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T J Simons

Publications and source records attributed to T J Simons.

At least 19 recordsLinked to original sources
PubMed1991

Intracellular free zinc and zinc buffering in human red blood cells.

Zn2+ has been allowed to equilibrate across the red cell membrane using two agents that increase membrane permeability to this ion: the ionophore A23187 and the specific carrier ethylmaltol. Extracellular free Zn2+ was controlled with EGTA (1,2-di(2-aminoethoxy)ethane-NNN'N'tetra-acetic acid] buffers, except in the case of ethylmaltol, which itself acts as a buffer. Measurement of cellular zinc content at different levels of free Zn2+ facilitated the study of intracellular Zn2+ binding. It was also possible to estimate intracellular free Zn2+ concentration in untreated cells using a "null-point" technique. Intracellular zinc was found to consist of an inexchangeable component of about 129 mumol/10(13) cells and an exchangeable component of 6.7 +/- 1.5 mumol/10(13) cells, with a free concentration of about 2.4 x 10(-11) M. The main component of Zn2+ buffering is hemoglobin, with a dissociation constant of about 2 x 10(-8) M.

Binding Sites↗
PubMed1991

Calcium-dependent zinc efflux in human red blood cells.

Zinc efflux from human red blood cells is largely brought about by a saturable mechanism that depends upon extracellular Ca2+ ions. It has a Vmax of about 35 mumol/10(13) cells hr, a Km for external Ca2+ of 1 x 10(-4) M, and a Km for internal Zn2+ of 1 x 10(-9) M. External Zn2+ inhibits with a K0.5 of 3 x 10(-6) M. Sr2+ is a substitute for external Ca2+, but changes in monovalent anions or cations have little effect on the Zn2+ efflux mechanism. It is unaffected by most inhibitors of red cell transport systems, although amiloride and D-600 (methoxyverapamil, a Ca2+ channel blocker) are weakly inhibitory. The transport is capable of bringing about the net efflux of Zn2+, against an electrochemical gradient, provided Ca2+ is present externally. This suggests it may be a Zn2+:Ca2+ exchange, which would be able to catalyze the uphill movement of Zn2+ at the expense of an inward Ca2+ gradient, which is itself maintained by the Ca2+ pump.

Biological Transport, Active↗
PubMed1991

Observations on the chemical nature of lead in human blood serum.

1. The binding of lead to human blood serum, and components of serum, was studied by titration with the addition of Pb(NO3)2 solution, monitoring the free Pb2+ concentration with a Pb2+ electrode, and by equilibrium dialysis. 2. In fresh serum, about 4999 out of 5000 parts of added lead were bound. This suggests that the free Pb2+ concentration is around 1/5000th of the total lead concentration in the serum of normal subjects, i.e. about 1 x 10(-12) mol/l. 3. About 60% of the binding of lead in serum is abolished by standing in air, by dialysis or by treatment with N-ethylmaleimide. This appears to be due to the presence of thiol compounds, mainly cysteine. The remaining 40% appears to be due to protein, mainly albumin.

Blood Proteins↗
PubMed1990

Anionic mechanisms of zinc uptake across the human red cell membrane.

1. Zinc is taken up into human red cells by two mechanisms that depend upon the presence of anions. One of these requires bicarbonate ions, is inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and appears to be catalysed by the anion exchanger. The second occurs in the presence of thiocyanate or salicylate ions and may represent transport of a neutral complex with Zn2+. 2. The initial rate of Zn2+ uptake via the anion exchanger is 64 +/- 13 mumol (10(13) cells x h)-1 microM-1 external Zn2+, in the presence of 5 mM-bicarbonate at pH 7.4 and 37 degrees C (+/- S.D.). This is about 1/250 of the corresponding rate of Pb2+ uptake by the anion exchanger. 3. The variation of transport with Zn2+ concentration, HCO3- concentration and pH suggests that the transported species may be ZnCO3Cl- or Zn(HCO3)Cl.OH-. 4. Zinc efflux could not be observed by either of the above routes. This observation suggests that the intracellular free Zn2+ concentration is below 3 nM.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1989

Lead contamination.

