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PubMed2000

Expression of smooth muscle myosin light chain 17 and unloaded shortening in single smooth muscle cells.

These experiments were performed to test the hypotheses that myosin light chain 17 (MLC(17)) a and b isoform expression varies between individual vascular smooth muscle (SM) cells and that their expression correlates with cell unloaded shortening velocity. Single SM cells isolated from rabbit aorta and carotid arteries were used to measure unloaded shortening velocity and subsequently were analyzed via RT-PCR for MLC(17) a and b mRNA ratio. The MLC(17b/a) mRNA and protein ratios from adjacent tissue sections correlate very well (R(2) = 0.68), allowing use of the mRNA ratio to predict the protein ratio. The rabbit MLC(17) isoform protein sequence was found to be similar to, but unique from, the swine, mouse, and chicken sequences. Isolated single SM cells from the aorta and carotid have resting lengths of 70-280 microm and shorten to 33-88 microm after contraction. Isolated cell maximum unloaded shortening velocity is highly variable (0.5-7.5 microm/s) but becomes more uniform when normalized to initial cell length (0.01-0.05 cell lengths/s). Carotid cells activated in the presence of okadaic acid (1 microm) have mean maximal unloaded shortening velocities not significantly different from carotid cells activated without okadaic acid (0.016 vs. 0.019 cell lengths/s). Resting cell length before activation is significantly correlated with final cell length after unloaded shortening. Neither initial cell length, final cell length, total cell length change, nor maximum unloaded shortening velocity (absolute or normalized) was significantly correlated with single-cell MLC(17b/a) mRNA ratio. These studies were performed in isolated single SM cells where unloaded shortening velocity and MLC(17b/a) mRNA ratios were measured in the same cell. In this preparation, the three-dimensional organization and milieu of the cell is kept intact, but without the intercellular heterogeneity concerns of multicellular preparations. These results suggest the MLC(17b/a) ratio is variable between individual SM cells from the same tissue, but it is not a determinant of unloaded shortening velocity in single SM cells.

Animals↗
PubMed1994

Isolation of a morphologically and functionally distinct smooth muscle cell type from the intimal aspect of the normal rat aorta. Evidence for smooth muscle cell heterogeneity.

Recent studies indicate that the neointima of injured rat arteries is composed of a subpopulation of smooth muscle cells (SMCs) distinct from medial smooth muscle cells. However, SMC diversity in normal adult aorta has remained elusive. This study characterizes two morphologically and functionally distinct SMC types isolated from different anatomic regions of the normal rat aorta. Rat aortic medial smooth muscle cells (MSMCs) were isolated from the media after removal of the intimal and adventitial cells. Rat aortic intimal smooth muscle cells (ISMCs) were isolated from the intimal aspect of everted rat aortas. The two cell types were characterized morphologically and immunohistochemically and were compared for their capacity to contract collagen gels in response to endothelin-1. MSMCs were spindle-shaped and grew in hills and valleys showing features previously described for vascular SMCs. Conversely, ISMCs displayed a polygonal and epitheloid shape, grew mainly as a monolayer, and had a higher proliferative rate. Both cell types expressed alpha-smooth muscle actin and were negative for Factor VIII-RAg. ISMCs produced large amounts of a laminin and type IV collagen-rich extracellular matrix which had a characteristic pericellular distribution. ISMCs, but not MSMCs, rapidly contracted collagen gels in response to endothelin-1. This study indicates that the normal rat aorta contains two types of SMCs located in anatomically distinct regions of the vessel wall. Because of their functional characteristics, the SMCs isolated from the intimal aspect of the aorta may play an important role in physiologic as well as pathologic conditions.

Animals↗
PubMed1988

The effects of vascular smooth muscle caldesmon on force production by 'desensitised' skeletal muscle fibres.

Smooth muscle caldesmon inhibits actomyosin MgATPase in the absence of Ca2+ and is the key regulatory component of Ca2+-regulated thin filaments. We now show that caldesmon can affect contractility as well. Glycerinated skeletal muscle fibres were treated so as to produce substantial contractions without Ca2+ as an activator. Addition of caldesmon caused reductions in force in a concentration- and time-dependent manner. Perfusion with caldesmon concentration for less than 5 min gave up to 48% reduction in isometric tension with a half-maximal effect at 1.5 micron caldesmon. Perfusion with 15 microM caldesmon for 30 min gave an irreversible tension drop.

