Biomedical subjects

W Van Driessche

Publications and source records attributed to W Van Driessche.

At least 19 recordsLinked to original sources

PubMed2000

Involvement of protein tyrosine kinase in osmoregulation of Na(+) transport and membrane capacitance in renal A6 cells.

Renal A6 cells have been reported in which hyposmolality stimulates Na(+) transport by increasing the number of conducting amiloride-sensitive 4-pS Na(+) channels at the apical membrane. To study a possible role of protein tyrosine kinase (PTK) in the hyposmolality-induced signaling, we investigated effects of PTK inhibitors on the hyposmolality-induced Na(+) transport in A6 cells. Tyrphostin A23 (a PTK inhibitor) blocked the stimulatory action of hyposmolality on a number of the conducting Na(+) channels. Tyrphostin A23 also abolished macroscopic Na(+) currents (amiloride-sensitive short-circuit current, I(Na)) by decreasing the elevating rate of the hyposmolality-increased I(Na). Genistein (another type of PTK inhibitor) also showed an effect similar to tyrphostin A23. Brefeldin A (BFA), which is an inhibitor of intracellular translocation of protein, blocked the action of hyposmolality on I(Na) by diminishing the elevating rate of the hyposmolality-increased I(Na), mimicking the inhibitory action of PTK inhibitor. Further, hyposmolality increased the activity of PTK. These observations suggest that hyposmolality would stimulate Na(+) transport by translocating the Na(+) channel protein (or regulatory protein) to the apical membrane via a PTK-dependent pathway. Further, hyposmolality also caused an increase in the plasma (apical) membrane capacitance, which was remarkably blocked by treatment with tyrphostin A23 or BFA. These observations also suggest that a PTK-dependent pathway would be involved in the hyposmolality-stimulated membrane fusion in A6 cells.

Animals↗

PubMed2000

Maitotoxin induces insertion of different ion channels into the Xenopus oocyte plasma membrane via Ca(2+)-stimulated exocytosis.

The activation of cation channels in oocytes of Xenopus laevis by the marine poison maitotoxin (MTX) was monitored as membrane current (I(m)), conductance (Gm) and membrane surface area determined by continuous measurements of membrane capacitance (Cm). When MTX (25 pM) was added to the bathing solution there was an abrupt, large increase in inward membrane currents. Current/voltage relationships (I/V curves) were linear and suggested activation of voltage-independent non-selective cation channels (NSCC). MTX-induced Ca(2+)-sensitive currents were mainly carried by Na+ and were suppressed by low (0 mM) or high (40 mM) external Ca2+ concentrations and removal of Na+. Gadolinium (Gd3+, 10-500 microM) also had inhibitory effects, demonstrating the possible involvement of stretch-activated cation channels (SACC). In a high concentration (500 microM), amiloride substantially reduced the MTX-activated current while lower amiloride concentrations (50-100 microM) stimulated the current further. Continuous measurements of Cm revealed that MTX induced exocytotic delivery and functional insertion of new channel proteins into the plasma membrane, indicated by a Ca(2+)-dependent increase in membrane surface area by around 28%. From these data we conclude that MTX activates NSCC that require relatively high concentrations of amiloride to be blocked. Furthermore, MTX possibly stimulates activation of Gd(3+)- and Ca(2+)-sensitive mechanosensitive cation channels. Stimulation of these channels is achieved by exocytotic delivery and functional insertion of new channels into the plasma membrane in a pathway that depends on the presence of extracellular Ca2+.

Amiloride↗

PubMed2000

Effects of extracellular Mg2+ on transepithelial capacitance and Na+ transport in A6 cells under different osmotic conditions.

