Reduction In Osmolality Research Articles

Inhibition by Cl? channel blockers of the volume-activated, diffusional mechanism of inositol transport in primary astrocytes in culture

[3H]Inositol accumulated by rat brain cultured astrocytes is released when cells swell by exposure to solutions of decreased osmolarity. Activation of inositol efflux was proportional to reductions in osmolarity from 30%-70%. This volume-activated inositol efflux pathway was increased (27%) in Na(+)-free medium and decreased (22%) in Cl(-)-free medium. It was independent of extracellular Ca2+ and was reduced (30%) in the presence of the intracellular chelator [1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl)-ester] (BAPTA-AM). The inositol efflux pathway was markedly inhibited by Cl- channel blockers, which at maximal inhibitory concentrations decreased inositol efflux by 70%-83%. The potency range of the drugs was: 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) > 1-9, dideoxyforskolin > 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS) > niflumic acid. Inositol efflux was strongly inhibited by the SH blocker N-ethyl maleimide (NEM), which at 100 microM abolished inositol release. Inositol efflux can be reversed by increasing its extracellular concentration, suggesting that the efflux is mediated by a diffusional pathway whose direction is given by the concentration gradient. The inhibition of volume-associated fluxes of inositol by Cl- channel blockers supports the suggestion of an anion channel as the common pathway for inorganic and organic osmolytes in cultured astrocytes.

The osmolality-sensitive taurine channel in flounder erythrocytes is strongly stimulated by noradrenaline under hypo-osmotic conditions

Stimulation of flounder erythrocytes by noradrenaline under isosmotic conditions (330 mosmol kg-1) and physiological Na+ concentration (113 mmol l-1) caused swelling of the cells. The EC50 of this cell swelling was 0.65 µmol l-1 noradrenaline. The effect of the noradrenaline-induced cell swelling on the taurine channel under isosmotic conditions was negligible. However, when the cells were stimulated by noradrenaline (1.0 µmol l-1) before, simultaneously with or after reduction of osmolality (255 mosmol kg-1), the volume regulatory efflux of taurine mediated by the taurine channel was transiently accelerated. The rate coefficient for taurine efflux was more than four times higher than in osmolality-stimulated cells not exposed to noradrenaline. The present paper deals with the accelerating effect of noradrenaline on the taurine channel under hypo-osmotic conditions and the lack of effect of noradrenaline-induced cell swelling on the channel under iso-osmotic conditions. Noradrenaline initiated the cell swelling by interacting with ß-receptors which appeared to be more related to the mammalian ß1-receptors than to the ß2-receptors. The receptor interaction activated the adenylate cyclase system and, in the presence of 1.0 µmol l-1 noradrenaline, the cellular cyclic AMP concentration increased about 23 times. Noradrenaline also stimulated the Na+/H+ and Cl-/HCO3- antiporters and this affected the extracellular pH as well as the cell volume. Depending on the extracellular Na+ concentration, the incubation medium was acidified (113 mmol l-1 Na+) or alkalized (2.7 mmol l-1 Na+). Under these two conditions, the accelerating effects of noradrenaline on the taurine efflux were of similar magnitude. Similar effects on the cell volume, the extracellular pH and the volume regulatory taurine efflux were obtained in the presence of the cyclic AMP analogue 8-bromo-cyclic AMP. Under hypo-osmotic conditions in the absence of noradrenaline, the cellular level of cyclic AMP was not elevated. There was no significant positive correlation between the water content of the cells (cell volume) under different conditions in the presence or absence of noradrenaline and the state of activation of the osmolality-sensitive taurine channel. We conclude that the mechanism(s) which activate(s) the osmolality-sensitive taurine channel in flounder erythrocytes is transiently and strongly accelerated by noradrenaline, but not triggered by the noradrenaline-induced events. The acceleration does not appear to be due to increased activity of the antiporters, but to increased cellular levels of cyclic AMP.

