Modulation of embryonic NA+-K+-ATPase activity and mouse preimplantation development in vitro in media containing high concentrations of potassium

The effect of various potassium concentrations (ranging from 1.4 mM to 30 mM K⁺) in modified Tyrode's medium on the culture of mouse zygotes obtained after in vitro fertilization to the blastocyst stage was examined. A clear dose-dependent negative effect of increasing K⁺ concentrations on the preimplantation embryonic development in vitro was found. We have previously shown that significantly more two-cell embryos reach the blastocyst stage when cultured during the second day postinsemination in medium supplemented with taurine. Because taurine, an amino acid that abounds in the reproductive tract, has been reported to inhibit the enzyme Na⁺-K⁺-adenosine triphosphatase (Na⁺-K⁺-AT-Pase), we used two other conditions known to inhibit the Na⁺-K⁺-ATPase to study their effect on mouse embryo development. Culturing embryos during a short period (the second day postinsemination) in low extracellular K⁺ concentrations (1.4 mM) or in medium supplemented with ouabain (50 μM) showed positive effects similar to those of culturing in medium with taurine (10 mM). This beneficial effect of ouabain was found in various K⁺ concentrations tested, including the high concentrations present in the oviduct. Although the effects of low K⁺ and taurine can possibly be ascribed to their other cellular effects, the effect of ouabain shows that inhibition of the Na⁺-K⁺-ATPase during the two-cell stage in the mouse is beneficial for further embryonic development to the blastocyst stage. © 1993 Wiley-Liss, Inc.     

Sodium-dependent amino acid transport in preimplantation mouse embryos. III. Na+-k+-atpase-linked mechanism in blastocysts.

Mouse blastocysts collapse in cytochalasin B (CB), reexpand (accumulate fluid) in control medium, but cannot reexpand in ouabain, an inhibitor of Na⁺K⁺-ATPases. These ATPases, then, seem to be necessary for fluid accumulation in blastocysts. Since intact blastocysts are relatively insensitive to ouabain, CB seems to make it possible for ouabain to reach the Na⁺K⁺-ATPases localized on the blastocoelic surface. CB-Collapsed blastocysts were found to transport alanine and lysine at the same rate as intact blastocysts, indicating that, in 1 hr, amino acids are transported into the cells of the intact blastocyst, and not into the fluid-filled blastocoel. Transport rates in CB-collapsed blastocysts do not exceed those in intact blastocysts, suggesting that hypothetical amino acid carriers are located only on the external blastocyst surface. Most important, ouabain strongly inhibits sodium-dependent alanine transport in CB-collapsed blastocysts, but not in intact blastocysts, providing strong evidence that Na⁺K⁺-ATPases, localized on the blastocoelic surface, are necessary for this transport. Ouabain does not inhibit sodium-independent lysine transport in CB-collapsed blastocysts. Thus, the dependency of both sodium-dependent amino acid transport and fluid accumulation upon Na⁺K⁺-ATPases, and the separate localization of amino acid carriers and these ATPases, provides functional evidence for an epithelial tissue type of mechanism for sodium-dependent amino acid transport in mouse blastocysts.     

Na+-dependent amino acid transport in preimplantation mouse embryos. II. Metabolic inhibitors and nature of the cation requirement.

