Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Primary ciliary muscle cell cultures derived from human donors (16-91 years) were established and characterized by comparing them with ciliary muscle in tissue sections using immunocytochemical and ultrastructural methods. Monoclonal antibodies against desmin, vimentin, alpha-actinin, smooth muscle (sm) specific alpha-actin and von Willebrand factor were used. In tissue sections of the ciliary body, ciliary muscle cells, vascular muscle cells, pericytes, endothelial cells and fibroblasts stain for vimentin. Both types of muscle cells and the pericytes stain for alpha-sm-actin, but only ciliary muscle cells stain for desmin. For tissue cultures, explants of the meridional and partly the reticular portion of the ciliary muscle were dissected and grown directly or after digestion of the explant with collagenase. Ten primary cell cultures with a typical hill-and-valley growth pattern similar to smooth muscle cells and two with a growth pattern similar to fibroblasts were established. All cultures could be subcultured up to the fifth passage. In fibroblast-like cultures 5-10% of the cells stained for alpha-sm-actin. Staining for desmin was not observed. In smooth muscle-like cultures, all cells stained positive for alpha-sm-actin. Desmin staining was not seen in growing non-confluent smooth muscle-like cultures. In confluent cultures, about 10% of the cells stained positive for desmin, preferentially in areas where the cells had formed hills. No culture stained for von Willebrand factor. Staining for alpha-actinin in smooth muscle-like cultures showed that the dense bands of the myofilaments were arranged in register, similar to the typical ciliary muscle cell morphology seen in tissue sections. Ultrastructurally, the smooth muscle-like cultures showed the typical morphology of cultured smooth muscle cells. We conclude that the smooth muscle-like cultures consist of ciliary muscle cells.
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PMID:Cell cultures of human ciliary muscle: growth, ultrastructural and immunocytochemical characteristics. 193 74

A spontaneously arising continuous cell line (Rb-1) derived from collagenase-elastase digested rabbit aorta has been propagated in vitro for over 100 passages. During this period, the Rb-1 cells remained spindle-shaped and formed regularly oriented parallel bundles. After Passage 50, Rb-1 cells were found to be serum-independent in their growth and reached higher saturation density than rabbit aorta smooth muscle cells. Alpha-actin and desmin filaments were detected by immunostaining in Rb-1 cells and early passage of rabbit aorta smooth muscle cells. The proportion of alpha-actin transcripts in Rb-1 cells was lower than that of transcripts for beta- and gamma-actins. The modal chromosome number was maintained at 44 between Passages 11 and 60, and two marker chromosomes were constantly present. Infection of Rb-1 cells with two strains of herpes simplex virus type 1 resulted in high titers of virus, whereas a herpes simplex virus type 2 temperature-sensitive mutant replicated only at the permissive temperature. The Rb-1 cell line could be used for the study of vascular smooth muscle cell proliferation and their interaction with viruses.
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PMID:Characterization of a continuous smooth muscle cell line derived from rabbit aorta. 268 Nov 30

Cerebral endothelium is being studied rather extensively in tissue culture, but no reports are available describing the tissue culture of cerebral microvascular smooth muscle. The present paper describes for the first time the isolation and culture of non-neoplastic mouse cerebral vascular smooth muscle. Microvessels from a dounce homogenate of mouse brain are plated onto plastic culture dishes in Dulbecco's modified Eagle media plus 20% fetal bovine serum and treated briefly with collagenase. Cells migrate from vessels and proliferate sufficiently to be transferred out of primary culture in 2 to 3 wk. Light microscopy reveals generally broad, polygonal cells that grow collectively in a "hill and valley" pattern. By transmission electron microscopy the cells possess many characteristics of smooth muscle: basal laminas, clusters of pinocytotic vesicles, and bundles of thin filaments. Several ill-defined cell-to-cell junctions are also present. Isoelectric focusing and sodium dodecyl sulfate-electrophoresis of cellular proteins on polyacrylamide gels after pulse labeling cultures with [S-35]methionine demonstrate that these cells actively synthesize a smooth-muscle-specific isoactin, alpha-actin. The identity of alpha-actin is confirmed by analysis of NH2-terminal peptides after actin digestion with trypsin and subsequent peptide cleavage with thermolysin. Both their morphology and active synthesis of alpha-actin strongly suggest that these cells are of smooth-muscle origin. Future studies of their metabolism and interactions with endothelium and astrocytes should provide a better understanding of the cerebral microcirculation.
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PMID:Cerebral microvascular smooth muscle in tissue culture. 623 74

