Metabolic factors influencing the sodium and potassium distribution in Ulva lactuca

1. Methods for the use of the marine green alga, Ulva lactuca, in studies on electrolyte metabolism are described. 2. The effect of illumination and iodoacetate on the potassium and sodium content, as well as the influence of light and running sea water on the iodoacetate effect was investigated. The rate of exchange of cellular potassium ion for K(42) under conditions of light and dark at 20 and 30 degrees C. was studied. 3. Ulva maintained in the dark for long periods loses some potassium and gains sodium, both effects being reversed upon illumination. The presence of 0.001 M iodoacetate in the dark causes a marked progressive loss of potassium and gain of sodium, phenomena which do not occur when the alga is illuminated. Evidence for the penetration of the inhibitor into the cell in the presence of light is presented. The iodoacetate effect on potassium and sodium content, once established, can be "washed out" of the alga when the plant is placed in light and running sea water without the inhibitor. Illumination and increased temperature each favor a more rapid exchange of tissue for environmental potassium ion. 4. In the interpretation of these findings it is emphasized that metabolic work, perhaps in the form of ion transports, must be done by the cell to compensate for the continual flow of potassium ion and sodium ion with their respective concentration gradients and thus maintain homeostasis within the cell. Evidence is presented which indicates separate mechanisms for the distribution of sodium and potassium in this organism. It is further suggested that the degradation of phosphoglyceric acid, an important glycolytic and photosynthetic intermediate, or one of the products of its metabolism supplied the energy for these ion transports(s). The role of permeability per se is considered.     

Sodium exclusion and potassium retention by the red marine alga,Porphyra perforata

Cells of the red marine alga, Porphyra perforata, accumulate potassium and exclude sodium, chloride, and calcium. Various metabolic inhibitors including dinitrophenol, anoxia, and p-chloromercuribenzoate partially abolish the cells' ability to retain potassium and exclude sodium. Iodoacetate induces potassium loss only in the dark; reduced sulfur compounds offer protection against the effects of p-chloromercuribenzoate; dinitrophenol stimulates respiration at concentrations which cause potassium loss and sodium gain. Following exposure to anoxia potassium accumulation and sodium extrusion take place against concentration gradients. These movements are retarded by sodium cyanide, but are stimulated by light. Sodium entry, following long exposure to 0.6 M sucrose, occurs rapidly with the concentration gradient, while potassium entry against the concentration gradient takes place slowly, and is prevented by cyanide.     

Microelectrode study of K+ accumulation by tight epithelia: II. Effect of inhibiting transepithelial Na+ transport on reaccumulation following depletion

The effects of restoring serosal potassium to potassium-depleted toad urinary bladders have been re-examined using double-barrelled microelectrodes. The data confirm the existence of a time-lag phenomenon, a dissociation between potassium reaccumulation and restoration of short-circuit current. Returning serosal potassium stimulates an increase in intracellular potassium activity 21-26 min before any increase can be detected in short-circuit current. The reaccumulation of potassium has been further studied using split frog skin, a far more suitable preparation for electrophysiologic study than toad bladder. Under baseline short-circuited conditions, potassium is accumulated against an electrochemical gradient of 22 +/- 4 mV. Reaccumulation of potassium by potassium-depleted tissues can be blocked by inhibiting the Na,K-exchange pump with high concentrations of ouabain. On the other hand, blocking apical sodium entry by the addition of 10(-4) M amiloride to the outer bathing medium does not interfere with reaccumulation of potassium. The data support the concept that the time-lag phenomenon of toad bladder reflects stimulation of potassium reaccumulation by the sodium pump in exchange for the extrusion of excess cell sodium collected during the period of potassium depletion. This reaccumulation of potassium can proceed before the entry of significant added amounts of sodium across the apical plasma membrane.     

