Cerebrocellular swelling in the presence of uraemic guanidino compounds: ameliorative effects of taurine.

Cell volumes (equilibrium non-inulin spaces) have been measured in slices of rat cerebral cortex incubated in the presence of uraemic guanidino compounds. Of 5 guanidino compounds tested, all but one caused significant cell swelling. This was most pronounced for guanidinosuccinic acid (GSA, 40 micromol/l)(+22%) and guanidine hydrochloride (G, 3 micromol/l)(+13%). Swelling was reduced by taurine in a dose-dependent manner, being completely abolished at 20 mmol/l. Swelling was also abolished by the antioxidants ascorbic acid (0.4 mmol/l) and butylated hydroxytoluene (0.5 mmol/l), the free radical scavenger N-acetyl-L-cysteine (10 mmol/l) and the lipid peroxidase inhibitor desmethyl tirilazad (100 micromol/l). The remission of swelling by 20 mmol/l taurine was reduced by 50% by the taurine transport inhibitor guanidinoethylsulphonate (GES, 1 mmol/l). This figure was not significantly altered when the concentration of GES was increased to 10 mmol/l. It was also reduced by 45% by the GABAA receptor antagonist bicuculline (100 micromol/l). It was completely abolished when both GES and bicuculline were present. It is suggested that guanidino compounds result in cells undergoing oxidative-nitrosative stress, and that taurine protects against the resultant cell swelling by 2 mechanisms One (intracellular) requires taurine transport and depends on its role as an antioxidant, with lipid peroxidation being probably a significant factor. The other (extracellular) is associated with activation of GABAA receptors.

Effects of ammonia and hepatic failure on the net efflux of endogenous glutamate, aspartate and taurine from rat cerebrocortical slices: modulation by elevated K+ concentrations.

Cerebrocortical minislices derived from control rats ("control slices") and from rats with thioacetamide (TAA)-induced hepatic failure showing moderate hyperammonemia and symptoms of hepatic encephalopathy (HE) ("HE slices"), were incubated with physiological saline in the absence or presence of 5 mM ammonium acetate ("ammonia"), at potassium ion (K+) concentrations ranging from 5 to 15 mM. The efflux of endogenous aspartate (Asp), glutamate (Glu) and taurine (Tau) to the incubation medium was assayed by HPLC. At 5 mM K+, perfusion of control slices with ammonia did not affect Glu and slightly depressed Asp efflux. Raising K+ concentrations in the incubation medium to 7.5 led to inhibition of Glu and Asp efflux by ammonia and the inhibitory effect was further potentiated at 10 mM K+. The inhibition was also significant at 15 mM K+. This suggests that, depression of excitatory neurotransmission associated with acute hyperammonemia is more pronounced under conditions of intense neuronal activity than in the resting state. HE moderately increased the efflux of Glu and Asp, and the stimulatory effect of HE on Glu and Asp efflux showed virtually no variation upon changing K+ concentration up to 15 mM. Ammonia strongly, and HE moderately, increased Tau efflux at 5 mM K+. However, both the ammonia- and HE-dependent Tau efflux decreased with increasing K+ concentration in the medium and was no longer significant at 10 mM concentration, indicating that intense neuronal activity obliterates the neuroprotective functions of this amino acid triggered by hyperammonemia.

Amino acid efflux and cell volume regulation in cerebrocortical minislices prepared from chronically hyponatraemic and hypernatraemic rats.

The rates of efflux of pre-loaded amino acids, and associated steady-state volumes, were measured in cells in cerebrocortical minislices prepared from chronically (4 day) hypo- and hypernatraemic rats. The findings were compared with those obtained when cells from normonatraemic rats were acutely exposed to comparable levels of anisosmotic stress. In the presence of 122 mmol/l Na+ cells from normal rats showed increases in the rates of efflux of D-aspartate and GABA, and significant swelling (both by comparison with levels in media containing 142 mmol/l Na+). Conversely there was no acceleration of efflux in cells from hyponatraemic rats (plasma Na+ = 119-126 mmol/l) and volumes were preserved at levels comparable with those in isomotically incubated cells from normal rats. In media containing 164 mmol/l Na+ amino acid efflux in cells from normal rats was retarded, and shrinkage occurred. In cells from chronically hypernatraemic rats (plasma Na+ = 160-166 mmol/l) the rates of efflux of D-aspartate and D-glutamate were accelerated by comparison with cells from normal rats, with volume preservation. However there was no increase in the rate of GABA or glycine efflux, and cell swelling was observed. It is concluded (i) that during chronic hyponatraemia the presence of D-aspartate or GABA is associated with cell volume preservation, (ii) during chronic hypernatraemia acidic, but not neutral, amino acids are also effective in this respect, and (iii) that the markedly differing patterns of efflux responses to acute and chronic anisosmotic stress are likely to reflect chronic volume-regulatory adaptations of the efflux mechanism(s).

