The effect of low-level activation on the mechanical properties of isolated frog muscle fibers

The mechanical properties, as revealed by minute length changes, of isolated twitch fibers of the frog have been studied at rest and during low-level activation. Resting tension is 77 ± 23 mN/cm² (mean ± SD) at 2.2 µm sarcomere length.¹ The slope of the tension curve (ΔP/ΔL) recorded during a constant-speed length change of a resting fiber is initially large. At length changes exceeding about 0.18 % of the initial length of the fiber ΔP/ΔL falls abruptly and remains close to zero during the rest of the length change. The amplitude of the tension response is reduced after a length change and returns to normal in about 3 min. Hypertonic sucrose-Ringer solutions cause a small, maintained rise in tension up to 1.4–1.6 times normal osmotic strength. Higher sucrose concentrations cause relatively large, transient tension responses. The initial ΔP/ΔL is increased in moderately hypertonic solutions; it may be reduced in more strongly hypertonic solutions. Elevated [K]_o (range 10–17.5 mM) causes a marked reduction in ΔP/ΔL. In this range of [K]_o the reduction is not accompanied by changes in resting tension. Addition of 1–1.5 mM caffeine to the Ringer solution affects the resting tension very little but also reduces ΔP/ΔL. The results suggest that stiffness and tension development are not related in a simple way.     

The effects of salt depletion on blood and tissue ion concentrations in the freshwater mussel,Ligumia subrostrata (Say)

Summary The extracellular and intracellular fluid volumes of pondwater acclimatedLigumia subrostrata are equal (3.9 ml/g dry tissue). Total blood solute is 47 mOsm and is composed primarily of Na (19.1 mM), Cl (10.6 mM), HCO₃ (12.7 mM), Ca (4.3 mM), and K (0.5 mM). Major intracellular solutes are K (14.0 mM), Na (7.0 mM) and Cl (2.4 mM).L. subrostrata continuously exposed to deionized water at 20°C exhibit a maximum decrease of 23% in extracellular fluid total solute within 30 days. The maximum [Na] and [Cl] losses are 40% and 76% respectively, while [Ca] and [HCO₃] increase by 44% and 37% respectively. No apparent change in extracellular [K] occurs. Intracellular [Na] decreases 53% and [Cl] decreases 79%, but [K] declines only 15%. Intracellular fluid volume, extracellular fluid volume, and total body water decrease 17%, 31%, and 22% respectively. Inulin clearance is 0.41 ml/g dry tissue·h for pondwater acclimated mussels and declines to 0.24 ml/g dry tissue·h during salt depletion. When salt depleted mussels are returned to solutions containing Na or Cl, they experience a net uptake of salt. The accumulated ions are about equally distributed in the extra- and intracellular compartments.     

Influence of the explantation milieu on intranuclear (Na), (K) and (Mg) of Chironomus thummi salivary gland cells

Intranuclear Na, K and Mg concentrations were determined in cells of salivary glands incubated for 1^h in selected NaCl/KCl/MgCl₂ media. By variation of the external milieu beyond “physiological” limits the intranuclear electrolytes can be shifted between ca 100 and 280 mM [K]ⁱ, between ca 8 and 100 mM [Na]ⁱ and between ca 5 and 75 mM [Mg]ⁱ. No significant competition or interactions of the 3 ionic species are apparent. The relationships [K]^e : [K]ⁱ and [Na]^e : [Na]ⁱ can best be described by a positive and linear, that between [Mg]^e : [Mg]ⁱ by a negative and exponential function. Regression parameters are given which permit a computation of intranuclear [Na], [K] and [Mg] as induced by NaCl/KCl/MgCl₂ in any binary or triple combination that is tolerated by the explanted gland without visible damage.     