Animals↗
PubMed1987

Analysis of alpha-smooth-muscle actin mRNA expression in rat aortic smooth-muscle cells using a specific cDNA probe.

We constructed two cDNA probes, the first of which hybridizes with all rat actin mRNAs while the second is specific for alpha-smooth muscle (SM) actin mRNA. Northern hybridization using these probes showed that, in normal rat aortic media, the proportion of alpha-SM actin mRNA expression increases during development, reaching about 90% of the total actin mRNA level in adult animals. As compared to the situation in normal aortic media, the proportion of alpha-SM actin mRNA was found to decrease significantly in intimal thickening 15 days after endothelial injury, i.e. when SM cells (SMCs) are actively replicating. At 60 days after injury, the SMCs were observed to have stopped dividing and to have recovered a normal content of alpha-SM actin mRNA. The content of alpha-SM actin mRNA was also selectively decreased (as compared to controls) in the hypotensive abdominal aortic media located below an aortic ligature, while it was not modified in the thoracic hypertensive segment above the same ligature. Primary cultures of rat aortic SMCs synthesize and contain low amounts of alpha-SM actin, but their alpha-SM actin mRNA content is similar to that of SMCs in vivo. As compared to primary cultures, the proportion of alpha-SM actin mRNA was found to be significantly decreased in SMCs at the fifth passage, at which stage it became comparable to the level of synthesized alpha-SM actin. Thus, the synthesis and expression of alpha-SM actin in SMCs appear to be regulated predominantly at the level of gene transcription in certain situations (e.g. aortic ligature in vivo and culture at the fifth passage), and predominantly at a post-transcriptional level in other situations (e.g. primary culture).

Actins↗
PubMed2007

The proteome and secretome of human arterial smooth muscle cell.

Smooth muscle cells (SMCs) play a crucial role in cardiovascular diseases. Proteomic analysis using two-dimensional gel electrophoresis (2DE) associated with mass spectrometry allows characterization of the proteome and secretome of human smooth muscle. The presence of a distinct SMC population in the arterial wall implies that under normal conditions, SMCs are phenotypically heterogeneous. Intracellular and secreted proteins from a primary culture of SMCs obtained from patients undergoing coronary bypass surgery were analyzed using 2DE in order to determine their specific features. The 2D reference maps show that SMCs are involved in a wide range of biological functions. They could constitute a useful tool for a wide range of investigators involved in vascular biology, allowing them to investigate SMC protein changes associated with cardiovascular disorders or environmental stimuli.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1997

Colonic smooth muscle cells possess a different subtype of somatostatin receptor from gastric smooth muscle cells.

Somatostatin (SS) alters colonic motility. To investigate whether SS has a direct effect on colonic smooth muscle cells, we prepared isolated muscle cells from the descending guinea pig colon and compared the effects of SS with those on isolated gastric smooth muscle cells. In gastric cells, SS had no effect on carbachol-induced contraction, whereas in colonic cells it caused inhibition. In colonic muscle cells, SS-28 caused >85% inhibition of contraction by cholecystokinin octapeptide (CCK-8), bombesin, 12-O-tetradecanoylphorbol-13-acetate, and ionomycin, whereas it had no effect on contraction by these agents in gastric cells. In gastric cells, SS inhibited relaxation. Three synthetic SS analogs had different relative affinities for causing effects in gastric and colonic cells. Pertussis toxin inhibited the action of SS-28 in each muscle cell type by 50-75%. SS-28 alone had a small contractile effect on cells from the circular layer of the colon. SS-28 inhibited carbachol-induced contraction in colonic cells from both the longitudinal and circular layers. These results demonstrate that the action of SS differs in colonic and gastric smooth muscle cells. SS inhibits contractants in colonic cells and relaxants in gastric cells. In colonic cells, SS has a weak contractile effect due to an effect on circular muscle cells and an inhibitory effect on cells from both longitudinal and circular layers. A different SS receptor subtype mediates the actions of SS in colonic and gastric muscle cells. In both cell types, the actions of SS are mediated by pertussis toxin-sensitive and -insensitive G proteins.

