Biomedical subjects

G Droogmans

Publications and source records attributed to G Droogmans.

At least 91 records · Page 5Linked to original sources

PubMed1997

Modulation of voltage-dependent properties of a swelling-activated Cl- current.

We used the patch-clamp technique to study the voltage-dependent properties of the swelling-activated Cl- current (ICl,swell) in BC3H1 myoblasts. This Cl- current is outwardly rectifying and exhibits time-dependent inactivation at positive potentials (potential for half-maximal inactivation of +75 mV). Single-channel Cl- currents with similar voltage-dependent characteristics could be measured in outside-out patches pulled from swollen cells. The estimated single-channel slope conductance in the region between +60 and +140 mV was 47 pS. The time course of inactivation was well described by a double exponential function, with a voltage-independent fast time constant (approximately 60 ms) and a voltage-dependent slow time constant (>200 ms). Recovery from inactivation, which occurred over the physiological voltage range, was also well described by a double exponential function, with a voltage-dependent fast time constant (10-80 ms) and a voltage-dependent slow time constant (>100 ms). The inactivation process was significantly accelerated by reducing the pH, increasing the Mg2+ concentration or reducing the Cl- concentration of the extracellular solution. Replacing extracellular Cl- by other permeant anions shifted the inactivation curve in parallel with their relative permeabilities (SCN- > I- > NO3- > Cl- >> gluconate). A leftward shift of the inactivation curve could also be induced by channel blockers. Additionally, the permeant anion and the channel blockers, but not external pH or Mg2+, modulated the recovery from inactivation. In conclusion, our results show that the voltage-dependent properties of ICl,swell are strongly influenced by external pH, external divalent cations, and by the nature of the permeant anion.

Calcium↗

PubMed1997

Ion channels in vascular endothelium.

The functional impact of ion channels in vascular endothelial cells (ECs) is still a matter of controversy. This review describes different types of ion channels in ECs and their role in electrogenesis, Ca2+ signaling, vessel permeability, cell-cell communication, mechano-sensor functions, and pH and volume regulation. One major function of ion channels in ECs is the control of Ca2+ influx either by a direct modulation of the Ca2+ influx pathway or by indirect modulation of K+ and Cl- channels, thereby clamping the membrane at a sufficiently negative potential to provide the necessary driving force for a sustained Ca2+ influx. We discuss various mechanisms of Ca2+ influx stimulation: those that activate nonselective, Ca(2+)-permeable cation channels or those that activate Ca(2+)-selective channels, exclusively or partially operated by the filling state of intracellular Ca2+ stores. We also describe the role of various Ca(2+)- and shear stress-activated K+ channels and different types of Cl- channels for the regulation of the membrane potential.

Animals↗

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

Swelling-activated efflux of taurine and other organic osmolytes in endothelial cells.

We used a combined biochemical, pharmacological, and electrophysiological approach to study the effects of hyposmotic swelling on organic osmolyte efflux in endothelial cells (EC). In [3H]taurine-loaded monolayers of calf pulmonary artery EC (CPAEC), hyposmolality activated time- and dose-dependent effluxes of [3H]taurine. Swelling-activated [3H]taurine efflux (Jtau swell)in CPAEC was inhibited by the anion channel blockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), fenamates, and also quinine (in a pH-dependent manner), ATP, and the phospholipase A2 inhibitor 4-bromophenacyl bromide. In contrast, Jtau swell was partly or totally insensitive to bumetanide, forskolin, phorbol 12-myristate 13-acetate, and staurosporine. Swelling also activated myo-[3H]inositol efflux that was blocked by tamoxifen, NPPB, DIDS, and niflumic acid. Moreover, the cellular content of taurine and other amino acids was significantly reduced in osmotically activated CPAEC. Finally, in whole cell patch-clamp experiments, taurine, glycine, aspartate, and glutamate exhibited significant permeability for swelling-activated anion channels. In conclusion, hyposmotic swelling activates efflux of taurine and other organic osmolytes in EC. In addition, our results suggest that anion channels may provide a pathway for swelling-activated efflux of organic osmolytes in EC.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1996

Annexin II modulates volume-activated chloride currents in vascular endothelial cells.

