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Biomedical subjects

T J Simons

Publications and source records attributed to T J Simons.

35 records · Page 2Linked to original sources
PubMed1984

Active transport of lead by human red blood cells.

Human red cells suspended in lead-citrate buffers (2.6 microM Pb2+) take up much less Pb than predicted from studies of equilibrium binding of Pb to haemolysates. Pb uptake is increased by ATP depletion, or by loading at 0 degrees C. Tracer studies with 203Pb indicate that the low uptake at 37 degrees C in the presence of substrate is not due to membrane impermeability to Pb. Cold-loaded cells extrude Pb against a concentration gradient at 37 degrees C when glucose is present. These results suggest that the cellular loading of Pb is dependent on the balance between an inward leak and an outward pump. The extrusion of Pb from the cells is possibly brought by the Ca pump.

Adenosine Triphosphate↗
PubMed1983

Characterization of sugar transport in the pigeon red blood cell.

Sugar transport in pigeon red blood cells is mediated by two pathways. One is saturable, shows competition between sugars, is inhibited by phloretin and cytochalasin B, and shows many of the properties of 'carrier-mediated' transport, characterized in the human red blood cell. The other is not saturable, and shows no competition between sugars. The saturable pathway is virtually absent from freshly drawn cells, but may be stimulated by pre-incubation with 2 mM-NaCN, or by Ca and the ionophore A 23187. The non-saturating pathway is stimulated only slightly by CN, but considerably by Ca and A 23187. The inhibition of sugar transport by cytochalasin B is antagonized competitively by sugars acting at the inner surface of the membrane. External sugars have no effect, as in the human red blood cell (Widdas, 1980). The binding of cytochalasin B to the cells shows a limited number of high-affinity sites. These are unrelated to inhibition of sugar transport as binding, but not transport, is prevented by the presence of cytochalasin E.

3-O-Methylglucose↗
PubMed1983

The role of calcium in the regulation of sugar transport in the pigeon red blood cell.

The saturable, 'carrier-mediated' pathway for sugar transport in pigeon red blood cells may be stimulated by metabolic depletion or by loading the cells with Ca by means of a high-voltage discharge or the ionophore A 23187. All of these methods for stimulating the saturable pathway of sugar transport also cause a drop in cellular ATP levels. The relationship between the stimulation of transport and the fall in ATP is very similar in metabolically depleted or Ca-loaded cells, but cells treated with Ca and A23187 show a greater stimulation of transport than would be expected from the decline in ATP. Altering free Ca2+ levels during metabolic depletion has little or no effect on stimulation of the saturable pathway. Conversely, metabolic depletion of fresh cells in Ca-free solutions has no detectable effect on intracellular free Ca2+ levels. These results suggest that Ca2+ ions are not involved in regulation of this pathway. The non-saturable pathway for sugar transport is stimulated by a rise in cell Ca. This process is probably stimulated half-maximally by about 10 microM-free Ca2+.

3-O-Methylglucose↗
PubMed1982

A method for estimating free Ca within human red blood cells, with an application to the study of their Ca-dependent K permeability.

Murphy, Coll, Rich and Williamson (J. Biol. Chem. 255:6600--6608, 1980) described a null-point method for estimating intracellular free Ca in liver cells. They used digitonin to lyse the cells in solutions of varying Ca concentration. This method has been adapted for use with human red cells. The values found are about 0.4 micron or micrometer Ca in fresh cells, and from 0.4 to 0.7 micron or micrometer Ca in blood-bank cells, at pH 7.2 and 37 degrees C. These are likely to be overestimates, and the errors and limitations of the method are discussed. Red cells may be loaded with Ca by metabolic depletion in Ca-containing solutions. Such cells have an elevated K permeability, and the relationships between free Ca, total Ca and K permeability were investigated, using 86Rb as a tracer for K. 86Rb flux studies show that the affinity of the K channel for Ca is the same in cells as in resealed ghosts where intracellular Ca can be controlled with Ca buffers, but the rate of tracer equilibration is 3-6 times faster in ghosts than in cells.

Calcium↗
PubMed1979

Actions of a carbocyanine dye on calcium-dependent potassium transport in human red cell ghosts.

