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Biomedical subjects

A C Hall

Publications and source records attributed to A C Hall.

At least 19 recordsLinked to original sources
PubMed2001

Regulatory volume decrease (RVD) by isolated and in situ bovine articular chondrocytes.

Articular chondrocytes in vivo are exposed to a changing osmotic environment under both physiological (static load) and pathological (osteoarthritis) conditions. Such changes to matrix hydration could alter cell volume in situ and influence matrix metabolism. However the ability of chondrocytes to regulate their volume in the face of osmotic perturbations have not been studied in detail. We have investigated the regulatory volume decrease (RVD) capacity of bovine articular chondrocytes within, and isolated from the matrix, before and following acute hypotonic challenge. Cell volumes were determined by visualising fluorescently-labelled chondrocytes using confocal laser scanning microscopy (CLSM) at 21 degrees C. Chondrocytes in situ were grouped into superficial (SZ), mid (MZ), and deep zones (DZ). When exposed to 180mOsm or 250mOsm hypotonic challenge, cells in situ swelled rapidly (within approximately 90 sec). Chondrocytes then exhibited rapid RVD (t(1/2) approximately 8 min), with cells from all zones returning to approximately 3% of their initial volume after 20 min. There was no significant difference in the rates of RVD between chondrocytes in the three zones. Similarly, no difference in the rate of RVD was observed for an osmotic shock from 280 to 250 or 180mOsm. Chondrocytes isolated from the matrix into medium of 380mOsm and then exposed to 280mOsm showed an identical RVD response to that of in situ cells. The RVD response of in situ cells was inhibited by REV 5901. The results suggested that the signalling pathways involved in RVD remained intact after chondrocyte isolation from cartilage and thus it was likely that there was no role for cell-matrix interactions in mediating RVD.

Animals↗
PubMed2001

The role of a swelling-activated taurine transport pathway in the regulation of articular chondrocyte volume.

Swelling articular chondrocytes by reducing osmolarity stimulates a taurine transport pathway, which is implicated in regulatory volume decrease (RVD) in various cell types. The present study investigated factors controlling the activity of this pathway in chondrocytes, in particular (1) the effects of the acute (seconds) and chronic (hours) exposure of chondrocytes to anisotonic media, and (2) whether there is a role for metabolites from the arachidonic acid cascade in activating the taurine transport pathway. For in situ and isolated chondrocytes, the point at which swelling-activated [14C]taurine efflux was stimulated (the "set-point") corresponded closely to the osmolarity of the incubation medium (180, 280 or 380 mosmol/l). However, the volume of chondrocytes isolated into these media and measured by confocal microscopy was not different ( congruent with 645 microm3). Activity of the swelling-activated taurine transport pathway was inhibited by REV5901 (an inhibitor of steps of the arachidonic acid cascade; K0.5 8+/-4 microM), NDGA (a general lipoxygenase inhibitor; K0.5 28+/-5 microM), or MK886 (an inhibitor of the 5-lipoxygenase-activating protein; 91% inhibition at 10 microM), but weakly by the more potent 5-lipoxygenase inhibitor REV5901 para (K0.5 350+/-100 microM). Addition of the leukotriene (LT) B4 or D4 receptor antagonists, CP-105,696 and L660,711 respectively, or of the leukotrienes LTB4, LTC4, LTD4 and LTE4 or lipoxins (hepoxylin A3 or B3) had no effect on the activity of the pathway in isotonic or hypotonic media. The role of the pathway in RVD was determined in isolated calcein-loaded chondrocytes using fluorescence imaging. RVD was observed and inhibited by REV5901 (50 microM) and by NDGA (75 microM). The data show that despite chronic exposure of chondrocytes to anisotonic media, the cells maintain a pre-determined volume that is the "set-point" for the activation of the taurine transport pathway following acute hypotonic challenge. This pathway appears to play a role in chondrocyte RVD, but its activation does not involve metabolites of the arachidonic acid cascade.

Biological Transport↗
PubMed2001

The osmotic sensitivity of isolated and in situ bovine articular chondrocytes.