Potassium reaccumulation by isolated frog epidermis

The involvement of potassium in transepithelial sodium transport was tested by studying net potassium reuptake by potassium-depleted frog skin epidermis. Normal potassium content in half-strength Ringer's (0.244 μequiv/mg dry weight) fell 43% after 16 h in K-free medium at 5°C. Reaccumulation, against an electrochemical potential gradient, to 83% of the initial tissue potassium content occurred following incubation for 4 h at 22°C in K-containing medium. Sodium was required in the solution bathing the inside, but not the outside surface of the skin, for net potassium reaccumulation. Ouabain caused an additional potassium loss from potassium-depleted epidermis, but did not have the same effect on potassium-depleted isolatedcells. Procaine, lithium and caffeine completely inhibited, antidiuretic hormone and cyclic AMP may partially inhibit and amiloride had no effect on potassium reaccumulation. In many cases decreases in sodium and water content were found to occur even in the absence of net potassium reaccumulation. The results suggest (1) potassium is actively transported into the epidermis, (2) this transport is not rigidly coupled to sodium extrusion or water loss, (3) potassium uptake is not rigidly coupled to transepithelial sodium transport, or only a small fraction is involved, (4) potassium diffusion is restricted in the extracellular space.     

Studies on the sodium and potassium transport in rabbit polymorphonuclear leukocytes

Rabbit polymorphonuclear leukocytes obtained from peritoneal exudates, incubated at 37°C. following exposure to 4°C., actively reaccumulate potassium while little or no net extrusion of sodium takes place. Preventing the utilization of oxidative metabolism with potassium cyanide, 2,4-dinitrophenol, or a nitrogen atmosphere does not inhibit the recovery process. Inhibitors blocking anaerobic glycolysis (sodium iodoacetate and sodium fluoride in low concentrations) completely abolish the capacity to reaccumulate potassium and cause a further dissipation of the sodium and potassium gradients. Water movements have been shown to be secondary to cation shifts. It is postulated that separate transport mechanisms exist for sodium and potassium and that the process of potassium reaccumulation relies on anaerobic glycolysis as a source of energy.     

Hepatocytes volume regulation

Cell volume regulation has been studied during isolated dog liver perfusion. In presence of ouabain (10(-4) M) rapid but quantitatively matched exchange of K for Na occurs and the cellular volume is maintained until (90 min later) intracellular K concentration falls below 80 mEq/litre. Additional mechanism of protection of cell volume as loss of intracellular anions should also play a r?le since ouabain produces rapidly a membrane depolarization and chloride gain. A similar sequence of events is obtained when inhibition of the sodium pump is produced by anoxia but in this case the chloride gain in excess of cation gain is particularly marked. Submitted to an hypotonic shock the hepatocytes swell but tend to partially recover their volume by loosing K, indeed when osmolarity is corrected the cells maintain a sub-normal volume. Ouabain inhibits (or masks?) this iso-osmotic regulation. When submitted to an hypertonic medium a reduced cell volume is obtained and maintained for hours even in presence of ouabain, which produces a Na/K exchange at the same rate as in normal conditions.     

Circadian Rhythms of Plasma Concentrations of Na+ and K+ and Their Urinary Excretion in Normal and Diabetic Rats

The effects of streptozotocin induced diabetes (50 mg/Kg) on the circadian rhythms in the excretion of sodium and potassium as well as their plasma concentration rhythms were investigated. Control (C) and diabetic (D) rats were studied during a light-dark (12h:12h) cycle and fed ad libitum. Statistically significant circadian rhythms were found for sodium and potassium excretion in C rats. The orthophases of both rhythms occurred in the dark phase, the potassium one occurring before that of sodium. In D rats there is increased excretion of both sodium and potassium with the rhythmicity maintained for sodium excretion only, which has an earlier orthophase than in the C rats. Plasma sodium and potassium concentrations showed a statistically significant circadian pattern in C rats, with orthophase in the light phase. This rhythmicity only appears in plasma potassium concentration for D rats, with orthophase at the end of the dark phase. The results in diabetic rats may suggest that the glomerular filtration rate (GFR) and/or tubular reabsorption rhythms are still contributing to the sodium excretory rhythm, and that the loss of the circadian rhythm in sodium plasma concentration has no influence on the sodium excretion rhythm. Nevertheless, the loss of the potassium excretion rhythm may suggest a disruption of the variations in the secretory process, as this excretion seems to be independent of the plasma potassium concentration rhythm, which is not lost in D rats.     

