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J C Ellory

Publications and source records attributed to J C Ellory.

At least 37 records · Page 2Linked to original sources
PubMed2000

Functional state of the plasma membrane Ca2+ pump in Plasmodium falciparum-infected human red blood cells.

The active Ca2+ transport properties of malaria-infected, intact red blood cells are unknown. We report here the first direct measurements of Ca2+ pump activity in human red cells infected with Plasmodium falciparum, at the mature, late trophozoite stage. Ca2+ pump activity was measured by the Co2+-exposure method adapted for use in low-K+ media, optimal for parasitised cells. This required a preliminary study in normal, uninfected red cells of the effects of cell volume, membrane potential and external Na+/K+ concentrations on Ca2+ pump performance. Pump-mediated Ca2+ extrusion in normal red cells was only slightly lower in low-K+ media relative to high-K+ media despite the large differences in membrane potential predicted by the Lew-Bookchin red cell model. The effect was prevented by clotrimazole, an inhibitor of the Ca2+-sensitive K+ (KCa) channel, suggesting that it was due to minor cell dehydration. The Ca2+-saturated Ca2+ extrusion rate through the Ca2+ pump (Vmax) of parasitised red cells was marginally inhibited (2-27 %) relative to that of both uninfected red cells from the malaria-infected culture (cohorts), and uninfected red cells from the same donor kept under identical conditions (co-culture). Thus, Ca2+ pump function is largely conserved in parasitised cells up to the mature, late trophozoite stage. A high proportion of the ionophore-induced Ca2+ load in parasitised red cells is taken up by cytoplasmic Ca2+ buffers within the parasite. Following pump-mediated Ca2+ removal from the host, there remained a large residual Ca2+ pool within the parasite which slowly leaked to the host cell, from which it was pumped out.

Animals↗
PubMed2000

The relationship between hypotonically-induced taurine and K fluxes in trout red blood cells.

Hypotonic swelling of teleost erythrocytes activates multiple transport systems leading to the regulatory decrease of cell volume. We have examined using pharmacological manipulation the swelling-induced taurine flux pathway in red blood cells of the rainbow trout and its relationship to swelling-induced K flux pathways. We show that the activation and deactivation of taurine flux is rapid and that the flux is a sigmoidal function of cell volume. N-ethylmaleimide (NEM) and the non-specific protein kinase inhibitor, staurosporine, both inactivated the hypotonically-induced taurine flux with concentrations eliciting half-maximal inhibition (IC50s) of 212 and 17 micromol(-1), respectively. The low taurine fluxes under isotonic conditions were unaffected. By contrast, the tyrosine kinase inhibitor, genistein, partially inhibited taurine flux under both isotonic and hypotonic conditions. The specific phosphatase inhibitor, calyculin A, had no inhibitory or stimulatory effect under either condition whilst the less-specific phosphatase inhibitor, ortho-vanadate, reduced taurine flux only under hypotonic conditions. In these respects the regulatory control of the taurine pathway differs from the Cl-dependent K flux. However, NEM and staurosporine also inhibited the Cl-independent K flux, both with similar IC50s to those observed for taurine fluxes. This supports the idea of the hypotonically-induced taurine flux and the Cl-independent K flux sharing the same transport pathway.

Animals↗
PubMed2000

Volume-sensitive KCI cotransport associated with human cervical carcinogenesis.

