Mechanisms and consequences of Na+,K+-pump regulation by insulin and leptin.

Hormonal control of the Na+,K+-pump modulates membrane potential in mammalian cells, which in turn drives ion coupled transport processes and maintains cell volume and osmotic balance. Na+,K+-pump regulation is particularly important in the musculoskeletal, cardiovascular and renal systems. Decreased Na+,K+-pump activity can result in a rise in intracellular Na+ concentrations which in turn increase Na+/Ca2+ exchange, thereby raising intracellular Ca2+ levels. In cardiac and skeletal muscle, this could interfere with normal contractile activity. Similarly, in vascular smooth muscle the result would be resistance to vasodilation. Inhibition of the Na+,K+-pump can also reduce the driving force for renal tubular Na+ reabsorption, elevating Na+ excretion. By virtue of decreasing the membrane potential, thus allowing more efficient depolarization of nerve endings and by increasing intracellular Ca2+, inhibition of the Na+,K+-pump can increase nervous tone. The ability of insulin to stimulate the Na+,K+-pump in various cells and tissues, and the physiological significance thereof, have been well documented. Much less is known about the effect of leptin on the Na+,K+-pump. We have shown that leptin inhibits Na+,K+-pump function in 3T3-L1 fibroblasts. Defects in insulin and leptin action are associated with diabetes and obesity, respectively, both of which are commonly associated with cardiovascular complications. In this review we discuss the mechanisms of Na+,K+-pump regulation by insulin and leptin and highlight how, when they fail, they may contribute to the pathophysiology of hypertension associated with diabetes and obesity.     

Two modes of inhibition of the Ca2+ pump in red cells by Ca2+

Two different and independent modes of inhibition of the Ca2+ pump by Ca2+ can be detected measuring active Ca2+ extrusion from resealed ghosts of human red cells: one requires extracellular and the other requires intracellular Ca2+. Ki for inhibition by extracellular Ca2+ is about 10 mM. Extracellular Mg2+ replaces Ca2+ in inhibiting Ca2+ transport but with an apparent affinity for inhibition about 3-times less than that for Ca2+. Inhibition by external Ca2+ is not affected by Na+ or K+ at both surfaces of the cell membrane, external EGTA, internal Ca2+ or ATP. The apparent affinity for external Ca2+ progressively raises as pH increases. The effects of extracellular Ca2+ and Mg2+ are consistent with the idea that for Ca2+ pumping to proceed, external sites in the pump must be protonated and not occupied by extracellular Ca2+ or Mg2+. Inhibition by intracellular Ca2+ takes place with a Ki of about 1 mM and is independent of external Ca2+. The inhibitory effects of intracellular Ca2+ can be accounted for if Ca2+ and CaATP were competitive inhibitors of the activation of the pump by Mg2+ and MgATP, respectively.     

Preserved function of the plasma membrane calcium pump of red blood cells from diabetic subjects with high levels of glycated haemoglobin

The activity of the plasma membrane Ca²⁺-pump decreases steeply throughout the 120 days lifespan of normal human red blood cells. Experiments with isolated membrane preparations showed that glycation of a lysine residue near the catalytic site of the pump ATPase had a powerful inhibitory effect. This prompted the question of whether glycation is the mechanism of age-related decline in pump activity in vivo. It is important to investigate this mechanism because the Ca²⁺ pump is a major regulator of Ca²⁺ homeostasis in all cells. Its impaired activity in diabetic patients, continuously exposed to high glycation rates, may thus contribute to varied tissue pathology in this disease. We measured Ca²⁺-pump activity as a function of red cell age in red cells from diabetics continuously exposed to high glucose concentrations, as documented by their high mean levels of glycated haemoglobin. The distribution of Ca²⁺-pump activities was indistinguishable from that in non-diabetics, and the pattern of activity decline with cell age in the diabetics’ red cells was identical to that observed in red cells from non-diabetics. These results indicate that in intact cells the Ca²⁺ pump is protected from glycation-induced inactivation.     

Trypsin activation of the red cell Ca2+-pump ATPase is calcium-sensitive

Stimulation of the calmodulin-independent activity of the red cell Ca2+-pump ATPase by trypsin treatment (of calmodulin free red cell membranes) is sensitive to Ca2+ in a concentration range near the KCa of the transport site. The Ca2+ requirement for this effect is absolute, whereas the calmodulin sensitivity of the ATPase can be abolished by sufficient trypsin attack in the absence of Ca2+, although Ca2+ accelerates inactivation. This indicates that the two effects of trypsin are due to at least two distinct cleavage sites in the pump protein.     