Circadian Rhythms of Plasma Concentrations of Na+ and K+ and Their Urinary Excretion in Normal and Diabetic Rats

The effects of streptozotocin induced diabetes (50 mg/Kg) on the circadian rhythms in the excretion of sodium and potassium as well as their plasma concentration rhythms were investigated. Control (C) and diabetic (D) rats were studied during a light-dark (12h:12h) cycle and fed ad libitum. Statistically significant circadian rhythms were found for sodium and potassium excretion in C rats. The orthophases of both rhythms occurred in the dark phase, the potassium one occurring before that of sodium. In D rats there is increased excretion of both sodium and potassium with the rhythmicity maintained for sodium excretion only, which has an earlier orthophase than in the C rats. Plasma sodium and potassium concentrations showed a statistically significant circadian pattern in C rats, with orthophase in the light phase. This rhythmicity only appears in plasma potassium concentration for D rats, with orthophase at the end of the dark phase. The results in diabetic rats may suggest that the glomerular filtration rate (GFR) and/or tubular reabsorption rhythms are still contributing to the sodium excretory rhythm, and that the loss of the circadian rhythm in sodium plasma concentration has no influence on the sodium excretion rhythm. Nevertheless, the loss of the potassium excretion rhythm may suggest a disruption of the variations in the secretory process, as this excretion seems to be independent of the plasma potassium concentration rhythm, which is not lost in D rats.     

An analysis of physiological states responsible for antero-posterior disintegration in Planaria dorotocephala

Summary Planaria were treated with equi-molal solutions of ammonium, potassium, sodium, magnesium, and calcium chlorides, made up in distilled water and the rates of cytolysis compared with cytolysis in distilled water. Potassium and ammonium accelerate cytolysis; some protection is afforded by sodium; still more by magnesium, and complete protection by calcium in the concentrations employed.In distilled water solutions of calcium chloride no cytolysis occurs in concentrations from M/500 to M/40,000; cytolysis is distinctly delayed in M/100,000. The protective action of M/1,000,000 is detectable.Potassium oxalate accelerates disintegration in hypotonic solutions.One per cent ethyl alcohol in distilled water causes cytolysis more rapidly than does distilled water alone, but in M/500 molal calcium chloride the alcohol solution is much less effective.Ringer's solution minus calcium affords no protection against death due to absence of calcium and death due to potassium oxalate but completely protects against cytolysis. Death in Ringer's solution minus calcium and in Ringer's solution with potassium oxalate occurs first in the anterior region and describes an antero-posterior gradient.Cytolysis in distilled water, in potassium oxalate solutions, in alcohol solutions, and in hypotonic calcium solutions of extreme dilution is initiated in the anterior end and describes an antero-posterior gradient within a zooid.Earlier work of the writer on the disintegrative action of lipoid solvents, heat, KNC, hyper- and hypotonic solutions is discussed. It is concluded that inPlanaria dorotocephala the antero-posterior gradient in cytolytic disintegration represents an antero-posterior differential in sensitivity to disturbance of the calcium-lipoid-water relation in the organism.     

Potassium-dependent sodium extrusion by cells of Porphyra perforata,a red marine alga

Potassium-free artificial sea water causes a loss of cell potassium and a gain of cell sodium in Porphyra perforata, which is not attributable to an inhibition of respiration. On adding KCl or RbCl to such low potassium, high sodium tissues, net accumulation of potassium or rubidium takes place, accompanied by net extrusion of sodium. Rates of potassium or rubidium accumulation and sodium extrusion are proportional to the amount of KCl or RbCl added only at low concentrations. Saturation of rates is evident at KCl or RbCl concentrations above 20-30 mM, suggesting the role of an ion carrier mechanism of transport. Evidence for and against mutually dependent sodium extrusion and potassium or rubidium accumulation is discussed.     

Lung liquid production by in vitro lungs from fetal guinea pigs: studies with metabolic inhibitors.

