Omission Of Sodium Research Articles

Impaired rat sciatic nerve sodium-potassium adenosine triphosphatase in acute streptozocin diabetes and its correction by dietary myo-inositol supplementation.

Nerve conduction impairment in experimental diabetes has been empirically but not mechanistically linked to altered nerve myo-inositol metabolism. The phospholipid-dependent membrane-bound sodium-potassium ATPase provides a potential mechanism to relate defects in diabetic peripheral nerve myo-inositol-phospholipid metabolism, impulse conduction, and energy utilization. Therefore, the effect of streptozocin-induced diabetes mellitus and dietary myo-inositol supplementation on rat sciatic nerve sodium-potassium ATPase was studied. ATPase activity was measured enzymatically in sciatic nerve homogenates from 4-wk streptozocin diabetic rats and age-matched controls either fed a standard or 1% myo-inositol supplemented diet. The sodium-potassium ATPase components were assessed by ouabain inhibition or the omission of sodium and potassium ions. Diabetes reduced the composite ATPase activity recovered in crude homogenates of sciatic nerve. The 40% reduction in the sodium-potassium ATPase was selectively prevented by 1% myo-inositol supplementation (which preserved normal nerve conduction). Thus, in diabetic peripheral nerve, abnormal myo-inositol metabolism is associated with abnormal sodium-potassium ATPase activity. The mechanism of the effect of dietary myo-inositol to correct diabetic nerve conduction may be through changes in a sodium-potassium ATPase, possibly via changes in myo-inositol-containing phospholipids.

Na+-dependent uptake of 4-azidophenylalanine by pig intestinal microvillus vesicles. Interaction with neutral amino acid uptake and labelling pattern.

A procedure for the synthesis of tritiated 4-azidophenylalanine in a radioactively homogeneous form is described. The characteristics of the 4-azidophenylalanine uptake by pig intestinal microvillar vesicles and its interaction with the neutral amino acid transport have been studied. 4-Azidophenylalanine is transported into an osmotically sensitive space and the transport is sodium dependent. It is demonstrated that 4-azidophenylalanine transport is inhibited by L-alanine and L-phenylalanine but not by L-lysine and D-glucose. Likewise 4-azidophenylalanine inhibited the initial transport of L-alanine and L-phenylalanine but the initial transport of L-lysine and D-glucose was unaffected. Treatment of microvillar vesicles with 4-azidophenylalanine in combination with photolysis irreversibly inactivated the L-alanine transport. Photolabelling in the presence of sodium using tritiated 4-azidophenylalanine resulted in labelling of 10 peptides, all found in the fraction insoluble in Triton X-100. Omission of sodium from the reaction mixture or addition of L-alanine did not specifically abolish labelling in any of the labelled components.

Reabsorption of dicarboxylic acids from the proximal convolution of rat kidney.

The transport of dicarboxylic acid in the late proximal convolution was investigated by measuring the 3.5 s efflux of 2-oxoglutarate from the tubular lumen of rats starved for 3 days. The 3.5 s efflux of 2-oxoglutarate comprises two components, one due to movement across the brush border, obeying Michaelis Menten kinetics with an apparent Km of 0.13 mmol/l and a Jmax of 0.41 pmol cm-1 s-1 and the other due to diffusional movement presumably via the paracellular pathway with a permeability of 23.0 microns2 s-1. Omission of sodium from the perfusion fluid reduced the transcellular efflux of 2-oxoglutarate by 76%, indicating a sodium-dependent transport system. Addition of 5 mmol/l lithium to the liminal and capillary perfusate reduced it by 56% indicating a specific inhibitory effect of lithium on dicarboxylic acid transport. Addition of 5 mmol/l H2DIDS to the luminal perfusate reduced 3.5 s transcellular 2-oxoglutarate efflux by 35%. The molecular specificity of the system was assessed by studying the inhibitory effects of a series of dicarboxylates, both aliphatic and aromatic, on the 3.5 s efflux of 2-oxoglutarate. Inhibitory constants (apparent Ki) were calculated for comparative purposes assuming competitive inhibition. From this, the system was found to have optimal affinity for dicarboxylates in the trans-configuration with a four or five carbon chain (i.e. succinate and glutarate). Substitution on the 2-carbon atom with CH3-, OH-, SH-, and O = resulted in little reduction in inhibitory potency as compared to succinate itself. However, 2-substitution with NH3+ (not with N-acetyl) as well as 2,3-disubstitution with CH3-, OH- or SH-, strongly reduced or abolished the inhibitory potency. Only with the exception of pyruvate all monocarboxylates tested, did not inhibit 2-oxoglutarate transport, but all tricarboxylates tested, i.e. citrate, isocitrate and tricarballate had an inhibitory effect. Citrate inhibition was higher at acidic than alkaline pH. A number of aromatic compounds was also tested. In most cases the inhibitory potency of the aromatic compounds was considerably weaker than that of the effective 4-5 carbon chain aliphatic compounds. Only benzene-1,4-dicarboxylate, benzene-2-nitro-1,4-dicarboxylate, and benzene-1,2-diacetate had a high inhibitory potency. In the case of the aromatic dicarboxylates the most important feature relating molecular structure to transport was the distance between the two carboxyl-groups in the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

