The stoicheiometry of the sodium pump

1. When resealed ghosts containing adenosine triphosphate (ATP), magnesium and sodium were incubated in a medium containing potassium, ATP was hydrolysed vigorously by a ouabain-sensitive mechanism. If the ghosts contained potassium instead of or in addition to sodium, and the external solution contained sodium but no potassium, there was little ouabain-sensitive hydrolysis of ATP. As it is known that the ouabain-sensitive ATPase in fragmented ghosts requires both sodium and potassium ions, these results show that the ATPase is activated by potassium externally and by sodium internally, and suggest that the ions activating the ATPase are the ions that are transported.2. Resealed ghosts containing ATP, magnesium and sodium were incubated in sodium-free media containing potassium, with and without ouabain, and the rate of loss of sodium and rate of hydrolysis of ATP were measured. The hydrolysis of 1 molecule of ATP by the ouabain-sensitive mechanism was accompanied by the ouabain-sensitive loss of about 3 sodium ions.3. (24)Na and (42)K were used to measure sodium efflux and potassium influx in identical batches of fresh red cells under the same conditions and at the same time. Each flux was measured in the presence and absence of ouabain. The ratio (ouabain-sensitive sodium efflux)/(ouabain-sensitive potassium influx) was significantly greater than 1 (1.20 +/- 0.01 and 1.35 +/- 0.01 in two experiments). If a small fraction of the potassium influx represented a ouabain-sensitive potassium: potassium exchange, the ratio of the numbers of ions moved in the sodium: potassium exchange catalysed by the pump must have been even further from unity.4. Resealed ghosts containing [gamma-(32)P]ATP, magnesium, (24)Na and orthophosphate were incubated in balanced salt solutions with and without potassium and with and without ouabain. A comparison of sodium efflux, estimated from (24)Na loss, with ATP hydrolysis, estimated from the formation of [(32)P]orthophosphate, showed that the sodium:sodium exchange in a potassium-free medium was accompanied by little or no ouabain-sensitive hydrolysis of ATP.5. Experiments on intact red cells loaded with (24)Na showed that both sodium:sodium exchange in a potassium-free medium, and sodium:potassium exchange in a medium containing potassium, were partially inhibited by oligomycin (1-10 mug/ml.). Inhibition of the sodium:potassium exchange was not affected by raising the external potassium concentration.     

Reversal of the potassium entry mechanism in red cells, with and without reversal of the entire pump cycle

1. (42)K has been used to study the ouabain-sensitive component of potassium efflux from intact human red cells.2. Ouabain-sensitive efflux of potassium was observed only in media containing either sodium ions in moderate or high concentration or potassium ions.3. The effects of sodium and potassium in the medium were not additive. Potassium ions always increased the ouabain-sensitive potassium efflux, but in media containing 4.2 mM-K an increase in sodium concentration from 4.5 to 131 mM had little effect.4. In potassium-free media, ouabain-sensitive potassium efflux increased roughly linearly as the external sodium concentration was increased from 35 to 155 mM.5. The sensitivity of potassium efflux to external potassium depended on the concentration of sodium in the medium. In a 5 mM-Na (choline) medium, ouabain-sensitive potassium efflux was half-maximal at about 0.27 mM-K. In a 150 mM-Na medium the effect of potassium was half-maximal at about 1 mM-K. The relation between external potassium concentration and ouabain-sensitive sodium efflux was similarly influenced by the concentration of sodium ions in the medium.6. Inosine greatly reduced the ouabain-sensitive efflux of potassium into both 5 mM-K and potassium-free media. It probably acted by reducing the intracellular phosphate concentration to a low level.7. Ouabain-sensitive potassium efflux was not affected by the concentration of inorganic phosphate outside the cells and was not associated with a ouabain-sensitive efflux of phosphate of comparable magnitude. A small associated efflux could not be excluded.8. Simultaneous measurements of sodium efflux and of potassium efflux from identical batches of cells incubated in potassium-free media showed that inosine reduced ouabain-sensitive potassium efflux at the same time as it increased ouabain-sensitive sodium efflux.9. When cells that had been largely depleted of energy stores by pre-incubation with 2-deoxyglucose were incubated in high-sodium media, with and without potassium, it was observed that potassium in the medium increased the ouabain-sensitive potassium efflux but reduced the ouabain-sensitive efflux of sodium.10. Simultaneous measurements of the ouabain-sensitive efflux of potassium and influx and efflux of sodium across the membranes of starved cells incubated in potassium-free media, with and without inosine, suggested that ouabain-sensitive potassium efflux was associated with a net ouabain-sensitive entry of sodium.11. The results are best explained by supposing that the ouabain-sensitive efflux of potassium does not reflect lack of discrimination by the mechanism responsible for sodium expulsion, but is brought about by the reversal of steps in the pump cycle normally responsible for potassium entry.     

