georg Schett Speakers bureau: AbbVie, BMS, Celgene, Janssen, Eli Lilly, Novartis, Roche and UCB, Jörg Distler Grant/research support from: Boehringer Ingelheim, Consultant of: Boehringer Ingelheim, Paid instructor for: Boehringer Ingelheim, Speakers bureau: Boehringer Ingelheim, Andreas Ramming Grant/research support from: Pfizer, Novartis, Consultant of: Boehringer Ingelheim, Novartis, Gilead, Pfizer, Speakers bureau: Boehringer Ingelheim, Roche, Janssen

Nucleoplasmic calcium regulation in rabbit aortic vascular smooth muscle cells

2003 ◽  
Vol 81 (3) ◽  
pp. 301-310 ◽  
Author(s):  
Bernard Abrenica ◽  
Grant N Pierce ◽  
James S.C Gilchrist
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Signal Intensity ◽  
Laser Scanning ◽  
Muscle Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Intracellular Ca

In this study, we investigated whether nucleoplasmic free Ca2+ in aortic vascular smooth muscle cells (VSMCs) might be independently regulated from cytosolic free Ca2+. Understanding mechanisms and pathways responsible for this regulation is especially relevant given the role of a numerous intranuclear Ca2+-sensitive proteins in transcriptional regulation, apoptosis and cell division. The question of an independent regulatory mechanism remains largely unsettled because the previous use of intensitometric fluorophores (e.g., Fluo-3) has been criticized on technical grounds. To circumvent the potential problem of fluorescence artifact, we utilized confocal laser scanning microscopy to image intracellular Ca2+ movements with the ratiometric fluorophore Indo-1. In cultured rabbit VSMCs, we found sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA) pumps and ryanodine receptor (RyR) Ca2+ channel proteins to be discretely arranged within a perinuclear locus, as determined by fluorescent staining patterns of BODIPY® FL thapsi gargin and BODIPY® FL-X Ry. When intracellular Ca2+ stores were mobilized by addition of thapsigargin (5 μM) and activatory concentrations of ryanodine (1 μM), Indo-1 ratiometric signals were largely restricted to the nucleoplasm. Cytosolic signals, by comparison, were relatively small and even then its spatial distribution was largely perinuclear rather homogeneous. These observations indicate perinuclear RyR and SERCA proteins are intimately involved in regulating VSMC nucleoplasmic Ca2+ concentrations. We also observed a similar pattern of largely nucleoplasmic Ca2+ mobilization upon exposure of cells to the immunosuppressant drug FK506 (tacrolimus), which binds to the RyR-associated immunophillin-binding proteins FKBP12 and FKBP12.6. However, initial FK506-induced nucleoplasmic Ca2+ mobilization was followed by marked reduction of Indo-1 signal intensity close to pretreatment levels. This suggested FK506 exerts both activatory and inhibitory effects upon RyR channels. The latter was reinforced by observed effects of FK506 to only reduce nucleoplasmic Indo-1 signal intensity when added following pretreatment with both activatory and inhibitory concentrations of ryanodine. These latter observations raise the possibility that VSMC nuclei represent an important sink of intracellular Ca2+ and may help explain vasodilatory actions of FK506 observed by others.Key words: Ca2+, RyR, SERCA, cell nucleus, FK506, thapsigargin, ryanodine.

Confocal laser scanning fluorescence microscopy of brain microvessels: Method and preliminary results

1991 ◽  
Vol 49 ◽  
pp. 158-159
Author(s):  
Shams Ghoneim ◽  
Gary Baumbach
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Laser Scanning ◽  
Muscle Cells ◽  
Confocal Laser ◽  
Chronic Hypertension ◽  
Predominant Component ◽  
Digestion Technique ◽  
Arteriolar Wall ◽  
Cellular Components

