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Biomedical subjects

A E Warner

Publications and source records attributed to A E Warner.

At least 37 records · Page 2Linked to original sources
PubMed1988

The control of chick myoblast fusion by ion channels operated by prostaglandins and acetylcholine.

Chick myoblast fusion in culture was investigated using prostanoid synthesis inhibitors to delay spontaneous fusion. During this delay myoblast fusion could be induced by prostaglandin E1 (PGE1), by raising extracellular potassium and by addition of carbachol. Carbachol-induced fusion, but not PGE-induced fusion, was prevented by the acetylcholine receptor blocker alpha-bungarotoxin. Fusion induced by any of these agents was prevented by the Ca channel blockers lanthanum and D600. The threshold for potassium-induced fusion was 7-8 mM; maximal fusion occurred at 16-20 mM. Low extracellular potassium inhibited spontaneous fusion. Intracellular potassium in fusion competent myoblasts was 101 m-moles/l cell. Calcium flux measurements demonstrated that high potassium increased calcium permeability in fusion-competent myoblasts. A 30-s exposure to high potassium or PGE1 was sufficient to initiate myoblast fusion. Anion-exchange inhibitors (SITS and DIDS) delayed spontaneous myoblast fusion and blocked fusion induced by PGE1 but not carbachol. Blocking the acetylcholine receptor shifted the dose-response relation for PGE-induced fusion to higher concentrations. PGE1-induced fusion required chloride ions; carbachol-induced fusion required sodium ions. Provided calcium channels were available, potassium always induced fusion. We conclude that myoblasts possess at least three, independent pathways, each of which can initiate myoblast fusion and that the PGE-activated pathway and the acetylcholine receptor-activated pathway act synergistically. We suggest that fusion competent myoblasts have a high resting membrane potential and that fusion is controlled by depolarization initiated directly (potassium), by an increase in permeability to chloride ions (PGE), or by activation of the acetylcholine receptor (carbachol); depolarization triggers a rise in calcium permeability. The consequent increase in intracellular calcium initiates myoblast fusion.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1988

A role for acetylcholine receptors in the fusion of chick myoblasts.

The role of acetylcholine receptors in the control of chick myoblast fusion in culture has been explored. Spontaneous fusion of myoblasts was inhibited by the nicotinic acetylcholine receptor antagonists alpha-bungarotoxin, Naja naja toxin and monoclonal antibody mcAb 5.5. The muscarinic antagonists QNB and n-methyl scopolamine were without effect. Atropine had no effect below 1 microM, where it blocks muscarinic receptors; at higher concentrations, when it blocks nicotinic receptors also, atropine inhibited myoblast fusion. The inhibitions imposed by acetylcholine receptor antagonists lasted for approximately 12 h; fusion stimulated by other endogenous substances then took over. The inhibition was limited to myoblast fusion. The increases in cell number, DNA content, the level of creatine phosphokinase activity (both total and muscle-specific isozyme) and the appearance of heavy chain myosin, which accompany muscle differentiation, followed a normal time course. Pre-fusion myoblasts, fusing myoblasts, and young myotubes specifically bound labeled alpha-bungarotoxin, indicating the presence of acetylcholine receptors. The nicotinic acetylcholine receptor agonist, carbachol, induced uptake of [14C]Guanidinium through the acetylcholine receptor. Myoblasts, aligned myoblasts and young myotubes expressed the synthetic enzyme Choline acetyltransferase and stained positively with antibodies against acetylcholine. The appearance of ChAT activity in myogenic cultures was prevented by treatment with BUDR; nonmyogenic cells in the cultures expressed ChAT at a level which was too low to account for the activity in myogenic cultures. We conclude that activation of the nicotinic acetylcholine receptor is part of the mechanism controlling spontaneous myoblast fusion and that myoblasts synthesize an endogenous, fusion-inducing agent that activates the nicotinic ACh receptor.

Animals↗
PubMed1988

Pulmonary removal of circulating endotoxin results in acute lung injury in sheep.

