Reduction Of Extracellular Osmolarity Research Articles

Cholesterol modulates the volume-regulated anion current in Ehrlich-Lettre ascites cells via effects on Rho and F-actin

The mechanisms controlling the volume-regulated anion current (VRAC) are incompletely elucidated. Here, we investigate the modulation of VRAC by cellular cholesterol and the potential involvement of F-actin, Rho, Rho kinase, and phosphatidylinositol-(4,5)-bisphosphate [PtdIns(4,5)P(2)] in this process. In Ehrlich-Lettre ascites (ELA) cells, a current with biophysical and pharmacological properties characteristic of VRAC was activated by hypotonic swelling. A 44% increase in cellular cholesterol content had no detectable effects on F-actin organization or VRAC activity. A 47% reduction in cellular cholesterol content increased cortical and stress fiber-associated F-actin content in swollen cells. Cholesterol depletion increased VRAC activation rate and maximal current after a modest (15%), but not after a severe (36%) reduction in extracellular osmolarity. The cholesterol depletion-induced increase in maximal VRAC current was prevented by F-actin disruption using latrunculin B (LB), while the current activation rate was unaffected by LB, but dependent on Rho kinase. Rho activity was decreased by approximately 20% in modestly, and approximately 50% in severely swollen cells. In modestly swollen cells, this reduction was prevented by cholesterol depletion, which also increased isotonic Rho activity. Thrombin, which stimulates Rho and causes actin polymerization, potentiated VRAC in modestly swollen cells. VRAC activity was unaffected by inclusion of a water-soluble PtdIns(4,5)P(2) analogue or a PtdIns(4,5)P(2)-blocking antibody in the pipette, or neomycin treatment to sequester PtdIns(4,5)P(2). It is suggested that in ELA cells, F-actin and Rho-Rho kinase modulate VRAC magnitude and activation rate, respectively, and that cholesterol depletion potentiates VRAC at least in part by preventing the hypotonicity-induced decrease in Rho activity and eliciting actin polymerization.

Molecular and Functional Characterization of the Melastatin-related Cation Channel TRPM3

Proteins of the mammalian TRP (transient receptor potential) family form a heterogenous group of cation channels important for cellular Ca2+ signaling and homeostasis. Here we present the full-length sequence of TRPM3, a member of the melastatin-like subfamily (TRPM) of TRP channels. TRPM3 expression was found in human kidney and brain. HEK293 cells transiently transfected with TRPM3 showed a constitutive Ca2+ and Mn2+ entry. Whole-cell patch clamp experiments confirmed the spontaneous activity of TRPM3 and revealed permeability ratios PCa/PNa of 1.57 and PNa/PCs of 0.75. In cell-attached patches, spontaneous inward and outward currents were observed. At negative membrane potentials and in the presence of either 140 mm Cs+, 140 mm Na+, or 100 mm Ca2+ in the pipette solution, the single channel conductance levels were 133, 83, and 65 pS, respectively. The Ca2+ entry in TRPM3-expressing HEK293 cells increased during treatment with hypotonic extracellular solution. The reduction of extracellular osmolarity was accompanied by cell swelling, suggesting volume-regulated activity of TRPM3. From its function and expression in human kidney, we propose a role of TRPM3 in renal Ca2+ homeostasis.

CDNA cloning and characterization of an osmotically sensitive TRP channel from ascidian eggs

The transient receptor potential (TRP) ion channels are thought to be involved in the entry of calcium ion into cells. In this study, we isolated a cDNA clone, HrTRPV, that shows high homology to Caenorhabditis elegans OSM-9, a TRPV subfamily member of the TRP family, from a Halocynthia roretzi fertilized egg cDNA library. We analyzed its properties using HrTRPV-transfected cells. Upon reduction of extracellular osmolarity, the intracellular calcium concentration was found to increase in HrTRPV-transfected cells. This increase in intracellular calcium concentration was dependent on the presence of extracellular calcium ion and was inhibited by treatment with gadolinium ion, a stretch-activated calcium channel blocker. Thus, these results indicate that ascidian egg HrTRPV is an osmotically sensitive TRP channel.

