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PubMed1984

The rat costo-uterine muscle: a preparation of smooth muscle containing a homogeneous population of beta-adrenoreceptors.

This paper describes some histological features of the costo-uterine muscle of the rat together with the effects of some sympathomimetic amines upon contractions evoked by field electrical stimulation of isolated preparations. Light and electron microscopy confirmed that the costo-uterine muscle of the rat consists of smooth muscle bundles, arranged longitudinally and interspersed with collagen. There were close contacts between individual cells within bundles. No axon profiles were observed. Histochemical techniques revealed only sparse catecholamine fluorescence at the border of the tissue in association with blood vessels. Isoprenaline, fenoterol, adrenaline, noradrenaline, salbutamol and phenylephrine consistently inhibited contractions evoked by field stimulation (40V, 30Hz, 2 ms for 5 sec every 200 sec). In contrast, tyramine was without effect upon electrically evoked contractions in concentrations of up to 200 mumol/l. The slopes of the log concentration-response curves of effective amines were similar, and all of these were capable of producing 100% inhibition of contraction. The potencies of the amines relative to isoprenaline = 100 were: fenoterol 135; salbutamol 16; adrenaline 16; noradrenaline 0.7; phenylephrine 0.1; tyramine less than 0.001. Propranolol, added to preparations in the absence of inhibitors of amine uptake and alpha-adrenoreceptors, competitively antagonised the effects of the amines. Schild plots had slopes which did not differ significantly from minus one, and the mean pA2 values fell within a narrow range, e.g. 8.65 with salbutamol; 9.20 with fenoterol. Mean pA2 values for propranolol with noradrenaline and isoprenaline were unaffected by the combined presence of phentolamine (10 mumol/l), cocaine (10 mumol/l), and corticosterone (10 mumol/l). The potencies of the agonists were also unaffected by the presence of these drugs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1997

An insert in the motor domain determines the functional properties of expressed smooth muscle myosin isoforms.

Smooth muscle myosin isoforms of the heavy chain and the essential light chain have been hypothesized to contribute to the different shortening velocities of phasic and tonic smooth muscles, and to their different affinities for MgADP. We used the baculovirus/insect cell system to express homogeneous heavy meromyosin molecules differing only in seven amino acid insert (QGPSFSY) in the motor domain near the active site, or in the type of essential light chain isoform. Myosin from tonic rabbit uterine smooth muscle lacks the heavy chain insert, while myosin from phasic chicken gizzard contains it. The properties of a mutant uterine heavy meromyosin with added insert, and a mutant gizzard heavy meromyosin with the insert deleted, were compared with their wild type progenitors. Phosphorylated heavy meromyosins with the insert have a twofold higher enzymatic activity and in vitro motility han heavy meromyosins without the insert. These functional properties were not altered by the essential light chain isoforms. The altered motility caused by the insert implies that it modulates the rate of ADP release, the molecular step believed to limit shortening velocity. The insert may thus account in part for both the lower sensitivity to MgADP and the higher shortening velocity of phasic compared to tonic smooth muscles.

Actins↗

PubMed1999

HSP27 in signal transduction and association with contractile proteins in smooth muscle cells.

Sustained smooth muscle contraction is mediated by protein kinase C (PKC) through a signal transduction cascade leading to contraction. Heat-shock protein 27 (HSP27) appears to be the link between these two major events, i.e., signal transduction and sustained smooth muscle contraction. We have investigated the involvement of HSP27 in signal transduction and HSP27 association with contractile proteins (e.g., actin, myosin, tropomyosin, and caldesmon) resulting in sustained smooth muscle contraction. We have carried out confocal microscopy to investigate the cellular reorganization and colocalization of proteins and immunoprecipitation of HSP27 with actin, myosin, tropomyosin, and caldesmon as detected by sequential immunoblotting. Our results indicate that 1) translocation of Raf-1 to the membrane when stimulated with ceramide is inhibited by vasoactive intestinal peptide (VIP), a relaxant neuropeptide; 2) PKC-alpha and mitogen-activated protein kinase translocate and colocalize on the membrane in response to ceramide, and PKC-alpha translocation is inhibited by VIP; 3) HSP27 colocalizes with actin when contraction occurs; and 4) HSP27 immunoprecipitates with actin and with the contractile proteins myosin, tropomyosin, and caldesmon. We propose a model in which HSP27 is involved in sustained smooth muscle contraction and modulates the interaction of actin, myosin, tropomyosin, and caldesmon.

