Potassium transport in erythrocytes from patients with gentamicin intolerance

A study was made of rubidium (potassium analog) influxes via ouabain-sensitive Na/K pump, bumetanide-sensitive cotransport and resistant to ouabain bumetanide membrane ion pathways and of the intracellular potassium and sodium contents in red blood cells from patients with high gentamicin sensitivity (GSP) and healthy patients (HP). It is found that red blood cells from two groups of donors do not differ in both intracellular potassium and sodium contents and pump-mediated rubidium influxes, however, bumetanide-sensitive rubidium influxes were twice as low in GSP as compared to HP (0.28 against 0.46 mumole per gram of hemoglobin). In the presence of gentamicin (10(-6) M) bumetanide-sensitive rubidium influxes were shown to decrease in red blood cells of GSP, being unchanged in erythrocytes of HP. It is suggested that the increased rates of hemolysis in response to hypotonicity in red blood cells of GSP may be due to a decreased activity of bumetanide-sensitive cotransport in plasma membrane of these cells.     

Ouabain-insensitive salt and water movements in duck red cells. II. Norepinephrine stimulation of sodium plus potassium cotransport

Catecholamines induce net salt and water movements in duck red cells incubated in isotonic solutions. The rate of this response is approximately three times greater than a comparable effect observed in 400 mosmol hypertonic solutions in the absence of hormone (W.F. Schmidt and T. J. McManus. 1977 a.J. Gen. Physiol. 70:59-79. Otherwise, these two systems share a great many similarities. In both cases, net water and salt movements have a marked dependence on external cation concentrations, are sensitive to furosemide and insensitive to ouabain, and allow the substitution of rubidium for external potassium. In the presence of ouabain, but the absence of external potassium (or rubidium), a furosemide-sensitive net extrusion of sodium against a large electrochemical gradient can be demonstrated. When norepinephrine-treated cells are incubated with ouabain and sufficient external sodium, the furosemide-sensitive, unidirectional influxes of both sodium and rubidium are half- maximally saturated at similar rubidium concentrations; with saturating external rubidium, the same fluxes are half-maximal at comparable levels of external sodium. In the absence of sodium, a catecholamine-stimulated, furosemide-sensitive influx of rubidium persists. In the absence of rubidium, a similar but smaller component of sodium influx can be seen. We interpret these results in terms of a cotransport model for sodium plus potassium which is activated by hypertonicity or norepinephrine. When either ion is absent from the incubation medium, the system promotes an exchange-diffusion type of movement of the co-ion into the cells. In the absence of external potassium, net movement of potassium out of the cell leads to a coupled extrusion of sodium against its electrochemical gradient.     

Dependence of ion transport across the plasma membrane on cell culture density. II. Active and passive cation transport during the growth of L cell cultures

Rubidium and lithium influxes as well as intracellular potassium and sodium contents were investigated in L cells during the culture growth. In sparse culture over the cell densities 0.5-3 X 10(4) cells/cm2 ouabain-sensitive rubidium influx is small and ouabain-resistant lithium influx in high. With the increase in culture density up to 4-5 X 10(4) cells/cm2 the active rubidium influx, mediated by ouabain-sensitive component, is enhanced, and ion "leakage" tested by lithium influx is diminished. Simultaneously with the exponential growth of culture the intracellular potassium content is increased and the intracellular sodium content is decreased resulting in the higher K/Na ratio in cell. During the further transition to dense culture and in stationary state (10-17 X 10(4) cells/cm2) the sodium content and lithium influx do not change significantly, but the potassium content is decreased. The decrease in intracellular potassium is correlated with that in the portion of cells in S-phase from 27-30 to 12%. Thus, in transformed cells the density-dependent alterations in membrane cation transport are observed.     

Nonsteady-State Three Compartment Tracer Kinetics: II. Sodium Flux Transients in the Toad Urinary Bladder in Response to Short Circuit

The theoretical approach presented in the previous paper provides an analytical method for determining the unidirectional, nonsteady-state fluxes in a three compartment system. Based on this a study was made of the sodium flux transients in the toad urinary bladder. A transient time-dependent state was generated by suddenly short-circuiting a bladder previously maintained in an open-circuited steady state. The sequence of experiments suggested by the theory provided the data required for the analysis. The results of these tracer experiments were consistent with the complex non-three compartmental structure of this tissue. As a result both of the inadequacy of the three compartment model in representing the tissue and of certain experimental difficulties, attempts at a quantitative solution were not entirely successful. Useful information was nevertheless obtained through a careful use of this model, and a qualitative analysis implied that the sodium influxes into the tissue at both of its surfaces are sensitive to changes in electrical potential while both effluxes are insensitive to this change. This suggests that both of the effluxes result from active processes while both influxes are associated with passive processes. The net transepithelial transport of sodium would then necessarily result from a more complex polarization than that proposed by Koefoed-Johnsen and Ussing.     

