Rho-associated kinase of chicken gizzard smooth muscle

Rho-associated kinase (Rho-kinase) from chicken gizzard smooth muscle was purified to apparent homogeneity (160 kDa on SDS-polyacrylamide gel electrophoresis) and identified as the ROKalpha isoform. Several substrates were phosphorylated. Rates with myosin phosphatase target subunit 1 (MYPT1), myosin, and the 20-kDa myosin light chain were higher than other substrates. Thiophosphorylation of MYPT1 inhibited myosin phosphatase activity. Phosphorylation of myosin at serine 19 increased actin-activated Mg+-ATPase activity, i.e. similar to myosin light chain kinase. Myosin phosphorylation was increased at higher ionic strengths, possibly by formation of 6 S myosin. Phosphorylation of the isolated light chain and myosin phosphatase was decreased by increasing ionic strength. Rho-kinase was stimulated 1.5-2-fold by guanosine 5'-O-3-(thio)triphosphate.RhoA, whereas limited tryptic hydrolysis caused a 5-6-fold activation, independent of RhoA. Several kinase inhibitors were screened and most effective were Y-27632, staurosporine, and H-89. Several lipids caused slight activation of Rho-kinase, but arachidonic acid (30-50 microM) induced a 5-6-fold activation, independent of RhoA. These results suggest that Rho-kinase of smooth muscle may be involved in the contractile process via phosphorylation of MYPT1 and myosin. Activation by arachidonic acid presents a possible regulatory mechanism for Rho-kinase.     

Effect of pressure on cultured smooth muscle cells

Recent studies indicate that smooth muscle cell (SMC) growth and morphology can be modulated by repetitive strain. However, there is very little known about the influence of pressure, without an associated cell stretch, on SMC phenotype. To study this, cultured bovine aortic SMC were grown on a rigid surface and placed in a custom-designed plexiglass pressure chamber with a carefully regulated 5% CO2/air environment. SMC were exposed to either atmospheric, 105 mm Hg or 120/90 mm Hg pressure at a frequency of 60 cycles/min (0.5 s systole, 0.5 s diastole). SMC number was determined on days 1, 3, 5, 7 and 9. SMC exposed to pressure were more elongated and displayed a significant increase in cell number by day 5 which persisted until day 9. Lactate dehydrogenase (LDH) in the conditioned media, an index of cytotoxicity, was not different between the groups at each time point. There was also no difference in pH or pCO2 of the media of SMC in any group. This is the first report of the effects of increased static and pulsatile pressure on SMC in vitro and indicates an increased proliferative rate. We hypothesize that the systemic pressure that the blood vessel is exposed to in vivo may have a significant regulatory influence on the phenotype of the smooth muscle cells which may affect the SMC response to injury or stimuli.     

Purification and properties of filamin, and actin binding protein from chicken gizzard

Filamin, a protein recently identified in chicken gizzard (Wang, K., Ash, F., and Singer, S. J. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 4483-4486), has been purified free of other components and its molecular properties have been examined. Filamin has a sedimentation constant (S020,w) of 8.86 S and a partial specific volume of 0.734 ml/g. Sedimentation equilibrium experiments give a value of 498,000 for the molecular weight of native filamin. From these data a frictional ratio of 2.32 has been calculated. On sodium dodecyl sulfate gel electrophoresis, filamin migrates as a single protein band with an estimated molecular weight of 240,000. Filamin is a soluble protein and under a variety of conditions tested does not by itself form filaments or precipitate from solution. However, filamin binds to rabbit skeletal muscle F-actin, and the complex is readily sedimented by centrifugation to yield a gelatinous pellet containing actin and filamin. These results indicate that filamin is a dimeric protein with a moderate degree of asymmetry that binds to actin. The results also suggest that the distribution of filamin in cells is derived from its interaction with polymerized actin.     

