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At least 37 records · Page 2Linked to original sources
PubMed2004

Agonist-induced association of tropomyosin with protein kinase Calpha in colonic smooth muscle.

Smooth muscle contraction regulated by myosin light chain phosphorylation is also regulated at the thin-filament level. Tropomyosin, a thin-filament regulatory protein, regulates contraction by modulating actin-myosin interactions. Present investigation shows that acetylcholine induces PKC-mediated and calcium-dependent phosphorylation of tropomyosin in colonic smooth muscle cells. Our data also shows that acetylcholine induces a significant and sustained increase in PKC-mediated association of tropomyosin with PKCalpha in the particulate fraction of colonic smooth muscle cells. Immunoblotting studies revealed that in colonic smooth muscle cells, there is no significant change in the amount of tropomyosin or actin in particulate fraction in response to acetylcholine, indicating that the increased association of tropomyosin with PKCalpha in the particulate fraction may be due to acetylcholine-induced translocation of PKCalpha to the particulate fraction. To investigate whether the association of PKCalpha with tropomyosin was due to a direct interaction, we performed in vitro direct binding assay. Tropomyosin cDNA amplified from colonic smooth muscle mRNA was expressed as GST-tropomyosin fusion protein. In vitro binding experiments using GST-tropomyosin and recombinant PKCalpha indicated direct interaction of tropomyosin with PKCalpha. PKC-mediated phosphorylation of tropomyosin and direct interaction of PKCalpha with tropomyosin suggest that tropomyosin could be a substrate for PKC. Phosphorylation of tropomyosin may aid in holding the slided tropomyosin away from myosin binding sites on actin, resulting in actomyosin interaction and sustained contraction.

Acetylcholine↗
PubMed1985

Adenosine and 5'-chloro-5'-deoxyadenosine inhibit the phosphorylation of phosphatidylinositol and myosin light chain in calf aorta smooth muscle.

Smooth muscle from calf aorta is homogenized and centrifuged. The insoluble material is subjected to sucrose density gradient centrifugation. When the heaviest fraction so obtained is incubated with radioactive ATP, two components incorporate most of the acid-insoluble radioactivity. One is a phosphoprotein with a molecular weight of 21,000. It has been identified as myosin light chain by its molecular weight, isoelectric point, and precipitation by antibody to calf aorta myosin. Its phosphorylation is strongly inhibited by EGTA, in agreement with published reports that myosin light chain kinase of smooth muscle is Ca2+ dependent. The other product is of low molecular weight, is extracted into acidic chloroform-methanol, and has been identified as phosphatidylinositol 4-phosphate. Adenosine and 5'-chloro-5'-deoxyadenosine, which are vasodilators, inhibit the phosphorylation of both substrates. Phosphorylation of phosphatidylinositol is inhibited at lower concentrations of the nucleosides than is the phosphorylation of myosin light chain. The inhibitory effects of the two nucleosides are not associated with changes in the concentration of cyclic AMP. The precise function of phosphatidylinositol phosphorylation in smooth muscle is not known, but correlations between smooth muscle contraction and increased turnover of phosphatidylinositol and its mono- and diphosphates have been reported. Myosin light chain is phosphorylated under conditions which favor smooth muscle contraction. We conclude that the inhibitory effects of adenosine described here are consistent with their physiological action as vasodilators.

Adenosine↗
PubMed1991

Contractile protein interactions in smooth muscle.

Smooth muscle tone and 'holding economy' depend on the rate constants governing the cross-bridge cycle. Thus, calcium activation via calmodulin-dependent myosin light chain phosphorylation may determine the apparent rate constant ('f') at which cross-bridges enter the force-generating state, forming actin-attached, strongly bound cross-bridges. This phosphorylation of the light chain may be inhibited in skinned fibers by a peptide mimic of the calmodulin recognition site of the myosin light chain kinase (RS 20) that relaxes smooth muscle. In smooth muscle, the apparent cross-bridge detachment rate constant ('g') also seems to be variable, a low constant allowing for a high holding economy and low shortening velocity in the 'latch state'. It may also account for force maintenance at low levels of myosin phosphorylation. Additionally, cross-bridge attachment may, however, be also controlled by other regulatory proteins such as calponin and caldesmon.

Actins↗
PubMed1978

The generation and conduction of activity in smooth muscle.