Rho family GTP binding proteins are involved in the regulatory volume decrease process in NIH3T3 mouse fibroblasts

The role of Rho GTPases in the regulatory volume decrease (RVD) process following osmotic cell swelling is controversial and has so far only been investigated for the swelling-activated Cl- efflux. We investigated the involvement of RhoA in the RVD process in NIH3T3 mouse fibroblasts, using wild-type cells and three clones expressing constitutively active RhoA (RhoAV14). RhoAV14 expression resulted in an up to fourfold increase in the rate of RVD, measured by large-angle light scattering. The increase in RVD rate correlated with RhoAV14 expression. RVD in wild-type cells was unaffected by the Rho kinase inhibitor Y-27632 and the phosphatidyl-inositol 3 kinase (PI3K) inhibitor wortmannin. The maximal rates of swelling-activated K+ (86 Rb+ as tracer) and taurine ([3H]taurine as tracer) efflux after a 30 % reduction in extracellular osmolarity were increased about twofold in cells with maximal RhoAV14 expression compared to wild-type cells, but were unaffected by Y-27632. The volume set points for activation of release of both osmolytes appeared to be reduced by RhoAV14 expression. The maximal taurine efflux rate constant was potentiated by the tyrosine phosphatase inhibitor Na(3)VO(4), and inhibited by the tyrosine kinase inhibitor genistein. The magnitude of the swelling-activated Cl- current (I(Cl,swell) ) was higher in RhoAV14 than in wild-type cells after a 7.5 % reduction in extracellular osmolarity, but, in contrast to 86Rb+ and [3H]taurine efflux, similar in both strains after a 30 % reduction in extracellular osmolarity. I(Cl,swell) was inhibited by Y-27632 and strongly potentiated by the myosin light chain kinase inhibitors ML-7 and AV25. It is suggested that RhoA, although not the volume sensor per se, is an important upstream modulator shared by multiple swelling-activated channels on which RhoA exerts its effects via divergent signalling pathways.

Arachidonic acid as a second messenger for hypotonicity-induced calcium transients in rat IMCD cells.

In rat inner medullary collecting duct (IMCD) cells in primary culture, hypotonic stress induces Ca2+ transients consisting of an early peak phase caused by a Ca2+ release from intracellular stores and a subsequent plateau phase that involves Ca2+ entry from the extracellular milieu. In the present study, the mechanisms by which cell swelling is transduced into the Ca2+ release were investigated. The free intracellular Ca2+ concentration ([Ca2+]i) was measured using the fluorescent dye fura-2 and cell volume using a confocal laser scanning microscope. In control experiments, after reduction of extracellular osmolarity from 600 to 300 mosmol/l, by omission of sucrose, [Ca2+]i rapidly increased from 106 +/- 9 nmol/l to a peak value of 405 +/- 22 nmol/l (P </= 0.05) and thereafter reached a steady-state of 230 +/- 23 nmol/l. In low-Ca2+ conditions (10 nmol/l), the reduction of osmolarity evoked only a transient increase of [Ca2+]i by 182 +/- 11 nmol/l (P </= 0.05), which reflected Ca2+ release from intracellular stores. Hyposmotic stress had no effect on inositol 1,4,5-triphosphate (IP3) production measured by a [3H]IP3 radioreceptor assay. Preincubation with 100 micromol/l ETYA (a non-metabolisible derivative of arachidonic acid) reduced the Ca2+ response to hyposmotic stress under high and low Ca2+ conditions (87 and 85% inhibition respectively) as well as the regulatory volume decrease (RVD). Extracellular application of arachidonic acid in isotonic medium led to an increase in [Ca2+]i under high and low Ca2+ conditions. Pretreatment of IMCD cells with 50 microg/ml D609 (a phosphatidylcholine-directed phospholipase C inhibitor) or with 200 micromol/l propranolol (a phosphatidate phosphohydrolase inhibitor) reduced the hypotonic Ca2+ response more strongly than pretreatment with 5 micromol/l BPhB (a phospholipase A2 inhibitor). The Ca2+ response was also suppressed after preincubation with 200 micromol/l RHC 80267 (a diacylglycerol lipase inhibitor). Preincubation with 50 ng/ml pertussis toxin (a G-protein inhibitor) reduced the transient component of the Ca2+ response partially. We conclude that G-proteins, phosphatidylcholine-directed phospholipase C, phospholipase A2, diacylglycerol lipase and arachidonic acid, but not IP3, are involved in the mechanisms by which Ca2+ is released from the intracellular stores during RVD in IMCD cells.