The electrophysiological characteristics of monolayers of cultured renal epithelial A6 cells were studied under short-circuit conditions. Replacing basolateral isosmotic (260 mOsm/kg H2O) media by hyposmotic (140 mOsm/kg H2O) solutions transiently increased the transepithelial capacitance (C(T)) by 57.3+/-2.3% after 16 min. The transepithelial Na+ current (I(Na)) increased concomitantly from 4.2+/-0.7 to 26.1+/-2.6 microA/cm2 with a time course that was noticeably slower, reaching its maximum after 60 min of hypotonicity. The transepithelial conductance (G(T)) increased synchronously with I(Na). Analysis of blocker-induced noise in I(Na), using the amiloride analogue 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC), showed that the hypotonic shock increased Na+ channel density (N(T)) at the apical border. The presence of 10 mM Mg2+ on both sides of the epithelium suppressed the hypotonicity-induced C(T) increase to 14.3+/-0.5%, whereas the I(Na) increase was even larger than without Mg2+. Both effects of Mg2+ were located at an extracellular, basolateral site, because apical administration was without effect, whereas the acute basolateral addition of Mg2+ at the moment of the hypotonic shock was sufficient. Interaction between Mg2+ and Ca2+ influenced the behaviour of C(T). At constant osmolality (200 mOsm/kg H2O) 10 mM Mg2+ increased I(Na), leaving C(T) unaffected, whereas 10 mM Ca2+ stimulated both I(Na) and CT. In the presence of 1 mM Mg2+, however, the Ca(2+)-induced CT increase was abolished. The failure of CT to increase during stimulation of I(Na) by Mg2+ suggests that the divalent cation activates pre-existing channels in the apical membrane. Noise analysis showed that the natriferic effects of Mg2+ were also mediated by an increase in NT. The moderate initial increase in CT in the presence of Mg2+ under hypotonic conditions, occurring in parallel with increases in GT and I(Na), reflects most likely Na+ channel insertion induced by the hypotonic treatment. However, the large, transient, Mg(2+)-sensitive increase in CT, not correlated with increases in GT and I(Na), seems to be unrelated to Na+ channel recruitment.

Animals↗

PubMed1999

Cell swelling activates stress-activated protein kinases, p38 MAP kinase and JNK, in renal epithelial A6 cells.

Osmotic shock is well recognized as one of the factors activating stress-activated protein kinases (SAPKs), p38 MAP kinase and c-Jun N-terminal kinases (JNKs). In renal epithelial A6 cells, hypo-osmotic shock transiently activated SAPKs with maximal activation at 5 min. A6 cells showed a regulatory volume decrease (RVD) after swelling when the cells were exposed to a hypo-osmotic solution. In contrast, activation of SAPKs was maintained over 90 min after hypo-osmotic shock in the presence of 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, a Cl(-) channel blocker), which completely blocked the RVD and kept the cells continuously swelling. Exposure of the cells to a high K(+) iso-osmotic solution containing nystatin, which induces continuous cell swelling, also continuously activated SAPKs. Furthermore, membrane deformation induced by chlorpromazine activated SAPKs. These results suggest that changes in membrane tension by cell swelling or chlorpromazine, but not osmolality, are important steps for activation of SAPKs in A6 cells.

Animals↗

PubMed1999

Transepithelial capacitance decrease reveals closure of lateral interspace in A6 epithelia.

A sine wave method was used to measure transepithelial capacitance (CT) at 4.1 kHz (CHFT ). Model calculations show that CHFT reflects the equivalent capacitance of the series arrangement of apical and basolateral membrane capacitance. Cell swelling induced by reducing the basolateral osmolality from 260 to 140 mosmol/kg H2O (NaCl or sucrose removal) transiently decreased CHFT. The decrease in CHFT (DeltaCHFT ) reached its maximum 30 s after the onset of cell swelling and a complete recovery of CHFT was attained within 3-4 min. DeltaCHFT could be diminished by manoeuvres that reduced the rate or amplitude of cell swelling, i.e. lowering the temperature or treatment with low concentrations of glutaraldehyde (0.025%). DeltaCHFT increased with the magnitude of the osmotic perturbation but saturated at large volume expansions. DeltaCHFT increased with culture time. Electron micrographs showed a clear correlation between time course of CHFT changes and the closure of the lateral interspace (LIS). A striking correlation between the occurrence of CHFT recovery and the ability of the cells to develop a regulatory volume decrease (RVD) was found: Gd3+ (0.5 mM) inhibited both phenomena. The frequency dependence of CT was obtained from impedance spectra recorded over the range of 4 Hz to 22 kHz. These data agree with model calculations in which the contribution of the access resistance to the lateral membrane was included. All observations are consistent with the idea that DeltaCHFT originates from the closure of the LIS during cell swelling. The latter phenomenon increases the access resistance to the lateral membrane, which results in a marked reduction of the basolateral membrane area detected at high frequencies with capacitance measurements.

Algorithms↗

PubMed1999

Forskolin increases apical sodium conductance in cultured toad kidney cells (A6) by stimulating membrane insertion.