Cellular Mechanism of the furosemide sensitive transport system in the kidney.

Experiments were performed in the distal tubule of the doubly-perfused kidney of Amphiuma to determine active and passive forces, involved in the transport processes of potassium, sodium and chloride. Ion-sensitive microelectrodes and conventional microelectrodes were applied to estimate intracellular ion activities, cell membrane potentials and net flux of potassium and chloride under control conditions and during inhibition of active transport. Sodium chloride cotransport, located in the luminal cell membrane is postulated, based on the following observations: Total omission of sodium from the tubular lumen inhibits furosemide sensitive chloride reabsorption, decreases the lumen positive transepithelial potential difference and leads to a dramatic decrease of intracellular chloride. The experiments further suggest that potassium ions are involved in the sodium chloride transport system because potassium reabsorption is inhibited by furosemide and because intracellular sodium falls significantly when potassium ions are removed from the tubular fluid. Furthermore, there is experimental evidence that the luminal potassium uptake mechanism is suppressed after potassium adaptation. Under these conditions potassium transport is found to be insensitive to furosemide. The data suggest a furosemide sensitive cotransport system for sodium, chloride and potassium, operative in the luminal cell membrane. The energy for this carrier-mediated transport process is provided by the large "downhill" gradient of sodium across the luminal cell membrane which is maintained by the sodium pump located in the peritubular cell membrane.

Resolution of monoamine uptake into binding and translocation components

Kinetic analysis of the energy-independent accumulation of noradrenaline as determined by either sodium omission or osmotic lysis, revealed two components: a) a low affinity system which may represent extraneuronal uptake and b) a high affinity component which was characteristic of the physiological re-uptake process. A comparison between intact and lysed synaptosomes suggested that this high affinity system may represent binding to the carrier protein which was not dependent on external sodium. Qualitatively similar results were observed for serotonin. The results are discussed in relation to models of neuronal re-uptake.

The neuronal and extraneuronal distribution of 3H(-)-noradrenaline in the perfused rat heart.

1. After inhibition of both COMT and MAO, rat hearts were perfused with 1 μM 3H-(−)-noradrenaline for 30 min. Cocaine-sensitive neuronal and corticosterone-sensitive extraneuronal compartments were of about equal importance for the distribution of the amine into the heart. The absence of calcium from the perfusion fluid increased the axoplasmic and decreased the extraneuronal distribution of noradrenaline. 2. Under the same experimental conditions, hearts were washed out after the initial perfusion, and the efflux curves were subjected to compartmental analysis. For the nerve endings three distribution compartments were identified (the axoplasmic compartments B and C, characterized by half times of 8 and 45 min, respectively, as well as the vesicular “bound fraction”), while there was only one important extraneuronal distribution compartment (compartment C with a half time of about 45 min). 3. Efflux from the axoplasmic compartment C was accelerated by unlabelled noradrenaline, not affected by cocaine, reduced by lowering of the temperature from 37° to 27° C. Moreover, it was greatly accelerated by the omission of sodium from the wash-out solution. 4. Efflux from the extraneuronal compartment C, on the other hand, was not affected by the presence of unlabelled noradrenaline, and impaired by lowering of the temperature as well as by corticosterone. The omission of sodium from the wash-out solution caused an acceleration of the extraneuronal efflux which was significantly smaller than that of axoplasmic efflux. 5. Other hearts were perfused with either 1 or 16.7 μM 3H-(−)-noradrenaline (plus 30 μM cocaine) for 30 min and then washed out with amine-free solution; in these experiments either MAO or COMT was inhibited. When MAO was inhibited, the efflux curves for normetanephrine indicated that the accumulation of the amine in the extraneuronal compartment C can easily exceed that concentration which saturates the intracellular COMT. When MAO was not inhibited, afflux curves for DOPEG failed to provide any evidence for saturation of extraneuronal MAO. These results are in good agreement with earlier determinations of the kinetic constants of the “extraneuronal metabolizing systems” of the rat heart (Fiebig and Trendelenburg, 1978b). 6. The results indicate that extraneuronal mechanisms play an important role in the rat heart, and also that the extraneuronal compartment C (for noradrenaline) corresponds to the compartment III (for isoprenaline described by Bonisch et al. (1974).

