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J C Ellory

Publications and source records attributed to J C Ellory.

At least 55 records · Page 3Linked to original sources
PubMed1998

Effects of urea and oxygen tension on K flux in sickle cells.

K influx and efflux (both ouabain- and bumetanide-resistant) in haemoglobin S-containing red cells (sickle cells) were markedly stimulated by urea (> 0.25 M). Stimulation was rapid and reversible. Volume-sensitive KCl cotransport in both HbA or HbS red cells is thought to be O2-dependent but we show here that urea-stimulated K fluxes in sickle cells were largely insensitive to O2 tension. Urea-stimulated K fluxes were not inhibited by lowering the external Ca concentration (with EGTA) but were abolished by Cl-substitution (with MeSO4 or NO3) or pretreatment of cells with the protein phosphatase inhibitor, calyculin A (0.1 muM). Results are consistent with a stimulatory action of urea on the KCl cotransporter, independent of oxygen tension, mediated via the phosphorylation cascade which regulates the transporter. The importance of this effect to the physiology and pathology of sickle cells is discussed.

Anemia, Sickle Cell↗
PubMed1998

Increased L-arginine transport in human erythrocytes in chronic heart failure.

1. Transport of L-arginine was investigated under zero-trans conditions in human erythrocytes from healthy donors and patients with heart failure. 2. Saturable influx of L-arginine was mediated by the classical cationic amino acid transport systems y+ and y+L. 3. The Vmax for L-arginine transport via system y+ increased from 292 to 490 mumol h-1 l-1 of cells in heart failure. 4. With system y+ inhibited by N-ethylmaleimide (0.2 mmol/l), the Vmax for the transport of L-arginine via system y+L was unaffected in erythrocytes from patients with heart failure. 5. The inhibition of L-arginine and L-leucine influx by NG-monomethyl-L-arginine was similar in erythrocytes from control and heart failure patients. 6. Plasma L-arginine levels were reduced in patients with heart failure (59 mumol/l) compared with controls (125 mumol/l). Plasma from patients with heart failure also contained the endogenous L-arginine analogue NG-monomethyl-L-arginine, which was undetectable in plasma from controls. 7. Intracellular concentrations of L-arginine and NG-monomethyl-L-arginine were significantly elevated in erythrocytes from patients with heart failure compared with controls, consistent with an increased transport capacity for L-arginine and NG-monomethyl-L-arginine. 8. The present study provides the first evidence that system y+ mediates the increased transport of L-arginine in human erythrocytes from patients with chronic heart failure. These findings are similar to our previous results obtained in patients with chronic renal failure. Since both pathologies seem to present with an increased synthesis of nitric oxide, studies of L-arginine transport in erythrocytes may provide a valuable paradigm to study abnormalities of the L-arginine-nitric oxide signalling pathway.

Amino Acids↗
PubMed1998

Swelling-induced arachidonic acid release via the 85-kDa cPLA2 in human neuroblastoma cells.

Arachidonic acid or its metabolites have been implicated in the regulatory volume decrease (RVD) response after hypotonic cell swelling in some mammalian cells. The present study investigated the role of arachidonic acid (AA) during RVD in the human neuroblastoma cell line CHP-100. During the first nine minutes of hypo-osmotic exposure the rate of 3H-arachidonic acid (3H-AA) release increased to 250 +/- 19% (mean +/- SE, n = 22) as compared with cells under iso-osmotic conditions. This release was significantly inhibited after preincubation with AACOCF3, an inhibitor of the 85-kDa cytosolic phospholipase A2 (cPLA2). This indicates that a PLA2, most likely the 85-kDa cPLA2 is activated during cell swelling. In contrast, preincubation with U73122, an inhibitor of phospholipase C, did not affect the swelling-induced release of 3H-AA. Swelling-activated efflux of 36Cl and 3H-taurine were inhibited after preincubation with AACOCF3. Thus the swelling-induced activation of cPLA2 may be essential for stimulation of both 36Cl and 3H-taurine efflux during RVD. As the above observation could result from a direct effect of AA or its metabolite leukotriene D4 (LTD4), the effects of these agents were investigated on swelling-induced 36Cl and 3H-taurine effluxes. In the presence of high concentrations of extracellular AA, the swelling-induced efflux of 36Cl and 3H-taurine were inhibited significantly. In contrast, addition of exogenous LTD4 had no significant effect on the swelling-activated 36Cl efflux. Furthermore, exogenous AA increased cytosolic calcium levels as measured in single cells loaded with the calcium sensitive dye Fura-2. On the basis of these results we propose that cell swelling activates phospholipase A2 and that this activation via an increased production of AA or some AA metabolite(s) other than LTD4 is essential for RVD.