Prostaglandins increase matrix metalloproteinase release from human ciliary smooth muscle cells

PURPOSE: To identify matrix metalloproteinases (MMPs) released by ciliary smooth muscle cells in vitro and to determine whether MMP release is altered by exposure to prostaglandins (PGs). METHODS: Human ciliary smooth muscle cells were grown to confluence in monolayer cultures and treated with PGF2 alpha, 11-deoxy-PGE1, or PhXA85 (the nonesterified analogue of PhXA41) for 12 to 72 hours. The activity of MMP in the medium was assayed using gelatin and casein zymography. Identification of the specific MMP associated with each band was made by Western blot analysis. Band intensity, which reflects activity, was measured with a scanning laser densitometer. RESULTS: Three major bands appeared in the gelatin zymographs at positions corresponding to molecular weights of 62 kDa, 68 kDa, and 97 kDa. A single band at 50 kDa predominated in the casein zymograms. Substitution of EDTA for calcium and zinc in the development solution eliminated the appearance of these bands, indicating that they reflect MMP activity. Immunoblotting, using MMP-specific antibodies, confirmed that the three bands in the gelatin zymographs were MMP-1, MMP-2, and MMP-9, respectively; the single band in the casein zymographs was MMP-3. Treatment with 200 nM PGF2 alpha, 11-deoxy-PGE1, or PhXA85 for 72 hours increased the combined density scores for MMP-1 and MMP-2 by 37%, 64%, and 27%; the density scores for MMP-9 by 268%, 253%, and 125%; and the density scores for MMP-3 by 35%, 71%, and 22%, respectively. CONCLUSIONS: These results indicate that ciliary smooth muscle cells can secrete MMP-1, MMP-2, MMP-3, and MMP-9. In addition, exposure to PGF2 alpha, 11-deoxy-PGE1, or PhXA85 increases production of all four MMPs. These observations support the hypothesis that increased MMP production by ciliary muscle cells has a role in increasing uveoscleral outflow facility after topical PG administration.     

Beta-adrenoceptors on smooth muscle, nerves and inflammatory cells

Although the bronchodilator action of beta 2-adrenoceptor agonists in asthma is largely due to relaxation of airway smooth muscle, these agents have other effects which may contribute to their anti-asthma action. Human airway smooth muscle contains only beta 2-receptors which, when stimulated, stimulate a rise in intracellular cAMP and activation of PKA (protein kinase A), which in turn phosphorylates several cellular proteins, resulting in relaxation. However, beta-agonists also influence membrane K+ channels and induce smooth muscle relaxation without a rise in cAMP, and this mechanism appears to be the major feature of bronchodilatation in asthma. There is also evidence that beta-agonists may modulate neurotransmission in airways via prejunctional receptors on airway nerves, both sensory and motor. Blockade of prejunctional beta 2-receptors in asthma patients may lead to marked rise in acetylcholine release, with severe bronchoconstriction. Although beta-agonists have little or no effect on the chronic inflammatory response which underlies chronic airway hyper-responsiveness, they do inhibit the release of histamine from mast cells in vitro. The presence of beta-receptors has also been detected not only on mast cells but also on eosinophils, macrophages, lymphocytes and neutrophils, but beta-agonists have little or no inhibitory action on the activities of all these cells due to rapid tachyphylaxis.     

Preparation of cultured smooth muscle cells from human myometrium for X-ray microanalysis

Methodological aspects of the use of X-ray microanalysis in physiological and pharmacological experiments on cultured myometrial cells were investigated. Cultured human myometrial cells were grown from biopsies after detaching the fibroblasts. Of the cultured cells, 95-98% showed desmin-like immunoreactivity. Transmission electron microscopy showed that subcultured cells were different from myometrial cells in situ. The effects of washing the cells to remove external salt-rich medium were investigated. All solutions removed the external medium, resulting in lower concentrations of Na and Cl. In the cells washed with 0.3 M mannitol, most of the elemental concentrations were significantly lower than in their unwashed counterparts and those washed in the other solutions. In cells washed in either 0.15 M ammonium acetate or distilled water, no significant differences in P and K compared with their unwashed counterparts were found. There were also no significant differences between cells washed in ammonium acetate and in distilled water. In subsequent experiments ammonium acetate was used. Incubation of cells in standard Ringer's solution resulted in an increase in Na and Cl, and a decrease in K, concomitantly with an increase in Ca. Although Ringer's solution per se can elicit changes in diffusible elements in the cells, physiological and pharmacological effects of oxytocin could still be detected in Ringer's solution. However, effects of oxytocin were different when the experiment was done in culture medium, instead of in Ringer's solution.     