The blocking effect of magnesium on the secretion of adrenal catecholamines induced by the omission of sodium from the extracellular medium.

The blocking action of Mg++ on catecholamine release induced by the substitution of extracellular Na+ by an osmotic equivalent amount of sucrose was studied in isolated, perfused bovine adrenal glands. Perfusing glands with 10 mM Mg++ produced at 51.1% inhibition on catecholamine release evoked by Na+ omission. Increasing the concentration of Mg++ to 20 mM this inhibitory effect was enhanced to 90.3%. D-600 (0.3 mM) promoted a marked blockade of acetylcholine-induced release of catechol hormones that was partially and significantly reverted increasing the concentration of Ca++ in the perfusion medium. D-600 (0.3 mM) failed to inhibit the catecholamine-releasing effect of Na+ deprivation. In adrenal glands previously perfused with D-600 (0.3 mM) and then exposed to a Locke solution containing D-600 (0.3 mM) + Mg++ (10 or 20 mM) the inhibition of the secretory responses evoked by the omission of Na+ was of the same magnitude as that obtained when the glands were perfused with Mg++ (10 or 20 mM) in the absence of D-600. These results are compatible with the view that the blocking effect of Mg++ may involve an intracellular site of action and that the access of Mg++ into the chromaffin cell may not be mediated through the Ca++ channels.

Noradrenaline transport by rat heart sympathetic nerves: a re-examination of the role of sodium ions.

The effect of sodium ion on 3H-(−)-noradrenaline (0.0875 to 0.5 μM) transport by rat heart atrial hemi-appendages incubated in vitro has been studied, and the following observations made: a) When sodium was omitted (choline and lithium substitution) there was no evidence for active noradrenaline transport, and only a component that did not show saturation kinetics up to 1 μM noradrenaline, remained. b) Omission of sodium or addition of 4×10−5 M desipramine inhibited noradrenaline transport to exactly the same extent, and their effects were not additive. Alprenolol did not reduce this sodium-independent transport, but tropolone lowered it somewhat. c) No evidence for corticosterone-sensitive noradrenaline transport (uptake-2) was found in this preparation at the low amine concentrations used. d) In control medium, the kinetic parameters of transport were: Km: 0.59 ± 0.063 μM and Vmax: 2.44 ± 0.43 (pmoles/mg protein/min). With 26 mM sodium and the rest substituted by choline, Km:2.26 ± 0.70 μM (P≤0.001) and Vmax: 2.74 ± 0.43 (pmoles/mg protein/min) (not significant). Also with 26 mM sodium, but with sucrose substitution, Km: 0.76 ± 0.13 μM (N.S.) and Vmax: 1.06 ± 0.13 (pmol/mg/min) (P<0.05). Such results indicate that sodium only modifies the affinity of the transport system for noradrenaline, without changing Vmax, and that changes in the latter are only a consequence of a reduction of the ionic strength. e) When noradrenaline transport was studied at different concentrations of external sodium, at constant ionic strength and with precautions to minimize the noradrenaline-releasing effect of low sodium, it was found that the data could be best represented by two hyperbolas placed in series. This suggests that the noradrenaline carrier has two sites for sodium, that do not interact with each other. When the same experiments were repeated in the absence of chloride, it was found that the noradrenaline transport system had lost virtually all its affinity for sodium. f) The effect of prolonged tissue incubation in the absence of sodium was found to produce a relatively small inactivation of noradrenaline transport. Such phenomenon was enhanced by raising the calcium concentration to 2 mM.