Effects of ammonia in vitro on endogenous taurine efflux and cell volume in rat cerebrocortical minislices: influence of inhibitors of volume-sensitive amino acid transport.

Rat cerebrocortical minislices were incubated with physiological saline in the absence or presence of 5 mM ammonium acetate ("ammonia") and/or inhibitors of osmosensitive amino acid transport: 50 microM niflumic acid and 100 microM N-ethyl-maleimide for 60 min, with medium changes after 20 min and 40 min. The efflux of endogenous taurine, glutamate and glutamine was assayed by high-performance liquid chromatography, and steady-state cell volumes were monitored in the slices with the [14C]inulin method. In the absence of ammonia, niflumic acid abolished taurine efflux but did not affect glutamate or glutamine efflux at all time-points, and increased cell volume at 20 min and 60 min. N-Ethyl-maleimide increased taurine, glutamine and glutamate efflux at 20 min and 40 min, inhibited taurine and glutamine efflux at 60 min, and increased cell volume at 20 min. Ammonia strongly stimulated taurine (by 380% at 20 min), and only moderately glutamate (30% at 20 min) or glutamine efflux (76% at 20 min). Ammonia increased cell volume above the control level at all time-points. Niflumic acid inhibited, but did not abolish ammonia-dependent taurine and glutamine efflux, and did not change glutamate efflux. The effects of ammonia + niflumic acid on cell volume did not differ from the effects of each compound separately. N-Ethyl-maleimide inhibited ammonia-dependent efflux of all three amino acids except for stimulation of glutamate efflux at 20 min. N-Ethyl-maleimide + ammonia decreased the cell volumes more than did each compound separately. It is concluded that although ammonia-induced taurine efflux is accompanied by an increase in cell volume, the underlying mechanism is not simply a cell volume regulatory response normally observed in hypoosmotic stress. Increased efflux of taurine, which is an inhibitory amino acid and a cell membrane protectant, may serve to counteract the deleterious effects of increased excitatory transmission accompanying acute hyperammonemic insult.

The role of taurine in the regulation of brain cell volume in chronically hyponatraemic rats.

Cells in slices prepared from the superficial cerebral cortex of normonatraemic rats underwent moderate swelling when exposed to low Na+ medium (122 mmol/l) accompanied by a large increase in the rate of efflux of preloaded taurine. In contrast, cells in slices from chronically (4 day) hyponatraemic rats did not increase in volume and the rate of taurine efflux was unchanged. The anion transport inhibitor 4,4'-diisothiocyanato-stilbene-2,2'-sulphonic acid (25 micromol/l) caused marked (-44%) reduction in taurine efflux in cells from normonatraemic rats; this response was strongly attenuated (-16%) by hyponatraemia. When slices from hyponatraemic rats were acutely exposed to medium containing 142 mmol/Na+ cells exhibited marked and paradoxical swelling. This response was completely abolished by the NaCl co-transport inhibitor bumetanide (50 micromol/l) and was not observed in slices that had not been pre-loaded with taurine. Forty eight hours after the start of the remission of hyponatraemia, cells from post-hyponatraemic rats displayed normal responses (i.e., moderate swelling and greatly accelerated taurine efflux) on exposure to 122 mmol/Na+. But at 24 h there was only partial restoration of the efflux response to 122 mmol/Na+, with an enhanced cell swelling response that was not significantly affected by bumetanide. It is concluded that (i) during chronic hyponatraemia, unlike acute hyposmotic stress, cortical cells preserve their volume and that this is not associated with any increase in the rate of taurine loss; there does however, appear to be a decrease in the anionic component of cellular taurine efflux; (ii) acute re-incubation of slices in medium containing 142 mmol/l Na+ is associated with cell swelling that may reflect up-regulation of Na/Cl/taurine co-transport; (iii) following restoration of normonatraemia the pattern of normal cellular response to acute hyposmotic stress is only gradually re-established.

