Smooth Muscle Contractile Apparatus Research Articles

Cerebrospinal fluid from subarachnoid haemorrhage patients causes excessive oxidative metabolism compared to vascular smooth muscle force generation.

Cerebrospinal fluid (CSF) from subarachnoid haemorrhage (SAH) patients can stimulate vascular smooth muscle to generate force in vitro. CSF from SAH patients suffering from delayed ischaemic neurological deficits due to cerebral vasospasm can generate near maximal force in vitro and previous experiments have ascribed this generation of force to be a calcium mediated event. The intracellular calcium concentration has been demonstrated to rise during the vasospastic process. Calcium also stimulates oxidative metabolism as does adenosine diphosphate (ADP), the product of adenosine triphosphate (ATP) hydrolysis. Significant alteration in high energy metabolites such as ATP, ADP and phosphocreatine have also been demonstrated in various models of SAH mediated vasospasm. Vascular smooth muscle predominantly uses oxidative metabolism for force generation and reserves glycolytic metabolism for ion homeostasis. A decrease in oxidative metabolism during force generation would imply failing mitochondria and increased glycolytic high-energy phosphate supply. Increased oxidative metabolism would imply a decreased efficiency of the contractile apparatus or mitochondria. The aim of this study was to see if SAH CSF stimulation of porcine carotid artery oxidative metabolism was altered during force generation when compared with incremental calcium stimulation with potassium chloride depolarisation. CSF from patients (n = 10) who had subarachnoid haemorrhage stimulated force generation but with a significant 'right shift' in oxygen consumption. This 'right shift' is indicative of an increased energy cost for contractile work. These results suggest that vascular smooth muscle contractile apparatus, when stimulated by subarachnoid cerebrospinal fluid, is consuming excess adenosine triphosphate during force generation.

The contractile apparatus and mechanical properties of airway smooth muscle.

The functional properties of airway smooth muscle are fundamental to the properties of the airways in vivo. However, many of the distinctive characteristics of smooth muscle are not easily accounted for on the basis of molecular models developed to account for the properties of striated muscles. The specialized ultrastructural features and regulatory mechanisms present in smooth muscle are likely to form the basis for many of its characteristic properties. The molecular organization and structure of the contractile apparatus in smooth muscle is consistent with a model of force generation based on the relative sliding of adjacent actin and myosin filaments. In airway smooth muscle, actomyosin activation is initiated by the phosphorylation of the 20 kDa light chain of myosin; but there is conflicting evidence regarding the role of myosin light chain phosphorylation in tension maintenance. Tension generated by the contractile filaments is transmitted throughout the cell via a network of actin filaments anchored at dense plaques at the cell membrane, where force is transmitted to the extracellular matrix via transmembrane integrins. Proteins bound to actin and/or localized to actin filament anchorage sites may participate in regulating the shape of the smooth muscle cell and the organization of its contractile filament system. These proteins may also participate in signalling pathways that regulate the crossbridge activation and other functions of the actin cytoskeleton. The length-dependence of active force and the mechanical plasticity of airway smooth muscle may play an important role in determining airway responsiveness during lung volume changes in vivo. The molecular basis for the length-dependence of tension in smooth muscle differs from that in skeletal muscle, and may involve mechano-transduction mechanisms that modulate contractile filament activation and cytoskeletal organization in response to changes in muscle length. The reorganization of contractile filaments may also underlie the plasticity of the mechanical response of airway smooth muscle. Changes in the structural organization and signalling pathways of airway smooth muscle cells resulting form alterations in mechanical forces in the lung may be important factors in the development of pathophysiological conditions of chronic airway hyperresponsiveness.

Transcriptional regulation of smooth muscle contractile apparatus expression.

The transcriptional regulatory mechanisms that control gene expression during differentiation and contractile protein accumulation are becoming well understood in skeletal and cardiac muscle lineages. Current understanding of smooth muscle-specific gene transcription is much more limited, though recent studies have begun to shed light on this topic. In this review, we summarize some of the themes emerging from these studies and identify transcriptional regulatory elements common to several smooth muscle genes. These include potential binding sites for serum response factor, Sp1, AP2, Mhox, and YY1, as well as a potential transforming growth factor-beta control element. We speculate that it may be possible to manipulate smooth muscle-specific gene expression in asthma or pulmonary arterial hypertension as an eventual therapy.

