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Biomedical subjects

R Whittam

Publications and source records attributed to R Whittam.

At least 19 recordsLinked to original sources
PubMed1985

The activation of the sodium pump in pig red blood cells by internal and external cations.

A study has been made with pig red blood cells of the activation of the sodium pump by internal and external cations. Cell Na and K concentrations were altered using a PCMBS cation loading procedure. The procedure was characterised for resultant ionic conditions, maintenance of ATP levels and fragility. The activation of the sodium pump by external K was measured in cells suspended in choline (Na-free) solutions. External Cs was used as a substitute for K and elicited lower rates of pump activity. Both the Vmax and apparent Km for 42K influx and 134Cs influx increased as internal Na concentration was raised (within the non-saturating range). Vmax/apparent Km ratios for cation influx were constant. Raising external Cs concentration exerted a similar influence on pump activation by internal Na: both the maximum pump velocity and the apparent Na-site dissociation constant (K'Na) increased. The results provide evidence for a transmembrane connection between cation binding sites on opposite faces of the membrane and are consistent with a consecutive model for the sodium pump in pig red blood cells.

4-Chloromercuribenzenesulfonate↗
PubMed1985

The change from symmetry to asymmetry of a sodium transport system in red cell membranes.

A study has been made with human red cells of sodium movements that are sensitive to the drug furosemide. The aim was to see if furosemide-sensitive movements that are symmetrical (exchange) became asymmetrical (net transport) on replacement of chloride with nitrate as the major external anion. Cells were incubated for 4 h at 37 degrees C with 140 mM sodium, and chloride or nitrate as the principal anion. Under a variety of conditions (presence and absence of ouabain or furosemide, or both) the cell sodium concentration was always higher when chloride was replaced with nitrate. The cells became leakier to sodium. Tracer studies indicated that, in contrast to the results in chloride medium, the decrease in sodium influx was greater than the fall in efflux when furosemide was added to cells in nitrate medium. The results confirm that the sensitivity of sodium efflux to furosemide depended on chloride. However, influx showed a different sensitivity in that furosemide still inhibited in cells incubated in nitrate medium. The stimulation of sodium influx with nitrate medium was independent of external potassium (10-50 mM) and the furosemide-sensitive influx was also constant. It is concluded that symmetrical transmembrane sodium movements with cells in chloride medium became downhill asymmetrical in nitrate medium, giving a net gain of cell sodium that was insensitive to ouabain and sensitive to furosemide. The drug thus partly retarded the gain of cell sodium that otherwise occurred in the somewhat leaky cells.

Biological Transport↗
PubMed1984

The effect of furosemide on sodium movements in human red blood cells.

A study has been made of Na movements in human red blood cells in order to test their sensitivity to inhibition by furosemide or bumetanide. Net changes as well as influx and efflux were measured. Unidirectional Na influx and efflux values confirmed previous evidence for components of both these fluxes sensitive to inhibition by furosemide or bumetanide. The difference between furosemide-sensitive unidirectional influx and efflux was determined at [Na]o 140 mM and shown to be non-significant, even when [Na]i was raised to about 45 mumol ml cells-1. No evidence was found for an influence of furosemide on net movements of Na in cells of either slightly elevated or physiological [Na]i incubated in a 140 mM-Na medium over 4 h. Incubation of cells, under similar conditions, for 12 h did not reveal a bumetanide-sensitive net Na movement. The results suggest that cells with normal or slightly elevated [Na]i, incubated in media of physiological Na concentration, do not possess a mechanism sensitive to inhibition by the loop diuretics that operates to regulate cell Na.

Biological Transport, Active↗
PubMed1976

The connexion between the ion-binding sites of the sodium pump.