Effect of astroglial cell swelling on pH of acidic intracellular compartments

A variety of pathological conditions lead to swelling of astrocytes, which in turn stimulates ion release by activation of ion channels at the plasma membrane. In the present study, acridine orange and fluorescein isothiocyanate coupled to dextran (FITC-dextran) have been used to examine the effect of cell swelling on pH in acidic compartments of cultured astroglial cells. Both NH 4Cl (2 mM) and chloroquine (10 μM), known to alkalinize acidic cellular compartments, led to the expected increase in acridine orange fluorescence intensity. Similar, albeit smaller, effects were elicited by a reduction of extracellular osmolarity (−80 mOsm) and treatment of the cells with glutamate (1 mM), manoeuvres which enhanced cell volume. Determination of changes in the FITC-dextran fluorescence ratio ( 485 440 nm ) allowed quantification of the pH changes in lysosomal compartments. Treatment with NH 4Cl, reduced extracellular osmolarity and glutamate increased lysosomal pH by 0.65 ± 0.07, 0.85 ± 0.14 and 0.25 ± 0.07, respectively. Measurement of cytosolic pH using 2′,7′-bis-(2-carboxyethyl)-5- (and -6) carboxyfluorescein (BCECF) demonstrated a pronounced acidification following cell swelling, observed with both reduced extracellular osmolarity (by 0.23 ± 0.05 pH units) and 1 mM glutamate (by 0.26 ± 0.02 pH units). In conclusion, pH within lysosomes and possibly other acidic cellular compartments of astrocytes is increased by cell swelling, which may have important consequences for astrocyte function.

Inhibition of transient potassium current in cultured and acutely dissociated mouse hippocampal neurons by GABAA receptor activation.

1. The regulation of A-current, one of several transient voltage-gated potassium currents, was studied using whole cell gigaohm seal voltage-clamp techniques on hippocampal pyramidal neurons that were either acutely dissociated from postnatal mouse brain or isolated from embryonic mouse brain and grown in dissociated culture. These neurons also express gamma-aminobutyric acid-A (GABAA) receptors, the activation of which can, under some circumstances, depolarize immature neurons and the dendrites of more mature neurons. 2. Application of GABA (50 microM) reduced the amplitude of A-current when potassium current amplitude was measured during a period of slow and incomplete desensitization of IGABA. A-current was reduced to 67 +/- 9% of control (mean +/- SD, n - 14) in acutely dissociated neurons, and to 64 +/- 11% of control (n = 15) in cultured neurons. Similar A-current reductions were seen in large outside-out membrane patches pulled from somata of cultured neurons, an observation suggesting that imperfect control of membrane voltage was not responsible for A-current inhibition. 3. A-current inhibition exhibited the sensitivity expected of a GABAA-sensitive process. It was mimicked by muscimol and blocked by bicuculline, picrotoxin, and reduction of [Cl-] in the external solution. Baclophen and phaclophen, effective as agonist and antagonist on GABAB receptors, did not affect A-currents or their inhibition. Reduction in extracellular osmolarity (to increase cell swelling as might occur with Cl- entry), or removal of external HCO3- (which might flow inward through GABAA channels and cause local external acidification), did not affect A-current or its inhibition. The mechanisms of inhibition is not clear at present. 4. We suggest that reduced A-current may favor GABA-induced depolarization and consequent activation of voltage-gated calcium channels.

Effects of arachidonic acid upon the volume-sensitive chloride current in rat osteoblast-like (ROS 17/2.8) cells.