Articular chondrocytes experience changes to matrix hydration during both physiological (static load) and pathophysiological (osteoarthrosis, OA) conditions. Such changes should alter chondrocytes' volume, which has been shown to modify matrix metabolism. However, the osmometric behaviour of chondrocytes is not well understood. Here, using confocal laser scanning microscopy (CLSM), we have investigated the 'passive' osmotic responses of fluorescent-labelled chondrocytes within, and isolated from, the matrix. The volume-regulatory pathways normally activated by cell shrinkage/swelling, were blocked by bumetanide/REV5901, respectively. Chondrocytes in situ were broadly grouped into superficial (SZ), mid (MZ) and deep (DZ) zones, and there was a significant increase in resting cell volume with depth into the cartilage. Variation in medium osmolarity (range 0-530 mOsm; corresponding to an extracellular osmolarity of approximately 150 to approximately 600 mOsm) caused a rapid and sustained change to in situ MZ chondrocytes' volume. Over the range 180-380 mOsm, the change to in situ or isolated chondrocytes' volume was similar. For MZ chondrocytes. ideal osmometric (Boyle-van't Hoff) behaviour was apparent over the extracellular osmolarity range of approximately 250 to approximately 600 mOsm. Chondrocytes within the SZ appeared to be more sensitive to reduced osmolarity, swelling more for a given reduction in osmolarity, than MZ or DZ chondrocytes. These data show that over wide variations in osmolarity, articular chondrocytes in situ were osmotically sensitive, and for MZ chondrocytes behaved as perfect osmometers with the extracellular matrix (ECM) not restraining cell volume changes. Changes to matrix hydration may therefore alter passive chondrocytes' volume and unless compensated by volume-regulatory pathways, could lead to changes in cell volume, and hence matrix metabolism.

Animals↗
PubMed2001

Hedgehog-dependent oligodendrocyte lineage specification in the telencephalon.

In the caudal neural tube, oligodendrocyte progenitors (OLPs) originate in the ventral neuroepithelium under the influence of Sonic hedgehog (SHH), then migrate throughout the spinal cord and brainstem before differentiating into myelin-forming cells. We present evidence that oligodendrogenesis in the anterior neural tube follows a similar pattern. We show that OLPs in the embryonic mouse forebrain express platelet-derived growth factor alpha-receptors (PDGFRA), as they do in more caudal regions. They first appear within a region of anterior hypothalamic neuroepithelium that co-expresses mRNA encoding SHH, its receptor PTC1 (PTCH) and the transcription factors OLIG1, OLIG2 and SOX10. Pdgfra-positive progenitors later spread through the forebrain into areas where Shh is not expressed, including the cerebral cortex. Cyclopamine inhibited OLP development in cultures of mouse basal forebrain, suggesting that hedgehog (HH) signalling is obligatory for oligodendrogenesis in the ventral telencephalon. Moreover, Pdgfra-positive progenitors did not appear on schedule in the ventral forebrains of Nkx2.1 null mice, which lack the telencephalic domain of Shh expression. However, OLPs did develop in cultures of Nkx2.1(-/-) basal forebrain and this was blocked by cyclopamine. OLPs also developed in neocortical cultures, even though Shh transcripts could not be detected in the embryonic cortex. Here, too, the appearance of OLPs was suppressed by cyclopamine. In keeping with these findings, we detected mRNA encoding SHH and Indian hedgehog (IHH) in both Nkx2.1(-/-) basal forebrain cultures and neocortical cultures. Overall, the data are consistent with the idea that OLPs in the telencephalon, possibly even some of those in the cortex, develop under the influence of SHH in the ventral forebrain.

Animals↗
PubMed2000

Axonal remodeling and synaptic differentiation in the cerebellum is regulated by WNT-7a signaling.

Synapse formation requires changes in cell morphology and the upregulation and localization of synaptic proteins. In the cerebellum, mossy fibers undergo extensive remodeling as they contact several granule cells and form complex, multisynaptic glomerular rosettes. Here we show that granule cells secrete factors that induce axon and growth cone remodeling in mossy fibers. This effect is blocked by the WNT antagonist, sFRP-1, and mimicked by WNT-7a, which is expressed by granule cells. WNT-7a also induces synapsin I clustering at remodeled areas of mossy fibers, a preliminary step in synaptogenesis. Wnt-7a mutant mice show a delay in the morphological maturation of glomerular rosettes and in the accumulation of synapsin I. We propose that WNT-7a can function as a synaptogenic factor.

Animals↗
PubMed1999

Circadian modulation of the ryanodine receptor type 2 in the SCN of rodents.

We examined the temporal modulation of intracellular calcium release channels in the suprachiasmatic nucleus (SCN). We found a circadian rhythm in [3H]ryanodine binding that was specific to the SCN. The peak in the rhythm occurred at CT 7 and was due to an increase in Bmax, which correlated well with immunoblots showing an increase in RyR-2 expression in the SCN. Double immunohistochemical studies showed that RyR-2 was expressed exclusively in neurons. Ryanodine and caffeine applied around CT 7-9 advanced the clock phase in a hamster brain slice preparation. No rhythm of IP3R was seen in any of the brain areas studied. Our results indicate that RyR-2 exhibits an endogenous rhythm, which influences the intracellular calcium dynamics and thus modulates SCN activity.