Effects of dipyridamole on the smooth muscle cells of the guinea-pig''s taenia coli

Passive film and accelerated corrosion tests were performed on both wrought AISI 316L stainless steel and wrought Vitallium (Haynes Stellite 25) Charnley Type cement restrictors. The morphology and incidence of corrosion at the wire-wire crevices and the methacrylate-metal crevices were determined by scanning electron microscopy. The results are interpreted in view of the previously-documented crevice corrosion susceptibility of these two alloys.     

Effect of nitroprusside on gap junctions of single tracheal smooth muscle cells excised from rats

To investigate the effect of nitroprusside on gap junctions of tracheal smooth muscle cells, we ionophoretically injected sodium nitroprusside into single tracheal smooth muscle cells excised from rats. The input resistance of the tracheal smooth muscle cells increased after the injection of nitroprusside. This results suggests that nitroprusside blocks gap junctions of tracheal smooth muscle cells.     

Mechanism of catecholamine-induced proliferation of vascular smooth muscle cells

BACKGROUND: Catecholamines have been shown to aggravate atherosclerosis in animals and humans, and abnormal proliferation of vascular smooth muscle cells (VSMC) is a key event in the early stage of atherosclerosis. Catecholamines may be involved in such cell growth. Therefore, a series of experiments using cultured VSMC was performed to elucidate their possible mitogenic effect. METHODS AND RESULTS: We examined the mitogenic effect of catecholamines using rat aortic smooth muscle cells (VSMC) by measuring [3H]thymidine incorporation, checking with flow cytometry, and counting the cell number directly. Furthermore, the catecholamine-activated signal transduction pathway was assessed by measurement of the formation of inositol 1, 4, 5-triphosphate, intracellular Ca2+ concentration, mitogen-activated protein kinase (MAPK) activity, and mitogenic gene expression. Norepinephrine (NE) and phenylephrine stimulated [3H]thymidine incorporation and cell growth. Clonidine and isoproterenol showed little of such effects. Prazosin was more effective than either yohimbine or propranolol in suppressing the mitogenic effect of NE, indicating that catecholamine-induced VSMC proliferation is mediated by alpha 1-adrenoceptors. The alpha 1-adrenoceptor activation was coupled to pertussis toxin-insensitive Gq-protein and triggered phosphoinositide hydrolysis with subsequent activation of protein kinase C and MAPK in VSMC. In response to NE, both 42- and 44-kD MAPK were activated and tyrosine was phosphorylated. alpha 1-Adrenoceptor stimulation with NE also caused accumulation of c-fos, c-jun, and c-myc mRNA. Chloroethylclonidine completely blocked the alpha 1-adrenoceptor-mediated mitogenesis. CONCLUSIONS: The effect of catecholamines appears to be mediated via the activation of the chloroethylclonidine-sensitive alpha 1-adrenoceptors that triggers the phosphoinositide hydrolysis and activates the MAPK pathway, leading to DNA synthesis and cell proliferation.     

