Comparative effects of methylene blue on vascular and tracheal smooth muscle.
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Adrenergic alpha-1 Receptor Antagonists↗
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Platelet-derived growth factor-BB and thrombin activate phosphoinositide 3-kinase and protein kinase B: role in mediating airway smooth muscle proliferation.
Proliferation of airway smooth muscle results from persistent inflammatory cytokine and growth factor stimulation and is a critical component of airway luminal narrowing in chronic asthma. Using primary cultures of bovine tracheal smooth muscle (BTSM) cells to examine the signaling basis of cell proliferation, platelet-derived growth factor (PDGF)-BB and thrombin (which act through distinct receptor types) were found to induce DNA synthesis in BTSM cells. Mitogen-induced DNA synthesis could be completely inhibited by LY294002, a selective phosphoinositide 3-kinase (PtdIns 3-kinase) inhibitor. Exposure of BTSM cells to PDGF-BB or thrombin resulted in rapid activation of PtdIns 3-kinase and accumulation of phosphoinositide-3,4,5-trisphosphate. Protein kinase B, a novel signaling protein kinase, was identified in BTSM cells and was activated by PDGF-BB and thrombin in a PtdIns 3-kinase-dependent manner; this may underlie mitogen-stimulated activation of p70(s6k). PD98059, a mitogen-activated protein kinase kinase 1 inhibitor, also partially inhibited PDGF-BB- and thrombin-stimulated DNA synthesis, indicating a modulatory role for mitogen-activated protein kinase in proliferation. GF109203X, Ro 31-8220, calphostin C, and chelerythrine (selective protein kinase C inhibitors) had no effect on PDGF-BB- or thrombin-stimulated DNA synthesis, suggesting that, despite abolishment of mitogen-stimulated protein kinase C activity, cell proliferation stimulated by PDGF-BB and thrombin is protein kinase C-independent. These data demonstrate that the PtdIns 3-kinase/protein kinase B pathway represents a key signaling route in airway smooth muscle proliferation, with the mitogen-activated protein kinase kinase 1/mitogen-activated protein kinase cascade providing a complementary signal required for the full mitogenic response.
3-Phosphoinositide-Dependent Protein Kinases↗
Calcitonin gene-related peptide stimulates cyclic AMP formation in rat aortic smooth muscle cells.
In rat aortic smooth muscle cells in culture, calcitonin gene-related peptide stimulated cAMP formation in a dose-dependent manner, half-maximally effective at 0.5 to 1 nM. There was no effect on formation of cGMP, which was increased 300-fold in the same experiments by atriopeptin or sodium nitroprusside. The vasodilator effect of CGRP in rat aorta requires an intact endothelium, indicating that increase in vascular smooth muscle cAMP is not in itself sufficient to bring about relaxation. cAMP is probably a mediator of CGRP action in vascular smooth muscle.
Animals↗
[Ca(2+)](i) signaling in renal arterial smooth muscle cells of pregnant rat is enhanced during inhibition of NOS.
Vascular resistance and arterial pressure are reduced during normal pregnancy, but dangerously elevated during pregnancy-induced hypertension (PIH), and changes in nitric oxide (NO) synthesis have been hypothesized as one potential cause. In support of this hypothesis, chronic inhibition of NO synthesis in pregnant rats has been shown to cause significant increases in renal vascular resistance and hypertension; however, the cellular mechanisms involved are unclear. We tested the hypothesis that the pregnancy-associated changes in renal vascular resistance reflect changes in contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) of renal arterial smooth muscle. Smooth muscle cells were isolated from renal interlobular arteries of virgin and pregnant Sprague-Dawley rats untreated or treated with the NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME; 4 mg. kg(-1). day(-1) for 5 days), then loaded with fura 2. In cells of virgin rats incubated in Hanks' solution (1 mM Ca(2+)), the basal [Ca(2+)](i) was 86 +/- 6 nM. Phenylephrine (Phe, 10(-5) M) caused a transient increase in [Ca(2+)](i) to 417 +/- 11 nM and maintained an increase to 183 +/- 8 nM and 32 +/- 3% cell contraction. Membrane depolarization by 51 mM KCl, which stimulates Ca(2+) entry from the extracellular space, caused maintained increase in [Ca(2+)](i) to 292 +/- 12 nM and 31 +/- 2% contraction. The maintained Phe- and KCl-induced [Ca(2+)](i) and contractions were reduced in pregnant rats but significantly enhanced in pregnant rats treated with L-NAME. Phe- and KCl-induced contraction and [Ca(2+)](i) were not significantly different between untreated and L-NAME-treated virgin rats or between untreated and L-NAME + L-arginine treated pregnant rats. In Ca(2+)-free Hanks', application of Phe or caffeine (10 mM), to stimulate Ca(2+) release from the intracellular stores, caused a transient increase in [Ca(2+)](i) and a small cell contraction that were not significantly different among the different groups. Thus renal interlobular smooth muscle of normal pregnant rats exhibits reduction in [Ca(2+)](i) signaling that involves Ca(2+) entry from the extracellular space but not Ca(2+) release from the intracellular stores. The reduced renal smooth muscle cell contraction and [Ca(2+)](i) in pregnant rats may explain the decreased renal vascular resistance associated with normal pregnancy, whereas the enhanced cell contraction and [Ca(2+)](i) during inhibition of NO synthesis in pregnant rats may, in part, explain the increased renal vascular resistance associated with PIH.