Potassium fluxes in dialyzed squid axons

Measurements have been made of K influx in squid giant axons under internal solute control by dialysis. With [ATP]_i = 1 µM, [Na]_i = 0, K influx was 6 ± 0.6 pmole/cm² sec; an increase to [ATP]_i = 4 mM gave an influx of 8 ± 0.5 pmole/cm² sec, while [ATP]_i 4, [Na]_i 80 gave a K influx of 19 ± 0.7 pmole/cm² sec (all measurements at ∼16°C). Strophanthidin (10 µM) in seawater quantitatively abolished the ATP-dependent increase in K influx. The concentration dependence of ATP-dependent K influx on [ATP]_i, [Na]_i, and [K]_o was measured; an [ATP]_i of 30 µM gave a K influx about half that at physiological concentrations (2–3 mM). About 7 mM [Na]_i yielded half the K influx found at 80 mM [Na]_i. The ATP-dependent K influx responded linearly to [K]_o from 1–20 mM and was independent of whether Na, Li, or choline was the principal cation of seawater. Substances tested as possible energy sources for the K pump were acetyl phosphate, phosphoarginine, PEP, and d-ATP. None was effective except d-ATP and this substance gave 70% of the maximal flux only when phosphoarginine or PEP was also present.     

Membrane permeability changes in amphibian eggs at ovulation

Electropotential differences between the cytoplasm and external medium have been compared in the mature R. pipiens occyte and the ovulated unfertilized egg as a function of [Na]_o, [K]_o, [Ca]_o and [Cl]_o. In solutions containing 1.0 mM Ca⁺⁺ the oocyte behaved as though it were predominantly permeable to K⁺ and Cl^?, i.e., like a KCl electrode. However, the steady potential decreased with decreasing [Ca]_o and in 5 × 10^?4 mM [Ca]_o the oocyte membrane behaved like a NaCl electrode. Studies on the steady potential as a function of [Na]_o, [K]_o and [Cl]_o in 1.0 mM Ca⁺⁺ or Ca-free solutions suggest that Ca⁺⁺ controls the passive permeability of the oocyte membrane to Na⁺ and Cl^?. In the ovulated unfertilized egg the K⁺ selectivity of the cell membrane disappeared and the system behaved like a NaCl electrode. No effect of external Ca⁺⁺ or K⁺ concentration changes on the steady potential was observed. These results indicate that the ion permeability properties of the ovulated egg are similar to that of the ovarian oocyte in Ca-deficient medium, and suggests that the mechanism of ovulation may involve the removal of Ca⁺⁺ regulation of ion permeability of the egg cell membrane.     

Villus and Crypt Cell Composition in the Secreting Mouse Jejunum Measured with X-ray Microanalysis

The response of the villus and crypt cells of the mouse jejunum to secretagogues has been assessed through measurements of cellular composition with x-ray microanalysis. In nonstimulated tissues the Na concentration ([Na]c) of the crypt cells was significantly less, and the K ([K]c) and Cl ([Cl]c) concentrations were significantly greater, than that of the villus cells. There was also a decreasing gradient of [Na]c and increasing gradient of [K]c from the villus tip to crypt base due to a greater number of cells with a high [Na]c and low [K]c in the upper regions of the villi. Theophylline (10 mmol L⁻¹) stimulated a sustained increase in bumetanide sensitive short circuit current (Isc) and significantly decreased the [Na]c of the villus cells. Similar, but smaller changes were seen in the crypt cells. Changes in villus cell [Na]c reflected a reduction in the number of cells with a high [Na]c. Inhibition of the apical Na/H exchanger (1 mmol L⁻¹ amiloride) had little effect on basal Isc and the subsequent addition of theophylline increased Isc to a comparable extent as seen without amiloride. However, after amiloride treatment the only change in cellular composition was a reduction in the [Cl]c of both crypt and villus cells, suggesting that both regions are involved in the secretory response. These data suggest that the dominant response of the jejunum to secretagogues is an inhibition of Na absorption via Na/H exchange in the villi and the secretory response is distributed throughout the crypt/villus axis. Received: 1 July 1997/Revised: 4 November 1997     

An Analysis of the Leakages of Sodium Ions into and Potassium Ions out of Striated Muscle Cells