Actins↗

PubMed1994

Biodegradable microspheres containing a colchicine analogue inhibit DNA synthesis in vascular smooth muscle cells.

BACKGROUND: Smooth muscle cell proliferation plays a major role in the genesis of restenosis after angioplasty or vascular injury. Local application of agents capable of modulating vascular responses, including smooth muscle cell proliferation, has been achieved, but difficulty in maintaining active levels locally has been a factor limiting the efficacy of such approaches. One strategy to maintain adequate levels is the local delivery of microspheres that release active agents over sustained time periods. METHODS AND RESULTS: We incorporated a colchicine analogue into biodegradable microspheres composed of a lactic acid/glycolic acid copolymer and characterized their drug release behavior as well as their effects on bovine aortic smooth muscle cells (BASMCs) in culture. Drug release was evaluated by spectrophotometric assay. Drug effects on DNA synthesis were measured by thymidine incorporation after addition of serum to subconfluent cells synchronized by serum withdrawal as well as in asynchronous cell populations. Polymeric microspheres incorporating 10% to 17% drug by weight and averaging 6 microns in size were found to release the colchicine analog in buffered saline solutions over more than several weeks. Drug-loaded particles inhibited DNA synthesis completely, with EC50 values ranging from 0.001 to 0.005 g% (wt/wt). Morphological changes suggesting microtubule depolymerization were observed after drug particle treatment, with similar EC50 values. Microspheres allowed to contact the cell surface demonstrated effects similar to those seen with microspheres suspended in the nutrient medium by porous polycarbonate filters, at EC50 values approximately fivefold lower. In contrast, control microspheres composed only of polymer with no incorporated active drug demonstrated no observable toxicity to BASMCs and < 40% inhibition of thymidine incorporation even in suspensions containing up to 0.5 g% particles. CONCLUSIONS: Biodegradable microspheres were fashioned that release a colchicine analogue and inhibit DNA synthesis in smooth muscle cells. Drug-loaded polymeric particles are candidates for local delivery at sites of arterial injury to decrease restenosis.

Animals↗

PubMed1994

Activation properties of myosin light chain kinase during contraction/relaxation cycles of tonic and phasic smooth muscles.

In intact smooth muscle, myosin light chain kinase (MLCK) is phosphorylated at its regulatory site by Ca2+/calmodulin-dependent protein kinase II resulting in an increase in the concentration of Ca2+/calmodulin required for half-maximal activation of the enzyme (KCaM). We investigated the physiological significance of MLCK phosphorylation during cycles of contraction and relaxation in tonic (tracheal) and phasic (uterine) smooth muscles. MLCK phosphorylation and dephosphorylation occurred at rates sufficient to modulate the Ca2+ sensitivity of light chain phosphorylation. In contractions of both smooth muscles (though using different sources of activating Ca2+), increases in [Ca2+]i preceded light chain phosphorylation; but, the rate of increase in light chain phosphorylation was significantly greater than the rate of increase in [Ca2+]i. The onset of MLCK phosphorylation with the resultant increase in KCaM coincided with the diminished rate of light chain phosphorylation. During spontaneous contractions of uterine smooth muscle, the Ca2+ transient was characterized by an initial rapid increase, a sustained plateau, and rapid decline. During the sustained phase of the Ca2+ transient, MLCK phosphorylation increased and coincided with dephosphorylation of light chain and relaxation. These results indicate that MLCK is sensitive to small increases in intracellular Ca2+ during the initiation of contraction and that the enzyme subsequently becomes desensitized to Ca2+/calmodulin, thereby limiting the extent of light chain phosphorylation.

Animals↗

PubMed2006

siRNA knock down of casein kinase 2 increases force and cross-bridge cycling rates in vascular smooth muscle.