Effect of gramicidin A and thallium ions on cation effluxes in frog sartorius muscle

The effluxes of potassium, rubidium, sodium and lithium from the sartorius muscle of Rana temporaria in magnesium-Ringer solution free of sodium and potassium have been studied with the flame-emission technique. The channel-forming antibiotic gramicidin A (2.5 X X10(-7)-1 X 10(-6) mol/l) enhanced the efflux of potassium and rubidium and increased the rate constants of these effluxes. Gramicidin had small if any effect on sodium and lithium effluxes and rate constants. After 60-100 min in a gramicidin-containing medium, the potassium efflux and the corresponding rate constant reached a steady-state level. This steady-state value depended on gramicidin concentration. Effect of gramicidin on both the potassium efflux and the rate constant was partially reversible. Thallium ions (2.5 X 10(-3) and 5 X 10(-3) mol/l) in sodium- and potassium- free magnesium Ringer solution caused a large increase in effluxes of all the cations examined (K+, Rb+ and Na+) both in presence and absence of gramicidin. Possible mechanisms of gramicidin and thallium effects are discussed.     

An analysis of the sodium and chloride fluxes in the flounderPlatichthys flesus

Summary The sodium, potassium and chloride influxes and effluxes through the euryhaline flounderPlatichthys flesus are analysed into active and passive components. It is demonstrated that for any aquatic animal the energy required for osmotic regulation is minimal when the potential difference between the animal's body fluids and the medium is the same as the diffusion potential for the ions. This is illustrated by reference to the flounder.This work was supported by N.E.R.C. grant No.: GR/3/1515. We are grateful to the Lancashire and Western Sea Fisheries Committee for supplying the flounders.     

Furosemide-sensitive cation transport in frog skeletal muscle fibers

Furosemide-inhibitable components in unidirectional cation fluxes have been identified in frog skeletal muscle. In sodium loaded muscles, placed in sodium-free rubidium lithium media, furosemide (1 mM) inhibits partially rubidium and lithium influxes as well as potassium and sodium outfluxes. The furosemide-inhibitable components were found to depend on the presence of ouabain. They were greatly diminished in sodium-free magnesium media and were present in chloride-free nitrate containing media. The dependence of furosemide-inhibitable sodium efflux on internal sodium content was also described.     

The influence of pH and membrane potential on passive Na+ and K+ fluxes in human red blood cells

Passive (ouabain-insensitive) Na+ and K+ effluxes from human red blood cells were measured over the range pHo 6.2-8.5. On raising pHo, Na+ efflux increased and this was mainly attributable to the piretanide-sensitive component: K+ efflux likewise but attributable to both piretanide-sensitive and piretanide-insensitive components. On replacing Cl- with non-penetrating anions (mainly gluconate), Na+ and K+ effluxes increased, mostly attributable to the piretanide-insensitive components. On restoring pHi either by reducing pHo or by applying DIDS, the influence of pHo on Na+ and K+ effluxes was diminished. These results suggest that pHi rather than Em is the dominant influence. Passive Na+ and K+ effluxes and influxes in the presence of bumetanide were tested fro conformity to the Ussing independence relationship. For K+, the calculated and observed ratios agreed, indicating that the sodium pump, 'cotransport' and leak wholly account for K+ fluxes in human red blood cells. For Na+, the ratios did not agree and a 1:1 Na+/Na+ exchange did not account for the discrepancy. Pathways for Na+ appear to be more numerous than for K+.     

Sodium flux ratio through the amiloride-sensitive entry pathway in frog skin

The sodium flux ratio of the amiloride-sensitive Na+ channel in the apical membrane of in vitro Rana catesbeiana skin has been evaluated at different sodium concentrations and membrane potentials in sulfate Ringer solution. Amiloride-sensitive unidirectional influxes and effluxes were determined as the difference between bidirectional 22Na and 24Na fluxes simultaneously measured in the absence and presence of 10(-4) M amiloride in the external bathing solution. Amiloride- sensitive Na+ effluxes were induced by incorporation of cation- selective ionophores (amphotericin B or nystatin) into the normally Na+- impermeable basolateral membrane. Apical membrane potentials (Va) were measured with intracellular microelectrodes. We conclude that since the flux ratio exponent, n', is very close to 1, sodium movement through this channel can be explained by a free-diffusion model in which ions move independently. This result, however, does not necessarily preclude the possibility that this transport channel may contain one or more ion binding sites.     

