The diffusion and electrogenic components of the membrane potential of hypoxic myocardium

The diffusion and electrogenic components of the resting potential of hypoxic ventricular muscle were separated by inhibition of the sodium pump with 10(-4) M ouabain. The response to varying external K concentrations (Ko) was studied. Arterially perfused rabbit hearts were submitted to 60 min hypoxia in Krebs solution containing 5 mM K throughout or to different external K concentrations during the last 20 min of hypoxia. For K concentrations between 1.5 and 10 mM, hypoxia did not change the resting potential except for a slight hyperpolarization in 7.5 mM K. The diffusion component of the resting potential did not differ from the resting potential at Ko less than 5 mM. An electrogenic potential of -3 to -6 mV was detectable at Ko values between 5 and 10 mM. The internal K concentration, Ki, was estimated from extrapolations to zero potential of the relation resting potential vs. Ko in normoxic and hypoxic hearts. These experiments revealed a decline of Ki of 16 mM with hypoxia. The variation of the diffusion potential with external K was fitted by a PNa:PK ratio five times lower than in normoxia. It has been concluded that an increase in K permeability and the persistence of electrogenic Na extrusion during hypoxia of rather short duration prevent membrane depolarization despite the myocardial K loss.     

Furosemide-sensitive Na and K fluxes in human red cells. Net uphill Na extrusion and equilibrium properties

This paper reports experiments designed to find the concentrations of internal and external Na and K at which inward and outward furosemide-sensitive (FS) Na and K fluxes are equal, so that there is no net FS movement of Na and K. The red cell cation content was modified by using the ionophore nystatin, varying cell Na (Nai) from 0 to 34 mM (K substitution, high-K cells) and cell K (Ki) from 0 to 30 mM (Na substitution, high-Na cells). All incubation media contained NaCl (Nao = 130 or 120 nM), and KCl (Ko = 0-30 mM). In high-K cells, incubated in the absence of Ko, there was net extrusion of Na through the FS pathway. The net FS Na extrusion increased when Nai was increased. Low concentrations of Ko (0-6 mM) slightly stimulated, whereas higher concentrations of Ko inhibited, FS Na efflux. Increasing Ko stimulated the FS Na influx (K0.5 = 4 mM). Under conditions similar to those that occur in vivo (Nai = 10, Ki = 130, Nao = 130, Ko = 4 mM, Cli/Clo = 0.7), net extrusion of Na occurs through the FS pathway (180-250 mumol/liter cell X h). The concentration of Ko at which the FS Na influx and efflux and the FS K influx and efflux become equal increased when Nai increased in high-K cells and when Ki was increased in high-Na cells. The net FS Na and K fluxes both approached zero at similar internal and external Na and K concentrations. In high-K cells, under conditions when net Na and K fluxes were near zero, the ratio of FS Na to FS K unidirectional flux was found to be 2:3. In high-K cells, the empirical expression (Nai/Nao)2(Ki/Ko)3 remained at constant value (apparent equilibrium constant, Kappeq +/- SEM = 22 +/- 2) for each set of internal and external cation concentrations at which there was no net Na flux. These results indicate that in the physiological region of concentrations of internal and external Na, K, and Cl, the stoichiometry of the FS Na and K fluxes is 2 Na:3 K. In high-Na cells under conditions when net FS Na and K fluxes were near zero, the ratio of FS Na to FS K unidirectional fluxes was 3:2 (1).(ABSTRACT TRUNCATED AT 400 WORDS)     

Modes of operation and variable stoichiometry of the furosemide- sensitive Na and K fluxes in human red cells

