Biomedical subjects

A C Hall

Publications and source records attributed to A C Hall.

At least 19 recordsLinked to original sources

PubMed1998

Oligodendrocyte population dynamics and the role of PDGF in vivo.

Oligodendrocyte progenitors originate near the floor plate of the spinal cord, then proliferate and migrate throughout the cord before giving rise to oligodendrocytes. Progenitor cell proliferation stops before birth because the cell cycle slows down, linked to an increase in differentiation and death. Experiments with transgenic mice show that platelet-derived growth factor (PDGF) drives progenitor cell division and suggest that slowing of and exit from the cycle reflects a decline in PDGF signaling. Overexpressing PDGF induces hyperproliferation of progenitor cells and excessive, ectopic production of oligodendrocytes. However, the superfluous oligodendrocytes die at an immature stage of differentiation, leaving a normal complement of myelin-forming cells. Therefore, cell survival controls override proliferation controls for determining the final number and distribution of mature oligodendrocytes.

Animals↗

PubMed1997

Characterization of the Na+, K(+)-ATPase in isolated bovine articular chondrocytes; molecular evidence for multiple alpha and beta isoforms.

We have used isoform-specific antibodies against the Na+, K(+)-ATPase alpha (alpha 1, alpha 2 and alpha 3) and beta (beta 1 and beta 2) subunit isoforms in order to establish their specific localization in isolated bovine articular chondrocytes. Immunoblotting confirmed the presence of the alpha 1 and alpha 3 isoforms, although alpha 1 expression was significantly greater than alpha 3 as assessed by immunofluorescence confocal laser scanning microscopy and PCR. A similar approach revealed the presence of the beta 1 and beta 2 isoforms in chondrocytes, although beta 2 immunostaining on the plasma membrane was more punctate than beta 1 which in contrast predominated in a subcellular compartment. The plasma membrane abundance of the Na+, K(+)-ATPase was found to be sensitive to the extracellular ionic concentration and long-term elevation of extracellular Na+ concentration significantly upregulated Na+, K(+)-ATPase density as measured by specific 3H-ouabain binding. Our observations suggest that the expression of alpha 3 and beta 2 is not restricted to excitable tissues as previously reported. The physiological relevance of alpha 3 expression in chondrocytes may be related to its low affinity for intracellular Na+ in an extracellular environment where Na+ concentration is unusually high (260-350 mM) compared to other cell types (140 mM). Glycoproteins and their branched carbohydrates have been implicated in cell recognition events, thus the beta 2 subunit glycoprotein may allow the chondrocyte to detect changes in its extracellular environment by physically interacting with components of the cellular cytoskeleton and matrix macromolecules.

Animals↗

PubMed1997

Biochemical and physiological characteristics of HlyA, a pore-forming cytolysin of Vibrio cholerae serogroup O1.

Among the various toxins produced by the bacterial species Vibrio cholerae is HlyA, a cytolytic protein commonly called the E1 Tor hemolysin. HlyA is synthesized and processed in a complex manner involving various processed or degraded forms, that may co-purify and complicate the interpretation of biochemical and physiological experiments. In this study a single form of HlyA was purified by gel filtration and chromatofocusing using fast protein liquid chromatography in the presence of protease inhibitors. A 45-fold purification was obtained, with a final recovery of 17% of pure 60,000 mol. wt HlyA. A significant improvement in specific activity to 8.5 x 10(6) Chinese hamster ovary tissue culture units per mg protein was obtained. Physiological activity studies indicated that cytolysis of erythrocytes (hemolysis) was inhibited by oxygen: storage of HlyA under oil, and experimentation in N2-flushed buffers maintained activity. HlyA-mediated lysis of human erythrocytes was characterized by a significant lag phase, followed by a rapid induction of hemolysis. Hemolysis was inhibited by sucrose, an osmotic protectant, suggesting that the initial action of HlyA on erythrocytes is to raise the basal cation permeability of the cell membrane. The most likely cytolytic mechanism is thus the formation of transmembrane lesions such as homopolymer pores in target cells, as has been found for toxins from numerous other bacterial pathogens.

