Isometric and isotonic relaxation in venous smooth muscle.

The time course of relaxation under isometric and isotonic conditions was studied in preparations of rat portal vein which maintained regular phasic contractions in solutions with increased [K-+] and [Ca-2+]. It was found that the smooth muscle during isotonic lengthening was able to carry the afterload for a period of time which was longer than expected from the control isometric response. The difference was largest for afterloads corresponding to about half the isometric peak force. The terminal decline in force was identical in afterloaded and purely isometric responses. Inhibitory influences of isotonic shortening causing increased rates of relaxation, as reported for striated muscles, were not observed. The differences in the course of relaxation between isotonic and isometric responses of portal vein could not be attributed to variations in membrane excitation. It also appears that the differences are not due to changes in release or uptake of activator calcium, or to presence of viscous elements in the tissue. It is suggested that the ability of the smooth muscle to carry load over a relatively long period during isotonic relaxation may be attributed to the characteristics of the instantaneous force-velocity relations in the force range above Po.     

h-Caldesmon in leiomyosarcoma and tumors with smooth muscle cell-like differentiation: its specific expression in the smooth muscle cell tumor

h-Caldesmon (h-CD) is a protein combined with actin and tropomyosin that regulates cellular contraction. h-CD has been thought to be expressed exclusively in vascular and visceral smooth muscle cells (SMC). We examined h-CD expression immunohistochemically in tumors with SMC and SMC-like differentiation to clarify whether h-CD is specifically expressed in SMC tumors. The tumors examined in this study were six leiomyomas (LM), two angioleiomyomas (ALM), six leiomyosarcomas (LMS), eight rhabdomyosarcomas (RMS), eight malignant fibrous histiocytomas (MFH), four desmoids, three glomus tumors (GT), and two inflammatory myofibroblastic pseudotumors (IMP) of urinary bladder. We found that LM, ALM, LMS, and GT showed intense and extensive immunoreactivity for h-CD, whereas other tumors were completely negative for h-CD. In addition, h-CD was not present in the vascular pericytes and myofibroblasts, in contrast to actin. Although myoepithelial cells were immunopositive for h-CD, neoplastic myoepithelial cells of myoepithelial tumors and mixed tumors of the salivary gland and skin were all negative. These findings indicate that h-CD is a specific marker of both SMC and its neoplasms and that immunohistochemical detection of h-CD may facilitate the differential diagnosis between LMS and other tumors with SMC-like differentiation, including myofibroblastic tumors.     

Ablation of smooth muscle caldesmon affects the relaxation kinetics of arterial muscle

Smooth muscle caldesmon (h-CaD) is an actin- and myosin-binding protein that reversibly inhibits the actomyosin ATPase activity in vitro. To test the function of h-CaD in vivo, we eliminated its expression in mice. The h-CaD-null animals appeared normal and fertile, although the litter size was smaller. Tissues from the homozygotes lacked h-CaD and exhibited upregulation of the non-muscle isoform, l-CaD, in visceral, but not vascular tonic smooth muscles. While the Ca²⁺ sensitivity of force generation of h-CaD-deficient smooth muscle remained largely unchanged, the kinetic behavior during relaxation in arteries was different. Both intact and permeabilized arterial smooth muscle tissues from the knockout animals relaxed more slowly than those of the wild type. Since this difference occurred after myosin dephosphorylation was complete, the kinetic effect most likely resulted from slower detachment of unphosphorylated crossbridges. Detailed analyses revealed that the apparently slower relaxation of h-CaD-null smooth muscle was due to an increase in the amplitude of a slower component of the biphasic tension decay. While the identity of this slower process has not been unequivocally determined, we propose it reflects a thin filament state that elicits fewer re-attached crossbridges. Our finding that h-CaD modulates the rate of smooth muscle relaxation clearly supports a role in the control of vascular tone.     

