Non-inulin Space Research Articles

Volume regulation and the efflux of amino acids from cells in incubated slices of rat cerebral cortex. I. Characteristics of transport mechanisms

The characteristics of amino acid efflux from pre-loaded cells in incubated slices of rat cerebral cortex have been investigated under basal conditions (isosmotic media, 315 mosmol/kg) and following mild hyposmotic shock (265 mOsmol/kg). Rates of efflux have been correlated with the extent of cell swelling in hyposmotic media. Hyposmolality accelerated the slow phase of cellular efflux of l-aspartate (+29%), γ-amino isobutyric acid (GABA) (+38%), l-glutamate (+28%) and glycine (+26%). The anion transport inhibitor 4,4′-di isothiocyanatostilbene-2,2′-sulfonic acid (DIDS, 25 or 100 μM) as well as trifluoperazine (TFP, 25 μM), an inhibitor of calmodulin activation, both retarded efflux in hyposmotic media, with associated cell swelling (increase in slice non-inulin space). The effects of DIDS and TFP were not additive. N-Ethylmaleimide (NEM, 100 μM) significantly retarded the efflux of neutral amino acids, with cell swelling: these effects were less pronounced in cells loaded with acidic amino acids. It is concluded that the hyposmotically-activated efflux of carboxylic amino acids, and associated cell swelling limitation, requires calmodulin activation and the presence of free sulfydryl groups.

Taurine efflux and the regulation of cell volume in incubated slices of rat cerebral cortex

The kinetics of efflux [ 3H]taurine have been examined in pre-loaded slices of rat cerebral cortex incubated in media of variable osmolality. Alterations in the rate of the slowest phase of efflux, considered to represent cellular loss, have been correlated with cell volumes, provisionally identified as the slice non-inulin space. Efflux was stimulated by reduction in medium osmolality, and impaired in hyperosmotic media; these variations were accompanied by moderate (non-osmometric) cell swelling and shrinkage, respectively. The rates of taurine efflux into media in which NaCl was partly replaced by sucrose, and measurement of the corresponding cell volumes, suggest that ionic strength, rather than osmolality or cell volume per se, may be a significant controlling factor. In both isosmolal and hyposmolal media efflux was significantly impaired by the anion transport inhibitor niflumic acid, with accompanying cell swelling, or by replacement of chloride by gluconate. In hyposmotic, but not isosmotic, media efflux was impaired, and cell volumes increased, in the presence of trifluoperazine or TMB-8, a reported blocker of intracellular calcium release, and the effects of niflumic acid and trifluoperazine on both variables were strongly additive. It is suggested that in both isosmotic and hyposmotic media taurine, efflux occurs by anionic transport, mainly through exchange with external chloride, whereas in hyposmotic media a second pathway is present, probably a volume-activated calmodulin-dependent channel.

Amiloride prevents the metabolic acidosis of a KCl load in nephrectomized rats.

1. Counter movements of K+ and H+ across cell membranes were studied in nephrectomized KCl-loaded rats. In one group of animals, the movements of K+ and H+ were determined during and after a KCl load, and in another group, amiloride was used in order to evaluate Na+ participation in K+/H+ exchange. 2. After a KCl load at constant PCO2, 79% of infused K+ left the inulin space, half of which was in exchange for H+. As a result, blood pH fell from 7.40 +/- 0.01 to 7.30 +/- 0.01 (mean +/- SEM; P less than 0.001). 3. During KCl infusion, the K+/H+ exchange ratio varied between 1.3 and 6.8, showing that the coupling ratio is not fixed. 4. Amiloride did not change blood pH and plasma [K+], but prevented the metabolic acidosis produced by the KCl load without affecting K+ entry into the non-inulin space. Therefore, K+ and H+ movements became completely dissociated. 5. The results indicate that KCl activates an amiloride-sensitive H+ extrusion from the cells. This finding is compatible with the view that Na+/H+ exchange participates in the metabolic acidosis produced by a KCl load.