We report in this paper different modes of Na and K transport in human red cells, which can be inhibited by furosemide in the presence of ouabain. Experimental evidence is provided for inward and outward coupled transport of Na and K, Ki/Ko and Nai/Nao exchange, and uncoupled Na or K efflux. The outward cotransport of Na and K was defined as the furosemide-sensitive (FS) component of Na and K effluxes into choline medium and as the Cl-dependent or cis-stimulated component of the ouabain-resistant (OR) Na and K effluxes. Inward cotransport of Na and K was defined by the stimulation by external Na (Nao) of the K influx and the stimulation by external K (Ko) of the Na influx in the presence of ouabain. Both effects were FS and Cl dependent. Experimental evidence for an FS Ki/Ko exchange pathway of the Na/K cotransport was provided by (a) the stimulation by external K of FS K influx and efflux, and (b) the stimulation by internal Na or K of FS K influx in the absence of external Na. Evidence for an FS Nai/Nao exchange pathway was provided by the stimulation of FS Na influx by internal Na from a K-free medium (130 mM NaCl). This pathway was four to six times smaller than the Ki/Ko exchange. In cells containing only Na or K, incubated in media containing only Na or K, respectively, there was FS efflux of the cation without simultaneous inward transport (FS uncoupled Na and K efflux). The stoichiometric ratio of FS outward cotransport of Na and K into choline medium varied with the ratio of Nai-to-Ki concentrations, and when Nai/Ki was close to 1, the ratio of FS outward Na to K flux was also 1. In choline media, FS Na efflux was inhibited by external K (noncompetitively), whereas FS k efflux was stimulated. The stimulation of FS K efflux was due to the stimulation by Ko of the Ki/Ko exchange pathway. Thus, the stoichiometry of FS Na and K effluxes also varied in the presence of external K. A minimal model for a reaction scheme of FS Na and K transport accounts for cis stimulation, trans inhibition, and trans stimulation, and for variable stoichiometry of the FS cation fluxes.     

Effect of carbachol on ouabain-sensitive uptake of 86Rb by dispersed lacrimal gland cells.

Uptake of ⁸⁶Rb was measured in dispersed rat exorbital lacrimal gland cells. The uptake was inhibited by ouabain (0.9 mM) and stimulated by carbachol (10^?5M). In the presence of quabain, in the absence of Ca, or in the presence of decreased extracellular Na, carbachol failed to stimulate ⁸⁶Rb uptake. Cellular concentrations of Na and K were also determined. Cells treated with carbachol had elevated Na content and decreased K content. Omission of external Ca prevented both the K loss and Na gain. Decreasing extracellular Na prevented the Na gain but only partially inhibited the loss of cellular K. The conclusions to be reached from these data are: (1) in the resting lacrimal cell, a quabain sensitive pump actively maintains the intracellular concentration of K high and that of Na low, (2) carbachol acts, through Ca, to increase the passive membrane permeability to Na and K as well as the activity of the pump, and (3) the stimulus for the activation of the pump may be a rise in the intracellular concentration of Na.     

Effects of hypoxia on the contractions and lactate release induced by high-K+, ouabain and epinephrine in guinea-pig aorta.

1. The 60 mM K+, 152 mM K+, Na-deficient medium and oubain-induced contractions of aorta were not so affected by severe hypoxia. 2. The 60 mM K+, 152 mM K+, Na(+)-deficient medium-induced responses were greatly reduced by deprivation of external Ca2+ in normoxia. 3. As the concentration of epinephrine increased, the remaining tensions which were expressed as a percentage of the original tensions became progressively greater in hypoxic condition. 4. The percentage of resistant components of the norepinephrine-induced contraction by the lower concentration was further reduced in Ca(2+)-free medium by severe hypoxic condition. 5. The tensions under normoxia and lactate release under severe hypoxia induced by 60 mM K+ or 2.5 x 10(-6) M epinephrine were of the same extent. 6. In conclusion, the inhibition of aortic response to epinephrine with severe hypoxia could not solely be explained by depression of the oxygen supply into the oxidative metabolism. Severe hypoxia did not affect Ca2+ influx through voltage-operated Ca2+ channels, but reduced both receptor-operated Ca2+ influx and intracellular Ca2+ release in the aorta.     