This study investigates the volume-sensitive KCI cotransporter (KCC) in various types of human cervical epithelial cell, testing the hypothesis that cervical malignancy is accompanied by differential expression of volume-sensitive KCC. Normal human cervical epithelial cells have KCCs which are quiescent in normal physiological conditions and are relatively refractory to hypotonic stress. By contrast, cervical cancer cells have KCCs which are also nearly quiescent in normal physiological conditions but high transport rates are observed in response to hypotonic challenge. Using isoform-specific primers, mRNA transcripts of KCC1, KCC3 and KCC4 were identified by reverse transcriptase polymerase chain reaction (RT-PCR) in several types of cervical cell, and confirmed by digestion with specific restriction endonucleases. By semiquantitative RT-PCR with beta-actin as the internal standard, the results indicate that cervical carcinogenesis is accompanied by the up-regulation of mRNA transcripts in KCC1, KCC3 and KCC4. [(Dihydroindenyl)oxy] alkanoic acid (DIOA), a KCC inhibitor, blocked both the regulatory volume decrease (RVD) process and volume-sensitive 86Rb+ efflux from cervical cancer cells in a dose-dependent manner. The volume-sensitive 86Rb+ efflux from cervical cancer cells was also blocked by two protein phosphatase inhibitors, calyculin A and okadaic acid, with IC50 values of 0.8 and 6 nM, respectively. Conversely, protein kinase inhibitors, chelerythrine and staurosporine, increased Cl- dependent 86Rb+ efflux. NEM (1 mM) led to a fivefold stimulation of 86Rb+ efflux which was totally Cl- dependent in cervical cancer cells. Hypotonicity could not stimulate any further 86Rb+ efflux after NEM treatment. These results indicate that the volume-sensitive KCC in cervical cancer cells plays a significant role in volume regulation and that the activities are modulated by a phosphorylation cascade. Taken together with our previous studies, we suggest the volume-regulatory ion channels and the co-transport systems work synergistically for volume regulation in human cervical cancer cells.

Carrier Proteins↗
PubMed2000

Effects of protein kinase and phosphatase inhibitors and anti-L antisera on K+ transport in LK sheep red cells.

We investigated the role of protein phosphorylation in the action of anti-L on low potassium (LK) sheep red cells. Anti-L stimulated the Na/K pump by four- to fivefold, but Na/K pump activity in anti-L-stimulated or in control cells was unaffected by protein kinase/protein phosphatase (PK/PP) inhibitors. KCl co-transport activity was inhibited by anti-L (about 50%). Co-transport was stimulated by staurosporine; and inhibited by calyculin A, okadaic acid, tyrphostin B46 and genistein; with a similar pattem in both control and anti-L-treated cells. O2 sensitivity of KCl co-transport was similar in control and anti-L-treated cells. Neither control nor anti-L-stimulated Na/K pump activities were O2 sensitive. Incubation with urea stimulated KCl co-transport in both control and anti-L-treated cells. Inhibition of co-transport by anti-L was unaffected by low concentrations of urea but was reduced at higher urea concentrations. Na/K pump activity of control cells was unaffected by incubation with urea, but that in cells stimulated by anti-L was reduced, though not significantly. Under high hydrostatic pressure, KCl co-transport was stimulated, and the inhibitory effects of PP inhibition (okadaic acid), anti-L or combinations of the two were reduced. Results suggest that anti-L does not affect K+ transport in LK sheep red cells via protein phosphorylation.

Animals↗
PubMed2000

Elevated temperatures enhance KCCl activity in sickle cells.

The maintenance of red blood cell volume is important in the pathophysiology of sickle cell disease. The KCl cotransporter (KCCl) is capable of mediating sickle cell dehydration. In this study, we have determined the effect of increased temperature (over the range 37-41 degrees C) on basal K+ transport and K+ transport following activation of KCCl by urea or N-ethylmaleimide (NEM). An increased temperature was found to have only a small effect (approximately a 20% increase) on basal K+ transport. In contrast, the increase was much greater (about 60%) after activation of KCCl by urea. Following activation of KCCl by NEM, the increase in K+ transport with increasing temperature was small (about 10%). This suggests that it is the signalling system rather than the transporter itself that is sensitive to temperature.

Anemia, Sickle Cell↗
PubMed2000

A comparison in normal individuals and sickle cell patients of reduced glutathione precursors and their transport between plasma and red cells.