Amino Acid Sequence↗

PubMed1997

Immunologic and autoradiographic localisation of the Na+, K(+)-ATPase in articular cartilage: upregulation in response to changes in extracellular Na+ concentration.

The maintenance of a relatively low intracellular Na+:K+ ratio is essential for the functioning of a wide range of cellular processes, and is achieved principally by the activity of the membrane-bound Na+, K(+)-ATPase. Chondrocytes, the cells of articular cartilage, exist in an ionic environment where the free extracellular [Na+] is higher (250-400 mM) than that of most other tissues (approximately 140 mM) owing to the fixed negative charges on glycosaminoglycans in the extracellular matrix. This can increase further during static joint loading when fluid expression occurs. To determine aspects of how chondrocytes regulate their ionic composition, in this study, the in situ distribution, pattern of isoform expression and density of the Na+, K(+)-ATPase within cartilage has been investigated. The density of the Na+, K(+)-ATPase was found to be high in the mid-zone, but lower in the surface and deep zones. Immunofluorescence microscopy using monoclonal antibodies to the catalytic alpha subunits of the Na+, K(+)-ATPase revealed the expression of isoforms alpha 1 and alpha 3. Alterations to the extracellular [Na+] (from 80-220 mM, or 120-220 mM) significantly elevated Na+, K(+)-ATPase density of in situ chondrocytes. The results indicate that the Na+, K(+)-ATPase is abundantly expressed in articular chondrocytes and its density is sensitive to the extracellular [Na+]. The expression of the alpha 3 isoform is surprising for a non-neuronal cell, and may indicate a physiological adaptation to the unusually high extracellular [Na+] to which chondrocytes are exposed in the extracellular matrix of cartilage.

Animals↗

PubMed1997

Four-dimensional imaging of living chondrocytes in cartilage using confocal microscopy: a pragmatic approach.

Regulation of cell volume is a fundamental cellular homeostatic mechanism in the face of osmotic stress. In normal articular cartilage, chondrocytes are exposed to a changing osmotic environment. We present a comprehensive protocol for studying the volume regulatory behavior of chondrocytes within intact cartilage tissue using confocal laser-scanning microscopy. Our data acquisition regime optimizes both signal-to-noise and cell viability during time-lapsed three-dimensional (3-D) (x, y, z, t) imaging. The porcine cartilage is treated as an integrated component of the imaging system, and we demonstrate methods for the direct assessment of tissue-induced axial attenuation and image distortion. Parameterized functions describing these two components of image degradation are used to correct experimental data. The current study also highlights the problems associated with the analysis and visualization of four-dimensional (4-D) images. We have devised two new types of data reconstruction. The first compresses each 3-D time point into a single quantitative view, termed a coordinate view. From these reconstructions we are able to simultaneously view and extract cell measurements. A second type, a 4-D reconstruction, uses color to represent relative changes in cell volume, again while maintaining the morphological and spatial information. Both these approaches of image analysis and visualization have been implemented to study the morphology, spatial distribution, and dynamic volume behavior of chondrocytes after osmotic perturbation. We have mapped chondrocyte shape, arrangement, and absolute volume in situ, which vary significantly from the tissue surface through to the underlying bone. Despite the rigid nature of the extracellular matrix, cartilage cells are osmotically sensitive and respond to stimulation of volume regulatory mechanisms. The combined techniques of confocal laser-scanning microscopy and vital cell labeling have enabled us to study, for the first time, the response of chondrocytes in situ to changes in interstitial osmotic pressure.

Animals↗

PubMed1997

Origins of spinal cord oligodendrocytes: possible developmental and evolutionary relationships with motor neurons.

Spinal cord oligodendrocytes develop from migratory glial progenitor cells that are generated by a small subset of neuroepithelial cells in the ventral part of the neural tube. Specification of these neuroepithelial oligodendrocyte precursors, in common with other ventral cells such as motor neurons, depends on morphogenetic signals from the notochord and/or floor plate. The ventrally derived signals can be mimicked in vitro by purified Sonic hedgehog (Shh) protein. Oligodendrocytes and motor neurons are induced over the same range of concentrations of Shh, consistent with the idea that Shh might specify a common precursor of motor neurons and oligodendrocytes. A lineage relationship between motor neurons and oligodendrocytes has previously been suggested by clonal analysis in the embryonic chick spinal cord. We propose a lineage diagram that connects oligodendrocytes and motor neurons and that takes into account the fact that motor neurons and oligodendrocyte precursors are generated at different times during development. Oligodendrocytes might originally have evolved to ensheath motor axons and facilitate a rapid escape response. If so, oligodendrocyte ontogeny and phylogeny might share a common basis.