Modulation of the calcium sensitivity of the smooth muscle contractile apparatus: molecular mechanisms, pharmacological and pathophysiological implications.

Smooth muscle contraction is the basis of the physiological reactivity of several systems (vascular, respiratory, gastrointestinal, urogenital ...). Hyperresponsiveness of smooth muscle may also contribute to a variety of problems such as arterial hypertension, asthma and spontaneous abortion. An increase in cytoplasmic calcium concentration ([Ca2+]i) is the key event in excitation-contraction coupling in smooth muscle and the relationship linking the [Ca2+]i value to the force of contraction represents the calcium sensitivity of the contractile apparatus (CaSCA). Recently, it has become evident that CaSCA can be modified upon the action of agonists or drugs as well as in some pathophysiological situations. Such modifications induce, at a fixed [Ca2+]i value, either an increase (referred to as sensitization) or a decrease (desensitization) of the contraction force. The molecular mechanisms underlying this modulation are not yet fully elucidated. Nevertheless, recent studies have identified sites of regulation of the actomyosin interaction in smooth muscle. Sensitization primarily results from the inhibition of myosin light chain phosphatase (MLCP) by intracellular messengers such as arachidonic acid or protein kinase C. In addition, phosphorylation of thin filament-associated proteins, caldesmon and calponin, increases CaSCA. Activation of small (monomeric) G-proteins such as rho or ras is also involved. Desensitization occurs as a consequence of phosphorylation of myosin light chain kinase (MLCK) by the calcium-calmodulin activated protein kinase II, or stimulation of MLCP by cyclic GMP-activated protein kinase. In the present review, examples of physiological modulation of CaCSA as well as pharmacological and pathophysiological implications are illustrated for some smooth muscles.

Mechanisms of platelet-induced angiospastic reactions: potentiation of calcium sensitivity

Platelet aggregation and adherence to the vessel lumina are common events in various physiological and pathological conditions. However, the mechanisms whereby aggregating platelets cause vasoconstriction remain unclear. We hypothesized that aggregating platelets may alter the Ca2+ sensitivity of the contractile apparatus in smooth muscle cells. We tested this hypothesis using rabbit common carotid arteries permeabilized with beta-escin. In these preparations, the receptor-effector coupling is functionally intact, while the intracellular ionic composition, in particular calcium concentration, may be clamped. Aggregating platelets in the presence of 100 microM GTP left-shifted the pCa-force curve. On average, the pD2 for calcium was 6.30 +/- 0.05 and 6.86 +/- 0.07 in the absence and presence of platelets, respectively (p < 0.01). This increase in calcium sensitivity was prevented by blockade of G-proteins with guanosine 5'-O-(2-thiodiphosphate). Platelets modulated calcium sensitivity at concentrations markedly lower than those that produce contractions of intact vessels. These data suggest that platelet activation in the vicinity of the vessel wall may sensitize the smooth muscle contractile apparatus to calcium via a G-protein-dependent mechanism. This phenomenon may enhance vessel responses to vasoactive substance that produce vessel contraction via elevation of intracellular calcium concentrations.

Involvement of botulinum C 3-sensitive GTP-binding proteins in α1-adrenoceptor subtypes mediating Ca 2+-sensitization

The mechanisms of α 1-adrenoceptor agonist-mediated sensitization of the contractile apparatus of smooth muscle to Ca 2+ were studied in β-escin-permeabilized thoracic artreal smooth muscle of rabbit. Addition of norepinephrine (10 μM) plus guanosine 5′-triphosphate (GTP, 50 μM) significantly enhanced Ca 2+ sensitivity as compared with the addition of 0.3 μM Ca 2+ alone (pCa6.5). In β-escin-skinned smooth muscle of chloroethylclonidine-treated tissues, the enhancement of Ca 2+-contraction produced by norepinephrine or clonidine was completely inhibited by guanosine 5′-O-(β-thiodiphosphate) (GDPβ-S, 1 mM). In addition, Clostridium botulinum C 3, which inactivates low molecular weight GTP-binding protein families, abolished norepinephrine- or clonide-induced Ca 2+-sensitization, but did not affect clonidine-induced of protein kinase C to the membrane. The norepinephrine-enhanced Ca 2+ sensitivity was partially reversed by a pretreatment with a selective myosin light chain kinase inhibitor (8R ∗, 9S ∗, 11S ∗-(−)-9-hydroxy-9- methoxycarbonyl-8-methyl-14- n-propoxy-2,3,9,10-tetrahydro-8,11-epoxy,1H,8H,11H-2,7b, 11a-triazadibenzo[a,g]cycloocta[cde]trinden-1- one (KT5926, 500 nM), but those of clonidine and in the chloroethylclonidine-treated tissues norepinephrine were not. These results suggest that Ca 2+-sensitization produced by the activation of the α 1-adrenoceptor subtypes is linked via a low molecular weight GTP-binding protein (Rho), and the regulations of phosphorylation in contractile elements.