Inhibitory mechanisms for cross-bridge cycling: the nitric oxide-cGMP signal transduction pathway in smooth muscle relaxation

Relaxation follows sequestration of Ca²⁺ mobilized by an excitatory stimulus in striated muscle. Removal of excitatory stimuli also relaxes smooth muscle in vitro after reductions in the myoplasmic [Ca²⁺] and dephosphorylation of the myosin regulatory light chains. However, there are several experimental procedures that produce relaxation in the presence of excitatory stimuli and elevated Ca²⁺-dependent cross-bridge phosphorylation. Of potential widespread physiological importance are treatments that increase myoplasmic [cGMP] owing to the ubiquity of nitric oxide (NO) as a signalling molecule for endothelial-mediated vasodilation and inhibitory nerves in most types of smooth muscle. Several mechanisms are implicated in the NO-cGMP mediated relaxation. Most studies support reductions in myoplasmic Ca²⁺. However, there is evidence that increases in cGMP also lower the Ca²⁺-sensitivity of cross-bridge phosphorylation. This would contribute to a decline in force through actions on the myosin light chain kinase/phosphatase system. In addition, changes in the dependence of force on phosphorylation are observed in tissues partially relaxed by treatments that elevate cGMP. This demonstrates that either the attachment and cycling of phosphorylated cross-bridges is impaired or blocked, or that the formation of dephosphorylated, force-generating cross-bridges (‘latch-bridges’) is reduced. Protein kinase G-catalysed phosphorylation of either a thin filament protein that blocks attachment of cross-bridges or a protein that inhibits myosin light chain phosphatase may explain the NO-induced relaxation with elevated cross-bridge phosphorylation.     

Maturational changes in airway smooth muscle shortening and relaxation. Implications for asthma

Greater airway responsiveness in healthy juveniles is considered a factor in the higher asthma prevalence at a young age compared with adults. Several studies on the contractile response of airway smooth muscle (ASM) from birth to adulthood have addressed the hypothesis that a maturation of ASM plays a role in juvenile airway hyperresponsiveness. Maturation of distinct ASM properties, i.e. force generation, shortening, and relaxation, has been reported, although the majority of the studies have focused on maturation of maximum force and/or sensitivity to contractile agonists. However, in most animal species maturation of the ability to generate force does not correlate with maturation of airway responsiveness. Ontogenesis of ASM shortening has been less extensively studied and the existing reports emphasize an increase during maturation of tissue passive forces opposing shortening. ASM spontaneous relaxation has been very minimally investigated. We have recently demonstrated that the ability of ASM to spontaneously relax during stimulation is sharply reduced in juvenile airway tissue. It remains to be determined the role of these ASM properties in the onset of childhood asthma and whether specific alterations are induced by the occurrence of obstructive airway diseases in young individuals.     

Some properties of the smooth muscle of rabbit portal vein

1. The morphology of the smooth muscle of the rabbit portal vein and its innervation were studied with fluorescence and electron microscopy. Two layers of smooth muscle were observed in the tunica media: an inner layer of circularly arranged muscle cells and an outer layer consisting of bundles of smooth muscle cells arranged in a near longitudinal direction. The membranes of neighbouring smooth muscle cells were occasionally fused to form ;tight junctions'.2. Bundles of non-myelinated nerve fibres were observed in the adventitia, and between bundles and layers of smooth muscle cells in the media. Studies on longitudinal sections with fluorescence microscopy revealed a network of varicose noradrenergic axons.3. Electrical and mechanical activity was recorded from longitudinal strips of smooth muscle from the media of the vein with a sucrose-gap apparatus.4. The preparation was spontaneously active under minimal resting tension (less than 150 mg) and at temperatures above 28 degrees C. Slow depolarizations led to a burst of spikes (multi-spike complexes), which corresponded to rhythmic contractions. In 10% of preparations, the interval between multi-spike complexes showed a slower depolarization, suggesting the record was from a pace-maker region.5. The frequency of spontaneous activity (3-27 beats/min) was very sensitive to changes in temperature and tension.6. Noradrenaline in low doses (0.01 mug) caused an increase in frequency of the multi-spike complexes. Higher doses (0.1-0.3 mug) initiated continuous high-frequency spiking, while very high doses (0.6-2.0 mug) caused maintained depolarization.7. Responses to repetitive electrical stimulation of the vein were qualitatively similar to those in response to exogenous noradrenaline. The relation between the mechanical response and the various parameters of stimulation was consistent with the stimulation of sympathetic nerve fibres in the wall of the vein.8. The actions of isoprenaline, phentolamine and propranolol indicated the presence of alpha ;excitatory' and beta ;inhibitory' adrenotrophic receptors on the smooth muscle.     