INTRODUCTION: Reduced glutathione is an important antioxidant in red cells whose depletion may contribute to the pathophysiology of sickle cell disease. The current study was designed to examine the availability of reduced glutathione precursors (glutamate, cysteine, glycine and possibly glutamine) together with the activity of the main transport pathways for their uptake (system ASC for cysteine and glycine; system gly for glycine). MATERIALS AND METHODS: Blood samples were obtained from normal (HbAA, HbA cells) and sickle cell disease patients (HbSS, HbS cells); amino acids were measured by HPLC; and transporter activity was measured by radioactive tracer fluxes (using serine and glycine for activity of system ASC; and glycine for that of system gly). RESULTS: Plasma concentrations of cysteine and glycine were increased and concentrations of all amino acids were elevated in HbS cells. The activity of system ASC was increased in HbS cells (both transport capacity and affinity were elevated for serine transport; transport capacity only for glycine). Activity of system gly was also increased (twofold increase in V(max) for glycine flux), though not significantly. Oxygenation also increased the activity of both transporters in normal and HbS cells. CO prevented deoxy-inhibition of glycine transport. Staurosporine (5 microM) inhibited O(2)-stimulated glycine transport through system ASC. It also inhibited the absolute magnitude of transport through system gly, but the O(2)-dependent flux was unaffected. CONCLUSION: Low reduced glutathione levels in HbS cells were not due to decreased substrate availability and O(2) stimulated transport of reduced glutathione precursors in both normal and HbS cells, through a mechanism that is likely to involve Hb and possibly protein phosphorylation.

Adult↗
PubMed2000

Oxygen-sensitive membrane transporters in vertebrate red cells.

Oxygen is essential for all higher forms of animal life. It is required for oxidative phosphorylation, which forms the bulk of the energy supply of most animals. In many vertebrates, transport of O(2) from respiratory to other tissues, and of CO(2) in the opposite direction, involves red cells. These are highly specialised, adapted for their respiratory function. Intracellular haemoglobin, carbonic anhydrase and the membrane anion exchanger (AE1) increase the effective O(2)- and CO(2)-carrying capacity of red cells by approximately 100-fold. O(2) also has a pathological role. It is a very reactive species chemically, and oxidation, free radical generation and peroxide formation can be major hazards. Cells that come into contact with potentially damaging levels of O(2) have a variety of systems to protect them against oxidative damage. Those in red cells include catalase, superoxide dismutase and glutathione. In this review, we focus on a third role of O(2), as a regulator of membrane transport systems, a role with important consequences for the homeostasis of the red cell and also the organism as a whole. We show that regulation of red cell transporters by O(2) is widespread throughout the vertebrate kingdom. The effect of O(2) is selective but involves a wide range of transporters, including inorganic and organic systems, and both electroneutral and conductive pathways. Finally, we discuss what is known about the mechanism of the O(2) effect and comment on its physiological and pathological roles.

Animals↗
PubMed1999

Passive Ca(2+) transport and Ca(2+)-dependent K(+) transport in Plasmodium falciparum-infected red cells.

Previous reports have indicated that Plasmodium falciparum-infected red cells (pRBC) have an increased Ca(2+) permeability. The magnitude of the increase is greater than that normally required to activate the Ca(2+)-dependent K(+) channel (K(Ca) channel) of the red cell membrane. However, there is evidence that this channel remains inactive in pRBC. To clarify this discrepancy, we have reassessed both the functional status of the K(Ca) channel and the Ca(2+) permeability properties of pRBC. For pRBC suspended in media containing Ca(2+), K(Ca) channel activation was elicited by treatment with the Ca(2+) ionophore A23187. In the absence of ionophore the channel remained inactive. In contrast to previous claims, the unidirectional influx of Ca(2+) into pRBC in which the Ca(2+) pump was inhibited by vanadate was found to be within the normal range (30-55 micromol (10(13) cells. hr)(-1)), provided the cells were suspended in glucose-containing media. However, for pRBC in glucose-free media the Ca(2+) influx increased to over 1 mmol (10(13) cells. hr)(-1), almost an order of magnitude higher than that seen in uninfected erythrocytes under equivalent conditions. The pathway responsible for the enhanced influx of Ca(2+) into glucose-deprived pRBC was expressed at approximately 30 hr post-invasion, and was inhibited by Ni(2+). Possible roles for this pathway in pRBC are considered.

Animals↗
PubMed1999

Neutral amino acid transport in bovine articular chondrocytes.