Smooth muscle, especially gastrointestinal smooth muscle, spontaneously generates oscillatory electrical activity that can control contractions in time and space by altering excitability. The origin and ionic mechanisms underlying these electrical control activities are still controversial, but they behave as coupled relaxation oscillators and they control muscle excitability. Normally, contractions are produced by the addition, during the depolarized phase of the oscillations, of further depolarization by acetylcholine or other means. Pharmacologists who wish to study drug actions on such muscles must be aware of the possibility that drug effects may be determined by these oscillations and may influence contractions by affecting these oscillations as well as by releasing, mimicking, or inhibiting the effects of nerve mediators or by affecting excitation-contraction coupling. Also the use of simplified organ bath preparations may eliminate or alter these control potentials so that results in vitro may not apply in vivo.

Animals↗
PubMed1991

Developmental changes in actin and myosin heavy chain isoform expression in smooth muscle.

Smooth muscle cells express isoforms of actin and myosin heavy chains (MHC). In early postnatal animals the nonmuscle (NM) actin and MHC isoforms in vascular (aorta) smooth muscle were present in relatively high percentages. More than 30% of the MHC and 40% of the actin isoforms were NM. The relative percentage of the NM isoforms decreased significantly as the animals reached maturity, with NM MHC less than 10% and NM actin less than 30% of the totals. Concurrent with this decrease in NM isoforms was an increase in the smooth muscle (SM) isoforms. The relative changes and time frame in which these changes occurred were very similar for the actin and MHC isoforms. In arterial tissue there were species differences for changes with development in the two SM MHC isoforms (SM1 and SM2). The ratio of SM1:SM2 in young rat aorta was approximately 0.5, while this same ratio was approximately 3 in young swine carotid. Both adult rats and swine had a SM1:SM2 MHC ratio of approximately 1.2. Rat bladder smooth muscle showed no significant change in NM vs SM ratio between young and old rats, while the SM1:SM2 ratio decreased from 2.7 to 1.7 between these age groups. The shifts in alpha and beta actin were similar to those in the vascular tissue, but of much smaller magnitude.

Actins↗
PubMed1998

[Novel mechanisms for increasing Ca2+ sensitivity of contractile apparatus in smooth muscle].

Smooth muscle contraction is primarily regulated not only by changes in cytosolic Ca2+ concentrations ([Ca2+]i) but also by changes in the force/[Ca2+]i ratio. The use of membrane-permeabilization technique facilitated demonstration of an increase in the level of force at constant [Ca2+]i (Ca2+ sensitization). It was clarified that Rho-associated kinase (Rho-kinase) is a novel mediator of Ca2+ sensitization of the smooth muscle contraction, by introducing the recombinant catalytic domain of Rho-kinase into the cytosol of vascular smooth muscle permeabilized with beta-escin. This review article focuses on novel mechanisms, by which activation of receptor-coupled G-protein(s) increases Ca2+ sensitivity of the contractile apparatus in smooth muscle: Rho-kinase and protein kinase C.

Animals↗
PubMed1997

Differential effects of a K+ channel agonist and Ca2+ antagonists on myosin light chain phosphorylation in relaxation of endothelin-1-contracted tracheal smooth muscle.

Smooth muscle contraction and relaxation are generally considered to be associated with phosphorylation and dephosphorylation of the 20-kDa regulatory myosin light chain (LC20). Thus, contractions of lamb tracheal smooth muscle induced by Bay K 8644 and relaxed by calcium channel blockers (verapamil, D-600 and nitrendipine) are accompanied by an increase and decrease, respectively, of LC20 phosphorylation. Similarly, endothelin-1 (ET-1) induces a sustained contraction, which is coupled with elevated LC20 phosphorylation and reversed by LC20 dephosphorylation after application of a potassium channel agonist (EMD 52692). In contrast, calcium channel blockers relax ET-1-induced contraction without any dephosphorylation of myosin light chains (MLC), suggesting that MLC phosphatase is inhibited in this case. Obviously, MLC dephosphorylation is not a prerequisite for smooth muscle relaxation. The variable relationship between MLC phosphorylation and force during relaxation suggests that there are mechanisms other than MLC phosphorylation that are important for regulation of contraction and relaxation in smooth muscle.

Animals↗
PubMed2005

Integrin-linked kinase is responsible for Ca2+-independent myosin diphosphorylation and contraction of vascular smooth muscle.

