Activation of K-Cl cotransport by mild warming in guinea pig red cells.

Unidirectional, ouabain-insensitive K+ influx rose steeply with warming at temperatures above 37 degreesC in guinea pig erythrocytes incubated in isotonic medium. The only component of ouabain-insensitive K+ influx to show the same steep rise was K-Cl cotransport (Q10 of 10 between 37 and 41 degrees C); Na-K-Cl cotransport remained constant or declined and residual K+ influx in hypertonic medium with ouabain and bumetanide rose only gradually. Similar results were obtained for unidirectional K+ efflux. Thermal activation of K-Cl cotransport-mediated K+ influx was fully dependent on the presence of chloride in the medium; none occurred with nitrate replacing chloride. The increase of K+ influx through K-Cl cotransport from 37 to 41 degrees C was blocked by calyculin A, a phosphatase inhibitor. The Q10 of K-Cl cotransport fully activated by hydroxylamine and hypotonicity was about 2. The time course of K+ entry showed an immediate transition to a higher rate when cells were instantly warmed from 37 to 41 degrees C, but there was a 7-min time lag in returning to a lower rate when cells were cooled from 41 to 37 degrees C. These results indicate that the steepness of the response of K-Cl cotransport to mild warming is due to altered regulation of the transporter. Total unidirectional K+ influx was equal to total unidirectional K+ efflux at 37-45 degrees C, but K+ influx exceeded K+ efflux at 41 degrees C when K-Cl cotransport was inhibited by calyculin or prevented by hypertonic incubation. The net loss of K+ that results from the thermal activation of isosomotic K-Cl cotransport reported here would offset a tendency for cell swelling that could arise with warming through an imbalance of pump and leak for Na+ or for K+.

On the trail of potassium in heat injury.

In both classical and exertional heatstroke and in various animal models of human heat injury, clinical manifestations have included observations of normokalemia, hyperkalemia, and hypokalemia. This review attempts to address these observations as well as the role of potassium and potassium depletion in heat injury with an emphasis on the integration of information from the level of transmembrane potassium transport mechanisms to systems physiology. Under moderate conditions of passive heat exposure or exercise in the heat, the adaptive capacity of the Na-K pump (Na+-K+ ATPase activity) and cotransport mechanisms can ordinarily accommodate the attendant increased efflux of intracellular K+ and influx of extracellular Na+ to maintain ionic equilibrium. Several factors affecting transmembrane K+ kinetics include protracted K+ deficiency, extreme hyperthermia, dehydration, and excessive exertion. These could elicit reduced membrane potentials and conductance, futile cycling of the Na-K pump with concomitant energy depletion and greatly increased metabolic heat production, reduced arteriolar vasodilation, altered neurotransmitter release, or cell swelling, each of which could contribute to the pathophysiology of heat injury. This review represents a preliminary attempt to link transmembrane K+ pathophysiology with clinical heat injury.

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.

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.

A comparison of effect of temperature on phosphorus metabolites, pH and Mg2+ in human and ground squirrel red cells.

1. 31P NMR spectra were obtained at temperatures ranging from 2 to 30 degrees C from freshly drawn human (cold-sensitive) and ground squirrel (cold-tolerant) red cells. The concentration of ATP was also determined by luciferin-luciferase assay over the same temperature range. 2. The concentration of ATP as determined by NMR or by the luciferin-luciferase assay did not change with temperature in either species. The absolute concentration of ATP in human cells determined by NMR was not significantly different from the total ATP determined enzymatically. 3. The concentration of 2,3-diphosphoglycerate was higher and that of pyridine nucleotides lower in human than in ground squirrel red cells. This species difference was independent of temperature. 4. Intracellular pH, as determined from the positions of the NMR peaks of 2- and 3-phosphates of diphosphoglycerate, became more alkaline as the temperature was lowered. 5. Free intracellular magnesium, determined from the difference in the positions of the peaks for alpha- and beta-phosphorus of ATP, increased in the ground squirrel red cells and decreased in the human red cells with cooling from 30 to 2 degrees C. Total magnesium, as determined by atomic emission spectroscopy, did not change with temperature in red cells of either species. 6. The intensities of all phosphorus metabolite signals from the ground squirrel cells increased with decreasing temperature, while those from the human cells were unaffected. Since chemical shift anisotropy in the presence of magnesium is a powerful spin-lattice relaxation mechanism for phosphates, this is additional evidence for the temperature dependence of free magnesium concentration in the ground squirrel cells. 7. We conclude that there is no difference in phosphorus metabolites or intracellular pH which could account for the differential cold sensitivity in human and ground squirrel red cells. We suggest that, in the cold-tolerant red cells from the ground squirrel, magnesium is released from binding sites as the temperature is lowered. The change in free intracellular Mg2+ may account at least in part for the unusually low temperature sensitivity of the Na(+)-K+ pump in the red cells of this species.