Lifespan of rabbit erythrocytes and activity of the reticulohistiocyte system

The survival rate was measured of differently aged rabbit erythrocytes that had been taken from young and old animals, tagged with ⁵¹Cr and injected into young and old animals. The survival rate of the donor erythrocytes depends mostly on the age of the recipient animals. The life span of the injected cells found in old animals was much lower than that found in young animals. On the other hand, the age of the donor animal also has an influence, although only a small one, on the life span of the red blood cells. The cells from young animals live rather longer than those from old animals.     

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.     

Physical and chemical reactions of phosphates in red cell membranes in relation to active transport

1. A study has been made of the chemical reactions associated with the active transport of sodium and potassium in human red cells, with special reference to the action of oligomycin and the labelling of cell membranes by radioactive ATP.2. Oligomycin had the same effects as ouabain on the various aspects of active transport in red cells. It inhibited sodium and potassium transport, the transport ATPase, and the lactate production that is controlled by the sodium pump.3. When cell membranes were incubated with radioactive ATP they became labelled. The labelling, however, was unrelated to the activity of the transport ATPase. Incubation of membranes and intact cells showed that the labelling was a result of physical adsorption of inorganic phosphate to the membranes.4. The results show that if there is a phosphate intermediate in the reaction of the transport ATPase in human red cell membranes, then it is present in amounts too small to be detectable by the methods that have been successfully used with organs such as brain and kidney.     

Synthesis of adenosine triphosphate at the expense of downhill cation movements in intact human red cells

1. When red cells that have been starved for about 6 hr are loaded with inorganic phosphate and incubated in high-sodium potassium-free media, the ouabain-sensitive efflux of potassium from the cells is accompanied by a ouabain-sensitive incorporation of inorganic phosphate into ATP.2. The magnitude of the incorporation varies roughly linearly with the concentration of sodium in the medium. The ratio (ouabain-sensitive potassium efflux)/(ouabain-sensitive ATP synthesis) is probably not much less than 2 nor much greater than 3.3. Potassium in the medium inhibits the ouabain-sensitive incorporation of phosphate. The concentration of potassium necessary for half-maximal inhibition is about the same as the concentration at which, under similar conditions, ouabain-sensitive potassium influx and the stimulation of ouabain-sensitive potassium efflux are both half-maximal.4. These observations suggest that the ouabain-sensitive efflux of potassium from red cells incubated in high-sodium potassium-free media is associated with a reversal of the entire pump cycle. In media containing sufficient potassium to saturate the pump, the efflux appears to involve the reversal of only part of the cycle.     

Age-related increase in erythrocyte oxidant sensitivity

Unlike erythrocytes from elderly humans, red blood cells from old mice are not more sensitive than are cells from young animals to lysis in hypotonic solutions, probably because the mean corpuscular volume decreases rather than increases with age in this species. However, when subjected to an oxidant stress (sodium ascorbate) red blood cells from old animals accumulate more methemoglobin and fewer remain intact than is the case with red blood cells from young mice. The data suggest that this increased vulnerability to oxidative damage is manifest relatively early in the lifespan of red blood cells from old animals and is not solely a property of the older cells. The pathogenesis of the decreased resistance to peroxidation is not known, but it does not appear to be the result of changes in reduced glutathione, NADH: methemoglobin reductase, superoxide dismutase, glutathione reductase, glutamic-oxaloacetic transaminase, or glucose 6-phosphodehydrogenase.     

