Biomedical subjects

W Van Driessche

Publications and source records attributed to W Van Driessche.

At least 37 records · Page 2Linked to original sources

PubMed1998

Hypotonicity activates a lanthanide-sensitive pathway for K+ release in A6 epithelia.

The nature of the pathway for K+ release activated during regulatory volume decrease (RVD) in A6 epithelia was investigated by measuring cell thickness (Tc) as an index of cell volume and by probing K+ efflux with 86Rb as tracer for K+ (RRb). Cell swelling was induced by sudden reduction of basolateral osmolality (from 260 to 140 mosmol/kgH2O). Experiments were performed in the absence of Na+ transport. Apical RRb was negligible in iso- and hyposmotic conditions. On the other hand, osmotic shock increased basolateral RRb (RblRb) rapidly, reaching a maximum 7 min after the peak in Tc. Quinine (0.5 mM) completely inhibited RVD and RblRb. Also verapamil (0.2 mM) impeded volume recovery considerably; lidocaine (0.2 mM) did not exert a noticeable effect. The K+ channel blocker Ba2+ (30 mM) delayed RVD but could not prevent complete volume recovery. Cs+ inhibited RVD noticeably at concentrations <40 mM. With large Cs+ concentrations (>40 mM), the initial osmometric swelling was followed by a gradual increase of Tc, suggesting activation of Cs+ influx. Chronic exposure of the basolateral surface to 0.5 mM La3+ or Gd3+ completely abolished RVD and RblRb. Acute administration of lanthanides at the time of osmolality decrease did not affect the initial phase of RVD and reduced RblRb only slightly. Apical Gd3+ exerted an inhibitory effect on RVD and RblRb. The effect of Gd3+ should therefore be localized at an intracellular site. The role of Ca2+ entry could be excluded by failure of extracellular Ca2+ removal to inhibit volume recovery. In contrast to lanthanides, chronically and acutely administered Mg2+ (0.5 mM) inhibited RVD and RblRb by approximately 50%. These data suggest that K+ excretion during RVD occurs through a rather poorly selective pathway that does not seem to be directly activated by membrane stretch.

Animals↗

PubMed1998

Swelling-activated cation-selective channels in A6 epithelia are permeable to large cations.

Effects of basolateral monovalent cation replacements (Na+ by Li+, K+, Cs+, methylammonium, and guanidinium) on permeability to 86Rb of volume-sensitive cation channels (VSCC) in the basolateral membrane and on regulatory volume decrease (RVD), elicited by a hyposmotic shock, were studied in A6 epithelia in the absence of apical Na+ uptake. A complete and quick RVD occurred only when the cells were perfused with Na+ or Li+ saline. With both cations, hypotonicity increased basolateral 86Rb release (RblRb), which reached a maximum after 15 min and declined back to control level. When the major cation was K+, Cs+, methylammonium, or guanidinium, the RVD was abolished. Methylammonium induced a biphasic time course of cell thickness (Tc), with an initial decline of Tc followed by a gradual increase. With K+, Cs+, or guanidinium, Tc increased monotonously after the rapid initial rise evoked by the hypotonic challenge. In the presence of K+, Cs+, or methylammonium, RblRb remained high during most of the hypotonic period, whereas with guanidinium blockage of RblRb was initiated after 6 min of hypotonicity, suggesting an intracellular location of the site of action. With all cations, 0.5 mM basolateral Gd3+ completely blocked RVD and fully abolished the RblRb increase induced by the hypotonic shock. The lanthanide also blocked the additional volume increase induced by Cs+, K+, guanidinium, or methylammonium. When pH was lowered from 7. 4 to 6.0, RVD and RblRb were markedly inhibited. This study demonstrates that the VSCCs in the basolateral membrane of A6 cells are permeable to K+, Rb+, Cs+, methylammonium, and guanidinium, whereas a marked inhibitory effect is exerted by Gd3+, protons, and possibly intracellular guanidinium.

Animals↗

PubMed1997

Insulin effects on ouabain binding in A6 renal cells.