Experiments were conducted in order to determine the energy source and nature of the cation dependency of [³H]methionine transport in preimplantation mouse embryos. The energy source of methionine transport was studied at the late four-cell and early blastocyst stages. The embryos, raised in vitro, were incubated for 1 hr in inhibitor(s) of energy metabolism and then transferred for 1 hr to medium that contained inhibitor(s) and ³H-methionine. These inhibitor studies suggest that respiration and glycolysis are needed to maintain uptake of methionine in early blastocysts. Late four-cell embryos seem to utilize respiration alone for transport.The cation dependency of methionine transport was studied at the late morula and early blastocyst stages. The kinetics of methionine uptake by early blastocysts in Na⁺-depleted media indicate a competitive type of inhibition. The uptake of methionine by early blastocysts is relatively resistant to ouabain and unaffected by K⁺-free medium. In contrast, methionine uptake by late morula-stage embryos is markedly inhibited by ouabain and K⁺-free medium in 1 hr. These results suggest that 1) Na⁺ serves to increase the affinity of methionine for the carrier in early blastocysts, 2) the cation gradients do not supply a major fraction of the energy required for methionine transport, and/or the gradients are difficult to perturb once the blastocyst has formed, and 3) putative Na⁺ pumps may be localized on the blastocoelic surface of the blastocysts.     

A possible involvement of cytoplasmic Ca2+ in sodium dependency of neurite outgrowth of rat pheochromocytoma PC12 cells

The effects of extracellular Na⁺, K⁺ and Cl^? on neurite outgrowth of PC12 pheochromocytoma cells were studied. Nerve growth factor (NGF)-induced neurite formation was inhibited upon substitution of choline chloride for NaCl under normal culture conditions. It was found that neurite formation increased proportionately with the concentration of Na⁺ in medium up to 150 mM. When PC12 cells were exposed to NGF in suspension culture followed by transfer to new dishes, they showed neurite extention in response to NGF in an RNA- and protein synthesis-independent manner. Under these conditions, neurite outgrowth occurred normally in 60–150 mM Na⁺, whereas it decreased significantly at lower concentrations of Na⁺. Na⁺ dependency was also observed for cyclic AMP-mediated neurite formation of PC12 cells. In contrast, neurite outgrowth was independent of K⁺ in the range 5–106 mM, suggesting that membrane potential did not play a role in this process. No alterations were observed in neurite outgrowth with Cl^? replaced by NO^?₃, SO^2?₄, or 2-hydroxyethanesulfonate. Thus, extracellular Na⁺ plays a role in controlling neurite formation of these cells. An attempt was made to relate this effect to a decrease in cytoplasmic Ca²⁺ concentration monitored by a fluorescent dye sensitive to Ca²⁺.     

An attempt to use isolated vacuoles to determine the distribution of sodium and potassium in cells of storage roots of red beet (Beta vulgaris L.)

Vacuoles isolated from red beet (Beta vulgaris L.) storage roots contain Na⁺ and K⁺ but their analysis does not give reliable information about the size of vacuolar pools of these ions in vivo. Analyses of isolated vacuoles indicated that between 53% and 90% of the Na⁺ was located in the vacuole and that the vacuolar concentrations of Na⁺ ranged between 4 and 45 mol m^-3. Calculated concentrations of K⁺ in the vacuoles varied between 32 and 72 mol m^-3 but, in contrast to Na⁺, only about 50% of the K⁺ was located in the vacuole. Considerations of the likely cytoplasmic concentrations of Na⁺ and K⁺ suggest that if these results indicate conditions in vivo a large proportion of these ions must be located in the extracellular space, where they would exert considerable osmotic pressure. To test this, the effect of washing on cell turgor (measured directly with a pressure probe) and on loss of Na⁺ and K⁺ was determined. Washing caused an increase in turgor of 5 bar but losses of Na⁺ and K⁺ were less than predicted by the experiments with isolated vacuoles. It is concluded that beet vacuoles leak Na⁺ and K⁺ when isolated resulting in an underestimation of the size of vacuolar pools of these cations in vivo. Nonetheless, the turgor measurements provide evidence for the presence of osmotically active solute in the extracellular space. The possible contribution of extracellular Na⁺ and K⁺ to the observed turgor reduction is calculated and the physiological importance of the accumulation of extracellular solutes is discussed.     