Chronic Hypertension alters both mechanical as well as compositional characteristics of cerebral blood vessels. We have previously determined that pial arterioles consist of two major cellular components : endothelium and smooth muscle. Smooth muscle cells are the predominant component in the arteriolar wall. The consensus of data published on the shape and dimension of vascular smooth muscle cells (VSM) is that the majority of cells are spindle shaped and of similar length and width. Changes in the morphological shape and dimension of VSM cells may occur during growth, aging or pathological conditions. In a previous study we demonstrated a digestion technique for SEM to view smooth muscle cells in arteriolar walls by removing basement membrane and arachnoid tissue covering and exposing the underlying cells. The technique proved technically unreliable, difficult to reproduce and resulted in damage to smooth muscle cells. The goal of this study was to determine if confocal microscopy techniques could offer a reliable, reproducible and safe substitute to digestion techniques in the study of cerebral smooth muscle cells.

scholarly journals The cytoskeletal and contractile apparatus of smooth muscle: contraction bands and segmentation of the contractile elements.

1990 ◽  
Vol 111 (6) ◽  
pp. 2463-2473 ◽  
Author(s):  
A Draeger ◽  
W B Amos ◽  
M Ikebe ◽  
J V Small
Keyword(s):  
Smooth Muscle ◽  
Laser Scanning ◽  
The Body ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Minimum Length ◽  
Contractile Apparatus ◽  
Isolated Cells ◽  
Dense Bodies ◽  
Cytoskeletal Elements

Confocal laser scanning microscopy of isolated and antibody-labeled avian gizzard smooth muscle cells has revealed the global organization of the contractile and cytoskeletal elements. The cytoskeleton, marked by antibodies to desmin and filamin is composed of a mainly longitudinal, meandering and branched system of fibrils that contrasts with the plait-like, interdigitating arrangement of linear fibrils of the contractile apparatus, labeled with antibodies to myosin and tropomyosin. Although desmin and filamin were colocalized in the body of the cell, filamin antibodies labeled additionally the vinculin-containing surface plaques. In confocal optical sections the contractile fibrils showed a continuous label for myosin for at least 5 microns along their length: there was no obvious or regular interruption of label as might be expected for registered myosin filaments. The cytoplasmic dense bodies, labeled with antibodies to alpha-actinin exhibited a regular, diagonal arrangement in both extended cells and in cells shortened in solution to one-fifth of their extended length: after the same shortening, the fibrils of the cytoskeleton that showed colocalization with the dense bodies in extended cells became crumpled and disordered. It is concluded that the dense bodies serve as coupling elements between the cytoskeletal and contractile systems. After extraction with Triton X-100, isolated cells bound so firmly to a glass substrate that they were unable to shorten as a whole when exposed to exogenous Mg ATP. Instead, they contracted internally, producing integral of 10 regularly spaced contraction nodes along their length. On the basis of differences of actin distribution two types of nodes could be distinguished: actin-positive nodes, in which actin straddled the node, and actin-negative nodes, characterized by an actin-free center flanked by actin fringes of 4.5 microns minimum length on either side. Myosin was concentrated in the center of the node in both cases. The differences in node morphology could be correlated with different degrees of coupling of the contractile with the cytoskeletal elements, effected by a preparation-dependent variability of proteolysis of the cells. The nodes were shown to be closely related to the supercontracted cell fragments shown in the accompanying paper (Small et al., 1990) and furnished further evidence for long actin filaments in smooth muscle. Further, the segmentation of the contractile elements pointed to a hierarchial organization of the myofilaments governed by as yet undetected elements.

