Biomedical subjects

A Draeger

Publications and source records attributed to A Draeger.

At least 19 recordsLinked to original sources

PubMed2006

Epithelial transformations in the establishment of the blood-gas barrier in the developing chick embryo lung.

The tall epithelium of the developing chick embryo lung is converted to a squamous one, which participates in formation of the thin blood-gas barrier. We show that this conversion occurred through processes resembling exocrine secretion. Initially, cells formed intraluminal protrusions (aposomes), and then transcellular double membranes were established. Gaps between the membranes opened, thus, severing the aposome from the cell. Alternatively, aposomes were squeezed out by adjacent cells or were spontaneously constricted and extruded. As a third mechanism, formation and fusion of severed vesicles or vacuoles below the aposome and their fusion with the apicolateral plasma membrane resulted in severing of the aposome. The atria started to form by progressive epithelial attenuation and subsequent invasion of the surrounding mesenchyme at regions delineated by subepithelial alpha-smooth muscle actin-positive cells. Further epithelial attenuation was achieved by vacuolation; rupture of such vacuoles with resultant numerous microfolds and microvilli, which were abscised to accomplish a smooth squamous epithelium just before hatching.

Actins↗

PubMed2006

Statin therapy induces ultrastructural damage in skeletal muscle in patients without myalgia.

Muscle pain and weakness are frequent complaints in patients receiving 3-hydroxymethylglutaryl coenzymeA (HMG CoA) reductase inhibitors (statins). Many patients with myalgia have creatine kinase levels that are either normal or only marginally elevated, and no obvious structural defects have been reported in patients with myalgia only. To investigate further the mechanism that mediates statin-induced skeletal muscle damage, skeletal muscle biopsies from statin-treated and non-statin-treated patients were examined using both electron microscopy and biochemical approaches. The present paper reports clear evidence of skeletal muscle damage in statin-treated patients, despite their being asymptomatic. Though the degree of overall damage is slight, it has a characteristic pattern that includes breakdown of the T-tubular system and subsarcolemmal rupture. These characteristic structural abnormalities observed in the statin-treated patients were reproduced by extraction of cholesterol from skeletal muscle fibres in vitro. These findings support the hypothesis that statin-induced cholesterol lowering per se contributes to myocyte damage and suggest further that it is the specific lipid/protein organization of the skeletal muscle cell itself that renders it particularly vulnerable.

Adult↗

PubMed2006

Regulation of ecto-5'-nucleotidase activity via Ca2+-dependent, annexin 2-mediated membrane rearrangement?

The spatial segregation of the plasma membrane plays a prominent role in distinguishing and sorting a large number of signals a cell receives simultaneously. The plasma membrane comprises regions known as lipid rafts, which serve as signal-transduction hubs and platforms for sorting membrane-associated proteins. Ca(2+)-binding proteins of the annexin family have been ascribed a role in the regulation of raft dynamics. Glycosylphosphatidylinositol-anchored 5'-nucleotidase is an extracellular, raft-associated enzyme responsible for conversion of extracellular ATP into adenosine. Our results point to a regulation of ecto-5'-nucleotidase activity by Ca(2+)-dependent, annexin-mediated stabilization of membrane rafts.

5'-Nucleotidase↗

PubMed2004

Increased cholesterol decreases uterine activity: functional effects of cholesterol alteration in pregnant rat myometrium.

Uterine quiescence is essential for successful pregnancy. Cholesterol and triglycerides are markedly increased in pregnancy. Cholesterol is enriched in microdomains of the plasma membrane known as rafts and caveolae. Both lipid rafts and caveolae have been implicated in cellular signaling cascades. The purpose of this work was to investigate whether manipulation of cholesterol content alters uterine contractility. Late pregnancy (19-21 days) rats were humanely euthanized and strips of longitudinal myometrium were then dissected. Force and Ca(2+) measurements were simultaneously recorded and cholesterol increased by the addition of 5 mg/ml cholesterol or 0.25 mg/ml low-density lipoproteins (LDLs) or reduced by 2% methyl-beta-cyclodextrin (MCD) or 2 U/ml cholesterol oxidase addition to the perfusate. Both LDLs and cholesterol profoundly inhibited spontaneous uterine force production and associated Ca(2+) transients; frequency, amplitude, and duration of contraction were all significantly reduced compared with preceding control contractions. Force and Ca(2+) were also reduced by cholesterol when 1 nM oxytocin was used to stimulate the myometrium. Uterine activity was significantly increased by cholesterol extraction with MCD or cholesterol oxidase treatment. Electron microscopy confirmed the lipid raft disrupting effect of MCD, as formerly electron microscopy-visible caveolae in the myometrial cell membrane all but disappeared after MCD treatment. These data show that uterine smooth muscle cell cholesterol content is critically important for functional activity. A novel finding of our study is that cholesterol is inhibitory for force generation. It may be one of the mechanisms operating to maintain uterine quiescence throughout gestation and may also contribute to difficulties in labor suffered by obese women.