Anion-coupled Na efflux mediated by the human red blood cell Na/K pump

The red cell Na/K pump is known to continue to extrude Na when both Na and K are removed from the external medium. Because this ouabain-sensitive flux occurs in the absence of an exchangeable cation, it is referred to as uncoupled Na efflux. This flux is also known to be inhibited by 5 mM Nao but to a lesser extent than that inhibitable by ouabain. Uncoupled Na efflux via the Na/K pump therefore can be divided into a Nao-sensitive and Nao-insensitive component. We used DIDS-treated, SO4-equilibrated human red blood cells suspended in HEPES-buffered (pHo 7.4) MgSO4 or (Tris)2SO4, in which we measured 22Na efflux, 35SO4 efflux, and changes in the membrane potential with the fluorescent dye, diS-C3 (5). A principal finding is that uncoupled Na efflux occurs electroneurally, in contrast to the pump's normal electrogenic operation when exchanging Nai for Ko. This electroneutral uncoupled efflux of Na was found to be balanced by an efflux of cellular anions. (We were unable to detect any ouabain-sensitive uptake of protons, measured in an unbuffered medium at pH 7.4 with a Radiometer pH-STAT.) The Nao-sensitive efflux of Nai was found to be 1.95 +/- 0.10 times the Nao-sensitive efflux of (SO4)i, indicating that the stoichiometry of this cotransport is two Na+ per SO4=, accounting for 60-80% of the electroneutral Na efflux. The remainder portion, that is, the ouabain-sensitive Nao-insensitive component, has been identified as PO4-coupled Na transport and is the subject of a separate paper. That uncoupled Na efflux occurs as a cotransport with anions is supported by the result, obtained with resealed ghosts, that when internal and external SO4 was substituted by the impermeant anion, tartrate i,o, the efflux of Na was inhibited 60-80%. This inhibition could be relieved by the inclusion, before DIDS treatment, of 5 mM Cli,o. Addition of 10 mM Ko to tartrate i,o ghosts, with or without Cli,o, resulted in full activation of Na/K exchange and the pump's electrogenicity. Although it can be concluded that Na efflux in the uncoupled mode occurs by means of a cotransport with cellular anions, the molecular basis for this change in the internal charge structure of the pump and its change in ion selectivity is at present unknown.     

Activation of potassium channels in erythrocytes of marine teleost Scorpaena porcus

To assess the possibility of stimulating Ca2+-activated K+ channels, marine fish erythrocytes were incubated at 20-22 degrees C in saline containing a Ca2+-ATPase inhibitor (orthovanadate), a Ca2+ ionophore (A23187), propranolol or Pb2+. Incubation of the cells for up to 2 h under control conditions or in the presence of 5 mM NH4VO3 and 1 mM Ca2+ did not affect the intracellular K+ and Na+ concentrations. About 50% cellular K+ was lost from erythrocytes incubated in the presence of 0.01 mM A23187, 1 mM EGTA and 0.4-1.0 mM Ca2+. There was a significant loss of cellular K+ after the addition of 0.05-0.2 mM propranolol to the incubation medium. The stimulatory effect of propranolol on the K+ efflux was independent of external Ca2+. Blockers of Ca2+ transport, verapamil and Co2+, caused only a small decrease in the K+ loss induced by propranolol. The treatment of erythrocytes with 1-2 microM Pb2+ led to a minor K+ loss, but at a Pb2+ concentration of 20-50 microM, about 70% cellular K+ was lost. The K+ efflux induced by propranolol or Pb2+ was completely blocked by 1 mM quinine. The induced K+ loss from the erythrocytes was accompanied by a slight increase in the intracellular Na+ concentration. These data indicate the possibility of inducing Ca2+- and Pb2+-activated potassium channels in erythrocytes of S. porcus. A distinctive feature of the cells is a high sensitivity to propranolol, which activates K+ channels in the absence of external Ca2+.     

Swelling and potassium uptake in cultured astrocytes

The intracellular water content of astrocytes in primary cultures shows a biphasic swelling pattern on exposure to various increased external K+ concentrations over the range of 1.5-100 mM. The two phases (physiological, 1.5-12 mM K+; pathological, 25-100 mM K+) are based on two different mechanisms. Both can be blocked by low Cl- solutions and involve intensive net uptake of K+. However, the physiological phase consists of the activation of a KCl + NaCl carrier, while the Na+ in turn is pumped out by Na+-K+ ATPase, with a resultant net accumulation of KCl. At pathological K+ concentrations the KCl + NaCl carrier is less active because the Na+ driving force, its energy source, is reduced (owing to depolarization by K+). However, the Donnan equilibrium across the cell membrane is heavily disturbed, which leads to passive KCl accumulation. The results suggest that volume changes in cultured glial cells during exposure to high K+ should be taken into consideration since they disguise K+ accumulation when only ion activity is measured.     

