The effect of harmaline on unidirectional potassium fluxes and ouabain binding in renal cell cultures.

Harmaline inhibits K+ influx into primary cell cultures of ground squirrel kidneys to a greater extent than either ouabain or furosemide. A concentration of 200 microM harmaline was required to inhibit half of the total K+ influx; this effect was also seen at low temperature (5 degrees C), and in another species (hamster). Although kinetic analysis of K+ influx indicates that harmaline does not compete with extracellular K+, harmaline did reduce the binding of [3H]ouabain to the cells. K+ efflux was also reduced. Therefore, harmaline may inhibit the furosemide-sensitive Na+/K+ cotransport system as well as the ouabain-sensitive Na+/K+ pump.

Cultured cells from renal cortex of hibernators and nonhibernators. Regulation of cell K+ at low temperature.

Cells were grown as primary monolayer cultures from kidney cortex of guinea pigs (nonhibernators), hamsters and ground squirrels (both hibernating species). When plates of cells were placed at 5 degrees C, cells of guinea pigs lost 37% of their K+ in 2 h and those of the hibernator lost about 10%. Uptake of 42K into the cells exhibited a simple, single exponential time course at both temperatures. Unidirectional efflux of K+ was equal to K+ influx in all cultures at 37 degrees C and, within limits of error, in hibernator cells at 5 degrees C. Efflux was 3-to 5-fold greater than influx in guinea pig cells at 5 degrees C. After 2 h in the cold the ouabain sensitive K+ influx remaining (7-15% of that at 37 degrees C) was about the same in the cells of the 3 species. Cells from active hamsters and from hibernating ground squirrels, however, exhibited significantly greater pump activity after 45 min in the cold (19 and 14%, respectively). The stimulation of K+ influx by increasing [K+] did not show an increase in Km+ at 5 degrees C in cells of guinea pigs and ground squirrels. Lowering [K+]c and/or raising [Na+]c by treatment in low- and high-K+ media caused only slight stimulation of K+ influx, except in cells of ground squirrels at 5 degrees C in which the stimulation was at least 11-times greater than at 37 degrees C or in cells of guinea pigs at either temperature. This altered kinetic response of K+ transport to cytoplasmic ion stimulation with cooling accounted for about one-third of the improved regulation of K+ at 5 degrees C in ground squirrel cells; the other two-thirds was attributable to a greater decrease in K+ leak with cooling. The inhibition of active transport by cold in all 3 species was much less severe than that previously seen in any (Na++K+)-ATPase of mammalian cells.

Temperature dependence of membrane function. Disparity between active potassium transport and (Na+ & K+)ATPase activity.

Ouabain-sensitive K+ influx in mammalian erythrocytes exhibits far less temperature sensitivity than the ((Na+ & K+)ATPase prepared by hypotonic lysis from the same population of cells. The results are not in accord with lipid phase change as the critical mechanism of cold inhibition of intact pumps.

Red cell amino acid transport. Evidence for the presence of system Gly in guinea pig reticulocytes.

Guinea pig reticulocytes are shown to possess an Na+-dependent glycine transporter which also requires Cl- for activity. Glycine transport by this route is saturable (apparent Km 98 microM; Vmax 24 mumol/g Hb per h) and inhibited by sarcosine. The properties of this carrier closely resemble those of System Gly previously demonstrated in pigeon and human erythrocytes. In contrast, no System Gly activity was detected in mature guinea pig erythrocytes.

Kinetics of the sodium pump in red cells of different temperature sensitivity.

Ouabain-sensitive K influx into ground squirrel and guinea pig red cells was measured at 5 and 37 degrees C as a function of external K and internal Na. In both species the external K affinity increases on cooling, being three- and fivefold higher in guinea pig and ground squirrel, respectively, at 5 than at 37 degrees C. Internal Na affinity also increased on cooling, by about the same extent. The effect of internal Na on ouabain-sensitive K influx in guinea pig cells fits a cubic Michaelis-Menten-type equation, but in ground squirrel cells this was true only at high [Na]i. There was still significant ouabain-sensitive K influx at low [Na]i. Ouabain-binding experiments indicated around 800 sites/cell for guinea pig and Columbian ground squirrel erythrocytes, and 280 sites/cell for thirteen-lined ground squirrel cells. There was no significant difference in ouabain bound per cell at 37 and 5 degrees C. Calculated turnover numbers for Columbian and thirteen-lined ground squirrel and guinea pig red cell sodium pumps at 37 degrees C were about equal, being 77-100 and 100-129 s-1, respectively. At 5 degrees C red cells from ground squirrels performed significantly better, the turnover numbers being 1.0-2.3 s-1 compared with 0.42-0.47 s-1 for erythrocytes of guinea pig. The results do not accord with a hypothesis that cold-sensitive Na pumps are blocked in one predominant form.

Resistance of erythrocytes of hibernating mammals to loss of potassium during hibernation and during cold storage.