1. A study has been made of K influx into human red blood cells in order to determine the influence of internal Na on the affinity of the Na pump for external K. Cells were prepared to contain minimal K and two Na concentrations (about 10 and 30 mueqiv/ml. cells) and incubated in solutions with a range of K concentrations. 2. In choline (Na-free) Ringer, activation of K influx by external K was hyperbolic. The Km for external K increased as the internal Na concentration was raised. The increase was greater than the increase in K influx with saturating external K. The Km for external K increased towards a limiting value as the internal Na concentration was raised. 3. In contrast, in Na-Ringer, the activation by external K was sigmoidal and the affinity for external K was independent of the internal Na concentration. 4. K influx was measured at two submaximal levels of external K with a range of internal Na. The affinity for internal Na fell as external K was raised. 5. The results suggest that in the absence of competition between Na and K on the same side of the membrane, there is a connexion between the ion-binding sites of the Na pump.

Binding Sites↗
PubMed1973

Reconstitution of the sodium pump from protein and phosphatidylserine: features of ouabain binding.

1. A study has been made of ATP splitting and ouabain binding to the sodium pump reconstituted from protein and phosphatidylserine.2. Ouabain was bound to protein alone, but when phosphatidylserine was added, binding was increased threefold. The stimulation resembled the course of activation of sodium-dependent ATPase activity.3. EGTA partly simulated the activation of ATPase by phosphatidylserine but did not enhance binding.4. The dissociation constant for the enzyme-ouabain complex was 3.5 x 10(-8)M. The turnover number (2,000 molecules of ATP per minute) and the number of receptor sites (3.8 x 10(13) per mg protein) were calculated.5. The results provide further evidence of the involvement of phosphatidylserine in the action of the sodium pump.

Adenosine Triphosphatases↗
PubMed1972

The temperature dependence of activation by phosphatidylserine of the sodium pump adenosine triphosphatase.

1. Treatment of rabbit brain homogenates with deoxycholate reduced ouabain-insensitive ATPase sixfold and subsequently adding phosphatidylserine had no effect. Ouabain-sensitive ATPase was made entirely latent but it was fully restored on adding phosphatidylserine.2. Temperature and pH were varied to see if the reconstituted system resembled that in the original membranes. Linear Arrhenius plots were always obtained with the homogenate, and the activation energy was higher for the ouabain-sensitive than for the ouabain-resistant enzyme.3. A break at about 15 degrees C was found in the Arrhenius plot of the reconstituted enzyme, but there was no break without added phosphatidylserine or when ouabain was added. The break suggests that the conformation and catalytic activity of the enzyme protein depended on the physical state of phosphatidylserine.

Activation Analysis↗
PubMed1972

The uptake and hydrolysis of p-nitrophenyl phosphate by red cells in relation to ATP hydrolysis by the sodium pump.

1. The hydrolysis of p-nitrophenyl phosphate has been studied in human red cells, ghosts and haemoglobin-free membranes to see whether hydrolysis was related to the functioning of the sodium pump.2. The cell membrane restricted p-nitrophenyl phosphate entry into cells and was rate-limiting for hydrolysis by the large amount of intracellular soluble phosphatase. The uptake was inversely proportional to the external chloride concentration, and inhibitors (phloretin and persantin) of inorganic phosphate uptake also reduced p-nitrophenyl phosphate uptake and hydrolysis. The entry mechanism of p-nitrophenyl phosphate appears to be similar to that of inorganic phosphate.3. p-Nitrophenyl phosphate was hydrolysed in cells almost entirely by ouabain-insensitive phosphatases, both soluble and membrane bound. In ghosts containing less soluble enzyme than cells there was still no component of p-nitrophenyl phosphate hydrolysis that was related to the sodium pump in being sensitive to external potassium or ouabain.4. Haemoglobin-free membranes lacking soluble p-nitrophenyl phosphatase required potassium for optimum p-nitrophenyl phosphate hydrolysis and this part was inhibited by ouabain as was also ATP hydrolysis by the sodium pump. The rates of potassium-dependent and potassium-independent hydrolysis were each increased about threefold on decreasing the electrolyte concentration from 150 to 25 mM. The response was found whether the main electrolyte was potassium chloride, sodium chloride, choline chloride or Tris chloride. In contrast, the changes were not found when the osmotic pressure was varied to the same extent with non-electrolytes. p-Nitrophenyl phosphate hydrolysis was thus activated on lowering the ionic strength. The soluble enzyme was similarly affected and the effect was reversible.5. The membrane ATPase activity was unaffected by the changes in ionic strength which markedly altered p-nitrophenyl phosphate hydrolysis.6. These results with red cell membranes show that the ionic strength under physiological conditions largely prevents the hydrolysis of p-nitrophenyl phosphate, but not of ATP. A possible mechanism is discussed in terms of the effect of different structures of water in electrolyte and non-electrolyte solutions on the enzymic activity of the sodium pump.