Cold activation of Na influx through the Na-H exchange pathway in guinea pig red cells.

Previous work showed that amiloride partially inhibits the net gain of Na in cold-stored red cells of guinea pig and that the proportion of unidirectional Na influx sensitive to amiloride increases dramatically with cooling. This study shows that at 37 degrees C amiloride-sensitive (AS) Na influx in guinea pig red blood cells is activated by cytoplasmic H+, hypertonic incubation, phorbol ester in the presence of extracellular Ca2+ and is correlated with cation-dependent H+ loss from acidified cells. Cytoplasmic acidification increases AS Na efflux into Na-free medium. These properties are consistent with the presence of a Na-H exchanger with a H+ regulatory site. Elevation of cytoplasmic free Mg2+ above 3 mM greatly increases AS Na influx: this correlates with a Na-dependent loss of Mg2+, indicating the presence of a Na-Mg exchanger. At 20 degrees C activators of Na-H exchange have little or no further stimulatory effect on the already elevated AS Na influx. AS Na influx is much larger than either Na-dependent H+ loss or AS Na efflux at 20 degrees C. The affinity of the AS Na influx for cytoplasmic H+ is greater at 20 degrees C than at 37 degrees C. Depletion of cytoplasmic Mg2+ does not abolish the high AS Na influx at 20 degrees C. Thus, elevation of AS Na influx with cooling appears to be due to increased activity of a Na-H exchanger (operating in a "slippage" mode) caused by greater sensitivity to H+ at a regulatory site.

ATP dependence of Na(+)-K+ pump of cold-sensitive and cold-tolerant mammalian red blood cells.

1. The ATP concentration of intact, cold-tolerant (ground squirrel) red cells and cold-sensitive (guinea-pig and human) red cells was monitored by use of the firefly tail, luciferin-luciferase assay. ATP kinetics of the pump in intact red blood cells was investigated by altering cell [ATP] by progressive depletion of ATP in the presence of 2-deoxy-D-glucose and then by measurement of ouabain-sensitive K+ influx at each level of [ATP] at various temperatures between 37 and 5 degrees C. Na(+)-K(+)-ATPase activity of broken membranes was also determined in parallel experiments using ouabain-sensitive release of 32P from [gamma-32P]ATP as a measure of activity. 2. Without depletion, there is no immediate decrease in [ATP] of intact cold-sensitive cells at low temperature (5 degrees C) at times when there are marked differences in the activities of the Na(+)-K+ pump of cold-tolerant and cold-sensitive cells. 3. At 37 degrees C Na(+)-K(+)-ATPase of all three species exhibited two components of ATP dependence at 37 degrees C, one with high velocity, low affinity, the other with low velocity, high affinity. Affinities of both components rose with cooling. 4. A similar, two component pattern was observed in intact guinea-pig and human red cells at 37 degrees C, except that the segment corresponding to the high affinity component had an apparent Km (Michaelis-Menten constant) 3- to 4-fold higher than that of the broken membrane preparation. 5. Cooling intact guinea-pig and human red cells decreased the apparent affinity of the high velocity, low affinity component for ATP, so that at 20 degrees C the value of Km approached or exceeded the levels of physiological ATP concentration. Below 20 degrees C only one component with values corresponding to that of the low velocity, high affinity component could be observed. 6. In intact ground squirrel cells only the low affinity, high velocity component was apparent between 37 and 5 degrees C. Its affinity for ATP rose with cooling between 37 and 5 degrees C.