Animals↗

PubMed2004

Membrane cholesterol regulates smooth muscle phasic contraction.

The regulation of contractile activity in smooth muscle cells involves rapid discrimination and processing of a multitude of simultaneous signals impinging on the membrane before an integrated functional response can be generated. The sarcolemma of smooth muscle cells is segregated into caveolar regions-largely identical with cholesterol-rich membrane rafts-and actin-attachment sites, localized in non-raft, glycerophospholipid regions. Here we demonstrate that selective extraction of cholesterol abolishes membrane segregation and disassembles caveolae. Simultaneous measurements of force and [Ca2+]i in rat ureters demonstrated that extraction of cholesterol resulted in inhibition of both force and intracellular Ca2+ signals. Considering the major structural reorganization of cholesterol-depleted sarcolemma, it is intriguing to note that decreased levels of membrane cholesterol are accompanied by a highly specific inhibition of phasic, but not tonic contractions. This implies that signalling cascades that ultimately lead to either phasic or tonic response may be spatially segregated in the plane of the sarcolemma. Replenishment of cholesterol restores normal contractile behavior. In addition, the tissue function is re-established by inhibiting the large-conductance K(+)-channel. Sucrose gradient ultracentrifugation in combination with Western blotting analysis demonstrates that its alpha-subunit is associated with detergent-resistant membranes, suggesting that the channel might be localized within the membrane rafts in vivo. These findings are important in understanding the complex signalling pathways in smooth muscle and conditions such as premature labor and hypertension.

Animals↗

PubMed2003

Membrane segregation and downregulation of raft markers during sarcolemmal differentiation in skeletal muscle cells.

Muscle contraction implies flexibility in combination with force resistance and requires a high degree of sarcolemmal organization. Smooth muscle cells differentiate largely from mesenchymal precursor cells and gradually assume a highly periodic sarcolemmal organization. Skeletal muscle undergoes an even more striking differentiation programme, leading to cell fusion and alignment into myofibrils. The lipid bilayer of each cell type is further segregated into raft and non-raft microdomains of distinct lipid composition. Considering the extent of developmental rearrangement in skeletal muscle, we investigated sarcolemmal microdomain organization in skeletal and smooth muscle cells. The rafts in both muscle types are characterized by marker proteins belonging to the annexin family which localize to the inner membrane leaflet, as well as glycosyl-phosphatidyl-inositol (GPI)-anchored enzymes attached to the outer leaflet. We demonstrate that the profound structural rearrangements that occur during skeletal muscle maturation coincide with a striking decrease in membrane lipid segregation, downregulation of annexins 2 and 6, and a significant decrease in raft-associated 5'-nucleotidase activity. The relative paucity of lipid rafts in mature skeletal in contrast to smooth muscle suggests that the organization of sarcolemmal microdomains contributes to the muscle-specific differences in stimulatory responses and contractile properties.

Annexins↗

PubMed2003

The expression levels of three raft-associated molecules in cultivated vascular cells are dependent on culture conditions.

Relaying a signal across the plasma membrane requires functional connections between the partner molecules. Membrane microdomains or lipid rafts provide an environment in which such specific interactions can take place. The integrity of these sites is often taken for granted when signalling pathways are investigated in cell culture. However, it is well known that smooth muscle and endothelial cells undergo cytoskeletal rearrangements during monolayer culturing. Likewise affected--and with potentially important consequences for signalling events--is the organization of the plasma membrane. The expression levels of three raft markers were massively upregulated, and raft-associated 5'-nucleotidase activity increased in conventional monolayer cultures as compared with a spheroidal coculture model, shown to promote the differentiation of endothelial cells. Our data point to a shift of raft components in monolayer cultures and demonstrate potential advantages of the spheroid coculture system for investigation of raft-mediated signalling events in endothelial cells.

5'-Nucleotidase↗

PubMed2002

Stress fibres-a Ca2+ -independent store for annexins?