The effects of insulin on the Na+-K+-ATPase pump of the basolateral membrane of tight epithelia were evaluated by measuring transepithelial transport and [3H]ouabain binding in cultured A6 kidney cells. [3H]Ouabain binding in epithelia incubated in either K+-containing or K+-free solutions was measured. Insulin induced increases in transepithelial sodium transport, as measured by the short-circuit current (Isc), and in the initial rate of [3H]ouabain binding determined when the preparation was bathed in K+-containing solutions. However, when initial [3H]ouabain binding in tissues incubated in K+-free solutions was measured the stimulation of the initial rate of [3H]ouabain binding caused by insulin was markedly reduced. Incubating the apical side of the epithelium with either amiloride or Na+-free solutions also reduced or abolished the increase in the initial rate of [3H]ouabain binding caused by insulin. Equilibrium binding measurements showed that insulin did not increase the maximum number of [3H]ouabain-binding sites in tissues incubated with either normal K+ or K+-free solutions. These results indicate that the increase in the initial rate of [3H]ouabain binding under transporting conditions is due to an effect on the binding kinetics of ouabain, probably related to an increased rate of Na+ entry, rather than to an increase in the number of Na+-K+-ATPases in the basolateral membrane. Cycloheximide inhibited both the increase in Isc and the increase in the initial rate of [3H]ouabain binding caused by insulin in epithelia incubated in K+-containing solutions. However, cycloheximide was without effect on the initial rate of [3H]ouabain binding in insulin-treated tissues incubated in K+-free solution. This finding suggests that the cycloheximide-sensitive step of the action of insulin is related to Na+ delivery to the pump.

Animals↗

PubMed1997

Synergism between hypotonically induced calcium release and fatty acyl-CoA esters induced calcium release from intracellular stores.

The non-mitochondrial Ca2+ stores in permeabilized A7r5 cells responded to a decrease in Mg-ATP concentration with a pronounced Ca2+ release if 20 microM CoA was present. This release was rather specific for the preincubation or removal of ATP. ATP gamma S was much less effective and AMP-PNP, GTP, ITP, CTP, UTP, ADP, AMP, adenosine and adenine had no effect. CoA activated with an EC50 of 6 microM. Dephospho-CoA was a less effective cofactor and desulfo-CoA was ineffective. The release induced by Mg-ATP removal did not occur in the presence of 2% fatty acid-free bovine serum albumin and did not develop at 4 degrees C. All these findings suggest that CoA had to be acylated by endogenous fatty-acyl-CoA synthetase to become effective. Myristoyl- and palmitoyl-CoA esters were identified as the most effective cofactors for the release. Ca2+ release induced by removing Mg-ATP did not occur if the osmolality of the medium was kept constant by addition of mannitol, sucrose, KCl, MgCl2 or Mg-GTP, indicating that the decrease in tonicity was the trigger for the release. Mg-ATP plus CoA also synergized with Ca2+ release induced by a hypotonic shock imposed by diluting the medium with H2O. Osmolality changes induced by decreasing the Mg-ATP concentration were more effective in releasing Ca2+ than equal decreases in concentration of all solutes. We conclude that fatty acyl-CoA esters sensitize the hypotonically induced Ca2+ release from the non-mitochondrial Ca2+ stores.

Acyl Coenzyme A↗

PubMed1997

Isovolumetric regulation of C6 rat glioma cells in hyperosmotic media.

Volume regulation of C6 glioma cells was studied in response to a gradual increase of extracellular osmolality from 300 to 440 mosmol/kgH2O at 37 degrees C. Maintenance of cell size depended on the rate of osmolality increase (CR): at CR of 3 mosmol.kg-1.min-1, cell volume was kept constant, whereas it decreased progressively at CR of 6 or 9 mosmol.kg-1.min-1. The ability of C6 cells to maintain their volume is termed isovolumetric regulation (IVR). Reducing temperature to 22 degrees C inhibited IVR significantly. Also, bumetanide and ouabain blocked the regulation, while 5-(N,N-dimethyl)amiloride (DMA) did not affect IVR: Extracellular acidification rate (EAR) was studied by microphysiometry. EAR gradually decreased in the presence and increased in the absence of IVR. Experiments with DMA show that these changes in EAR were related to the activity of the Na+/H+ exchanger. It was stimulated by cell shrinkage but not by hyperosmolality itself. Our data demonstrate that C6 glioma cells are able to prevent volume decrease at a low rate of elevation of external osmolality and at 37 degrees C. This process requires electrolyte uptake by the Na(+)-K(+)-2Cl cotransporter and Na+/K+ pump.