Kinetics of inhibition of the plasma membrane calcium pump by vanadate in intact human red cells.

The lack of specific inhibitors of the plasma membrane Ca2+ pump (PMCA) has made vanadate (VO3-), a non-specific inhibitor, an invaluable tool in the study of PMCA function. However, three important properties of vanadate as an inhibitor of the PMCA in intact cells, namely its speed of action in different experimental conditions, the reversibility of its inhibitory effects at different doses, and its dose-response, had never been characterized, despite extensive use. We report here the speed, reversibility and dose-response of PMCA inhibition by vanadate in intact human red cells. Near maximal inhibitory concentrations (1mM) in the red cell suspension blocked almost instantly the uphill Ca2+ extrusion by the PMCA, regardless of the intracellular Ca2+ concentration, cation composition of the external media, membrane potential or volume-stability of the cell. PMCA inhibition by vanadate, at concentrations of 10mM and 1mM, was not reversed by washing, resuspending, and incubating the cells for up to 2h in vanadate-free media. Vanadate inhibited PMCA-mediated Ca2+ efflux in intact red cells with a K1/2 of approximately 3 microM, a value similar to that described for the Ca2+-ATPase in isolated red cell membranes.     

Characteristics of the Ca2+ pump and Ca2+-ATPase in the plasma membrane of rat myometrium.

A plasma membrane-enriched fraction from rat myometrium shows ATP-Mg2+-dependent active calcium uptake which is independent of the presence of oxalate and is abolished by the Ca2+ ionophore A23187. Ca2+ loaded into vesicles via the ATP-dependent Ca2+ uptake was released by extravesicular Na+. This showed that the Na+/Ca2+ exchange and the Ca2+ uptake were both occurring in plasma membrane vesicles. In a medium containing KCl, vanadate readily inhibited the Ca2+ uptake (K1/2 5 microM); when sucrose replaced KCl, 400 microM-vanadate was required for half inhibition. Only a slight stimulation of the calcium pump by calmodulin was observed in untreated membrane vesicles. Extraction of endogenous calmodulin from the membranes by EGTA decreased the activity and Ca2+ affinity of the calcium pump; both activity and affinity were fully restored by adding back calmodulin or by limited proteolysis. A monoclonal antibody (JA3) directed against the human erythrocyte Ca2+ pump reacted with the 140 kDa Ca2+-pump protein of the myometrial plasma membrane. The Ca2+-ATPase activity of these membranes is not specific for ATP, and is not inhibited by mercurial agents, whereas Ca2+ uptake has the opposite properties. Ca2+-ATPase activity is also over 100 times that of calcium transport; it appears that the ATPase responsible for transport is largely masked by the presence of another Ca2+-ATPase of unknown function. Measurements of total Ca2+-ATPase activity are, therefore, probably not directly relevant to the question of intracellular Ca2+ control.     

Effects of lanthanum on calcium-dependent phenomena in human red cells.

Lanthanum (0.25 mM) does not penetrate into fresh or Mg2+-depleted cells, whereas it does into ATP-depleted or ATP + 2,3-diphosphoglycerate-depleted cells, into cells containing more than 3 mM calcium, or cells stored for more than 4 weeks in acid/citrate/dextrose solution. In fresh cells loaded with calcium, extracellular lanthanum blocks the active Ca2+-efflux completely and inhibits (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity to about 50%. In Mg2+-depleted cells Ca2+-Ca2+ exchange is inhibited by lanthanum. Ca2+-leak is unaffected by lanthanum up to 0.25 mM concentration; higher lanthanum concentrations reduce leak rate. In NaCl medium Ca2+-leak +/ S.D. amounts to 0.28 +/ 0.08 mumol/1 of cells per min, whereas in KC1 medium to 0.15 +/ 0.04 mumol/1 of cells per min at 2.5 mM [Ca2+]e and 0.25 mM [La3+]e pH 7.1. Lanthanum inhibits Ca2+-dependent rapid K+ transport in ATP-depleted and propranolol-treated red cells, i.e. whenever intracellular calcium is below a critical level. The inhibition of the rapid K+ transport can be attributed to protein-lanthanum interactions on the cell surface, since lanthanum is effectively detached from the membrane lipids by propranolol. Lanthanum at 0.2--0.25 mM concentration has no direct effect on the morphology of red cells. The shape regeneration of Ca2+-loaded cells, however, is blocked by lanthanum owing to Ca2+-pump inhibition. Using lanthanum the transition in cell shape can be quantitatively correlated to intracellular Ca2+ concentrations.     