Effects of ouabain on proliferation of human endothelial cells correlate with Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase activity and intracellular ratio of Na<Superscript>+</Superscript> and K<Superscript>+</Superscript>

Side-by-side with inhibition of the Na⁺,K⁺-ATPase ouabain and other cardiotonic steroids (CTS) can affect cell functions by mechanisms other than regulation of the intracellular Na⁺ and K⁺ ratio ([Na⁺]_i/[K⁺]_i). Thus, we compared the doseand time-dependences of the effect of ouabain on intracellular [Na⁺]_i/[K⁺]_i ratio, Na⁺,K⁺-ATPase activity, and proliferation of human umbilical vein endothelial cells (HUVEC). Treatment of the cells with 1-3 nM ouabain for 24-72 h decreased the [Na⁺]_i/[K⁺]_i ratio and increased cell proliferation by 20-50%. We discovered that the same ouabain concentrations increased Na⁺,K⁺-ATPase activity by 25-30%, as measured by the rate of ⁸⁶Rb⁺ influx. Higher ouabain concentrations inhibited Na⁺,K⁺-ATPase, increased [Na⁺]_i/[K⁺]_i ratio, suppressed cell growth, and caused cell death. When cells were treated with low ouabain concentrations for 48 or 72 h, a negative correlation between [Na⁺]_i/[K⁺]_i ratio and cell growth activation was observed. In cells treated with high ouabain concentrations for 24 h, the [Na⁺]_i/[K⁺]_i ratio correlated positively with proliferation inhibition. These data demonstrate that inhibition of HUVEC proliferation at high CTS concentrations correlates with dissipation of the Na⁺ and K⁺ concentration gradients, whereas cell growth stimulation by low CTS doses results from activation of Na⁺,K⁺-ATPase and decrease in the [Na⁺]_i/[K⁺]_i ratio.     

Determination of Ion Content and Ion Fluxes in the Halotolerant Alga Dunaliella salina

A method to determine intracellular cation contents in Dunaliella by separation on cation-exchange minicolumns is described. The separation efficiency of cells from extracellular cations is over 99.9%; the procedure causes no apparent perturbation to the cells and can be applied to measure both fluxes and internal content of any desired cation. Using this technique it is demonstrated that the intracellular averaged Na⁺, K⁺, and Ca²⁺ concentrations in Dunaliella salina cultured at 1 to 4 molar NaCl, 5 millimolar K⁺, and 0.3 millimolar Ca²⁺ are 20 to 100 millimolar, 150 to 250 millimolar, and 1 to 3 millimolar, respectively. The intracellular K⁺ concentration is maintained constant over a wide range of media K⁺ concentrations (0.5-10 millimolar), leading to a ratio of K⁺ in the cells to K⁺ in the medium of 10 to 1,000. Severe limitation of external K⁺, induces loss of K⁺ and increase in Na⁺ inside the cells. The results suggest that Dunaliella cells possess efficient mechanisms to eliminate Na⁺ and accumulate K⁺ and that intracellular Na⁺ and K⁺ concentrations are carefully regulated. The contribution of the intracellular Na⁺ and K⁺ salts to the total osmotic pressure of cells grown at 1 to 4 molar NaCl, is 5 to 20%.     

Kinetic Comparisons of Heart and Kidney Na(+),K(+)-ATPases

Most kinetic measurements of the partial reactions of Na⁺,K⁺-ATPase have been conducted on enzyme from mammalian kidney. Here we present a kinetic model that is based on the available equilibrium and kinetic parameters of purified kidney enzyme, and allows predictions of its steady-state turnover and pump current in intact cells as a function of ion and ATP concentrations and the membrane voltage. Using this model, we calculated the expected dependence of the pump current on voltage and extracellular Na⁺ concentration. The simulations indicate a lower voltage dependence at negative potentials of the kidney enzyme in comparison with heart muscle Na⁺,K⁺-ATPase, in agreement with experimental results. The voltage dependence is enhanced at high extracellular Na⁺ concentrations. This effect can be explained by a voltage-dependent depopulation of extracellular K⁺ ion binding sites on the E2P state and an increase in the proportion of enzyme in the E1P(Na⁺)₃ state in the steady state. This causes a decrease in the effective rate constant for occlusion of K⁺ by the E2P state and hence a drop in turnover. Around a membrane potential of zero, negligible voltage dependence is observed because the voltage-independent E2(K⁺)₂ → E1 + 2K⁺ transition is the major rate-determining step.     