Salt excretion by the perfused head of trout adapted to sea water and its inhibition by adrenaline

Summary Isolated heads of trout (Salmo gairdneri) were used to study the unidirectional flux of sodium and chloride across the gills in salt water.Two perfusion techniques were employed. Under constant pressure perfusion, the addition of adrenaline during the perfusion causes an increase in the flow-rate. Under constant flow-rate conditions, adrenaline provokes a decrease in pressure. A comparison of influx determination made with these two techniques of perfusion shows that variations in flow-rate of perfusion do not affect the assessment of these fluxes.A net efflux of sodium, but not of chloride, is demonstrated in sea water. The effluxes of sodium and chloride observed in sea water are decreasedd about 50% during a rapid transfer to fresh water. The addition of potassium to this medium stimulates the effluxes of sodium and chloride, suggesting a Na/K exchange participating in the chloride excretion.Adrenaline causes an inhibition of sodium and chloride efflux in sea water which persists after transfer to fresh water and the addition of potassium. Only the influx of chloride is inhibited at a concentration of 10^–5 M whereas the sodium influx is unaffected. The presence of adrenaline results in a net influx of both sodium and chloride.The differential action of adrenaline on the influxes of sodium and chloride suggests that the hemodynamic modifications provoked by this catecholamine occur independently of its aforementioned ion exchange effects.     

Application of Non-invasive Microsensing System to Simultaneously Measure Both H+ and O2 Fluxes Around the Pollen Tube

Various ionic and molecular activities in the extracellular environment are vital to plant cell physiological processes. A noninvasive microsensing system (NMS) based on either the scanning ion-selective electrode technique (SIET) or the scanning polarographic electrode technique (SPET) is able to obtain information regarding the transportation of various ions/molecules in intact samples under normal physiological conditions. The two-probe simultaneous test system (2STS) is an integrated system composed of SIET,SPET, and a Xu-Kunkel sampling protocol. In the present study, 2STS was able to simultaneously measure fluxes of H+ and O2 of the lily (Lilium Iongiflorum Thunb. cv. Ace) pollen tube while avoiding interference between the two probes. The results indicate that the proton fluxes were effluxes, whereas the oxygen fluxes were influxes, and they were closely correlated to each other surrounding the constitutive alkaline band region. Specifically, when the proton effluxes increased, the oxygen influxes also increased. Therefore,the hypothesis of condensed active mitochondria existing in the alkalized area of the pollen tube proposed by Hepler's group is supported.     

Application of Non-invasive Microsensing System to Simultaneously Measure Both H^＋ and O2 Fluxes Around the Pollen Tube

Various Ionic and molecular activities in the extraceUular environment are vital to plant cell physiological processes. A noninvasive microsensing system （NMS） based on either the scanning ion-selective electrode technique （SIET） or the scanning polarographlc electrode technique （SPET） is able to obtain information regarding the transportation of various Ions/molecules in Intact samples under normal physiological conditions. The two-probe simultaneous test system （2STS） Is an Integrated system composed of SIET, SPET, and a Xu-Kunkel sampling protocol. In the present study, 2STS was able to simultaneously measure fluxes of H^＋ and O2 of the Uly （Lillum Iongiflorum Thunb. cv. Ace） pollen tube while avoiding interference between the two probes. The results Indicate that the proton fluxes were effluxes, whereas the oxygen fluxes were Influxes, and they were closely correlated to each other surrounding the constitutive alkaline band region. Specifically, when the proton effluxes increased, the oxygen Influxes also increased. Therefore, the hypothesis of condensed active mitochondria existing in the alkalized area of the pollen tube proposed by Hepler＇s group is supported.     