1. The sodium-dependent amino acid transport systems responsible for proline, glycine and glutamine transport, together with the sodium-independent systems for leucine and tryptophan, have been investigated in isolated bovine chondrocytes by inhibition studies and ion replacement. Each system was characterized kinetically. 2. Transport via system A was identified using the system-specific analogue alpha-methylaminoisobutyric acid (MeAIB) as an inhibitor of proline, glycine and glutamine transport. 3. Uptake of proline, glycine and glutamine via system ASC was identified by inhibition with alanine or serine. 4. System Gly was identified by the inhibition of glycine transport with excess sarcosine (a substrate for system Gly) whilst systems A and ASC were inhibited. This system, having a very limited substrate specificity and tissue distribution, was also shown to be Na+ and Cl- dependent. Evidence for expression of the system Gly component GLYT-1 was obtained using the reverse transcriptase-polymerase chain reaction (RT-PCR). 5. System N, also of narrow substrate specificity and tissue distribution, was shown to be present in chondrocytes. Na+-dependent glutamine uptake was inhibited by high concentrations of histidine (a substrate of system N) in the presence of excess MeAIB and serine. 6. System L was identified using the system specific analogue 2-aminobicyclo(2,2, 1)heptane-2-carboxylic acid (BCH) and D-leucine as inhibitors of leucine and tryptophan transport. 7. The presence of system T was tested by using leucine, tryptophan and tyrosine inhibition. It was concluded that this system was absent in the chondrocyte. 8. Kinetic analysis showed the Na+-independent chondrocyte L system to have apparent affinities for leucine and tryptophan of 125 +/- 27 and 36 +/- 11 microM, respectively. 9. Transport of the essential amino acids leucine and tryptophan into bovine chondrocytes occurs only by the Na+-independent system L, but with a higher affinity than the conventional L system.

Amino Acids↗
PubMed1999

Effect of the substituted benzaldehyde 12C79 on Cl--dependent K+ influx in human red blood cells.

Ouabain- and bumetanide-resistant K+ influx, and haemoglobin (Hb) O2 saturation, were measured in HbA red cells over a range of oxygen tensions (PO2 values) in the presence and absence of 12C79 (5 mM), a substituted benzaldehyde which increases the O2 affinity of Hb. PO2 values for half-maximal O2 saturation declined from 29+/-2 mmHg (mean +/-SEM, n=3) in control cells to 7+/-1 mmHg with 12C79. In control cells, Cl--dependent K+ influx (indicative of KCl cotransport activity) was fully O2 dependent, i.e. inactive at low PO2 values. By contrast, in the presence of 12C79, KCl cotransport was largely resistant to inactivation at low PO2 values. Substantial cotransport activity was still present (>60% of that at high PO2 values) in N2, although O2 saturation was low (about 10%). In all cases, Cl--independent K+ influxes were low [<0.25 mmol (l cells h)-1] and unaffected by PO2 or 12C79. The significance of these results is discussed.

Antisickling Agents↗
PubMed1999

Regulatory volume response of erythrocytes exposed to a gradual and slow decrease in medium osmolality.

A sudden decrease in external medium osmolality (90 mosmol/kg) causes an immediate swelling of trout erythrocytes, followed by a regulatory volume decrease (RVD) due to activation of both a KCl cotransporter and a taurine transport pathway. Here, we determined how trout red cells respond when they are exposed to a gradual and slow decrease in medium osmolality (80 mosmol/kg at a rate of 0.7 mosmol/kg per min). Erythrocytes were unable to regulate their volume efficiently when swollen gradually and it increased continuously throughout the experimental period (120 min). As long as volume was increased slowly by 15-25%, regulatory pathways remained essentially inactivated, erythrocytes losing no significant amount of intracellular osmotically active solutes. Above this swelling threshold, a response was triggered but the quantity of solutes lost via the regulatory pathways was still not sufficient to counterbalance the continuous entry of water due to the slow and gradual decrease in medium tonicity.

Animals↗
PubMed1999

Identification of system y+L as the high-affinity transporter for L-arginine in human platelets: up-regulation of L-arginine influx in uraemia.