Adenosine Triphosphatases↗
PubMed1971

The control by internal calcium of membrane permeability to sodium and potassium.

1. A study has been made of the relationship between the concentration of internal calcium and the permeability of human red cell membranes to sodium and potassium.2. Fresh red cells contain very little calcium, but after being depleted of ATP by ageing they took up calcium from Ringer solution. The entry was unaffected by external sodium and potassium but was markedly pH dependent. When supplied with energy, calcium-loaded cells actively extruded calcium by a saturable process which was also unaffected by the distribution of sodium and potassium across the membranes. The activity of the calcium pump was sufficient to maintain the low internal concentration found under physiological conditions.3. Raising internal calcium in metabolically poor cells caused a loss of cell potassium which was greater than the concomitant sodium gain. These changes were reversed when ATP was supplied. External calcium had no effect. The increase in permeability to sodium and potassium was enhanced by the simultaneous addition of fluoride, and, even more so, of iodoacetate. These inhibitors had no effect on membrane permeability unless calcium was also present. Inosine potentiated the action of fluoride and iodoacetate in causing potassium loss, by allowing more calcium to enter the cells.4. The results suggest that the permeability of human red cell membranes to sodium and potassium is regulated by internal calcium, which in turn is controlled by a calcium pump that utilizes ATP.

Adenosine Triphosphate↗
PubMed1971

The influence of the chloride gradient across red cell membranes on sodium and potassium movements.

1. A study has been made to see whether active and passive movements of sodium and potassium in human red blood cells are influenced by changing the chloride gradient and hence the potential difference across the cell membrane.2. Chloride distribution was measured between red cells and isotonic solutions with a range of concentrations of chloride and non-penetrating anions (EDTA, citrate, gluconate). The cell chloride concentration was greater than that outside with low external chloride, suggesting that the sign of the membrane potential was reversed. The chloride ratio (internal/external) was approximately equal to the inverse of the hydrogen ion ratio at normal and low external chloride, and inversely proportional to external pH. These results show that chloride is passively distributed, making it valid to calculate the membrane potential from the chloride ratio.3. Ouabain-sensitive (pump) potassium influx and sodium efflux were decreased by not more than 20 and 40% respectively on reversing the chloride gradient, corresponding to a change in membrane potential from -9 to +30 mV. In contrast, passive (ouabain-insensitive) movements were reversibly altered - potassium influx was decreased about 60% and potassium efflux was increased some tenfold. Sodium influx was unaffected by the nature of the anion and depended only on the external sodium concentration, whereas ouabain-insensitive sodium efflux was increased about threefold. When external sodium was replaced by potassium there was a decrease in ouabain-insensitive sodium efflux with normal chloride, but an increase in low-chloride medium.4. Net movements of sodium and potassium were roughly in accord with the unidirectional fluxes.5. The results suggest that reversing the chloride gradient and, therefore, the sign of the membrane potential, had little effect on the sodium pump, but caused a marked increase in passive outward movements of both sodium and potassium ions.

Biological Transport, Active↗