Resting load regulates cytosolic calcium-force relationship of the contraction of bovine cerebrovascular smooth muscle.

1. We determined the effects of a change in preload, or resting load, on the development of force and on cytosolic calcium concentration ([Ca2+]i) during the contraction induced by high K+ depolarization and by the stable analogue of thromboxane A2, U-46619, using front-surface fluorometry and medial strips of bovine middle cerebral artery loaded with fura-2. 2. Increase in resting load of strips from 0.495 mN (low load; 0.85L(max)) to 1.98 mN (optimal load; L(max)) elevated resting levels of [Ca2+]i slightly, and, significantly, after a delay of a few seconds. The force developed by high K+ depolarization at resting load of 1.98 mN was much greater than that at a resting load of 0.495 mN (191.8% of that at 0.495 mN); however, there was no difference in [Ca2+]i elevation induced by high K+ depolarization between resting loads of 0.495 and 1.98 mN. 3. Both in the presence and absence of extracellular Ca2+, the force developed by U-46619 (0.1 microM) at resting load of 1.98 mN was also much greater than that at a resting load of 0.495 mN. Both in the presence and absence of extracellular Ca2+, there was no difference in the [Ca2+]i transients induced by U-46619 between two different resting loads. 4. The [Ca2+]i-force relationship during the contraction induced by high K+ depolarization was shifted to the left when the resting load was increased from 0.495 to 1.98 mN. At 0.495 mN resting load, this Ca(2+)-force relationship was shifted to the left by U-46619. This left-side shift of the curve by U-46619 was further enhanced by the increase in resting load from 0.495 to 1.98 mN. 5. The augmentation of force development induced by the change in resting load (from 0.495 to 1.98 mN) was not affected by a relatively specific inhibitor of protein kinase C, 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydro-chloride (H-7; 10 microM). 6. We conclude that (1) at a given degree of [Ca2+]i elevation, the developed force induced by U-46619 was greater than that induced by high K+ depolarization, and (2) preload, or resting load (below optimal load), regulates contractile responsiveness of cerebrovascular smooth muscle to various stimulations, mainly by modulating the [Ca2+]i-force relationship, without affecting the extent of [Ca2+]i elevation. The results suggest the possibility that Ca(2+)-insensitive pathways may be involved in the stretch-dependent regulation of Ca2+ sensitivity of the contractile apparatus in smooth muscle.

The Structure of the Contractile Apparatus in Ultrarapidly Frozen Smooth Muscle: Freeze-Fracture, Deep-Etch, and Freeze-Substitution Studies

The structure of the smooth muscle contractile apparatus was studied using ultrarapid freezing followed by freeze substitution or by longitudinal freeze-fracture, deep-etch, and platinum-carbon replication. Freeze substitution minimises the detrimental effects of chemical fixation and freeze fracture eliminates them entirely whilst revealing the ultrastructure in three dimensions. Unidirectionally shadowed freeze-fracture replicas of ultra-rapidly frozen, relaxed, intact smooth muscle showed a well-preserved actin filament structure; the 5.5-nm repeat of the actin subunits was clearly observed. In transversely fractured tissue the thick filaments were revealed, with a distribution comparable to that seen in transverse sections of freeze-substituted muscle. Relaxed muscle permeabilised using Triton X-100 showed a similar structure to that of intact tissue after ultrarapid freezing and examination both by freeze fracture and by freeze substitution; the ratios of actin to myosin were also comparable. In permeabilised, rigorised tissue the structure of the actomyosin complex was revealed in detail; this was especially clear in freeze-substituted muscle. A cross-bridge spacing of 38 nm was measured in freeze-fractured, deep-etched tissue. The structural detail revealed is compatible with a side polar model of the actomyosin interaction and with the sliding filament mechanism of muscle contraction.

The effects of a novel vasodilator, LP-805, on cytosolic Ca2+ concentrations and on tension in rabbit isolated femoral arteries.