Arachidonic Acid↗
PubMed1997

Transport of L-arginine and the nitric oxide inhibitor NG-monomethyl-L-arginine in human erythrocytes in chronic renal failure.

1. Transport of L-arginine and the nitric oxide synthase inhibitors NG-monomethyl-L-arginine and NG-nitro-L-arginine was investigated in human erythrocytes from healthy donors and uraemic patients on haemodialysis. 2. Although K(m) values for total L-arginine influx were not significantly different in erythrocytes freshly isolated from controls or uraemic patients, uraemia was associated with an increase in the Vmax for transport (826 compared with 1176 mumol h-1 l-1 of cells) which was reduced to control values after dialysis. 3. Saturable influx of L-arginine was mediated by the classical cationic amino acid transport system y+ and system y+L, known to transport cationic and neutral amino acids with higher affinity. 4. Under zero-trans conditions, the Vmax for L-arginine transport via system y+increased from 271 to 700 mumol h-1 l-1 of cells in uraemia, while K(m) values increased from 44 to 94 mumol/l. Dialysis had no significant effect on the kinetic parameters altered by uraemia. 5. Under zero-trans conditions, and with system y+ inhibited by N-ethylmaleimide (0.2 mmol/l), transport of L-arginine via system y+L was unaffected by uraemia. 6. Saturable influx of NG-monomethyl-L-arginine was also mediated by systems y+ (K(m) = 56 mumol/l, Vmax = 353 mumol h-1 l-1 of cells) and y+L (K(m) = 17 mumol/l, Vmax = 51.3 mumol h-1 l-1 of cells) and, as with L-arginine, uraemia increased the transport capacity for NG-monomethyl-L-arginine. 7. Influx of the neutral nitric oxide synthase inhibitor NG-nitro-L-arginine was not readily saturable. 8. Intracellular concentrations of L-arginine and NG-monomethyl-L-arginine were significantly increased in erythrocytes from uraemic patients when compared with controls, consistent with an increased transport capacity for L-arginine and NG-monomethyl-L-arginine. 9. The present study provides evidence that system y+ mediates the increased transport of L-arginine and NG-monomethyl-L-arginine in human erythrocytes from patients with chronic renal failure. Our findings may have implications for the activity of the L-arginine-nitric oxide signalling pathway in vascular endothelial and smooth-muscle cells in uraemia.

Adult↗
PubMed1997

Do HbSS erythrocytes lose KCl in physiological conditions?

KCl cotransporter activity in sickle (HbSS) red blood cells (RBCs) was measured in cells suspended in 'simple' physiological saline, saline augmented with inorganic salts, and autologous plasma. Our results showed that the transporter was only functioning at 20% of the level of cells in saline when cells were resuspended in autologous plasma. Kinetic analysis of the data showed that plasma decreased both Vmax and Km for K+ of the transporter. The plasma factor(s) responsible was heat-stable and dialysable (i.e. size < 10 kD). Adding magnesium, calcium, inorganic phosphate or bicarbonate to 'simple' saline to mimic the effect of plasma revealed that Mg2+ and Ca2+ had no significant effect at physiological concentrations. Pi was not effective at 1.1 mM, but did inhibit significantly (42+/-2%) at 5.6 mM. HCO3- had a major inhibitory effect on K+ influx when added to saline, and was identified as the principal candidate for the plasma effect. We suggest bicarbonate may play a significant role in modifying KCl cotransport, and hence HbSS cell volume in vivo. It acts by altering the set point of the transporter via the signalling systems involved in its regulation.

Adolescent↗
PubMed1996

KCl cotransport activation in human erythrocytes by high hydrostatic pressure.