ALBUMIN CAN REVERSE THE RELEASE OF POTASSIUM FROM HUMAN ERYTHROCYTES TREATED WITH THE NON-IONIC DETERGENT,BRIJ 58

Exchange of erythrocyte intracellular (i/c) K⁺for extracellular (e/c) Na⁺in human erythrocytes treated with sub-CMC concentrations of the non-ionic detergent Brij 58 can be stopped by reincubation in serum or albumin containing solutions. The progressive equilibration of the K⁺contents of detergent-treated human erythrocytes with the incubation medium was reversed by an albumin-mediated withdrawal of detergent molecules from the cell. Re-establishment of near normal [K⁺] in terms of K⁺/kg water proceeds in two ways: (i) a metabolism-dependent net accumulation of K⁺ions; and (ii) a metabolism-independent shrinkage of erythrocytes, this being the more significant factor.     

The sided action of Na+ and of K+ on reconstituted shark (Na+ + K+)-ATPase engaged in Na+−Na+ exchange accompanied by ATP hydrolysis. I. The ATP activation curve

The ATP hydrolysis dependent Na⁺-Na⁺ exchange of reconstituted shark (Na⁺ + K⁺)-ATPase is electrogenic with a transport stoichiometry as for the Na⁺-K⁺ exchange, suggesting that translocation of extracellular Na⁺ is taking place via the same route as extracellular K⁺. The preparation thus offers an opportunity to compare the sided action of Na⁺ and of K⁺ on the affinity for ATP in a reaction in which the intermediary steps in the overall reaction seems to be the same without and with K⁺. With Na⁺ but no K⁺ on the two sides of the enzyme, the ATP-activation curve is hyperbolic and the affinity for ATP is high. Extracellular K⁺ in concentrations of 50 μM (the lowest tested) and up gives biphasic ATP activation curves, with both a high- and a low-affinity component for ATP. Cytoplasmic K⁺ also gives biphasic ATP-activation curves, however, only when the K⁺ concentration is 50 mM or higher (Na⁺ + K⁺ = 130 mM). The different ATP-activation curves are explained from the Albers-Post scheme, in which there is an ATP-dependent and an ATP-independent deocclusion of E₂(Na₂⁺) and E₂(K₂⁺), respectively, and in which the dephosphorylation of E₂-P is rate limiting in the presence of Na⁺ (but no K⁺) extracellular, whereas in the presence of extracellular K⁺ it is the deocclusion of E₂(K₂⁺) which is rate limiting.     

Dependence of the intracellular concentrations of univalent ions and hydrogenase activity on the salt composition and pH of the medium in the haloalkaliphilic sulfate-reducing bacterium <Emphasis Type="Italic">Desulfonatronum thiodismutans</Emphasis>

It has been shown that the intracellular concentrations of Na⁺, K⁺, and Cl^? ions in Desulfonatronum thiodismutans depend on the extracellular concentration of Na⁺ ions. An increase in the extracellular concentration of Na⁺ results in the accumulation of K⁺ ions in cells, which points to the possibility that these ions perform an osmoprotective function. When the concentration of the NaCl added to the medium was increased to 4%, the concentration gradient of Cl^? ions changed insignificantly. It was found that D. thiodismutans contains two forms of hydrogenase—periplasmic and cytoplasmic. Both enzymes are capable of functioning in solutions with high ionic force; however they exhibit different sensitivities to Na⁺, K⁺, and Li⁺ salts and pH. The enzymes were found to be resistant to high concentrations of Na⁺ and K⁺ chlorides and Na⁺ bicarbonate. The cytoplasmic hydrogenase differed significantly from the periplasmic one in having much higher salt tolerance and lower pH optimum. The activity of these enzymes depended on the nature of both the cationic and anionic components of the salts. For instance, the inhibitory effect of NaCl was less pronounced than that of LiCl, whereas Na⁺ and Li⁺ sulfates inhibited the activity of both hydrogenase types to an equal degree. The highest activity of these enzymes was observed at low Na⁺ concentrations, close to those typical of cells growing at optimal salt concentrations.     