Further characterization of the sorbitol permease in PAP-HT25 cells.

The sorbitol permease enables the efflux of sorbitol from cultured rabbit papillary cells (PAP-HT25) in response to a reduction in osmolality. The anion transport inhibitor 5-nitro-2-(3-phenylpropylamino)benzoate (100 microM) inhibited efflux by 92%, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (0.5 mM) reduced efflux by 40%. 2,4-Dinitrobenzenesulfonate and p-chloromercuriphenylsulfonic acid had no effect. The protease trypsin (0.05 mg/ml) reduced sorbitol efflux by 53%, pronase (0.01 mg/ml) by 47%, and papain (0.1 mg/ml) by 49%; chymotrypsin had no effect. Sugars and sugar alcohols at different concentrations (10-200 mM) in the bathing solution did not influence sorbitol efflux. Determination of the osmotically induced influx of sugar alcohols showed that xylitol uptake was faster than that of sorbitol; 6-deoxysorbitol was slower; L-sorbitol, arabitol, galactitol, and 2-deoxysorbitol entered at the same rate as sorbitol; and maltitol did not enter the cells. Sorbitol and 6-deoxysorbitol at 9 mM competitively inhibited [14C]sorbitol influx by 24 and 32%, respectively, whereas xylitol, taurine, betaine, and myo-inositol showed no inhibition. We conclude that 1) a specific inhibitor of the permease was not found, 2) the sorbitol permease or associated regulator is a protein, and 3) the C-6 atom of sorbitol is important in the selectivity of the permease.

Intracellular Ca2+ release and Ca2+ influx during regulatory volume decrease in IMCD cells.

Volume changes and cytosolic Ca2+ concentration ([Ca2+]i) of inner medullary collecting duct (IMCD) cells under hypotonic stress were monitored by means of confocal laser scanning microscopy and fura 2 fluorescence, respectively. Reduction of extracellular osmolality from 600 to 300 mosmol/kgH2O by omission of sucrose led to an increase in cell volume within 1 min to 135 +/- 3% (n = 9), followed by a partial regulatory volume decrease (RVD) to 109 +/- 2% (n = 9) within the ensuring 5 min. In parallel, [Ca2+]i rose from 145 +/- 9 to 433 +/- 16 nmol/l (n = 9) and thereafter reached a lower steady state of 259 +/- 9 nmol/l. Under low-Ca2+ conditions (10 nmol/l) RVD was not impeded and reduction of osmolality evoked only a transient increase of [Ca2+]i by 182 +/- 22 nmol/l (n = 6). Preincubation with 100 mumol/l 8-(N,N-diethylamino)octyl-3,4,5-trimethoxy-benzoate hydrochloride (TMB-8) or 20 mmol/l caffeine, both effective inhibitors of Ca2+ release from intracellular stores, in low Ca2+ as well as in high Ca2+, inhibited the Ca2+ response and abolished RVD. The temporal relationship between Ca2+ release from intracellular stores and Ca2+ entry was analyzed by determining fura 2 quenching, using Mn2+ as a substitute for external Ca2+. Intracellular Ca2+ release preceded Mn2+ influx by 17 +/- 3 s (n = 10). Mn2+ influx persisted during the whole period of exposure to hypotonicity, indicating that there is no time-dependent Ca2+ channel inactivation. Preincubation with TMB-8 or caffeine reduced Mn2+ influx to the control level, indicating that activation of Ca2+ channels in the plasma membrane occurs via intracellular Ca2+ release.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular calcium in primary cultures of rat renal inner medullary collecting duct cells during variations of extracellular osmolality.