Effects of depletion of K+, Na+, or Ca2+ on DNA synthesis and cell cation content in chick embryo fibroblasts.

Decreasing the K+ concentration of the medium from 5 mM to 0.59 mM decreased the K+ content of chick embryo fibroblasts to 22% of control values and increased the Na+ content to 820% of control values. The alteration of monovalent cation content occurred within two hours but had no effect on the rate of DNA synthesis, as measured by 3H-thymidine incorporation, for at least 16 hours. By decreasing the Na+ concentration in the medium, a 50% reduction in cellular Na+ could be obtained with no effect on thymidine incorporation. Since these changes in cellular Na+ or K+ are much larger than any known to occur under physiological conditions but have no effect on thymidine incorporation, we conclude that Na+ and K+ do not play a critical role in determining multiplication rate. Addition of 1.8 mEGTA to cells in media containing 1.7 mM Ca2+ and 0.8 mM Mg2+ inhibited thymidine incorporation and sharply decreased cellular K+ and increased cellular Na+ content. However, there was no reduction in total cellular Ca2+ levels. Likewise, decreasing the Ca2+ concentration of the medium below 0.01 mM inhibited thymidine incorporation, decreased cellular K+ and Mg2+, and increased cellular Na+ but did not affect total cellular Ca2+ levels. Inhibition of DNA synthesis, therefore, could not be correlated with changes in cellular Ca2+ levels.     

The use of HgCl2 to evaluate the cosubstrate: amino acid transport stoichiometry in Ehrlich ascites tumor cells

Recent investigations have indicated that cellular rheogenic properties may interfere with the correct estimation of Na+ and amino transport stoichiometry. We have reevaluated the stoichiometry of Na+ and alpha-aminoisobutyric acid (alpha-AIB) cotransport in Ehrlich ascites tumor cells depleted of Na+ and ATP by incubation in Na+-free HEPES-buffered medium (pH 7.2) containing 160 mM K+ and 2.5 microM valinomycin. Transfer of the cells to a medium with 10 mM 22Na+, 10 mM 3H-AIB, and 150 mM K+ resulted in an enhancement of Na+ flux above basal levels, which represents 0.6 of the AIB uptake. Under these conditions the membrane potential, -7.0 +/- 0.1 mV (SEM), does not change with the addition of AIB, -7.3 +/- 0.6 mV (SEM). HgCl2 (10 microM) added to the medium inhibited AIB flux and AIB-stimulated Na+ flux by 45-50% but did not change the coupling ratio. HgCl2 (10 microM) does not inhibit the basal Na+ flux nor does it affect cellular Na+ or K+ content. In physiological medium cotransport is electrogenic. The membrane potential of Ehrlich cells in physiological medium is -22.3 +/- 0.8 mV (SEM) and depolarizes to -16.7 +/- 0.7 mV (SEM) upon addition of AIB. Under these conditions the coupling ratio was highly variable but the ratio of codepression is 0.90 +/- 0.02 (SEM) in the presence of HgCl2 (10 microM). These results are consistent with a model (Smith and Robinson, 1981) in which the stoichiometry is one cosubstrate molecule per molecule of alpha-AIB. We suggest that H+ provides the alternative cosubstrate in this low Na+ environment and that in high Na+ medium the Na+:AIB stoichiometry approaches 1:1.     

Atrioventricular block in low potassium and K dependence of the nodal resting potential

A progressive conduction block leading to atrioventricular dissociation develops in perfused rabbit hearts within 20-30 min of exposure to Krebs containing 0.5 mM potassium (low K). A decrease in potassium permeability resulting in membrane depolarization (as seen in Purkinje fibers) could be responsible for the loss of excitability in nodal cells. We investigated the K dependence of the resting potential and the long-term effects of low K perfusion on the resting and action potentials of nodal cells in rabbit hearts. The resting potential of atrial, atrionodal, and nodal cells varied by 52, 41, and 34 mV per decade of change in Ko within the range of 5-50 mM K. Hyperpolarization of the resting membrane, a progressive decline in action potential amplitude, and a decrease in maximum rate of rise were observed in nodal fibers when exposed to low K. Loss of propagated activity occurred in the middle node within 20-30 min while the cells remained hyperpolarized. There was no evidence of electrogenic Na extrusion and it seems that the low nodal resting potential results from a high resting PNa/PK permeability ratio. The early decrease in rate of rise in low K probably reflects an increase in K-dependent outward currents, whereas the progressive deterioration and final loss of conducted electrical activity may result from an accumulation of internal Na and Ca overload produced by low K inhibition of the Na pump.     