Uptake and metabolism of cyclic AMP in rabbit choroid plexus in vitro

The uptake and fate of labelled cyclic nucleotides in isolated rabbit choroid plexus were studied in vitro. Radioactivity accumulated almost linearily for 240 min, reaching tissue/medium ratios of 89 ± 2.4 for cyclic AMP and 83 ± 2.5 for cyclic GMP. The uptake process was saturable at substrate concentrations ranging from 1.0 μ m to 10 m m. The uptake of [ 3H]-cyclic AMP was significantly inhibited by lowering of temperature, anoxia, ouabain and omission of sodium as well as by cyclic GMP, 5'-AMP, probenecid and several other organic acids but not by neutral or basic compounds. Thin layer Chromatographie identification of the intracellular radioactivity revealed that after 10 min incubation less than 10% was attributable to cyclic AMP itself, whereas about 45% was associated with ATP + ADP and 2% with the acid insoluble fraction of the choroid plexus. Inhibition of phosphodiesterase activity by theophylline increased the intracellular cyclic AMP fraction of total radioactivity accumulated, but decreased the tissue/medium ratio by about two thirds. The finding of a cyclic AMP generating system sensitive to histamine and prostaglandin E 1, but not to noradrenaline, in the choroid plexus suggests a functional role of cyclic AMP in this tissue.

Amino acid transport by two glial cell lines and by proliferating glia

Glial membrane function was evaluated by investigating the transport of 12 amino acids by two glial cell lines in culture and in a model of glial proliferation. C-6 and C-2 1 glia had very similar profiles of amino acid accumulation. Uptake velocities were greatest for glutamic acid and least for γ-aminobutyric acid in both cell types. Culture of C-6 glia in higher concentrations of fetal bovine serum resulted in altered cell and culture morphology, glial proliferation, and selective increases in amino acid uptake. Leucine, phenylalanine, and valine uptake velocities increased fivefold. Uptake of aspartic and glutamic acids, lysine, and serine more than doubled, whereas that of the other amino acids showed no change. Accumulation of amino acids into C-6 glia was saturable and temperature sensitive. The individual transports were distinct in their response to substrate inhibition and to sodium omission. Uptake of the putative neurotransmitters did not manifest the marked sodium dependence found in central nervous system preparations. We conclude that glial cells have characteristic membrane processes for amino acid transport which can interact with the microenvironment. These systems undergo profound but selective changes during glial proliferation induced by serum. Similar alterations may occur during gliosis, resulting in disturbance of the microenvironment and neural function.

Hereditary variation in uric acid transport by avian kidney slices.

Uric acid transport in renal cortical slices from a selected line of hyperuricemic chickens was investigated. Slices from the hyperuricemic (HUA) line accumulated less than half as much uric acid as slices from a control (LUA) line when uric acid in the medium varied from 0.01 to 5 mM. Uric acid uptake by both lines increased as the uric acid concentration in the medium was raised from 0.1 to 0.5 mM, but was markedly inhibited in the HUA line at 3-5 mM. Omission of sodium or potassium from the incubation medium inhibited uric acid uptake by slices from both lines. Ouabain inhibited uric acid uptake in the LUA line. The sodium and potassium requirements for initiation of uric acid uptake were higher, and the potassium requirement for maximal uptake was lower, for slices of the HUA line. No genetic differences in potassium or sodium contents of slices were observed when the potassium content of the incubation medium was altered or when the medium contained ouabain. These studies indicate that hereditary hyperuricemia in chickens may be due to a qualitative change in renal uric acid transport which involves the interaction of cations in the transport process.

The different effects of D-600 (methoxyverapamil) on the release of adrenal catecholamines induced by acetylcholine, high potassium or sodium deprivation.

1 Bovine adrenal glands were perfused with Locke solution and catecholamine release was induced by acetylcholine, by a depolarizing concentration of potassium, or by omission of sodium from the perfusion fluid. 2 D-600 (methoxyverapamil) at the concentration of 30 muM produced a 23% inhibition of catecholamine release evoked by acetylcholine (0.1 mM) in the presence of physostigmine (10 muM). 3 A concentration of D-600 of 0.3 mM produced 86% and 85% inhibition in the output of catecholamines in response to acetylcholine and high potassium respectively. 4 D-600 (0.3 mM) failed to block the release of catecholamines evoked by sodium deprivation. 5 The results suggest the involvement of intracellular calcium in the exocytotic release of catecholamines induced by sodium omission.

Chloride-dependent transmural potential in the rectal wall of Schistocerca gregaria

Rectum transmural potential (PD) and short-circuit current ( I sc) of the desert locust, Schistocerca gregaria, have been studied in vitro, with everted rectal wall preparations in solutions of different ionic composition. Initially, a PD of about 35 mV (lumen positive) and a I sc of about 300 μA cm −2 were recorded. Omission of sodium or potassium (Tris as substitute), from the luminal side or from both sides led to an increase of 4 to 6 mV in PD (lumen more positive) together with an increase in I sc. In the absence of chloride alone (sulphate as substitute) the PD quickly dropped to nearly zero. In each case the control values were recovered on replacing the corresponding ions. Neither the PD nor the I sc changed when substitutions affected only the haemocoelic solution. These findings corroborate the assumption that active transport of chloride ions from lumen to haemolymph is the major factor for transmural PD and account for the short-circuit current in the rectal wall of desert locust. A working scheme is given to explain the influence of sodium, potassium, and chloride ions on the PD.