There is ample evidence of calcium being an intracellular second messenger during volume regulatory processes in various cells including inner medullary collecting duct (IMCD) cells. Therefore, we measured intracellular calcium concentrations (Cai) under anisotonic conditions in primary cultures of IMCD cells using the Fura-2 technique. Basal steady-state calcium at 600 mosmol/l was found to be 110 +/- 4 nmol/l; n = 119. Exposure to hypotonic medium (300 mosmol/l, reduction of sucrose) resulted, within 1 min, in a strong increase in calcium to 563 +/- 87 nmol/l (n = 7; P < 0.01), followed by a decrease over 4-6 min to twice the initial values. The calcium increase was smaller (260 +/- 14 nmol/l; n = 5; P < 0.05) when the osmotic pressure was decreased by reducing NaCl instead of sucrose. Stepwise reduction of osmolarity to either 500 or 400 mosmol/l increased calcium by a significantly smaller extent, suggesting a threshold for calcium influx between 400 and 300 mosmol/l. In hypotonic calcium-free solutions no significant increase in calcium was observed. Verapamil (40 mumol/l), D-600 (40 mumol/l), diltiazem (40 mumol/l), and nifedipine (40 mumol/l) inhibited the hypotonically induced calcium influx in decreasing order of potency. Lanthanum (La3+) and gadolinium (Gd3+) had no effect. Membrane depolarization by incubation in potassium-rich solution diminished calcium influx. Preincubation with cytochalasin B (50 mumol/l for 30 min) resulted in a lower basal calcium level and attenuated the calcium increase during hypotonic shock. These results demonstrate an increased calcium influx during hypotonic shock in IMCD cells in culture mediated by channels whose nature (stretch activated and/or voltage dependent) remains to be determined. The transient increase in Cai in turn may trigger inorganic and organic osmolyte fluxes observed previously.

Diabetes-induced reduction of neuronal survival in hypotonic environments in culture

Dorsal root ganglion (DRG) neurons from streptozotocin (STZ)-induced diabetic and normal C57BL mice were exposed to three different hypotonic environments ( 1 2 , 1 4 , and 1/8 osmolar solutions). After rapid applications of these hypotonic solutions to the neurons, the cell volume autoregulatory mechanism operated in 1 2 osmolar solution but was disrupted in superhypotonic solutions below 1 4 osmolar in both kinds of mice. None of the neurons could survive 12 h after treatment with superhypotonic solutions. On the other hand, a gradual reduction of osmolarity of the culture medium enabled neurons in the normal mice to survive in 1 2 and 1 4 osmolar solutions as well as i an isotonic solution. However, this reduction of osmolarity increased neuronal cell death in the diabetic mice. These results suggest that the ability of DRG neurons to survive in hypotonic environments in culture may be lost in diabetes.

The Na(+)-independent taurine influx in flounder erythrocytes and its association with the volume regulatory taurine efflux.

95% of the Na(+)-independent influx of taurine in flounder erythrocytes at normal osmolality (330 mosmol kg-1) and 0.30 mmol l-1 taurine was mediated by a saturable system (Vmax = 0.689 nmol g-1 dry mass min-1; Km = 0.47 mmol l-1). The influx was inhibited by taurine analogues, but was not significantly affected by reduced osmolality. This saturable influx of taurine was probably mediated by the so-called Na(+)-dependent influx system for taurine operating in the 0 Na+: 1 taurine mode. The remaining 5% of the Na(+)-independent influx was mediated by a diffusional pathway (Kd = 0.050 microliter g-1 dry mass min-1), since it did not show saturation kinetics, was not inhibited by taurine analogues and did not mediate counter-exchange. This non-saturable influx system for taurine was strongly, but transiently, stimulated by reduction of osmolality. The time course for this stimulatory effect was the same as that for the system that mediates the volume regulatory efflux of taurine. The relative inhibitory effect of bumetanide, furosemide, DIDS and quinine on the fluxes mediated by these two transport systems were also the same. We suggest that these unidirectional fluxes of taurine were mediated by only one transport system: a taurine channel. The effect of reduction of osmolality on the rate coefficient for efflux of beta-alanine was equal to the effect on the efflux of taurine, but greater than the effect on the efflux of choline. This difference probably reflects structural and/or electrical restrictions on the substrates to be transported by the taurine channel. The volume regulatory efflux of taurine was inhibited in the presence of the anti-calmodulin drug trifluoperazine and, in a Ca(2+)-free medium, added EGTA. The 5-lipoxygenase inhibitor nordihydroguaiaretic acid completely blocked the volume regulatory efflux of taurine. We suggest that both Ca2+/calmodulin and leukotrienes contribute to the control of the transport mediated by the taurine channel.