Animals↗
Mitogenic actions of endothelin-1 and epidermal growth factor in cultured airway smooth muscle.
1. Hyperplasia of airway smooth muscle contributes to the increase in bronchomotor responsiveness that characterizes asthma. We have investigated the mitogenic potential of endothelin-1 (ET-1) and epidermal growth factor (EGF) in guinea-pig cultured airway smooth muscle and the relationship of these actions to tyrosine phosphorylation and increases in intracellular calcium (Ca2+i). 2. ET-1 stimulated DNA and protein synthesis and also increased cell number, but these mitogenic actions were small compared with those of EGF. 3. ET-1 and EGF increased the level of tyrosine phophorylation of a range of proteins with apparent molecular weights between 80 and 100 kDa and also increased phosphorylation of a single protein of 33 kDa. 4. Ca2+i levels were increased by both ET-1 and EGF. However, concentrations of EGF three orders of magnitude higher than those having mitogenic actions or increasing protein tyrosine phosphorylation were required. ET-1 was a more potent stimulant of increases in intracellular calcium than of mitogenesis. 5. We conclude that elevation of Ca2+i is unlikely to be an important signal for the mitogenic action of EGF. It is suggested that stimulants at the EGF receptor (EGF and transforming growth factor alpha) may play a role in the airway smooth muscle hyperplasia in asthma.
Animals↗
Omega-3 polyunsaturated fatty acids inhibit migration of human vascular smooth muscle cells in vitro.
Arterial smooth muscle cell migration from the media to the intima is a crucial process in the pathogenesis of atherosclerosis. Platelet-derived growth factor (PDGF) has been proposed to play a key role in the development of advanced atherosclerotic lesions by stimulating the migration and proliferation of vascular smooth muscle cells. Polyunsaturated fatty acids (PUFA) of the omega-3 series, extracted from fish oil has been shown to have beneficial effects on atherosclerosis. In this study, we evaluated the effects of omega-3 PUFA on the migration of human aortic smooth muscle cell (hASMC) in vitro. The migration assay was performed according to the Capsoni's method using transwell culture plates. PDGF, fibrinogen or 10% FCS significantly stimulated hASMC migration, however, omega-3 PUFA significantly inhibited PDGF-induced migration of hASMC. These results suggest that the inhibitory effect of omega-3 PUFA on cell migration may be an important aspect by which omega-3 PUFA exerts its antiatherosclerotic influence.
Cell Movement↗
Heterogeneity of myosin antigenic expression in vascular smooth muscle in vivo.
Rabbit antisera elicited against purified human nonmuscle (platelet) and smooth muscle (uterine myometrium) myosins identified distinct species of myosin when frozen sections of a variety of mammalian tissues were examined by immunofluorescence microscopy. Antiplatelet myosin antiserum specifically stained several nonmuscle cell types including epithelial, some connective tissue, and all vascular endothelial (arterial, venous, capillary) cells. Antismooth muscle myosin antiserum stained only smooth muscle and no other cell types. Neither antiserum reacted with rat cardiac (ventricular) or skeletal muscle cells. Antismooth muscle myosin antiserum staining was detectable in medial vascular smooth muscle in all vessels examined from rat, bovine, human, and guinea pig sources (including elastic and muscular arteries, arterioles, venules, and veins). Although antiplatelet myosin antiserum did not stain nonvascular smooth muscle or vascular smooth muscle in muscular arteries, arterioles, venules, or veins, it did uniformly and specifically stain medial vascular smooth muscle in elastic arteries. This staining of elastic arteries was abolished by absorption of antiplatelet myosin antiserum with purified platelet myosin but not uterine myosin. Similarly, the reactivity of antismooth muscle myosin antiserum was abolished by incubation with uterine but not platelet myosin. The differences in staining patterns observed with antiplatelet myosin antiserum and antismooth muscle myosin antiserum in elastic arteries versus other blood vessels suggests a heterogeneity of antigenic expression in vascular smooth muscle myosin. The most likely explanations for this heterogeneity are the presence of different gene products (myosin isozymes) or a posttranslational alteration (possibly conformational) of a single myosin species. Heterogeneity in this important component of the contractile apparatus of vascular smooth muscle may have significant implications for the physiology and pathophysiology of the vessel wall.