Mechanism of apical K+ channel modulation in principal renal tubule cells. Effect of inhibition of basolateral Na(+)-K(+)-ATPase

The effects of inhibition of the basolateral Na(+)-K(+)-ATPase (pump) on the apical low-conductance K+ channel of principal cells in rat cortical collecting duct (CCD) were studied with patch-clamp techniques. Inhibition of pump activity by removal of K+ from the bath solution or addition of strophanthidin reversibly reduced K+ channel activity in cell-attached patches to 36% of the control value. The effect of pump inhibition on K+ channel activity was dependent on the presence of extracellular Ca2+, since removal of Ca2+ in the bath solution abolished the inhibitory effect of 0 mM K+ bath. The intracellular [Ca2+] (measured with fura-2) was significantly increased, from 125 nM (control) to 335 nM (0 mM K+ bath) or 408 nM (0.2 mM strophanthidin), during inhibition of pump activity. In contrast, cell pH decreased only moderately, from 7.45 to 7.35. Raising intracellular Ca2+ by addition of 2 microM ionomycin mimicked the effect of pump inhibition on K+ channel activity. 0.1 mM amiloride also significantly reduced the inhibitory effect of the K+ removal. Because the apical low-conductance K channel in inside-out patches is not sensitive to Ca2+ (Wang, W., A. Schwab, and G. Giebisch, 1990. American Journal of Physiology. 259:F494-F502), it is suggested that the inhibitory effect of Ca2+ is mediated by a Ca(2+)-dependent signal transduction pathway. This view was supported in experiments in which application of 200 nM staurosporine, a potent inhibitor of Ca(2+)- dependent protein kinase C (PKC), markedly diminished the effect of the pump inhibition on channel activity. We conclude that a Ca(2+)- dependent protein kinase such as PKC plays a key role in the downregulation of apical low-conductance K+ channel activity during inhibition of the basolateral Na(+)-K(+)-ATPase.     

Effects of glycyrrhizin and glycyrrhetinic acid on (Na+ + K+)-ATPase of renal basolateral membranes in vitro

Studies were made on the direct effects of glycyrrhizin and its aglycone, glycyrrhetinic acid on the activities of (Na+ + K+)-ATPase and (Ca2+ + Mg2+)-ATPase, a membrane bound Na+ and Ca2+-extrusion pump enzyme of the basolateral membranes (BLM) of canine kidney. Glycyrrhetinic acid inhibited the activity of the Na+-pump enzyme dose-dependently (IC50 = 1.5 x 10(-4) M), but had no effect on that of the Ca2+-pump enzyme of kidney BLM and homogenates. Glycyrrhizin also inhibited the Na+-pump enzyme activity but had less effect (IC50 = 2 x 10(-3) M). The effects of these compounds were due to competitive inhibition with ATP binding to the enzyme (Ki = 12 microM) and so were different from that of ouabain, which inhibits the Na+-pump by binding to its extracellular K+-binding site. The direct effect of glycyrrhetinic acid on the membrane may be important role in the multiple actions of licorice.     

Detection and localization of a Ca2+-ATPase activity in Toxoplasma gondii

Toxoplasma gondii, the agent causing toxoplasmosis, is an obligate intracellular protozoan parasite. A calcium signal appears to be essential for intracellular transduction during the active process of host cell invasion. We have looked for a Ca2+-transport ATPase in tachyzoites and found Ca2+-ATPase activity (11-22 nmol Pi liberated/mg protein/min) in the tachyzoite membrane fraction. This ATP-dependent activity was stimulated by Ca2+ and Mg2+ ions and by calmodulin, and was inhibited by pump inhibitors (sodium orthovanadate or thapsigargin). We used cytochemistry and X-ray microanalysis of cerium phosphate precipitates and immunolabelling to find the Ca2+, Mg2+-ATPase. It was located mainly in the membrane complex, the conoid, nucleus, secretory organelles (rhoptries, dense granules) and in vesicles with a high calcium concentration. Thus, Toxoplasma gondii possesses Ca2+-pump ATPase (Ca2+, Mg2+-ATPase) as do eukaryotic cells.     