1. 3,3'-Diethylthiadicarbocyanine iodide (diS-C2-(5)), a fluorescent dye widely used to estimate membrane potentials, inhibits the Ca-dependent K transport system of human red cells and ghosts. It does not affect non-specific cation leaks, net chloride movements or the Ca-activated ATPase. 2. The nature of the inhibitory effect depends upon the conditions under which the dye is applied. When Ca-containing ghosts with 100 mM internal K are suspended in a choline medium at 37 degrees C, 0.3 microM-diS-C2-(5) causes virtually complete inhibition of K transport within 1 min (haematocrit 0.3%). Inhibition cannot be reversed by washing the ghosts to remove bound dye, although partial recovery occurs on subsequent incubation at 37 degrees C. 3. Inhibition is less marked when ghosts are suspended in solutions containing 100 mM-K, and is largely reversed by washing. The degree of inhibition varies with the dye concentration: 50% inhibition occurs with a free diS-C2-(5) concentration of 0.7 microM, and about 5 x 10(6) dye molecules are bound per ghost. 4. Changes in internal and external K concentrations, keeping internal Ca constant, have no effect on the dye concentration required for 50% inhibition of K efflux. 5. When internal Ca is varied, keeping internal and external K constant, the dye concentration needed for 50% inhibition of K efflux varies inversely with the K efflux in the absence of dye. That is, diS-C2-(5) is most effective as an inhibitor when K efflux is at a maximum. This suggests that the dye reacts preferentially with the transport system in the Ca-activated state. 6. The effects of several related molecules were examined, using Rb influx as a measure of K permeability. Inhibition is obtained in some cases, but is not directly related to the ability of the ghosts to bind the dyes. An anionic dye, diS-C3SO3--(5) inhibits Rb influx only when present inside the ghosts. 7. The implications of these findings are discussed.

Biological Transport↗
PubMed1976

The preparation of human red cell ghosts containing calcium buffers.

1. Ca buffers may be introduced into human red cells by reversible haemolysis. The resealed ghosts retain Ca and chelating anions in the same ratio as in the haemolysing solution, enabling the intracellular Ca2+ concentration to be calculated simply. 2. The passive permeability of the ghosts to Na and Cl is unaffected by intracellular Ca2+ concentrations in the 10(-8)-10(-4) M range, whereas the K permeability is greatly increased at concentrations above 10(-7) M. 3. These preparations enable Ca-dependent K movements to be studied under stable conditions. When the ghosts contain about 5 X 10(-6) M-Ca2+, over 96% of K transport occurs via the Ca-sensitive route.

Buffers↗
PubMed1976

Calcium-dependent potassium exchange in human red cell ghosts.

1. The properties of the Ca-dependent K transport system of human red cell ghosts have been examined under equilibrium exchange conditions. 2. K transport is stimulated half-maximally by about 0-4muM-Ca2i+ or 5muM-Sr2i+, but much higher concentrations of Ba2i+ give only slight stimulation. Mg is a weak antagonist to Ca. 3. The free Ca2+ concentration in human red cells is estimated to be below 0-25muM. 4. The curve relating the rate of K transport to the intracellular Ca2+ concentration is complicated and suggests that internal Ca acts at three or more sites. 5. K, Rb and possibly Cs ions are transported by the Ca-dependent system. Under comparable conditions the relative rates are 1(K):1-5(Rb): less than 0-05(Cs). 6. No Ca-dependent transport of Na, Li or choline could be detected. If Na is transported, it must be at less than 1/40 of the rate of K. 7. The rate of K transport is almost linearly related to the K concentration in the 0-200 mM range, but the curve is sigmoid close to the origin. 8. Intracellular, but not extracellular Na inhibits K transport, in a way that suggests competition with K at more than one site. 9. These results suggest that the transport system has a complex mechanism.

Barium↗
PubMed1975

The interaction of ATP-analogues possessing a blocked gamma-phosphate group with the sodium pump in human red cells.