Effects of urea on taurine efflux and cell volume in incubated rat cerebral cortical minislices.

The effects of urea on the rate of efflux of preloaded taurine and volume regulation have been examined in incubated minislices from rat superficial cerebral cortex. As external urea was increased in the range 0-100 mmol/L, there was a concentration-dependent slowing of cellular taurine efflux. Cell volumes progressively increased over the range 0-50 mmol/L urea, but decreased slightly in 100 mmol/L. Urea had no effect on cell volume in the absence of taurine. Retardation of efflux, and cell swelling in the presence of 50 mmol/L urea were entirely abolished by trimethylamine (100 mumol/L). TMA had no effect on either variable in the absence of urea. It is suggested that impaired loss of taurine and accompanying cell swelling may be factors contributing to the neurological disturbances accompanying uremia.

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%), gamma-aminoisobutyric acid (GABA) (+ 38%), L-glutamate (+ 28%) and glycine (+ 26%). The anion transport inhibitor 4,4'-diisothiocyanatostilbene-2,2'-sulfonic acid (DIDS, 25 or 100 microM) as well as trifluoperazine (TFP, 25 microM), 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 microM) 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.

Volume regulation and the efflux of amino acids from cells in incubated slices of rat cerebral cortex. II. Dependence on Ca2+ ions.

The efflux of gamma-aminoisobutyric acid (GABA) and L-glutamate from pre-loaded cells in rat cerebral cortical slices has been studied during interventions designed to affect the availability of intracellular Ca2+ during hyposmotic swelling and membrane depolarization due to raised extracellular K+. Calmodulin-dependent acceleration of amino acid efflux in hyposmotic media, with cell swelling less than would be predicted on the basis of perfect osmometric behaviour (see Ref. [1]), was unaffected by Ca-ionophore in the presence of external Ca2+ or by the omission of external Ca2+, but was suppressed by pre-exposure of slices to thapsigargin (2 microM), which is reported to deplete cytosolic Ca2+, and by TMB-8 (0.5 mM), which blocks release of Ca2+ from internal stores. TMB-8 also led to significant cell swelling. The effects of TMB-8 were reversed by Ca-ionophore. Raised external K+ (54 mM) led to accelerated amino acid efflux which required calmodulin activation and was blocked by (i) omission of external Ca2+, (ii) the voltage-sensitive Ca2+ channel blocker nifedipine (1 microM), (iii) the anion transport inhibitor DIDS (25 microM for GABA, 100 microM for L-glutamate, see Ref. [1]), and (iv) the -SH group acetylator N-ethylmaleimide. TMB-8 was without effect in high K+ media. These results suggest that while enhanced amino acids efflux probably occurs through the same population of Ca/calmodulin-dependent, DIDS-sensitive pathways following hyposmotic shock or membrane depolarization, the source of Ca2+ ions required for the activation of these pathways may depend upon which of these acceleratory stimuli is applied.

The role of sulphydryl groups in efflux of taurine and GABA from cerebral cortical cells.

1. Effluxes of taurine and GABA from pre-loaded cells in slices of rat cerebral cortex in isosmotic media is enhanced by the -SH reagent p-chloromercuriphenylsulphonic acid (pCMPS) (100 microM) accompanied by moderate swelling. Those effects were more pronounced with GABA than with taurine. N-ethylmaleimide (NEM) (100 microM) had only slight affects on these variables. 2. The acceleration of effluxes that occurs when medium osmolality is reduced from 315 to 265 mosmol/kg is blocked by NEM and by pCMPS, with pronounced cell swelling. 3. The inhibitory effects of these reagents on efflux is abolished when cell swelling is prevented by the addition of 25 mM sucrose to hyposmotic incubation media (with equimolar reduction in NaCl concentration). 4. Pre-exposure of slices to dithiothreitol (DTT) (100 microM) blocks the effects of NEM and pCMPS on GABA efflux in hyposmotic media, but has no effect on taurine efflux. 5. The cell membrane and cytoskeletal responses which may underlie these effects are briefly discussed.

Taurine efflux and cell volume regulation in cerebral cortical slices during chronic hypernatraemia.