A method for culturing canine tracheal smooth muscle cells in vitro: Morphologic and pharmacologic observations

A method of culturing canine tracheal smooth muscle cells in vitro is described. The morphology of these cells is monitored up to 60 days in culture and selected stages are illustrated. The characteristics of these cells are numerous mechanical attachments, the presence of thick filaments in suitably processed cells, and their contractile response to in vitro administration of carbachol, a cholinomimetic drug. They also possess nexus formations and both thin (actin) filaments and 10-nm filaments. Mitosis is found in the nonconfluent preparations up to 16 days after culturing. Cultures of 2 to 8 days appear to be most useful as pharmacological test vehicles. This system will be used to explore the phenomenon of adrenergic beta-2 receptor desensitization in airway smooth muscle, to attempt to localize these receptor sites and to determine how receptor affinity and/or number may be regulating cell response to pharmacologic agents.     

Mechanics of canine iliac artery smooth muscle in vitro.

Transmural pressure and external diameter were continuously recorded from intact segments of canine iliac artery and used to determine wall responses to maximal smooth muscle (SM) activation by norepinephrine (NE). Three different approaches were used:a) step inflations from low pressure following NE, B) slow, continuous inflation from low pressure following NE, and c) direct isometric and isobaric responses to NE. Passive pressure-diameter data were determined. The results show that active wall stress and diameter responses to NE determined using direct isometric and isobaric responses and from slow inflation responses (less than 1 mmHg/s) were not significantly different. Pressure-diameter and stress-strain curves determined using continuous (0.2 mmHg/s) and step pressure inflations after NE were not signficantly different. Pressure-diameter curves obtained at different inflation rates were not those expected from a simple viscoelastic material. The results suggest that slow inflation of iliac arteries with activated SM is a reasonable method for assessing contractile properties of SM. Furthermore, they also suggest that activation of iliac artery SM at high contractile element length (L greater than Lo) does not produce attenuated constriction responses.     

VIP inhibition of vascular smooth muscle: complementary to β2-adrenoceptor mediated relaxation in the isolated rat portal vein

Exogenous VIP caused a concentration dependent inhibition of the spontaneous mechanical activity in the isolated rat mesenteric-portal vein preparation via a mechanism which was completely independent of the propranolol-blocked β-adrenoceptor, of high K⁺ in the medium and of exogenous bovine pancreatic polypeptide, neurotensin and opioids. The potency of VIP ((pD₂=7.52±0.18, n=6) was about 30 times higher than that of isoprenaline in the atropine and phentolamine-blocked preparation. The isoprenaline inhibition was mediated via a β₂-type of adrenoceptor with low apparent affinity for noradrenaline (intrinsic activity (a) = 0.27±0.01, n=8). Opposite effects of exogenous VIP and noradrenaline were on the other hand observed in the atropinized and β-blocked preparation. These results suggest that in the rat portal vein neuronal VIP and circulating adrenaline may be complementary in their antagonism of the α-adrenoceptor mediated increase in contractility.     

beta-Adrenergic relaxation and cAMP kinase activation in coronary arterial smooth muscle

If beta-adrenergic relaxation of smooth muscle is partly mediated by the adenosine 3',5'-cyclic monophosphate (cAMP) system, then beta-stimulation should be correlated to activation of cAMP-dependent protein kinase (cPK). Studies were performed with bovine coronary arterial strips to identify isozymic forms of cPK and to determine if beta-relaxation is correlated to activation of cPK (reflected by elevated ratios of cPK activity without cAMP to cPK activity with cAMP). Both ion exchange chromatography and a new electrophoretic technique revealed two cPK isozymes (types I and II). No change in cPK activity occurred in strips contracted with 30 mM KCl. In contrast, dose- and time-dependent relaxation during beta-stimulation with isoproterenol was highly correlated to parallel increases in cPK activity. Increased cPK activity was inhibited in assays performed with a specific inhibitor of cPK. Both relaxation and activation of cPK were abolished during beta-adrenergic blockade with propranolol. Relaxation by KCl removal or the ionophore R02-2985, unlike beta-mediated relaxation, did not increase cPK activity. These findings show that beta-mediated relaxation of isolated coronary arterial strips specifically activates cPK, and they support the hypothesis that beta-induced relaxation of vascular smooth muscle involves the cAMP system.     