Endotoxemia has often been associated with the development of the adult respiratory distress syndrome. Our previous studies have shown that sheep, a popular animal model for adult respiratory distress syndrome, have abundant resident pulmonary intravascular macrophages that rapidly remove inorganic particles and live bacteria from the circulating blood. In this study, we examine the fate of circulating endotoxin in sheep and correlated the site of uptake with early morphologic evidence of tissue injury. Mature sheep and rats received intravenous 125I-labeled lipopolysaccharide (LPS). The dose was 0.8 microgram/kg in sheep and 17.0 micrograms/kg in rats. 125I-LPS clearance from the blood was assayed by gamma-counting of blood samples drawn over 1 hour and was correlated with circulating leukocyte numbers. The distribution of 125I-LPS was determined by gamma-counting of samples of major organs and tissues at time of necropsy. Lungs and liver were examined morphologically in both species. The half-life of circulating 125I-LPS was 2.38 minutes in sheep, and 12.39 minutes in rats. The endotoxin content of the lungs after injection was 77.58% of the total recovered dose in sheep, but only 2.02% in rats. Neutrophil margination occurred in the lungs of both species. In sheep, almost 25 minutes elapsed before peripheral neutrophil numbers decreased by 50%, much longer than the time required for LPS sequestration in the lungs. Rapid LPS uptake by the sheep lungs was associated with early (10-minute) ultrastructural changes including signs of pulmonary intravascular macrophage activation and microvascular congestion. By 60 minutes, many capillaries were occluded with neutrophils, platelets, and fibrin. There was interstitial edema, and endothelial cells showed evidence of severe injury. We conclude that in contrast to the rat, the sheep clears circulating endotoxin more rapidly and preferentially sequesters it in the lungs. Subsequent release of mediators by activated pulmonary intravascular macrophages may then lead to influx of other inflammatory cells and cascading injury.

Animals↗
PubMed1987

Gap junctional communication and compaction during preimplantation stages of mouse development.

The ability of gap junction antibodies to block dye transfer and electrical coupling was examined in the compacted 8-cell mouse zygote. In control zygotes, Lucifer yellow injected into 1 cell transferred to the rest of the embryo. When antibodies raised against the major protein extracted from gap junctions were co-injected with Lucifer yellow, dye transfer failed in 86% of the zygotes tested and electrical coupling was almost completely inhibited. Subsequently, the antibody-containing cells were extruded. When the antibodies were injected into 1 cell at the 2-cell stage, 82% of the zygotes divided normally to the 8-cell stage. Cells containing gap junction antibodies were uncompacted, but continued to divide. We conclude that these antibodies inhibit gap junctional communication in the early mouse zygote and that communication through gap junctions may be involved in the maintenance of compaction.

Animals↗
PubMed1987

The use of antibodies to gap junction protein to explore the role of gap junctional communication during development.

Antibodies raised against the major 27 kDa protein electrophoretically eluted from isolated gap junctions and affinity purified against the antigen have been used to explore the role of communication through gap junctions in the early amphibian and mouse embryos. In both species, injection of the antibodies into one cell completely blocks both dye transfer and electrical coupling between cells connected by gap junctions. In the amphibian embryo the generation of a communication-incompetent clone of cells leads to patterning defects in the region derived from the antibody-injected cell. In the mouse embryo, blocking cell-to-cell communication leads to decompaction of the communication-incompetent cells. The possible significance of these findings in relation to development in general and to the organization of the first transporting epithelia to appear during development is discussed.

Animals↗
PubMed1987

Uptake of bloodborne bacteria by pulmonary intravascular macrophages and consequent inflammatory responses in sheep.

Bacteria are primarily removed from the bloodstream of most species by mononuclear phagocytic cells in the liver and spleen. We have recently described large numbers of pulmonary intravascular macrophages in several ruminant species that also remove inert particles from the bloodstream. To determine the role of these cells in removal of bacteria from the bloodstream, sheep and rats were injected intravenously with a single dose of Pseudomonas aeruginosa, and kinetics of clearance, relative uptake among organs, and morphologic changes in the lungs and liver 1 h after injection were compared. In rats, bacteria were removed primarily by the liver, and pathologic changes were found in hepatic sinusoids but not in the lungs. However, in sheep, bacteria were removed primarily by the lungs, and pulmonary capillaries became filled with neutrophils, platelets, and fibrin deposits. We propose that uptake of bacteria by pulmonary intravascular macrophages and subsequent release of inflammatory mediators are central to the pathologic changes produced in ruminant models of sepsis-induced adult respiratory distress syndrome.