Contraction of smooth muscle involves myosin light chain (MLC) kinase catalyzed phosphorylation of the regulatory MLC, activation of myosin, and the development of force. However, this cannot account for all aspects of a smooth muscle contraction, suggesting that other regulatory mechanisms exist. One potentially important technique to study alternative sites of contractile regulation is the use of small interfering RNA (siRNA). The goal of this study was to determine whether siRNA technology can decrease the levels of a specific protein and allow for the determination of how that protein affects contractile regulation. To achieve this goal, we tested the hypothesis that casein kinase 2 (CK2) is part of the complex regulatory scheme present in vascular smooth muscle. Using intact strips of swine carotid artery, we determined that siRNA against CK2 produced a tissue that resulted in a approximately 60% knockdown after 4 days in organ culture. Intact strips of vascular tissue depleted of CK2 produced greater levels of force and exhibited an increased sensitivity to all stimuli tested. This was accompanied by an increase in cross-bridge cycling rates but not by a change in MLC phosphorylation levels. alpha-Toxin-permeabilized vascular tissue depleted of CK2 also showed an increased sensitivity to calcium compared with control tissues. Our results demonstrate that siRNA is a viable technique with which to study regulatory pathways in intact smooth muscle tissue. Our results also demonstrate that CK2 plays an important role in the mechanism(s) responsible for the development of force and cross-bridge cycling by a MLC phosphorylation-independent pathway.

Actinin↗

PubMed1975

Vibration-induced inhibition of vascular smooth muscle contraction.

Vascular smooth muscle is known to be exposed to an oscillating strain under physiological and patho-physiological conditions as well as in different occupational and environmental situations. The effect of vibrations of smooth muscle seems to be largly unknown. In the present experiments on isolated preparations of the rat portal vein and the rabbit thoracic aorta, imposed sinusoidal changes in length were found to cause prompt reduction in active force, the extent of which was dependent on amplitude (1-10% of tissue length, peak to peak, i.e. approximately plus or minus 50-500 mum) and frequency of vibration (1-400 Hz) as well as on the prevailing level of active and passive forces. Vibration caused only small and inconsistant reductions of passive force of vascular smooth muscle. The results are in accordance with the hypothesis that vibrations exert a direct action on the contractile process by causing an increased rate of detachment of actin-myosin cross-links. It is suggested that, in vivo, vibrations may affect the diameter of conduit arteries locally in the case of turbulent blood flow as seen in post-stenotic dilation and arterio-venous anastomosis. Possibly, even the normal pulse pressure oscillations may sometimes tend to inhibit the smooth muscle activity in such arteries and thereby influence their diameters.

Animals↗

PubMed1989

Pharmacological characterization of [Leu-13-psi-CH2NH-Leu14]-bombesin as a specific bombesin receptor antagonist on isolated smooth muscle cells.

Isolated smooth muscle cells from guinea pig stomach were used to study the pharmacological characteristics of a newly synthetized bombesin analog, [Leu13-psi-CH2NH-Leu14]-bombesin (psi 13,14-BN) to function as antagonist of bombesin-induced contractile response. The antagonism caused by this new analog was compared to that obtained with the substance P analog [D-Arg1,D-Pro2,D-Trp7,9,Leu11]Substance P [( APTTL]SP), which has been used until now to characterize bombesin receptors on smooth muscle cells. psi 13,14-BN resulted to be more potent than [APTTL]SP as antagonist of bombesin action on smooth muscle. Comparing the IC50, psi 13,14-BN (IC50 70 nM) was 8 times more potent than [APTTL]SP (IC50 600 nM). In contrast to [APTTL] SP, the action of psi 13,14-BN was shown to be specific toward bombesin receptors in that it does not interfere with receptors for other agents (i.e., cholecystokinin, acetylcholine or substance P). The antagonism induced by both compounds was competitive inasmuch as the slope of the regression lines obtained by Schild plot analysis were not significantly different from the unity. The apparent affinity for the bombesin receptor was 0.8 nM for psi 13,14-BN and 7.8 nM for [APTTL]SP. These results indicate that psi 13,14-BN acts on isolated gastric smooth muscle cells as a competitive bombesin receptor antagonist, with a higher affinity and specificity than the substance P analog used previously.

Animals↗

PubMed1994

Regulation of vascular smooth muscle tone.