Animals↗
PubMed1987

Studies on isolated smooth muscle cells. IX. Application of papain for isolation of single smooth muscle cells from guinea-pig taenia coli.

To prepare single smooth muscle cells from the taenia coli of guinea pig, the application of papain to the enzymatic solution was examined under two conditions: 1) the isolation in a modified Tyrode solution (containing 0.18 mM Ca2+: 0.18 mM Ca2+-Tyrode solution) and 2) the isolation in a high-K+ Tyrode solution (Na+ was replaced by K+, and Ca2+ was not added: high-K+ Tyrode solution). The presence of papain during collagenase digestion reduced contamination of broken cells and cell debris. In the case of the high-K+ Tyrode solution, papain increased the yield of single cells significantly. The cells were contracted in a dose-dependent manner by Ca2+ in the high-K+ Tyrode solution and by carbachol in 0.18 mM Ca2+-Tyrode solution; furthermore, the contractions were antagonized by verapamil and atropine, respectively. Treatment with papain did not affect cell sensitivity to the stimulants. Therefore, our results suggest that the addition of papain is useful for the isolation of single cells to investigate the physiological and pharmacological characteristics of smooth muscle.

Animals↗
PubMed2000

Smooth muscle alpha-actin CArG elements coordinate formation of a smooth muscle cell-selective, serum response factor-containing activation complex.

Previous studies have shown that multiple serum response factor (SRF)-binding CArG elements were required for smooth muscle cell (SMC)-specific regulation of smooth muscle (SM) alpha-actin expression. However, a critical question remains as to the mechanisms whereby a ubiquitously expressed transcription factor such as SRF might contribute to SMC-specific expression. The goal of the present study was to investigate the hypothesis that SMC-selective expression of SM alpha-actin is due at least in part to (1) unique CArG flanking sequences that distinguish the SM alpha-actin CArGs from other ubiquitously expressed CArG-dependent genes such as c-fos, (2) cooperative interactions between CArG elements, and (3) SRF-dependent binding of SMC-selective proteins to the CArG-containing regions of the promoter. Results demonstrated that specific sequences flanking CArG B were important for promoter activity in SMCs but not in bovine aortic endothelial cells. We also provided evidence indicating that the structural orientation between CArGs A and B was an important determinant of promoter function. Electrophoretic mobility shift assays and methylation interference footprinting demonstrated that a unique SRF-containing complex formed that was selective for SMCs and, furthermore, that this complex was probably stabilized by protein-protein interactions and not by specific interactions with CArG flanking sequences. Taken together, the results of these studies provide evidence that SM alpha-actin expression in SMCs is complex and may involve the formation of a unique multiprotein initiation complex that is coordinated by SRF complexes bound to multiple CArG elements.

Actins↗
PubMed1994

Regulation of actomyosin and contraction in smooth muscle.

Unlike striated muscle cells, smooth muscle cells do not have an organized sarcomeric structure. However, all smooth muscle cells contain the contractile proteins, myosin, actin, and tropomyosin. Polymorphism of the myosin heavy chain exists in smooth muscle cells. Two myosin heavy chain (MHC) isoforms, SM1 (204 kDa) and SM2 (200 kDa), are present in smooth muscle cells; however, their ratios vary in smooth muscles from different sources. The hypertrophy of the urinary bladder induced by partial outlet obstruction in rabbits is associated with an alteration of the SM1-to-SM2 ratio from 1:3 to 1:1. Both heavy chains react with polyclonal antibody against smooth muscle myosin; however, antibody prepared against a peptide from the C-terminal region of the SM2 heavy chain cross-reacts only with the SM2 heavy chain. Removal of the obstruction reverses the bladder to normal mass with a concomitant change in the SM1-to-SM2 ratio back to 1:3. The expression of the SM1 mRNA is increased in response to obstruction-induced hypertrophy, and it also returns to normal upon removal of the obstruction. Urinary bladder smooth muscle contains predominantly gamma-actin. Obstruction-induced hypertrophy of the bladder smooth muscle is associated with an increase in the gamma-actin at both protein and mRNA levels. The beta-non-muscle actin is decreased and the alpha-smooth muscle actin is unchanged in response to obstruction-induced bladder hypertrophy. Contraction of all smooth muscles involves similar mechanisms. This review describes our current understanding of the mechanisms regulating contraction of the smooth muscle of the urinary bladder.