Pulmonary arterial microvascular smooth muscle function governs many aspects of lung physiology and pathophysiology. Acutely, microvascular smooth muscle cells (SMC) modulate pulmonary vascular resistance; chronically, they contribute to vascular remodeling. Recent work has also suggested a possible immune function for pulmonary smooth muscle through cytokine-stimulated nitric oxide production. To facilitate study of the mechanisms underlying these functions, we have developed methods for isolating pulmonary arterial microvessels from the rat and culturing SMC from these vessels. The pulmonary arterial circulation was filled with a suspension of iron oxide in agar, and a subpleural tissue sample was obtained. The vessels were cleared of surrounding lung parenchyma by partial collagenase digestion, and the iron-containing arteries were separated magnetically. The diameter of the harvested arteries confirmed an intraacinar origin, and the cultured cells expressed smooth muscle isoforms of alpha-actin and myosin but did not take up acetylated low density lipoprotein. To assess a possible immune effector role for these cells, confluent monolayers were stimulated with cytokines and endotoxin. At 24 h, immunofluorescent staining for inducible nitric oxide synthase was prominent within these cells. Nitric oxide production, as measured by nitrite levels in the cell-conditioned medium, was also markedly elevated but reduced by adding NG-monomethyl-L-arginine. We conclude that rat pulmonary arterial microvascular SMC can be obtained by the iron oxide infusion method and that these cells express an inducible nitric oxide synthase after cytokine stimulation.
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PMID:Culture of pulmonary microvascular smooth muscle cells from intraacinar arteries of the rat: characterization and inducible production of nitric oxide. 751 71

Neoplastic transformation mediated by ras oncogenes is associated with deregulated expression of genes encoding, for example, various proteases, lysyl oxidase, and smooth-muscle alpha-actin. To define the role of these genes in the initiation or maintenance of the ras-transformed state, we compared their steady-state mRNA levels in two different sets of preneoplastic fibroblast lines, ras-transformed clones, and phenotypic revertants derived from them. Compared with the preneoplastic fibroblasts, the ras-transformed derivatives exhibited elevated levels of cathepsin L (major excreted protein), transin (stromelysin I, matrix metalloproteinase-3), and collagenase I (matrix metalloproteinase-1) mRNA but undetectable levels of lysyl oxidase mRNA. Partial restoration of lysyl oxidase transcription was observed in four of five phenotypic revertants derived from rat FE-8 and NIHpEJcl3 cells. The elevated levels of transin mRNA found in NIHpEJcl3 cells were diminished to the pretransformation level in interferon revertants but were not reduced in phenotypic rat FE-8 revertants expressing a high level of the ras oncoprotein. High steady-state levels of collagenase I mRNA were dependent on ras expression but were not closely associated with the transformed phenotype. High levels of cathepsin L mRNA were associated with neither high ras expression nor neoplastic transformation. The downregulation of smooth-muscle alpha-actin, characteristic of transformed cell lines, was not reversible in phenotypic revertants.
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PMID:Partial restoration of pre-transformation levels of lysyl oxidase and transin mRNAs in phenotypic ras revertants. 772 41