ETA and ETB receptors on vascular smooth muscle cells from mesenteric vessels of spontaneously hypertensive rats

1. To investigate the contribution of ETA and ETB receptors, calcium responses to the ETB agonist, IRL-1620, to endothelin-1 (ET-1) and to the ETA antagonist, BQ-123, were examined in primary cultured unpassaged vascular smooth muscle cells (VSMC) from mesenteric vessels of 3, 9 and 17 week old spontaneously hypertensive rats (SHR), Wistar and Wistar-Kyoto (WKY) rats using Fura-2 methodology. 2. IRL-1620 (10(-7) mol/L) and ET-1 (10(-9) mol/L) increased [Ca2+]i in all strains and ages. Responses to ET-1 and IRL-1620 were blunted in 17 week SHR. BQ-123 significantly reduced ET-1-stimulated [Ca2+]i. In endothelium-denuded mesenteric vessels, ET-1 and IRL-1620 induced significant [Ca2+]i responses. 3. Binding of ET-1 was significantly lower in mesenteric artery membranes from 17 week SHR compared to controls. 4. Thus, ETA and ETB receptors are present in rat mesenteric VSMC. In adult SHR, a reduced density of ET receptors results in decreased responses to IRL-1620 and to ET-1.     

Oxidized collagen stimulates proliferation of vascular smooth muscle cells

We have examined the use of presurgical morphine-midazolam combination in 80 children aged 2-10 y undergoing repair of hypospadias. They were allocated randomly, in a double-blind study, to receive one of four morphine-midazolam combination doses (n = 20 each); (group I: 75 microg/kg each) [corrected] (group II: 75 microg/kg [corrected] morphine, 50 microg/kg [corrected] midazolam); (group III: 50 microg/kg [corrected] morphine, 75 microg/kg [corrected] midazolam); (group IV: 50 microg/kg [corrected] each). Drugs were given after induction of anesthesia and before the start of surgery. Observational scoring system, using crying, movement, agitation, posture and localization of pain as scoring criteria, was used to assess the children during their stay in the recovery room together with their sedative and/or analgesic requirement. Pre-surgical morphine-midazolam administration produced stable hemodynamic variables with satisfactory postoperative analgesia suggesting 75 microg/kg [corrected] dose of both morphine and midazolam as upper permissible dose, and 50 microg/kg [corrected] each as lower effective dose.     

Growth of rabbit aortic smooth muscle cells in serum from patients with juvenile diabetes

The effect of human diabetic serum on the growth of rabbit arterial smooth muscle cell cultures was studied in the stationary phase of growth. The serum was obtained from young, male, non-obese, juvenile diabetics and non-diabetics. The experiments were carried out using dialysed as well as non-dialysed serum. The concentration of cholesterol and triglycerides were equal in normal and diabetic serum. Media supplemented with diabetic serum from both short term and long term diabetics stimulated the outgrowth of the smooth muscle cells significantly (2p less than 0.01). A statistically significantly stimulation of growth was also observed using dialysed human diabetic serum (2p less than 0.05). Autoradiographic studies showed that the number of 3H-thymidine labelled cells and of cells in mitosis increased appreciably after incubation in diabetic human serum (2p less than 0.005). The present data show that human serum from juvenile diabetics contains a factor or factors which promote an excessive growth of arterial medial cells. The factor(s) is not lipids as hyperlipemia was not present nor is it glucose, aminoacids, fructose or ketones, as the growth effect remained after dialysis.     

血管平滑肌细胞的增殖因素及机制

血管平滑肌细胞(VSMC)增殖已成为目前心血管疾病研究领域里的热点,VSMC增殖、迁移及凋亡在冠状动脉粥样硬化(AS)、冠状动脉搭桥术、经皮冠状动脉介入治疗(PCI)术后血管再狭窄的发生和发展过程中起重要作用.本文作者对VSMC增殖及促增殖因素和机制进行综述.     