Explore the source record for details and available documents.

Drug Contamination↗
PubMed1988

Calcium and neuronal function.

Calcium is unique among metals because its ions have a very large concentration gradient across the plasma membrane of all cells, from 10(-3) M Ca2+ outside, to 10(-7) M Ca2+ inside. This gradient is maintained by the use of metabolic energy through ion pumping, and its existence allows cells to use transient increases in the intracellular Ca2+ concentration as signals, which regulate cell function. In neurones these Ca signals are initiated by electrical activity (action potentials) which open voltage-dependent Ca channels in the plasma membrane, allowing Ca to enter the cell. Intracellular Ca signals can also be produced by transmitters at synapses, which open Ca channels, either directly, or indirectly by causing local depolarization and the opening of voltage-dependent Ca channels. The main effects of Ca signals on neurones are to alter their electrical activity, by modifying the opening and closing of Na and K channels, and to stimulate the release of transmitter substance. Ca has a host of other effects, such as the regulation of metabolic activity, the regulation of cell growth, and the long-term modification of synaptic efficiency, and it is even implicated in the destruction of neurones.

Animals↗
PubMed1988

Active transport of lead by the calcium pump in human red cell ghosts.

1. Resealed human red cell ghosts containing lead buffers bring about a net transfer of lead from the cell interior to the outside. This transfer is ATP dependent. 2. The active transport of lead is characterized by a Vmax (maximum velocity) of 11 mmol/(l cells.h) and a KM (Michaelis constant) of 5 x 10(-8) M for internal Pb2+, at pH 6.8 and 37 degrees C. 3. Lead efflux is antagonized by internal calcium, and is inhibited by vanadate with the same IC50 (inhibition constant) with which vanadate inhibits calcium pumping. 4. It is concluded that lead is transported by the calcium pump.

Adenosine Triphosphate↗
PubMed1987

Effects of lead ions on events associated with exocytosis in isolated bovine adrenal medullary cells.

Lead buffers (citrate and Tiron) were used to investigate the effects of low concentrations (0.1-6 microM) of Pb2+ on stimulus-secretion coupling in isolated bovine chromaffin cells. Nicotinic agonists and high K elicit secretion by enhancing Ca2+ influx into chromaffin cells. Pb2+ inhibited the catecholamine secretion in response to 500 microM carbachol and 77 mM K+ depolarization but was without significant effect on basal secretion. Pb2+ also inhibited the influx of 45Ca occurring in response to these agents. The K0.5 values for inhibition suggest that the carbachol-evoked flux is more sensitive to Pb2+ than influx in response to a direct depolarization. When extracellular calcium was lowered in the absence of Pb2+, both secretion and 45Ca entry were reduced. The effects of Pb2+ were comparable to those of lowered Ca2+. 22Na influx through nicotinic receptor-mediated channels, measured in the presence of tetrodotoxin (2 microM) and ouabain (50 microM), was inhibited by Pb2+. The results suggest that Pb2+ inhibits exocytotic catecholamine secretion by inhibiting Ca2+ influx. The differential sensitivity to Pb2+ of K- and carbachol-evoked 45Ca flux, coupled with the 22Na measurements, indicates that Pb2+ inhibits the movement of ions through acetylcholine-induced channels as well as through voltage-sensitive calcium channels.

Acetylcholine↗
PubMed1987

Lead enters bovine adrenal medullary cells through calcium channels.