The membrane-associated, microfilament-binding protein annexin II is abundantly expressed in endothelial cells from calf pulmonary artery (CPAE cells). We have analyzed its role in the regulation of volume-activated chloride currents (ICl, vol) by loading the cells via the patch pipette with a peptide comprising the N-terminal 14 residues of annexin II. This sequence harbors the binding site for the intracellular annexin II ligand, p11, and the peptide interferes with the annexin II-p11 complex formation. Loading of a CPAE cell with this peptide caused a gradual decrease in the amplitude of ICl, vol during repetitive stimulations with a 28% hypotonic extracellular solution. This run down of the current was virtually absent in untreated cells and in cells that were loaded with a mutated 14-amino acid peptide, which has a single amino acid replacement known to result in a more than 1000 times reduced affinity for binding to p11. We conclude that annexin II-p11 complex formation is either directly or indirectly involved in the activation of ICl, vol in endothelial cells.

Animals↗

PubMed1996

Membrane currents and the resting membrane potential in cultured bovine pulmonary artery endothelial cells.

1. We have used the whole-cell patch-clamp technique to characterize the ionic conductances that determine the resting membrane potential in cultured endothelial cells from calf pulmonary artery (CPAE cells). 2. Resting membrane potentials were scattered between -88 and +5 mV with a mean +/- S.E.M. of -26 +/- 3 mV (n = 104). 3. The most prominent membrane current in resting cells was an inwardly rectifying K+ current. This current showed Na(+)-dependent inactivation and was efficiently blocked by external Ba2+ (EC50 = 2.2 microM), but was relatively insensitive to quinine, quinidine and TEA. 4. Hypertonic cell shrinkage inhibited an outwardly rectifying Cl- current, which was also efficiently blocked by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB; 100 microM), quinine (500 microM) and quinidine (500 microM). 5. A linear, time-independent background current remained after elimination of these two currents. This current was dependent on extracellular monovalent cations with a permeability sequence of Cs+ > Na+ > Li+ >> N-methyl-D-glucamine. It was partially blocked by millimolar concentrations of the divalent cations Ca2+, Ni2+ and Ba2+. Gd3+ (200 microM) had no significant effect on this background current. 6. Continuous measurements of the membrane potential confirm that the three described conductances are the major determinants of the membrane potential. Due to the low slope conductance in the region between -70 and 0 mV, small changes in one of the current components can evoke large depolarizations or hyperpolarizations, which explains the large scattering of the resting membrane potentials.

Animals↗

PubMed1996

The chloride current induced by expression of the protein pICln in Xenopus oocytes differs from the endogenous volume-sensitive chloride current.

1. Phenotypical similarities between ICl,swell, the cell-swelling-induced chloride current and ICln, the nucleotide-sensitive chloride current induced by expression of mammalian pICln in Xenopus oocytes, have led to models which identify pICln either as the volume-sensitive chloride channel or as a cytosolic regulator thereof. 2. To investigate critically the relationship between ICl,swell and pICln two-microelectrode voltage clamp experiments were performed on Xenopus oocytes in which either human pICln was expressed or endogenous ICl,swell was activated. 3. Several criteria that clearly differentiated ICln from ICl,swell were detected. Outward rectification and the discrimination between NO3- and Cl- were more pronounced for ICln. Cyclamate blocked ICln but not ICl,swell. In contrast to ICl,swell, inactivation kinetics of ICln were pH independent and extracellular cAMP blocked only the outward ICln component. Finally, ICln was readily expressed in collagenase-defolliculated oocytes and was not modulated by extracellular hypotonicity, whereas ICl,swell could only be triggered in follicle-enclosed or manually defolliculated oocytes. 4. We therefore conclude that ICln and ICl,swell are two different chloride currents. Consequently, any model which invokes a crucial role for pICln in ICl,swell should be critically reviewed.

Animals↗

PubMed1996

Kinetics of the non-specific calcium leak from non-mitochondrial calcium stores in permeabilized A7r5 cells.