To explore the role of perivascular cells in angiogenesis and vasomotricity, placental cultures of perivascular cells were performed from calibrated villi excised from term placentas. Microvessels were isolated using repeated digestion of villi by collagenase-dispase and purification by Percoll gradients. Plated on Petri dishes, the microvessels became adherent to the gelatin matrix permitting to cells to proliferate. Cells were harvested and subcultured. Endothelial and pericyte cell lines were identified by phase contrast microscopy. Pericyte number predominated rapidly, the endothelial cells remaining visible. After seven days, cells started to cluster, thus piled up and built numerous nodules. Medium-size oval endothelial cells were stained by anti-von Willebrand factor and anti-IgG coupled to fluorescein. Large cells with irregular border reacted to smooth muscle anti-alpha-actin and anti-IgG coupled to fluorescein. There was no cross-reaction of these two cell types with the antibodies. In contrast, nodules were stained by both immunostainings. Endothelial cells reacting to von Willebrand factor antibody were frequently associated to the nodule. The isolation of microvessels from the human placenta described in this study allowed the establishment of cultures of endothelial cells and pericytes that show: i) rapid predominance of pericytes over endothelial cells, ii) formation of nodules, iii) participation of endothelial cells and pericytes to nodules formation.
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PMID:Mixed culture of pericytes and endothelial cells from fetal microvessels of the human placenta. 778 33

Cells from the muscular layer of neonatal (3-day-old) rabbit urinary bladders were dissociated with collagenase, and cultured in M199 supplemented with 10% fetal bovine serum and antibiotic-antimycotic. Cells in culture were of two types: long and short. The short cells were thick and spindle-shaped, and the long cells were flat and elongated. The long cells can be about 15 times longer than the short cells. The short cells do not divide, but the long cells divide readily. Expressions of smooth muscle and non-muscle myosins, alpha-smooth muscle actin, vimentin, and h-caldesmon were determined by immuno-fluorescence microscopy using specific antibodies. Both types of cells react strongly with antibodies against smooth and non-muscle myosins. Unlike the short cells, the long cells also contain alpha-actin and vimentin. The expression of h-caldesmon was very weak in both cell types. Also, cells dissociated from the smooth muscle layers of adult (6-month-old) rabbit bladder were cultured under the same conditions as the cells from the neonatal bladders to see if the heterogeneity of smooth muscle cells, exhibited by cells from neonatal rabbits, is also shown by cells from adult bladder. Two types of cells were also identified. The cells were then fixed and examined with the same panel of antibodies that we used for the neonatal cells. The long cells from adult bladder muscle express similar proteins to those in the neonatal long cells, and the short cells were stained positively with smooth muscle myosin, non-muscle myosin, alpha-smooth muscle actin, and lightly with caldesmon. Although the absence of vimentin in the short cells from adults is similar to that from neonatal, the strong expression of alpha-actin in the adult short cells is unlike the short cells from neonatal rabbits, in which their expression is barely detectable.
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PMID:Identification of two types of smooth muscle cells from rabbit urinary bladder. 870 36

Recent advances in biomedical sciences have led to the development of various methods for the evaluation of the physiopathology of respiratory diseases. This study reports morphologic and functional features of cells isolated by a new method from bronchial biopsies of normal and asthmatic subjects. Both epithelial and fibroblastic cells were isolated from the same biopsies using collagenase. The cells were cultured for several passages and stored frozen. Two selective culture media were used in order to obtain pure epithelial and fibroblastic cell populations. Immunofluorescence analysis of intermediate filaments, keratins, and vimentin confirmed the type of the isolated cells. The proportions of alpha-actin-expressing cells varied among the fibroblastic cell populations isolated from normal and asthmatic subjects. Interestingly, the population containing high numbers of alpha-actin-expressing cells and presenting the fastest collagen contraction kinetic was isolated from bronchial biopsies of an asthmatic subject. Moreover, the fibroblastic cells that showed the best contractile properties 24 h after their seeding in floating collagen gels were isolated from bronchial biopsies of asthmatic patients having PC20 values below 1 mg/ml. On the basis of these data, we propose a new approach to isolate, culture and characterize human bronchial cells in vitro.
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PMID:Morphologic and functional properties of bronchial cells isolated from normal and asthmatic subjects. 881 Jun 34