Agents that stimulate secretion also accelerate the rate of Pb uptake into adrenal medullary cells. For example, when cells are suspended in a medium containing 5 microM Pb2+, depolarization by 77 mM K increases the rate of Pb uptake from 12 +/- 1 to 47 +/- 5 mumol/(L cells X min). K-induced Pb uptake has an apparent Km for Pb2+ of 2.6 microM, and is antagonized by Ca2+ with a K0.5 of 1.4 mM. The Ca channel blocker D-600 inhibits Pb entry with a K0.5 of 0.4 microM. Pb uptake is also stimulated by the Ca channel agonist BAY K 8644. These observations suggest that Pb passes through Ca channels. The permeability of the channels to Pb appears to be at least 10 times the permeability to Ca.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1986

Passive transport and binding of lead by human red blood cells.

The uptake of Pb into human red blood cells has been studied using Pb buffers. Passive Pb movements can be studied conveniently when the cells are depleted of adenosine 5'-triphosphate (ATP), to eliminate active transport, and of inorganic phosphate, to prevent precipitation of lead phosphate. Pb can cross the membrane passively in either direction. Influx and efflux show similar properties. Passive Pb transport is strongly stimulated by HCO3-, and is reduced by replacing Cl- with ClO4-. It is inhibited by low concentrations of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS) and 4,4'-diisothiocyanostilbene-2.2'-disulphonic acid (DIDS), characteristic inhibitors of anion transport. Pb uptake is unaffected by varying the external concentrations of Na+, K+ and Ca2+. When Pb enters the cell, it binds mainly to haemoglobin. The ratio of bound Pb:free Pb2+ in the cytosol is estimated to be 6000:1. Pb binding to haemoglobin is unaffected by oxygenation. Binding to albumin is quantitatively similar to binding to haemoglobin. The implications of these results for the transport and binding of Pb in the blood are discussed.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1986

The role of anion transport in the passive movement of lead across the human red cell membrane.

Passive Pb transport across the red cell membrane has been studied by measuring Pb uptake from Pb-buffered solutions into resealed ghosts containing EGTA. Over 90% of Pb uptake occurs by a pathway which is inhibited by drugs which block anion transport. The order of effectiveness is 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS) greater than phloretin greater than furosemide and bumetanide. Ouabain and cytochalasin B are ineffective. This implicates the anion-exchange mechanism in Pb uptake. The rate of Pb uptake by this route is directly proportional to external Pb2+ and HCO3- concentrations, and inversely proportional to the H+ concentration. These findings suggest that Pb transport depends on the formation of PbCO3 in solution. Pb transport depends upon the presence of a second anion. In the presence of HCO3-, the rate is stimulated in the order ClO4- less than NO3- and CH3CO2- less than F- less than Cl- less than Br- less than I-. The temperature dependence of Pb uptake is similar to that of HCO3-(-)Cl- exchange. Changes in membrane potential appear to influence Pb transport. The effects are small and somewhat variable, but in general a negative internal potential accelerates uptake and reduces exit. A positive internal potential reduces uptake and accelerates exit. These results suggest that Pb is transported on the anion exchanger. Exchange of PbCO3 for a monovalent anion best fits the experimental data, although transport of a ternary PbCO3(-)anion- complex is a possibility.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1985

Influence of lead ions on cation permeability in human red cell ghosts.

Intracellular Pb2+ ions can replace Ca2+ ions in stimulating the Ca-dependent K permeability of human red blood cells. In metabolically depleted resealed ghosts, the threshold for stimulation of 86Rb efflux by internal Pb2+ is around 5 X 10(-10) M, and stimulation is half-maximal at about 2 X 10(-9) M, and maximal at 10(-8) M Pb2+. There is no effect on 22Na efflux in this concentration range. 86Rb efflux is antagonized by internal Mg2+ ions, and by the channel-blocking drugs quinidine and diS-C2(5), as observed for the Ca-dependent K permeability in red cells. In ghosts containing EDTA, which prevents any internal effects of Pb2+ ions, external Pb2+ increases both 22Na and 86Rb permeability when its concentration exceeds 6 X 10(-7) M. This effect is seemingly unrelated to the Ca-dependent K permeability. This work makes extensive use of Pb2+ ion buffers, and gives information about their preparation and properties.

Buffers↗
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↗