不同前负荷条件下去神经平滑肌的应力松弛与膜电流特征

BACKGROUND: Muscle stretch test is a method for testing mechanical properties of denervated muscle under diverse loads. OBJECTIVE: To observe changes of spontaneous tension wave in myography and membrane electric current in aorta smooth muscle samples under several preload conditions. METHODS: Denervated intact smooth muscle samples were taken from the Kunming mouse aorta and urinary bladder wall. Smooth muscle samples were fixed on a micro positioning device and the first stretch was induced for a passive tension up to 1 g, this position was determined as the initial length (L0). Intermittently, with increasing sample length from L0 every 5 minutes, the 1st and 10th stretch were recorded as low- (L0+1) and high-preload (L0+10). 3% CaCl2 and 0.05% nitrendipine were dropped on the samples before L0+1 and L0+10 stretch were recorded. Membrane current changes were evaluated by glass microelectrodes and MultiClamp 700B Amplifier and pClamp 10 analyzer software. The membrane current changes after L0+1 and L0+10 were analyzed. RESULTS AND CONCLUSION: Prolonged stress relaxation phase was shortened with increasing preload in smooth muscle preparations. The stress relaxation phase in smooth muscle samples from urinary bladder wall was shorter than aorta samples. It was revealed that there were compliance differences between two kinds of smooth muscle samples. In aorta samples, myogenic spontaneous contraction amplitude during stress relaxation and membrane current were significantly increased with increasing preload. Increased amplitude and frequency of membrane current in aorta smooth muscle samples under high calcium concentration (3% CaCl2) were inhibited by L-type calcium channel blockade (0.05% nitrendipine). Collectively, increased preload in smooth muscle samples induced a directly decrease of compliance with an enlargement of myogenic spontaneous contraction amplitude. It appeared vital in particular in aorta samples. Membrane current significantly enhanced in spontaneous contraction period suggested the mechanical stretching ionic inward flow involved in this period. Membrane current amplitude was increased by high-preload under elevated calcium conditions; however, this change was suppressed by L-type calcium channel blockade. This study indicates quick stretching influences not only mechanically-gated channels in smooth muscle, but also the activation of L-type calcium channel. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

h-Caldesmon as a specific marker for smooth muscle tumors. Comparison with other smooth muscle markers in bone tumors

Caldesmon is a protein widely distributed in smooth and non-smooth muscle cells and is thought to regulate cellular contraction. Its isoform, high-molecular-weight caldesmon (h-CD), was demonstrated to be specific for smooth muscle cells and smooth muscle tumors of the soft tissue and to never be expressed in myofibroblasts. We performed an immunohistochemical study to examine h-CD expression in the following bone tumors: conventional and non-conventional osteosarcoma, 13; malignant fibrous histiocytoma of bone, 5; giant cell tumors of bone, 5; chondroblastoma, 3; metastatic leiomyosarcoma, 2; and rhabdomyosarcoma, 1. Frequent immunoreactivity for muscle actin (alpha-smooth muscle actin or muscle-specific actin) was seen in 11 of 13 osteosarcomas and all other tumors, whereas h-CD was expressed intensely only in 2 leiomyosarcomas. h-CD is considered a specific and useful marker to distinguish smooth muscle tumor from bone tumors with myoid differentiation.     

Reversal of alpha 1-receptor mediated relaxation in intestinal smooth muscle

The alpha 1-receptor agonist phenylephrine relaxed longitudinal rabbit jejunal muscle contracted in vitro by low concentrations of barium ions (1 mM). When the Ba2+ concentration was increased to 10-15 mM the response to phenylephrine was a contraction, and at Ba2+ concentrations in between the high and low range this response was biphasic--a relaxation followed by a contractile phase. The alpha 2-receptor agonist clonidine did not affect the tone of the Ba2+ contracted preparation. When the muscle preparation was contracted by Sr2+ (1-20 mM) in the presence of Ca2+ (2.5 mM), phenylephrine relaxed it, and no contractile response to phenylephrine was observed. In the absence of extracellular Ca2+, 5 mM Ba2+ caused a contraction. Under these conditions phenylephrine had no effect on the tissue tone. When Ca2+ was added in a low concentration (0.2-2 mM), phenylephrine elicited a gradually increasing contractile response. At 5 mM Ca2+ the contractile response was replaced by the normal relaxation. The contractile response to phenylephrine in the presence of 5 mM Ba2+ and 2.5 mM Ca2+ was partially blocked by low concentrations of verapamil. In higher concentrations verapamil abolished the tissue tonus completely. The contractile response to phenylephrine in the presence of 5 mM Ba2+ and 2.5 mM Ca2+ could be reverted to the normal relaxation by the addition of 20 mM Mg2+. Increasing the K+ concentration from the normal 5.9 to 62.9 mM blocked the phenylephrine-induced relaxation. No contractile response to phenylephrine occurred. It is concluded that Ba2+ could reverse the response of alpha 1 receptor stimulation in rabbit jejunum from a relaxation to a contraction and that this contractile response was dependent on the presence of Ca2+.     