Transport and oxidation of glycine in mammalian pancreatic islets with reference to the mechanism of amino acid-induced insulin release.

Microdissected pancreatic islets of obese-hyperglycemic mice were used for studying the uptake, oxidation and insulin-releasing ability of glycine. Glycine did not influence insulin release either in the presence or absence of glucose. It was rapidly taken up by the islet cells, reaching distribution ratios much higher than unity. This concentrative uptake was depressed by the omission of sodium from the medium. L-Alanine, L-leucine, D-glucose, or glibenclamide also reduced the glycine uptake, whereas diazoxide had no effect. The production of radioactive CO&lt;sub&gt;2&lt;/sub&gt; from uniformly &lt;sup&gt;14&lt;/sup&gt;C-labeled glycine remained insignificant even when tested at concentrations as high as 20 mM. It is suggested that the &lt;i&gt;β&lt;/i&gt;-cells transport glycine by the alanine-preferring A-system. The data are consistent with our previous concept that this system does not represent a trigger site for insulin release from mature mouse-&lt;i&gt;β&lt;/i&gt;-cells. Glycine appears to be of minor importance as a fuel for the &lt;i&gt;β&lt;/i&gt;-cells.

Transport of -aminoisobutyric acid in mammalian pancretic -cells.

Transport of α-aminoisobutyric acid (AIB) was studied in microdissected pancreatic islets of obesehyperglycaemic mice. These islets are characterized by an unusually high proportion of insulin-secretingβ-cells. The following main observations were made. 1. The islet cells exhibited a concentrative uptake of AIB, yielding distribution ratios much higher than unity. The uptake was depressed by anoxia and by the omission of sodium from the medium. 2. L-Alanine and L-methionine, but not L-leucine, reduced the accumulation of AIB in islets which had been preincubated in the absence of exogenous amino acid. After preloading with L-alanine there was an enhanced uptake of AIB. 3. D-Glucose, D-galactose, diazoxide, dibutyryl cyclic 3′,5′-AMP, and D-mannoheptulose had minor or insignificant effects on the uptake of AIB. 4. Efflux of AIB from the islet cells was stimulated by exogenous AIB or L-alanine. It is concluded that the pancreaticβ-cells contain a mechanism for transport of neutral amino acids similar to the A-system of many other cells. The stimulating effect of glucose on insulin biosynthesis does not seem to be mediated by this system. Co-transport of amino acid and sodium is probably not enough to elicit a discharge of insulin from matureβ-cells.

Effect of changes in ionic environment on the action of bradykinin on the guinea-pig taenia coli

The effect of changes in the concentration of sodium and calcium in the medium and the effect of adrenalin on the mechanical and electrical responses of the guinea-pig taenia coli to bradykinin were investigated using an organ bath and a sucrosce-gap method. In normal Krebs solution the response to bradykinin was mostly biphasic: an acute initial phase characterized by spike inhibition and decreased isometric tension, followed by a second phase with an increased frequency and amplitude of the spikes and membrane depolarization, accompanied by an increase in isometric tension. Omission of sodium at normal or twice the normal calcium concentration strenghtened and lengthened the initial phase and supressed the second phase. Lowering the calcium concentration anolished the initial phase of bradykinin, while the second phase, especially the increased electrical activity, was enhanced. After lowering both the calcium and the sodium concentration the spontaneous spike activity ceased almost immediately. Under these conditions bradykinin only caused slight hyperpolarization. The effect of substituting strontium for calcium was similar to that of lowering the calcium concentration. However, after a long period of exposure to strontium the intial phase of the response to bradykinin reappeared and was enhanced by doubling the strontium concentration. After a preceding dose of adrenalin, 5 μg, the initial phase of the bradykinin effect was lengthened, while the second phase was not significantly affected. It is suggested that bradykinin acts no more than one site at the cell membrane. The spike inhibition in the initial phase may be explained if it is assumed that bradykinin prevents the release of calcium superficially bound to the cell membrane and involved in the spike generation. Spike stimulation accompanied by depolarization in the second phase is thought to be due to an action of bradykinin at another site in the membrane.