1. The (Na++K+)-ATPase of red cell membranes is unable to hydrolyse ATP-analogues in which the oxygen atom linking the beta- and gamma-phosphate groups is replaced by a minusCH2minus or minusNH-bridge. 2. In resealed ghosts both these ATP-analogues support K:K exchange but not Na:K exchange. ATP supports both modes of operation of the sodium pump, whereas neither occurs without any nucleotide. 3. These results support the hypothesis that ATP is needed as a cofactor for K:K exchange to occur, and make it extremely unlikely that phosphorylation from ATP is involved.

Adenine Nucleotides↗
PubMed1974

Potassium: potassium exchange catalysed by the sodium pump in human red cells.

1. When red cells were so depleted of Na that Na:K exchange had almost ceased, the ouabain-sensitive K efflux seen in K-containing media was accompanied by an almost equal ouabain-sensitive K influx.2. This suggests that the Na pump in these cells was carrying out a one-for-one K:K exchange across the erythrocyte membrane.3. 30-40% of the (42)K efflux from resealed ghosts was sensitive to ouabain when the ghosts contained 1 mM-ATP, 2 mM orthophosphate, 10 mM-K and less than 1 mM-Na, and the suspending medium contained 10 mM-K and 0-Na, choline being the predominant cation.4. In resealed ghosts, the rate of K:K exchange saturated as internal K was increased, and was half-maximal at about 10 mM-K.5. When internal ATP was maintained with a phosphocreatine:creatine phosphokinase regenerating system, K:K exchange saturated as internal ATP was increased, and was half-maximal at about 100 muM-ATP.6. The rate of K:K exchange did not depend on whether the ADP concentration was roughly the same as the ATP concentration or very much less, suggesting that ADP did not affect the rate of K:K exchange.7. GTP, ITP and UTP were unable to substitute for ATP in supporting K:K exchange. CTP was a poor substitute.8. There was no evidence to support the hypothesis that K:K exchange is accompanied by a ouabain-sensitive hydrolysis of ATP.9. Internal Na was a strong inhibitor of ouabain-sensitive K efflux from ghosts containing 9 mM-K. 4 mM-Na was sufficient to produce 90% inhibition.10. The rate of K:K exchange depended on the orthophosphate concentration inside the ghosts (confirming Glynn, Lew & Lüthi, 1970). The curve obtained suggested that the rate was half-maximal at about 1.7 mM orthophosphate.11. These experiments suggested that inhibition by internal K is an important factor affecting the Na efflux from intact red cells. Experiments measuring Na:K exchange as a function of internal Na in low-K ghosts supported this hypothesis.12. The significance of these findings is discussed.

Adenosine Triphosphate↗
PubMedDate not supplied

Lead-calcium interactions in cellular lead toxicity.

The interaction of Pb and Ca with cellular sites depends upon the concentration of free ions present (Pb2+, Ca2+). The ability of Pb2+ to form complexes with simple anions such as Cl- and OH-, the formation of precipitates such as Pb(OH)2 and Pb3(PO4)2, and the ubiquity of Pb as a contaminant in laboratory reagents implies that particular care is needed in order to define the Pb2+ concentration of a solution. The free Pb2+ concentration may be controlled with Pb2+ buffers, and measured with a Pb2+ selective electrode, a fluorescent dye, fura-2, or an NMR indicator, 19F-BAPTA. Pb(2+)-Ca2+ interactions occur in three main situations at the cellular level. Pb2+ and Ca2+ compete at the plasma membrane for transport systems which effect their entry or exit, such as Ca2+ channels, and the Ca2+ pump. Intracellular Ca2+ is buffered to around 10(-7) M by proteins, endoplasmic reticulum and mitochondria. Pb2+ disturbs intracellular Ca2+ homeostasis. Ca(2+)-Pb2+ interactions at mitochondria have been described, but other mechanisms have not yet been explored. Increases in intracellular [Ca2+] act as a signal (or second messenger). Pb2+ interacts with a number of Ca(2+)-dependent effector mechanisms, such as calmodulin (a Ca2+ receptor protein which couples to several enzymes e.g., phosphodiesterase, protein kinases), protein kinase C, Ca(2+)-dependent K+ channels in the plasma membrane and neurotransmitter release. The actions of Pb2+ on neurotransmission may be relevant to Pb(2+)-induced human neuropathy and encephalopathy.

Animals↗