In two species of hibernators, hamsters and ground squirrels, erythrocytes were collected by heart puncture and the K content of the cells of hibernating individuals was compared with that of awake individuals. The K concentration of hamsters did not decline significantly during each bout of hibernation (maximum period of 5 days) but in long-term bouts in ground squirrels (i.e. more than 5 days) the K concentration of cells dropped significantly. When ground squirrels were allowed to rewarm the K content of cells rose toward normal values within a few hours. Erythrocytes of both hamsters and ground squirrels lose K more slowly than those of guinea pigs (nonhibernators) when stored in vitro for up to 10 days at 5 degrees C. In ground squirrels the rate of loss of K during storage is the same as in vivo during hibernation, and stored cells taken from hibernating ground squirrels also lose K at the same rate. The rate of loss of K from guinea pig cells corresponded with that predicted from passive diffusion unopposed by transport. The actual rate of loss of K from ground squirrel cells was slower than such a predicted rate but corresponded with it when glucose was omitted from the storage medium or ouabain was added to it. Despite the slight loss of K that may occur in hibernation, therefore, the cells of hibernators are more cold adapted than those of a nonhibernating mammal, and this adaptation depends in part upon active transport.

Temperature adaptation of active sodium-potassium transport and of passive permeability in erythrocytes of ground squirrels.

Unidirectional active and passive fluxes of (42)K and (24)Na were measured in red blood cells of ground squirrels (hibernators) and guinea pigs (nonhibernators). As temperature is lowered, "active" (ouabain-sensitive) K influx and Na efflux were more greatly diminished in guinea pig cells than in those of ground squirrels. The fraction of total K influx which is ouabain sensitive in red blood cells of ground squirrels was virtually constant at all temperatures, whereas it decreased abruptly in guinea pig cells as temperature was lowered. All the passive fluxes (i.e., Na influx, K efflux, and ouabain-insensitive K influx and Na efflux) decreased logarithmically with decrease in temperature in both species, but in ground squirrels the temperature dependence (Q(10) 2.5-3.0) was greater than in guinea pig (Q(10) 1.6-1.9). Thus, red blood cells of ground squirrel are able to resist loss of K and gain of Na at low temperature both because of relatively greater Na-K transport (than in cells of nonhibernators) and because of reduced passive leakage of ions.

Active and passive transport of sodium and potassium ions in erythrocytes of severely malnourished Jamaican children.

Erythrocytes of normal and malnourished children, both marasmic and oedematous (kwashiorkor), were equilibrated in standard incubation medium and their ion transport via the Na/K pump and the pathways of passive permeation were measured as unidirectional fluxes of 86Rb (as a congener of K) and 22Na. Cells of children with kwashiorkor exhibited a 65 per cent higher ouabain-sensitive K(Rb) influx ('pump rate') than those of normal or marasmic children. When allowance was made for cytoplasmic Na concentration, the pump rate was slower in younger (12 months and under) normal children than in older children. Judged by the same criterion, cells of older marasmic children also had slower steady-state pump activity. The passive permeation of K through the residual 'leak' pathway (ie, ouabain-and-bumetanide-insensitive influx) and Na permeation (ouabain-and-bumetanide-insensitive Na efflux) were greater in malnourished children than in normal children by a factor of two or more. During treatment for malnutrition, both Na-pump activity and ouabain binding increased rapidly in marasmic children. Passive permeation did not return to normal levels in malnourished children during the period of hospitalization.

The absence of adapted sodium and potassium transport in erythrocytes of cerebral palsied children with secondary malnutrition.

Previous studies of erythrocyte ion (potassium and sodium) transport during marasmus and kwashiorkor have indicated increased passive permeation to both ions in both syndromes, and increased Na,K pump activity in kwashiorkor and reduced activity in marasmus. Children with severe cerebral palsy (CP) frequently suffer secondary protein energy malnutrition (PEM). Unlike marasmus and kwashiorkor, this PEM is uncomplicated by micronutrient deficiency, parasitism and infections. Because of deformities classification of PEM cannot be performed in these children by stature-based anthropometry, therefore we used triceps skinfold thicknesses less than the fifth percentile and absence of weight gain in the previous year as criteria for malnutrition. K influx data from well- and malnourished CP children, and from well-nourished controls reveal that ouabain-sensitive K influx is highest in malnourished CP, followed by well-nourished CP (P = 0.02), and lowest in controls (P less than 0.001, vs. malnourished). Determinations of ouabain-sensitive Na efflux, though less precise and therefore more variable, were consistent with this finding of no decrease of Na,K pump activity occurring during the development of this malnutrition. There were no statistically significant differences in ouabain-insensitive fluxes of either Na or K. Ion transport in undernourished CP children thus resembles that found in kwashiorkor rather than in marasmus; but oedema is rarely seen in this form of secondary PEM.

Cold tolerance in mammalian cells.