Depolarization Increases Chloride‐Dependent Glutamate Sequestration in Synaptic Membranes of Rat Cerebral Cortex

To assess the functions of Cl- -dependent glutamate "binding" (Cl- -dependent glutamate uptake) in synaptic membranes, possible effects of depolarization on the uptake were examined. When rat cerebral cortical slices were preincubated with depolarizing agents such as veratrine (7 micrograms/ml), 10 microM aconitine, 56 mM K+, and 50 microM monensin, [3H]glutamate uptake by the crude synaptic membranes, which were subsequently prepared from the pretreated slices, was increased by 60-85%. Stimulation of the glutamate uptake by predepolarization was dependent on Na+ but not on Ca2+. The bindings of gamma-[3H]aminobutyric acid and 5-[3H]hydroxytryptamine were not significantly affected by the predepolarization. Veratrine pretreatment increased the maximal density of the glutamate uptake sites without affecting the affinity for glutamate. Several characteristics of the uptake sites increased by the veratrine pretreatment coincided with those of Cl- -dependent glutamate uptake sites. Na+-dependent glutamate binding (Na+-dependent glutamate uptake) to the membranes was not affected by pretreatment with veratrine. The content of endogenous glutamate and the noninulin space in the membrane fractions were not changed by the predepolarization. The increase in the glutamate uptake induced by pretreatment with high K+ was reversible: it returned to the control level after a second incubation of the slices in control medium. These results suggest that the Cl- -dependent glutamate sequestration system in synaptic membranes is regulated by the membrane potential.

N-methylaspartate-activated calcium channels in rat brain cortex slices. Effect of calcium channel blockers and of inhibitory and depressant substances

N-Methyl- dl-aspartate, l-glutamate, kainate and dl-homocysteate were found to increase the initial rate and the maximal uptake of 45Ca into the non-inulin space of rat brain cortex slices incubated in vitro. The N-methylaspartate-stimulated calcium uptake was blocked by cadmium and cobalt ions, but not by the organic calcium channel blocker nifedipine or by tetrodotoxin, both of which stimulated the N-methylaspartate-independent calcium influx, γ-Aminobutyrate increased the spontaneous calcium influx, and also reduced that stimulated by N-methylaspartate to the same level, as found with γ-aminobutyrate alone. Adenosine (1–100 μM), ethanol (0.1 M), pentobarbital (10–100 μM) and morphine (0.2 mM), were unable to inhibit the N-methylaspartate-activated calcium influx. Ethanol (0.1 M), had no effect on the glutamate- or kainate-activated calcium influx. These findings suggest that the excitatory amino acids, because of their neuronal depolarizing action in brain cortex, lead to the opening of voltage-sensitive calcium channels, which may be blocked by cadmium, but not by the organic calcium channel antagonist, nifedipine. The activation of calcium channels by the excitatory amino acid N-methylaspartate, was entirely unaffected by the depressants ethanol, pentobarbital or morphine, or by the endogenous inhibitory substance, adenosine, thus suggesting that their inhibitory or depressant effects occur through interference with a neuronal mechanism unrelated to the one studied here. γ-Aminobutyrate, on the other hand, considerably inhibited N-methylaspartate-induced calcium uptake, an effect interpreted as due to a γ-aminobutyrate-induced increase in chloride conductance, that “clamps” the membrane potential and does not allow further depolarization by N-methylaspartate.

Kainate, N-methylaspartate and other excitatory amino acids increase calcium influx into rat brain cortex cells in vitro

Kainate (0.62–5 mM) was found to increase the initial rate of influx of 45Ca and of 22Na into the non-inulin space of rat thin brain cortex slices incubated in vitro, and to shorten the equilibration time for both these ions. N-methyl- dl-aspartate (50–1000 μM), l-glutamate (0.62–5 mM), dl-homocysteate (0.62–2.5 mM), and ibotenate (6–170 μM) also significantly increased the influx of 45Ca into the non-inulin space of this preparation, while the non-neurotoxic acidic amino N-acetyl- l-aspartate, and α-methy- dl-aspartate (both 1.25–5 mM), did not increase such influx. We suggest that enhanced calcium uptaje may represent the basis for the neurotoxic effects of these compounds.

Ions and water in the epithelial cells of rabbit descending colon.