Characterization of the Na+-dependent taurine influx in flounder erythrocytes

About 92% of the taurine influx in flounder erythrocytes at physiological conditions in vitro (330 mosmol·l-1, 145 mmol·l-1 Na+, 0.30 mmol·l-1 taurine) is Na+-dependent. This influx is highly specific for taurine. The β-amino compounds hypotaurine and β-alanine were the only compounds which mimicked the inhibitory effect of taurine on influx of [14C]taurine, the former more than the latter. Counterexchange of taurine was also mediated by the taurine transporters. Reduction of osmolality per se did not affect the activity of these transporters. Non-linear regression analysis of the influx values revealed the presence of two different influx systems: a system with high affinity and low capacity and another with low affinity and high capacity. However, we cannot exclude the possibility that this influx of taurine was mediated by only one transporter which operated in different modes depending on the extracellular Na+ concentration. On the assumption that the Na+-dependent influx was mediated by two separate systems, the maximal velocity of the low capacity system was 2.55 nmol·g dry weight-1·min-1 at 145 mmol·ll-1 extracellular Na+. This capacity was about 50% lower than that of the high capacity system. The Michaelis constants were 0.013 and 1.34 mmol·l-1, respectively. Reduction of the extracellular Na+ concentration reduced maximal velocity and the affinity to taurine of both transport systems. At 10 mmol·l-1 Na+ or lower concentrations the high capacity system did not seem to operate. The activation method suggested that each taurine molecule transported by the high capacity system was accompanied by two Na+. The stoichiometry of the low capacity system was 1 taurine: 1 Na+. The Hill-coefficient for both transport systems was 1.00.

Role of calcium in organic osmolyte efflux when MDCK cells are shifted from hypertonic to isotonic medium.

Madin-Darby canine kidney (MDCK) cells accumulate the nonperturbing osmolytes myo-inositol and betaine when grown in hypertonic medium. When returned to isotonic conditions, there is a transient basolateral efflux of these osmolytes, contributing to regulatory volume decrease. Using fura-2 fluorescence, we estimated intracellular calcium concentrations after switching MDCK cells from 500 to 300 mosM medium. Cell calcium increased 565 +/- 93 nM within 5 min. Lowering extracellular calcium inhibited the increase in cell calcium and osmolyte efflux when cells were shifted from 500 to 300 mosM medium. The calcium channel blockers lanthanum and nifedipine also inhibited osmolyte efflux after the shift from 500 to 300 mosM. In the absence of change in medium tonicity, increasing cell calcium by exposure to 1 microM ionomycin did not alter osmolyte efflux. As in PAP-HT25 cells, the cytochrome P-450 inhibitors ketoconazole and SKF-525A inhibited the efflux of both osmolytes caused by a reduction in osmolarity. Thus an early rise in cell calcium that is dependent on extra-cellular calcium and a pathway blocked by inhibitors of cytochrome P-450 oxidase are critical in regulation of osmolyte efflux when MDCK cells are shifted from hypertonic to isotonic medium.