Acids↗

PubMed1997

Downregulation of volume-activated Cl- currents during muscle differentiation.

We have used the whole cell configuration of the patch-clamp technique to investigate volume-activated Cl- currents in BC3H1 and C2C12 cells, two mouse muscle cell lines that can be switched from a proliferating to a differentiated musclelike state. Reducing the extracellular osmolality by 40% evoked large Cl- currents in proliferating BC3H1 and C2C12 cells. These currents were outwardly rectifying and had an anion permeability sequence as follows: I- > Br- > Cl- >> gluconate. They were inhibited by >50% by flufenamic acid (500 microM), niflumic acid (500 microM), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 microM) but were relatively insensitive to tamoxifen (100 microM). A reduction in the serum concentration in the culture medium induced growth arrest in both cell lines, and the cells started to differentiate into spindle-shaped nonfusing muscle cells (BC3H1) or myotubes (C2C12). This differentiation was accompanied by a drastic decrease in the magnitude of the volume-activated Cl- currents. The close correlation between volume-activated Cl- currents and cell proliferation suggests that these currents may be involved in cell proliferation.

Animals↗

PubMed1997

Isovolumetric regulation in a distal nephron cell line (A6).

The effects of gradually reducing the osmolality of the basolateral solution (pi b) were examined in a renal epithelial cell line (A6). pi b was linearly decreased with time from 260 to 140 mosmol/ kgH2O. Cell volume did not change when pi b was gradually decreased at dilution rates (D(r)) of 1-1.5 mosmol. kgH2O-1. min-1. Increasing D(r) to 3 or 6 mosmol. kgH2O-1. min-1 abolished this isovolumetric regulation (IVR). Replacing Cl- by NO3- or SCN- inhibited IVR markedly, whereas Br- substituted perfectly for Cl-. On the other hand, with all these anions, the regulatory volume decrease (RVD) was completely developed. Ba2+ (30 mM) markedly slowed down RVD but improved IVR. The discrepancies between RVD and IVR suggest that different mechanisms are used to control cell volume during gradual and shockwise hyposmotic perturbations. During gradual and shockwise reductions of pi b, cellular K+ content was reduced to the same extent; 86Rb efflux was only partially inhibited by Ba2+. The amount of intracellular K+ depletion could account for 70% of the cationic osmolyte loss, which suggests that K+ is the major cation excreted during both types of perturbations.

Animals↗

PubMed1997

K+ transport and capacitance of the basolateral membrane of the larval frog skin.

Skin from larval bullfrogs was mounted in an Ussing-type chamber in which the apical surface was bathed with a Ringer solution containing 115 mM K+ and the basolateral surface was bathed with a Ringer solution containing 115 mM Na+. Ion transport was measured as the short-circuit current (Isc) with a low-noise voltage clamp, and skin resistance (Rm) was measured by applying a direct current voltage pulse. Membrane impedance was calculated by applying a voltage signal consisting of 53 sine waves to the command stage of the voltage clamp. From the ratio of the Fourier-transformed voltage and current signals, it was possible to calculate the resistance and capacitance of the apical and basolateral membranes of the epithelium (Ra and Rb, Ca and Cb, respectively). With SO4(2-) as the anion, Rm decreased rapidly within 5 min following the addition of 150 U/ml nystatin to the apical solution, whereas Isc increased from 0.66 to 52.03 microA/cm2 over a 60-min period. These results indicate that nystatin becomes rapidly incorporated into the apical membrane and that the increase in basolateral K+ permeability requires a more prolonged time course. Intermediate levels of Isc were obtained by adding 50, 100, and 150 U/ml nystatin to the apical solution. This produced a progressive decrease in Ra and Rb while Ca and Cb remained constant. With Cl- as the anion, Isc values increased from 2.03 to 89.57 microA/cm2 following treatment with 150 U/ml nystatin, whereas with gluconate as the anion Isc was only increased from 0.63 to 11.64 microA/cm2. This suggests that the increase in basolateral K+ permeability produced by nystatin treatment, in the presence of more permeable anions, is due to swelling of the epithelial cells of the tissue rather than the gradient for apical K+ entry. Finally, Cb was not different among skins exposed to Cl-, SO4(2-), or gluconate, despite the large differences in Isc, nor did inhibition of Isc by treatment with hyperosmotic dextrose cause significant changes in Cb. These results support the hypothesis that increases in cell volume activate K+ channels that are already present in the basolateral membrane of epithelial cells.