Route,mechanism, and implications of proton import during Na+/K+ exchange by native Na+/K+-ATPase pumps

A single Na⁺/K⁺-ATPase pumps three Na⁺ outwards and two K⁺ inwards by alternately exposing ion-binding sites to opposite sides of the membrane in a conformational sequence coupled to pump autophosphorylation from ATP and auto-dephosphorylation. The larger flow of Na⁺ than K⁺ generates outward current across the cell membrane. Less well understood is the ability of Na⁺/K⁺ pumps to generate an inward current of protons. Originally noted in pumps deprived of external K⁺ and Na⁺ ions, as inward current at negative membrane potentials that becomes amplified when external pH is lowered, this proton current is generally viewed as an artifact of those unnatural conditions. We demonstrate here that this inward current also flows at physiological K⁺ and Na⁺ concentrations. We show that protons exploit ready reversibility of conformational changes associated with extracellular Na⁺ release from phosphorylated Na⁺/K⁺ pumps. Reversal of a subset of these transitions allows an extracellular proton to bind an acidic side chain and to be subsequently released to the cytoplasm. This back-step of phosphorylated Na⁺/K⁺ pumps that enables proton import is not required for completion of the 3 Na⁺/2 K⁺ transport cycle. However, the back-step occurs readily during Na⁺/K⁺ transport when external K⁺ ion binding and occlusion are delayed, and it occurs more frequently when lowered extracellular pH raises the probability of protonation of the externally accessible carboxylate side chain. The proton route passes through the Na⁺-selective binding site III and is distinct from the principal pathway traversed by the majority of transported Na⁺ and K⁺ ions that passes through binding site II. The inferred occurrence of Na⁺/K⁺ exchange and H⁺ import during the same conformational cycle of a single molecule identifies the Na⁺/K⁺ pump as a hybrid transporter. Whether Na⁺/K⁺ pump–mediated proton inflow may have any physiological or pathophysiological significance remains to be clarified.     

Metabolic depression and Na+/K+ gradients in the aestivating Australian goldfields frog,Neobatrachus wilsmorei

During aestivation the metabolic rate of the Australian goldfields frog Neobatrachus wilsmorei was reduced by 80% from its standard metabolic rate. The in vitro rate of oxygen consumption of isolated muscle and skin from aestivating frogs was up to 50% lower than that of the non-aestivating frogs. This in vitro rate of oxygen consumption was maintained for 6–12 h, indicating an intrinsic metabolic depression of tissues during aestivation. Frogs became dehydrated during aestivation. Muscle, skin and liver also became dehydrated during aestivation, but brain and kidney did not. Na⁺ and K⁺ contents and extracellular space measurement for muscle indicated that ion gradients were maintained across the muscle cell membrane during aestivation. Increases in plasma concentrations of Na⁺ and K⁺ were matched with similar increases in muscle intracellular ion concentrations. Extracellular space measurements were unsuccessful in the other tissues, but K⁺ content in all tissues (per dry weight) was maintained during aestivation, and the concentration of plasma K⁺ did not increase above that which can be accounted for by dehydration, indicating that K⁺ gradients were maintained.Abbreviations bm body mass - DPM disintegrations per minute - dw dry weight - MR metabolic rate - vO₂ rate of oxygen consumption - ww wet weight     

Characterization of monovalent ion transport systems in an insect cell line (Manduca sexta embryonic cell line che)