Net sodium influx under K-free conditions was independent of the intracellular sodium ion concentration, [Na]_i, and was increased by ouabain. Unidirectional sodium influx was the sum of a component independent of [Na]_i and a component that increased linearly with increasing [Na]_i. Net influx of sodium ions in K-free solutions varied with the external sodium ion concentration, [Na]_o, and a steady-state balance of the sodium ion fluxes occurred at [Na]_o = 40 mM. When solutions were K-free and contained 10^-4 M ouabain, net sodium influx varied linearly with [Na]_o and a steady state for the intracellular sodium was observed at [Na]_o = 13 mM. The steady state observed in the presence of ouabain was the result of a pump-leak balance as the external sodium ion concentration with which the muscle sodium would be in equilibrium, under these conditions, was 0.11 mM. The rate constant for total potassium loss to K-free Ringer solution was independent of [Na]_i but dependent on [Na]_o. Replacing external NaCl with MgCl₂ brought about reductions in net potassium efflux. Ouabain was without effect on net potassium efflux in K-free Ringer solution with [Na]_o = 120 mM, but increased potassium efflux in a medium with NaCl replaced by MgCl₂. When muscles were enriched with sodium ions, potassium efflux into K-free, Mg⁺⁺-substituted Ringer solution fell to around 0.1 pmol/cm²·s and was increased 14-fold by addition of ouabain.     

Ion transport and permeability in the mouse egg

Sodium, potassium, and chloride unidirectional fluxes have been studied in the mature mouse egg. Their relationship to cell membrane potential and conductance has been investigated. Unidirectional Na efflux is composed of a ouabain sensitive component, presumably representing an active Na efflux, an external Na-dependent component and a diffusional component. The data indicate that the external Na-dependent component represents a Na:Na exchange mechanism. There also exists an ouabain-sensitive component of K influx. The stoichiometry of the ouabain-sensitive fluxes is approx. 2.7:1 (Na to K). From the diffusional components of Na and K flux, the membrane permeability to these cations has been estimated. P_Na and P_K are 1.2 × 10⁻⁷ cm sec⁻¹ and 0.8 × 10⁻⁷ cm sec⁻¹ respectively. These permeabilities, in conjunction with the internal exchangeable fractions of Na and K and the external concentrations, predict an egg membrane potential of −11 mV (inside negative). Microelectrode measurements yield an egg membrane potential of −14 ± 0.4 mV, indicating that the cell membrane potential is predominantly a result of the Na and K permeabilities and distributions. Internal exchangeable Cl is 67 ± 3 mM in standard medium, as determined from ³⁶Cl distribution. The chloride equilibrium potential is therefore −15 mV, which is not significantly different from the egg membrane potential. This suggests that Cl distributes passively across the egg membrane, reflecting the egg membrane potential. Hyperpolarization of the egg membrane potential to −27 ± 1.5 mV by reduction of external Na results in an exchangeable internal Cl of 49 ± 8 mM. This yields a Cl equilibrium potential of −24 mV, indicating that the Cl distribution shifts in the predicted manner upon a change in cell membrane potential. Tracer flux data indicate that Cl conductance comprises the bulk of the total membrane conductance with Na and K sharing the remainder in approximately equal amounts.     

Electrolyte Metabolism in HeLa Cells

Methods have been developed to study cellular Na, K, and Cl concentrations in HeLa cells. Cell [Na] and [K] are functions of the age of the culture. As the culture grows [K], expressed in mmols/liter cell H₂O, rises from an initial value of 121 to a peak of 206 at about 4 days, and thereafter falls until it has almost returned to the initial value by the 9th day. [Na] falls as [K] rises, but there is no fixed relationship between the cellular concentrations of the two cations. There is, however, a correlation between generation time and cellular [K]. Measurements of net K uptake and net Na extrusion were carried out during 1 hour incubation at 37°C of low K cells. Both net K uptake and net Na extrusion took place against chemical concentration gradients, so that at least one transport system must be active; if the Cl distribution is passive both net K uptake and net Na extrusion are active. Studies with inhibitors of respiration and glycolysis lead to the conclusion that respiration is not required for these net transports, which appear to derive their energy from glycolytic sources.     