Animals↗

PubMed1996

The cAMP-regulated and 293B-inhibited K+ conductance of rat colonic crypt base cells.

We have shown previously that secretagogues acting via the second messenger adenosine 3',5'-cyclic monophosphate (cAMP) activate, besides their marked effect on the luminal Cl- conductance, a K+ conductance in the basolateral membrane of colonic crypt cells. This conductance is blocked by the chromanol 293B. This K+ conductance is examined here in more detail in cell-attached (c.a.) and cell-excised (c.e.) patch- clamp studies. Addition of forskolin (5 micromol/l) to the bath led to the activation of very small-conductance (probably < 3 pS) K+ channels in c.a. patches (n = 54). These channels were reversibly inhibited by the addition of 0.1 mmol/l of 293B to the bath (n = 21). Noise analysis revealed that these channels had fast kinetics and produced a Lorentzian noise component with a corner frequency (fc) of 308 +/- 10 Hz (n = 30). The current/voltage curves of this noise indicated that the underlying ion channels were K+ selective. 293B reduced the power density of the noise (So) to 46 +/- 8.7% of its control value and shifted fc from 291 +/- 26 to 468 +/- 54 Hz (n = 8). In c.e. patches from cells previously stimulated by forskolin, the same type of current persisted in 3 out of 18 experiments when the bath solution was a cytosolic-type solution without adenosine 5'-triphosphate (ATP) (CYT). In 15 experiments the addition of ATP (1 mmol/l) to CYT solution was necessary to induce or augment channel activity. In six experiments excision was performed into CYT + ATP solution and channel activity persisted. 293B exerted a reversible inhibitory effect. The channel activity was reduced by 5 mmol/l Ba2+ and was completely absent when K+ in the bath was replaced by Na+. These data suggest that forskolin activates a K+ channel of very small conductance which can be inhibited directly and reversibly by 293B.

Animals↗

PubMed1996

Volume regulation in rat brain glial cells: lack of a substantial contribution of free amino acids.

Volume regulation of C6 glioma cells was studied while the bath osmolality was reduced from 300 to 150 mosmol/kg. Exposure to a hyposmotic challenge elicited a typical regulatory volume decrease (RVD). No regulatory volume increase was observed upon restoration of isosmotic conditions. During a second subsequent hyposmotic challenge, the cells did not respond with RVD. High extracellular K+ concentration and the K+ channel blockers Ba2+ and quinine inhibited the RVD. RVD was abolished after Cl- was replaced by gluconate and by the Cl- channel blocker 5-nitro-2(3-phenylpropylamino)benzoic acid. Amino acid (AA) concentration in cell and perfusate was determined. In control, cell content was only 26 mmol/l. Hypotonicity increased the efflux of AA from 0.14 to 0.60 mmol/min. During the second hyposmotic challenge, the release was 0.32 mmol/min. The data show that C6 cells adjust their volume under hyposmotic conditions but lose the ability to restore their volume during a subsequent hyposmotic treatment. K+ and Cl- are the main osmolytes involved in volume adjustment through conductive pathways. AA do not contribute substantially to cell volume regulation.

Amino Acids↗

PubMed1996

Electrolyte transport mechanisms involved in regulatory volume increase in C6 glioma cells.