Ultrastructural observations of the human uterine smooth muscle cells during gestation

At term pregnancy, the myometrium consists of bundles of smooth muscle cells bound together by varying amounts of connective tissue. Each bundle contains both dark and light muscle cells. During uterine contractions it is believed that the smooth muscle cells become darker, decrease in volume, and exhibit changes in diameter. This is accompanied by widening of the interspaces and by a decrease in the areas of cellular contact. Between contractions, there are more light cells which become arranged closer to each other and exhibit large areas of interdigitation. The significance of these observations in the mechanism of uterine contraction and retraction is discussed. Cell believed to be modified smooth muscle cells occupy the myoendometrial junction and the decidua basalis. They are irregular in shape, poor in myofilament content, and rich in other cytoplasmic organelles and form a loosely arranged layer of cells between the myometrium and the trophoblast. The possible functional significance of these cells is also discussed.     

Inhibitory effects of L-ENK on the proliferation of cultured smooth muscle cells of rats

A case of Di Guglielmo's syndrome passed through the three stages of chronic erythromyelosis, erythroleukemia and acute myeloid leukemia (AML). According to the FAB classification the subsequent stages of this syndrome were refractory anemia (RA), RA with excess of blasts (RAEB), AML-M6, AML-M2 and undifferentiated AML-MO as the end-stage disease. Light- and electronmicroscopice findings on peripheral blood and bone marrow slides showed a pronounced trilineage myelodysplastic syndrome (MDS) during the RA, RAEB, AML-M6 and M2 phases of the disease, i.e. dysplastic erythropoiesis with PAS-positive erythroblasts, agranular and hypogranular neutrophils and dysplastic megakaryocytes. It is concluded that this case of Di Guglielmo's syndrome with chronic erythromyelosis, erythroleukemia and AML appears to be a continuum of trilineage MDS, AML-M6 and M2 with dyserythropoiesis which evolved into AML-M0.     

Inhibitory action of insulin-sensitizing agents on calcium channels in smooth muscle cells from resistance arteries of guinea-pig

Herein is presented the sequence of a catfish full-length p53 cDNA obtained from a cloned B cell line cDNA library. Southern blot analyses determined that a restriction fragment linked polymorphism (RFLP) existed with PstI among outbred catfish. Western blot analyses demonstrated that, when compared to PBLs, the catfish leukocyte lines express higher levels of p53 protein. Additionally, the results of Western blot analyses and in vitro translation experiments suggest that the catfish leukocyte lines may produce truncated forms of p53 due to internal initiation.     

The effects of changing intracellular pH on calcium and potassium currents in smooth muscle cells from the guinea-pig ureter

Effect of weight training exercise and treadmill exercise on postexercise oxygen consumption. Med. Sci. Sports Exerc., Vol. 30, No. 4, pp. 518-522, 1998. To compare the effect of weight training (WT) and treadmill (TM) exercise on postexercise oxygen consumption (VO2), 15 males (mean +/- SD) age = 22.7 +/- 1.6 yr; height = 175.0 +/- 6.2 cm; mass = 82.0 +/- 14.3 kg) performed a 27-min bout of WT and a 27-min bout of TM exercise at matched rates of VO2. WT consisted of performing two circuits of eight exercises at 60% of each subject's one repetition maximum with a work/rest ratio of 45 s/60 s. Approximately 5 d after WT each subject walked or jogged on the TM at a pace that elicited an average VO2 matched with his mean value during WT. VO2 was measured continuously during exercise and the first 30 min into recovery and at 60 and 90 min into recovery. VO2 during WT (1.58 L.min-1) and TM exercise (1.55 L.min-1) were not significantly (P > 0.05) different; thus the two activities were matched for VO2. Total oxygen consumption during the first 30 min of recovery was significantly higher (P < 0.05) as a result of WT (19.0 L) compared with that during TM exercise (12.7 L). However, VO2 values at 60 (0.32 vs 0.29 L.min-1), and 90 min (0.33 vs 0.30 L.min-1) were not significantly different (P > 0.05) between WT and TM exercise, respectively. The results suggest that, during the first 30 min following exercise. WT elicits a greater elevated postexercise VO2 than TM exercise when the two activities are performed at matched VO2 and equal durations. Therefore, total energy expenditure as a consequence of WT will be underestimated if based on exercise VO2 only.