Animals↗
PubMed1987

Lithium inhibits morphogenesis of the nervous system but not neuronal differentiation in Xenopus laevis.

Xenopus embryos treated with 100 mM-lithium from the 2- to 4-cell stage to the early blastula stage (4h) failed to neurulate and developed without a discernible anteroposterior axis. The internal structure of defective embryos was grossly disorganized, but immunohistochemical staining with cell-type-specific antibodies revealed differentiated nerve and muscle cells. Quantitative assay in tissue cultures from control and acutely abnormal lithium-treated embryos showed that neural differentiation was enhanced and muscle differentiation unaffected. The embryos took up about 0.5 mM-lithium at threshold, maximal effects resulted at 2-3 mM. Most of the lithium was extruded from the cells into the blastocoel fluid, where lithium reached 17 mM. The threshold intracellular concentration was about 150 microM. Lithium uptake rose steeply as the osmotic/ionic strength of the bathing medium increased. Sodium, potassium and lithium were equally able to increase the permeability of the embryo. However, sodium ions enhanced, while potassium ions interfered with, the uptake of lithium. Treatment with lithium at progressively later stages reduced the developmental defects and neural differentiation returned to normal levels. The uptake of lithium did not decline concomitantly. We conclude that lithium does not inhibit neural induction, but interferes with dorsal patterning. The sensitivity of the embryo to lithium is determined by developmental stage. The very low, effective intracellular concentrations may be important in understanding the mechanism of lithium-generated defects.

Animals↗
PubMed1986

Intravascular pulmonary macrophages: a novel cell removes particles from blood.

Clearance of radiolabeled gold colloid from the blood and relative organ distribution of retained colloid and magnetic iron oxide particles were compared between rats and calves. When unanesthetized calves 1 wk of age were injected intravenously with these two particle types, uptake was predominantly pulmonary. In contrast, in the rat there was overwhelming hepatic uptake. Similar pulmonary localization of injected particulate material was found in adult goats. When lung tissue was examined by electron microscopy, injected iron oxide was found within intravascular pulmonary macrophages. The mononuclear phagocyte system removes particulate material of both exogenous and endogenous origin from the circulation; hepatic and splenic uptake of such material usually predominates. We found that, unlike the species previously studied, ruminants have actively phagocytic cells within the pulmonary vasculature. Thus pulmonary intravascular macrophages constitute an important part of the mononuclear phagocyte system in some species.

Animals↗
PubMed1986

Pulmonary intravascular macrophages in sheep. Morphology and function of a novel constituent of the mononuclear phagocyte system.

Macrophages resident in the pulmonary capillaries of sheep avidly remove injected particles from the circulating blood. Both sheep and rats were injected intravenously with radiolabeled gold colloid and magnetic iron oxide particles. One hour later, particle uptake in various organs was quantified by gamma counting and magnetometry. Organ localization of both gold and iron oxide particles was predominantly hepatic in rats. In marked contrast, sheep had predominantly pulmonary uptake. Ultrastructural morphology showed that pulmonary iron oxide uptake was by intravascular macrophages. Pulmonary intravascular macrophages were present in ruminant lungs in large numbers. Lungs of sheep given no particles were fixed by intratracheal instillation of glutaraldehyde; randomly chosen tissue samples were routinely processed for electron microscopy and studied with stereological methods. We found that these macrophages occupied 15.3% of the intravascular volume, and had 15.9 m2 of free surface available for contact with blood. Intravascular macrophages were closely applied to 7.1% of the endothelial surface, including numerous short segments with 12 to 15 nm of membrane interspace, increased subplasmalemmal cytoplasmic density, and intercellular electron-dense material. We conclude that pulmonary intravascular macrophages in sheep comprise an important component of their mononuclear phagocyte system. Furthermore, we suggest that these macrophages, through phagocytic uptake of bacteria or endotoxin, may contribute to pulmonary inflammation and injury.