Volume regulation of C6 glioma cells was studied with an automatic system for monitoring cell thickness, while increasing bath osmolality from 300 to 440 mosmol/kgH2O. At 37 degrees C, tissues incubated in solutions containing active substances (inositol, D-biotin, hydrocortisone, prostaglandin E1, insulin, transferrin, sodium selenite, and 3,5,3'-triiodothyronine) responded to hyperosmotic challenge with a typical regulatory volume increase (RVI). Lowering temperature or removing the active substances inhibited osmoregulation. Bumetanide, amiloride, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, or ouabain significantly reduced RVI. Ion substitutions of Na+, Cl-, NaCl, or HCO3- also importantly affected the process. Extracellular acidification rate (EAR) was studied by microphysiometry. Hyperosmotic shock induced an increase in EAR with a time course that matched volume recovery. This increase in EAR was prevented by amiloride. The data show that under hyperosmotic conditions C6 cells are able to regulate their volume. Ion substitutions and application of blockers demonstrate that Na+/H+ and Cl-/HCO3- exchangers and Na(+)-K(-)-2Cl- cotransporter are involved in RVI. The rise in EAR is due to the enhanced activity of Na+/H+ antiporter, which seems to be volume dependent but not osmotic dependent.

Animals↗

PubMed1995

Volume regulation in a toad epithelial cell line: role of coactivation of K+ and Cl- channels.

1. We have measured changes in cell volume, membrane potential and ionic currents in distal nephron A6 cells following a challenge with hypotonic solutions (HTS). 2. The volume increase induced by HTS is compensated by a regulatory volume decrease (RVD), which is inhibited by both 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and quinine. Quinine (500 microM) completely blocked RVD, whereas 100 microM NPPB delayed and attenuated RVD. 3. The resting potential in A6 cells was -52.3 +/- 4.8 mV (n = 53), and shifted to -35.1 +/- 2.2 mV (n = 33) during HTS. 4. Resting membrane current in A6 cells was 0.35 +/- 0.12 pA pF-1 at -80 mV and 0.51 +/- 0.16 pA pF-1 at +80 mV (n = 5). During cell swelling these values increased to 11.5 +/- 1.1 and 29.3 +/- 2.8 pA pF-1 (n = 29), respectively. 5. Quinine (500 microM) completely blocked the HTS-activated current at -15 mV, the reversal potential for Cl- currents, but exerted only a small block at -100 mV (K+ equilibrium potential). NPPB (100 microM) inhibited the current at both potentials almost to the same extent. The HTS-induced net current reversed at -41 +/- 2.5 mV (n = 15), which is close to the measured resting potential during HTS. 6. The quinine-insensitive current reversed near the Cl- equilibrium potential. The quinine-sensitive current reversed near the K+ equilibrium potential. The respective conductances activated by HTS at the zero-current potential were 2.1 +/- 0.7 nS for K+ and 5.2 +/- 1.3 nS for Cl- (n = 15). 7. Single channel analysis unveiled activation of at least two different channels during HTS. A 36 pS channel reversing at the Cl- equilibrium potential showed increased open probability at depolarized potentials. HTS also activated a K+ channel with a 29 pS conductance in high-K+ extracellular solutions (130 mM) or 12 pS in 2.5 mM K+. 8. This coactivation of K+ and Cl- channels shifts the membrane potential towards a value between EK and ECl (the reversal potentials for K+ and Cl-), where a net efflux of Cl- (Cl- inward current) and K+ (K+ outward current) under zero-current conditions occurs. Block of either the K+ or the Cl- conductance will shift the zero-current potential towards the equilibrium potential of the unblocked channel, preventing net efflux of osmolytes and RVD. This coactivation of K+ and Cl- currents causes a shift of osmolytes out of the cells, which almost completely accounts for the observed RVD.

Animals↗

PubMed1995

Effect of dexamethasone on sodium channel block and densities in A6 cells.

The association (ON) and dissociation (OFF) rates of either positively charged amiloride or its uncharged analogue, CDPC (6-chloro-3, 5-diaminopyrazine-2-carboxamide), with the apical Na+ channel protein of renal A6 cells were analysed during exposure to the synthetic glucocorticoid, dexamethasone, using noise analysis. These rates were further used to reach specific conclusions about single-channel current, channel density and open probability of the channel in the absence of the blocker. Short-term exposure (3 h) to 10(-7) mol/l dexamethasone at the basolateral side increased the short-circuit current, Isc by 85%, without a change in the ON and OFF rates of the interaction between amiloride and the Na+ channel. A longer incubation (24 h) with dexamethasone tripled the current with a notable increase in the ON rate of the interaction between amiloride and the and channel. The OFF rate remained constant. The effects of dexamethasone on the rate constants of the reaction of amiloride with the channel did not match with the expected changes in membrane potential. On the other hand, ON and OFF rates of the interaction between neutral CDPC and the channel were not influenced by a 24-h incubation with dexamethasone. Further calculations disclosed that the gain in macroscopic current after a 24-h incubation with dexamethasone might be explained by an increase in Na+ channel density, and, to a lesser extent, by a rise in single-channel current. This all occurred without a change in the fraction of time spent by the channel in the conducting state in the absence of the blocker.