Intracellular Ca2+ transients in HT29 cells induced by hypotonic cell swelling

Cell swelling induced by hypotonic solution led to an osmolality-dependent increase in intracellular Ca2+ activity ([Ca2+]i) in HT29 cells. At moderate reductions in osmolality from 290 to 240 or 225 mosmol/l in most cases only a small monophasic increase of [Ca2+]i to a stable plateau of 10-20 nmol/l above resting [Ca2+]i was observed. Lower osmolalities resulted in a triphasic increase of [Ca2+]i to a peak value. In a first phase after the volume change, lasting 20-40 s, [Ca2+]i increased slowly by about 30 nmol/l. Thereafter [Ca2+]i increased more rapidly within 20-30 s to a peak value. This peak was 189 +/- 45 nmol/l (190 mosmol/l, n = 9) and 243 +/- 41 nmol/l (160 mosmol/l, n = 20) above resting [Ca2+]i. The peak was then followed by a decline of [Ca2+]i over the next 2-3 min to a stable plateau value of 28 +/- 6 (n = 6) and 32 +/- 11 nmol/l (n = 11) above resting [Ca2+]i at 190 and 160 mosmol/l, respectively. The plateau lasted as long as the hypotonic solution was present. Under Ca(2+)-free bath conditions the peak value for the cell-swelling-induced [Ca2+]i transient was reached significantly later (60-100 s, compared to 40-60 s under control conditions). The peak values under Ca(2+)-free conditions were not significantly lower. This indicates that the [Ca2+]i peak was mostly of intracellular origin. No [Ca2+]i plateau phase was observed under Ca(2+)-free bath conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Volume-activated Rb+ transport in astrocytes in culture

The involvement of K+ on the volume regulatory process in astrocytes was investigated by characterizing the hyposmolarity-induced efflux of K+ using 86Rb as a tracer. About 70 and 30% of the intracellular content of 86Rb was released after reductions in osmolarity from 320 to 160 or 220 mosM, respectively, during the time in which cells exhibit a volume regulatory response subsequent to swelling. No significant increase in 86Rb efflux was observed with lower reductions in osmolarity. The 86Rb efflux was Ca2+ independent and insensitive to temperature. It was inhibited by furosemide but not by bumetanide and was unaffected when nitrate, but not gluconate, replaced intracellular Cl-. The efflux was markedly inhibited by quinidine and by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Quinidine also prevented the volume regulatory decrease of cells, and this effect was overcome when a large cation permeability was imposed by gramicidin. In isosmotic conditions 86Rb efflux was not activated by N-ethylmaleimide, but this drug strongly inhibited the hyposmolarity-activated release. These findings suggest that 86Rb efflux from astrocytes associated to cell swelling is not mediated by an electroneutral cotransporter and rather favor the implication of a conductive exit pathway that may be a Ca(2+)-independent K+ channel.

Neurons respond to hyposmotic conditions by an increase in intracellular free calcium.

The effect of hyposmotic conditions on the concentration of intracellular free calcium ([Ca2+]i) was studied in cultured cerebellar granule cells and cerebral cortical neurons after loading of the cells with the fluorescent Ca2+ chelator Fluo-3. It was found that in both types of neurons exposure to media with a decrease in osmolarity of 20 to 50% of the osmolarity in the isosmotic medium (320 mOsm) led to a dose dependent increase in [Ca2+]i with a time course showing the highest value at the earliest measured time point, i.e. 40 s after exposure to the hyposmotic media and a subsequent decline towards the basal level during the following 320 s. The response in the cortical neurons was larger than in the granule cells but both types of neurons exhibited a similar increase in [Ca2+]i after exposure to 50 mM K+ which was of the same magnitude as the increase in [Ca2+]i observed in the cortical neurons exposed for 40 s to a medium with a 50% reduction in osmolarity. In both types of neurons the blocker of voltage gated Ca2+ channels verapamil had no effect on the hyposmolarity induced increase in [Ca2+]i. On the contrary, this increase in [Ca2+]i was dependent upon external calcium and could be inhibited partly or completely by the inorganic blockers of Ca2+ channels Mg2+ and La3+. Dantrolene which prevents release of Ca2+ from internal stores had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypotonically induced changes in the plasma membrane of cultured mammalian cells