Animals↗
Response of equine airway smooth muscle to acetylcholine and electrical stimulation in vitro.
Smooth muscle strips from the midcervical portion of the trachea and bronchial smooth muscle strips from third-generation airways of horses were placed in tissue baths, and isometric contractile force was measured. Active force was measured in response to electrical stimulation and exogenous acetylcholine. Square-wave electrical stimuli were applied at various voltages (10, 12, 15, 18, 20, 25 V), frequencies (3, 5, 10, 15, 20, 25, 30 Hz), and pulse durations (0.2, 0.5, 1.0, 1.5, 2.0 ms). Isometric contractile force increased as voltage, frequency, and pulse duration increased. Maximal contractile response to electrical stimulation was obtained at 18 V, 25 Hz, and 0.5 ms. Atropine (10(-6)M) or tetrodotoxin (3 x 10(-6)M) blocked the contraction, indicating that the contractile response was attributable to the release of neurotransmitter from cholinergic nerves. Cumulative concentration-response curves to acetylcholine (10(-9)M through 10(-4)M) were determined. Isometric contractile force increased as acetylcholine concentration increased. There was a significant (P less than 0.05) difference in the 50% effective dose for acetylcholine in tracheal smooth muscle and bronchial smooth muscle. The mean (+/- SD) contractile response to maximal electrical stimulus was 89% (+/- 7.4%) of that in response to 10(-4)M acetylcholine in tracheal smooth muscle and was 68% (+/- 10.4%) of the response to 10(-4)M acetylcholine in bronchial smooth muscle.
Acetylcholine↗
Spatial pattern of smooth muscle differentiation is specified by the epithelium in the stomach of mouse embryo.
Smooth muscle induction in the stomach of mouse embryo was analyzed experimentally by tissue recombination and organ culture technique. When observed immunohistochemically with anti-alpha-smooth muscle actin antibody, smooth muscle myoblasts first differentiated and formed the circular muscle layer at 13 days of gestation. The longitudinal muscle layer appeared at 15 days in the forestomach and at neonatal period in the glandular stomach. The lamina muscularis mucosae was observed by the neonatal period. When the undifferentiated 11-day stomach was cultivated in vitro for 3 days, an alpha-smooth muscle actin-positive cell layer was formed and the explant exhibited rhythmical contraction. The mesenchyme alone remained as an undifferentiated cell mass. When the mesenchyme was recombined with the epithelium, a distinct smooth muscle layer was formed. These demonstrated that the alpha-smooth muscle actin expression was induced by the epithelium. Next, we examined by heterologous recombination the roles of the epithelium in formation of the organ-specific spatial pattern of smooth muscle differentiation. The stomach mesenchyme recombined with the tracheal epithelium exhibited a partially tracheal type of alpha-smooth muscle actin expression. The uterine epithelium induced only a small number of smooth muscle myoblasts and the spatial organization was disordered. Finally, we assayed if the mode of smooth muscle induction by the stomach epithelium changes during development. Heterochronic recombination proved that the 14-day forestomach epithelium induced a muscularis mucosae-like smooth muscle layer that was contiguous to the epithelium, whereas the 11-day stomach and the 13-day forestomach epithelium induced a circular muscle-like smooth muscle layer that was distant from the epithelium. As a whole, our data suggest that the spatial pattern of smooth muscle differentiation is regulated by organ-specific and stage-specific influences from the epithelium.
Actins↗
Histopathological study of stromal smooth muscle cells in fibroadenoma of the breast.
Smooth muscle cells are extremely rare stromal components of fibroadenoma. Eighty-five cases of fibroadenoma were reviewed in order to investigate the frequency of smooth muscle cells and to discuss its origin. Of the 85 cases, four (4.7%) cases showed smooth muscle cells in the stroma. Distribution in terms of age and tumor size did not show any difference from other cases. Three cases were classified as an intracanalicular subtype, and one was a mastopathic subtype. The cells possessed most of the ultrastructural characteristics of smooth muscle cells, but poor indentation of nuclear contours suggested the last remnant of fibroblasts. It was therefore easy to recognize the cells derived from fibroblasts. In two of four cases, smooth muscle cells were observed in the stroma, which had marked hyalinization and calcification. Therefore, it was supposed that smooth muscle cells could appear in the stroma of long-standing tumors.