Growth and cellular ion content of a salt-sensitive symbiotic system Azolla pinnata-Anabaena azollae under NaCl stress

Salinity, at a concentration of 10 mM NaCl affected the growth of Azolla pinnata-Anabaena azollae association and became lethal at 40 mM. Plants exposed up to 30 mM NaCl exhibited longer roots than the control, especially during the beginning of incubation. Average root number in plants exposed to 10 and 20 mM NaCl remained almost the same as in control. A further rise in NaCl concentration to 30 mM reduced the root number, and roots shed off at 40 mM NaCl. Presence of NaCl in the nutrient solution increased the cellular Na+ of the intact association exhibiting differential accumulation by individual partners, while it reduced the cellular Ca2+ level. However, cellular K+ content did not show significant change. Cellular Na+ based on fresh weight of respective individual partners (host tissues and cyanobiont) remained higher in the host tissues than the cyanobiont, while reverse was true for K+ and Ca2+ contents. The contribution of A. azollae in the total cellular ion content of the association was a little because of meagre contribution of the cyanobiont mass (19-21%). High salt sensitivity of Azolla-Anabaena complex is due to an inability of the association to maintain low Na+ and high Ca2+ cellular level.     

Cavitation in the mouse preimplantation embryo: Na/K-ATPase and the origin of nascent blastocoele fluid

This study tested the proposition that Na/K-ATPase activity could be involved in the morphogenetic aspects of mouse blastocyst formation by facilitating the localization of certain organelles to apposed borders, the production of nascent blastocoele fluid, and cavitation. It was assumed that such Na/K-ATPase activity would be sensitive to varying concentrations of external K (Ko)--which would alter plasma membrane potentials--and to ouabain--which would directly alter Na/K-ATPase function. Morulae were cultured for 40 hr in varying concentrations of Ko and/or ouabain and were observed for their ability to form blastocoeles (cavitate) and to localize mitochondria to apposed cell borders. Cavitation was accelerated when Ko was decreased from the control value of 6.0 to 0.6 mM and was delayed when Ko was increased to 25 mM. With Ko at 6.0 mM, 10(-5) M ouabain accelerated cavitation while 10(-4) M ouabain delayed cavitation and reduced the total number of embryos that cavitated by the end of the 40-hr culture period. With Ko at 0.6 mM, 10(-5) M ouabain now delayed cavitation while 10(-4) M ouabain almost completely inhibited it. When Ko was increased to 25 mM, 10(-5) M ouabain again accelerated cavitation while 10(-4) M ouabain delayed-rather than inhibited--cavitation. Morphometric analyses at the electron microscopic level showed changes in the distances of mitochondria from apposed cell borders with conditions that accelerated or delayed cavitation and these changes differed for inside and outside cells of the morula. These observations are consistent with the proposition that Na/K-ATPase activity could be involved in the localization of organelles to apposed cell borders, the production of nascent blastocoele fluid, and in cavitation during mouse blastocyst development.     

ADP+orthophosphate (P(i)) stimulates an Na/K pump-mediated coefflux of P(i) and Na in human red blood cell ghosts