Peripheral effects of thyroid hormones: Alteration of intracellular Na-concentration,ouabain-sensitive Na-transport,and Na-Li countertransport in human red blood cells

Summary To investigate the effect of thyroid hormones on erythrocyte cation transport systems and intracellular electrolyte content we have measured the activity of Na-K ATPase, Na-Li countertransport, as well as red cell sodium and potassium contents in patients with hyperthyroidism and in euthyroid controls. Intracellular Na- and K-concentrations were determined in erythrocytes washed three times in isotonic MgCl₂ solution. Ouabain-sensitive Na-transport was estimated as the increase of Na before and after addition of ouabain in an erythrocyte suspension in isotonic Na-free medium. Na-Li countertransport was measured according to the method described by Canessa et al. [2]. The patients with hyperthyroidism exhibited a significantly elevated intracellular sodium content as well as a highly increased Na-K ATPase activity. Intracellular potassium content was not altered in the hyperthyroid subjects, but Na-Li countertransport was markedly decreased as compared to the controls.The results indicate that different ion transport systems of the erythrocyte membrane are influenced by thyroid hormones. We suggest that the elevation of Na-K ATPase activity might be due to the increased intracellular sodium concentration which is caused by the diminished countertransport pathway. Furthermore, the activity of Na-K ATPase, Na-Li countertransport, and intracellular sodium content in erythrocytes might be a useful peripheral indicator of thyroid hormone excess.Supported by the Bundesministerium für Forschung und Technologie (MMT 27)     

Erythrocyte membrane cation carrier in mania.

Erythrocyte sodium and potassium concentrations, erythrocyte membrane ATPase (Na-K specific and non-specific) and the rate of potassium influx into erythrocytes (ouabain-sensitive and insensitive) were estimated in a group of female patients suffering from mania and repeated on about two thirds of them when they had recovered. With recovery there was a statistically significant increase in the erythrocyte ouabain-sensitive potassium influx. The other parameters showed no significant overall change with recovery but the initial severity correlated significantly and negatively with the change in erythrocyte Na-K ATPase with recovery. The changes that occurred in the erythorcyte sodium concentration and Na-K ATPase activity were not random since they correlated significantly with changes in the active potassium influx.     

Effect of age on some properties of mice erythrocytes

The hematological parameters of young (2-month-old) and old (2-year-old) mice were compared. No differences could be detected with the exception of an increased percentage of reticulocytes in the old animals suggesting that anemia in senescent mice does not occur. Red blood cell mean half-life in old mice was 8 +/- 0.8 days compared to 12 +/- 1 days in young mice. This reduced survival of red blood cell is not due to a different rate of cell phagocytosis in the reticulohistiocytic system of young and old animals since erythrocytes from young mice have the same mean half-life when injected both in young and old animals and vice versa. Thus, the old mice have a reduced red cell life-span but the same hematocrit of the young, suggesting that old animals possess a chronologically younger population of erythrocytes than do young animals. This has been confirmed by measuring the specific activities of some red blood cell age-dependent enzymes (hexokinase, glucose-6-phosphate dehydrogenase, pyruvate kinase) that were found to be higher in the older animals, and by the separation of erythrocytes into different density (age) groups by Percoll/albumin density gradient centrifugation. However, the erythrocytes osmotic fragility, and the cellular contents of adenine and pyridine nucleotides, as well as the content of 2,3-diphosphoglycerate and reduced glutathione, show that circulating erythrocytes in old animals constitute an heterogeneous cell population whose properties cannot be explained on the basis of a chronologically younger erythrocyte population. Furthermore, evaluation of cell components in hemopoietic tissues have shown an increased porportion of erythroid precursor cells in old animals confirming that old mice compensate for reduced red cell survival with an increased erythropoiesis.     

Phagocytic activity of the reticulohistiocyte system in rabbits after splenectomy and activation with ink

The survival rate was measured of differently aged rabbit erythrocytes that had been taken from young animals, tagged with ⁵¹Cr and injected into young and old animals. The recipient animals had either been splenectomized, or had an ink-activated reticulohistiocyte system, or both. In young as well as in old ink-treated animals the injected cells lived for a much shorter time than in the non-treated animals. In splenectomized animals the red blood cells lived a little longer than those in the control group. In animals which had been both splenectomized and ink-treated the ⁵¹Cr-tagged cells again lived for a shorter time than those in the control group, but longer than in the ink-treated animals.     