Elevated [Cl-]i, and [Na+]i inhibit Na+, K+, Cl- cotransport by different mechanisms in squid giant axons

Bumetanide-sensitive (BS) unidirectional fluxes of (36)Cl- or (22)Na+ were measured in internally dialyzed squid giant axons while varying the intra- or extracellular concentrations of Na+ and/or Cl-. Raising either [Cl-]i or [Na+]i resulted in a concentration-dependent reduction of the BS influx of both (36)Cl- and (22)Na+. Raising [Cl-]i above 200 mM completely blocked BS influxes. However, raising [Na+]i to 290 mM resulted in saturable but incomplete inhibition of both BS Na+ influx and BS Cl- influx. The consequences of varying intracellular Cl- on cotransporter effluxes were complex. At lower [Cl-]i values (below 100 mM) intracellular Cl- activated cotransporter effluxes. Surprisingly, however, raising [Cl-]i levels > 125 mM resulted in a [Cl-]i-dependent inhibition of BS effluxes of both Na+ and Cl-. On the other hand, raising [Na+]i resulted only in the activation of the BS Na+ efflux; intracellular Na+ did not inhibit BS efflux even at 290 mM. The inhibitory effects of intracellular Na+ on cotransporter-mediated influxes, and lack of inhibitory effects on BS effluxes, are consistent with the trans-side inhibition expected for an ordered binding/release model of cotransporter operation. However, the inhibitory effects of intracellular Cl- on both influxes and effluxes are not explained by such a model. These data suggest that Cl may interact with an intracellular site (or sites), which does not mediate Cl transport, but does modulate the transport activity of the Na+, K+, Cl- cotransporter.     

Dependence of ion transport across the plasma membrane on the density of the cell culture. I. Ion flows and the potassium and sodium content in 3 Chinese hamster cell lines (CHO)

Cation transport has been investigated in three lines of Chinese ovary cells CHO-K1 during the cell culture growth. With the increase in the cell density potassium and sodium contents decreased from 1.2 to 0.8-0.5 and from 0.5 to 0.15-0.1 mmole/g protein, respectively. The time courses of potassium and sodium changes were different, and the increase in intracellular K/Na ratio from 1.5-2.0 to 5-10 with the increase in cell density was revealed. The rubidium influx was found to decrease during the culture growth mainly due to the decrease in ouabain-inhibitable and (ouabain + furosemide)- non-inhibitable influxes. The changes in cation fluxes and cation contents were observed in transformed cells without contact inhibition of division and were considered as a manifestation of density-dependent alterations of plasma membrane.     

Il-2-regulated expression of Na+,K(+)-ATPase in activated human lymphocytes

Expression of Na+,K(+)-ATPase alfal-subunit and of oubain-sensitive rubidium influxes has been investigated in human peripheral blood lymphocytes. Isolated lymphocytes were stimulated by phytogemagglutinin (PHA) or interleukin-2 (IL-2). It has been shown that during the early stage of the PHA-activation the alfal-subunit abundance in the membrane fractions of the human blood lymphocytes does not change, whereas at the late stages of Go-->G1-->S transition (16-48 h) the alfa1 protein content increases. A translation inhibitor cycloheximide was found to prevent the late increase in alfa1-subunit expression. An immunosuppressant cyclosporin A decreases both IL-2-dependent T-lymphocyte progression and alfa1-subunit abundance by 48 h of PHA-induced lymphocyte activation. In the lymphocytes pretreated by PHA in submitogenic concentration (0.8-1.0 microg/ml) exogenous IL-2 (100 U/ml) induces a proliferative response as well as alfal-protein accumulation. A decrease in alfa1-protein accumulation in the presence of specific inhibitors of separate signal transduction pathways enables us to conclude that protein kinases ERK1/2 (MAPK pathway) and JAK3 (JAK-STAT pathway) mediate the IL-2-dependent regulation of Na+,K(+)-ATPase expression during lymphocyte transition from resting stage to proliferation. A correlation between changes in ouabain-sensitive rubidium influxes and the alfal-subunit amount has been demonstrated. It is concluded that IL-2-dependent-progression of normal human lymphocytes from quiescence to proliferation is accompanied by the increase in Na+,K(+)-ATPase alfa1-subunits expression, and the enhanced transport activity of a sodium pump during the prereplicative stage is provided by the increased number of functional pump units in plasma membrane.     

Activation of the potassium uptake system during fermentation in Saccharomyces cerevisiae.

Fermentable sugars activated the K+ uptake system, increasing the Vmaxs of Rb+, Na+, and Li+ influxes, but sugars did not affect the effluxes of these cations. This activation seems to be a direct effect of fermentation and not the consequence of the H+ pump ATPase activation or internal pH decrease produced by fermentation.     