Amiloride↗

PubMed1995

Regulatory volume decrease in a renal distal tubular cell line (A6). I. Role of K+ and Cl-.

Changes in volume of A6 epithelial cells were monitored by recording cell thickness (Tc). The response of Tc to a reduction of the basolateral osmolality from 260 to 140 mosmol/kg was recorded while transepithelial Na+ transport was inhibited by 20 microM amiloride. With Cl--containing bathing media, this osmotic challenge elicited a rapid rise in Tc followed by a regulatory volume decrease (RVD). Substitution of SO4(2-) or gluconate for Cl- markedly reduced the RVD, whereas cells completely maintained their ability to regulate their volume after replacing Cl- by NO3(-). A conductive pathway for Cl- excretion is suggested, which is insensitive to NPPB [5-nitro-2-(3-phenylpropylamino)benzoic acid], an inhibitor of some types of Cl- channels. Ba2+ (5 or 20 mM) reduced the RVD. A more pronounced inhibition of the RVD was obtained with 500 microM quinine, a potent blocker of volume-activated K+ channels. K+-induced depolarization of the basolateral membranes of tissues incubated with SO4(2-)-containing solutions completely abolished the RVD. Noise analysis in the presence of Ba2+ showed the activation of an apical K+ conductive pathway. These results demonstrate that cell volume regulation is controlled by processes involving Cl- and K+ excretion through conductive pathways.

Animals↗

PubMed1995

Regulatory volume decrease in a renal distal tubular cell line (A6). II. Effect of Na+ transport rate.

A6 epithelia, a cell line originating from the distal tubular part of the kidney of Xenopus laevis, were cultured on permeable supports and mounted in an Ussing-type chamber. Cell thickness (Tc), short-circuit current (Isc) and transepithelial conductance (Gt) were recorded while tissues were bilaterally incubated in NaCl solutions and the transepithelial potential was clamped to zero. Effects of inhibition and stimulation of transepithelial Na+ transport on cell volume and on its regulation during a hyposmotic challenge were investigated. Under control conditions a slow spontaneous decrease of Tc described by a linear baseline was recorded. The reduction of the apical osmolality from 260 to 140 mosmol/kg did not alter cell volume significantly, demonstrating a negligible water permeability of the apical barrier. The inhibition of Na+ uptake by replacing apical Na+ by N-methyl-d-glucamine (NMDG+) did not affect cell volume under isotonic conditions. An increase of Tc by 12.1% above the control baseline was recorded after blocking active transport with ouabain for 60 min. The activation of Na+ transport with insulin or oxytocin, which is known to activate the apical water permeability in other epithelia, did not alter cell volume significantly. The insensitivity of cell volume to alterations in apical Na+uptake or Na+ pump rate confirms the close coupling between apical and basolateral transport processes. The blockage of basolateral K+ channels by 5 mM Ba2+ elicited a significant increase in Tc of 16.3% above control. Quinine, a potent blocker of volume-activated K+ channels, did not change Tc significantly. Basolateral hypotonicity elicited a rapid rise in Tc followed by a regulatory volume decrease (RVD). An RVD was also recorded after blocking apical Na+ uptake as well as after stimulating apical Na+ uptake with oxytocin or insulin. Inhibition of active transport with ouabain as well as blocking K+ efflux at the basolateral side with Ba2+ or quinine abolished the RVD. The inhibition of the RVD by ouabain seems to be caused by a depletion of cellular K+, whereas the effects of Ba2+ and quinine are most likely due to the blockage of the basolateral K+ pathway.

Animals↗

PubMed1995

Regulatory volume decrease in cultured kidney cells (A6): role of amino acids.