The Na/K pump in human red blood cells that normally exchanges 3 Nai for 2 Ko is known to continue to transport Na in a ouabain-sensitive and ATP-dependent manner when the medium is made free of both Nao and Ko. Although this Na efflux is called "uncoupled" because of removal of ions to exchange with, the efflux has been shown to be comprised of a coefflux with cellular anions. The work described in this paper presents a new mode of operation of uncoupled Na efflux. This new mode not only depends upon the combined presence of ADP and intracellular orthophosphate (P(i))i but the Na efflux that is stimulated to occur is coeffluxed with (P(i))i. These studies were carried out with DIDS- treated resealed red cell ghosts, suspended in buffered (NMG)2SO4, that were made to contain, in addition to other constituents, varying concentrations of ADP and P(i) together with Na2 SO4, MgSO4 and hexokinase. While neither ADP nor P(i) was effective alone, ouabain- sensitive uncoupled Na efflux, (measured with 22Na) could be activated by [ADP+P(i)] where the K0.5 for ADP in the presence of 10 mmol (P(i))i/liter ghosts was 100-200 mumol/liter ghosts and the K0.5 for (P(i))i, in the presence of 500 mumol ADP/liter ghosts was 3-4 mmol/liter ghosts. [ADP+P(i)] activation of this Na efflux could be inhibited by as little as 2 mumol ATP/liter ghosts but the inhibition could be relieved by the addition of 50 mM glucose, given entrapped hexokinase. While ouabain-sensitive Na efflux was found to be coeffluxed with P(i) (measured with entrapped [32P]H3PO4), this was not so for SO4 (measured with 35SO4). The stoichiometry of Na to P(i) efflux was found to be approximately 2 to 1. Na efflux as well as (P(i))i efflux were both inhibited by 10 mM Nao (K0.5 approximately equal to 4 mM). But, whereas 20 mM Ko (K0.5 approximately equal to 6 mM) inhibited the efflux of (P(i))i, as would be expected from previous work, Na efflux was actually increased. When Ko influx was measured in this situation there was a 1 for 1 exchange of Nai for Ko, that is, of course, downhill with respect to the gradient of each ion. Surprisingly AsO4 was unable to replace P(i) for activation of Na efflux but Na efflux could be inhibited by vanadate and oligomycin. In terms of mechanism, it is likely that ADP acts to promote the formation of the phosphoenzyme (EP) by (P(i))i that would otherwise be inhibited by Nai.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of internal solute concentrations of marine Vibrio alginolyticus in response to external NaCl concentration.

Slightly halophilic marine Vibrio alginolyticus grown in the range of NaCl from 0.2 to 1.5 M maintained the total internal solute concentration always higher than the external medium by about 0.25 osM. The concentrations of macromolecules such as DNA, RNA, and protein were little affected by the increase in medium NaCl. The internal K+ concentration was kept to about 400 mM in the range of medium NaCl from 0.4 to 0.8 M; it rose to 510 mM when the bacterium was grown in 1.5 M NaCl, indicating that K+ increased only slightly in response to the large increase in medium NaCl. Thus, in contrast to the case of nonhalophilic and extremely halophilic bacteria, K+ was unlikely to act as a major component to regulate the internal solute concentration of marine V. alginolyticus. The internal Na+ and Cl- concentrations were maintained always lower than those in the growth medium, but they increased in response to the increase in medium NaCl. The concentration of internal Na+ was close to that of K+ at the concentration of medium NaCl that supports the optimal growth of this organism. The total amino acid content of V. alginolyticus increased from 76 to 413 mM by the increase in medium NaCl from 0.2 to 1.5 M. The concentrations of glutamic acid and prolined were 254 and 72 mM, respectively, when grown in 1.5 M NaCl. These results indicated that Na+, Cl- and amino acids, especially glutamic acid and proline, contributed to the regulation of internal solute concentration of V. alginolyticus in response to the increased external NaCl.     

The recovery from the relaxant effect by ouabain and the metabolic dependency of K+ contractions in guinea-pig taenia coli.