Lungs from fetal guinea pigs (62 +/- 2 days of gestation) were supported in vitro for 3 h, and lung liquid production was measured by dye dilution. Eighteen untreated preparations produced fluid at 1.76 +/- 0.30 mL.kg-1 body weight.h-1 during the first hour, with no significant changes in later hours. When inhibitors of respiratory processes were placed in the outer saline during the middle hour, production changed significantly, as follows: (a) sodium iodoacetate at 10(-3) M stopped production (87.2 +/- 10.3 and 100% reductions, successive hours; n = 6), at 10(-4) M it reduced production (60.0 +/- 10.3 and 63.4 +/- 9.3% reduction, successive hours; n = 12); (b) sodium fluoride, 10(-3) M, almost stopped production (93.2 +/- 12.1 and 89.5 +/- 9.3% reductions, successive hours; n = 6); (c) sodium cyanide at high concentration (10(-3) M) reduced production slowly (35.5 +/- 12.3 and 73.1 +/- 22.4%; successive hours; n = 6); (d) sodium azide, 10(-3) M, also reduced production (67.6 +/- 14.2 and 59.7 +/- 14.0%, successive hours; n = 6); total lactate lost rose 1.8 +/- 0.5 fold; (e) dinitrophenol produced strong reabsorptions; at 10(-3) M, production fell 115.4 +/- 15.9 and 113.1 +/- 47.3%, successive hours (n = 4), and at 2 x 10(-4) M it fell 143.8 +/- 33.8 and 153.4 +/- 26.7%, successive hours (n = 6); total lactate lost rose 2- to 3-fold. Control preparations showed no significant changes. The results suggest that lung liquid production requires glycolysis and aerobic metabolism. However, reabsorption appears to continue on glycolysis alone, a particularly useful situation for neonates suffering respiratory distress.     

Diurnal Rhythms of Plasma Ions and Metabolites Levels in Thoroughbred Racehorses

The purposes of this study were to observe the presence of diurnal rhythms in plasma ions and metabolites levels in Thoroughbred racehorses under physical training, and to determine the time of blood sampling in clinical investigations. Plasma calcium, phosphorus, potassium, sodium, chloride, magnesium, iron, glucose, cholesterol, triglycerides, and total proteins levels were studied over a 72-h period. Blood samples were taken every 4 hours from five male and five female Thoroughbred racehorses under physical training. COSINOR analyses (P = 0.05) were done. Plasma potassium and triglycerides showed significant diurnal rhythms, with its acrophases occurring at dark period. No significant diurnal rhythms of other variables were found. It was concluded that, in Thoroughbred racehorses, the optimum time for potassium, and triglycerides sampling seems to be light period. And for other variables, time of diagnosis is not important.     

Diurnal Rhythms of Plasma Ions and Metabolites Levels in Thoroughbred Racehorses

The purposes of this study were to observe the presence of diurnal rhythms in plasma ions and metabolites levels in Thoroughbred racehorses under physical training, and to determine the time of blood sampling in clinical investigations. Plasma calcium, phosphorus, potassium, sodium, chloride, magnesium, iron, glucose, cholesterol, triglycerides, and total proteins levels were studied over a 72-h period. Blood samples were taken every 4 hours from five male and five female Thoroughbred racehorses under physical training. COSINOR analyses (P = 0.05) were done. Plasma potassium and triglycerides showed significant diurnal rhythms, with its acrophases occurring at dark period. No significant diurnal rhythms of other variables were found. It was concluded that, in Thoroughbred racehorses, the optimum time for potassium, and triglycerides sampling seems to be light period. And for other variables, time of diagnosis is not important.     

Effect of ethylenediamine-tetra-acetate, sodium, iodoacetate and potassium cyanide on the release of cobalophilins from polymorphonuclear granulocytes during phagocytosis