Kinetic studies of L-arginine transport in human platelets have identified a high-affinity, low-capacity transport system [Michaelis-Menten constant (K(m)) about 10 microM] for cationic amino acids that also transports neutral amino acids with high affinity in the presence of Na+ but not K+. These characteristics, together with our kinetic cis-inhibition studies, indicate that saturable L-arginine transport in human platelets is mediated via the system y+L and not the classic cationic transporter system y+. We present here the first evidence that L-arginine transport via system y+L is increased twofold in platelets from patients with chronic renal failure. System y+L has been described in human erythrocytes, peripheral blood mononuclear cells and placenta, and up-regulation of system y+L activity in human platelets could explain the paradox of increased nitric oxide (NO) production by uraemic platelets under conditions of decreased plasma L-arginine and elevated NG-monomethyl-L-arginine (L-NMMA) concentrations.

Amino Acid Transport Systems, Basic↗
PubMed1999

Characterization of the K+ (Na+)/H+ monovalent cation exchanger in the human red blood cell membrane: effects of transport inhibitors.

The (ouabain + bumetanide + EGTA)-insensitive K+ influx (defined as residual K+ influx) in the human erythrocyte was investigated with respect to the characterization of the recently identified K+(Na+)/H+ exchanger (Richter et al. 1997). In particular, the effects of selected ion transport inhibitors on this flux in physiological ionic strength (high ionic strength, HIS) as well as low ionic strength (LIS) solutions were qstudied. The stimulation of the K+ influx observed in LIS medium was further enhanced when DIDS, phloretin, eosin-5-maleimide, furosemide, DIOA, NPPB, or DCDPC was present at a concentration of 0.1 mmol/l. This paradoxical, inhibitor-induced increase of the K+ influx was more pronounced in LIS media where chloride (7.5 mmol/l) was replaced by nitrate. For DNDS, niflumic acid, and MK-196 (0.1 mmol/l) an enhanced K+ transport could only be observed in nitrate-containing LIS solution. Bumetanide and purine riboside, at a concentration of 0.1 mmol/l, did not cause significant changes of the K+ influx in either chloride- or nitrate-containing LIS media. Dipyridamole and ruthenium red (0.1 mmol/l), which are positively charged, significantly reduced the K+ influx in both chloride- and nitrate-containing LIS media. In nitrate-containing HIS solution only dipyridamole inhibited the K+ influx. The residual K+ influx in LIS solution was significantly increased by removing internal [Mg2+], and decreased by quinacrine (1 mmol/l). In HIS solution, no effect of altering intracellular Mg2+ occurred but a stimulation of the flux by quinacrine was observed. The results are discussed in terms of a more general surface charge effect of the used inhibitors on the K+(Na+)/H+ exchanger.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1998

Differential oxygen sensitivity of the K+-Cl- cotransporter in normal and sickle human red blood cells.

1. K+ influx and efflux were measured in normal (HbA) and sickle (HbS) red blood cells to investigate the interaction of swelling, H+ ions and urea with O2 (0 to 150 mmHg O2) in the presence of ouabain and bumetanide (both 100 microM). 2. In HbA cells, K+-C1- cotransport was O2 dependent. At low oxygen tensions (PO2s) the transporter was inactive and refractory to low pH, swelling or urea. 3. C1--independent K+ influxes in sickle cells were elevated at low PO2s, as previously reported. C1--dependent K+ influxes were large at both high and low PO2s, whether stimulated by swelling, H+ ions or urea. In the absence of O2, C1--dependent K+ influxes were similar in magnitude to those measured at high PO2s. The minimum for C1--dependent K+ influx was observed at PO2s of about 40-70 mmHg. 4. K+ efflux from HbS cells was stimulated by the addition of urea (500 mM). The rate constants were of similar magnitude whether measured at high PO2 or in the absence of O2, and were predominantly C1- dependent under both conditions. 5. In HbS red blood cells, reduction of extracellular Ca2+, addition of 1 mM Mg2+ or nitrendipine (10 microM) to the saline had no effect. Inhibitors of K+-C1- cotransport, [(dihydroindenyl)oxy] alkanoic acid (DIOA; 100 microM) or calyculin A (0.1 microM), inhibited influxes by a similar magnitude to C1- substitution. 6. Results are significant for the pathophysiology of sickle cell disease. Low pH and urea are able to stimulate KC1 loss from sickle cells, leading to cellular dehydration, even in regions of low PO2.

Anemia, Sickle Cell↗
PubMed1998

Inhibition of Na+, K+-ATPase activates swelling-induced taurine efflux in a human neuroblastoma cell line.