Transport of monovalent thallium across the membrane of oocyte of the lamprey <Emphasis Type="Italic">Lampetra fluviatilis</Emphasis>

Mechanisms of transport of monovalent thallium across the membrane of oocyte of the lamprey Lampetra fluviatilis were studied by using ²⁰⁴Tl. The Tl⁺ transport in lamprey oocytes has been shown to be realized by at least two pathways: through Na/K-pump and by mechanisms of Na,K,Cl-cotransport. In the standard Ringer solution (mM): 4 KCl, 140 NaCl, 0.5 CaCl₂, 5 glucose, 10 Tris-HCl-in the presence of ouabain, the coefficient of the ²⁰⁴Tl stationary distribution (cell/medium) was within the range of 2.3–2.5, while the time necessary to reach its 50% value amounted to 40–45 min at 20°C. In potassium-free media, transport of ²⁰⁴Tl via Na/K-pump was described by simple kinetics with saturation and was characterized by the value V _max = 520 pmol/(cell h) and K _M = 0.3 mM. In the presence of 4 mM K⁺ and 0.1 mM/1 Tl⁺, the ouabain-sensitive Tl⁺ flux decreased to 75 pmol/(cell h). At activation of the mechanism of Na,K,Cl-cotransport by the outer Na⁺ (in Na-NMDG media of different composition) the total influx of Tl⁺ reached 193 ± 20 pmol/(cell h), while the bumetanide-sensitive component—119 ± 12 pmol/(cell h) with K _M for Na⁺ about 20 mM. In the incubation media with variable concentration of chloride ions (replacement of Cl⁻ by NO₃) the total Tl⁺ flux reached 220 ± 21, while via the mechanisms of Na,K,Cl-cotransport—87 ± 8 pmol/(cell h). Under our experimental conditions, mechanisms of active transport and Na,K,Cl-cotransport accounted for 94% of the Tl⁺ influx. The potassium channels that usually are permeable also to monovalent thallium ions were not revealed.     

Metabolism of purine bases, nucleosides and alkaloids in theobromine-forming Theobroma cacao leaves

We examined the purine alkaloid content and purine metabolism in cacao (Theobroma cacao L.) plant leaves at various ages: young small leaves (stage I), developing intermediate size leaves (stage II), fully developed leaves (stage III) from flush shoots, and aged leaves (stage IV) from 1-year-old shoots. The major purine alkaloid in stage I leaves was theobromine (4.5 μmol g^–1 fresh weight), followed by caffeine (0.75 μmol g^–1 fresh weight). More than 75% of purine alkaloids disappeared with subsequent leaf development (stages II–IV). In stage I leaves, ¹⁴C-labelled adenine, adenosine, guanine, guanosine, hypoxanthine and inosine were converted to salvage products (nucleotides and nucleic acids), to degradation products (ureides and CO₂) and to purine alkaloids (3- and 7-methylxanthine, 7-methylxanthosine and theobromine). In contrast, ¹⁴C-labelled xanthine and xanthosine were not used for nucleotide synthesis. They were completely degraded, but nearly 20% of [8-¹⁴C]Xanthosine was converted in stage I leaves to purine alkaloids. These observations are consistent with the following biosynthetic pathways for theobromine: (a) AMP → IMP → 5′-xanthosine monophosphate → xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine; (b) GMP → guanosine → xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine; (c) xanthine → 3-methylxanthine → theobromine. Although no caffeine biosynthesis from ¹⁴C-labelled purine bases and nucleosides was observed during 18 h incubations, exogenously supplied [8-¹⁴C]Theobromine was converted to caffeine in young leaves. Conversion of theobromine to caffeine may, therefore, be slow in cacao leaves. No purine alkaloid synthesis was observed in the subsequent growth stages (stages II–IV). Significant degradation of purine alkaloids was found in leaves of stages II and III, in which [8-¹⁴C]Theobromine was degraded to CO₂ via 3-methylxanthine, xanthine and allantoic acid. [8-¹⁴C]Caffeine was catabolised to CO₂ via theophylline (1,3-dimethylxanthine) or theobromine.     