Volume regulation was studied in A6 epithelia grown on permeable supports by measuring cell thickness (Tc) while simultaneously recording short circuit current (ISC) and transepithelial conductance (Gt). Lowering the tonicity of the basolateral solution (pi b) from 250 or 215 to 140 mOsm/kg elicited a rapid rise in Tc followed by a regulation of the cell volume towards control. This decrease in Tc displays the characteristics of the regulatory volume decrease (RVD). Upon restoring the isoosmotic conditions, Tc decreased rapidly below its control value. A post RVD regulatory volume increase (RVI) as described for other cell types was not observed. The subsequent reduction of the basolateral osmolality increased Tc to the level recorded at the end of the first hypoosmotic pulse. Because cell content was not altered during the isoosmotic period the second hypoosmotic challenge was isotonic with the cell and did therefore not evoke an RVD. However, the cell did not lose its ability to volume regulate since an RVD could be elicited by further reduction of pi b from 140 to 100 mOsm/kg. The possibility of an involvement of amino acids in the RVD was tested. The amount of amino acids in the cell as well as excreted in the bath was determined by amino acid analysis. Millimolar concentrations of threonine, serine, alanine, glutamate, glycine and aspartate were found in the cell extract. The cellular amino acid concentration was 28.8 +/- 0.4 mM. The amounts of glycine, aspartate and glutamate excreted from the cell during the hypotonic treatment were significantly larger than in control conditions. The excretion of these amino acids during hypotonicity decreased the cellular amino acid concentration by 8.4 +/- 0.2 mM. This quantity cannot completely account for the RVD during the first hypotonic challenge. The addition of glycine, aspartate and glutamate to the bathing solutions, although used at concentrations higher than intracellularly, did not reduce RVD. On the contrary, this maneuver increased the amplitude of the RVD following both hypoosmotic pulses. This result suggests a stimulatory role of the amino acids on the processes responsible for the RVD.

Amino Acids↗

PubMed1994

Effect of insulin on area and Na+ channel density of apical membrane of cultured toad kidney cells.

1. The stimulation of transepithelial Na+ transport caused by insulin in A6 cultured toad kidney cells was investigated by determination of membrane capacitance (Cm), short circuit current (Isc) and current fluctuation analysis. Values of Cm are proportional to membrane area while blocker-induced current fluctuation analysis provides an estimate of the number of active amiloride-sensitive Na+ channels in the apical membrane. 2. Insulin simultaneously increased Cm, Isc and Gt (transepithelial conductance) in epithelia incubated with Na(+)-containing solutions on both sides. 3. Analysis of 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC)-induced noise showed that insulin increased the number of active Na+ channels in the apical membrane, without altering the single channel current. 4. When nystatin was used to permeabilize the apical membrane the impedance data revealed the presence of a second time constant. Analysis of these data indicated that the basolateral membrane capacitance (Cb) is much larger than the apical membrane capacitance (Ca). Insulin administered to nystatin-treated epithelia increased the values for both capacitances. 5. We suggest that the stimulation of transepithelial Na+ transport caused by insulin may be associated with the exocytotic delivery of transporters to the apical membrane.

Amiloride↗

PubMed1994

Cell swelling activates a poorly selective monovalent cation channel in the apical membrane of toad urinary bladder.

We measured the effects of conditions that increase cell volume on ion movements through the Ca(2+)-blockable poorly selective monovalent cation channel of the apical membrane of the toad urinary bladder. Three conditions were studied: dilution of the basolateral solution, basolateral perfusion with solutions prepared with solutes of low reflection coefficient and dilution of the apical solution in bladders treated with oxytocin. All three procedures markedly increased K+ movements and elevated the plateau of the Lorentzian component of the power spectrum by enhancing ion currents through the Ca(2+)-blockable pathway. Simultaneously there was a large increase in Ca(2+)-sensitive conductance. The magnitude of this increased conductance strongly suggests that the stimulation of ion flow is due to increased ion permeability and not solely to increases in driving force across the apical membrane. We were not able to detect an increase in the movements of alkali-earth ions induced by the conditions that increase cell volume. We speculate that activation of the Ca(2+)-blockable channel may play an important role in the regulation of cell volume and/or in K+ homeostasis.

Animals↗