Age-dependence of polyadenylate stimulation of nuclear-envelope nucleoside triphosphatase

Nuclear envelopes of mammalian cells contain a nucleoside triphosphatase which is probably involved in mRNA transport through the nuclear membrane. The activity of the enzyme, studied in RNA-depleted nuclear ghosts, can be stimulated by poly(A) or by poly(A) (+)mRNA. Using nuclear ghost preparations from mature (8–10 months' old) and old (40–42 months' old) Wistar rats, it was shown that in “old” preparations the basal activity of the enzyme is significantly reduced (by 15%). In addition, the enzyme from old animals responds only very little to poly(A) or poly(A) (+)mRNA, compared to preparations from mature animals. Using a concentration of 6.8 × 10¹¹ poly(A) (+)mRNA molecules per μg of enzyme preparation, the nucleoside triphosphatase from mature animals is stimulated by 77% and the enzyme from old animals by only 26%.

Binding studies of poly(A) to pore laminae revealed that the number of binding sites in unphosphorylated preparations from old animals is significantly reduced (by 24%) compared to “mature” preparations. As a consequence of in vitro phosphorylation, no difference is observable in the number of binding sites between the two age groups. The values for half-maximal saturation binding constants for poly(A) are identical in unphosphorylated and phosphorylated pore-laminae preparations, irrespective of the age group studied. The results presented indicate that in old animals the pathway from the phosphorylated to the dephosphorylated nuclear-envelope protein which is controlled by poly(A) is impaired in the proposed cycle for mRNA efflux from nuclei.     

Epidermal Langerhans cell density and contact sensitivity in young and aged BALB/c mice

The loss of tissue and organ function with age may depend on the inability of old cells to carry out specialized functions. Like other systems in the body, the immune system deteriorates with age. Over the past 10 years it has become clear that the skin can play an active role in immunological processes. In this report we evaluated changes in murine cutaneous immunity with age. Studies in humans had shown a decreased Langerhans cell density with age, but it is difficult to control for the effect of ultraviolet light in human studies. Since ultraviolet light has a significant effect on Langerhans cells, we chose to evaluate the effect of age on Langerhans cell density using inbred mice not exposed to ultraviolet light. Cutaneous immunity was examined phenotypically by studying Langerhans cell density and functionally by studying allergic contact sensitivity. Langerhans cell density was assessed in epidermal sheets prepared from ear skin of mice and examined by ATPase histochemistry and fluoresceinated anti-Ia staining. With both methods, aged (18 months old) mice had approximately two-thirds the number of Langerhans cells that young (10-12 weeks old) animals did. Allergic contact sensitivity response to trinitrochlorobenzene (TNCB) was compared between aged and young animals. Although the aged animals demonstrated increased variability in their responsiveness, there was no overall difference in this example of cutaneous immunoreactivity between the two age groups.     

Thyroid hormone regulation of myosin heavy chain isoform composition in young and old rats,with special reference to IIX myosin

The effects of 4 weeks of thyroid hormone (3,5,3′-triiodothyronine, T₃) treatment on the myosin heavy chain (MHC) composition were compared in the slow-twitch soleus and the fast-twitch extensor digitorum longus (EDL) muscles from young (3–6 months) and old (20–24 months) male albino rats. Four MHC isoforms were separated on silverstained 6% sodium dodecyl sulphate polyacrylamide gel electrophoresis. According to immunoblotting experiments with specific MHC monoclonal antibodies, the four MHCs corresponded to types I, IIB, IIX and IIA. In the soleus, the type I MHC content was higher in the old than in the young animals, and the type IIA content lower. Type IIX myosin was observed in some young control soleus, but not in old ones. After T₃ treatment, the content of type I MHC decreased substantially in both young and old animals and that of type IIA increased. After T₃ treatment, type IIX myosin was observed in both young and old animals, with a slighty higher IIX myosin content in old age, but the age-related different in the contents of types I and IIA was diminished. In EDL, the type IIX MHC content was significantly higher in the old animals, at the significantly by T₃ treatment in EDL, either in young or old animals. In conclusion, an age-related motor unit transformation is observed in both the slow-twich soleus and the fast-twitch EDL and the capacity for MHC isoform switching in response to T₃ treatment is not impaired in old age.     