Animals↗
PubMed1985

Metastasis of bile ductule carcinoma in a cow.

An 8-year-old Holstein cow was referred with a history of weight loss, poor milk production, and hyperfibrinoginemia. Laboratory evaluation showed high gamma-glutamyltransferase activity, prolonged sulfobromophthalein clearance half-time, and prolonged prothrombin and partial thromboplastin times. Multiple firm nodules with histologic evidence of bile ductule carcinoma were found on exploratory laparotomy and liver biopsy. Pulmonary and lymph node tumor metastases were extensive. Tumor development in this case could not be associated with any of the known contributing factors in ruminants. This case demonstrates the extensive metastatic potential of this tumor and nonspecific signs with which bovine hepatic disease can be manifested.

Adenocarcinoma↗
PubMed1985

The role of gap junctions in amphibian development.

The possibility that communication through gap junctions may be important during embryonic development has often been raised since gap junctions were first described between early embryonic cells. It is now known that this direct cell-to-cell communication pathway disappears between groups of embryonic cells with different developmental fates as the embryo progresses through development, suggesting that transfer through the gap junctional pathway may play some part in controlling events during development. Supportive evidence for a role for gap junctions comes from experiments demonstrating that the properties of gap junctions differ at the border separating each segment in insect epidermis. Recently it has been shown that the ability to exchange small dyes between cells in the amphibian embryo depends on the position of each cell with respect to the grey crescent. When communication through gap junctions is prevented, by injecting antibodies to gap junctions protein, pattern formation is severely disturbed in the non-communicating region. The paper describes experiments on the pattern of junctional communication at early stages of development of the amphibian embryo and illustrates how anti-gap junction antibodies are being used to determine when and where communication through gap junctions may play an important role during development.

Amphibians↗
PubMed1983

Muscle activity and the loss of electrical coupling between striated muscle cells in Xenopus embryos.

The gap junctions between embryonic striated muscle cells are lost during development. The time course of their elimination has been examined with electrophysiological techniques in myotomes of Xenopus laevis embryos. Gap junctions were detected by the passage of electronic current or the fluorescent dye, Lucifer Yellow, from one muscle cell to another. These tracers only spread to neighboring cells when injected intracellularly. All the muscle cells are electrically coupled at stage 24 when neuromuscular transmission begins, but normally many cells become uncoupled during the next 48 hr. In contrast, the muscle cells remain electrically coupled if neuromuscular transmission is blocked during that period with tricaine or alpha-bungarotoxin. When muscle activity recovers, the loss of coupling resumes. Once the coupling has disappeared, neuromuscular blockade does not restore it. Muscle contraction is blocked during development in a mutant of Xenopus, even though the muscle cells remain electrically excitable. After stage 32 in these immobile embryos, the muscle cells are stimulated repeatedly by regular bursts of neural activity. Although they never contract, the mutant muscle cells become uncoupled at the same time as the muscle cells in normal embryos. The results suggest that some consequence of repeated cholinergic activation, other than contraction, stimulates the loss of gap junctions between striated muscle cells during development. The elimination of gap junctions may be required for neural control of subsequent muscle differentiation.

Aminobenzoates↗
PubMed1982

Permeability of gap junctions at the segmental border in insect epidermis.

We have examined cell-cell communication between epidermal cells of fifth-instar larvae of the milkweed bug Oncopeltus fasciatus and those of maggots of the blowfly Calliphora erythrocephala. Ionic coupling and the transfer of injected Lucifer Yellow (molecular weight 450) and lead-EDTA (molecular weight 374) were used to map the pattern of communication. All epidermal cells, regardless of their position with respect to the segmental border, were ionically coupled. In both species Lucifer yellow was transferred freely between cells lying in the same segment--that is, in the same developmental compartment as defined by cell lineage. Dye injections close to the segmental border showed that Lucifer Yellow was not transferred between cells in adjacent segments--that is, across the compartmental border. In Calliphora failure of Lucifer Yellow transfer at the segmental border was always observed; in Oncopeltus Lucifer Yellow was not transferred in 90% of preparations examined. Injections of PbEDTA2- in Calliphora showed that this anion was transferred freely from cell to cell and did not respect the segmental boundary. Previous studies of the distribution of gap junctions at and away from the segmental border make it unlikely that the failure of Lucifer Yellow to cross from segment to segment is due to reduced number of gap-junctional channels at the border. We conclude that gap junctions at the segmental borders may have different permeability properties from those between cells in the same segment.