1. Ouabain (2.5 x 10(-5) M) inhibited preferentially the tonic response to 40 mM K+ medium (containing enough Na+) without affecting the phasic in taenia coli. When 11 mM lactate was added to the medium (pH 6.5) in the presence of ouabain, the tonic phase to 40 mM K+ recovered markedly. 2. Ouabain (2.5 x 10(-5) M) did not affect the tonic tension in 152 mM K+ medium (Na+ 0 mM). However, ouabain inhibited the recovered tension by the addition of 50 mM Na+ in the 152 mM K+ medium. But ouabain failed to inhibit the marked recovered tension by the addition of 11 mM lactate which utilized, even in the absence of external Na+, in 152 mM K+ medium. 3. Ouabain partly inhibited the shortening to 40 mM K+ (containing enough Na+) at light load; however, it inhibited markedly the shortening at heavy load. 4. There is a possibility that ouabain inhibits active transport of glucose depending on external Na+ in taenia coli of smooth muscle. Ouabain could not inhibit the tension by lactate which utilized under conditions of independent on Na+. Furthermore, it is suggested that ouabain inhibits the contraction which depends on aerobic metabolism; however, it has only a slight effect on contraction which depends on aerobic metabolism; however, it has only a slight effect on contraction which was not so dependent on aerobic metabolism.     

Apparent affinity of the Na/K pump for ouabain in cultured chick cardiac myocytes. Effects of Nai and Ko

The measured apparent affinity (K0.5) of the Na/K pump for ouabain has been reported to vary over a wide range. In a previous report we found that changing Nai could alter apparent affinity by at least an order of magnitude and that the model presented predicted this variability. To increase our understanding of this variability, isolated cells or two- to three-cell clusters of cardiac myocytes from 11-d embryonic chick were used to measure the effects of Nai and Ko on the K0.5 of the Na/K pump for ouabain. Myocytes were whole-cell patch clamped and Na/K pump current (Ip) was measured in preparations exposed to a Ca-free modified Hank's solution (HBSS) that contained 1 mM Ba, 10 mM Cs, and 0.1 mM Cd. Under these conditions there are no Ko-sensitive currents other than Ip because removal of Ko in the presence of ouabain had no effect on the current-voltage (I-V) relation. The I-V relation for Ip showed that in the presence of 5.4 mM Ko and 51 mM Nai, Ip has a slight voltage dependence, decreasing approximately 30% from 0 to -130 mV. Increasing Nai in the patch pipette from 6 to 51 mM (Ko = 5.4 mM) caused Ip to increase from 0.46 +/- 0.07 (n = 5) to 1.34 +/- 0.08 microA/cm2 (n = 13) with a K0.5 for Nai of 17.4 mM and decreased the K0.5 for ouabain from 18.5 +/- 1.8 (n = 4) to 3.1 +/- 0.4 microM (n = 3). Similarly, varying Ko between 0.3 and 10.8 mM (Nai = 24 mM) increased Ip from 0.13 +/- 0.01 (n = 5) to 0.90 +/- 0.05 microA/cm2 (n = 5) with a K0.5 for Ko of 1.94 mM and increased K0.5 for ouabain from 0.56 +/- 0.14 (n = 3-6) to 10.0 +/- 1.1 microM (n = 6). All of these changes are predicted by the model presented. A qualitative explanation of these results is that Nai and Ko interact with the Na/K pump to shift the steady-state distribution of the Na/K pump molecules among the kinetic states. This shift in state distribution alters the probability that the Na/K pump will be in the conformation that binds ouabain with high affinity, thus altering the apparent affinity. In intact cells, the measured apparent affinity represents a combination of all the rate constants in the model and does not equate to simple first-order binding kinetics.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphate from the phosphointermediate (EP) of the human red blood cell Na/K pump is coeffluxed with Na, in the absence of external K