The influence of ethylenediamine-tetra-acetate (EDTA, 10(-3) M, 10(-5) M and 10(-7) M), sodium iodoacetate (CH2 . I. COONa, 10(-4) M and 10(-6) M) and potassium cyanide (KCN, 10(-2) M, 10(-3) M and 10(-5) M) on the release of cobalophilins (vitamin B12 binding proteins) from polymorphonuclear granulocoytes (PMN) was studied. The agents mentioned above reduced the release of cobalophilins from resting and functionally stimulated granulocytes. This effect increased with the growth of concentration of these agents in the sample. The inhibitory effect of EDTA, CH2 . I. COONa and KCN on phagocytosis-activated cobalophilins release occurred irrespective of the time of granulocytes stimulation. This could be observed in these experiments, where granulocytes were first affected by these chemical agents and then stimulated functionally, as well as in those samples where EDTA, CH2 . I . COONa, and KCN influenced the cells after incubation with latex particles. The inhibitory effect of EDTA was diminished in the presence of a higher concentration of calcium ions in an incubation medium. On the contrary, CH2 . I . COONa reduced the release of cobalophilins from PMN during phagocytosis irrespective of the concentration of calcium ions in the medium.     

Effect of fluoride and other metabolic inhibitors on intracellular sodium and potassium concentrations in L cells

Fluoride, iodoacetate, oxamate, 2,4-dinitrophenol, cycloheximide, and ouabain were studied to determine if any of these inhibitors affected the intracellular concentration of sodium and potassium in an L cell strain of mouse fibroblasts and to determine if the changes observed in these parameters could be correlated with growth rate. The results indicated that (1) the intracellular concentration of sodium and potassium could not be correlated with growth rate, (2) fluoride, iodoacetate, oxamate, 2,4-dinitrophenol, and cycloheximide at concentrations having an equal effect on growth had a similar effect on the intracellular sodium and potassium concentration These changes were not as great as those seen with ouabain, which at a concentration which did not inhibit growth, had an equal or greater effect on the intracellular sodium and potassium concentration.     

The cytoprotective properties of prostaglandin E2 against the toxic effects of actinomycin C on embryonic neural retina cells.

Prostaglandins (PGs) have been shown to cytoprotect various tissue types against the toxic effects of many chemicals. The mechanism of this protection is poorly understood, but the involvement of cAMP is often implied. Only one previous study examined nervous tissue and PG protection. The present study was designed to determine if PGE2 affords cytoprotection to a more specific nervous tissue (embryonic neural retina) from the toxicity of actinomycin C (AMC) using a trypan blue exclusion assay. The lowest concentration of PGE2 (2 x 10(-5)M) had no effect, but as the concentration increased (3 x 10(-5)M and 5 x 10(-5)M), PGE2 did afford protection against AMC in a dose dependent fashion. Theophylline treated cells were not protected, suggesting that cAMP may not be the primary mechanism of protection.     

Delayed Neuronal Death After Brief Histotoxic Hypoxia In Vitro

Abstract: The effect of three metabolic inhibitors—iodoacetate, potassium cyanide, and potassium arsenate—on neuronal viability was studied in primary rat cortical and hippocampal CA1 neuronal cultures. Iodoacetate (0.1 m M ) applied for 5 min to 8-day-old cultures resulted in delayed neuronal death within 3–24 h in cortical and hippocampal CA1 neurons. Neuronal degeneration was preceded by transient inhibition of energy metabolism to ∼40% and a permanent inhibition of protein synthesis to ∼50%. The inhibition of protein synthesis and the neuronal death were prevented by the free radical scavenger vitamin E but not by the glutamate antagonist MK-801. Removal of calcium during iodoacetate exposure could not protect against toxicity, and there was no increase of intracellular calcium concentration during and shortly after iodoacetate treatment. Cyanide and arsenate produced only partial neuronal degeneration, even at a dose of 10 m M . These observations demonstrate that brief exposure of neurons to low concentrations of iodoacetate produces a delayed type of neuronal death that is not mediated by either calcium or glutamate. The therapeutic effect of vitamin E points to a free-radical mediated injury and suggests that this type of pathology may also be involved in delayed neuronal death after transient energy depletion in vivo.     