Vascular smooth muscle tone is regulated primarily by the sarcoplasmic free Ca2+ concentration, which determines the level of myosin phosphorylation. Stimulation of the muscle results in an increase in free [Ca2+], whereupon Ca2+ binds to calmodulin, inducing a conformational change enabling calmodulin to interact with and activate myosin light chain kinase. The active Ca2+.calmodulin.myosin light chain kinase complex catalyses the phosphorylation of serine-19 of the two 20-kDa light chains of myosin; this triggers cross-bridge cycling and the development of force. Relaxation follows restoration of free [Ca2+] to the resting level, whereupon calmodulin dissociates from myosin light chain kinase, which is thereby inactivated, and myosin is dephosphorylated by myosin light chain phosphatase and remains detached from actin. Overwhelming evidence now exists in favour of the central role of myosin phosphorylation-dephosphorylation in smooth muscle contraction-relaxation. However, considerable evidence supports the existence of additional, secondary mechanisms that can modulate the contractile state of smooth muscle either by altering the Ca2+ sensitivity of the contractile response or otherwise modulating one of the molecular events occurring downstream of the Ca2+ signal, e.g., the interaction of phosphorylated myosin heads with actin. The interplay of several regulatory elements confers on the contractile response of vascular smooth muscle the high degree of flexibility and adaptability required for the effective regulation of blood pressure.

Amino Acid Sequence↗

PubMedDate not supplied

Transcriptional profile of mechanically induced genes in human vascular smooth muscle cells.

Vascular smooth muscle cells must monitor and respond to their mechanical environment; however, the molecular response of these cells to mechanical stimuli remains incompletely defined. By applying a highly uniform biaxial cyclic strain to cultured cells, we used DNA microarray technology to describe the transcriptional profile of mechanically induced genes in human aortic smooth muscle cells. We first identified vascular endothelial growth factor (VEGF) as a mechanically induced gene in these cells; VEGF served as a positive control for these experiments. We then used a DNA microarray with 5000 genes with putative functions to identify additional mechanically induced genes. Surprisingly, relatively few genes are mechanically induced in human aortic smooth muscle cells. Only 3 transcripts of 5000 were induced >2.5-fold: cyclooxygenase-1, tenascin-C, and plasminogen activator inhibitor-1. Downregulated transcripts included matrix metalloproteinase-1 and thrombomodulin. The transcriptional profile of mechanically induced genes in human aortic smooth muscle cells suggests a response of defense against excessive deformation. These data also demonstrate that in addition to identifying large clusters of genes that respond to a given stimulus, DNA microarray technology may be used to identify a small subset of genes that comprise a highly specific molecular response.

Cells, Cultured↗

PubMed1991

Expression of thrombomodulin by smooth muscle cells in culture: different effects of tumor necrosis factor and cyclic adenosine monophosphate on thrombomodulin expression by endothelial cells and smooth muscle cells in culture.

Thrombomodulin (TM), a critical component of the protein C anticoagulant pathway, has previously been localized to endothelial cells (EC), but not smooth muscle cells (SMC) of the blood vessel wall. We demonstrate that cultured rat, bovine, as well as human SMC, but not blood vessel wall smooth muscle tissue, possess significant functional levels of TM and TM mRNA. Cyclic adenosine monophosphate stimulates TM expression in cultured SMC, but not EC, while tumor necrosis factor suppresses TM expression in EC but not cultured SMC. We postulate that following acute or chronic EC injury, luminal SMC can express TM, and are therefore able to protect the damaged blood vessel from thrombosis.

Animals↗

PubMed1983

Cross-bridge elasticity in single smooth muscle cells.

In smooth muscle, a cross-bridge mechanism is believed to be responsible for active force generation and fiber shortening. In the present studies, the viscoelastic and kinetic properties of the cross-bridge were probed by eliciting tension transients in response to small, rapid, step length changes (delta L = 0.3-1.0% Lcell in 2 ms). Tension transients were obtained in a single smooth muscle cell isolated from the toad (Bufo marinus) stomach muscularis, which was tied between a force transducer and a displacement device. To record the transients, which were of extremely small magnitude (0.1 microN), a high-frequency (400 Hz), ultrasensitive force transducer (18 mV/microN) was designed and built. The transients obtained during maximal force generation (Fmax = 2.26 microN) were characterized by a linear elastic response (Emax = 1.26 X 10(4) mN/mm2) coincident with the length step, which was followed by a biphasic tension recovery made up of two exponentials (tau fast = 5-20 ms, tau slow = 50-300 ms). During the development of force upon activation, transients were elicited. The relationship between stiffness and force was linear, which suggests that the transients originate within the cross-bridge and reflect the cross-bridge's viscoelastic and kinetic properties. The observed fiber elasticity suggests that the smooth muscle cross-bridge is considerably more compliant than in fast striated muscle. A thermodynamic model is presented that allows for an analysis of the factors contributing to the increased compliance of the smooth muscle cross-bridge.