Diversities of transport of sodium in rodent red cells.

1. Na-K pumps of rodent red cells reveal variations among species in terms of kinetic properties such as ouabain sensitivity, Na/K coupling and temperature sensitivity and variations within an individual organism related to such physiological challenges as K deficiency, calorie deficiency and seasonal changes in temperature. 2. Passive Na entry among rodents collectively occurs through the same routes as in red cells of other mammals, but red cells of hamsters, rats and thirteen-lined ground squirrels lack or are deficient in an amiloride-sensitive, shrinkage-activated Na-H exchange. 3. In guinea-pig this pathway appears to be both activated and uncoupled by cooling from 37 to 20 degrees C. 4. Red cells of rodents in general and hamsters in particular are rich in a Na-Mg exchange pathway. In hamsters, this appears to be the only amiloride-sensitive pathway in simple media. 5. In hamster cells, Na entry through the amiloride-sensitive Mg-activated pathway exhibits the same kinetics as previously shown for Na activation of Mg extrusion.

Seasonal changes in cation transport in red blood cells of grey squirrel (Sciurus carolinensis) in relation to thermogenesis and cellular adaptation to cold.

1. Unidirectional influx of 42K was measured in red cells of grey squirrels at seasonal intervals over two years. 2. Na/K pump-related (i.e. ouabain-sensitive) K influx at 37 degrees C was maximal in cells collected in January and was more than three times greater than cells collected in summer. Na/K pump activity, maximized by loading the cells with Na, exhibited a similar difference. 3. At 5 degrees C in fresh cells, ouabain-sensitive K influx, expressed as per cent of that at 37 degrees C, was highest in March. In Na-loaded cells it was lowest in summer. 4. Passive "leak" K influx (i.e., the residual influx remaining in presence of ouabain and bumetanide) was highest in October, and declined progressively to the summer months, when it was only 27% of that in October. 5. Cotransport (i.e., bumetanide-sensitive K influx) exhibited the same seasonal pattern as Na/K pump activity in fresh cells. 6. Net gain of Na in cells stored at 5 degrees C for three days in March was less than half of that in January or summer. 7. High transport activity in January may correlate with a requirement for increased non-shivering thermogenesis. However, red cells of grey squirrels exhibit maximum resistance to low temperature in March and at this time resemble the red cells of hibernating mammals.

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.

Membrane transport of sodium ions in erythrocytes of the American black bear, Ursus americanus.

1. Membrane transport of Na ions was investigated in red blood cells of bears by methods of measurement of unidirectional isotopic fluxes. 2. Like red blood cells of dogs, bear red cells contain a high Na concentration and low concentrations of K and ATP. 3. As in dog red cells, Na efflux from bear cells was not inhibited by ouabain but was activated by the presence of Ca in the medium, possibly indicating the presence of a Na-Ca exchange mechanism. 4. ATP depletion of cells was accelerated by Ca in the medium, consistent with the presence of a strong ATP-dependent Ca pump. 5. As in other carnivore red cells, Na influx into bear cells was strongly activated by shrinkage and inhibited by swelling. Shrinkage-activated influx was blocked by amiloride. 6. Amiloride-sensitive influx was activated by cytoplasmic Ca and also correlated with the presence of a Na-dependent, amiloride-sensitive H loss. 7. Amiloride-sensitive Na influx exhibited a strong seasonal cycle with a minimum in the middle of the hibernation period, suggesting a possible avenue of cellular energy conservation.

Membrane transport of potassium ions in erythrocytes of the American black bear, Ursus americanus.