Smooth muscle contraction is activated by phosphorylation at Ser-19 of LC20 (the 20 kDa light chains of myosin II) by Ca2+/calmodulin-dependent MLCK (myosin light-chain kinase). Diphosphorylation of LC20 at Ser-19 and Thr-18 is observed in smooth muscle tissues and cultured cells in response to various contractile stimuli, and in pathological circumstances associated with hypercontractility. MLCP (myosin light-chain phosphatase) inhibition can lead to LC20 diphosphorylation and Ca2+-independent contraction, which is not attributable to MLCK. Two kinases have emerged as candidates for Ca2+-independent LC20 diphosphorylation: ILK (integrin-linked kinase) and ZIPK (zipper-interacting protein kinase). Triton X-100-skinned rat caudal arterial smooth muscle was used to investigate the relative importance of ILK and ZIPK in Ca2+-independent, microcystin (phosphatase inhibitor)-induced LC20 diphosphorylation and contraction. Western blotting and in-gel kinase assays revealed that both kinases were retained in this preparation. Ca2+-independent contraction of calmodulin-depleted tissue in response to microcystin was resistant to MLCK inhibitors [AV25 (a 25-amino-acid peptide derived from the autoinhibitory domain of MLCK), ML-7, ML-9 and wortmannin], protein kinase C inhibitor (GF109203X) and Rho-associated kinase inhibitors (Y-27632 and H-1152), but blocked by the non-selective kinase inhibitor staurosporine. ZIPK was inhibited by AV25 (IC50 0.63+/-0.05 microM), whereas ILK was insensitive to AV25 (at concentrations as high as 100 microM). AV25 had no effect on Ca2+-independent, microcystin-induced LC20 mono- or di-phosphorylation, with a modest effect on force. We conclude that direct inhibition of MLCP in the absence of Ca2+ unmasks ILK activity, which phosphorylates LC20 at Ser-19 and Thr-18 to induce contraction. ILK is probably the kinase responsible for myosin diphosphorylation in vascular smooth muscle cells and tissues.

Animals↗
PubMed1989

What is latch? New ideas about tonic contraction in smooth muscle.

Smooth muscles have traditionally been classified as phasic or tonic, the tonic muscles being those which maintain a steady tension indefinitely with a low consumption of energy. Until ten years ago it was considered that the differences between smooth muscle types reflected different innervation or excitation-contraction coupling. However, recent work makes it clear that the contractile apparatus itself is adapted in tonic muscles.

Animals↗
PubMed1981

Cyclic-AMP mediated relaxation of chemically skinned fibers of smooth muscle.

Smooth muscle from guinea pig taenia coli was chemically skinned with Triton X-100 and stored in ATP-salt solution containing 50% glycerol at -20 degrees C. Fiber bundles were relaxed at Ca2+-concentrations below 10(-7) M, but contracted at 10(-6) M Ca2+. The isometric tension developed could be partly relaxed by the addition of c-AMP (in the presence of NaF), and it could also be inhibited following preincubation with the catalytic subunit of c-AMP dependent protein kinase. The inhibitory effect was much more pronounced at intermediate Ca2+-concentrations (e.g. 10(-6)) than at concentrations producing a maximum contraction, suggesting that Ca-sensitivity had been lowered. Sodium fluoride which was required to potentiate the c-AMP effects was found to have a slight relaxing effect per se. The c-AMP effect may be mediated through activation of cyclic AMP-dependent kinase, producing, phosphorylation of the myosin light chain kinase which, according to Adelstein et al. (1978), may result in a net dephosphorylation of the myosin light chains and a concomittant inhibition of the contractile response.

Adenosine Triphosphatases↗
PubMed2000

Kinetic and spectroscopic evidence for three actomyosin:ADP states in smooth muscle.

Smooth muscle myosin II undergoes an additional movement of the regulatory domain with ADP release that is not seen with fast skeletal muscle myosin II. In this study, we have examined the interactions of smooth muscle myosin subfragment 1 with ADP to see if this additional movement corresponds to an identifiable state change. These studies indicate that for this myosin:ADP, both the catalytic site and the actin-binding site can each assume one of two conformations. Relatively loose coupling between these two binding sites leads to three discrete actin-associated ADP states. Following an initial, weakly bound state, binding of myosin:ADP to actin shifts the equilibrium toward a mixture of two states that each bind actin strongly but differ in the conformation of their catalytic sites. By contrast, fast myosins, including Dictyostelium myosin II, have reciprocal coupling between the actin- and ADP-binding sites, so that either actin or nucleotide, but not both, can be tightly bound. This uncoupling, which generates a second strongly bound actomyosin ADP state in smooth muscle, would prolong the fraction of the ATPase cycle time that this actomyosin spends in a force-generating conformation and may be central to explaining the physiologic differences between this and other myosins.