Efflux of cellular taurine from pre-loaded cerebral cortical slices incubated in hypo- and hyperosmotic media has been studied in normal and chronically hypernatraemic rats. Significant differences in transport mechanisms between the two groups has been noted. Hyperosmotic media retard efflux in cells from normal animals, with associated cell shrinkage, but accelerate efflux in cells from hypernatraemic rats, in which cell volumes are well maintained at pre-hypernatraemic levels. In hypernatraemic rats an anionic component of taurine efflux, present in normal animals, is lacking. Conversely, a distinct, calmodulin-dependent component which in normal rats is stimulated only in hypo-osmotic media, is present in both hypo- and hyperosmotically incubated slices from hypernatraemic rats, and inhibition of calmodulin-activation leads to cell swelling. This altered pattern of efflux and cell volume-regulation persists for at least 5 h following recovery from hypernatraemia, but remits by 30 h, indicating slow down-regulation of the hypernatraemically activated calmodulin-dependent efflux pathway and re-expression of anionic taurine transport.

Effects of extracellular bicarbonate ions and pH on volume-regulatory taurine efflux from rat cerebral cortical slices in vitro: evidence for separate neutral and anionic transport mechanisms.

Net efflux of [3H]taurine from cells in pre-loaded slices of rat cerebral cortex has previously been shown to occur via an anionic pathway, believed to consist of exchange of anionic taurine for extracellular Cl-, and operating under both isomotic and hypoosmotic conditions, and a calmodulin-dependent mechanism, activated by hypoosmotic stress: the latter may comprise conductive channels (Law, R.O. (1994) Biochim. Biophys. Acta 1221, 21-28). Experiments have now been performed to examine two inter-related problems; firstly, how anion/anion exchange (assuming 1:1 stochiometry) could contribute to the regulation of brain cell volume, and secondly, whether the hypoosmotically-activated component of efflux represents a second anionic transport process or loss of neutral taurine. The former process has been shown to be strongly dependent upon extracellular pH and bicarbonate concentration, being accelerated by low pH (7.0) and high (60 mmol/l) bicarbonate, and retarded by alkalinization (pH 7.8) and low (2.5 mmol/l) bicarbonate. Taurine efflux is inhibited by acetazolamide, with accompanying cell swelling in both isoosmotic and hypoosmotic media. It is hypothesized that inwardly directed bicarbonate transport, in exchange for intracellular Cl-, operates in parallel with efflux of anionic taurine in exchange for extracellular Cl-, and it is the subsequent conversion of bicarbonate to CO2 and water, under the influence of carbonic anhydrase, that effects a volume-regulatory decrease in internal osmotic potential. The dependence of taurine efflux upon pH and bicarbonate persists in the presence of trifluoperazine (an inhibitor of calmodulin activation) but is abolished in hypoosmotic media by the anion transport inhibitor niflumic acid. Cell depolarization in high K+ has no effect on taurine efflux, which is envisaged as involving parallel electroneutral anion exchange processes (taurine/Cl- and Cl-/bicarbonate) augmented, in hypoosmotic media, by diffusive efflux of neutral taurine.

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.

Regulation of mammalian brain cell volume.

Maintenance of brain cell volume is of crucial importance for normal central nervous system (CNS) function. This review considers volume regulation primarily in response to disturbances of body fluid osmolality. Brain cells counter the tendency to swell or shrink by appropriate adjustment of their internal osmotic potential. This is achieved by loss or uptake of inorganic ions and low molecular weight organic solutes (osmolytes). The latter comprise mainly amino acids, myoinositol, choline, and methylamines. Taurine may be of particular importance in volume control, especially in young animals. Brain cell volume regulation, however, is only one contributory factor to maintenance of constant brain volume (water content), and operates in parallel with important alterations in bulk fluid and electrolyte movement across the blood-brain barrier and between the interstitium and cerebrospinal fluid, which themselves moderate the requirement for transient alteration in cell volume during acute osmotic imbalance. Although altered cerebral content of inorganic ions and osmolytes are usually regarded as responses, respectively, to acute and chronic osmotic disturbances, osmolytes (especially taurine) may also participate in short-term cell volume regulation.

Efflux of potassium (86Rb+) attenuates the volume-restorative effect of sodium-amino acid cotransport in rat renal inner medullary cells shrunken by exposure to hyperosmotic media.