POTASSIUM-INDUCED RELEASE OF [3H]GABA AND OF [3H]NORADRENALINE FROM NORMAL AND RESERPINIZED RAT BRAIN CORTEX SLICES. DIFFERENCES IN CALCIUMDEPENDENCY, AND IN SENSITIVITY TO POTASSIUM IONS

The release of [³H]GABA induced by elevated extracellular potassium (K)_o, from thin rat brain cortex slices, has been compared with that of [³H]noradrenaline ([³H]NA), released by the same procedures, both from normal slices, and from slices pre-treated with reserpine and nialamide, [³H]NA being predominantly a vesicular component in the former situation, and a soluble substance in the latter one. 46 mM-(K)_o released considerably more [³H]NA from normal than from drug-treated slices, while the release of GABA was about two thirds of the latter. When 4min ‘pulses’ of increasing concentrations of potassium were applied, it was observed that the release of GABA and of [³H]NA from drug-treated slices increased in proportion to (K)_o, up to 36-46 mM and then declined considerably with higher (K)_o. The dependency of potassium-induced release on the concentration of calcium in the medium, indicated that release of [³H]NA from normal slices was proportional to calcium up to 1.5-2 mM, while that of [³H]NA from drug-treated slices increased up to 0.5 mM-Calcium, and then declined with higher concentrations. GABA release also increased up to 0.5 mM-calcium, but no further changes were observed at higher concentrations. The calcium antagonist D-600 inhibited high (K)_o-induced release of [³H]NA from normal slices to a greater extent than that of [³H]GABA or of [³H]NA from drug-treated slices. These results, in which elevated (K)_o-induced release of [³H]GABA resembles considerably that of soluble NA, but differs from that of NA present in synaptic vesicles, suggest that release of [³H]GABA also occurs from the soluble cytoplasmic compartment, and that the partial calcium requirement that is found is unrelated to that of transmitter secretion. These findings are also a further indication of the lack of specificity of elevated (K)_o as a stimulus for inducing transmitter secretions.     

The structures of six urinary oligosaccharides that are characteristic for a patient with morquio syndrome type b

Morquio syndrome type B is an inherited, lysosomal storage disease characterised by a marked deficiency in acid β-d-galactosidase, while the 2-acetamido-2-deoxy-β-d-galactose 6-sulphate sulphatase activity is normal. Urinary oligosaccharides were studied in order to evaluate the effect of the diminished β-d-galactosidase activity on the catabolism of glycoconjugates and to compare their structures with those excreted by patients with GM₁-gangliosidosis. The following oligosaccharides were isolated: β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→6)-β-d-Manp-(1→4)- d-GlcpNAc (1), β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→6)-[α-d-Manp- (1→3)]-β-d-Manp-(1→4)-d-GlcpNAc (2a), β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)- α-d-Manp-(1→3)-[α-d-Manp-(1→6)]-β-d-Manp-(1→4)-d-GlcpNAc (2b), β-d-Galp- (1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→3)-[β-d-Galp-(1→4)-β-d-GlcpNAc-(1→ 2)-α-d-Manp-(1→6)]-β-d-Manp-(1→4)-d-GlcpNAc (3), β-d-Galp-(1→4)-β-d-Glcp- NAc-(1→2)-α-d-Manp-(1→3)-{β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-[β-d-Galp- (1→4)-β-d-GlcpNAc-(1→6)]-α-d-Manp-(1→6)}-β-d-Manp-(1→4)-d-GlcpNAc (4), β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→3)-[β-d-GlcpNAc-(1→4)]-[β- d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→6)]-β-d-Manp-(1→4)-d-Glcp- NAc (5). Significant differences between Morquio syndrome type B and GM₁-gangliosidosis have been observed, with regard to the excretion rate and the specific structures of urinary oligosaccharides. Compounds 2a, 2b, and 5 are novel members of the series of oligosaccharides isolated from the urine of patients with inherited, lysosomal storage diseases.     