1. The effects of arachidonic acid upon the volume-sensitive Cl- current present in cultured osteoblastic cells (ROS 17/2.8) was studied using the whole-cell patch-clamp technique. 2. Arachidonate produced two distinct phases of inhibition, a rapid phase occurring within 10-15 s of application, preceding a slower phase that occurred 2 min after onset of arachidonate superfusion. Accompanying the slower inhibitory phase was an acceleration of the time-dependent inactivation exhibited by the current at strongly depolarized potentials (> + 50 mV). The half-maximal inhibitory concentrations (IC50) were 177 +/- 31 and 10 +/- 4 microM for the two phases, respectively. 3. Arachidonate was still effective in the presence of inhibitors of cyclo-oxygenase (indomethacin, 10 microM), lipoxygenase (nordihydroguaretic acid, 10-100 microM) and cytochrome P450 (SKF525A, 100 microM; ethoxyresorufin, 10 microM; metyrapone, 500 microM; piperonyl butoxide, 500 microM; cimetidine, 1 mM). The effects of arachidonate could not be produced by another cis unsaturated fatty acid, oleic acid. 4. Measurements of cell volume showed that arachidonate effectively inhibited the regulatory volume decrease elicited by ROS 17/2.8 cells in response to a reduction in extracellular osmolarity. 5. It is concluded that the volume-sensitive Cl- conductance in ROS 17/2.8 cells is directly modulated by arachidonate and may represent a physiological mechanism by which volume regulation can be controlled in these cells.

Calcium entry stimulated by swelling of Madin-Darby canine kidney cells.

Cell swelling in Madin-Darby canine kidney (MDCK) cells by reduction of extracellular osmolarity (omission of 70 ad 150 mmol/l mannitol, respectively) leads to the activation of anion of channels and Ca2+ sensitive K+ channels. The K+ channel activation leads to an initial transient hyperpolarization of the cell membrane potential (PD) followed by a sustained depolarization due to activation of anion channels. The present study elucidates the role of intracellular calcium (Ca2+i) in regulatory cell volume decrease (RVD) of MDCK cells. While reduction of extracellular osmolarity by omitting 70 mmol/l mannitol did not lead to a detectable change in Ca2+i, severe cell swelling by omitting 150 mmol/l mannitol led to a transient rise in Ca2+i. PD changes, on the other hand, were not different under either condition. In addition, the response of PD to cell swelling was not altered by treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate diester, pertussis toxin or cholera toxin. In the nominal absence of extracellular Ca2+, reduction of extracellular osmolarity did not lead to an increase in Ca2+i and no initial transient hyperpolarization was observed, whereas addition of 10 mumol/l ATP still led to a significant hyperpolarization. Omission of extracellular Ca2+ was followed by a strong decrease in cell membrane resistance (Rm) due to activation of a depolarizing cation conductance. Subsequent readdition of Ca2+ caused a marked increase in Ca2+i due to Ca2+ influx. This Ca2+ entry was further stimulated by cell swelling. RVD was significantly blunted in the absence of extracellular Ca2+. The results suggest that cell swelling stimulates a Ca2+-permeable pathway in the cell membrane favoring Ca2+ entry into the cell with subsequent activation of Ca2+-sensitive K+ channels.

Mechanosensitive Single Channel Calcium Currents in Rat Mesangial Cells

Mechanical stimulation of mesangial cells enhances expression of early response protooncogenes is an extracellular Ca2+-dependent manner. This has lead investigators to postulate that a mechanosensitive Ca2+ channel may exist in these cells. At the present time, however, very little is known about single channel Ca2+ currents in mesangial cells. To examine this question, cell-attached patches of rat mesangial cells in early passages were studied with 100 mM BaCl2 patch-clamp pipets. Our studies identified a 21 pS, Co2+-sensitive channel that responded with an increase in open probability to mechanical stretch, applied through the patch pipet. The channel was also activated by a reduction in extracellular osmolarity. In both instances, channel responses underwent spontaneous adaptation despite continuous stimulation. This Ca2+ channel exhibited electrophysiological and kinetic properties that distinguished it from other cation-selective channels previously reported by us and other investigators on rat mesangial cell membranes.

Effect of cell volume on Acridine Orange fluorescence in hepatocytes.