Annexins belong to a family of lipid-binding proteins that are implicated in membrane organization. Several members are capable of binding to actin and, in smooth muscle cells, annexin 6 is known to form a Ca(2+)-dependent, plasmalemmal complex with actin filaments. Annexins can also associate with F-actin containing stress fibres within cultured smooth muscle cells or fibroblasts in a Ca(2+)-independent manner. Depolymerization of stress-fibre systems with cytochalasin D leads to the translocation of actin-bound annexin 2 from the cytoplasm to the plasma membrane at high intracellular levels of Ca(2+). This type of Ca(2+)-dependent annexin mobility is observed only in cells of mesenchymal phenotype, which have a well-developed stress-fibre system; not in epithelial cells.

Annexin A2↗

PubMed2001

Diacylglycerol kinase theta is translocated and phosphoinositide 3-kinase-dependently activated by noradrenaline but not angiotensin II in intact small arteries.

Diacylglycerol (DG) kinase (DGK) phosphorylates the lipid second messenger DG to phosphatidic acid. We reported previously that noradrenaline (NA), but not angiotensin II (AII), increases membrane-associated DGK activity in rat small arteries [Ohanian and Heagerty (1994) Biochem. J. 300, 51-56]. Here, we have identified this DGK activity as DGKtheta, present in both smooth muscle and endothelial cells of these small vessels. Subcellular fractionation of artery homogenates revealed that DGKtheta was present in nuclear, plasma membrane (and/or Golgi) and cytosolic fractions. Upon NA stimulation, DGKtheta translocated towards the membrane and cytosol (155 and 153% increases relative to the control, respectively) at 30 s, followed by a return to near-basal levels at 5 min; AII was without effect. Translocation to the membrane was to both Triton-soluble and -insoluble fractions. NA, but not AII, transiently increased DGKtheta activity in immunoprecipitates (126% at 60 s). Membrane translocation and DGKtheta activation were regulated differently: NA-induced DGKtheta activation, but not translocation, was dependent on transient activation of phosphoinositide 3-kinase (PI 3-K). In addition, DGK activity co-immunoprecipitated with protein kinase B, a downstream effector of PI 3-K, and was increased greatly by NA stimulation. The rapid and agonist-specific activation of DGKtheta suggests that this pathway may have a physiological role in vascular smooth-muscle responses.

Androstadienes↗

PubMed2001

MMP-19: cellular localization of a novel metalloproteinase within normal breast tissue and mammary gland tumours.

Matrix metalloproteinases (MMPs) are instrumental in promoting and facilitating the spread of malignant diseases and in the de novo formation of blood vessels. This study has mapped the immunoreactivity of a novel, angiogenesis-related metalloproteinase--MMP-19--in normal breast tissue and in benign and malignant breast lesions and compared this pattern of expression with that of MMP-2. In the normal resting mammary gland, MMP-19 was strongly expressed in the myoepithelial layer of the ductal system; the alveolar and ductal epithelia displayed considerable, but lobule-specific, variations in labelling intensity. MMP-19 was also present within the smooth muscle and endothelial layers of large and medium-sized blood vessels, as well as within capillary walls. In benign lesions, all tumour cells and their surrounding vasculature were uniformly and strongly immunoreactive for MMP-19. Progression towards an invasive phenotype and neoplastic dedifferentiation led to the disappearance of MMP-19 from tumour cells and blood vessels and a concomitant rise in the levels of MMP-2. In vitro experiments conducted with isolated smooth muscle cells cultivated on a solid substratum, or within the interstices of a collagen matrix, indicated that the expression of MMP-19 is influenced by the architecture of the surrounding extracellular matrix.

Adolescent↗

PubMed2000

Annexins in cell membrane dynamics. Ca(2+)-regulated association of lipid microdomains.