1. Pressure induced a 4- to 5-fold stimulation of the residual (i.e. oubain-bumetanide insensitive) 86Rb+ influx across the human red cell membrane. This enhancement showed a broad pHo dependence with a maximum stimulation around pHo 7. 2. At atmospheric pressure, the protein kinase inhibitors staurosporine and chelerythrine stimulated a normally silent component of 86Rb+ influx in a dose-dependent manner with a half-maximum stimulatory concentration at about 550 nM and 140 microM, respectively. The component stimulated by staurosporine was entirely Cl- dependent, but part of the chelerythrine effect was Cl- independent. 3. Staurosporine (3 microM), chelerythrine (200 microM) and N-ethylmaleimide (1 mM) stimulated further the increased residual 86Rb+ influx in cells at high pressure. 4. The serine/threonine protein phosphatase inhibitors okadaic acid, cantharidin and calyculin A inhibited the stimulatory pressure effect in a dose-dependent manner with half-maximum inhibitory concentrations of 70 nM, 2.5 microM and 3.3 nM, respectively. In contrast, deltamethrin, a specific protein phosphatase type 2B inhibitor, did not affect the stimulation by pressure, up to a concentration of 10 microM. 5. Decreasing the internal ionized magnesium concentration ([Mg2+]i) with A23187 and EDTA stimulated the increased residual 86Rb+ influx in cells at high pressure. On the other hand, increasing the [Mg2+]i nearly abolished the stimulatory pressure effect. 6. Decreasing the [Mg2+]i produced a marked change in the pHo dependence curve, with a linear increase of the 86Rb+ influx at higher pHo values. 7. We demonstrate that high pressure stimulates the normally silent component of 86Rb+ influx by modifying the phosphorylation/dephosphorylation ratio of the KCl cotransporter.

Cell Membrane↗
PubMed1996

Volume-activated taurine permeability in cells of the human erythroleukemic cell line K562.

The effects of hypotonic shock on cell volume, taurine influx and efflux were examined in the human erythroleukemic cell line K562. Cells exposed to hypotonic solutions exhibited a regulatory volume decrease (RVD) following rapid increases in cell volume. Cell swelling was associated with a increased taurine influx and efflux. The volume-activated taurine pathway was Na(+)-independent, and increased in parallel with increasing cell volume. The chloride channel blocker, 2,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC), completely blocked the volume-activated taurine influx and efflux, while [dihydroindenyl)oxy]alkanoic acids (DIOA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), an anion exchanger and anion channel blocker, respectively, also inhibited significantly. These results suggest that taurine transport is increased in response to hypotonic stress, which may be mediated via a volume-activated, DCDPC-sensitive anion channel.

Anions↗
PubMed1996

The role of swelling-induced anion channels during neuronal volume regulation.

Regulation of cell volume is an essential function of most mammalian cells. In the cells of the central nervous system, maintenance of cell osmolarity and, hence, volume, is particularly crucial because of the restrictive nature of the skull. Cell volume regulation involves a variety of pathways, with considerable differences between cell types. One common pathway activated during hypo-osmotic stress involves chloride (Cl-) channels. However, hypo-osmotically stimulated anion permeability can be regulated by a diverse array of second messengers. Although neuronal swelling can occur in a number of pathological and nonpathological conditions, our understanding of neuronal volume regulation is limited. This article summarizes our current understanding of the role of anion channels during neuronal volume regulation.

Adenosine Triphosphate↗
PubMed1996

Human erythrocyte choline uptake in uraemia: the role of intracellular substrate and an investigation into the effects of haemodialysis.

1. Erythrocyte choline transport was studied in 10 haemodialysis patients immediately before and after a haemodialysis session and in 10 control subjects. Choline uptake was measured in erythrocytes from normal and uraemic patients after washing in vitro and subsequent incubation in autologous plasma. Amines present in uraemic plasma were examined for their effect on choline transport in normal erythrocytes. 2. NMR spectroscopy was used to measure choline, trimethylamine and dimethylamine in erythrocyte extracts from nine control subjects, 32 subjects with renal impairment and nine samples from haemodialysis patients. 3. The increased choline influx in uraemic erythrocytes is significantly decreased by prior haemodialysis (mean Vmax pre-dialysis 146 +/- 20 mumol h-1 l-1, post-dialysis 113 +/- 13 mumol h-1 l-1 (P < 0.005). After in vitro washing there is a fall in Vmax, and no longer any significant difference between pre- and post-dialysis samples. There remains a significant difference in the erythrocyte choline Vmax between samples from patients with chronic renal failure and from normal subjects (P < 0.005). 4. Human plasma was found to contain factors capable of increasing choline uptake. Trimethylamine and dimethylamine were found to inhibit choline uptake. Trimethylamine and trimethylamine-N-oxide trans-stimulated choline efflux, but the major transport substrate present in erythrocyte extracts from all groups was choline, which was higher in those with renal impairment (71 +/- 10 mumol/l) than in haemodialysis patients (47 +/- 10 mumol/l) and control subjects with normal renal function (40 +/- 9 mumol/l). 5. Our data suggest that erythrocyte choline transport is increased in uraemia as a consequence of increased transporter number or activity, rather than the presence of intracellular substrate.

Choline↗
PubMed1996

Accumulation of the endogenous L-arginine analogue NG-monomethyl-L-arginine in human end-stage renal failure patients on regular haemodialysis.