We have investigated the detailed kinetics of the passive Ca2+ leak from non-mitochondrial Ca2+ stores in permeabilized A7r5 cells. The decrease in the content of stored Ca2+ in the presence of 2 microM thapsigargin deviated from a single-exponential curve in the initial phase of the efflux. The deviation persisted after correcting this efflux for passively bound Ca2+. The non-single-exponential nature of the spontaneous release also occurred when the initial store Ca2+ content was reduced to 40% of its original value by pretreatment with 200 nM inositol 1,4,5-trisphosphate (InsP3). The passive Ca2+ leak could be modelled by two exponential curves with discrete rate constants of 0.06 min-1 and 0.98 min-1, and not by any other type of non-exponential decay. We concluded that individual store units are heterogeneous with respect to their passive Ca2+ permeability. This non-exponential nature of the passive Ca2+ release is unrelated to the non-single-exponential InsP3-induced Ca2+ release.

Animals↗

PubMed1996

Do voltage-gated Kv1.1 and inward rectifier Kir2.1 potassium channels form heteromultimers?

Possible heteromultimer formation between Kv- and Kir-type K+ channels was investigated, in connection with the known functional diversity of K+ channels in vivo. Voltage-clamp experiments were performed on Xenopus oocytes, either injected with concatenated Kir2.1-Kv1.1 mRNA, or co-injected with Kv1.1 and Kir2.1 mRNA. K+ currents could be approximated by the algebraic sum of the 2 K+ current types alone. The tandem construct did not show functional expression, although it could be detected by Western blotting. We conclude that Kv1.1 and Kir2.1 alpha-subunit proteins fail to assemble and do not contribute functional diversity to K+ channels.

Animals↗

PubMed1996

Hypotonically induced calcium release from intracellular calcium stores.

Osmotic cell swelling induced by hypotonic stress is associated with a rise in intracellular Ca2+ concentration, which is at least partly due to a release of Ca2+ from internal stores. Since osmotic influx of water dilutes the cytoplasmic milieu, we have investigated how nonmitochondrial Ca2+ stores in permeabilized A7r5 cells respond to a reduction in cytoplasmic tonicity. We now present experimental evidence for a direct Ca2+ release from the stores when exposed to a hypotonic medium. The release is graded, but does not occur through the inositol trisphosphate or the ryanodine receptor. Ca2+ seems to be released through the passive leak pathway, and this phenomenon can be partially inhibited by divalent cations in the following order of potency: Ni2+ = Co2+ > Mn2+ > Mg2+ > Ba2+. This release also occurs in intact A7r5 cells. This novel mechanism of hypotonically induced Ca2+ release is therefore an inherent property of the stores, which can occur in the absence of second messengers. Intracellular stores can therefore act as osmosensors.

Animals↗

PubMed1996

The ubiquitously expressed pICln protein forms homomeric complexes in vitro.

Endothelial cells contain a cell-volume sensitive chloride conductance with biophysical properties similar to those of a ubiquitously expressed chloride current that is activated by cell swelling. The latter current has been associated with the ICln protein (pICln) which may be the chloride channel itself or, alternatively, a channel regulator. We were therefore interested in whether pICln is involved in the endothelial volume-sensitive chloride current. As a first step, we have cloned human pICln and studied its expression at the protein level. Using a polyclonal antiserum raised against human pICln we found a widespread expression of pICln, both in endothelial cells and in other cell lines. A characteristic feature of pICln is its anomalous migration during denaturing polyacrylamide gel electrophoresis. We also demonstrate that bacterially expressed pICln forms homomeric complexes in vitro.

Base Sequence↗

PubMed1996

The volume-activated chloride current in endothelial cells from bovine pulmonary artery is not modulated by phosphorylation.