The role of membrane traffic in the stimulation of apical Na+ permeability caused by increases in cytoplasmic cyclic AMP was assessed by measuring the effects of forskolin on transepithelial capacitance (CT), transepithelial conductance (GT), and short-circuit current (Isc) in A6 cultured toad kidney cells. Apical water permeability was probed by recording cell volume changes after reducing the osmolality of the apical bath. We found that forskolin does not increase the osmotic water permeability of the apical membrane of A6 cells, and thus does not stimulate the insertion of water channels. Comparison of the effects of forskolin and insulin on Na+ transport demonstrated that both agents produce reversible increases in CT, GT and Isc. GT and CT increased proportionally during the rising phase of the insulin response. However, a non-linear relationship between both parameters was recorded when forskolin was given in NaCl Ringer's solution. The relationship between CT and GT became linear after the effects of forskolin on Cl- conductances were eliminated by substituting Cl- by an impermeant anion. In contrast, in Cl--containing Na+-free solutions, the non-linearity became more pronounced. Successive additions of insulin and forskolin caused additive increases in CT. Because increases in CT and Na+ transport occurred in the absence of stimulation of water permeability and increases of CT and GT were directly proportional when Na+ was the major permeating ion across the apical membrane, we suggest that the increase in apical Na+ permeability in the presence of either forskolin or insulin is due to the insertion of channels residing in intracellular pools. In contrast, the increased Cl- permeability caused by forskolin may be related to the activation of channels already present in the membrane.

Animals↗

PubMed1999

Capacitance measurements reveal different pathways for the activation of CFTR.

We used the Xenopus laevis oocyte expression system to characterize adenosine 3',5'-cyclic monophosphate (cAMP) activation of the cystic fibrosis transmembrane conductance regulator (CFTR). With conventional two-microelectrode voltage-clamp techniques, we recorded transmembrane conductance (Gm) and membrane current (Im). Using five different sine wave frequencies, we also monitored changes of the plasma membrane surface area by recording continuously membrane capacitance (Cm) under voltage-clamp conditions. Impedance spectra recorded in the frequency range 0.1-500 Hz showed that, at least up to 200 Hz, Cm is independent of the frequency. In control oocytes, cAMP (100 microM) treatment did not affect Gm or Im but evoked a small, slowly occurring increase in Cm, probably mediated by cAMP-stimulated exocytosis. However, in oocytes expressing CFTR, large simultaneous increases of Gm, Im and Cm occurred after stimulation with cAMP. Oocytes injected with the delta F508 CFTR mutant behaved like control oocytes and cAMP had no additional effects on Gm, Im or Cm. In oocytes injected with wild-type CFTR, adenosine 5'-triphosphate (ATP, 100 microM) did not activate the cAMP-induced augmentation of Im, Gm or Cm further. On the other hand, cAMP-induced increases in Cm were reduced significantly by the specific blockers of protein kinase A (PKA) KT5720 and N-[2-(methylamino-9-ethyl]-5-isoquinolinesulphonamide hydrochloride (H8), whereas the increases in Gm and Im were essentially unaffected by these agents. Reducing intracellular Ca2+ by injection of a Ca2+ chelator 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) prevented PKA-dependent exocytosis while activation of Im and Gm of already-inserted CFTR still could be detected. The specific cAMP antagonist adenosine 3',5'-cyclic monophosphothioate Rp diastereomer (RpcAMPS) completely suppressed the effects of cAMP on all parameters. These findings are consistent with the concept of different pathways of CFTR activation by cAMP: already-inserted CFTR Cl- channels are activated directly by cAMP, while traffic of CFTR proteins from an intracellular pool to the plasma membrane and functional insertion into the plasma membrane occurs via cAMP- and Ca(2+)-dependent PKA-mediated exocytosis.

Adenosine Triphosphate↗

PubMed1999

Frequency-dependent capacitance of the apical membrane of frog skin: dielectric relaxation processes.

Impedance analysis of the isolated epithelium of frog skin (northern Rana pipiens) was carried out in the frequency range between 0.1 Hz and 5.5 kHz while Na+ transport was abolished. Under these conditions, the impedance is determined almost completely by the dielectric properties of the apical membranes of the cells and the parallel shunt resistance. The modeling of the apical membrane impedance function required the inclusion of dielectric relaxation processes as originally described by. J. Chem. Phys. 9:341-351), where each process is characterized by a dielectric increment, relaxation frequency, and power law dependence. We found that the apical plasma membrane exhibited several populations of audio frequency dielectric relaxation processes centered at 30, 103, 2364, and 6604 Hz, with mean capacitive increments of 0.72, 1.00, 0.88, and 0.29 microF/cm2, respectively, that gave rise to dc capacitances of 1.95 +/- 0.06 microF/cm2 in 49 tissues. Capacitance was uncorrelated with large ranges of parallel shunt resistance and was not changed appreciably within minutes by K+ depolarization and hence a decrease in basolateral membrane resistance. A significant linear correlation existed between the dc capacitance and Na+ transport rates measured as short-circuit currents (Cadc = 0.028 Isc + 1.48; Isc between 4 and 35 microA/cm2) before inhibition of transport by amiloride and substitution of all Na+ with NMDG (N-methyl-D-glucamine) in the apical solution. The existence of dominant audio frequency capacitive relaxation processes complicates and precludes unequivocal interpretation of changes of capacitance in terms of membrane area alone when capacitance is measured at audio frequencies.