Animals↗

PubMed1997

Suprachiasmatic nucleus neurons are glucose sensitive.

The suprachiasmatic nucleus (SCN) in the hypothalamus serves as the pacemaker for mammalian circadian rhythms. In a hamster brain slice preparation, the authors were able to record spontaneous activity from SCN cells for up to 4 days in vitro and verify a self-sustained rhythm in firing. The phase of this rhythm was altered by the concentration of glucose in the bathing medium, with time of peak firing advanced for a 20 mM glucose condition and slightly delayed for a 5 mM glucose condition, relative to 10 mM. The advancing effect of 20 mM glucose and the delaying effect of 5 mM glucose were not maintained during a 2nd day in vitro after changing the bathing medium back to 10 mM glucose, thus indicating the effect was not a permanent phase shift of the underlying oscillation. In experiments recording from cell-attached membrane patches on acutely dissociated hamster SCN neurons, exchanging the bathing medium from high (20 mM) to zero glucose increased potassium (K+)-selective channel activity. With inside-out membrane patches, the authors revealed the presence of a glybenclamide-sensitive K+ channel (190 pS) and a larger conductance (260 pS) Ca(2+)-dependent K+ channel that were both reversibly inhibited by ATP at the cytoplasmic surface. Furthermore, 1 mM tetraethylammonium chloride was demonstrated to advance peak firing time in the brain slice in a similar manner to a high concentration of glucose (20 mM). The authors interpret the result to imply that SCNs are sensitive to glucose, most probably via ATP modulation of K+ channel activity in these neurons. Tonic modulation of K+ channel activity appears to alter output of the pacemaker but does not reset the phase.

Adenosine Triphosphate↗

PubMed1997

Membrane attack induced by HlyA, a pore-forming toxin of Vibrio cholerae.

Determining the activity of purified toxins has generally provided the basis for establishing their role in the host-pathogen relationship. The bacterial genus Vibrio produces a number of exotoxins in addition to cholera toxin, including haemolysin A (HlyA; Vibrio cholerae) and thermostable direct haemolysin (TDH; Vibrio parahaemolyticus), both of which possess membrane-targeting cytolytic activity. The action of HlyA has been analyzed using protocols previously applied to TDH: lysis and flux experiments on human erythrocytes showed that HlyA similarly causes lysis after cell swelling (by colloid osmosis) due to an elevation of cation permeability. However, kinetic measurements of flux, haemolysis and cation selectivity showed that HlyA and TDH form pores with distinct and characteristic features.

Adult↗

PubMed1996

Nature of the cation leak induced in erythrocyte membranes by Kanagawa haemolysin of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is an important enteric pathogen that produces an exotoxin prepared as Kanagawa haemolysin (KH). Isotope flux techniques were used to analyse toxin action on the basal permeability of human erythrocytes. KH induced a cation leak that was (i) rapid in onset (lag phase < 1 min), (ii) 'pore-like' in terms of kinetic characteristics, and (iii) of high magnitude initially (first 10 min) and then subsequently lower (but still raised with reference to control cells). The susceptibilities of the induced flux pathway to washout in initial and later periods suggested a protracted binding time course for toxin action. Neuraminidase treatment of erythrocytes enhanced both haemolysis and flux induced by KH, suggesting that the affinity of the toxin for the membrane had increased, possibly as a result of additional toxin receptors being unmasked by this enzyme. These results show that KH elevates the basal permeability of human erythrocytes in a complex manner, a process that probably underlies the deleterious effects of this toxin on cellular function.

Bacterial Toxins↗

PubMed1996

Changes to erythrocyte membrane cation permeability induced by a bacterial toxin.