When the osmolality of the bathing medium was increased from 710 to 2000 mosmol/kg H2O, cells in incubated slices of rat renal inner medulla lost water and K+, and the rate of efflux of preloaded 86Rb+ (a tracer for K+) was significantly depressed. Addition of 2-aminoisobutyric acid (AIB, 10 mmol/l) partly restored cell water content but without re-accumulation of K+; the rate of 86Rb+ efflux was greatly increased. The presence of Ba2+ (1 mmol/l) or trifluoperazine (50 mumol/l) led to complete recovery of cell volume and K+ contents, with markedly reduced efflux of 86Rb+. Neither additive had any significant effect upon these variables in the absence of AIB or in media of 710 mosmol/kg. Efflux of 86Rb+ was pH-sensitive within the physiological range, and was depressed when external AIB was reduced below approx. 5 mmol/l. When external Na+ was increased from 145 to 500 mmol/l (total osmolality 350 to 2500 mosmol/kg) efflux was retarded only slightly if AIB was present, but markedly if AIB was omitted. Inner medullary cells may contain a class of Ba(2+)-inhibitable, calmodulin-dependent K+ conductive pathway which is activated in strongly hyperosmotic media by the operation of an inwardly-directed Na(+)-amino acid symport (cf. Law, R.O. (1988) Pflügers Arch. 413, 43-50) and which serves to moderate the volume-restorative effect of this membrane mechanism.

Sodium-dependent potassium (86Rb+) efflux moderates volume regulation by cells in rat renal inner medullary slices exposed to strongly hyperosmotic media.

The possibility has been examined that Na(+)-dependent K(+)-conductive pathways, known to exist in certain excitable cell membranes and inhibitable by the drug R56865, may also be present in cells of rat renal inner medulla, and that their activation may explain aspects of volume regulation by these cells in tissue slices exposed to strongly hyperosmotic media, as reflected by the rate of efflux of preloaded 86Rb+ (a marker for K+) and steady-state cell volumes and K+ contents. Cells incubated in media of 2000 mosmol/kg (400 mM Na+, 1172 mM urea) shrink and lose K+ by comparison with those in 720 mosmol/kg (203 mM Na+, 266 mM urea). If 2-aminisobutyric acid (10 mM) is added there is partial restoration of cell volume due to inwardly directed Na(+)-amino acid cotransport, but 86Rb+ efflux is accelerated and cells fail to regain net K+. R56865 (5 microM) completely blocks the increase in efflux and causes marked increases in cell volume and K+ contents, but only in strongly hyperosmotic media and in the presence of both Na+ and amino acid. In mildly hyperosmotic media, or media of 2000 mosmol/kg from which Na+ or amino acid is omitted, R56865 is without effect on these variables.

Efflux and accumulation of amino nitrogen in relation to the volume of rat renal inner medullary cells exposed to media of variable osmolality.

The rate of efflux of 2-amino[14C]isobutyric acid (AIB) from pre-loaded slices of rat renal inner medulla has been followed during incubation in media whose osmolality was varied between 350 and 2500 mosmol/kg H2O by adjustment of NaCl and urea concentrations. Efflux was biphasic, and it was assumed that the second, slower phase represented mainly cellular loss of AIB. As a function of cell volume (water content) the mean net rate of 2nd phase efflux declined far more abruptly (-36%) during an increase in external osmolality from 350 to 720 mosmol/kg than during further increase to 2500 mosmol/kg, over which range the rate of efflux fell by only a further 12%. Conversely, relative decrements of steady-state cell water contents during these two transitions were -17% and -37%, respectively. It is probable that in strongly hyperosmolal media (above 720 mosmol/kg) reduction in the rate of amino acid catabolism, with resultant cellular accumulation, becomes more important than passive efflux as a determinant of cell amino nitrogen content, and that this is caused by the enzyme-destabilizing effect of high intracellular concentrations of permeant urea. This interpretation is supported by the finding in the present study that trimethylamine N-oxide, which is known to counteract the destabilizing effect of urea, completely inhibited the accumulation of amino nitrogen (ninhydrin-positive substances) in media stronger than 720 mosmol/kg, as well as leading to further reduction of steady-state cell water contents, but was without effect on either variable in more dilute media. It is proposed that, under the conditions of this investigation, amino acids contribute to cell volume maintenance mainly by efflux and by metabolic accumulation under mildly and strongly hyperosmolal conditions, respectively, and that this interpretation is consistent with recent findings on the fluctuations in medullary levels of Na+, urea and total amino nitrogen in the intact kidneys of rats during acute water diuresis and oliguria (Law, R.O. (1991) Pflügers Arch. 418, 442-446).