1. Isolated sheets of rabbit descending colon epithelial cells stripped from their underlying muscle coats were incubated in chambers at 37 degrees C with oxygenated media, and their non-inulin space water, sodium, potassium and chloride contents were subsequently determined. 2. With sodium Ringer bathing both surfaces, amiloride, 10(-4) M, decreased non-inulin space sodium content by 76 mmol/kg dry wt. Ouabain, 10(-3) M, caused loss of non-inulin space potassium which was not completely compensated for by uptake of sodium over 30 min incubation. Chloride and water, therefore, decreased. Amiloride, 10(-4) M, inhibited but did not prevent this uptake of sodium after ouabain. 3. Tissues exposed to sodium-free choline Ringer rapidly exchanged non-inulin space sodium for choline and, more slowly, lost potassium, chloride and water. The equilibration of sodium in the non-inulin space when sodium Ringer was restored to the mucosal medium alone was largely amiloride-insensitive. For restoration of non-inulin space potassium to normal levels, sodium was required in the serosal but not the mucosal medium. 4. Neither the absence of glucose nor the absence of chloride from the mucosal medium affected the non-inulin space sodium content when sodium was restored to the mucosal medium bathing sodium-depleted tissues. 5. It is argued that, whereas non-inulin space potassium and water contents are synonymous with their cellular values, only about one third of non-inulin space sodium is cellular when sodium Ringer bathes both surfaces, and the concentration of the sodium within the cellular transport pool approximated 20 mmol/kg H2O, consistent with estimates obtained from other techniques.

The sodium transport pool.

The sodium transport pool in epithelial cells represents sodium involved in active transport across the epithelium. There has been much controversy about the size of such a pool and even about its existence. Techniques for estimating the size of this pool are described. By analysis of toad bladder epithelial cells scraped from hemibladders mounted in chambers under a variety of conditions it has proved possible to detect and to quantify a sodium transport pool. Only about 20 percent of non-inulin space sodium measured flame photometrically is contained in this pool. This represents the total sodium entering cells from the mucosal medium, is in good agreement with the cellular sodium measured by the electron microprobe, and averages some 10-16 mmol/kg tissue H2O. Measurements of the pool in other tissues are considered.

Diphenylhydantoin and movement of radioactive sodium into electrically stimulated cerebral slices

Thin slices prepared from guinea pig cerebral cortex responded to electrical stimulation for 1.5–10 min at 1000 c/sec or 100 c/sec with a drop in content of creatine phosphate, ATP and K + in the non-inulin space. Non-inulin Na + rose. 22Na uptake also rose from an initial rate of about 480 μEq/g initial wt./hr to about 1360 μEq/g initial wt./hr. All of the above changes were diminished by diphenylhydantoin at concentrations of 1–2.7 × 10 −4 M. These results suggest that diphenylhydantoin diminishes the ability of neuronal elements to depolarize in response to electrical excitation.

Penicillin-Induced Metabolic Alterations in Isolated Cerebral Cortex

Thin slices prepared from guinea pig cerebral cortex were incubated in the presence of varying concentrations of penicillin G sodium. Early effects included a fall in levels of creatine phosphate and adenosine triphosphate. Lactate production and respiration were inhibited. Slices lost potassium ions from the noninulin space, but this effect occurred more slowly than the effects on high-energy phosphates. Penicillin appears to interfere primarily with energy production in cerebral tissues. Its epileptogenic properties could, however, result from leakage of potassium ions from some elements with secondary effects on others.

The site of the aldosterone induced stimulation of sodium transport

The content and concentration of major ions within the scraped mucosal epithelial cells of the urinary bladder of the toad Bufo marinus were determined using [ 14C]- and [ 3H]-inulin to correct for adherent extracellular fluid. In aldosterone treated hemi-bladders the radioactive sodium content and concentration increased significantly as compared with paired control hemi-bladders when both tissues were exposed to [ 24Na] in the mucosal bathing medium. Changes in total non-inulin space sodium were not detected but the stimulation of transepithelial sodium transport by aldosterone in this set of observations was small, averaging only some 30% above control. The results are compatible with our hypothesis that the major action of aldosterone is to facilitate the entry of sodium from the mucosal medium across the apical permeability barrier of the transporting mucosal cells.