Intravascular pressure regulates local and global Ca2+ signaling in cerebral artery smooth muscle cells

2001 ◽  
Vol 281 (2) ◽  
pp. C439-C448 ◽  
Author(s):  
Jonathan H. Jaggar
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Laser Scanning ◽  
Muscle Cells ◽  
Membrane Depolarization ◽  
Wave Frequency ◽  
Current Frequency ◽  
Frequency Wave ◽  
Intracellular Ca ◽  
Intravascular Pressure

The regulation of intracellular Ca2+ signals in smooth muscle cells and arterial diameter by intravascular pressure was investigated in rat cerebral arteries (∼150 μm) using a laser scanning confocal microscope and the fluorescent Ca2+ indicator fluo 3. Elevation of pressure from 10 to 60 mmHg increased Ca2+spark frequency 2.6-fold, Ca2+ wave frequency 1.9-fold, and global intracellular Ca2+ concentration ([Ca2+]i) 1.4-fold in smooth muscle cells, and constricted arteries. Ryanodine (10 μM), an inhibitor of ryanodine-sensitive Ca2+ release channels, or thapsigargin (100 nM), an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase, abolished sparks and waves, elevated global [Ca2+]i, and constricted pressurized (60 mmHg) arteries. Diltiazem (25 μM), a voltage-dependent Ca2+ channel (VDCC) blocker, significantly reduced sparks, waves, and global [Ca2+]i, and dilated pressurized (60 mmHg) arteries. Steady membrane depolarization elevated Ca2+ signaling similar to pressure and increased transient Ca2+-sensitive K+ channel current frequency e-fold for ∼7 mV, and these effects were prevented by VDCC blockers. Data are consistent with the hypothesis that pressure induces a steady membrane depolarization that activates VDCCs, leading to an elevation of spark frequency, wave frequency, and global [Ca2+]i. In addition, pressure induces contraction via an elevation of global [Ca2+]i, whereas the net effect of sparks and waves, which do not significantly contribute to global [Ca2+]i in arteries pressurized to between 10 and 60 mmHg, is to oppose contraction.

Characterization of [3H]nifedipine binding to intact vascular smooth muscle cells

1988 ◽  
Vol 254 (1) ◽  
pp. C45-C52 ◽  
Author(s):  
K. Sumimoto ◽  
M. Hirata ◽  
H. Kuriyama
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Binding Capacity ◽  
Specific Binding ◽  
Smooth Muscles ◽  
Muscle Cells ◽  
Scatchard Analysis ◽  
Intact Cells ◽  
Porcine Coronary Artery ◽  
Control Value

Specific binding of the dihydropyridine Ca2+ antagonist [3H]nifedipine to dispersed smooth muscle cells of the porcine coronary artery was investigated and the findings were compared with the binding to microsomes of smooth muscles. Specific binding to intact cells was saturable and reversible. The dissociation constant was 1.93 +/- 0.42 nM and the maximal binding capacity was 59.6 +/- 12.4 fmol/10(6) cells, as assessed by Scatchard analysis of the equilibrium binding at 25 degrees C. The Kd value with intact cells was slightly higher than that observed with microsomes. Specific binding of [3H]nifedipine to intact cells was completely displaced by unlabeled dihydropyridine derivatives. Among other Ca2+ antagonists, verapamil and d-cis-diltiazem partially and flunarizine completely inhibited the binding. In the case of microsomes, d-cis-diltiazem stimulated the binding of [3H]nifedipine. These results suggest that there may be multiple binding sites for different subclasses of Ca2+ antagonists. Polyvalent cations had no effect on the binding to intact cells. In the case of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-treated microsomes, the addition of CaCl2 and BaCl2 increased the Bmax, but the Kd value remained unchanged. MnCl2 and CdCl2 had stimulatory or inhibitory effects, depending on the concentrations, whereas LaCl3 had no effect. The effect of membrane depolarization on the binding was also examined. When the intact cells were incubated in high [K+]o solution for 60 min, the Kd was lowered to 1.4 nM from the control value of 2.0 nM, thereby indicating that [3H]nifedipine binds to Ca2+ channels, with a higher affinity, at depolarized states.