Adolescent↗
Tin ethyl etiopurpurin significantly inhibits vascular smooth muscle cell proliferation in vivo.
Smooth muscle cell proliferation is a major component of restenosis following angioplasty. Hematoporphyrin derivative and other photosensitive compounds inhibit proliferation by causing cellular necrosis upon light activation (photodynamic therapy). Other photosensitive compounds, such as benzoporphyrin derivative, have been suggested as having non-cytotoxic antiproliferative effects without photodynamic therapy, although other studies using benzoporphyrin derivative were negative. Inhibition of smooth muscle cell proliferation was examined in an in vivo rabbit model of vascular injury using a novel synthetic chlorin derivative, tin ethyl etiopurpurin, and benzoporphyrin derivative without photodynamic therapy. Tin ethyl etiopurpurin and benzoporphyrin derivative inhibited smooth muscle cell proliferation by 50-90% of control (p < or = 0.05) without toxic side effects. These results suggest that tin ethyl etiopurpurin and benzoporphyrin derivative without photodynamic therapy may provide a novel and potent antiproliferative therapy that might be useful in the treatment of restenosis.
Animals↗
Mechanisms of smooth muscle contraction.
Work performed with differentiated contractile smooth muscle tissue over the last two decades has made clear that covalent modification of myosin by phosphorylation of the 20-kDa myosin light chains is a significant mode of regulation of contractile activity in smooth muscle, particularly in regard to the generation of phasic contractions and the initial development of tonic contractions. This regulatory mechanism appears to be of unique importance in smooth muscle compared with striated muscle. It is equally clear, however, that there is an important role for protein kinase C in the regulation of smooth muscle tone maintenance, particularly in vascular smooth muscle. Several possible signal transduction cascades involving protein kinase C are outlined. Increasing evidence suggests a link between protein kinase C and actin-based regulatory mechanisms. This review places emphasis on relating up-to-date biochemical facts to the physiological realities of the smooth muscle cell.
Animals↗
Regulation of renin-like enzyme in cultured human vascular smooth muscle cells.
Cultured human arterial smooth muscle cells produced an immunologically specific renin-like enzyme. The renin-like enzyme in the culture medium was mostly an inactive form; the proportion of the active form in the cell was 30 to 75%. Phorbol 12-myristate 13-acetate, N'-O'-dibutylyladenosine 3', 5'-monophosphate and isoproterenol with theophylline increased the renin-like enzyme in the medium and in the cell, dose dependently. Endothelial cell growth supplement also increased the renin-like enzyme produced by cultured vascular smooth muscle cells, and heparin promoted the effects of endothelial cell growth supplement. The existence of the regulation of the renin-like enzyme produced by cultured vascular smooth muscle cells strongly suggests the existence of a local renin angiotensin system in human vascular walls.
Cells, Cultured↗
Mechanisms of mechanical strain memory in airway smooth muscle.
We evaluated the hypothesis that mechanical deformation of airway smooth muscle induces structural remodeling of airway smooth muscle cells, thereby modulating mechanical performance in subsequent contractions. This hypothesis implied that past experience of mechanical deformation was retained (or "memorized") as structural changes in airway smooth muscle cells, which modulated the cell's subsequent contractile responses. We termed this phenomenon mechanical strain memory. Preshortening has been found to induce attenuation of both force and isotonic shortening velocity in cholinergic receptor-activated airway smooth muscle. Rapid stretching of cholinergic receptor-activated airway smooth muscle from an initial length to a final length resulted in post-stretch force and myosin light chain phosphorylation that correlated significantly with initial length. Thus post-stretch muscle strips appeared to retain memory of the initial length prior to rapid stretch (mechanical strain memory). Cytoskeletal recruitment of actin- and integrin-binding proteins and Erk 1/2 MAPK appeared to be important mechanisms of mechanical strain memory. Sinusoidal length oscillation led to force attenuation during oscillation and in subsequent contractions in intact airway smooth muscle, and p38 MAPK appeared to be an important mechanism. In contrast, application of local mechanical strain to cultured airway smooth muscle cells induced local actin polymerization and cytoskeletal stiffening. It is conceivable that deep inspiration-induced bronchoprotection may be a manifestation of mechanical strain memory such that mechanical deformation from past breathing cycles modulated the mechanical performance of airway smooth muscle in subsequent cycles in a continuous and dynamic manner.
Adaptation, Physiological↗
Force development with inosine triphosphate and uridine triphosphate in chemically skinned vascular smooth muscle.