COMPARTMENTATION OF THE INULIN SPACE IN MOUSE BRAIN SLICES

(1) Mouse cerebrum slices swell in tris-buffered Krebs-Ringer medium. Swelling is rapid at first, then slows to a more or less constant rate. Even after 3 hr incubation, water content/g of tissue dry wt. shows no sign of an asymptotic limit. Swelling is the same at 37 degrees and at 0 degree. (2) Tissue water measured by incubation with tritiated water is equal to total tissue water measured by drying slices. Equilibration between tritiated water and tissue water is complete within 2 min. (3) Tissue liquid can be divided into three phenomenologically distinguishable compartments: first inulin space, which is the compartment permeable to inulin at both 0 degree and 37 degrees; second inulin space, which is the compartment permeable to inulin at 37 degrees but not at 0 degree; and 37 degrees non-inulin space, which is the compartment impermeable to inulin at both 0 degree and 37 degrees. The evidence for this is: (a) Penetration of inulin into tissue is greater at 37 degrees than at 0 degree. After the first 20 min the rate of penetration at 0 degree is approximately equal to the rate of penetration at 37 degrees, and only slightly less than the rate of increase of total tissue water. Therefore the smaller inulin space observed at 0 degree cannot be due to slower entry of inulin. (b) The inulin content of slices incubated in inulin-containing medium at 37 degrees and cooled to 0 degree in the same medium is the same as the inulin content of tissue incubated at 37 degrees without subsequent cooling. In contrast, the inulin content of tissues preincubated in inulin-free medium at 37 degrees and then incubated in inulin-containing medium at 0 degree is the same as the inulin content of tissues incubated in inulin-containing medium at 0 degree without preincubation at 37 degrees. Therefore the smaller inulin space at 0 degree than at 37 degrees can be due neither to a reversible temperature-dependent change in the size of one single inulin space nor to an irreversible, greater swelling of a single inulin space at the higher temperature, but is due to some portion of the 37 degrees inulin space becoming impermeable to inulin at 0 degree. (c) Some inulin is retained by tissue incubated with inulin at 37 degrees, then transferred to inulin-free medium at 0 degree; the amount of retained inulin is equal to the difference between inulin content of tissue incubated with inulin at 37 degrees and tissue incubated with inulin at 0 degree This confirms 3b above and in addition shows that inulin which has entered the second inulin space at 37 degrees is trapped there when this space becomes impermeable to inulin at 0 degree. (4) The penetration of the amino acids, L-lysine and D-glutamate at 0 degree is equal to the penetration of inulin at 37 degrees. This confirms the real existence of the 37 degrees inulin space at 0 degree, and shows that the barrier at 0 degree between the first and second inulin spaces does not exist for these substances. (5) The amino acids L-leucine and glycine penetrate total tissue water at 0 degree. L-leucine is actively transported at this temperature. (6) The amino acids alpha-aminoisobutyric acid, L-leucine, and L-lysine at 2 mM have no effect at 37 degrees on either the inulin space or the non-inulin space. (7) The inulin space is insensitive at 37 degrees to physiologically significant changes in the medium. In contrast, the non-inulin space is quite sensitive to these changes. Addition of D-glutamate greatly increases the non-inulin space; addition of ouabain or cyanide, or omission of glucose, increases the non-inulin space slightly; and replacement of Na+ ion by choline+ ion greatly decreases this space. These changes are independent and roughly additive.