The Photometabolism of Acetate by Chlorella pyrenoidosa

Chlorella pyrenoidosa can utilize sodium acetate as a carbonsource for growth in the light. Growth proceeds under aerobicconditions both in the presence and in the absence of carbondioxide, but under anaerobic conditions only in its presence.The assimilation of acetate does not result from oxidation tocarbon dioxide followed by photosynthetic fixation because theproducts of ¹⁴C-acetate assimilation are different from theproducts of ¹⁴CO₂ fixation in the presence of unlabelled acetate. In aerobic conditions 10-⁶ M DCMU induces a pattern of acetateassimilation in the light similar to that in the dark. Thus,in the presence of DCMU in the light, less acetate carbon isincorporated into cells, particularly into lipids, polysaccharide,and protein, and more is released as carbon dioxide than inits absence. The effect of 4 x 10^-3 M MFA on acetate assimilationin the presence of 10^-6 M DCMU is the same in light and dark.Acetate assimilation is unaffected by desaspidine and sodiumbisulphite. The mean generation time of C. pyrenoidosa growing on acetatein the light under aerobic conditions is 20 hours. When 10^-5M DCMU is added the mean generation time is 60 hours, the sameas that for Chlorella growing on acetate in the dark. The activityof the enzymes of the glyoxylate cycle, isocitrate lyase (E.C.4.1.3.1.)and malate synthetase (E.C.4.1.3.2.) is repressed in the light,but activity of both enzymes increases markedly when DCMU isadded.     

SODIUM AND CHLORIDE FLUXES IN SYNAPTOSOMES IN VITRO

Synaptosomes incubated in a physiological saline extrude sodium and take up potassium. As would be expected this process is completely blocked by metabolic inhibitors such as cyanide and iodoacetate. However, when metabolic inhibitors are replaced by ouabain (100 μM) there is an increase in the steady state intrasynaptosomal sodium and chloride content even though there is no change in the potassium content. The increases are prevented when synaptosomes are incubated with metabolic inhibitors in addition to ouabain. There is therefore a ouabain-insensitive process that transports sodium, chloride and concomitantly water into synaptosomes. It appears not to function when the supply of metabolic energy is inhibited. The diuretic furosemide (1 mM) in the presence of ouabain inhibits the entry of sodium and chloride without affecting the intrasynaptosomal potassium concentration. Ethacrynic acid (1 mM) has a somewhat similar effect but in addition appears to damage the synaptosome membrane. Kinetic measurements were made of the uptake of sodium, potassium and chloride under conditions of metabolic inhibition and the permeability constants of the membrane determined. Values of 0.068, 0.117 and 0.032 × 10^-6 (cm s^-1) were found for the permeability constants of the membrane to (respectively) sodium, potassium and chloride. Measurements of the rate of uptake in the presence of ouabain revealed an inwardly directed sodium and chloride flux of 5-20 pmol cm^-2 s^-1. Calculation of the fluxes from the steady state ion concentrations also reveals an inwardly directed sodium and chloride flux, though of lesser magnitude. The influx of water is less than would be expected to preserve osmotic equality suggesting that the translocation of sodium and chloride is the primary event. Although its function remains uncertain the flux has a considerable effect on the ion content of synaptosomes.     

Energy Supply System for the Gliding Movement of Hormogonia of the Cyanobacterium Nostoc cycadae

The effects of selected metabolic inhibitors and light on thegliding movement of the hormogonia of the cyanobacterium Nostoccycadae were examined. Respiratory poisons (sodium cyanide,sodium azide) stopped the movement in the dark, but not in light.DCMU had little effect on the movement in light. The inhibitoryeffect of monoiodoacetic acid (MIA) on the gliding movementin light was restored by adding DCMU, suggesting that the glidingmovement in light is linked to a cyclic electron flow involvingphotosystem I, not to a noncyclic electron flow involving photosystemII. This was then proved by experiments on the effects of selectedwavelengths of light on the movement; light absorbed by chlorophyllwas found to accelerate the movement, but that absorbed by phycobilinsdid not. The proton-conducting uncouplers carbonyl cyanide m-chlorophenylhydrazone(CCCP) and 2,4-dinitrophenol (DNP) immediately abolished themovement. Arsenate or N, N'-dicyclohexylcarbodiimide (DCCD),known inhibitors of ATP synthesis, had little effect on themovement. Light irradiation caused acidification of the cellsuspension. From these results, the direct driving force for the glidingmovement of this organism seems to be the trans-cell membraneelectrochemical potential difference of protons, generated bya respiratory chain in the dark and a cyclic photoelectron flowsystem in light. (Received April 14, 1986; Accepted March 12, 1987)