The monovalent ion transport systems of an immortalized insect cell line (CHE) have been investigated. These cells are unusual in that unlike most vertebrate cells, their normal extracellular environment consists of high potassium and low sodium concentrations. CHE cells maintained high intracellular [K⁺] through both a furosemide-inhibitable and a vanadate-inhibitable transport system. Intracellular exchangeable [Na⁺] was slightly lower than the extracellular [Na⁺] and was maintained at this level through a vanadate-sensitive transport system. Na⁺ uptake was also inhibited by furosemide: however, the stoichiometry of furosemide-sensitive Na⁺ uptake when compared with furosemide-sensitive K⁺ uptake indicated that these cations are not cotransported. 4,4′-Diisothiocyano-2,2′-disulfonic acid stilbene (DIDS) inhibited Na⁺, K⁺, and Cl^? uptake. Vanadate and furosemide decreased cytoplasmimic pH, while cytoplasmic pH increased in the presence of DIDS. A model is presented explaining how Na⁺, K⁺, Cl^?, H⁺ and HCO₃ ^? fluxes are regulated in these cells.     

Alanine and leucine transport in unfertilized pig oocytes and early blastocysts

Amino acid transport is facilitated by specific transporters within the plasma membrane of the cell. In mouse oocytes and cleavage-stage conceptus Na⁺-dependent L-alanine and L-leucine transport are nearly undetectable. Sodium-dependent transport via system B^O,+ in the mouse conceptus increases greatly between the 8-cell and blastocyst stages. By contrast, data presented here for the pig show that L-alanine and L-leucine transport is mainly Na⁺-dependent in the oocyte; this Na⁺-dependent component of transport becomes undetectable by the blastocyst stage. The Na⁺-dependent component of transport in oocytes is inhibited by BCH (2-aminoendo-bicyclo[2.2.1] hexane-2-carboxylic acid) and L-lysine and thus could be a form of system B^O,+. In both oocytes and blastocysts Na⁺-independent L-leucine transport is inhibited by BCH, which is consistent with the presence of system L. The dramatic decrease in Na⁺-dependent amino acid transport activity could occur in pig conceptuses in association with the onset of RNA synthesis during the 4-cell stage. Regardless of the precise time during development at which it occurs, however, this dramatic, developmentally regulated decrease in Na⁺-dependent alanine and leucine transport activity contrasts sharply with the large increase in Na⁺-dependent system B^O,+ activity that occurs during preimplantation development of murine conceptuses. Elucidation of the molecular mechanisms by which these changes occur should contribute to an understanding of regulation of gene expression during early development. © 1993 Wiley-Liss, Inc.     

An improved culture medium for mouse blastocysts

Summary Eagle's basal medium, modified to contain essential amino acids at the concentrations optimal for mouse blastocyst hatching, attachment, and outgrowth, supported in vitro development of the mouse blastocyst better than other tissue culture media tested. This medium was improved for growth and differentiation of the inner cell mass by doubling the concentration of amino acids and glucose and by adding uridine (10⁻⁵ M) and β-mercaptoethanol (10⁻⁵ M). In this improved medium nearly all blastocysts grown from the two-cell stage hatched and formed trophoblast outgrowths, and 62% developed into two-layer egg cylinders. This work was supported by the U.S. Department of Energy.     