Influence of membrane potential on the sodium-dependent uptake of gamma-aminobutyric acid by presynaptic nerve terminals: Experimental observations and theoretical considerations

Summary Sodium, potassium and veratridine were tested for their effects on the uptake of gamma-aminobutyric acid (GABA) by pinched-off presynaptic nerve terminals (synaptosomes). As noted by previous investigators, the uptake from media containing 1 m GABA (high-affinity uptake) is markedly Na-dependent; the uptake averaged 65 pmoles/mg synaptosome protein × min, with [Na]₀=145mm and [K]₀=5mm, and declined by about 90% when the external Na concentration ([Na]₀) was reduced to 13mm (Na replaced by Li). The relationship between [Na]₀ and GABA uptake was sigmoid, suggesting that two or more Na⁺ ions may be required to activate the uptake of one GABA molecule. Thermodynamic considerations indicate that with a Na⁺/GABA stoichiometry of 21, the Na electrochemical gradient, alone, could provide sufficient energy to maintain a maximum steady-state GABA gradient ([GABA] _i /[GABA]₀) of about 10⁴ across the plasma membrane of GABA-nergic terminals.In Ca-free media with constant [Na]₀, GABA uptake was inhibited, without delay, by increasing [K]₀ or by introducing 75 m veratridine; the effect of veratridine was blocked by 200nm tetrodotoxin. The rapid onset (within 10 sec) of the veratridine and elevated-K effects implies that alterations in intra-terminal ion concentrations are not responsible for the inhibition. The uptake of GABA was inversely proportional to log [K]₀. These observations are consistent with the idea that the inhibitory effects of both veratridine and elevated [K]₀ may be a consequence of their depolarizing action. The data are discussed in terms of a barrier model (Hall, J. E., Mead, C.A., Szabo, G. 1973.J. Membrane Biol. 11:75) which relates carrier-mediated ionic flux to membrane potential.     

Two new 4′,4′′-disubstituted dipyrazolylpyridine derivatives, and the structures and spin states of their iron(II) complexes

Reaction of 2 equiv of the sodium salt of ethyl pyrazole-4-carboxylate, with 1 equiv of 2,6-dibromopyridine, in diglyme at 130 °C for 5 days yields 2,6-di[4-(ethylcarboxy)pyrazol-1-yl]pyridine (L¹), with 2-bromo-6-[4-(ethylcarboxy)pyrazol-1-yl]pyridine (L²) as a significant byproduct. Reduction of L¹ with excess NaBH₄ in thf affords 2,6-di[4-(hydroxymethyl)pyrazol-1-yl]pyridine (L³) in low yield. The crystalline complex [Fe(L¹)₂][BF₄]₂ · 2CF₃CH₂OH is low-spin at 150 K, while bulk samples with this formula are approximately 10% high-spin and 90% low-spin at room temperature. This ratio does not vary significantly on cooling from its magnetic susceptibility, suggesting that the material might be contaminated by a second, minor high-spin phase. Single crystals of [Fe(L³)₂][BF₄]₂·1.4CH₃CN have a mixed spin-state population, with the low-spin state predominating at 150 K. The [Fe(L³)₂(BF₄)]⁺ moieties in the lattice associate into 1-D chains through intermolecular O-H?O and O-H?F hydrogen bonding. Bulk samples of [Fe(L³)₂][BF₄]₂ · H₂O are fully low-spin below 200 K, but the magnetic data imply the onset of a gradual thermal spin-transition centred above room temperature. DSC and TGA measurements imply that this transition is centred at 322 K, and involves loss of lattice water. Both complexes undergo spin-crossover in (CD₃)₂CO solution, with transition midpoints near 250 K.     