1. LP-805, 8-tert-butyl-6,7-dihydropyrrolo-[3,2-e]-5-methylpyrazolo- [1,5a]-pyrimidine-3-carbonitrile, is a newly synthesized potent vasodilator. To investigate the cellular mechanisms of vasorelaxation induced by LP-805, we simultaneously determined the effects of LP-805 on cytosolic Ca2+ concentrations ([Ca2+]i) and on tension of smooth muscle of rabbit femoral arterial strips, with or without the endothelium, using front-surface fluorometry and fura-2. 2. In the absence of the endothelium, LP-805, in a concentration-dependent manner, decreased [Ca2+]i and tension during the contraction induced by K(+)-depolarization, at relatively low concentrations ([K+]o < or = 30 mM). The decreases in [Ca2+]i and tension were fully antagonized by treatment with 2 x 10(-6) M glibenclamide. The [Ca2+]i-tension relationship in the LP-805-induced relaxation was similar to that of K(+)-depolarization-induced contractions. 3. LP-805, in a concentration-dependent manner (IC50 for inhibition of tension; 1.7 x 10(-6) M), decreased both [Ca2+]i and tension during the steady-state of contractions induced by 1 x 10(-7) M noradrenaline (NA) in the strips without the endothelium. Glibenclamide completely inhibited these reductions of [Ca2+]i and tension. At the steady-state of relaxation induced by LP-805 during NA-induced contraction, [Ca2+]i-tension relation was shifted to the left of that obtained with high K(+)-induced contraction. 4. NA induced transient increases in [Ca2+]i and tension in the absence of extracellular Ca2+. LP-805 (up to 3 x 10(-6) M) had no effect on these intracellular Ca2+ mobilisation and tension development induced by NA. 5. In strips with an intact endothelium, LP-805 decreased both [Ca2+]i and tension during contraction induced by 1 x 10(-7) M NA. The concentration-response curve for inhibition of [Ca2+]i and tension obtained in the presence of the endothelium was shifted to the left from that obtained in the absence of endothelium. IC50 for the inhibition of tension obtained in the strips with the endothelium was 4.0 x 10(-7) M. Treatment with 1 x 10(-4) M NG-nitro-L-arginine (L-NOARG) attenuated reductions of both [Ca2+]i and tension induced by LP-805 and the concentration-response curve shifted to the right and overlapped that obtained in the absence of the endothelium. Treatment with glibenclamide almost fully overcame the reduction of [Ca2+]i induced by LP-805, while the reversion of tension was 50% at most. 6. In the presence of the endothelium with L-NOARG, LP-805 reduced the tension to the extent of that expected from the reduction of [Ca2'ji, as based on the [Ca2+]i-tension relationship obtained with LP-805 in the absence of endothelium. On the contrary, in the presence of the endothelium without L-NOARG, LP-805 induced a greater reduction of tension than expected from the reduction of [Ca2+J1.This effect became more apparent after treatment with glibenclamide.7. These results suggest that: (1) LP-805 relaxes smooth muscle mainly by activating ATP-sensitive K+channels of smooth muscle and by releasing endothelium-derived relaxing factor (EDRF). (2) Activation of ATP-sensitive K+ channels decrease [Ca2+]i and thereby relax smooth muscle with no effect on Ca2"-sensitivity of the contractile apparatus of smooth muscle or on the agonist-induced Ca2"-release process. (3) EDRF induced by LP-805 relaxes smooth muscle not only by decreasing [Ca2+]i but also decreasing Ca2+-sensitivity of the contractile apparatus of smooth muscle. In the presence of an intact endothelium, a decrease in Ca2+-sensitivity of the contractile apparatus may play an important role in LP-805-induced relaxation.

Diosmin‐induced increase in sensitivity to Ca2+ of the smooth muscle contractile apparatus in the rat isolated femoral vein

The effect of diosmin, a flavone derivative, on the Ca2+ sensitivity of the venous contractile apparatus was investigated in chemically (beta-escin) skinned strips from the rat isolated femoral vein. Diosmin (0.5-10 microM) shifted to the left the concentration-response curve to Ca2+ (0.05-5 microM). The maximal effect was observed in the presence of 1 microM diosmin which increased the contractile response evoked by 0.15 microM Ca2+ from 26.3% to 78.9% of the maximal Ca(2+)-induced response. This work demonstrates that the venotonic action of diosmin involves an increase in the Ca2+ sensitivity of the contractile apparatus. Such a mechanism of action could represent a new and important means of therapeutic control of vasomotor activity.