Actomyosin↗
PubMed1994

T lymphocytes adhere to airway smooth muscle cells via integrins and CD44 and induce smooth muscle cell DNA synthesis.

Asthma is a disease of airway inflammation and hyperreactivity that is associated with a lymphocytic infiltrate in the bronchial submucosa. The interactions between infiltrating T lymphocytes with cellular and extracellular matrix components of the airway and the consequences of these interactions have not been defined. We demonstrate the constitutive expression of CD44 on human airway smooth muscle (ASM) cells in culture as well as in human bronchial tissue transplanted into severe combined immunodeficient mice. In contrast, basal levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expression are minimal but are induced on ASM by inflammatory mediators such as tumor necrosis factor alpha (TNF-alpha). Activated, but not resting T cells, adhere to cultured ASM; stimulation of the ASM with TNF-alpha enhanced this adhesion. Adhesion was partially blocked by monoclonal antibodies (mAb) specific for lymphocyte function-associated antigen 1 (LFA-1) and very late antigen 4 (VLA-4) on T cells and ICAM-1 and VCAM-1 on ASM cells. The observed integrin-independent adhesion was mediated by CD44/hyaluronate interactions as it was inhibited by anti-CD44 mAb 5F12 and by hyaluronidase. Furthermore, the adhesion of activated T lymphocytes induced DNA synthesis in growth-arrested ASM cells. Thus, the interaction between T cells and ASM may provide insight into the mechanisms that induce bronchial inflammation and possibly ASM cell hyperplasia seen in asthma.

Animals↗
PubMed2006

Cytokine-induced differentiation of multipotent adult progenitor cells into functional smooth muscle cells.

Smooth muscle formation and function are critical in development and postnatal life. Hence, studies aimed at better understanding SMC differentiation are of great importance. Here, we report that multipotent adult progenitor cells (MAPCs) isolated from rat, murine, porcine, and human bone marrow demonstrate the potential to differentiate into cells with an SMC-like phenotype and function. TGF-beta1 alone or combined with PDGF-BB in serum-free medium induces a temporally correct expression of transcripts and proteins consistent with smooth muscle development. Furthermore, SMCs derived from MAPCs (MAPC-SMCs) demonstrated functional L-type calcium channels. MAPC-SMCs entrapped in fibrin vascular molds became circumferentially aligned and generated force in response to KCl, the L-type channel opener FPL64176, or the SMC agonists 5-HT and ET-1, and exhibited complete relaxation in response to the Rho-kinase inhibitor Y-27632. Cyclic distention (5% circumferential strain) for 3 weeks increased responses by 2- to 3-fold, consistent with what occurred in neonatal SMCs. These results provide evidence that MAPC-SMCs are phenotypically and functionally similar to neonatal SMCs and that the in vitro MAPC-SMC differentiation system may be an ideal model for the study of SMC development. Moreover, MAPC-SMCs may lend themselves to tissue engineering applications.

Animals↗
PubMed1994

Volatile anesthetics and agonist-induced contractions in porcine coronary artery smooth muscle and Ca2+ mobilization in cultured immortalized vascular smooth muscle cells.