Effect of metabolic depletion on the furosemide-sensitive Na and K fluxes in human red cells

Summary We report in this paper the effect of metabolic depletion on several modes of furosemide-sensitive (FS) Na and K transport in human red blood cells. The reduction of ATP content below 100 mol/liter cells produced a marked decrease in the maximal activation (V _max) of the outward. FS transport of Na and K into choline medium in the presence of ouabain (0.1 mM) and 1 mM MgCl₂. TheK _0.5 for internal Na to activate the FS Na efflux was not altered by metabolic depletion. However, metabolic depletion markedly decreased the K _i for external K (K _o ) to inhibit the FS Na efflux into choline medium (from 25 to 11 mM). Repletion of ATP content by incubation of cells in a substraterich medium recovered control levels ofV _max of the FS Na and K fluxes and of K _i for external K to inhibit FS Na efflux. TheV _max of FS Na and K influxes was also markedly decreased when the ATP content dropped below 100 mol/liter cells. This was mainly due to a decrease in the inward-coupled transport of K and Na (Na _o -stimulated K influx and the K _o -stimulated Na influx). The FS K _i /K _o exchange pathway of the Na–K cotransport, estimated from the FS K influx from choline-20 mM K _o medium into cells containing 22 mmol Na/liter cells, was also reduced by starvation. Starvation did not inhibit the FS Na _i /Na _o exchange pathway, estimated as FS Na influx from a medium containing 130 mM NaCl into cells containing 22 mmol Na/liter cells. The unidirectional FS²²Na efflux and influx were also measured in control and starved cells containing 22 mmol Na/liter cells, incubated in a Na medium (130 mM) at varying external K (0 to 20 mM). In substrate-fed cells, incubated in the absence of external K, FS Na efflux was larger than Na influx. This FS net Na extrusion (400 to 500 mol/liter cells·hr) decreased when external K was increased, approaching zero around 15 mM K _o . In starved cells the net Na extrusion was markedly decreased and it approached zero at lower K _o than in substrate-fed cells. Our results indicate that the FS Na and K fluxes, and their major component, the gradient driven Na–K–Cl cotransport system, are dependent on the metabolic integrity of the cells.     

Influence of Some Ions on the Membrane Potential of Ascaris Muscle

The influence of several ions on the membrane potential of the somatic muscle of Ascaris has been investigated by changing their concentration in the surrounding solution. When [K]_o is increased at the expense of [Na]_o leaving [Cl]_o constant, the membrane potential is first seen to increase. [K]_o higher than 45 mM reduces the membrane potential with a slope of 23 mv for a tenfold change in [K]_o. However, when [K]_o is increased keeping [Na]_o and [Cl]_o low and constant, the line relating the membrane potential with log [K]_o has a slope of almost 50 mv. If [Cl]_o is reduced in the absence of external Na, after the [K]_o is increased to 45 mM, the membrane potential decreases with a slope of 59 mv per tenfold change in [Cl]_o in close agreement with the Nernst equation. If Cl^- is replaced by SO₄ ^2-, a depolarization is produced, while chloride replacement by NO₃ ^-, Br^-, and I^- results in a hyperpolarization of the membrane. Removal of the external Na⁺ ions increases the average membrane potential by 17 mv.     

Ionic dependence of bumetanide binding to the rabbit parotid Na/K/Cl cotransporter