The sarcolemma of smooth muscle cells is composed of alternating stiff actin-binding, and flexible caveolar domains. In addition to these stable macrodomains, the plasma membrane contains dynamic glycosphingolipid- and cholesterol-enriched microdomains, which act as sorting posts for specific proteins and are involved in membrane trafficking and signal transduction. We demonstrate that these lipid rafts are neither periodically organized nor exclusively confined to the actin attachment sites or caveolar regions. Changes in the Ca(2+) concentration that are affected during smooth muscle contraction lead to important structural rearrangements within the sarcolemma, which can be attributed to members of the annexin protein family. We show that the associations of annexins II, V, and VI with smooth muscle microsomal membranes exhibit a high degree of Ca(2+) sensitivity, and that the extraction of annexins II and VI by detergent is prevented by elevated Ca(2+) concentrations. Annexin VI participates in the formation of a reversible, membrane-cytoskeleton complex (Babiychuk, E.B., R.J. Palstra, J. Schaller, U. Kämpfer, and A. Draeger. 1999. J. Biol. Chem. 274:35191-35195). Annexin II promotes the Ca(2+)-dependent association of lipid raft microdomains, whereas annexin V interacts with glycerophospholipid microcompartments. These interactions bring about a new configuration of membrane-bound constituents, with potentially important consequences for signaling events and Ca(2+) flux.

Annexin A2↗

PubMed2000

[Contribution to the differentiation of cross-reacting antibodies in brucellosis serology--1. Reactions with various Yersinia serotypes and antibody avidity].

Brucella and Yersinia enterocolitca O:9 (Y.e.O:9) have antigenic determinants in common causing cross-reactions which interfere strongly in Brucella serology. Hoffmann (1988) observed cross-reactivity between Y.e.O:9, Y.e.O:5, and Y.e.O:6 and used an ELISA for the differentiation between Brucella- and Yersinia antibodies since Y.e.O:5 and Y.e.O:6 produced only low-level cross-reactions with Brucella antigen. The observations of Hoffmann were confirmed and led to the construction of an ELISA with 4 antigens. Sera from natural outbreaks of brucellosis and from artificial infections with Brucella and Y.e.O:9 were properly identifiable by this system. Furthermore, the test material has been used to measure the antibody avidity by a specially designed ELISA. This test produced a further divergence between specific and non-specific reactions.

Animals↗

PubMed2000

Smooth muscle actomyosin promotes Ca2+-dependent interactions between annexin VI and detergent-insoluble glycosphingolipid-enriched membrane domains.

The mechanical link coupling cytoskeletal and contractile proteins to the sarcolemma of smooth muscle cells is essential for transmitting tension from the cell's interior to exterior. In addition to the well-characterized actin-integrin associations present in adhaerens junctions, our recent work has postulated the existence of a reversible annexin-dependent membrane-cytoskeleton complex, forged in response to a rise in intracellular Ca2+ concentration following smooth muscle cell stimulation (Babiychuk et al., J. Biol Chem. 1999, 274, 35191-35195). Detailed biochemical characterization of the interactions responsible for the formation of this complex revealed that annexins II and VI interact with actomyosin, or detergent-insoluble glycosphingolipid-enriched membrane domains (rafts) purified from smooth muscle, in a concentration- and Ca2+-dependent manner. Annexin II interacted with lipid rafts with high Ca2+-sensitivity, while for annexin VI this interaction required non-physiologically high concentrations of free Ca2+. However, the Ca2+-sensitivity of the latter interaction strongly increased in the presence of purified smooth muscle actomyosin. The detailed biochemical analysis of the interactions occurring between annexin II, annexin VI, actomyosin and rafts suggests that annexins regulate sarcolemmal organization during smooth muscle cell contraction.

Actomyosin↗

PubMed1999

Sorting of murine vascular smooth muscle cells during wound healing in the chicken chorioallantoic membrane.

The vascular wall is built up of a heterogeneous population of smooth muscle cells, which exhibit not only morphological distinctions but also important differences in the composition of their structural and contractile proteins. "Epithelioid" smooth muscle cells correspond to an intimal-like type and display features associated with immaturity, whereas "spindle-shaped" cells closely resemble the more typical medial smooth muscle population. We have investigated the integration of these two cell types into the vascular architecture of an in vivo wound-healing model. Stably transfected with the beta-galactosidase gene, intima- and media-like cells were injected intravenously into the chicken chorioallantoic membrane, within which superficial foci of granulation tissue had been created by thermal or chemical injury. At 24 to 72 h after injection, cells had honed in on the lesion sites and were observed in juxtaposition to the endothelial lining of the capillaries. They began to deposit laminin, thereby indicating an impending role in the formation of the vascular wall. Intima- and media-like smooth muscle cells did not differ in their capacity to associate with capillaries, and, in so doing, their biochemical lineage characteristics became indistinguishable from one another. However, intima-like cells also penetrated the adventitial and medial layers of arteries. These findings reveal vascular smooth muscle cells to possess an extraordinary degree of plasticity, being able to adapt flexibly to changes in functional demands.