BACKGROUND: These experiments addressed four specific questions. Do isoflurane and halothane (0.5-3.0% in the gas phase) inhibit contractions evoked in isolated porcine coronary artery rings (without endothelium) by the specific Ca2+ mobilizing agonists serotonin, endothelin-1, and F-? Are contractions evoked by phorbol-activated protein kinase C inhibited by the anesthetics? In a well-characterized vascular smooth muscle cell culture model (A7r5 and A10), do the anesthetics attenuate serotonin- and endothelin-induced Ca2+ mobilization? Do the anesthetics inhibit intracellular Ca2+ mobilization via facilitated cAMP formation? METHODS: Tension was measured in rings suspended in organ chambers. Apparent intracellular Ca2+ was estimated in cells using indo-1 and flow cytometry. Cyclic AMP was measured by radioimmunoassay. RESULTS: At the anesthetic concentrations examined, isoflurane attenuated contractions evoked by serotonin and F- but not those induced by endothelin-1 or phorbol dibutyrate. In cells, isoflurane 2% attenuated 3 x 10(-5) M serotonin-induced Ca2+ mobilization by about 26%, whereas Ca2+ responses evoked by endothelin 10(-8) M were more resistant to anesthetic inhibitory effect. Halothane attenuated contractions in rings evoked by serotonin, endothelin, and F- but lacked effect on phorbol-induced responses. In cells, halothane 2% inhibited Ca2+ mobilization induced by serotonin by about 43% and that induced by endothelin by about 31%. Neither anesthetic facilitated cAMP formation. CONCLUSIONS: Isoflurane and halothane variably attenuated contractions evoked by Ca2+ mobilizing agonists--by a cellular action beyond the receptor level--but did not inhibit phorbol activated protein kinase C. Serotonin- and endothelin-induced Ca2+ mobilization was inhibited by isoflurane and halothane--but the mechanism does not depend upon increased cAMP.

Aluminum Compounds↗
PubMed1999

Hyperplasia in multiple smooth muscle tissues in transgenic mice expressing a temperature-sensitive SV40 T-antigen under the control of smooth muscle alpha-actin regulatory sequences.

Control of smooth muscle cell (SMC) proliferation is of fundamental importance in the development and pathology of the vasculature. To derive vascular SMC with conditional inactivation of negative cell cycle regulatory proteins in the context of smooth muscle protein expression, a 3.4 kb fragment of the mouse SMC alpha-actin promoter was used to target a temperature-sensitive mutant SV40 T antigen (tsA58) to smooth muscle in transgenic mice. Mice with this genotype display a heritable phenotype of abnormal SMC proliferation in the central tail artery, vasa deferentia, seminal vesicles, prostate, and uterus, with the latter resembling uterine leiomyomatosis and prostatic hypertrophy. Neither the aorta nor other viscera manifested abnormal proliferation. Cultures from aorta, vas deferens, seminal vesicle, and kidney tissue were characterized with regard to protein expression, stability, and matrix remodelling capacity. The alpha-actin content/cell was up to 3-4-fold higher, as well as more stable than in primary SMC cultures, suggesting successful selection for propagation of cells expressing this differentiation marker. All cells displayed enhanced growth at the permissive temperature. As an initial functional assessment, the cells were compared to non-transformed mouse aortic SMC with respect to the ability to remodel collagen gel matrices, and demonstrated conservation of this physiologic function. This in vivo analysis of the SMC alpha-actin promoter supports a broader range of smooth muscle-directed expression activity than previously recognized, and establishes the feasibility of its use to direct transgene expression to vascular as well as genito-urinary smooth muscle. The targeted expression of the tsA58 T antigen has yielded transgenic animals with several manifestations of smooth muscle hyperplasia; these animals have in turn permitted the derivation of several murine SMC lines with phenotypic stability and conditionally-modulated proliferation. These cells will allow expansion of derivative transfected smooth muscle cell lines under permissive conditions, as well as oncogene inactivation at the restrictive temperature when desired for functional studies.

Actins↗
PubMed2005

IP3-mediated Ca2+ increases do not involve the ryanodine receptor, but ryanodine receptor antagonists reduce IP3-mediated Ca2+ increases in guinea-pig colonic smooth muscle cells.