Summary The Na/K/Cl-dependent component of the binding of the loop diuretic bumetanide to basolateral membrane vesicles from the rabbit parotid is studied. A Scatchard analysis indicates that this binding is due to a single high-affinity site withK _D =3.2±0.3 m (n=9) at 100mm sodium, 100mm potassium and 5mm chloride. When KCl-dependent²²Na transport and tracer [³H]-bumetanide binding are monitored simultaneously as a function of (unlabeled) bumetanide concentration it is found that theK _0.5 for bumetanide inhibition of both processes are identical indicating that the high-affinity bumetanide binding site studied here is identical with a bumetanide-inhibitory site on the Na/K/Cl cotransport system previously identified in this preparation (R.J. Turner, J.N. George and B.J. Baum,J. Membrane Biol. 94:143–152, 1986). High-affinity bumetanide binding exhibits a hyperbolic dependence on both [Na] and [K] consistent with Na/bumetanide and K/bumetanide binding stoichiometries of 11 andK _0.5 values of approximately 33mm for sodium and 23mm for potassium. In contrast, the dependence on [Cl] is biphasic, with bumetanide binding increasing from 0 to 5mm chloride and decreasing toward baseline levels thereafter. Scatchard analysis of this latter inhibitory effect of chloride indicates a competitive interaction with bumetanide in agreement with earlier indications that bumetanide inhibits Na/K/Cl cotransport at a chloride site. However, studies of the effects of various anions on bumetanide binding and²²Na transport show a poor correlation between the specificities of these two processes, suggesting that the inhibitory chloride site is not a chloride transport site.     

Na and Cl transport and short-circuit current in rabbit ileum

Summary Na and Cl fluxes and short-circuit current (I _sc) in rabbit ileum have been studied as a function of ionic concentrations in HCO₃-free solutions. Both net Na flux (J _net ^Na ) andI _sc show similar saturation functions of [Na] at fixed [Cl]. They show no significant difference between zero and 112mm Na but at 140mm NaI _sc is significantly greater than theJ _net ^Na . Net Cl transport, secretion, is observed only at 140mm Na and is approximately equivalent to the difference between theI _sc andJ _net ^Na . The transcellular mucosa-to-serosa Na fluxes measured at 140 and 70mm Na do not differ significantly from the correspondingI _sc. The net Cl flux varies with [Cl] at fixed [Na] whileI _sc is virtually not affected by [Cl]. These results suggest that the absorptive Na transport process is electrogenic and responsible for theI _sc and that the secretory fluxes of Na and Cl are coupled, require high [Na], vary with [Cl], and do not contribute toI _sc. K-free solution abolishes theI _sc after a prolonged lag. Finally, the effect of a low resistance shunt pathway on active Na absorption is examined with a four-compartment model.Deceased (October 16, 1974).     

Vanadate binding to the (Na + K)-ATPase

A particulate (Na + K)-ATPase preparation from dog kidney bound [⁴⁸V]-ortho-vanadate rapidly at 37°C through a divalent cation-dependent process. In the presence of 3 mM MgCl₂ theK _d was 96 nM; substituting MnCl₂ decreased theK _d to 12 nM but the maximal binding remained the same, 2.8 nmol per mg protein, consistent with 1 mol vanadate per functional enzyme complex. Adding KCl in the presence of MgCl₂ increased binding, with aK _0.5 for KCl near 0.5 mM; the increased binding was associated with a drop inK _d for vanadate to 11 nM but with no change in maximal binding. Adding NaCl in the presence of MgCl₂ decreased binding markedly, with anI ₅₀ for NaCl of 7 mM. However, in the presence of MnCl₂ neither KCl nor NaCl affected vanadate binding appreciably. Both the nonhydrolyzable, ,-imido analog of ATP and nitrophenyl phosphate, a substrate for the K-phosphatase reaction that this enzyme also catalyzes, decreased vanadate binding at concentrations consistent with their acting at the low-affinity substrate site of the enzyme; the presence of KCl increased the concentration of each required to decrease vanadate binding. Oligomycin decreased vanadate binding in the presence of MgCl₂, whereas dimethyl sulfoxide and ouabain increased it. With inside-out membrane vesicles from red blood cells vanadate inhibited both the K-phosphatase and (Na + K)-ATPase reactions; however, with the K-phosphatase reaction extravesicular K⁺ (corresponding to intracellular K⁺) both stimulated catalysis and augmented vanadate inhibition, whereas with the (Na + K)-ATPase reaction intravesicular K⁺ (corresponding to extracellular K⁺) both stimulated catalysis and augmented vanadate binding.