Hepatic proteolysis is inhibited by cell swelling following a variety of experimental manoeuvres, such as reduction of extracellular osmolarity, concentrative uptake of amino acids, or blockade of K+ channels by barium. On the other hand, proteolysis is known to be accomplished by pH-sensitive lysosomal proteases. Accordingly, NH3/NH4+ inhibits proteolysis by intralysosomal alkalinization. The present study has been performed to test for an effect of cell volume on the pH of acidic intracellular compartments, as assessed by Acridine Orange fluorescence at > 520 nm (F > 520). F > 520 is enhanced by NH3/NH4+ (2 and 20 mmol/l respectively), by glutamine (2 mmol/l), by the K(+)-channel blocker barium (10 mmol/l) and by reduction of extracellular osmolarity (by 20 and 80 mosmol/l respectively). The observations point to release of Acridine Orange from acidic cellular compartments, which is indicative of alkalinization of these compartments during cell swelling. This effect may contribute to the regulation of proteolysis.

Cell shrinkage stimulates bradykinin-induced cell membrane potential oscillations in NIH 3T3 fibroblasts expressing the ras-oncogene.

In NIH 3T3 fibroblasts expressing the Ha-ras oncogene (+ ras) bradykinin leads to sustained oscillations of cell membrane potential due to oscillations of intracellular Ca2+ with subsequent activation of Ca(2+)-sensitive K+ channels. In cells not expressing the oncogene (-ras), bradykinin leads only to a single transient hyperpolarization of the cell membrane. The present study has been performed to elucidate the possible interaction of cell volume, intracellular pH and bradykinin-induced oscillations of the cell membrane potential. Bradykinin leads to cell shrinkage and intracellular alkalinization of both + ras cells and -ras cells. Inhibition of Na+/H+ exchanger by HOE 694 abolishes the bradykinin-induced alkalinization but does not significantly interfere with the bradykinin-induced oscillations of cell membrane potential. In contrast, prevention of bradykinin-induced cell shrinkage by simultaneous reduction of extracellular osmolarity blunts the oscillations. Thus, cell shrinkage stimulates bradykinin-induced oscillations of cell membrane potential. On the other hand, cell shrinkage alone does not elicit oscillations unless, in addition, Ca2+ entry is stimulated by ionomycin.

Volume regulation in liver: Further characterization by inhibitors and ionic substitutions

The present study has been performed to elucidate the mechanisms of volume regulation in isolated perfused liver. Reduction of extracellular osmolarity by 80 mOsm/L leads to a release of potassium and a sustained alkalinization of effluent. Reexposure to isotonic perfusate leads to reuptake of potassium by the liver and acidification of effluent. Part of the alkalinization could be due to release of bicarbonate parallel to potassium release. Carboanhydrase inhibition and replacement of bicarbonate/CO2 by HEPES buffer, however, do not significantly modify volume regulatory potassium release or reuptake. Reduction of perfusate chloride to 37 mmol/L by replacement of NaCl with raffinose leads to a decrease of liver weight indicative of shrinkage of liver cells. Subsequent omission of 180 mmol/L raffinose leads to potassium and chloride release and to alkalinization of effluent. Volume regulatory release of potassium is impaired in 1 mmol/L quinidine, 1 mmol/L SITS and 5 mmol/L barium. Volume regulatory reuptake of potassium is impaired by 1 mmol/L amiloride. Volume regulatory release of potassium is not appreciably affected by either; 1 mmol/L furosemide, 1 mumol/L verapamil, 1 mmol/L amiloride or 1 mmol/L barium and volume regulatory potassium reuptake proved insensitive to 1 mmol/L furosemide or 1 mmol/L barium. The data suggest that the cells release potassium and chloride during regulatory volume decrease by quinidine, SITS and weakly barium-sensitive transport systems and that regulatory volume increase is accomplished by activation of Na/H exchange.