Dystrophin (Xp21), a New Phenotype Marker of Cultured Rat Aortic Myocytes

Dystropbin is a low-abundance cytoskeletal protein involved in the maintenance of membrane integrity in striated muscle. Very little is known about its role in smooth muscle. Utrophin (a dystropbin-related protein) is an ubiquitous protein whose role is still unclear. Changes in the expression of both proteins (if any) during phenotypic modulation of smooth muscle have not yet been reported. In contrast, modulated expression of heavy-molecular-weight caldesmon (h-CaD), a well-known specific regulatory protein of the contractile apparatus in smooth muscle, is well documented, along with its nonmuscle isoform, low-molecular-weight caldesmon (l-CaD), and other cytoskeletal proteins. We investigated three properties of cultured rat aortic smooth muscle cells: morphology, contractile ability, and expression of dystrophin, utrophin, h-CaD, and l-CaD. Cells were grown either in serum substitute supplemented medium (U-medium), where they reexpressed contractility, or in fetal calf serum-supplemented medium (F-medium), where they did not. It was found that only cultures grown in U-medium continued expressing dystrophin, even during the proliferation phase, contrary to cells grown in F-medium. However, when F-medium was changed for U-medium the cells recovered their contractility and reexpressed dystrophin. Expression of utrophin, h-CaD, and l-CaD was similar in both culture types. Dystrophin was demonstrated to be a true phenotype marker of cultured rat aortic smooth muscle cells, particularly with respect to their actual contractility.

Cytosolic Ca 2+ transients in endothelium-dependent relaxation of pig coronary artery, and effects of captopril

To determine the mechanism of endothelium-dependent relaxation by bradykinin, we simultaneously measured changes in cytosolic calcium concentrations ([Ca 2+] i) and force of fura-2-loaded strips of porcine coronary artery. We also examined effects of captopril, an angiotensin converting enzyme inhibitor, on bradykinin-induced relaxation. Bradykinin, in a concentration-dependent manner (10 −10 to 10 −7 M), decreased both [Ca 2+] i and force to resting levels, during 10 −5 M prostaglandin F 2α-induced contractions, only when endothelium was intact. Treatment with 10 −5 M captopril enhanced the bradykinin-induced decreases in [Ca 2+] i and force and shifted the concentration-response bradykinin induced a greater relaxation than that expected from the reduction in [Ca 2+] i. Captopril had no effects on the relationship between reduction in [Ca 2+] i and relaxation induced by bradykinin. Bradykinin relaxes porcine coronary artery in an endothelium-dependent manner, by decreasing [Ca 2+] i and also by controlling the Ca 2+ sensitivity of the contractile apparatus of smooth muscle. Captopril enhanced the bradykinin-induced relaxation, with no apparent direct effect on Ca 2+ sensitivity of the contractile apparatus.

Nitric oxide does not mediate the attenuated pulmonary vascular reactivity of chronic pneumonia

Chronic Pseudomonas pneumonia is associated with decreased acute hypoxic pulmonary vasoconstriction. However, it is not known whether this is a result of a generalized reduction in contractile responsiveness. We therefore examined the effect of chronic Pseudomonas pneumonia on in vitro pulmonary vascular responsiveness to agonists. We then investigated the role of nitric oxide (NO) in the altered pulmonary vascular contractility. Control rats or rats infected with Pseudomonas (pneumonia) were killed, and small intrapulmonary arteries (100-200 microns effective lumen radius) were removed. In the pneumonia group, arteries were harvested from the pneumonic area of the lung. Vascular responsiveness was assessed in vitro by obtaining cumulative dose-response curves to contractile agonists [phenylephrine (PE), 5-hydroxytryptamine (5-HT), prostaglandin F2 alpha (PGF2 alpha), and KCl]. KCL-induced (voltage-operated) contractions were not significantly depressed in small pulmonary arteries from pneumonic lungs, suggesting that the smooth muscle contractile apparatus in these arteries was preserved. Contractile responses to the three receptor-operated agonists (PE, 5-HT, and PGF2 alpha) were significantly depressed in arteries subserving the pneumonic lobe of infected rats. NG-monomethyl-L-arginine, which blocks the synthesis of NO, caused a shift toward the left in the dose-contraction curves to PE, PGF2 alpha, and 5-HT in vessels from the sterile control lungs, but it had little effect on arteries from the pneumonic lungs. Chronic Pseudomonas pneumonia is associated with depressed pulmonary vascular contractility in vitro, particularly affecting the receptor-mediated contractile responses. Excessive NO release does not contribute to this attenuated vascular contractility.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ethanol on contractile response of gall bladder isolated from guinea pig