Allantois↗

PubMed1999

Annexin VI participates in the formation of a reversible, membrane-cytoskeleton complex in smooth muscle cells.

The plasmalemma of smooth muscle cells is periodically banded. This arrangement ensures efficient transmission of contractile activity, via the firm, actin-anchoring regions, while the more elastic caveolae-containing "hinge" regions facilitate rapid cellular adaptation to changes in cell length. Since cellular mechanics are undoubtedly regulated by components of the membrane and cytoskeleton, we have investigated the potential role played by annexins (a family of phospholipid- and actin-binding, Ca(2+)-regulated proteins) in regulating sarcolemmal organization. Stimulation of smooth muscle cells elicited a relocation of annexin VI from the cytoplasm to the plasmalemma. In smooth, but not in striated muscle extracts, annexins II and VI coprecipitated with actomyosin and the caveolar fraction of the sarcolemma at elevated Ca(2+) concentrations. Recombination of actomyosin, annexins, and caveolar lipids in the presence of Ca(2+) led to formation of a structured precipitate. Participation of all 3 components was required, indicating that a Ca(2+)-dependent, cytoskeleton-membrane complex had been generated. This association, which occurred at physiological Ca(2+) concentrations, corroborates our biochemical fractionation and immunohistochemical findings and suggests that annexins play a role in regulating sarcolemmal organization during smooth muscle contraction.

Actomyosin↗

PubMed1999

Intima-like smooth muscle cells: developmental link between endothelium and media?

The presence of non-contractile smooth muscle cells within the arterial wall raises questions as to their origin and function. These cells abound within the aortae of murine and porcine neonates, but are also present within the intimal and medial layers of adult arteries. They are largely devoid of smooth muscle-associated proteins and manifest an epithelioid form. Their morphological resemblance to endothelial cells prompted us to explore this potential relationship and to investigate their angiogenic properties in three-dimensional collagen gels. Using well-characterized smooth muscle cell lines, displaying either the intima-like (epithelioid) or media-like (spindle-shaped) morphology, we were able to show that intima-like cells share several features in common with endothelial ones and can transform into a media-like phenotype, whereby they irreversibly lose their characteristic pattern of protein expression. Intima-like, but not media-like, vascular smooth muscle cells are capable of forming capillary tubes, and, in co-cultures, can induce media-like ones to participate in this process. Such capillaries consist of a randomly-organized, mixed population of endothelial cells with intima-like or media-like smooth muscle ones. The functional significance of this diversity in smooth muscle cell type is not well understood, but phenotypic plasticity could conceivably figure as an important adaptive response to changes in the local environment.

Animals↗

PubMed1999

[The effect of annexins on the contractile activity of the smooth muscles in the rat portal vein].

This study examines the effects of the family of calcium-dependent phospholipids binding proteins (annexins) on the contractile properties of rat portal vein. We developed the new method of annexin's purification from pig stomach muscle. It found that in nanomolar concentrations annexins caused dose-dependent effects on phasic and tonic component of rat portal vein contractile activity. The frequency of spontaneous contractions was increased whereas the amplitude was decreased. At the same time we observed the rise of basal tone level of contractions. We suggested that annexins may change the contractile properties of vascular smooth muscle.

Animals↗

PubMed1998

Expression of smooth muscle markers in the developing murine lung: potential contractile properties and lineal descent.

Contractile cells in the mammalian lung develop in close association with the outgrowing stem bronchi. Fully differentiated smooth muscle cells are typically found in proximal regions, residing in the substantial muscular walls of the major airways and blood vessels. More distally, cells expressing markers of differentiated smooth muscle cells to a variable degree, and which may therefore possess contractile properties, are to be found scattered around the interstitium. We have investigated the temporal and spatial distribution of smooth muscle lineage markers (smooth muscle myosin mRNA) and of those indicative of contractile function (metavinculin mRNA) in the murine lung. In the smooth muscle layers of the bronchi and major blood vessels, these genes are expressed from the onset of pulmonary budding, concurrently with the appearance of alpha-smooth muscle actin and calponin proteins. During fetal development, smooth muscle-associated genes and proteins are restricted to this committed smooth muscle population. The first signs of myofibroblast or pericyte differentiation become manifest perinatally, when their expression of alpha-smooth muscle actin escalates. In the adult lung, such cells may be readily pin-pointed by their positive reaction for metavinculin mRNA, but, at maturity, they do not always coexpress alpha-smooth muscle actin.

Actins↗