Physiologic rate of carrier-mediated Ca2+ entry matches active extrusion in human erythrocytes.

The intracellular Ca2+ concentration of nearly all cells is kept at submicromolar levels. The magnitudes of transmembrane Ca2+ movement that maintain this steady state in the human red blood cell have long been debated. Although there is agreement that the physiologic extrusion of Ca2+ by the well-characterized Ca2+. ATPase amounts to 45 mumol/liter cells per h (1982. Nature (Lond.). 298:478-481), the reported passive entry rates in physiological saline (2-20 mumol/liter cells per h) are all substantially lower. This discrepancy could be due to incomplete inhibition of the pump in the previous measurements of Ca2+ entry. We therefore examined both rate and mechanism of entry after completely inactivating the pump. This required pretreatment with iodoacetamide (to lower the intracellular ATP concentration) and vanadate (to inhibit any residual Ca2+ pump activity). The rate of Ca2+ entry (53 mumol/liter cells per h) was now found to be comparable to the accepted extrusion rate. Entry closely obeyed Michaelis-Menten kinetics (Vmax = 321 +/- 17 nmol Ca/g dry wt per h, Km = 1.26 +/- 0.13 mM), was competitively inhibited by external Sr2+ (Ki = 10.8 +/- 1.2 mM), and was accelerated by intracellular Ca2+. 45Ca2+ efflux from these pump-inactivated cells was also accelerated by either external Ca2+ or Sr2+. These accelerating effects of divalent cations on the opposite (trans) face of the membrane rule out a simple channel. Substrate-gated channels are also ruled out: cells equilibrated with 45Ca2+ lost the isotope when unlabeled Ca2+ or Sr2+ was added externally. Thus, passive Ca2+ movements occur predominantly by a reversible carrier-mediated mechanism for which Sr2+ is an alternate substrate. The carrier's intrinsic affinity constants for Ca2+ and Sr2+, 1.46 and 0.37 mM-1, respectively, indicate that Ca2+ is the preferred substrate.     

Investigation of properties of the Ca2+ influx and of the Ca2+-activated K+ efflux (Gárdos effect) in vanadate-treated and ATP-depleted human red blood cells

In this study the properties of the 45Ca2+ influx in human red blood cells (RBC) induced by NaVO3 or ATP-depletion were compared. Both NaVO3-induced and ATP-depletion-induced 45Ca2+ influxes were in the range 10(-6)-10(-5) mol Ca2+ x l(-1)cells x h(-1). The saturatability of ATP-depletion-induced 45Ca2+ influx with Ca2+ was much less pronounced than that of NaVO3-induced 45Ca2+ influx. The NaVO3-induced Ca2+ influx was sensitive to nifedipine (IC50 = 50 micromol/l) and Cu2+ (IC50 = 9 micromol/l) but these inhibitors had only a marginal effect when ATP-depletion was used as the Ca2+ influx inducer. On the other hand, polymyxin B (PXB) (1-5 mg/ml) strongly stimulated the ATP-depletion-induced 45Ca2+ influx whereas its effect on the NaVO3-induced Ca2+ influx was biphasic, with about 10% stimulation at lower PXB concentrations and an inhibition of 40% at higher concentrations. SDS-PAGE revealed that both NaVO3 and PXB induced changes in the protein phosphorylation pattern in the presence of Ca2+. NaVO3 stimulated the phosphorylation of several proteins and this effect was counteracted by PXB. The comparison of the kinetics and temperature dependencies of the Gárdos effect induced by NaVO3 and the ATP-depletion showed marked differences. The ability of NaVO3 to induce the Gárdos effect dramatically increased in ATP-depleted cells. These findings indicate that the 45Ca2+ influxes preceding the activation of the Ca2+-activated K+ efflux (Gárdos effect) stimulated by NaVO3 and by ATP-depletion, are mediated by different transport pathways. In addition, obtained results demonstrate that ATP-depletion and NaVO3-treatment exert additive action in triggering the Gárdos effect.     