We measured plasma levels of L-arginine, its analogue NG-monomethyl-L-arginine (L-NMMA), and related amino acids in normal subjects and uraemic patients (n = 31), before and after haemodialysis. Plasma levels of L-arginine were reduced to less than half of normal values in uraemic subjects compared with controls, and were not affected by haemodialysis. L-NMMA was not detectable in non-uraemic subjects, but markedly elevated in uraemic patients. In parallel, we used human red blood cells as a model to study the effect of L-NMMA upon the transport of L-arginine. L-NMMA trans-stimulated L-arginine transport significantly, confirming that L-arginine and L-NMMA share common transport pathways. Our results suggest altered L-arginine metabolism and the presence of an increased concentration of a NO synthase inhibitor in uraemia. We propose that alterations in plasma L-arginine levels and increased production of L-arginine analogues will alter NO synthesis and may help to explain some pathological changes seen in uraemia.

Adult↗
PubMed1996

Swelling-activated amino acid efflux in the human neuroblastoma cell line CHP-100.

1. The effects of hypoosmotic stress on cell volume and amino acid efflux were evaluated in the human neuroblastoma cell line CHP-100 with the Coulter Counter Multisizer and radiolabeled amino acid efflux, respectively. 2. CHP-100 cells swelled by approximately 35 +/- 5% (means +/- SE) when the osmolarity of the solution was decreased from 290 to 190 mOsm/kg H2O. The rapid swelling was followed by a biphasic regulatory volume decrease (RVD). 3. In cells loaded with 14C-taurine, hypoosmotic stress induced a 300 +/- 22% (n = 23, P < 0.05) increase in taurine efflux compared with controls. This efflux was inhibited by the chloride channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), 4,4'-diisothio-cyanostilbene-2,2'-disulfonic acid (DIDS), niflumic acid and by the volume-activated anion channel blocker tamoxifen. In addition, the swelling-activated taurine efflux was dependent upon extracellular calcium. 4. Similarly, in cells loaded with 14C-glycine, hypoosmotic stress significantly increased glycine efflux, which was also sensitive to NPPB. In contrast, efflux of 3H-glutamate was not significantly altered after hypoosmotic stress. 5. With the use of patch clamp recording techniques, Cl- channels were activated in cell attached patches after exposure to hypoosmotic solutions. 6. In nystatin perforated patches, permeability of the hypoosmotically activated anion channel was observed to be SCN- > I- > Br- > Cl- >> Glutamate. 7. It is concluded that in CHP-100 cells, anion channels are activated during hypoosmotic stress and these channels represent a pathway for efflux of amino acids.

Amino Acids↗
PubMed1995

Activation of a novel organic solute transporter in mammalian red blood cells.

1. Suspending human red blood cells in isotonic sucrose (low ionic strength, LIS) medium induces a significant increase in membrane transport of glutamine, glutamate, lactate, histidine, taurine, glycine, serine, choline and carnitine but not sorbitol or sucrose. 2. Progressive lowering of ionic strength by sucrose or NaCl replacement gave a similar activation profile for taurine influx as found earlier for residual K+(86Rb+) flux. 3. The induced taurine transport could be measured as enhanced influx and efflux. Influx was linear with external concentration up to 10 mM, largely insensitive to alteration in cell volume, and did not vary with red blood cell age. 4. Unlike previous results for residual K+ transport, altering transmembrane potential with gluconate or glucuronate media led to an increase in taurine influx similar to that observed in LIS media. Varying medium pH confirmed the effect was not due to alteration in pH. 5. The LIS-induced flux was sensitive to a variety of 'classical' anion transport inhibitors in the order of potency DNDS, DIDS, NPPB, DIOA, niflumic acid, furosemide (frusemide), glibenclamide, nitrendipine and bumetanide. 6. The taurine flux showed a temperature dependence similar to that of the LIS-induced residual K+ flux. High hydrostatic pressure (40 MPa), however, inhibited taurine flux but stimulated residual K+ influx in LIS media. 7. A significant enhanced taurine flux could be demonstrated in red blood cells of other species, including horse, cattle, pig and high and low potassium type sheep. 8. It is concluded that lowering ionic strength activates a transport pathway for organic molecules sharing some similarities with background Cl- channels and LIS-induced residual K+ fluxes. In the latter context, however, there are certain significant differences (effect of transmembrane potential; volume; pressure sensitivity; species distribution) which may be important, and the unequivocal identity of the two transport processes remains to be confirmed.

Animals↗
PubMed1995

Swelling-induced chloride currents in neuroblastoma cells are calcium dependent.