Animals↗
PubMed1999

Differential effects of hydrostatic pressure on cation transport pathways of isolated articular chondrocytes.

Articular cartilages are exposed to significant loads in vivo, which by their effects on chondrocyte metabolism can alter the mechanical properties of the extracellular matrix. The mechanism(s) by which chondrocytes sense and respond to load are not well understood. One component of load, hydrostatic pressure, can be studied independently of the other factors that change during load. In this study, the effects of pressure have been investigated on three K transport pathways in isolated bovine articular chondrocytes. Pressure inhibited the Na/K pump (ouabain-sensitive), Na/K/2Cl cotransporter (bumetanide-sensitive), and residual (ouabain- and bumetanide-insensitive) pathways; however, the response of each system was different. Both pressure level and duration were important in determining the extent of inhibition. There was marked suppression of the Na/K pump, particularly when pressure (2.5-50 MPa) was maintained for the full incubation period (usually 10 min). The Na/K/2Cl cotransporter was more pressure-sensitive, with only a short application (20 sec) of a low pressure (7.5 MPa) being sufficient for inhibition. Over the higher range (20-50 MPa), pressure had little further effect. The inhibitory action on the Na/K pump was dependent on the [Na]i. Thus, when the [Na]i was set to values above or below those normally present, the inhibitory effect was reduced or abolished. The suppressive effect of pressure on Na/K pump and residual pathways was reversed at atmospheric pressure. The pressure dependence of inhibition of the K flux through the residual pathway was similar to that reported for lipid bilayers. These results indicate that hydrostatic pressure may act directly on chondrocyte membrane transporters. Alterations to matrix synthesis resulting from the application of load might therefore result in part from variations to the intracellular ionic/osmotic composition of chondrocytes arising from changes to the activity of membrane transport pathways.

Animals↗
PubMed1999

Lithium and synaptic plasticity.

Lithium, a small cation, has been used in the treatment of bipolar disorders since its introduction in the 1950s by John Cade. Extensive research on the mechanism of action of lithium has revealed several possible targets. For some time, the most widely accepted action of lithium was its inhibitory effect on the synthesis of inositol, resulting in depletion of inositol with profound effects on neuronal signal transduction pathways. However, several studies show that some effects of lithium are not mediated through inositol depletion. Recent findings demonstrate that lithium directly inhibits, in a non-competitive fashion, the activity of glycogen synthase kinase (GSK)-3beta, a serine/threonine kinase highly expressed in the central nervous system. Interestingly, inhibition of GSK-3beta has been shown to regulate neuronal plasticity by inducing axonal remodelling and increasing the levels of synaptic proteins. These findings raise the possibility for developing new therapeutic approaches for the treatment of bipolar disorders.

Antimanic Agents↗
PubMed1999

Modulation of Na+ x H+ exchange by hydrostatic pressure in isolated bovine articular chondrocytes.

The effects of increased hydrostatic pressure on Na+ x H+ exchange activity in bovine articular chondrocytes have been characterized. Chondrocytes were isolated from the cartilage matrix and the cells were loaded with the pH-sensitive fluorophore BCECF. Cells were acidified by ammonium rebound and the rate of recovery of pHi back to control levels was determined using cuvette fluorimetry. The application of hydrostatic pressure (1-300 atm) to cells within the fluorimeter was found to stimulate the rate of recovery from acidification, recorded as proton fluxes, in MOPS buffered media. This increase was dependent on the presence of extracellular Na+ ions and was inhibited by the Na+ x H+ exchange inhibitor EIPA. The pressure-stimulated increase in H+ flux is therefore mediated completely by Na+ x H+ exchange. In addition, the stimulation could be abolished by the kinase inhibitor staurosporine, was not additive with the stimulation of Na+ x H+ exchange elicited by the addition of serum and was unaffected by low concentrations of the myosin light chain kinase inhibitor ML-7. We therefore conclude that hydrostatic pressure activates Na+ x H+ exchange in this cell type by a pathway which involves direct phosphorylation of the transporter protein itself. This is the first demonstration of the activation of Na+ x H+ exchange by hydrostatic pressure and the relevance of this finding to the biology of cartilage tissue is discussed.

Acid-Base Equilibrium↗
PubMed1999

Role of membrane conductances and protein synthesis in subjective day phase advances of the hamster circadian clock by neuropeptide Y.