We employed the patch-clamp technique to investigate the effects of various phosphorylation pathways on activation and modulation of volume-activated Cl- currents (ICl,vol) in cultured endothelial cells from bovine pulmonary arteries (CPAE cells). Half-maximal activation of ICl,vol occurred at a hypotonicity of 27.5+/-1.2%. Run-down of the current upon repetitive activation was less than 15% within 60 min. Stimulation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) or by (-)-indolactam did not affect ICl,vol. Down regulation of PKC activity by a 24-h preincubation of the cells with 0.2 micromol/l PMA, or its inhibition by loading the cells with the specific inhibitory 19-31 pseudosubstrate peptide, did not influence ICl,vol. Trifluoperazine and tamoxifen fully blocked ICl,vol with concentrations required for half-maximal inhibition of 3.0 and 2.4 micromol/l respectively. This inhibitory effect is probably not mediated by the calmodulin-antagonistic action of these compounds, because it occurs at free intracellular [Ca2+] of 50 nmol/l, which are below the threshold for calmodulin activation. The tyrosine kinase inhibitor herbimycin A (1 micromol/l) and genistein (100 micromol/l) did not affect ICl,vol. Exposing CPAE cells to lysophosphatidic acid (1 micromol/l), an activator of p42 MAPkinase and the focal adhesion kinase p125(FAK) in endothelial cells, neither evoked a Cl- current nor affected ICl,vol. Neither wortmannin (10 micromol/l), an inhibitor of MAP kinases and of PI-3 kinase, nor rapamycin (0.1 mmol/l), which interferes with the p70S6 kinase pathway, affected ICl,vol. Exposure of CPAE cells to heat or Na-arsenite, both activators of a recently discovered stress-activated tyrosine phosphorylation pathway, neither activated a current nor affected the hypotonic solution-induced Cl- current. We conclude that none of the studied phosphorylation pathways is essential for the activation of the Cl- current induced by hypotonicity.

Animals↗

PubMed1996

Activation of the volume-sensitive chloride current in vascular endothelial cells requires a permissive intracellular Ca2+ concentration.

Combined patch clamp and Ca2+-measurements (Fura-2) were used to study the dependence of volume-activated Cl--currents (ICl,vol) of endothelial cells from bovine pulmonary artery on the intracellular Ca2+-concentration [Ca2+]i. Loading the cells with high concentrations of EGTA or BAPTA via ruptured membrane patches or by preincubating them with 50 microM BAPTA-AM caused a substantial decrease of ICl,vol. This reduction was independent of the activation state of the current: the current amplitude was not only diminished if [Ca2+]i was lowered at the peak of the volume-activated current, but this low Ca2+-concentration also prevented activation of the current by a second hypotonic challenge.ICl,vol is already maximally activated at intracellular Ca2+-concentrations between 50 and 100 nmol/l, a further increase of [Ca2+]i does not affect the size of ICl,vol.These results indicate that a sustained full activation of ICl,vol in endothelial cells requires submicromolar concentrations of Ca2+, and that changes in [Ca2+]i do not modulate the current.

Animals↗

PubMed1996

Characterization of volume-activated chloride currents in endothelial cells from bovine pulmonary artery.

We have measured the kinetic and pharmacological properties of volume-activated Cl- currents (ICl, vol) in endothelial cells, and tried to correlate them with those of the already described volume-activated current ICln. Both conductances show a similar permeability sequence for monovalent anions, and they are blocked by extracellular ATP. In the present report, we demonstrate by Western blot and RT-PCR that cultured endothelial cells from bovine pulmonary artery (CPAE) contain pICln. The expression of this protein has been shown to be closely associated with the ICln current. ICl, vol showed however, in contrast with ICln, no striking inactivation at positive potentials. This property is also at variance with that of the volume-activated current related to MDR-1. Activation of ICl, vol at potentials more negative than -80 mV was not time dependent, which excludes a major contribution of a ClC-2 related current. The antiviral nucleoside analogue AZT (3'-azido-3'-deoxythymidine) inhibited ICl, vol by 21 +/- 2.7% (n = 10), at a concentration of 100 microM. Another antiviral drug, acyclovir (ACV, 9-[2-hydroxyethoxy) methyl]guanine) blocked ICl, vol by 27 +/- 6.2% at 100 microM (n = 11). Both blocking effects are much smaller than those reported for ICln. The phenol derivative gossypol, which blocks ICln-related currents, efficiently inhibited ICl, vol in CPAE cells (67 +/- 2.1% at 1 microM, n = 7, KI = 0.4 microns). The presence of pICln in CPAE cells and the similar qualitative pharmacological profile of ICl, vol and ICln support the hypothesis that pICln is a good molecular candidate for ICl, vol in endothelial cells. The discrepant kinetic properties may indicate that these time-dependent currents at high positive or negative potentials are not intrinsic properties of the channels, but are caused by time-dependent depletion/accumulation phenomena due to the large amplitudes of these currents.