This study is concerned with Na/K pump-mediated phosphate efflux that occurs during uncoupled Na efflux in human red blood cells. Uncoupled Na efflux is known to be a ouabain-sensitive mode of the Na/K pump that occurs in the absence of external Nao and Ko. Because this efflux (measured with 22Na) is also inhibited by 5 mM Nao, the efflux can be separated into a Nao-sensitive and a Nao-insensitive component. Previous work established that the Nao-sensitive efflux is actually comprised of an electroneutral coefflux of Na with cellular anions, such as SO4 (as 35SO4). The present work focuses on the Nao-insensitive component in which the principal finding is that orthophosphate (P(i)) is coeffluxed with Na in a ouabain-sensitive manner. This P(i) efflux can be seen to occur, in the absence of Ko, in both DIDS-treated intact cells and resealed red cell ghosts. This efflux of P(i) was shown to be derived directly from the pump's substrate, ATP, by the use of resealed ghosts made to contain both ATP and P(i) in which either the ATP or the P(i) were labeled with, respectively, [gamma-32P]ATP or [32P]H3PO4. (These resealed ghosts also contained Na, Mg, P(i), SO4, Ap5A, as well as an arginine kinase/creatine kinase nucleotide regenerating system for the control of ATP and ADP concentrations, and were suspended usually in (NMG)2SO4 at pH 7.4.) It was found that 32P was only coeffluxed with Na when the 32P was contained in [gamma-32P]ATP and not in [32P]H3PO4. This result implies that the 32P that is released comes from ATP via the pump's phosphointermediate (EP) without commingling with the cellular pool of P(i). Ko (as K2SO4) inhibits this 32P efflux as well as the Nao-sensitive 35SO4 efflux, with a K0.5 of 0.3-0.4 mM. The K0.5 for inhibition of P(i) efflux by Ko is not influenced by Nao, nor can Nao act as a congenor for Ko in any of the flux reactions involving Ko. The stoichiometry of Na to SO4 and Na to P(i) efflux is approximately 2:1 under circumstances where the stoichiometry of Na effluxed to ATP utilized is 3:1. From these and other results reported, it is suggested that there are two types of uncoupled Na efflux that differ from each other on the basis of their sensitivity to Nao, the source (cellular vs substrate) and kind of anion (SO4 vs P(i)) transported.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of changes in sodium electrochemical potential gradient on p-aminohippurate transport in newt kidney

1. The relation between p-aminohippurate uptake and the electrochemical potential gradient of Na+ (delta muNa+) across the peritubular membrane was examined in newt (Triturus pyrrhogaster) kidney. The delta muNa+ was modified by changing cellular Na+ concentration and/or lowering the electrical potential difference across the peritubular membrane (peritubular membrane potential) 2. Elevation of external K+ concentration or addition of alanine at 40 mM to the medium decreased the delta muNa+ mainly through the depolarization of the cells. Addition of 1 mM ouabain resulted in a decrease in the peritubular membrane potential and increase in cellular Na+ concentration, thus decrease in the delta muNa+. 3. p-Aminohippurate uptake decreased in proportion to the decrease in the delta muNa+ under all experimental conditions, indicating that the maintenance of the delta muNa+ is required for p-aminohippurate transport. 4. All three different experimental conditions, high medium K+ concentration, 40 mM alanine or 1 mM ouabain, increased the apparent Michaelis constant, Kt, without affecting the maximal uptake rate, V, for p-aminohippurate. These results suggests that the delta muNa+, largely the peritubular membrane potential, may affect the association and/or dissociation of p-aminohippurate and Na+ at both interfaces of the peritubular membrane of the proximal tubular cells.     

Sodium pump-mediated ATP:ADP exchange. The sided effects of sodium and potassium ions

Resealed human red cell ghosts containing caged ATP (Kaplan et al., 1978) and [3H]ADP were irradiated at 340 nm. The photochemical release of free ATP initiated a rapid transphosphorylation reaction (ATP:ADP exchange), a component of which is inhibited by ouabain. The reaction rate was measured by following the rate of appearance of [3H]ATP. The sodium pump-mediated ATP:ADP exchange reaction showed high-affinity stimulation by Mg ions (less than 10 microM) and was inhibited at higher levels. At optimal [Mg], extracellular Na (Nao) had a biphasic effect. Nao progressively inhibited the reaction rate between 0 and 10 mM and stimulated at higher levels. Intracellular Na (Nai) activated the reaction; the rate was maximal when Nai was 1 mM and remained unaltered up to 115 mM Nai at constant Nao. Extracellular K ions (Ko) inhibited the reaction; at high Nao, half-maximal inhibition was observed with 0.9 mM Ko. Lio inhibited the exchange rate with a lower affinity than Ko; half-maximal inhibition was produced by approximately 50 mM Lio. Intracellular K ions were without dramatic effect on the reaction rate in the concentration range where Ko inhibited completely. The relationship between these observations and previous studies on porous preparations is discussed, as well as the extent to which these observations support the hypothesis that the sodium pump-mediated ATP:ADP exchange reaction accompanies the Na:Na exchange transport mode of the sodium pump.