Cells from three cell lines were electrorotated in media of osmotic strengths from 330 mOsm to 60 mOsm. From the field-frequency dependence of the rotation speed, the passive electrical properties of the surfaces were deduced. In all cases, the area-specific membrane capacitance (Cm) decreased with osmolality. At 280 mOsm (iso-osmotic), SP2 (mouse myeloma) and G8 (hybridoma) cells had Cm values of 1.01 +/- 0.04 microF/cm2 and 1.09 +/- 0.03 microF/cm2, respectively, whereas dispase-treated L-cells (sarcoma fibroblasts) exhibited Cm = 2.18 +/- 0.10 microF/cm2. As the osmolality was reduced, the Cm reached a well-defined minimum at 150 mOsm (SP2) or 180 mOsm (G8). Further reduction in osmolality gave a 7% increase in Cm, after which a plateau close to 0.80 microF/cm2 was reached. However, the whole-cell capacities increased about twofold from 200 mOsm to 60 mOsm. L-cells showed very little change in Cm between 280 mOsm and 150 mOsm, but below 150 mOsm the Cm decreased rapidly. The changes in Cm correlate well with the swelling of the cells assessed by means of van't Hoff plots. The apparent membrane conductance (including the effect of surface conductance) decreased with Cm, but then increased again instead of exhibiting a plateau. The rotation speed of the cells increased as the osmolality was lowered, and eventually attained almost the theoretical value. All measurements indicate that hypo-osmotically stressed cells obtain the necessary membrane area by using material from microvilli. However, below about 200 mOsm the whole-cell capacities indicate the progressive incorporation of "extra" membrane into the cell surface.

Osmolarity reduction transiently increases K+ conductance of confluent rat hepatocytes in primary culture.

Rat hepatocytes in confluent primary cultures were impaled with conventional microelectrodes. Reducing extracellular osmolarity by 80 mosmol/l leads to a transient hyperpolarization of cell membranes (maximum after 5 min) from -40 +/- 4 to -51 +/- 2 mV (n = 7). This hyperpolarization is blocked by 1 mmol/l Ba2+ and 0.5 mmol/l quinine. In ion substitution experiments, increasing K+ 10-fold (from 2.7 to 27 mmol/l) depolarizes membrane voltage by 9 +/- 2 mV in normosmotic solutions. In hyposmotic solutions this depolarization is increased to 20 +/- 1 mV at the time of maximum hyperpolarization and decreases thereafter to 8 +/- 2 mV (n = 4). Cable analysis reveals a transient decrease of specific membrane resistance that exactly parallels the increase in membrane voltage response to high K+. In addition, electrical coupling between cells continuously decreases under hyposmotic conditions, indicating that intercellular communication is affected. Reducing Cl- 100-fold (from 116.5 to 1.2 mmol/l; HCO(3-)-free solutions) depolarizes hepatocytes by 24 +/- 3 mV under normosmotic conditions. In hyposmotic solutions, this effect is increased to 39 +/- 4 mV at maximum hyperpolarization and decreases again to 26 +/- 3 mV (n = 8). This transient increase in the voltage response to Cl- removal is abolished by 0.5 mmol/l quinine (n = 5) and 1 mmol/l Ba2+ (n = 5), suggesting that it is indirect via changes in K+ conductance. This concept is corroborated by ion substitution experiments (HCO(3-)-free conditions), which show that under hyposmotic conditions voltage response to high K+ is considerably decreased in low Cl- solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

The ionic basis of the hypo-osmotic depolarization in neurons from the opisthobranch mollusc Elysia chlorotica.