Extracellular Allosteric Na Binding to the Na,K-ATPase in Cardiac Myocytes

Whole-cell patch-clamp measurements of the current, I_p, produced by the Na⁺,K⁺-ATPase across the plasma membrane of rabbit cardiac myocytes show an increase in I_p over the extracellular Na⁺ concentration range 0–50 mM. This is not predicted by the classical Albers-Post scheme of the Na⁺,K⁺-ATPase mechanism, where extracellular Na⁺ should act as a competitive inhibitor of extracellular K⁺ binding, which is necessary for the stimulation of enzyme dephosphorylation and the pumping of K⁺ ions into the cytoplasm. The increase in I_p is consistent with Na⁺ binding to an extracellular allosteric site, independent of the ion transport sites, and an increase in turnover via an acceleration of the rate-determining release of K⁺ to the cytoplasm, E2(K⁺)₂ → E1 + 2K⁺. At normal physiological concentrations of extracellular Na⁺ of 140 mM, it is to be expected that binding of Na⁺ to the allosteric site would be nearly saturated. Its purpose would seem to be simply to optimize the enzyme’s ion pumping rate under its normal physiological conditions. Based on published crystal structures, a possible location of the allosteric site is within a cleft between the α- and β-subunits of the enzyme.     

The Third Sodium Binding Site of Na,K-ATPase Is Functionally Linked to Acidic pH-Activated Inward Current

Sodium- and potassium-activated adenosine triphosphatases (Na,K-ATPase) is the ubiquitous active transport system that maintains the Na⁺ and K⁺ gradients across the plasma membrane by exchanging three intracellular Na⁺ ions against two extracellular K⁺ ions. In addition to the two cation binding sites homologous to the calcium site of sarcoplasmic and endoplasmic reticulum calcium ATPase and which are alternatively occupied by Na⁺ and K⁺ ions, a third Na⁺-specific site is located close to transmembrane domains 5, 6 and 9, and mutations close to this site induce marked alterations of the voltage-dependent release of Na⁺ to the extracellular side. In the absence of extracellular Na⁺ and K⁺, Na,K-ATPase carries an acidic pH-activated, ouabain-sensitive “leak” current. We investigated the relationship between the third Na⁺ binding site and the pH-activated current. The decrease (in E961A, T814A and Y778F mutants) or the increase (in G813A mutant) of the voltage-dependent extracellular Na⁺ affinity was paralleled by a decrease or an increase in the pH-activated current, respectively. Moreover, replacing E961 with oxygen-containing side chain residues such as glutamine or aspartate had little effect on the voltage-dependent affinity for extracellular Na⁺ and produced only small effects on the pH-activated current. Our results suggest that extracellular protons and Na⁺ ions share a high field access channel between the extracellular solution and the third Na⁺ binding site.     

Embryo metabolism during the expansion of the bovine blastocyst

Embryo metabolism was evaluated during re‐expansion of in vitro produced bovine blastocysts collapsed with cytochalasin D (CCD) and incubated in the presence and absence of ouabain, a specific inhibitor of the Na⁺, K⁺ pump. Day 8 expanded blastocysts were treated for 2 to 4 hr with 20 μg/ml CCD. Four conditions were tested: untreated embryos and embryos collapsed with CCD and allowed to re‐expand for 4 hr in the presence of 0 M, 1 nM, or 1 μM ouabain. Incubation of collapsed embryos for 4 hr in the presence of 1 nM or 1 μM ouabain significantly inhibited blastocyst re‐expansion. Glucose, pyruvate, and amino lactate uptake/release were not significantly affected by ouabain treatment and did not correlate with the degree of blastocyst re‐expansion. Few variations in the uptake/release of amino acids by the embryos were observed. Ouabain treatment significantly decreased oxygen uptake which directly correlated with the degree of blastocyst re‐expansion. For embryos allowed to re‐expand in the presence or absence of ouabain, a direct correlation was observed between the uptake of oxygen and of glucose. One mM cyanide or 2,4 dinitrophenol inhibited blastocyst re‐expansion although 0.01 and 0.1 mM were ineffective. This study indicates a role for oxidative metabolism in providing the energy necessary for blastocoel expansion in the bovine. Nevertheless, blastocyst expansion is relatively insensitive to inhibition of oxidative phosphorylation indicating the ability of the bovine blastocyst to adapt to hypoxic conditions. Mol. Reprod. Dev. 53:171–178, 1999. © 1999 Wiley‐Liss, Inc.