Vibrio parahaemolyticus secretes an exotoxin prepared as Kanagawa haemolysin (KH) which causes marked alterations to the function of epithelial, cardiac and other cell types, but whose cellular mode of action is poorly understood. Using human red cells as a model system with radiotracer flux techniques, we have shown that KH (1) markedly elevated the basal leak to K+, (2) raised Ca2+ influx, and as a consequence of this, (3) stimulated the Ca2+-activated K+ channel. These results suggest that an important deleterious effect of this toxin is to elevate cation permeability, which will have both direct and indirect actions on the behaviour of a variety of cell types in vivo.

Adult↗

PubMed1996

Effects of inhalational general anaesthetics on native glycine receptors in rat medullary neurones and recombinant glycine receptors in Xenopus oocytes.

1. Glycine responses were studied under voltage clamp in Xenopus oocytes injected with cDNA encoding mammalian glycine receptor subunits and in rat medullary neurones. Bath application of glycine gave strychnine-sensitive currents which reversed close to the expected equilibrium potentials for chloride ions. The peak currents for the receptors expressed in oocytes fitted a Hill equation with EC50 = 215 +/- 5 microM and Hill coefficient nH = 1.70 +/- 0.05 (means +/- s.e. means). The peak currents from the receptors in medullary neurones fitted a Hill equation with EC50 = 30 +/- 1 microM and Hill coefficient nH = 1.76 +/- 0.08. The current-voltage relationship for the receptors expressed in oocytes showed strong outward rectification (with Vrev = -21 +/- 2 mV), while that for the glycine responses from the medullary neurones in symmetrical Cl- was linear (with Vrev = 3.2 +/- 0.6 mV). 2. Inhalational general anaesthetics, at concentrations close to their human minimum alveolar concentrations (MACs), potentiated responses to low concentrations of glycine. The potentiation observed with the recombinant receptors (between 60-22%) was approximately twice that found with the medullary neurones (between 40-80%). For both the recombinant receptors and the receptors in medullary neurones, the degree of potentiation increased in the order of methoxyflurane approximately sevoflurane < halothane approximately isoflurane approximately enflurane. There was no significant difference between the potentiations observed for the two optical isomers of isoflurane. 3. For both the recombinant and native receptors, isoflurane potentiated the currents in a dose-dependent manner at low concentrations of glycine, although at high glycine concentrations the anaesthetic had no significant effect on the glycine-activated responses. The major effect of isoflurane was to cause a parallel leftward shift in the glycine concentration-response curves. The glycine EC50 concentration for the recombinant receptors decreased from a control value of 215 +/- 5 microM to 84 +/- 7 microM glycine at 610 microM isoflurane, while that for the medullary neurones decreased from a control value of 30 +/- 1 microM to 18 +/- 2 microM glycine at the same concentration of isoflurane. The potentiation was independent of membrane potential. 4. Isoflurane also potentiated responses to taurine, a partial agonist at the glycine receptor. This was observed for receptors expressed in oocytes at both low and saturating concentrations of taurine. The EC50 concentration decreased from a control value of 1.6 +/- 0.2 to 0.9 +/- 0.1 mM taurine in the presence of 305 microM isoflurane, while the maximum response to taurine increased from 47 +/- 2 to 59 +/- 2% of the maximum response to glycine. 5. Glycine receptors, like other members of the fast ligand-gated receptor superfamily, are sensitive to clinically relevant concentrations of inhalational general anaesthetics. Effects at these receptors may, therefore, play some role in the maintenance of the anaesthetic state.

Administration, Inhalation↗

PubMed1996

The cellular physiology of articular cartilage.

The cells of articular cartilage, or chondrocytes, live in an unusual and constantly changing physicochemical environment. They receive poorly understood signals during the loading of the tissue and produce, through a balance between macromolecular synthesis and degradation, a mechanically resilient extracellular matrix. Matrix turnover is influenced by changes to the intracellular composition (cell volume, pH and ionic content) of chondrocytes, and there are suggestions that this is altered in the disease process of osteoarthrosis. However, there is little information on the fundamental aspects of articular cartilage cellular physiology, which is essential if the factors controlling cartilage integrity in health and disease are to be understood. The present short review focuses on some of the membrane transporters of chondrocytes involved in volume regulation, electrophysiology and the regulation of intracellular pH.