Animals↗

PubMed1996

Volume-activated Cl- channels.

1. An increase in cell volume activates, in most mammalian cells, a Cl- current, ICl,vol. This current is involved in a variety of cellular functions, such as the maintenance of a constant cell volume, pH regulation, and control of membrane potential. It might also play a role in the regulation of cell proliferation and in the processes that control transition from proliferation to differentiation. This review focuses on various aspects of this current, including its biophysical characterisation and its functional role for various cell processes. 2. Volume-activated Cl- channels show all outward rectification. Iodide is more permeable than chloride. In some cell types, ICl,vol inactivates at positive potentials. Single channel conductance can be divided mainly into two groups: small (< 5 pS) and medium conductance channels (around 50 pS). 3. The pharmacology and modulation of these channels are reviewed in detail, and suggest the existence of an heterogeneous family of multiple volume-activated Cl- channels. 4. Molecular candidates for this channel (i.e. ClC-2, a member of the ClC-family of voltage-dependent Cl- channels, the mdr-1 encoded P-glycoprotein, the nucleotide-sensitive pICln protein and phospholemman) will be discussed.

Animals↗

PubMed1996

Potent block of volume-activated chloride currents in endothelial cells by the uncharged form of quinine and quinidine.

1. The effects of quinine and quinidine on the volume-activated chloride current (ICl(vol)) in cultured endothelial cells from bovine pulmonary artery were studied by use of the whole-cell patch-clamp technique. 2. At pH 7.4 both quinine and quinidine induced a fast and reversible block of ICl(vol)) with Ki values of 20 +/- 4 microM and 30 +/- 10 microM, respectively. 3. The blocking efficiency of both drugs increased dramatically with increasing extracellular pH, indicating that the blockade is mediated by the uncharged form of quinine and quinidine. 4. These results suggest a hydrophobic interaction with high affinity between volume-activated chloride channels and uncharged quinine and quinidine within the membrane bilayer of endothelial cells.

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

Volume-activated chloride currents are not correlated with P-glycoprotein expression.

It has been proposed that P-glycoprotein, the product of the human MDR1 gene, may function not only as a drug transporter but, depending on the conditions, as a volume-activated Cl- channel [Valverde, Diaz, Sepúlveda, Gill, Hyde and Higgins (1992) Nature (London) 355, 830-833; Gill, Hyde, Higgins, Valverde, Mintenig and Sepúlveda (1992) Cell 71, 23-32]. To verify this hypothesis, we have compared volume-activated Cl- currents with the level of MDR1 mRNA and its protein product in the human KB3 (epitheloid lung cancer) and HeLa cell lines. The related MDR2 was also included to find out whether it could account for observed discrepancies between Cl- current and MDR1 expression. A 40% decrease in osmolarity evoked a Cl- current in both cell types (at +80 mV: 50.3 +/- 4.3 pA/pF in KB3, n = 13; 28.2 +/- 3.3 pA/pF in HeLa, n = 16). The blocking of this current in both cell types by 5-nitro-2-(3-phenylpropylamino)-benzoic acid and by 1,9-dideoxyforskolin is similar to that of the presumed P-glycoprotein associated Cl- channel. As measured by reverse-transcriptase polymerase chain reaction, KB3 cells expressed only an extremely small amount of the messengers for MDR1 and MDR2. The signal observed for MDR1 in HeLa cells was at least an order of magnitude more intense than in KB3 cells, while MDR2 mRNA was undetectable. A clear difference in MDR1 expression between KB3 and HeLa was also observed at the protein level. These data are difficult to reconcile with the hypothesis that in HeLa and KB3 cells MDR1- or MDR2- encoded P-glycoproteins are associated with volume-activated Cl- channels.

ATP Binding Cassette Transporter, Subfamily B, Mem↗