The effects of ethanol treatment in vitro and in vivo on gall bladder contraction were investigated using gall bladder strips isolated from guinea pigs. In vitro pretreatment of the strips with ethanol at a concentration of over 50 mM significantly attenuated the reactivity and sensitivity of contractile responses to KCl, acetylcholine and histamine in a concentration-dependent manner. Indomethacin treatment or removal of extracellular calcium remarkably reduced gall bladder contractile response to acetylcholine. The depressive effect of ethanol in vitro on gall bladder contraction was also noted in the presence of indomethacin or absence of calcium in the medium. The concentration-response curve of calcium-induced contraction in 40 mM KCl-depolarized gall bladder strip shifted to the right on pretreatment with ethanol. In the case of strips following the chronic administration of 3% ethanol solution ad libitum for 4 weeks, contractile responses to KCl, acetylcholine and histamine did not differ, compared to those in the pair-fed group. This chronic ethanol administration induced tolerance to the acute inhibitory effect of ethanol on gall bladder contractile responses to the agonists. Ethanol is thus shown to exert direct inhibitory action on gall bladder contraction by lowering the calcium sensitivity of the contractile apparatus of smooth muscle; it is unlikely that ethanol consumption would affect gall bladder motility in vivo, owing to the tolerance produced toward the acute inhibitory action of ethanol.

G protein-mediated inhibition of myosin light-chain phosphatase in vascular smooth muscle.

The mechanism of G protein-mediated sensitization of the contractile apparatus of smooth muscle to Ca2+ was studied in receptor-coupled alpha-toxin-permeabilized rabbit portal vein smooth muscle. To test the hypothesis that Ca2+ sensitization is due to inhibition of myosin light-chain (MLC) phosphatase activity, we measured the effect of guanosine 5'-[gamma-thio]triphosphate and phenylephrine on the rate of MLC dephosphorylation in muscles preactivated with Ca2+ and incubated in Ca(2+)- and ATP-free solution containing 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9) to block MLC kinase activity. Guanosine 5'-[gamma-thio]triphosphate alone (300 microM) or in combination (3 microM) with phenylephrine decreased the rates of relaxation and dephosphorylation of MLC to about half of control values; this inhibition is sufficient to account for maximal G protein-mediated Ca2+ sensitization of MLC phosphorylation. The rate of thiophosphorylation of MLC with adenosine 5'-[gamma-thio]-triphosphate was not affected by guanosine 5'-[gamma-thio]triphosphate. We suggest that inhibition of protein phosphatase(s) by G protein(s) may have important regulatory functions.

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

Supercontraction and segmentation of the contractile apparatus of smooth muscle

Supercontraction and segmentation of the contractile apparatus of smooth muscle

Direct inhibition of chicken gizzard smooth muscle contractile apparatus by caffeine

The mechanism of the inhibitory effect of caffeine on smooth muscle contraction was examined using chicken gizzard. Caffeine (0.1-5 mmol/l) inhibited the KCl-induced contraction of the muscle with an IC50 of 1.1 mmol/l. Forskolin (0.01-10 mumol/l) also inhibited KCl-induced contraction. The inhibitory effect of caffeine was potentiated by a low concentration of forskolin (0.3 mumol/l) and the inhibitory effect of forskolin was potentiated by a low concentration of caffeine (0.1 mmol/l). Although caffeine and forskolin increased tissue cyclic AMP levels, caffeine inhibited the KCl-induced contraction more strongly than forskolin at a given cyclic AMP level. Caffeine (1-40 mmol/l) inhibited the contractions induced by 3 mumol/l Ca2+ in Triton X-100-permeabilized muscle. Caffeine (1-40 mmol/l) inhibited the phosphorylation of 20 kDa myosin light chain (MLC) in native actomyosin preparation and the inhibition was enhanced by decreasing the ATP concentration in the reaction medium. Calmodulin (CaM) activity, as monitored by Ca2+/CaM-dependent erythrocyte membrane (Ca2+ + Mg2+)-ATPase, was not affected by 20 mmol/l caffeine. Time-dependent dephosphorylation of MLC upon removal of Ca2+, an indicator of phosphate activity, was not affected by caffeine. Caffeine also inhibited the Ca2(+)-independent contraction in thiophosphorylated permeabilized muscle. These results indicate that caffeine inhibits smooth muscle contraction by a direct inhibition of MLC kinase and actin-myosin interaction. A part of the inhibitory effect may be mediated by cyclic AMP-dependent mechanism(s).