Animals↗

PubMed1995

Evidence for a protein S receptor(s) on human vascular smooth muscle cells. Analysis of the binding characteristics and mitogenic properties of protein S on human vascular smooth muscle cells.

The presence of specific binding sites for the coagulation factor protein S (PS) on the surface of human vascular smooth muscle cells (HVSMC) is described. The binding characteristics of 125I-PS to HVSMC were studied and found to be saturable, reversible and, as described by the Hill equation, co-operative (h 1.74; Kd 0.33 nM). Autoradiographic analysis of detergent extracts of HVSMC chemically cross-linked with 125I-PS and separated by SDS/PAGE revealed radioactivity associated with two proteins with apparent molecular masses of 220 and 230 kDa respectively. The mitogenic activity of PS on HVSMC was also investigated. Protein S was shown to stimulate DNA synthesis of growth-arrested HVSMC and to support their proliferation under low-serum conditions in a sustained and dose-dependent manner.

Adolescent↗

PubMed1991

Localization of the DMDL gene-encoded dystrophin-related protein using a panel of nineteen monoclonal antibodies: presence at neuromuscular junctions, in the sarcolemma of dystrophic skeletal muscle, in vascular and other smooth muscles, and in proliferating brain cell lines.

mAbs have been raised against different epitopes on the protein product of the DMDL gene, which is an autosomal homologue of the X-linked DMD gene for dystrophin. These antibodies provide direct evidence that DMDL protein is localized near acetylcholine receptors at neuromuscular junctions in normal and mdx mouse intercostal muscle. The primary location in tissues other than skeletal muscle is smooth muscle, especially in the vascular system, which may account for the wide tissue distribution previously demonstrated by Western blotting. The DMDL protein was undetectable in the nonjunctional sarcolemma of normal human muscle, but was observed in nonjunctional sarcolemma of Duchenne muscular dystrophy patients, where dystrophin itself is absent or greatly reduced. The expression of DMDL protein is not restricted to smooth and skeletal muscle, however, since relatively large amounts are present in transformed brain cell lines of both glial and Schwann cell origin. This contrasts with the low levels of DMDL protein in adult brain tissue.

Animals↗

PubMed2001

Adenylyl cyclase 3 mediates prostaglandin E(2)-induced growth inhibition in arterial smooth muscle cells.

Arterial smooth muscle cell (SMC) proliferation contributes to a number of vascular pathologies. Prostaglandin E(2) (PGE(2)), produced by the endothelium and by SMCs themselves, acts as a potent SMC growth inhibitor. The growth-inhibitory effects of PGE(2) are mediated through activation of G-protein-coupled membrane receptors, activation of adenylyl cyclases (ACs), formation of cAMP, and subsequent inhibition of mitogenic signal transduction pathways in SMCs. Of the 10 different mammalian AC isoforms known today, seven isoforms (AC2-7 and AC9) are expressed in SMCs from various species. We show that, despite the presence of several different AC isoforms, the principal AC isoform activated by PGE(2) in human arterial SMCs is a calmodulin kinase II-inhibited AC with characteristics similar to those of AC3. AC3 is expressed in isolated human arterial SMCs and in intact aorta. We further show that arterial SMCs isolated from AC3-deficient mice are resistant to PGE(2)-induced growth inhibition. In summary, AC3 is the principal AC isoform activated by PGE(2) in arterial SMCs, and AC3 mediates the growth-inhibitory effects of PGE(2). Because AC3 activity is inhibited by intracellular calcium through calmodulin kinase II, AC3 may serve as an important integrator of growth-inhibitory signals that stimulate cAMP formation and growth factors that increase intracellular calcium.