The sensitivity of the sodium pump to external sodium

1. When red cells are incubated in potassium-free solutions, ouabain-sensitive sodium efflux is nearly absent with 5 mM-Na externally, but increases as the external sodium concentration is reduced from 5 mM to zero. This increase suggests that the transport mechanism is very sensitive to small amounts of sodium at the outside surface of the cell membrane. Further evidence for such sensitivity has been obtained from the effects of external sodium on the relation between potassium influx and external potassium concentration.2. With 5 mM-[K](o), potassium influx is rather insensitive to [Na](o) but at low potassium concentrations even low levels of sodium inhibit.3. With 140 mM-[Na](o) the potassium influx curve is S-shaped below 1 mM [K](o). At much lower sodium concentrations, the S-shaped region and the value of [K](o) for which potassium influx is half-maximal are both shifted progressively towards zero. At 10 muM-[Na](o), potassium influx is half maximal at 0.14 mM-[K](o) and the curve is close to a rectangular hyperbola down to 22 muM-[K](o); there seems to be a trace of inflexion at about 15 muM-[K](o).4. When [Na](o) is reduced from 5 mM to zero, removal of the inhibitory effect of external sodium ions on sodium: potassium exchange could lead to an increase in sodium efflux into nominally potassium-free solutions if these solutions did in fact contain traces of potassium. Such traces could arise by leakage from the cells, but, in a number of experiments, direct measurements showed that [K](o) was too low to account in this way for all of the observed ouabain-sensitive sodium efflux. A further reason for rejecting this explanation is that ouabain-sensitive potassium loss into nominally (Na+K)-free solutions was unaffected by adding 5 mM-Na. (A slight increase in ouabain-resistant loss was observed.)5. The ouabain-sensitive efflux of sodium into (Na+K)-free solutions therefore seems to represent a mode of behaviour of the transport mechanism distinct both from the sodium: potassium exchange that occurs under physiological conditions and from the sodium: sodium exchange that occurs in K-free, Na-rich media.     

Treatment with dehydroepiandrosterone (DHEA) stimulates oxidative energy metabolism in the cerebral mitochondria. A comparative study of effects in old and young adult rats

The content of the neurosteroids, dehydroepiandrosterone (DHEA) in the brain decreases with aging. Also the oxidative energy metabolism is known to decrease with aging. Hence we examined the effects of treatment with DHEA (0.2 or 1.0 mg/kg body weight for 7 days) on oxidative energy metabolism in brain mitochondria from old and young adult rats. State 3 respiration rates in brain mitochondria from old animals were considerably lower than those in young adults. Treatment with DHEA stimulated state 3 and state 4 respiration rates in both the groups of the animals in a dose-dependent manner. In the old rats following DHEA treatment, the state 3 respiration rates became comparable to or increased beyond those of untreated young adults. In contrast to the old rats, stimulatory effect of DHEA treatment was of greater magnitude in the young adults. However, at higher dose (1.0 mg) the effect declined. Cytochrome aa3 content in the brain mitochondria from old rats was significantly low but the content of cytochrome b was unchanged while the content of cytochromes c+c1 had increased. Treatment with DHEA increased the content of cytochrome aa3 and b in old as well as in young adult animals. Higher dose of DHEA (1.0 mg) had adverse effect on the content of cytochrome c+c1. DHEA treatment stimulated ATPase activity in a dose-dependent manner in young adult rats whereas in the old rats the effect on ATPase activity was marginal. Dehydrogenases activities were somewhat lower in the old rats. DHEA treatment stimulated mitochondrial dehydrogenases activities in both the groups. Results of our studies suggest that judicious use of DHEA treatment can improve oxidative energy metabolism parameters in brain mitochondria from young adult as well as old rats.     