Calcium signaling mechanisms were examined in vessel segments and dispersed single smooth muscle cells (SMC) of interlobular arteries and afferent arterioles (< 50 microns diameter) from the rat kidney. These resistance vessels were isolated from rat kidneys, using an iron oxide-sieving technique with subsequent collagenase digestion. Individual cells were identified by their characteristic oval appearance and positive staining for smooth muscle-specific alpha-actin and heavy chain myosin SM-1 and SM-2. Cytosolic calcium concentration ([Ca2+]i) was measured using fura 2 ratiometric fluorescence at 340 and 380 nm wavelength with a microscope-based photometer. Angiotensin II (ANG II) and arginine vasopressin (AVP), at concentrations of 10(-10)-10(-6) M, produced dose-dependent increases in [Ca2+]i; maximum increases were 221 +/- 49 nM for ANG II and 237 +/- 49 nM for AVP. The temporal response patterns for both agonists were characterized by a square-shaped, immediate step increase in [Ca2+]i to a near maximum level that was maintained through the recording period of 150-200 s. Responses of individual dispersed SMC and short vessel segments were similar. Losartan antagonized the action of ANG II, indicating mediation by AT1 receptors on preglomerular arteriolar SMC. The V1-selective antagonist [d(CH2)5Tyr(Me)2Tyr(NH2)9]AVP completely inhibited AVP-induced [Ca2+]i changes. The importance of calcium entry in hormone-induced changes in [Ca2+]i was demonstrated by the finding that neither ANG II nor AVP elicited a [Ca2+]i response in media rendered nominally calcium free by addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Calcium entry occurred primarily through L-type, voltage-gated calcium channels as the dihydropyridine, nifedipine, completely prevented or reversed [Ca2+]i changes normally elicited by either hormone. Our results provide new information about the similarity of calcium signaling in single SMC and short segments freshly isolated from renal interlobular arteries and afferent arterioles. The observations indicate that AT1 and V1 receptors are coupled to signal transduction pathways leading to rapid changes in [Ca2+]i. Calcium mobilization appears to play a minor to nonexistent role under the experimental conditions. The predominant mechanism involves calcium entry through dihydropyridine-sensitive, voltage-gated calcium channels in single SMC from these resistance vessels.
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PMID:ANG II and vasopressin stimulate calcium entry in dispersed smooth muscle cells of preglomerular arterioles. 953 Feb 66

Single smooth muscle cells isolated from guinea pig ileum using collagenase and papain produce contractile response to muscarinic agents, while the cultured cells do not. Using fluo-3/AM and a confocal laser scanning fluorescence microscope, it was observed that carbachol, a muscarinic agent, caused an increase in the intracellular Ca2+ of both single and cultured cells. SDS-PAGE and Western Blot analyses revealed the expression of myosin heavy chain isoforms of SM1 (204 kDa) and SM2 (200 kDa) in single smooth muscle cells, and non muscle isoform (196 kDa) of myosin heavy chain only in the cultured cells. With respect to actin isoforms, alpha-actin was predominant in single cells and beta-actin was major in the cultured cells. Two types of tropomyosin monomer, 39 kDa and 41 kDa, were detected in single cells, while the 41 kDa monomer was lost in cultured cells. These differences in contractile protein profiles between single and cultured cells were collaborated with the observation of cells using immunofluorescence microscope with responsible antibodies to isoforms of myosin heavy chain, actin and tropomyosin. These results suggest that the loss of contractility in cultured smooth muscle cells is profoundly related to changes in contractile protein profiles from smooth muscle type to non muscle type.
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PMID:Contractile protein isoforms of single and cultured smooth muscle cells from guinea pig ileum. 1037 28


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