Animals↗
PubMed1982

Intracellular sodium and the differentiation of amphibian embryonic neurones.

1. Experiments have been done to examine the mechanism of the inhibition of neural differentiation produced by inhibiting the sodium pump with cardiac glycosides during the mid-neural fold stages of development of the amphibian embryo. Neural differentiation was assessed quantitatively by counting the number of neurones that undergo primary differentiation in tissue culture, as a proportion of the total number of differentiated cells.2. Inhibition of the sodium pump by lowering the extracellular potassium concentration ([K(o)]) to 0 during the mid-neural fold stages inhibited neural differentiation.3. Raising the extracellular calcium concentration to 10 mM during treatment with strophanthidin protected differentiating neurones from the effects of the sodium pump inhibitor. Lowering [Ca](o) to 0.05 mM potentiated the effect of low doses of glycoside.4. In the presence of high extracellular calcium and 5 x 10(-6) M-strophanthidin the membrane potential of neural plate cells remained close to the levels recorded at the beginning of neurulation; the normal increase in resting potential was not restored.5. Addition of 10 mM-Sr(2+) to the bathing medium also protected nerve cells against the inhibition produced by strophanthidin; Sr(2+) was less effective than Ca(2+).6. Addition of either 10 mM-Mg(2+) or Mn(2+) had no effect on the inhibition of differentiation produced by strophanthidin.7. Addition of Mn(2+) along with high Ca(2+) prevented calcium from exerting its protective effect.8. The eyes of embryos treated with high Ca(2+) together with strophanthidin during neurulation and then allowed to grow into tadpoles developed normally. When Mn(2+) was present together with Ca(2+) and strophanthidin the eyes were disrupted similarly to those of embryos treated with strophanthidin alone.9. Replacement of extracellular sodium with equimolar amounts of choline or lithium prevented the cardiac glycoside from inhibiting neural differentiaion.10. The protection afforded by lowering [Na](o) was abolished when [Ca](o) was simultaneously lowered to 0.05 mM.11. Tadpoles from embryos treated with low extracellular sodium together with strophanthidin during neurulation had normal eyes compared to those treated with strophanthidin alone.12. Measurement of the intracellular sodium concentration ([Na](i)) with sodium-sensitive micro-electrodes put [Na](i) at about 30 mM before the neural folds lift. As the sodium pump is activated (stages 14(1/2)-15)[Na](i) in the neural plate falls; by the end of the mid-neural fold stage it is less than 10 mM.13. Addition of 5 x 10(-6)M-strophanthidin to the bathing fluid before activation of the sodium pump prevented the fall in [Na](i); in embryos where [Na](i) had begun to drop strophanthidin produced a rise to about 30 mM.14. When 10 mM-calcium was present along with strophanthidin [Na](i) fell to about 17 mM during neurulation, despite inhibition of the sodium pump.15. It is concluded that it is unlikely that either abolition of the normal increase in resting potential or a fall in gap junction permeability is responsible for the reduction in neural differentiation produced by blocking the sodium pump during neurulation.16. The results are consistent with the view that strophanthidin achieves its effect by preventing the fall in [Na](i) that occurs during normal neurulation because of activation of the sodium pump. They are discussed in the light of this suggestion.

Ambystoma mexicanum↗
PubMed1980

Free calcium in Xenopus embryos measured with ion-selective microelectrodes.

A regulatory role for intracellular free calcium has been suggested in cell division and intercellular communication via gap junctions. Previous measurements of cytoplasmic free Ca2+ during cell division and uncoupling have been made with the Ca2+-sensitive photoprotein aequorin. We now report experiments using Ca2+-sensitive microelectrodes to monitor free Ca2+ in the cells and intercellular fluid of early embryos of the amphibian Xenopus laevis. In addition to measuring basal levels, we have looked for changes in free Ca2+ during cell division and during electrical uncoupling of the normally coupled embryonic cells, induced by acidification of the intracellular medium.