The contraction of vascular smooth muscle is thought to be regulated by reversible phosphorylation of the 20,000 dalton light chains of myosin, catalyzed by myosin light chain kinase that is dependent on calcium and calmodulin. With phosphorylation, there is a coincident increase in the actin-activated myosin NTPase activity, cross bridge interaction and contractile activity. However, this myosin phosphorylation mechanism may not be the sole factor controlling actin-myosin interaction in vascular smooth muscle. Other mechanisms may function in addition to this myosin-linked regulation. A calcium-insensitive regulation of contraction was observed in helical strips of chemically skinned (Triton X-100) arterial smooth muscle. Millimolar concentrations of inosine triphosphate and uridine triphosphate supported concentration dependent force development in the absence of calcium. Force development was a function of the MgNTP concentration. At high free calcium concentrations, an additional component of force was observed. ITP and UTP, in contrast to ATP, are less effective substrates for the myosin light chain kinase, and their effect on actin-myosin interaction is thus less than that of ATP. They are, however, utilized by the myosin NTPase after treatment by ATP-gamma-S. The efficacy of the substrate for the activated NTPase is greater for UTP than ITP than for ATP.
Acid Anhydride Hydrolases↗
Tyrphostin A9 and wortmannin perturb the Golgi complex and block proliferation of vascular smooth muscle cells.
To proliferate, vascular smooth muscle cells first convert from a contractile to a synthetic phenotype. Earlier studies indicate that this process is supported by fibronectin and accelerated by platelet-derived growth factor (PDGF). Here, the mechanisms in this transition were further explored. Isolated rat aortic smooth muscle cells were treated with tyrphostin A9, a PDGF receptor tyrosine kinase inhibitor, and wortmannin, a phosphoinositide 3-kinase inhibitor. Electron microscopy did not show any effect on the reorganization of the cells during the first days in culture, i.e. the loss of actin filaments and the formation of a large secretory apparatus. Conversely, both drugs caused hypertrophy of the Golgi complex, with large and partly vacuolized cisternal stacks. Nevertheless, a juxtanuclear staining pattern for the Golgi enzyme mannosidase II, the coat protein beta-COP, and the PDGF beta-receptor was retained. Moreover, the serum-induced proliferation of the cells was blocked. These findings suggest that signaling via PDGF receptor tyrosine kinases and phosphoinositide 3-kinases is not necessary for the shift of the smooth muscle cells from a contractile to a synthetic phenotype. On the other hand, these enzymes apparently carry out important functions in the control of intracellular membrane traffic and cell division.
Androstadienes↗
Nerve growth factor activation of Erk-1 and Erk-2 induces matrix metalloproteinase-9 expression in vascular smooth muscle cells.
In response to vascular injury, smooth muscle cells migrate from the media into the intima, where they contribute to the development of neointimal lesions. Increased matrix metalloproteinase (MMP) expression contributes to the migratory response of smooth muscle cells by releasing them from their surrounding extracellular matrix. MMPs may also participate in the remodeling of extracellular matrix in vascular lesions that could lead to plaque weakening and subsequent rupture. Neurotrophins and their receptors, the Trk family of receptor tyrosine kinases, are expressed in neointimal lesions, where they induce smooth muscle cell migration. We now report that nerve growth factor (NGF)-induced activation of the TrkA receptor tyrosine kinase induces MMP-9 expression in both primary cultured rat aortic smooth muscle cells and in a smooth muscle cell line genetically manipulated to express TrkA. The response to NGF was specific for MMP-9 expression, as the expression of MMP-2, MMP-3, or the tissue inhibitor of metalloproteinase-2 was not changed. Activation of the Shc/mitogen-activated protein kinase pathway mediates the induction of MMP-9 in response to NGF, as this response is abrogated in cells expressing a mutant TrkA receptor that does not bind Shc and by pretreatment of cells with the MEK-1 inhibitor, U0126. Thus, these results indicate that the neurotrophin/Trk receptor system, by virtue of its potent chemotactic activity for smooth muscle cells and its ability to induce MMP-9 expression, is a critical mediator in the remodeling that occurs in the vascular wall in response to injury.
Animals↗
Effects of cyclic GMP on smooth muscle relaxation.
Cyclic GMP levels within smooth muscle are affected then by a number of different pathways. Physiologically NO and ANF are probably the two most important regulators for smooth muscle function, but a variety of other mediators and pharmacological agents may also influence this system. Because of the important role that cyclic GMP plays in the control of smooth muscle tone, which clearly includes vascular smooth muscle, it is now and will continue to be in the future an important physiological and biochemical target for research and a pharmacological target for therapeutic agents.
Animals↗