Hemolysate increases calcium-inhibition of the Na+,K+ pump of resealed human red cell ghosts

The Na+,K+ pump of resealed human red cell ghosts is more sensitive to inhibition by intracellular Ca (Cai) when they contain diluted hemolysate compared to ghosts without hemolysate. The activity of the Na+,K+ pump was assessed by measuring ouabain-sensitive 22Na efflux in ghosts that, in addition to the presence or absence of hemolysate, also contained arsenazo III to measure free Cai and a regenerating system to maintain a constant concentration of ATP. Incorporating hemolysate diluted 20-fold compared to in situ conditions doubled the inhibitory effects of 1-50 microM free Cai on the Na+,K+ pump and caused 50% inhibition to occur between 5 and 10 microM free Cai. Increased inhibition in the presence of the hemolysate was not due to a cytoplasm-induced decrease in the ATP content of the ghosts. These findings are consistent with the suggestion that the cytoplasm of human red cells contains a factor which increases the sensitivity of the Na+,K+ pump to inhibition by Cai.     

Purified red blood cell Ca2+-pump ATPase: evidence for direct inhibition by presumed anti-calmodulin drugs in the absence of calmodulin

A variety of presumed anti-calmodulin (anti-CaM) drugs was tested for their potential inhibitory effects on the isolated, purified and reconstituted Ca2+-pump ATPase of human red blood cell membranes. Anti-CaM drugs inhibited the Ca2+-pump ATPase both in the absence and presence of added CaM. Qualitatively similar inhibition was observed in two different ATPase assay systems. In asolectin vesicles in the absence of added CaM trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalene- sulfonamide (W-7), vinblastine, dibucaine, imipramine, propranolol and dimethylpropranolol (UM-272) were all inhibitory. Potency of anti-CaM drugs was generally greater on the enzyme reconstituted in asolectin vesicles than on the enzyme reconstituted in phosphatidylcholine vesicles, either in the presence or absence of CaM. The results emphasize that anti-CaM drugs have actions other than to bind to CaM. Possible direct interaction of amphipathic cationic anti-CaM drugs with the Ca2+-pump ATPase and/or its lipid environment is suggested.     

Decreased intracellular Na+ concentration is an early event in murine erythroleukemic cell differentiation

A decrease in Na+/K+-pump activity is an early event of Friend murine erythroleukemic (MEL) cell differentiation along the erythroid pathway. This decreased Na+/K+-pump activity has been proposed to be an essential step in differentiation which would cause a rise in intracellular Na+ concentration and then, by means of Na+/Ca2+ exchange, an increase in intracellular Ca2+. An increase in intracellular Ca2+ has been proposed to be essential for induction of differentiation. A critical prediction of this Na+-Ca2+ hypothesis is the rise in intracellular Na+. To test this prediction we have measured intracellular Na+ using a novel triple isotope method involving 3H2O, [14C]sucrose, and 22Na to measure total water, extracellular fluid, and Na+, respectively. 22Na equilibration occurred in less than 10 min. In uninduced cells, intracellular Na+ was 15.2 +/- 2.2 mM (S.D., n = 22); after induction for 14-16 h with dimethyl sulfoxide, intracellular Na+ decreased significantly (p less than 0.0001) to 8.4 +/- 1.4 mM (n = 21). The time course of the decline in intracellular Na+ paralleled that of the decrease in the Na+/K+-pump activity. These results are in direct contradiction to the Na+-Ca2+ hypothesis and suggest that observed changes in Na+/K+-pump activity can be explained solely on the basis of changes in intracellular Na+. The drop in intracellular Na+ is due to a decrease in Na+ influx. We suggest, however, that the decrease in the Na+ influx is not itself an essential event of differentiation, but may be induced by a change in the flux of another ion coupled to Na+.     

The nature of the Ca2+-pump defect in the red blood cells of patients with cystic fibrosis

The reduction in (Ca2+ + Mg2+)-ATPase activity in the cystic fibrosis red blood cells can be attributed to a reduction in the number of active Ca2+ pumps per red blood cell and an altered interaction of calcium ions with the pump. Despite this, the normal free intracellular [Ca2+] is preserved due to a lower rate of passive calcium entry.     