THE CATIONIC COMPOSITION OF INCUBATED CEREBRAL CORTEX SLICES

1. A new type of cutting table is described. It makes use of the elastic properties of a nylon thread, 0.08 mm thick, in which longitudinal vibrations greatly increase its ability to cut through soft tissue. Two slices of cerebral cortex may thus be obtained within 3-4 min after the death of an animal. 2. The extent of the swelling of a brain slice, as well as the ionic shift, is directly related to the amount of oxygen in the incubating fluid. Under the best conditions of oxygen supply, the swelling of a first slice was close to 12.2 +/- 4.3 per cent after 5 hr of incubation. The corresponding values for the Na+ and K+ contents were respectively 119.3 +/- 6.9 and 70.9 +/- 5.0 microequiv./g of final fresh weight (P2). 3. Incubation in complete anoxia leads to a considerable shift in the cation content of the slice, which acquires a composition close to that of the incubating fluid. This suggests that a large part of the cell population is still metabolically active when oxygen is present. 4. The inulin space represents 47.1 +/- 5.8 per cent of the initial fresh weight. It is independent of the amount of fluid taken up by the slice as well as of anoxia. 5. The cation content of the non-inulin space, calculated by assuming that the inulin space has the same composition as the incubation medium, was 77.7 microequiv./ml and 160.3 microequiv./ml for Na+ and K+ respectively. 6. The meaning of the inulin space, as well as the physico-chemical state of the cations in the slice, are discussed.

THE SODIUM, POTASSIUM AND CHLORIDE OF CEREBRAL TISSUES: MAINTENANCE, CHANGE ON STIMULATION AND SUBSEQUENT RECOVERY.

1. Cerebral tissues were prepared for incubation by cutting them from the brain rapidly and in situ, and the calcium concentration in the incubating medium was altered from the customary 2.8mm to 0.75mm. This provided incubated cerebral cortex with fluid and ion content more closely resembling that of the brain in vivo than hitherto obtained. 2. From a systematic difference in size between inulin spaces of slices with one and those with two cut surfaces, it was estimated that cutting directly affected a layer 0.02mm. thick. On the basis of the volume of this layer, it was calculated that the portion of the tissue not affected by cutting had an inulin space of 258 mul./g. initial wt., and during the process of preparation and incubation had gained 30muequiv. of sodium and 17 muequiv. of chloride/g. and had lost 14 muequiv. of potassium/g. 3. Several aspects of the ion content of the incubated tissue were compatible with the observed membrane potential of -60mv between cellular and extracellular phases. 4. In response to electrical stimulation, sodium of the non-inulin space increased from 28 to 57 muequiv./g., potassium decreased from 68 to 48 muequiv./g. and chloride increased from 16 to 22 muequiv./g. in the non-inulin space. These changes were complete in about 6min., and thereafter the concentrations remained steady during continued stimulation. Initial rates of change were 460 muequiv./g./hr. for sodium and 480 muequiv./g./hr. for potassium. 5. After stimulation was stopped the ionic composition of the tissue returned completely to its pre-stimulation state within 10min. Initial rates for extrusion of sodium and gain of potassium were 160 and 230 muequiv./g./hr. respectively.

MOVEMENTS OF RADIOACTIVE SODIUM IN CEREBRAL-CORTEX SLICES IN RESPONSE TO ELECTRICAL STIMULATION.

1. Sodium exchange was measured with (24)Na in incubated guinea-pig cerebral-cortex slices maintained under adequate metabolic conditions with a steady content of fluid and ions resembling that of brain in vivo. 2. Evidence was obtained indicating that Na(+) ions behaved in the inulin space as if they were extracellular, and that their entry into the non-inulin space of unstimulated tissue was about 10 times slower and could be separated, on the basis of complete exchangeability, into two components, a ;fast' one, which reacted to electrical stimulation, and a ;slow' one, exchanging at a rate of about 8muequiv./g./hr., which was not affected by stimulation. 3. The average rate of sodium turnover in unstimulated slices was 175-275muequiv./g./hr., whereas that for stimulated slices was approx. 4-6 times this, or 1050-1180muequiv./g./hr. The stimulated rate was equivalent to a turnover of 32% of the sodium in the non-inulin space/min., or 3mmuequiv./g./impulse. 4. Response to the onset of stimulation appeared to be immediate, but after cessation of stimulation increased sodium movements persisted for several minutes before return to unstimulated values. 5. Calculations based on electrochemical gradients suggested that about one-quarter of the energy available from respiration was required for sodium and potassium transport at maximal rates in both unstimulated and stimulated cerebral-cortex slices.