Amino Acid Transport in Kidney: HETEROGENEITY OF α-AMINOISOBUTYRIC UPTAKE

Abstract 1. More than one mode of transport can be observed for concentrative uptake of α-aminoisobutyric acid (AIB) by rat kidney cortex slices when the initial concentration of AIB in the medium is varied between 0.2 and 16 mm. 2. The relative contribution from both systems to the total observed uptake at various concentrations of AIB was calculated. The Km values for the two systems were 3.87 mm and 24.8 mm. 3. Sodium-dependent inhibitory reactions influence the uptake of 0.8 mm AIB more than of 8.0 mm AIB; inhibitors affecting —SH groups have the opposite effect. 4. l-Alanine is an effective competitive inhibitor of AIB transport at high substrate concentrations; more than one Ki value for l-alanine can be derived depending on the concentration of AIB. External l-proline enhances the affinity of AIB for uptake at low substrate concentrations; an inhibitory effect is observed at high substrate concentrations. Although omission of sodium from the medium abolishes concentrative uptake of AIB at all concentrations, it is restored at low substrate concentration by the addition of 4 mm l-proline to the external sodium-free medium. l-Proline placed internally stimulates AIB accumulation at low substrate concentrations only in the presence of sodium. 5. A site at which a ternary complex between proline or sodium and the AIB-carrier complex may be formed is proposed for AIB transport at low concentrations; another system with different properties is used predominantly for AIB transport at high concentrations.

Hexose and Glycerol Absorption by Some Trypanosomatidae

SYNOPSIS. Culture forms of 9 species of Trypanosomatidae were studied. Of these, Crithidia fasciculata, Leishmania brasiliensis, L. donovani, L. tropica, Trypanosoma conorhini, T. cruzi and T. rangeli consumed significantly less glycerol than glucose, both when the 2 substrates were offered singly or simultaneously. On the other hand, T. gambiense and T. rhodesiense consumed as much glycerol as glucose when the 2 substrates were given separately. When both substrates were offered simultaneously, more glycerol than glucose was consumed, but the sum of glucose + glycerol carbon taken up approximated closely that taken from glucose alone. This held for species consuming only little glycerol, indicating that in all cases some mutual inhibition took place. Atttempts to adapt T. cruzi to glycerol consumption were unsuccessful. When T. gambiense and T. rhosiense were cultivated in the presence of glycerol, their normrapid glycerol consumption remained also unchanged but, curiothey consumed more glucose than before “adaptation.” Iodoacetami and NaF strongly inhibited glucose and glycerol absorption of 7 gambiense; phloridzin, deoxygalactose and deoxyglucose gave nonificant inhibition. KCN slightly stimulated glucose absorption, weakly inhibited glycerol absorption. Omission of sodium from medium was without effect, while lack of CO2 markedly inhiglucose and glycerol uptake. Galactose was not a satisfactory sub strate for T. gambiense and T. rhodesiense, nor were deoxygalactos and deoxyglucose. These 3 hexoses were consumed in small amonly and did not allow maintenance of motility.

Effects of ouabain, glutamate and cations on phosphate incorporation in brain slices

Guinea-pig brain cortex slices were incubated in media in which the concentrations of sodium, potassium, calcium, glutamate and ouabain were varied, and the absolute levels and rate of phosphate incorporation into ATP and 5 acid-insoluble phosphate fractions were measured. The level of inorganic phosphate and of the acid-insoluble phosphate fractions, except for phosphoprotein, did not vary significantly under different conditions. ATP levels were lowered by ouabain, glutamate or omission of sodium from the medium. The rate of phosphate incorporation into all acid-insoluble phosphate fractions was lowered by 75% when sodium was replaced by choline and by 60% by glutamate. Incorporation was stimulated by 35% by the omission of calcium from the medium and by up to 20% on raising the potassium concentration from 2.5 to 7.5 mM, except for phospholipid, incorporation into which was reduced by 35% by raised potassium. Ouabain at 10 μM caused a 35% inhibition of incorporation in the sodium medium in both rat and guinea-pig cortex slices. Lower concentrations of ouabain gave smaller inhibitions. The rise in incorporation caused by omission of calcium and the fall caused by glutamate, were abolished by ouabain. The fall in incorporation into phospholipid, and the rise in all other acid-insoluble fractions, caused by raised potassium, were both reversed by ouabain. All effects of glutamate, calcium and ouabain were abolished when sodium was replaced by choline.