Actins↗
PubMed2004

Caldesmon phosphorylation is catalyzed by two kinases in permeabilized and intact vascular smooth muscle.

Smooth muscle contraction is initiated by myosin light chain (MLC) phosphorylation catalyzed by the Ca(2+) dependent MLC kinase. However, many aspects of smooth muscle contraction cannot be accounted for by MLC phosphorylation. One hypothesis that has received experimental support involves the thin filament protein caldesmon. Caldesmon inhibits myosin ATPase activity; phosphorylation of caldesmon relieves this inhibitory effect. The primary candidates for catalysis of caldesmon phosphorylation are the p42/p44 ERK MAP kinases. However, we and others have shown that inhibition of the ERK MAP kinases has no effect on many smooth muscles. The goal of this study was to determine if evidence for a second endogenous caldesmon kinase may be obtained. We used Triton X-100 skinned and intact tissues of the swine carotid artery to address this goal. Caldesmon phosphorylation was evident in resting and Ca(2+) stimulated Triton X-100 skinned fibers. Ca(2+)-dependent caldesmon phosphorylation was partially sensitive to the ERK MAP kinase inhibitor PD98059, whereas all caldesmon phosphorylation was sensitive to the general kinase inhibitor, staurosporine. Histamine increased caldesmon phosphorylation levels in intact swine carotid artery, which was sensitive to both PD98059 and staurosporine. Histamine increased ERK MAP kinase activity, which was reversed by PD98059, staurosporine, and EGTA. Histamine-induced contractions were inhibited by staurosporine but not by PD98059. We interpret these results to suggest that although ERK MAP kinases catalyze caldesmon phosphorylation, a second staurosporine sensitive kinase is also important in caldesmon phosphorylation and it is this pathway that may be more important in contractile regulation.

Adenosine Triphosphate↗
PubMed2005

Extracellular signal-regulated kinase1/2 in contraction of vascular smooth muscle.

Smooth muscle contractility is regulated by both intracellular Ca2+ concentration ([Ca2+]i) and Ca2+ sensitivity of the contractile apparatus. Extracellular signal-regulated kinases1/2 (ERK1/2) have been implicated in modulating Ca2+ sensitivity of smooth muscle contraction but mechanisms of action remain elusive. This study investigated the roles of ERK1/2 in modulating [Ca2+]i, calcium sensitivity and the 20-kDa myosin light chain (MLC20) phosphorylation during contraction activated by alpha1-adrenoceptor agonist phenylephrine and thromboxane A2 mimetic U46619 in rat tail artery strips. A specific inhibitor for ERK1/2 activation, U0126, inhibited phenylephrine- and U46619-induced contraction, shifting both concentration-response curves rightward. During phenylephrine-stimulated contraction, U0126 exhibited concentration-dependent inhibition towards force but significant decreases in [Ca2+]i were detected only at higher concentration. Both phenylephrine and U46619 induced a transient activation of ERK1/2 which was abolished by U0126 but unaffected by a general tyrosine kinase inhibitor genistein or Rho kinase inhibitor Y27632 at concentrations inhibiting more than 50% force. Interestingly, U0126 had no effect on steady-state MLC20 phosphorylation levels stimulated by both receptor agonists. These results indicated that during contraction of rat tail artery smooth muscle activated by alpha1-adrenoceptor agonist or thromboxane A2 analogue, ERK1/2 increase Ca2+ sensitivity that does not involve the modulation of MLC20 phosphorylation.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2002

Characterization of a voltage-dependent Na(+) current in human esophageal smooth muscle.

Smooth muscle contraction is critical to peristalsis in the human esophagus, yet the nature of the channels mediating excitation remains to be elucidated. The objective of this study was to characterize the inward currents in human esophageal smooth muscle cells (HESMCs). Esophageal tissue was isolated from patients undergoing surgery for cancer and grown in primary culture, and currents were recorded using patch-clamp electrophysiology. Depolarization elicited inward current activating positive to -40 mV and peaking at 0 mV and consisting of transient and sustained components. The transient current was half activated at -16 mV and half inactivated at -67 mV. The transient current was abolished by removal of bath Na(+) or application of TTX (IC(50) ~20 nM), whereas it persisted in the absence of bath Ca(2+) or the presence of Cd(2+). These data provide evidence that cultured HESMCs express voltage-dependent Na(+) channels. RT-PCR revealed mRNA transcripts for Na(x), the "atypical" Na(+) channel isoform, as well as Na(v)1.4. These studies provide the first evidence of Na(v)1.4 in smooth muscle and contribute to a model of excitation in HESMCs.