The Na+ pump (Na+, K+-ATPase) has been implicated in the regulation of many cellular functions, including cell volume regulation. The effects of inhibiting Na+ pump activity on cell volume and taurine efflux were evaluated in the human neuroblastoma cell line CHP-100. Cell volume changes monitored with the Coulter Multisizer technique and confocal microscopy showed that neuroblastoma cells exposed to ouabain swelled by 22 +/- 4% (n = 5). The rapid cell swelling was followed by regulatory volume decrease (RVD). In cells treated with ouabain, 14C-taurine efflux increased by 183 +/- 11% compared with controls. However, cells exposed simultaneously to ouabain and hypoosmotic solution resulted in a 14C-taurine efflux of 207 +/- 18%. Western blot and immunofluorescence microscopy with specific monoclonal antibodies for the catalytic alpha isoforms of Na+, K+-ATPase demonstrated high levels of the ubiquitously expressed alpha1 and the neuronal-specific alpha3. Ouabain-binding data showed that CHP-100 cells express approximately 3 x 10(5) pump units/cell. The present data indicate that efflux of taurine may be involved during volume recovery subsequent to blockade of Na+, K+-ATPase in CHP-100 cells.

Biological Transport↗
PubMed1998

Expression of cation exchanger NHE and anion exchanger AE isoforms in primary human bone-derived osteoblasts.

The authors used isoform-specific antibodies against cation (NHE) and anion (AE) exchange isoforms in order to establish their specific expression and localization in dispersed human bone-derived cells. Immunocytochemical preparations of permeabilized osteoblasts probed with polyclonal antibodies were optically analysed by conventional immunofluorescence and con-focal laser scanning microscopy. These techniques demonstrated the abundant presence of epitopes of the cation exchangers NHE1 and NHE3 and the anion exchanger AE2 in these cells. The NHE1 and NHE3 isoform proteins were predominantly located in subplasmalemmal and nucleoplasmic vesicles. The AE2 isoform was densely localized to a subcellular location characteristic of the Golgi complex. The molecular identity of the AE and NHE isoforms was investigated by RT-PCR that confirmed the presence of NHE1 and NHE3 transcripts in addition to NHE4. RT-PCR and diagnostic restriction analysis of amplified AE cDNA established preferential AE2 expression. Since AE2 has been shown to act as a sulfate transporter at low pH, it is possible that it performs this function in the osteoblast Golgi complex where sulfation reactions occur post-translationally on numerous extracellular matrix macromolecules prior to secretion and mineralization. The Na(+)/H(+)exchanger proteins are regulated by mitogenic and non-mitogenic stimuli in the osseus environment and are involved in the large fluxes of ions and protons that necessarily occur during bone formation and resorption and thus play an important role in intracellular ion homeostasis in osteoblasts.

Alkaline Phosphatase↗
PubMed1998

Ca2+-dependent capacitance increases in rat basophilic leukemia cells following activation of store-operated Ca2+ entry and dialysis with high-Ca2+-containing intracellular solution.

Ca2+-dependent vesicular fusion was studied in single whole-cell patch-clamped rat basophilic leukemia (RBL) cells using the capacitance technique. Dialysis of the cells with 10 microM free Ca2+ and 300 microM guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S]) resulted in prominent capacitance increases. However, dialysis with either Ca2+ (225 nM to 10 microM) or GTP[gamma-S] alone failed to induce a capacitance change. Under conditions of weak Ca2+ buffering (0.1 mM EGTA), activation of Ca2+-release-activated Ca2+ (CRAC) channels by dialysis with inositol 1,4,5-trisphosphate (InsP3) failed to induce a capacitance increase even in the presence of GTP[gamma-S]. However, when Ca2+ATPases were inhibited by thapsigargin, InsP3 and GTP[gamma-S] led to a pronounced elevation in membrane capacitance. This increase was dependent on a rise in intracellular Ca2+ because it was abolished when cells were dialysed with a high level of EGTA (10 mM) in the recording pipette. The increase was also dependent on Ca2+ influx because it was effectively suppressed when external Ca2+ was removed. Our results demonstrate that ICRAC represents an important source of Ca2+ for triggering a secretory response.

Animals↗