The resting potential of identified cells (Parker cells) in the abdominal ganglion of Elysia chlorotica (Gould) depolarizes by about 30 mV in response to a 50% reduction in osmolality and returns to the original potential in 20 min. Cell volume recovery requires approximately 2 h. Thus, recovery of the resting potential is not dependent on recovery of cell volume. The hypo-osmotic depolarization persists following inhibition of the electrogenic Na+/K(+)-ATPase with ouabain, and the levels of extracellular K+ and Cl- have little effect on the magnitude of the depolarization, while decreasing extracellular Na+ concentration produces a depolarization of only 10 mV. This suggests that the hypo-osmotic depolarization in Parker cells results mostly from increased relative permeability to Na+. Following transfer from 920 to 460 mosmol kg-1, Na+, Cl- and proline betaine leave the cells while intracellular K+ is conserved. Loss of intracellular Na+ and conservation of intracellular K+ are dependent on active transport by the Na+/K(+)-ATPase. Na+ and proline betaine leave the cells with a time course that is much longer than that of the hypo-osmotic depolarization. Unlike the other solutes, most of the reduction in intracellular Cl- concentration occurs coincidentally with the hypo-osmotic depolarization. However, unlike the hypo-osmotic depolarization, bulk loss of Cl- does not require the reduction in osmolality, only the reduction in extracellular ion concentrations. There is no apparent relationship between membrane depolarization and the regulation of intracellular osmolytes in Elysia neurons following hypo-osmotic stress.

Effect of osmolality on cytosolic free calcium and aldosterone secretion

Alterations in extracellular osmolality have powerful inverse effects on basal and potassium- and angiotensin-stimulated aldosterone secretion. With the use of bovine glomerulosa cells grown in primary culture, the effects of alterations in osmolality on cytosolic calcium concentration ([Ca2+]c), efflux and uptake of 45Ca2+, and aldosterone secretion were determined. Alterations in osmolality, independent of sodium concentration, have inverse effects on aldosterone secretion, which are correlated with simultaneous changes in [Ca2+]c measured using fura-2. Reductions in osmolality cause dose-dependent biphasic increases in [Ca2+]c different from the monophasic increases in [Ca2+]c produced by increases in potassium concentration. Like potassium- and angiotensin-stimulated increases in [Ca2+]c, hypotonically induced increases in [Ca2+]c are associated with an increase in 45Ca2+ efflux. Reductions in osmolality also increased the uptake of 45Ca2+, an effect apparent at 2 min and persistent for at least 30 min. In the absence of extracellular calcium, reductions in osmolality, as increases in potassium concentration but not angiotensin, fail to increase [Ca2+]c, efflux of 45Ca2+, or aldosterone secretion. In conclusion, osmolality-induced alterations in aldosterone secretion are associated with parallel changes in [Ca2+]c, effects caused by alteration in the influx of extracellular calcium. On the basis of these and previous studies, we hypothesize that osmolality affects calcium influx by activating voltage-dependent or stretch-activated calcium channels.

Iodinated Contrast Media: State of the Art and the Thorny Path toward Reduction of Osmolality

Iodinated Contrast Media: State of the Art and the Thorny Path toward Reduction of Osmolality

Red cell volume regulation: the pivotal role of ionic strength in controlling swelling-dependent transport systems

A volume increase of trout erythrocytes can be induced either by β-adrenergic stimulation of a Na +/H + antiport in an isotonic medium (isotonic swelling) or by suspending red cells in an hypotonic medium (hypotonic swelling). In both cases cells regulate their volume by a loss of osmolytes via specific pathways. After hypotonic swelling several volume-dependent pathways were activated allowing K +, Na +, taurine and choline to diffuse. All these pathways were fully inhibited by furosemide and inhibitors of the anion exchanger (DIDS, niflumic acid), and the K + loss was mediated essentially via a ‘Cl −-independent’ pathway. After isotonic swelling, the taurine, choline and Na + pathways were practically not activated and the K + loss was strictly ‘Cl −-dependent’. Thus cellular swelling is a prerequisite for activation of these pathways but, for a given volume increase, the degree of activation and the degree of anion-dependence of the K + pathway depend on the nature of the stimulus, whether hormonal or by reduction of osmolality. It appears that the pattern of the response induced by hormonal stimulation is not triggered by either cellular cAMP (since it can be reproduced in the absence of hormone by isotonic swelling in an ammonium-containing saline) or by the tonicity of the medium in which swelling occurs since after swelling in an isotonic medium containing urea, the cells adopt the regulatory pattern normally observed after hypotonic swelling. We demonstrated that the stimulus is the change in cellular ionic strength induced by swelling: when ionic strength drops, the cells adopt the hypotonic swelling pattern; when ionic strength increases, the isotonic swelling pattern is activated. To explain this modulating effect of ionic strength a speculative model is proposed, which also allows the integration of two further sets of experimental results: (i) all the volume-activated transport systems are blocked by inhibitors of the anion exchanger and (ii) a Cl −-dependent, DIDS-sensitive K + pathway can be activated in static volume trout red cells (i.e., in the absence of volume increase) by the conformational change of hemoglobin induced by the binding of O 2 or CO to the heme.