Animals↗

PubMed1998

Apical Cl- channels in A6 cells.

Short-circuit current (Isc), transepithelial conductance (Gt), electrical capacitance (CT) and the fluctuation in Isc were analyzed in polarized epithelial cells from the distal nephron of Xenopus laevis (A6 cell line). Tissues were incubated with Na+- and Cl--free solutions on the apical surface. Basolateral perfusate was NaCl-Ringer. Agents that increase cellular cAMP evoked increases in Gt, CT, Isc and generated a Lorentzian Isc-noise. The responses could be related to active, electrogenic secretion of Cl-. Arginine-vasotocin and oxytocin caused a typical peak-plateau response pattern. Stimulation with a membrane-permeant nonhydrolyzable cAMP analogue or forskolin showed stable increases in Gt with only moderate peaking of Isc. Phosphodiesterase inhibitors also stimulated Cl- secretion with peaking responses in Gt and Isc. All stimulants elicited a spontaneous Lorentzian noise, originating from the activated apical Cl- channel, with almost identical corner frequency (40-50 Hz). Repetitive challenge with the hormones led to a refractory behavior of all parameters. Activation of the cAMP route could overcome this refractoriness. All agents caused CT, a measure of apical membrane area, to increase in a manner roughly synchronous with Gt. These results suggest that activation of the cAMP-messenger route may, at least partly, involve exocytosis of a vesicular Cl- channel pool. Apical flufenamate depressed Cl- current and conductance and apparently generated blocker-noise. However, blocking kinetics extracted from noise experiments could not be reconciled with those obtained from current inhibition, suggesting the drug does not act as simple open-channel inhibitor.

Animals↗

PubMed1998

Cu2+ reveals different binding sites of amiloride and CDPC on the apical Na channel of frog skin.

The effect of Cu2+ ions, present in the mucosal bathing solution, on the transepithelial short-circuit current (Isc) and conductance (Gt) and on the blocker-induced noise of apical Na channels, was studied on the isolated ventral skin of the frog Rana temporaria. Cu2+ effects were concentration-dependent, the full effect being reached at 50 micromol/l. Cu2+ increased Isc and Gt; this effect was eliminated by high concentrations of amiloride (30 micromol/l) and of CDPC (150 micromol/l). Cu2+ markedly reduced the corner frequency (fc) of the Na channel noise, while having virtually no effect on the fc of CDPC-induced noise. Cu2+ reduces the association rate constant of amiloride to the Na channel to one third; this effect is interpreted as indicating competition between Cu2+ and amiloride for the same (negatively charged) binding site on the channel, while CDPC appears to bind on a different site.

Amiloride↗

PubMed1998

Na+ dependence of single-channel current and channel density generate saturation of Na+ uptake in A6 cells.

In high-resistance, salt-absorbing epithelia the apical amiloride-sensitive Na+ channel is the key site for regulation of salt and water balance. The saturation of macroscopic Na+ transport through these channels was investigated using A6 epithelial monolayers. The relation between transepithelial Na+ transport (INa) and apical Na+ concentration ([Na+]ap) under short-circuit conditions was studied. Michaelis-Menten analysis of the saturable short-circuit current (Isc) yielded an apparent Michaelis-Menten constant (KmI) of 5 mmol/l and a maximal current (Imax) of 8 microA/cm2. The microscopic parameters underlying INa, namely the single-channel current (i) and the open channel density (No), were investigated by the analysis of current fluctuations induced by the electroneutral amiloride analogue CDPC (6-chloro-3, 5-diaminopyrazine-2-carboxamide). A two-state model analysis yielded the absolute values of i (0.18 +/- 0.01 pA) and No (65.38 +/- 9.57 million channels/cm2 of epithelium) at [Na+]ap = 110 mmol/l containing 50 mumol/l CDPC. Our data indicate that in A6 cells both i and No depend on [Na+]ap. Between 3 and approximately 20 mmol/l the density of conducting pores, No, decreases sharply and behaves again as an almost [Na+]ap-independent parameter at higher [Na+]ap. The single-channel current clearly saturates with an apparent Michaelis-Menten constant, Kmi, of approximately 17 mmol/l. Thus, the [Na+]ap dependence of No as well as the limited transport capacity of the amiloride-sensitive Na+ channel are both responsible for the saturation of INa.

Amiloride↗

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↗