The effect of hypoosmolarity on the electrical properties of Madin Darby canine kidney cells

The present study has been performed to test for the effect of hypotonic extracellular fluid on the electrical properties of Madin Darby canine kidney (MDCK)-cells. The volume of suspended MDCK-cells is 1,892 +/- 16 fl (n = 8) in isotonic (298.7 mosmol/l) extracellular fluid. Exposure of the cells to hypotonic (230.7 mosmol/l) extracellular fluid is followed by cellular swelling to 2,269 +/- 18 fl (n = 4) and subsequent volume regulatory decrease to 2,052 +/- 22 fl (n = 4) within 512 s. Volume regulatory decrease is abolished by quinidine (1 mmol/l) and by lipoxygenase inhibitor nordihydroguaiaretic acid (50 mumol/l). The potential difference across the cell membrane averages -53.6 +/- 0.9 mV (n = 49) in isotonic extracellular perfusates. Reduction of extracellular osmolarity depolarizes the cell membrane by +25.7 +/- 0.8 mV (n = 67), reduces the apparent potassium selectivity of the cell membrane, from 0.55 +/- 0.07 (n = 9) to 0.09 +/- 0.01 (n = 26), and increases the apparent chloride selectivity from close to zero to 0.34 +/- 0.02 (n = 21). Potassium channel blocker barium (1 mmol/l) depolarizes the cell membrane by +15.2 +/- 1.1 mV (n = 13). In the presence of barium, reduction of extracellular osmolarity leads to a further depolarization by +14.0 +/- 1.4 mV (n = 12). Addition of chloride channel blocker anthracene-9-COOH (1 mmol/l) leads to a hyperpolarization of the cell membrane by -6.7 +/- 2.2 mV (n = 11). In the presence of anthracene-9-COOH, reduction of the extracellular osmolarity leads to a depolarization by +22.4 +/- 1.7 mV (n = 11).(ABSTRACT TRUNCATED AT 250 WORDS)

Volume-sensitive taurine transport in fish erythrocytes.

Taurine plays an important role in cell volume regulation in both vertebrates and invertebrates. Erythrocytes from two euryhaline fish species, the eel (Anguilla japonica) and the starry flounder (Platichthys stellatus) were found to contain high intracellular concentrations of this amino acid (approximately equal to 30 mmol per liter of cell water). Kinetic studies established that the cells possessed a saturable high-affinity Na+-dependent beta-amino-acid transport system which also required Cl- for activity (apparent Km (taurine) 75 and 80 microM; Vmax 0.85 and 0.29 mumol/g Hb per hr for eel (20 degrees C) and flounder cells (10 degrees C), respectively. This beta-system operated with an apparent Na+/Cl-/taurine coupling ratio of 2:1:1. A reduction in extracellular osmolarity, leading to an increase in cell volume, reversibly decreased the activity of the transporter. In contrast, low medium osmolarity stimulated the activity of a Na+-independent nonsaturable transport route selective for taurine, gamma-amino-n-butyric acid and small neutral amino acids, producing a net efflux of taurine from the cells. Neither component of taurine transport was detected in human erythrocytes. It is suggested that these functionally distinct transport routes participate in the osmotic regulation of intracellular taurine levels and hence contribute to the homeostatic regulation of cell volume. Volume-induced increases in Na+-independent taurine transport activity were suppressed by noradrenaline and 8-bromoadenosine-3', 5'-cyclic monophosphate, but unaffected by the anticalmodulin drug, pimozide.

The effect of dextran 40 on serum electrolyte concentration in the dog.

LOW molecular weight dextran (LMWD) has been advocated in the treatment of numerous clinical conditions in which local or generalized rheological disturbances play a role. In some of these conditions, such as acute lower extremity ischemia or acute myocardial infarction, the rheological disturbance may last for days or weeks and effective therapy must be prolonged. Gelin and co-workers 1 studied the electrolyte changes in humans after a single injection of LMWD in dextrose. He concluded that LMWD caused a marked diuresis during infusion with an increase of potassium excretion and decrease of sodium and chloride excretion in the urine. He postulated that LMWD caused a mobilization of electrolytes from the extracellular fluid with reduction of extracellular osmolarity. He also theorized that LMWD may act as an osmotic diuretic, a belief effectively challenged by Bergentz et al 2 and Rabelo et al. 3 We have been concerned that continued LMWD therapy