Involvement of the cytoskeleton in calcium-dependent stress relaxation of rat aortic smooth muscle

Rat aortic smooth muscle exhibits a remarkable capacity for stress relaxation, the release of tension following tissue stretch. Stress relaxation was markedly enhanced in contracted aortic rings compared with unstimulated tissue. The magnitude of stress relaxation in contracted aortic rings correlated well with the passive tension imposed on the tissue by stretching, but showed little relationship to changes in tissue length or to the level of tension developed in response to agonist stimulation prior to stretch. The enhancement of stress relaxation in precontracted tissue was not affected by intimal rubbing or treatment with L-NAME. By comparison, the removal of extracellular calcium markedly attenuated stress relaxation. In addition, the use of cytochalasin B to block actin polymerization inhibited stress relaxation, whereas colchicine, a drug used to cause microtubule disassembly, had no effect on the phenomenon. The results indicate that the enhanced stress relaxation in contracted tissue is a calcium-dependent process and is not due to passive tissue elastic properties. We suggest that stress relaxation may not involve cross-bridge formation but could be explained by the remodelling of a portion of the tension-bearing actin cytoskeleton. This revised version was published online in September 2006 with corrections to the Cover Date.     

Adenosine potentiates neurally- and histamine-induced contraction of canine airway smooth muscle

We studied the effect of adenosine on airway reactivity of isolated canine bronchial smooth muscle under isometric conditions in vitro. Administration of adenosine and its analogs increased the contractile responses of bronchial segments to electrical field stimulation in a dose-dependent fashion, where the rank order potency was N-ethylcarboxamideadenosine greater than adenosine greater than N-cyclohexyladenosine, but had no effect on those to exogenous acetylcholine. This potentiation was more pronounced at relatively low than at high stimulus frequencies, the maximal increase from the baseline responses being 56.3 +/- 9.6% at 1 Hz (mean +/- SE, p less than 0.01). Adenosine also increased the histamine-induced contraction causing a leftward shift of the histamine dose-response curves, an effect that was abolished in the presence of atropine. These results suggest that adenosine potentiates airway responsiveness to vagal stimulation and to histamine through the activation of prejunctional A2 receptor, probably involving the accelerated release of acetylcholine from the cholinergic nerve terminals.     

Receptors on smooth muscle cells: characterization by contraction and specific antagonists

Smooth muscle cells were isolated from the stomach of the guinea pig, and the kinetics, stoichiometry, and specificity of contraction in response to the C-terminal octapeptides of cholecystokinin (CCK-OP), gastrin-17, and acetylcholine were examined. All three agonists elicited dose-dependent peak contraction that did not depend on the presence of extra-cellular calcium. The potencies of CCK-OP and gastrin-17 were equal (D50, 10(-11) M) and 10 times greater than the potency of acetylcholine (D50, 10(-10) M). A combination of low doses of acetylcholine and CCK-OP was synergistic; however, its effect did not exceed the maximal responses to either agonists alone or to high extracellular concentrations of calcium. The specificity of the receptors was established by the use of atropine and the two CCK-receptor antagonists dibutyryl cGMP and proglumide. The span of the dose-response curves was wide, suggesting the existence of receptor heterogeneity. It is concluded that gastric smooth muscle cells of the guinea pig possess distinct, high-affinity receptors for CCK-gastrin and acetylcholine; the receptors mediate contraction that is not immediately dependent on the presence of extracellular calcium.