Cell volume regulation by trout erythrocytes: characteristics of the transport systems activated by hypotonic swelling.

1. An osmolality reduction of the suspending medium leads to osmotic swelling of trout erythrocytes, which is followed by a volume readjustment towards the original level. The regulatory volume decrease (RVD) was not complete after 1 h. 2. During RVD the cells lost K+ and Cl- but gained Na+. This entry of Na+, which is about half the K+ loss, explains the incomplete volume recovery (it was complete when Na+ was replaced by impermeant N-methyl-D-glucamine). The cells also lose large quantities of taurine, which accounts for about 53% of the volume recovery. In addition RVD is accompanied by the activation of a pathway allowing some large organic cations which are normally impermeant, such as choline or tetramethyl-ammonium, to rapidly penetrate the cells. 3. The swelling-activated K+ loss is not significantly affected by replacement of Cl- by NO3-, indicating that K+ moves through a Cl(-)-independent K+ pathway. Furosemide, DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) and niflumic acid inhibit the K+ loss. From experiments performed in high-K(+)-containing media, it appears that these compounds block the K+ flux, not by inhibiting Cl- movements but by interfering with the K+ pathway. 4. All the volume-activated pathways (K+, Na+, taurine, choline) are fully inhibited by furosemide and by inhibitors of the anion exchanger such as DIDS and niflumic acid. The concentration required for 50% inhibition (IC50) of both inorganic cations and taurine appears to be similar. It is proposed that DIDS interacts with a unique target which controls all the volume-sensitive transport systems.

Osmolarity-sensitive release of free amino acids from cultured kidney cells (MDCK).

The amino acid pool of MDCK cells was essentially constituted by alanine, glycine, glutamic acid, serine, taurine, lysine, beta-alanine and glutamine. Upon reductions in osmolarity, free amino acids were rapidly mobilized. In 50% hyposmotic solutions, the intracellular content of free amino acids decreased from 69 to 25 mM. Glutamic acid, taurine and beta-alanine were the most sensitive to hyposmolarity, followed by glycine, alanine and serine, whereas isoleucine, phenylalanine and valine were only weakly reactive. The properties of this osmolarity-sensitive release of amino acids were examined using 3H-taurine. Decreasing osmolarity to 85, 75 or 50% increased taurine efflux from 0.6% per min to 1.6, 3.5 and 5.06 per min, respectively. The time course of 3H-taurine release closely follows that of the regulatory volume decrease in MDCK cells. Taurine release was unaffected by removal of Na+, Cl- or Ca2+, or by treating cells with colchicine or cytochalasin. It was temperature dependent and decreased at low pH. Taurine release was unaffected by bumetanide (an inhibitor of the Na+/K+/2Cl- carrier); it was inhibited 16 and 67 by TEA and quinidine (inhibitors of K+ conductances), unaffected by gadolinium or diphenylamine-2-carboxylate (inhibitors of Cl- channels) and inhibited 50% by DIDS. The inhibitory effects of DIDS and quinidine were additive. Quinidine but not DIDS inhibited taurine uptake by MDCK cells.