Adenylyl Cyclases↗

PubMedDate not supplied

Isolation and characterization of a novel short chain alcohol dehydrogenase-like isozyme by differential display of distinct smooth muscle cell phenotypes.

Gastrointestinal smooth muscle development proceeds by the linear differentiation of distinct smooth muscle cell phenotypes. In an effort to identify specific gene products associated with distinct smooth muscle cell phenotypes, we performed differential display on smooth muscle myoblasts versus immature smooth muscle myocytes. This analysis identified a novel short-chain alcohol dehydrogenase-like isozyme which was preferentially expressed in smooth muscle myoblasts over immature and mature smooth muscle myocytes. We postulate that this novel short-chain alcohol dehydrogenase-like isozyme may play a role in potentiating the dedifferentiation of smooth muscle cells in vitro.

Alcohol Dehydrogenase↗

PubMed1962

Action of the calcium antagonists cocaine and ethanol on contraction and potassium efflux of smooth muscle.

Isolated longitudinal smooth muscle from guinea pig ileum exposed to a high potassium depolarizing medium exhibited a sustained increase in muscle tone and an increase in potassium efflux. When the concentration of calcium ion in the medium was elevated the increase in muscle tone was enhanced, but the change in potassium efflux was reduced slightly. Lowering the calcium concentration diminished the increase in muscle tone. Both cocaine and ethanol completely inhibited the sustained contraction of potassium-depolarized fibers. Addition of excess calcium ion reversed these inhibitions. Cocaine acted primarily like a competitive antagonist; and ethanol, like an indirect antagonist of calcium, ion. Under certain conditions acetylcholine potentiated the reversal by calcium ion of the drug-induced inhibitions. The two inhibitory drugs had dissimilar effects on potassium efflux from smooth muscle fibers immersed in Tyrode solution. Cocaine depressed and ethanol enhanced this membrane process. However, the increase in potassium efflux induced by acetylcholine was inhibited by ethanol. This inhibition also was reversed by increasing the concentration of calcium ion in the medium. The data suggested that calcium activates and cocaine and ethanol inhibit a cellular reaction which occurs beyond the point of membrane depolarization and is essential for smooth muscle contraction. Furthermore, calcium serves to depress membrane excitability, but appears to have a specific stimulatory role in the acetylcholine-induced increase in potassium efflux from longitudinal fibers.

Acetylcholine↗

PubMed1999

Effects of nifedipine on anorectal smooth muscle in vitro.

INTRODUCTION: Glyceryl trinitrate reduces anal resting pressure and aids the healing of anal fissures. However, some patients develop tachyphylaxis and the fissure fails to heal, suggesting that other agents are needed. This study assesses the effects of nifedipine (a calcium channel antagonist) in modulating resting tone and agonist-induced contractions in human internal anal sphincter (IAS) and rectal circular muscle. METHODS: Smooth muscle strips from the IAS and rectal circular muscle from ten patients undergoing surgical resection were mounted for isometric tension recording in a superfusion organ bath. The effects of noradrenaline and carbachol were assessed in the presence of various perfusates. RESULTS: LAS strips developed tone and spontaneous activity. Noradrenaline produced dose-dependent contractions. In calcium-free Krebs solution, tone and activity were abolished and no contractions were elicited in response to noradrenaline. Nifedipine also abolished tone and spontaneous activity, but contractions to noradrenaline were only slightly attenuated. In contrast, rectal smooth muscle strips developed spontaneous activity but no resting tone and contracted in response to carbachol. In calcium-free Krebs solution, the spontaneous activity and carbachol contractions were abolished. Addition of nifedipine to the perfusate abolished spontaneous activity and greatly reduced contractions. DISCUSSION: These data suggest that spontaneous activity and resting tone are dependent on extracellular calcium and flux across the cells. Agonist-induced contraction in the IAS is attributable mainly to the release of calcium from intracellular stores, whereas rectal circular smooth muscle depends principally on extracellular calcium entering the cell for contraction. The attenuation of contractions in both tissues and the abolition of resting tone in the IAS suggest that nifedipine may be useful in the management of patients with anorectal disorders.

Adult↗