Adult↗

PubMed1996

Pathways for K+ transport across the bovine articular chondrocyte membrane and their sensitivity to cell volume.

The contributions of various K+ transport pathways in bovine chondrocytes isolated from articular cartilage and their responses to changes in cell volume have been studied. K+(86Rb+) uptake mediated by the Na(+)-K(+) pump and Na(+)-K(+)-2Cl- cotransporter were stimulated by cell shrinkage, the latter as part of the regulatory volume increases (RVI) response, the former as an indirect effect resulting from the rise in intracellular Na+ concentration during RVI. For both transporters, there was an increase in the maximum velocity with no detectable effect on the Michaelis constant. There was no evidence for volume-sensitive K+ transport mediated by the K(+)-Cl- cotransporter, or Ca(2+)-activated K+ channels. However, chondrocyte swelling stimulated a ouabain- and bumetanide-insensitive K+ flux sensitive to pimozide and other drugs, which exhibited some of the properties of the relatively nonspecific volume-sensitive "osmolyte" channel described in other cell types.

Animals↗

PubMed1996

Effects of hydrostatic pressure on matrix synthesis in different regions of the intervertebral disk.

The intervertebral disk is routinely subjected to compressive loads that alter with posture and muscle activity and can produce pressures > 2 MPa in human lumbar disks in vivo (A. Nachemson and G. Elfstrom. Scand. J. Rehabil. Med. 2, Suppl. 1:1-40, 1979; A. Nachemson and J. M. Morris. J. Bone Jt. Surg. Am. Vol. 46A: 1077-1092, 1964). We measured the effect of load on hydrostatic pressures in bovine caudal disks. With increase in applied load, pressure increased linearly in the nucleus and inner annulus. The resting pressure measured after slaughter (0.19 +/- 0.05 MPa) and the pressure at failure (34 MPa, estimated from the vertebrae/disk segment failure load of 7,430 +/- 590 N) define the limits that can occur in vivo. Because hydrostatic pressure influences matrix synthesis in articular cartilage, we have examined the effects of pressures in the range 1-10 MPa applied for 20 s or 2 h on proteoglycan synthesis in bovine caudal and human lumbar intervertebral disks in vitro. In the nucleus pulposus and inner annulus of bovine disks, application of hydrostatic pressure in the range of 1-7.5 MPa for only 20 s stimulated matrix synthesis over the following 2 h at atmospheric pressure. The maximum stimulation in the bovine disks was seen in the inner annulus after application of 2.5 MPa, where proteoglycan synthesis rates doubled. Exposure to 2.5 MPa also stimulated synthesis in the nucleus pulposus of human disks taken at surgery, whereas 7.5 MPa inhibited synthesis in five out of six specimens. With 2-h continuous exposure to the same levels of pressure, no stimulation was seen in the nucleus of bovine disks, and significant stimulation was only observed at 5.0 MPa in the inner annulus. Exposure to 10 MPa for either 20 s or 2 h inhibited proteoglycan synthesis in these regions of the disks. In contrast, in the outer annulus, where loading does not lead to a rise in hydrostatic pressure in vivo, there was no significant response to hydrostatic pressure over the range of 1-10 MPa in bovine or human disks.

Animals↗

PubMed1996

Ciclazindol inhibits ATP-sensitive K+ channels and stimulates insulin secretion in CR1-G1 insulin-secreting cells.

Ciclazindol, an anorectic drug, was shown to inhibit ATP-sensitive K+ (K(ATP)) channel currents and stimulate insulin secretion from CRI-G1 insulin-secreting cells. In contrast, the structurally related anorectic agent mazindol and the amphetamine-based anorectic compounds diethylpropion, fenfluramine, and phentermine had no effect on K(ATP) channel activity in this cell line. Similarly, cicliazindol elicited insulin secretion from CRI-G1 cells, whereas mazindol had no secretagogue action. The mechanism by which ciclazindol acts to inhibit K(ATP) channel activity is different than that of the sulfonylureas as ciclazindol is effective after procedures that decouple the sulfonylurea receptor from the K(ATP) channel. In agreement with this finding, ciclazindol failed to displace [3H]glibenclamide from CRI-G1 microsomal membranes. Further experiments demonstrated that ciclazindol has no significant effect on voltage-activated currents in this cell line.