Aging and developmental transitions in the B cell lineage

One explanation for the deterioration of the humoral immune response in elderly individuals is that B lymphopoiesis declines with increasing age. Recent studies report a dramatic decline in pre-B cell numbers in old mice. Surprisingly, the number of mature B cells does not decline with age. To determine if new B cells are made in aged animals despite the drop in pre-B cells, we used 5'-bromo-2-deoxyuridine labeling to determine the production rate of B cells in the bone marrow and spleen of young and old mice. Because of the great variability in the number of early B lineage cells in old mice, we acquired data on >60 young and 50 old mice throughout these experiments. The transitional and mature B cell compartments in the spleen have slower labeling kinetics in old mice as compared to young. By the end of 4 weeks of labeling, an average of only 15% of the mature B cell compartment consists of newly made cells compared to 30% in young mice. However, in contrast to an earlier report, our results indicate that there is no statistical difference in the rate of production of new immature B cells in the marrow of young and old animals. In total, our results confirm previous work showing that mature B cells in old mice have a slower turnover, but more importantly suggest that the defect in mature B cell turnover is not due to a decline in B lymphopoiesis, but rather an inability of the newly made cells to replenish the peripheral compartments.     

Age-related change in brush borders of rat kidney cortex

Age-related change of rat renal brush borders was examined with electron microscopy and biochemical procedures. Total activity of renal brush border enzymes, such as alkaline phosphatase and leucine aminopeptidase in the homogenate, was significantly decreased with age. Acid phosphatase activity and protein content were not significantly changed with age. Specific activity of leucine aminopeptidase in brush border fraction was significantly decreased at a later stage of age. Protein content of brush border fraction was decreased significantly with age. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, some of the proteins disappeared during aging. Electron microscopic observations of kidney cortex showed that microvilli of renal brush borders from old rats were observed to be fewer than those from the young; epithelial cells in young rats have more densely packed brush border projections than those in old rats. From these results, it is suggested that during aging renal brush borders are degraded, and that protein components of the brush borders were different between old and young.     

Age-related modifications of leucine uptake in brush-border membrane vesicles from rat jejunum.

Leucine uptake in brush-border membrane vesicles purified from rat jejunum is sodium-dependent, sensitive to the membrane electrical potential difference and enhanced by the intravesicular presence of potassium. This last effect is not mediated by the genesis of an electrical potential difference, since potassium activation and electrical potential effects are additive. Sodium-dependent leucine Vmax (1568 +/- 91 pmol/mg per 3 s, is higher in young rats than in adult and old animals. The diffusion component of leucine transport decreases with increasing age. Preloading the vesicles with 100 mM KCl increases leucine Vmax 200% in young animals, 100% in adult and 44% in old animals. The potassium activation is a saturation function of the cation concentration. Leucine uptake in brush border membrane from old animals is less sensitive to the electrical potential difference than in membranes from adult and young animals.     

The distribution of potassium,sodium and chloride across the apical membrane of renal tubular cells: effect of acute metabolic alkalosis

Studies were undertaken to define the effect of acute metabolic alkalosis (hypertonic sodium bicarbonate i.v.) on the chemical gradients for potassium, sodium and chloride across the apical membrane of individual renal tubule cells. Electron microprobe analysis was used on freeze-dried cryosections of the rat renal cortex to measure electrolyte concentrations in proximal tubule cells and in the various cell types of the superficial distal tubule. Analyses were also performed in fluid samples obtained by micropuncture from proximal and early and late distal collection sites. Compared with the appropriate controls (hypertonic sodium chloride i.v.), administration of sodium bicarbonate resulted only in small and mostly insignificant increases in cell potassium concentrations and induced only minor alterations in the cell/tubule fluid potassium concentration gradient for all cell types analysed. This observation suggests that under this condition factors other than an increase in cell potassium concentration are important in modulating potassium transfer across the apical membrane of potassium secreting cells. Nevertheless, since in alkalosis phosphorus and cell dry weight were decreased, and hence cell volume increased, in all but the intercalated cells, actually the potassium content of most tubular cells was higher under this condition. In comparison with animals infused with isotonic saline at low rates (hydropenic controls), infusion of either hypertonic sodium chloride or sodium bicarbonate led to a sharp increase in distal tubule fluid sodium concentrations and in the sodium concentrations of distal convoluted tubule, connecting tubule and principal cells, indicating that under both conditions the primary event causing enhanced transepithelial sodium absorption is stimulation of the sodium entry step. The ensuing rise in cell sodium concentration shold lead secondarily to stimulation of active basolateral sodium extrusion. Intercalated cell sodium concentration was higher only in alkalosis which supports the notion that this cell type is not involved in transepithelial sodium transport.