WATER DISTRIBUTION IN INCUBATED SLICES OF BRAIN AND OTHER TISSUES

Slices of rat cerebral cortex swell about 40% during one hour's aerobic incubation in bicarbonate-buffered medium. Thiocyanate or sucrose added to the medium equilibrate with part of the tissue fluid leaving a "non-thiocyanate" or "non-sucrose" space equal to about 45% of the total fluid which was present in the fresh unincubated slice. This space does not change with the extent of swelling or the time during which the tracer is present. The swelling thus appears to be extracellular. Inuilin in the medium equilibrates with little more than the water of swelling and docs not appear to enter the rest of the extracellular fluid. The swelling thus seems to involve uptake of fluid which is not continuous with the true tissue fluids. With glutamate or high potassium in the medium, swelling is increased and the non-thiocyanate, non-sucrose, and non-inulin spaces increase correspondingly. This increase is apparently intracellular. Swelling is also increased under anaerobic conditions. Results with sucrose and inulin indicate that this increase is due to intracellular swelling. The space occupied by thiocyanate increases with the time during which the thiocyanate is present in the medium under anaerobic conditions. Swelling can be decreased but not prevented by colloids in the medium. Medium rendered hypertonic with sucrose apparently causes cell shrinkage, as judged by the non-thiocyanate or non-sucrose space, without preventing uptake of "swelling water". Liver and kidney slices swell about 10%. Thiocyanate occupies the whole of the tissue fluid while inulin leaves a constant non-inulin space, equal to about 60% of the total fluid of the fresh unincubated slice. Sectors of diaphragm swell about 15%. Thiocyanate and inulin occupy about equal spaces. The non-thiocyanate and non-inulin spaces are about 49% of the total fluid of the fresh tissues. Diaphragm cut into strips parallel to the circumference swells more and thiocyanate and inulin penetrate most of the fluid. Sciatic nerve swells nearly as much as brain. The swelling is reduced if the ends are tied. Thiocyanate penetrates the tissue progressively with time. Inulin seems to occupy little more than the water of swelling, as in brain.

WATER DISTRIBUTION IN INCUBATED SLICES OF BRAIN AND OTHER TISSUES

Slices of rat cerebral cortex swell about 40% during one hour's aerobic incubation in bicarbonate-buffered medium. Thiocyanate or sucrose added to the medium equilibrate with part of the tissue fluid leaving a "non-thiocyanate" or "non-sucrose" space equal to about 45% of the total fluid which was present in the fresh unincubated slice. This space does not change with the extent of swelling or the time during which the tracer is present. The swelling thus appears to be extracellular. Inuilin in the medium equilibrates with little more than the water of swelling and docs not appear to enter the rest of the extracellular fluid. The swelling thus seems to involve uptake of fluid which is not continuous with the true tissue fluids. With glutamate or high potassium in the medium, swelling is increased and the non-thiocyanate, non-sucrose, and non-inulin spaces increase correspondingly. This increase is apparently intracellular. Swelling is also increased under anaerobic conditions. Results with sucrose and inulin indicate that this increase is due to intracellular swelling. The space occupied by thiocyanate increases with the time during which the thiocyanate is present in the medium under anaerobic conditions. Swelling can be decreased but not prevented by colloids in the medium. Medium rendered hypertonic with sucrose apparently causes cell shrinkage, as judged by the non-thiocyanate or non-sucrose space, without preventing uptake of "swelling water". Liver and kidney slices swell about 10%. Thiocyanate occupies the whole of the tissue fluid while inulin leaves a constant non-inulin space, equal to about 60% of the total fluid of the fresh unincubated slice. Sectors of diaphragm swell about 15%. Thiocyanate and inulin occupy about equal spaces. The non-thiocyanate and non-inulin spaces are about 49% of the total fluid of the fresh tissues. Diaphragm cut into strips parallel to the circumference swells more and thiocyanate and inulin penetrate most of the fluid. Sciatic nerve swells nearly as much as brain. The swelling is reduced if the ends are tied. Thiocyanate penetrates the tissue progressively with time. Inulin seems to occupy little more than the water of swelling, as in brain.