Cadmium↗
PubMed1975

Thick filaments in vertebrate smooth muscle.

Smooth muscle cells of the mouse vas deferens fixed with 5% glutaraldehyde contained three types of filaments, namely, thin (50-80) A) filaments, intermediate (100 A) filaments and thick (120-180 A) filments. However, in 2 out of 16 experiments, under identical conditions, the cells did not contain thick filaments. With OSO4 fixation, thin filaments were not prominent, the most obvious being thick (120-250 A) and intermediate (100 A) filaments. After soaking in a modified Ringer solution under no applied tension for one hour, thick filaments (120-180 A) appeared prominently in smooth muscle cells of the mouse vas deferens and thin filaments were in ordered bundles. By 4 hours, thick filaments had increased in size and density, with thin filaments distributed randomly around them. After 8 hours in Ringer, thin filaments were diffuse and difficult to discern, while thick filaments were large (up to 300 A) and electron-dense. Intermediate (100 A) filaments were present in association with dark bodies. Physiological experiments indicated that the intracellular components responsible for the development of a mechanical response were still functional at this time. The presence of "thick filaments" is also reported in degenerating smooth muscle cells of the guinea-pig vas deferens in tissue culture.

Actins↗
PubMed1990

Hypertrophy of visceral smooth muscle.

Smooth muscles of viscera undergo a large increase in volume when there is a chronic, partial obstruction impairing the flow of lumenal contents. Hypertrophy of smooth muscle occurs in various medical conditions and several methods are available for inducing it experimentally in laboratory animals, especially in urinary bladder, small intestine and ureter. The hypertrophic response differs somewhat with the type of organ, the animal species, the age of the subject, and the experimental procedure. Ten- to fifteen-fold increases in muscle volume develop within a few weeks in the urinary bladder or the ileum of adult animals, a growth that would not have occurred in the lifespan of the animal without the experimental intervention. The general architecture of the muscle and the boundaries with adjacent tissues are well preserved. In intestinal hypertrophy, muscle cells increase in number: mitoses are found in mature, fully differentiated muscle cells. Cell division by full longitudinal splitting of muscle cells may also occur. Enlargement of muscle cells accounts for most of the muscle hypertrophy. The hypertrophic muscle cell has an irregular profile with deep indentations of the cell membrane, bearing caveolae and dense bands; however, the cell surface grows less than the cell volume (reduction of surface-to-volume ratio). The nucleus is crenated and is much less enlarged than the cell (reduction of the nucleo-plasmatic ratio). Mitochondria grow in number but in some muscles their spatial density decreases; intermediate filaments increase more than myofilaments. The spatial density of sarcoplasmic reticulum is generally increased. In the hypertrophic intestine, gap junctions increase in number and size; in the bladder, gap junctions are absent both in control and in hypertrophy. Thus the hypertrophic muscle cell is not only larger than a control cell, but has a different pattern of its structural components. Extensive neo-angiogenesis maintains a good blood supply to the hypertrophic muscle. The density of innervation is much decreased in the hypertrophic intestine, whereas it appears well maintained in the bladder. Neuronal enlargement is found in the intramural ganglia of the intestine and in the pelvic ganglion. The mechanisms involved in hypertrophic growth are unknown. Three possible factors, mechanical factors, especially stretch, altered nerve discharge, and trophic factors are discussed.

Animals↗
PubMed1993

[A comparative study of the structural state of skeletal muscle and smooth muscle fiber tropomyosin in ghost skeletal muscle fibers by a fluorescent probe method].

The structural state of skeletal muscle and smooth muscle fiber tropomyosins in ghost fibers of skeletal muscles has been studied by means of polarization microfluorimetry. Tropomyosins and F-actin of ghost fibers were labelled with N-(iodoacetyl)-N-(1-naphthyl-5-sulfo)-ethylenediamine (1,5-IA-EDANS) or the phalloidin-rhodamine complex, respectively. It has been found that skeletal tropomyosin bound to ghost muscle fibers is more flexible in comparison with smooth muscle tropomyosin. The flexibility of the thin filaments of the ghost fibers labelled in F-actin by the phalloidin-rhodamine complex and containing smooth muscle tropomyosin is higher than that of the thin filaments containing the bound skeletal muscle protein.

Animals↗