The effects of osmotic stress on chloride (CI-) currents in the human neuroblastoma cell line CHP-100 were evaluated. Following exposure to hypoosmotic solution, an increase in whole-cell CI- current was observed. This current was blocked by the CI- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB). In cells loaded with the CI- permeability marker 125I, exposure to hypoosmotic solution increased 125I efflux by 197 +/- 14% (n = 41, p < 0.05) over controls. This increase was sensitive to NPPB. Hypoosmotic stress also increased cytosolic calcium levels (Ca2+) in fura-2-loaded cells. Pretreatment with EGTA inhibited the increase in cytosolic Ca2+, 125I efflux, and whole-cell CI- current produced by hypoosmotic solution. Antagonists of N-, L-, and T-type Ca2+ channels did not alter stimulation in 125I efflux or cytosolic Ca2+ levels during osmotic stress. However, omega-conotoxin MVIIC, a P-type Ca2+ channel blocker, inhibited hypoosmotically activated whole-cell CI- currents and increases in cytosolic Ca2+. It is concluded that a Ca(2+)-dependent change in CI- permeability is activated in CHP-100 cells in response to osmotic stress.

Barium Compounds↗
PubMed1994

Transport of diverse substrates into malaria-infected erythrocytes via a pathway showing functional characteristics of a chloride channel.

Following infection by the malaria parasite, Plasmodium falciparum, human erythrocytes show increased permeability to a variety of low molecular weight solutes. In this study a number of anion transport blockers were identified as potent inhibitors of the transport of a wide range of solutes into human erythrocytes infected in vitro with P. falciparum. 5-Nitro-2-(3-phenyl-propylamino)benzoic acid (NPPB), furosemide, and niflumate blocked the malaria-induced transport of monovalent cations, neutral amino acids, sugars, nucleosides, and monovalent anions. For all of the substrates tested the order of potency of these three inhibitors was the same (NPPB > furosemide > niflumate) and dose-response curves for the effect of these inhibitors on malaria-induced choline transport were similar to those for malaria-induced thymidine transport. The data suggest that much, if not all, of the high capacity (non-saturable) transport of low molecular weight solutes into P. falciparum-infected erythrocytes is via a single type of pathway. The broad specificity of the pathway, its non-saturability in the physiological concentration range, and its failure to distinguish between stereoisomers (L- and D-alanine) are consistent with its being a type of pore or channel. For those substrates for which quantitative influx measurements were made the magnitude of the malaria-induced (inhibitor-sensitive) transport was in the order: Cl- > lactate > thymidine, adenosine > carnitine > choline > K+. The pathway is therefore anion-selective. The pharmacological and substrate-selectivity properties of the pathway show marked similarities to those of chloride channels in other cell types; this raises the possibility that the high capacity transport of small organic solutes may be an important and, as yet, largely unrecognized role for such channels in other tissues.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Additive in vitro effects of anti-sickling drugs.

To study the effect of anti-sickling drugs on cellular dehydration induced by entry of Ca, sickle cells were subjected to cyclical oxygenation-deoxygenation for 15 h in Ca-containing buffer. The consequential loss of cation (K) via the Ca-dependent K efflux (Gardos) channel caused cell dehydration and loss of deformability. Inhibition of a specific fraction of Ca entry by verapamil had no rheologically protective effect, whereas inhibition of the Gardos channel by clotrimazole or nitrendipine had a marked protective effect. When Gardos channel inhibition (by either clotrimazole or nitrendipine) was combined with stabilization of the oxy-conformation of sickle haemoglobin (by the substituted benzaldehyde 12C79), an additive protective rheological effect was achieved with 60-78% reduction in clogging rate of 5 microns diameter pores when compared with no drug. Therapeutic use of anti-sickling compounds in combination may achieve increased efficacy with lower toxicity.

Anemia, Sickle Cell↗
PubMed1994

Specific inhibition of Ca-activated K channels in red cells by selected dihydropyridine derivatives.

1. Thirty two dihydropyridine derivatives were screened as potential inhibitors of the Ca-activated K-channel in human red cells. 2. Three derivatives (26, 29, 32 see Tables 1 and 2) with high activity were then characterized in detail, and also tested against the smooth muscle Ca-channel and shown to have varying potencies. 3. One of the more potent derivatives (32) and nitrendipine were also tested on the Ca-activated K-channel, Maxi-K channel, from mouse pancreatic beta-cells. 4. We conclude from our results that it may be possible to develop selective Gardos-channel inhibitors based on these molecules, which may be of benefit in the treatment of sickle cell disease.

Calcium↗