Smooth muscle responds to IP3-generating (sarcolemma acting) neurotransmitters and hormones by releasing Ca2+ from the sarcoplasmic reticulum (SR) via IP3 receptors (IP3Rs). This release may propagate as Ca2+ waves. The Ca2+ signal emanating from IP3 generation may be amplified by its activating further Ca2+ release from ryanodine receptors (RyRs) in the process of Ca2+-induced Ca2+ release (CICR). Evidence for this proposal has relied largely on the use of blocking drugs such as ryanodine, tetracaine and dantrolene, reportedly specific inhibitors of RyRs. Here we have examined whether or not Ca2+ released via IP3Rs subsequently activates RyRs. In addition, the specificity of the blocking agents has been assessed by determining the extent of their ability to block IP3-mediated Ca2+ release under conditions in which RyRs were not activated. IP3-evoked Ca2+ release and Ca2+ waves did not require or activate RyRs. However, the RyR blocking drugs inhibited IP3-mediated Ca2+ signals at concentrations thought to be selective for RyRs. In single colonic smooth muscle cells, voltage clamped in the whole cell configuration, carbachol (CCh) evoked propagating Ca2+ waves which were not inhibited by ryanodine when the sarcolemma potential was -70 mV. At -20 mV, at which potential the SR Ca2+ content was increased and RyRs activated, ryanodine inhibited the Ca2+ waves. Photolysed caged IP3 increased [Ca2+]c; ryanodine, by itself, did not reduce the IP3-evoked [Ca2+]c increase when the sarcolemma potential was maintained at -70 mV. However, after activation of RyRs by caffeine, in the continued presence of ryanodine, the IP3-evoked [Ca2+]c increase was inhibited. In other experiments, RyRs were activated (as evidenced by the occurrence of spontaneous transient outward currents) by depolarizing the sarcolemma to -20 mV and again ryanodine was effective in inhibiting IP3-evoked Ca2+ increase. Thus while ineffective by itself, ryanodine inhibited IP3-evoked Ca2+ increases, presumably by causing persistent opening of the channel and depleting the SR of Ca2+, after RyRs were activated. These experiments establish that IP3-evoked Ca2+ release and Ca2+ waves do not activate RyRs; had they done so ryanodine would have inhibited the Ca2+ increase. However, under conditions where ryanodine was ineffective against the IP3-evoked Ca2+ transient (i.e. when RyRs were not activated, e.g. at a membrane potential of -70 mV) tetracaine and dantrolene each blocked IP3-evoked Ca2+ increases. The results show that although IP3-mediated Ca2+ release does not activate RyRs, RyR blockers can inhibit IP3-mediated Ca2+ signals.

Animals↗
PubMed2002

Identification of albumin precursor protein, Phi AP3, and alpha-smooth muscle actin as novel components of redox sensing machinery in vascular smooth muscle cells.

Aerobic organisms are continually subjected to environmental stressors that compromise redox homeostasis and induce cellular injury. In vascular smooth muscle cells (vSMCs), the activation/repression of redox-regulated genes after environmental stress often involves protein binding to cis-acting antioxidant response elements (AREs). The present study was conducted to identify proteins that participate in redox-regulated protein binding to human c-Ha-ras and mouse glutathione S-transferase A1 AREs in vSMCs after oxidant injury. Challenge of vSMCs with 0.3 or 3 microM hydrogen peroxide, 3-methylcholanthrene, benzo[a]pyrene-7,8-diol, 3-hydroxy benzo[a]pyrene, and benzo[a]pyrene-3,6-quinone induced concentration-related increases in ARE protein binding. The profiles of ARE complex assembly were comparable, but exhibited chemical specificity. Pretreatment with 0.5 mM N-acetylcysteine inhibited activation of ARE protein binding in hydrogen peroxide-treated cells. Preparative electrophoretic mobility shift assays coupled to Western analysis identified NF-E2-related proteins 1 and 2 and JunD in complexes assembled on AREs. Polyethylenimine affinity and sequence-specific serial immobilized DNA affinity chromatography followed by N-terminal sequencing identified albumin precursor protein, phi AP3, and alpha-smooth muscle actin as members of the ARE signaling pathway. Sequence analysis of albumin protein revealed homology to the redox-regulated transcription factors Bach1 and 2, as well as cytoskeletal and molecular motor proteins. These results implicate albumin precursor protein, phi AP3, and alpha-smooth muscle actin as participants in redox sensing in vSMCs, and suggest that protein complex assembly involves interactions between leucine zipper and zinc finger transcription factors with cytoskeletal proteins.

Actins↗