Adenosine Triphosphate↗

PubMed1995

Volume-sensitive taurine transport in bovine articular chondrocytes.

1. The swelling of bovine articular chondrocytes isolated from, or in situ within, cartilage by hypotonic shock rapidly activated the efflux or influx of radiolabelled taurine, an amino acid involved in volume regulation in a range of other cell types. 2. When chondrocytes were isolated by the use of collagenase into media of 280 or 380 mosmol l-1, the activation of taurine efflux was at about the osmolarity of the isolating medium, but it was more marked for a given hypotonic shock in the cells isolated at the lower osmolarity. The volume of chondrocytes following isolation in these two osmolarities was the same, suggesting that the cells possess volume regulatory capacity. 3. In isolated chondrocytes, the induced pathway had some of the characteristics of a volume-activated channel, i.e. no transport saturation with increasing substrate concentration, and lack of trans acceleration. The pattern of inhibition of the volume-activated pathway by pharmacological blockers (e.g. pimozide, [(dihydro-indenyl)oxy]alkanoic acid (DIOA) and tamoxifen) differed from that described for a similar pathway in other cell types. 4. The transport of other potential osmolytes (uridine, sorbitol and inositol) was stimulated by cell swelling, independent of sodium and inhibited by pimozide with a selectivity ratio of taurine, 1.00; uridine, 0.84; sorbitol, 0.66; and inositol, 0.38. The selectivity of taurine: inositol was not altered at different cell volumes. 5. The intracellular taurine concentration of chondrocytes within cartilage was low (about 2 mmol (l cell water)-1) showing a negligible role for taurine as an osmolyte during recovery from cell swelling. The swelling-induced loss of taurine from chondrocytes in situ was largely inhibited by pimozide and other drugs, showing that despite the rigid nature of cartilage, the chondrocytes were osmotically sensitive within the extracellular matrix.

Animals↗

PubMed1995

Control of matrix synthesis in isolated bovine chondrocytes by extracellular and intracellular pH.

The effects of extracellular and intracellular pH on matrix synthesis by isolated bovine chondrocytes were studied using radioisotope incorporation (35SO4 and 3H proline) and fluorescence techniques. Matrix synthesis exhibited a bimodal relation with decreased extracellular pH; with slight reductions (7.4 > pH > 7.1), synthesis increased (by up to 50%), whereas in more acidic media (pH < 7.1), synthesis was inhibited by up to 75% of control levels. The pHi was largely unchanged with extracellular acidity over the range producing stimulation of matrix synthesis but fell when exposed to the more acidic media shown to have an inhibitory action on matrix synthesis. The inhibition of matrix synthesis by lactic acid addition was unaffected by the lactic acid transporter alpha-CHC, suggesting H+ transport by this pathway is small. Direct imposition of a sustained intracellular acidosis (pHi = 6.65) using ammonium prepulse with amiloride inhibited matrix synthesis by about 20%. These results show that matrix synthesis by chondrocytes was affected by extracellular pH, an action which could not be entirely explained by changes to pHi.

Animals↗

PubMed1995

Stimulation of KCl co-transport in equine erythrocytes by hydrostatic pressure: effects of kinase/phosphatase inhibition.

The effects of hydrostatic pressure on the KCl co-transporter of equine erythrocytes were studied to determine factors involved in its regulation. Pressure (0.1-40MPa) increased Cl-dependent K+ transport; in the presence of the putative kinase inhibitor N-ethylmaleimide (NEM) which stimulates the transporter, or the phosphatase inhibitor calyculin A, pressure had no significant effect. The sequential application of NEM and calyculin A clamped the transporter at about 30% of maximal flux compared to NEM alone; pressure also had no further effect. These results suggest that pressure acts on the phosphorylation status of the transporter or regulatory peptide, rather than on the ion flux per se. Since the activation of the KCl co-transporter by pressure occurs without an apparent change in cell volume these results have implications for any universal model for the regulation of KCl co-transport.

Animals↗