Neurons of the mammalian circadian pacemaker in the hypothalamic suprachiasmatic nuclei exhibit a rhythm in firing rate that can be reset by neuropeptide Y. We recorded the effects of neuropeptide Y on Na+ and K+ conductances of hamster suprachiasmatic nuclei neurons using whole-cell, perforated-patch and cell-attached patch-clamp recordings, both in dissociated and brain slice preparations. While neuropeptide Y had no effect on voltage-gated Na+ currents, neuropeptide Y activated a leak K+ current. Neuropeptide Y phase advances in the suprachiasmatic nuclei brain slice preparation were blocked by a number of K+ channel blockers (tetraethylammonium chloride, dendrotoxin-I, glybenclamide). However, a K+ ionophore, valinomycin, did not shift the rhythm. The inhibition by tetraethylammonium chloride did not persist in the presence of glutamatergic receptor blockers. We have previously shown that glutamate can oppose neuropeptide Y phase-shifting actions, suggesting that K+ channel inhibition acts by inducing glutamate release. Protein synthesis inhibitors had no effect on clock phase when applied during the subjective day, and had no influence on neuropeptide Y-induced phase shifts. On the other hand, glutamate's ability to inhibit neuropeptide Y shifts was abolished by protein synthesis inhibition. Thus, while neuropeptide Y phase shifts do not require protein synthesis, glutamate blocks neuropeptide Y shifts via increased gene expression during the subjective day, at a time when it does not reset the clock. These results indicate that neuropeptide Y phase shifts via a mechanism that does not involve changes in membrane conductance or protein synthesis.

2-Amino-5-phosphonovalerate↗
PubMed1999

Defective oligodendrocyte development and severe hypomyelination in PDGF-A knockout mice.

There is a class of oligodendrocyte progenitors, called O-2A progenitors, that is characterized by expression of platelet-derived growth factor &agr;-receptors (PDGFR(&agr;)). It is not known whether all oligodendrocytes are derived from these PDGFRalpha-progenitors or whether a subset(s) of oligodendrocytes develops from a different, PDGFR alpha-negative lineage(s). We investigated the relationship between PDGF and oligodendrogenesis by examining mice that lack either PDGF-A or PDGF-B. PDGF-A null mice had many fewer PDGFR alpha-progenitors than either wild-type or PDGF-B null mice, demonstrating that proliferation of these cells relies heavily (though not exclusively) on PDGF-AA homodimers. PDGF-A-deficient mice also had reduced numbers of oligodendrocytes and a dysmyelinating phenotype (tremor). Not all parts of the central nervous system (CNS) were equally affected in the knockout. For example, there were profound reductions in the numbers of PDGFR alpha-progenitors and oligodendrocytes in the spinal cord and cerebellum, but less severe reductions of both cell types in the medulla. This correlation suggests a close link between PDGFRalpha-progenitors and oligodendrogenesis in most or all parts of the CNS. We also provide evidence that myelin proteolipid protein (PLP/DM-20)-positive cells in the late embryonic brainstem are non-dividing cells, presumably immature oligodendrocytes, and not proliferating precursors.

Animals↗
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↗
PubMed1998

Histamine phase shifts the hamster circadian pacemaker via an NMDA dependent mechanism.

The SCN acts as the central pacemaker for circadian rhythms in mammals. Histamine has been shown to affect circadian rhythms both in vivo and in vitro. We investigated the mechanism by which histamine phase shifts circadian rhythms in vitro. Hypothalamic slices containing the SCN were prepared from golden hamsters, and spontaneous firing rates of individual cells were recorded on the second day in vitro. Application of histamine (1 microM-10 mM) at the extrapolated time of 2 h after lights off (ZT 14) on day 1 in vitro delayed the time of peak firing in a dose-dependent manner. Pre-exposure to the N-methyl-D-aspartate (NMDA) receptor antagonist (+/-)-2-amino-5-phosphonopentanoic acid (AP-5; 100 microM-1 mM) 5 min before histamine (1 microM) was applied to the slice blocked the phase-delaying effects of histamine. Application of the H1 blocker mepryamine (100 nM) or the H2 blocker cimetidine (10 microM) followed by histamine had no effect on the phase delay induced by histamine. In whole cell recordings from acutely dissociated neurons of hamster SCN, histamine (50 microM) was shown to potentiate NMDA-evoked currents by 52 +/- 12%. These experiments demonstrate that histamine phase shifts of the circadian clock are dependent on NMDA receptor activation and that histamine can directly potentiate NMDA currents in SCN neurons. Histamine may alter circadian clock function by acting directly on NMDA receptors, possibly via binding to the polyamine site.

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↗