The Kinetics of Ouabain Inhibition and the Partition of Rubidium Influx in Human Red Blood Cells

In the development of ouabain inhibition of rubidium influx in human red blood cells a time lag can be detected which is a function of at least three variables: the concentrations of external sodium, rubidium, and ouabain. The inhibition is antagonized by rubidium and favored by sodium. Similar considerations could be applied to the binding of ouabain to membrane sites. The total influx of rubidium as a function of external rubidium concentration can be separated into two components: (a) a linear uptake not affected by external sodium or ouabain and not requiring an energy supply, and (b) a saturable component. The latter component, on the basis of the different effects of the aforementioned factors, can be divided into three fractions. The first is ouabain-sensitive, inhibited by external sodium at low rubidium, and requires an energy supply; this represents about 70–80% of the total uptake and is related to the active sodium extrusion mechanism. The second is ouabain-insensitive, activated by external sodium over the entire range of rubidium concentrations studied, and dependent on internal ATP; this represents about 15% of the total influx; it could be coupled to an active sodium extrusion or belong to a rubidium-potassium exchange. The third, which can be called residual influx, is ouabain-insensitive, unaffected by external sodium, and independent of internal ATP; this represents about 10–20% of the total influx.     

Proton-activated rubidium transport catalyzed by the sodium pump

Although the sodium pump normally exchanges three sodium for two potassium ions, experiments with inside-out red cell membrane vesicles show that the stoichiometry is reduced when the cytoplasmic sodium concentration is decreased to less than 1 mM. The present study was designed to gain insight into the question whether other monovalent cations, particularly protons, can act as sodium congeners in effecting pump-mediated potassium transport (ATP-dependent rubidium efflux from inside-out vesicles). The results show that at low cytoplasmic sodium concentration, an increase in proton concentration effects a further reduction in sodium:rubidium stoichiometry, to a value less than the minimal expected (1Na+:3Rb+). Furthermore, when vesicles containing 86RbCl are incubated in nominally sodium-free medium. ATP-dependent net rubidium efflux (normal influx) occurs when the pH is reduced from approximately 7.0 to 6.2 or less. This efflux is inhibited by strophanthidin and vanadate. These experiments support the notion that the sodium pump can operate as an ATP-dependent proton-activated rubidium (potassium) pump without obligatory countertransport of sodium ions.     

Transmembrane effects in the sodium pump system. I. The effect of external potassium and rubidium on the dependence of sodium efflux on sodium concentration in the frog muscle

The dependence of sodium efflux on intracellular sodium content with various potassium and rubidium concentration in the external medium has been studied on frog sartorious muscle. In potassium-sodium-free magnesium medium ouabain-sensitive sodium efflux was shown to be proportional to internal sodium concentration. In the presence of external ribidium (0.5--5.0 mM) the efflux concentration relations are non-linear, being closely described by assuming that 3 Na+ are transported per pump cycle. In sodium loaded muscles the efflux concentration curve was found to be dependent on the external rubidium concentration, becoming linear instead of S-shaped with the decrease in internal rubidium concentration from 5.0--2.5 to 1.0--0.5 mM. The apparent affinity constant for the internal sodium pump site increased with increasing the external rubidium (potassium) concentration. The data obtained may contribute to the kinetic evaluation of the type of Na-K pump mechanism, being more consistent with simultaneous model of pump operation.     

CALCULATIONS OF BIOELECTRIC POTENTIALS : VI. SOME EFFECTS OF GUAIACOL ON NITELLA

Values have been calculated for apparent mobilities and partition coefficients in the outer non-aqueous layer of the protoplasm of Nitella. Among the alkali metals (with the exception of cesium) the order of mobilities resembles that in water and the partition coefficients (except for cesium) follow the rule of Shedlovsky and Uhlig, according to which the partition coefficient increases with the ionic radius. Taking the mobility of the chloride ion as unity, we obtain the following: lithium 2.04, sodium 2.33, potassium 8.76, rubidium 8.76, cesium 1.72, ammonium 4.05, ½ magnesium 20.7, and ½ calcium 7.52. After exposure to guaiacol these values become: lithium 5.83, sodium 7.30, potassium 8.76, rubidium 8,76, cesium 3.38, ammonium 4.91, ½ magnesium 20.7, and ½ calcium 14.46. The partition coefficients of the chlorides are as follows, when that of potassium chloride is taken as unity: lithium 0.0133, sodium 0.0263, rubidium 1.0, cesium 0.0152, ammonium 0.0182, magnesium 0.0017, and calcium 0.02. These are raised by guaiacol to the following: lithium 0.149, sodium 0.426, rubidium 1.0, cesium 0.82, ammonium 0.935, magnesium 0.0263, and calcium 0.323 (that of potassium is not changed). The effect of guaiacol on the mobilities of the sodium and potassium ions resembles that seen in Halicystis but differs from that found in Valonia where guaiacol increases the mobility of the sodium ion but decreases that of the potassium ion.