Effect of compound 48/80 and ruthenium red on the Ca2+-ATPase of sarcoplasmic reticulum

The effects of the condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde (compound 48/80) and ruthenium red on the partial reactions of the catalytic cycle of the sarcoplasmic reticulum Ca2+-ATPase of skeletal muscle were studied. The ATPase activity and both Ca2+ and Sr2+ uptake were inhibited by compound 48/80 when oxalate was used as a precipitating agent. The degree of inhibition decreased when oxalate was replaced by orthophosphate as the precipitating anion. Both the fast Ca2+ efflux and the synthesis of ATP observed during reversal of the Ca2+ pump were inhibited by compound 48/80. Inhibition of the reversal of the Ca2+ pump was caused by a competition between compound 48/80 and orthophosphate for the phosphorylation site of the enzyme. The fast Ca2+ release promoted by arsenate was impaired by compound 48/80. Ruthenium red competes with Ca2+ for the high affinity binding site of the Ca2+-ATPase, but did not interfere with the binding of Ca2+ to the low affinity binding site of the enzyme. In presence of Ca2+ concentrations higher than 5 microM, ruthenium red in concentrations up to 200 microM had no effect on both ATPase activity and Ca2+ uptake. However, the fast Ca2+ efflux promoted by arsenate and the fast Ca2+ efflux coupled with the synthesis of ATP observed during the reversal of the Ca2+ pump were inhibited by ruthenium red, half-maximal inhibition being attained in presence of 10-20 microM ruthenium red. In contrast to the effect of compound 48/80, ruthenium red did not inhibit the phosphorylation of the enzyme by orthophosphate. The ATP in equilibrium with Pi exchange catalyzed by the Ca2+-ATPase in the absence of transmembrane Ca2+ gradient was also inhibited by ruthenium red.     

Inhibition of membrane Ca2+-ATPase in vitro by mating pheromone in Rhodosporidium toruloides, a heterobasidiomycetous yeast

Direct addition of physiological concentrations of rhodotorucine A, a lipopeptide mating pheromone of Rhodosporidium toruloides, to the particulate fraction of the target cell strongly inhibited Ca2+-ATPase activity. The pheromone effect was mating-type specific. Membrane Ca2+-ATPase solubilized by a nonionic detergent and further purified by calmodulin-affinity chromatography was also inhibited by the pheromone. Rhodotorucine A S-oxide, a biologically inactive analogue, had no effect on Ca2+-ATPase. The results suggested that the inhibition of membrane Ca2+-ATPase is a critical event in the signaling of mating pheromone and the inhibition of membrane Ca2+-pump could be responsible for the pheromone-induced rapid raise of intracellular Ca2+ concentration reported.     

Modulation of human erythrocyte Ca2+-ATPase activity by glycerol: The role of calmodulin

The effect of an intracellular cryoprotectant glycerol on human erythrocyte Ca2+-ATPase activity and possible involvement of calmodulin in the regulation of Ca2+-pump under these conditions were investigated. The experiments were carried out using saponin-permeabilized cells and isolated erythrocyte membrane fractions (white ghosts). Addition of rather low concentrations of glycerol to the medium increased Ca2+-ATPase activity in the saponin-permeabilized cells; the maximal effect was observed at 10% glycerol. Subsequent increase in glycerol concentrations above 20% was accompanied by inhibition of Ca2+-ATPase activity. Lack of stimulating effect of glycerol on white ghost Ca2+-ATPase may be attributed to removal of endogenous compounds regulating activity of this ion transport system. Inhibitory analysis using R24571 revealed that activation of Ca2+-ATPase by 10% glycerol was observed only in the case of inhibitor administration after modification of cells with glycerol; in the case of inhibitor addition before erythrocyte contact with glycerol, this phenomenon disappeared. These data suggest the possibility of regulation of human erythrocyte Ca2+-ATPase by glycerol; this regulatory effect may be attributed to both glycerol-induced structural changes in the membrane and also involvement of calmodulin in modulation of catalytic activity of the Ca2+-pump.     

A calcium-activated potassium channel present in foetal red cells of the sheep but absent from reticulocytes and mature red cells.

Red cells of adult sheep, like those of other ruminants, lack the calcium-activated potassium channel which is present in the membrane of human red cells. Since the activities of other transport systems in the sheep red cell are known to decrease during maturation of the cell or during development of the animal it was investigated whether the K+ channel is present in red cells from younger animals or in reticulocytes. Using the divalent cation ionophore A23187 to increase the intracellular Ca of intact cells, it was found that the K+-selective channel is present in foetal red cells from the foetus or newborn animal but not in reticulocytes. The presence of the channel showed no dependence on the K+ genotype of the sheep and was not associated with either "high K+"- or "low K+"-type Na+ pump. No Ca2+-dependent change in K+ permeability was found in red cells from either newborn or adult donkeys suggesting that its presence in the red cells of the foetus may not be general. The role of the K+ channel in the mammalian red cell and the relationship between the K+ channel and the Na+ pump are discussed.