Desensitization of swine arterial smooth muscle to transplasmalemmal Ca2+ influx.

1. The effect of transplasmalemmal Ca2+ influx on the [Ca2+]i dependence of smooth muscle contraction was evaluated by measuring intracellular [Ca2+] (as estimated by aequorin), myosin phosphorylation, and isometric stress in swine carotid media. 2. Extracellular Ca2+ was removed by incubation in physiological saline with 1 mM-EGTA and no added CaCl2 for 20 min (termed EGTA treatment). In some preparations, intracellular Ca2+ was released by a brief (5 min) histamine stimulation while in this Ca2(+)-free EGTA solution (termed histamine treatment). 3. Restoration of extracellular CaCl2 to EGTA and histamine-treated preparations in the continued presence of histamine was associated with an initial large aequorin light transient. However, this light transient was not initially associated with an increase in myosin phosphorylation or rapid stress development, suggesting that the contractile apparatus was desensitized to aequorin-estimated myoplasmic [Ca2+]. The desensitization was temporary, and resolved by 10 min after restoration of extracellular CaCl2. 4. The light transient observed upon restoration of extracellular CaCl2 was smaller in preparations only EGTA treated when compared to preparations treated with both EGTA and histamine, suggesting that histamine treatment further desensitized the contractile apparatus. 5. The stress development rate was not slowed when histamine and extracellular CaCl2 were simultaneously added to EGTA-treated preparations, suggesting that the desensitization was only to transplasmalemmal Ca2+ influx (from extracellular CaCl2 readdition), and not intracellular Ca2+ release (from the histamine stimulation). 6. In EGTA and histamine-treated preparations, restoration of extracellular CaCl2 in the presence of 109 mM-KCl was associated with a larger aequorin light signal than was observed upon readdition of CaCl2 in the presence of histamine, suggesting that depolarization also further desensitized the contractile apparatus. 7. Depolarization of EGTA-treated preparations did not increase [Ca2+] or stress, suggesting that depolarization did not release intracellular Ca2+ stores. 8. No significant light transient was observed upon addition of extracellular LaCl3, suggesting that tissue damage or leakage of aequorin into the extracellular space was not the cause of the Ca2(+)-reintroduction light signal. 9. These data suggest that removal of extracellular CaCl2 desensitizes the contractile apparatus of smooth muscle to transplasmalemmal Ca2+ influx. This desensitization is only to readdition of extracellular Ca2+; the contractile apparatus still responds to intracellular Ca2+ release. The desensitization is increased by prior depolarization or brief histamine treatment (potentially by depleting intracellular Ca2+). The source of activator Ca2+ appears to affect the relationship between aequorin light and phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)

Periodic organization of the contractile apparatus in smooth muscle revealed by the motion of dense bodies in single cells.

To study the organization of the contractile apparatus in smooth muscle and its behavior during shortening, the movement of dense bodies in contracting saponin skinned, isolated cells was analyzed from digital images collected at fixed time intervals. These cells were optically lucent so that punctate structures, identified immunocytochemically as dense bodies, were visible in them with the phase contrast microscope. Methods were adapted and developed to track the bodies and to study their relative motion. Analysis of their tracks or trajectories indicated that the bodies did not move passively as cells shortened and that nearby bodies often had similar patterns of motion. Analysis of the relative motion of the bodies indicated that some bodies were structurally linked to one another or constrained so that the distance between them remained relatively constant during contraction. Such bodies tended to fall into laterally oriented, semirigid groups found at approximately 6-microns intervals along the cell axis. Other dense bodies moved rapidly toward one another axially during contraction. Such bodies were often members of separate semirigid groups. This suggests that the semirigid groups of dense bodies in smooth muscle cells may provide a framework for the attachment of the contractile structures to the cytoskeleton and the cell surface and indicates that smooth muscle may be more well-ordered than previously thought. The methods described here for the analysis of the motion of intracellular structures should be directly applicable to the study of motion in other cell types.