As whole organisms, most mammals have a poor tolerance for hypothermia. But their cells may have a capacity for a far wider cold tolerance, which may be expressed in peripheral tissues, sporadically in core tissue and in cultured cells. Against this background the cold resistance of cells of deep hibernators may be seen as the extreme of a continuum and is complicated by the consideration that the voluntary hypothermia of hibernation is probably in most cases a metabolic adaptation to forestall starvation. Similarly, cold resistance of peripheral tissues may in diving animals be confounded by the need to be adapted to hypoxia as well. Hence, attempts to analyse cold resistance by comparisons of absolute rates of arbitrarily chosen reactions may be misleading. A more useful approach is analysis of maintenance of balance: balance between ATP synthesis and utilization, balance between macromolecule synthesis and degradation and balance between pumps and leaks. Cation pumps and leaks constitute a major component of energy utilization and are central to other cell functions, even during minimal metabolism. Hence, the maintenance of ion gradients is a central issue in understanding adaptation not only to hypothermia but also to starvation and hypothermia. Of the three hypometabolic states, hypothermia has been best studied in this regard. In most cases, passive permeability is more reduced at low temperature in cold-tolerant cells than in cold-sensitive ones. In some cases there is also a difference in Na-K pump activity and perhaps in ATP dependent Ca-pump activity. Pump activities and probably the maintenance of minimal leak require ongoing metabolism. The question of whether, in cold-sensitive cells, energy supplies are adequate at low temperature was once the focus of this field, but has been ignored for a decade without having been fully resolved. There are many instances of less temperature sensitivity of specific metabolic activities (mitochondrial respiration, etc.) in hibernators than in non-hibernators, without any verification of whether this is essential for survival at low body temperature. Certainly, robust pumping has been found in some failing cold-sensitive cells at low temperature, suggesting no shortage of ATP in these cases, but in other cases the issue may be a more complex one than just that of ATP availability.(ABSTRACT TRUNCATED AT 400 WORDS)

Sodium transport through the amiloride-sensitive Na-Mg pathway of hamster red cells.

Previous work showed that in hamster red cells the amiloride-sensitive (AS) Na+ influx of 0.8 mmol/liter cells/hr is not mediated by Na-H exchange as in other red cells, but depends upon intracellular Mg2+ and can be increased by 40-fold by loading cells with Mg2+ to 10 mM. The purpose of this study was to verify the connection of AS Na+ influx with Na-dependent, amiloride-sensitive Mg2+ efflux and to utilize AS Na+ influx to explore that pathway. Determination of unidirectional influx of Na+ and net loss of Mg2+ in parallel sets of cells showed that activation by extracellular [Na+] follows a simple Michaelis-Menten relationship for both processes with a Km of 105-107 mM and that activation of both processes is sigmoidally dependent upon cytoplasmic [Mg2+] with a [Mg2+]0.5 of 2.1-2.3 mM and a Hill coefficient of 1.8. Comparison of Vmax for both sets of experiments indicated a stoichiometry of 2 Na:1 Mg. Amiloride inhibits Na+ influx and Mg2+ extrusion in parallel (Ki = 0.3 mM). Like Mg2+ extrusion, amiloride-sensitive Na+ influx shows an absolute requirement for cytoplasmic ATP and is increased by cell swelling. Hence, amiloride-sensitive Na+ influx in hamster red cells appears to be through the Na-Mg exchange pathway. There was no amiloride-sensitive Na+ efflux in hamster red cells loaded with Na+ and incubated with high [Mg2+] in the medium with or without external Na+, nor with ATP depletion. Hence, this is not a simple Na-Mg exchange carrier.

Elevating intracellular free Mg2+ preserves sensitivity of Na(+)-K+ pump to ATP at reduced temperatures in guinea pig red blood cells.

Red cells of hibernating species have a higher relative rate of Na(+)-K+ pump activity at low temperature than the red cells of a mammal with a typical sensitivity to cold. The kinetics of ATP stimulation of the Na(+)-K+ pump were determined in guinea pig and ground squirrel red cells at different temperatures between 5 and 37 degrees C by measuring ouabain-sensitive K+ influx at different levels of ATP. In guinea pig cells, elevation of intracellular free Mg2+ to 2 mmol.1-1 by use of the divalent cation ionophore A23187 caused the apparent affinity of the pump for ATP to increase with cooling to 20 degrees C, rather than to decrease, as occurs in cells not loaded with Mg2+. In ground squirrel cells raising intracellular free Mg2+ had little effect on apparent affinity of the pump for ATP at 20 degrees C. ATP affinity rose slightly with cooling both in Mg(2+)-enriched and in control ground squirrel cells. Increased intracellular free Mg2+ in guinea pig cells stimulated Na(+)-K+ pump activity so that at 20 degrees C the pump rate was the same in the Mg(2+)-enriched guinea pig and control ground squirrel cells. Pump activity in Mg(2+)-enriched guinea pig cells at 5 degrees C was significantly improved but still lower than pump activity in control cells from ground squirrel. Thus, loss of affinity of the Na(+)-K+ pump for ATP that occurs with cooling in cold-sensitive guinea pig red cells can be, at least partially, prevented by elevating cytoplasmic free Mg2+. Conversely, in ground squirrel red cells natural rise of free Mg2+ may in part account for the preservation of the ATP affinity of their Na(+)-K+ pump with cooling.