1. Membrane transport of K ions was investigated in red blood cells of bears by methods of measurement of unidirectional isotopic fluxes. 2. Unlike red cells of dogs, red cells of bears exhibited a significant, though small, component of ouabain-sensitive K influx. 3. Ouabain-insensitive K influx, as in other carnivore cells, was activated by swelling and inhibited by shrinkage. Swelling-induced K influx was dependent upon presence of chloride ions but was not inhibited by furosemide or bumetanide. 4. Ouabain-sensitive K influx was largest with ATP and with high concentration of Na in the cell, but it persisted in the absence of cytoplasmic Na or ATP. It was also resistant to the drug, harmaline, at a concentration that in other cells fully inhibits ouabain-sensitive K influx. 5. It was concluded that under such adverse conditions ouabain-sensitive K influx represents another mode of the Na/K pump not fully described elsewhere. 6. Also, as in low K red cells of sheep and goat, apparent absence of Na/K pump activity in carnivore red cells may represent suppression rather than elimination of activity. 7. Ouabain-insensitive K influx showed a seasonal pattern with minima occurring in early winter, earlier than for the minimum observed in Na influx. 8. Ouabain-sensitive K influx tended to be lower in the hibernation season of the bear, but the seasonal pattern was not consistent.

Maintenance of cation gradients in cold-stored erythrocytes of guinea pig and ground squirrel: improvement by amiloride.

Red blood cells of ground squirrel, a hibernator, gain Na at one-third the rate of guinea pig red blood cells when stored in saline medium at 5 degrees C for several days. This result correlates with the known slower loss of K during storage in ground squirrel cells. In ground squirrel cells Na gain is balanced by K loss, so that there is no net gain of solute; in guinea pig cells the total cation content rises progressively. Amiloride, a drug which inhibits Na entry, retards Na uptake in cells of both species. Surprisingly, amiloride also slowed K loss and, in guinea pig red cells, the decline of ATP content. In guinea pig cells amiloride reduced the gain of total cation by half. The results substantiate the difference in cold sensitivity of ion regulation of red blood cells of these two species and demonstrate the possible usefulness of amiloride-type drugs in nonfreezing preservation of red blood cells.

Differential effects of cooling in hibernator and nonhibernator cells: Na permeation.

Unidirectional Na influx is less inhibited by cooling in guinea pig red blood cells than in ground squirrel cells. To isolate the source of this difference, component pathways of 24Na entry were estimated by use of selective inhibitors (ouabain, bumetanide, amiloride). Amiloride slightly inhibited influx in ground squirrel cells at every temperature between 37 and 5 degrees C. Amiloride did not consistently inhibit Na influx at 37 degrees C in guinea pig cells but caused a 44% inhibition at 25 degrees C and a 35% inhibition at 5 degrees C. Cytoplasmic acidification caused an increase in amiloride-sensitive influx in guinea pig cells, which was greater at 20 degrees C than at 37 degrees C; cytoplasmic acidification decreased amiloride-sensitive Na influx in ground squirrel cells at 37 degrees C and had no effect at 20 degrees C.

Reduced ion transport in erythrocytes of male Sprague-Dawley rats during starvation.

Although several studies have suggested that the reduced activity of the Na+-K+ pump during starvation is a source of energy conservation, the hypothesis has not been tested in intact cells, nor has the contribution of passive permeability been considered in a controlled animal study. In this study three components of K+ influx (Na+-K+ pump = ouabain sensitive, cotransport = bumetanide sensitive and leak = both ouabain and bumetanide insensitive) and Na+ influx were measured with 42K+ and 24Na+ in intact red blood cells of adult male rats. During starvation rats lost an average of 28% of their body weight; pump K+ influx in cells stabilized for 2 h in incubation medium fell from 7.03 +/- 0.74 (SEM) to 4.82 +/- 0.25 mueq/(mL cells.h) with cell [Na+] of 6.4 +/- 0.9 and 4.4 +/- 0.2 mmol/L cells, respectively. Maximized Na+-K+ pump activity in Na+-loaded cells was also lower in cells of starved rats than in those of controls and was inversely correlated with extent of weight loss in the starved rats. Leak K+ influx was reduced from 0.73 +/- 0.08 to 0.47 +/- 0.03. Lower Na+ influx in cells of starved rats was not significant statistically, although alteration in passive Na+ transport was apparent. The results indicate decreases in both active and passive components of ion turnover of erythrocytes of rats during starvation.

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.

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)