Animals↗
PubMed1980

Intracellular pH in early Xenopus embryos: its effect on current flow between blastomeres.

1. Electrophysiological techniques were used to monitor the flow of electric current from one cell to the next in Xenopus laevis embryos between the 4-cell and early blastula stages of development. Intracellular pH and blastocoel pH were determined using pH-sensitive micro-electrodes. 2. The resting intracellular pH was 7.74+/-0.02 (S.E. of mean, n = 29); there were no systematic differences between developmental stages. Blastocoel cavity pH was 8.4+/-0.06 (S.E. of mean, n = 10). The intracellular buffer value was 18 m-equiv. H+/pH unit per litre. 3. In embryos treated with bicarbonate buffered Holtfreter solution equilibrated with 100% CO2 the intracellular pH fell to 6.3+/-0.17 (S.D., n = 8). The membrane potential fell and the input resistance increased. The size of the effect on membrane potential and input resistance varied. 4. From the 32-cell stage onwards current flow from one cell to the next was abolished when the intracellular pH fell to below 6.5; the effect was rapid in onset and completely reversible. At cleavage stages of development lowering intracellular pH with CO2 had no effect on current flow from cell to cell. 5. The relationship between intracellular pH and current flow from cell to cell was sigmoid and covered between 0.2 and 0.4 pH units. The pH at which current flow was completely abolished ranged from 6.85 to 6.4. 6. Alterations in extraembryonic pH over the range 5.8-7.5 had no effect on any parameter measured. 7. We conclude that lowering the intracellular pH increases the resistance of both non-junctional junctional membranes. The data do not allow us to extract the pH junctional conductance relationship. 8. Variations in intracellular pH may provide a useful tool for the study of the functional role of direct cell to cell communication in both adult organs and early embryos.

Animals↗
PubMed1979

The function of the sodium pump during differentiation of amphibian embryonic neurones.

1. A method has been developed for studying the differentiation in tissue culture of ectoderm and mesoderm derivatives, dissected from amphibian embryos which have just completed neurulation. 2. Neurones, striated muscle cells and pigment cells, together with other unidentifiable cell types, differentiated as a monolayer with approximately the same time course as in the whole embryo. The proportion of different cell types in the cultures was measured quantitatively by cell counting. 3. Treatment of embryos during neurulation with the cardiac glycoside strophanthidin reduced the number of neurones which subsequently differentiated in culture. Other cell types were not affected. 4. The relationship between inhibition of neural differentiation and strophanthidin concentration was sigmoid, with maximum inhibition at 10(-5) M-strophanthidin and the mid-point at 5 X 10(-7) M-strophanthidin. 35% of neurones differentiating in culture were not affected by glycoside treatment. 5. The glycoside hexahydroscillaren A had no effect on neural differentiation. 6. Increasing extracellular potassium to 100 nM during strophanthidin treatment completely protected differentiating neurones from the inhibitory effect of strophanthidin. 7. Treatment of embryos with 100 mM-KCl during neurulation had no effect on the subsequent differentiation of neurones. 8. Treatment of cultures with an antibody to mouse salivary gland Nerve Growth Factor reduced the number of neurones by 30%. 9. Exposure to strophanthidin while the embryo moved from the early neural fold stage to the late neural fold stage was as effective in reducing subsequent neural differentiation as treatment throughout neurulation. 10. The proportion of nerve cells in the cultures was not affected if strophanthidin treatment ended before the early neural fold stage or did not begin until the late neural fold stage. 11. Embryos treated with strophanthidin during neurulation and then allowed to grow into tadpoles developed abnormal nervous systems. 10(-6) M-strophanthidin had little effect on the volume of grey matter, but reduced the white matter by 50%. 12. The results are consistent with the view that strophanthidin achieves its effect on neural differentiation by inhibiting the sodium pump. They are discussed in the light of the suggestion that activation of the sodium pump is an essential part of nerual differentiation.

Ambystoma↗