Molecular diversity of KVα- and β-subunit expression in canine gastrointestinal smooth muscles

1999 ◽  
Vol 277 (1) ◽  
pp. G127-G136 ◽  
Author(s):  
Anne Epperson ◽  
Helena P. Bonner ◽  
Sean M. Ward ◽  
William J. Hatton ◽  
Karri K. Bradley ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Smooth Muscles ◽  
Muscle Cells ◽  
Pcr Analysis ◽  
Transcriptional Level ◽  
Gi Tract ◽  
Excitable Cells ◽  
Subunit Expression ◽  
Molecular Components

Voltage-activated K+(KV) channels play an important role in regulating the membrane potential in excitable cells. In gastrointestinal (GI) smooth muscles, these channels are particularly important in modulating spontaneous electrical activities. The purpose of this study was to identify the molecular components that may be responsible for the KV currents found in the canine GI tract. In this report, we have examined the qualitative expression of eighteen different KV channel genes in canine GI smooth muscle cells at the transcriptional level using RT-PCR analysis. Our results demonstrate the expression of KV1.4, KV1.5, KV1.6, KV2.2, and KV4.3 transcripts in all regions of the GI tract examined. Transcripts encoding KV1.2, KVβ1.1, and KVβ1.2 subunits were differentially expressed. KV1.1, KV1.3, KV2.1, KV3.1, KV3.2, KV3.4, KV4.1, KV4.2, and KVβ2.1 transcripts were not detected in any GI smooth muscle cells. We have also determined the protein expression for a subset of these KV channel subunits using specific antibodies by immunoblotting and immunohistochemistry. Immunoblotting and immunohistochemistry demonstrated that KV1.2, KV1.4, KV1.5, and KV2.2 are expressed at the protein level in GI tissues and smooth muscle cells. KV2.1 was not detected in any regions of the GI tract examined. These results suggest that the wide array of electrical activity found in different regions of the canine GI tract may be due in part to the differential expression of KV channel subunits.

scholarly journals Detection of differentially regulated subsarcolemmal calcium signals activated by vasoactive agonists in rat pulmonary artery smooth muscle cells

2014 ◽  
Vol 306 (7) ◽  
pp. C659-C669 ◽  
Author(s):  
Krishna P. Subedi ◽  
Omkar Paudel ◽  
James S. K. Sham
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Laser Scanning ◽  
Muscle Cells ◽  
Laser Scanning Confocal Microscopy ◽  
Cellular Functions ◽  
Permeabilized Cells ◽  
Scanning Confocal Microscopy ◽  
Subcellular Compartments ◽  
Transient Elevation

Intracellular calcium (Ca2+) plays pivotal roles in distinct cellular functions through global and local signaling in various subcellular compartments, and subcellular Ca2+ signal is the key factor for independent regulation of different cellular functions. In vascular smooth muscle cells, subsarcolemmal Ca2+ is an important regulator of excitation-contraction coupling, and nucleoplasmic Ca2+ is crucial for excitation-transcription coupling. However, information on Ca2+ signals in these subcellular compartments is limited. To study the regulation of the subcellular Ca2+ signals, genetically encoded Ca2+ indicators (cameleon), D3cpv, targeting the plasma membrane (PM), cytoplasm, and nucleoplasm were transfected into rat pulmonary arterial smooth muscle cells (PASMCs) and Ca2+ signals were monitored using laser scanning confocal microscopy. In situ calibration showed that the Kd for Ca2+ of D3cpv was comparable in the cytoplasm and nucleoplasm, but it was slightly higher in the PM. Stimulation of digitonin-permeabilized cells with 1,4,5-trisphosphate (IP3) elicited a transient elevation of Ca2+ concentration with similar amplitude and kinetics in the nucleoplasm and cytoplasm. Activation of G protein-coupled receptors by endothelin-1 and angiotensin II preferentially elevated the subsarcolemmal Ca2+ signal with higher amplitude in the PM region than the nucleoplasm and cytoplasm. In contrast, the receptor tyrosine kinase activator, platelet-derived growth factor, elicited Ca2+ signals with similar amplitudes in all three regions, except that the rise-time and decay-time were slightly slower in the PM region. These data clearly revealed compartmentalization of Ca2+ signals in the subsarcolemmal regions and provide the basis for further investigations of differential regulation of subcellular Ca2+ signals in PASMCs.

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