Biomedical subjects

B Nilius

Publications and source records attributed to B Nilius.

At least 19 recordsLinked to original sources

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↗

PubMed1995

Drug-transport and volume-activated chloride channel functions in human erythroleukemia cells: relation to expression level of P-glycoprotein.

The characteristics of volume-activated chloride currents, drug transport function and levels of P-glycoprotein (PgP) expression were compared between two human chronic erythroleukemia cell lines: a parental (K562) cell line and a derivative obtained by vinblastine selection (K562 VBL400). Parental K562 cells showed no detectable P-glycoprotein expression, measured at the protein level (immunofluorescence labeling with monoclonal antibodies), and had very low levels of MDR-1 mRNA expression (RT-PCR analysis), when compared with levels measured in K562 VBL400. Differences in Pgp-mediated transport were estimated by comparing the rates of Fluo3 accumulation. The higher drug-transport function of K562 VBL400 cells (e.g., lower Fluo3 accumulation) correlated with their elevated levels of MDR-1. The rate of dye transport was sensitive to verapamil but was not affected by the tonicity of the extracellular medium. In contrast to the clear differences in transport function, the characteristics of chloride currents induced by cell swelling were indistinguishable between the two cell lines. Currents measured in the whole-cell configuration were outwardly rectifying, had a higher permeability to iodide than to chloride (SCN- > I- > Cl- > gluconate), were potently blocked by NPPB and were unresponsive to verapamil. The percentage of responding cells and the mean current density were nearly identical in both cell lines. In addition, activation of the volume-sensitive current was not prevented during whole-cell recordings obtained with pipettes containing high concentration of cytotoxic drugs (vincristine or vinblastine). These results do not lend support to the previously reported association between Pgp expression and volume-sensitive chloride channels, and suggest that a different protein is responsible for this type of chloride channel in K562 cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Lack of correlation between mdr-1 expression and volume-activation of cloride-currents in rat colon cancer cells.

Correlation between expression of the mdr-1 genes (a and b) at the mRNA and protein level and volume-activation of chloride-channels was studied in rat colon cancer CC531 cells by means of RT-PCR, Western blotting and patch clamp, respectively. Three different kinds of cell lines were used: CC531-PAR, CC531-COL and CC531-REV. At the mRNA level, the parental cell line CC531-PAR showed significantly less mdr-1a expression in comparison with CC531-COL, a drug-resistant cell line induced from the parental CC531 cells by growth in the presence of colchicin. The third cell line, CC531-REV, was a spontaneous revertant of the drug-resistant cell line to a drug-sensitive one, but with a maintained level of mdr-1a mRNA. In none of the three cell lines, mdr-1b mRNA could be detected. At the protein level, a clear difference in mdr1 expression between CC531-PAR/REV and CC531-COL was observed. Although the amount of mdr-1a mRNA detected in CC531-REV was comparable to that found in CC531-COL, the amount of mdr-1 encoded protein in CC531-REV was remarkably reduced. In all three cell types, cell swelling activated chloride-currents which could be blocked by NPPB.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Immunological stimulation of single rat basophilic leukemia RBL-2H3 cells co-activates Ca(2+)-entry and K(+)-channels.

The relationship between type 1 Fc epsilon-receptor (Fc epsilon RI) mediated cell stimulation, Ca(2+)-signals and membrane currents was studied in rat mucosal mast cells, subline RBL-2H3 by combining patch-clamp, Fura-2, 45Ca(2+)-uptake and secretory response measurements. Cells were stimulated by Fc epsilon RI clustering either with IgE and antigen or by an IgE specific monoclonal antibody. Both stimuli induced a biphasic increase in the free intracellular Ca(2+)-concentration ([Ca2+]i). Fc epsilon RI clustering in Ca(2+)-free solution induces a transient increase in [Ca2+]i reflecting Ca2+ release from the Ins(1,4,5)P3 sensitive stores. Mn2+ applied to a nominally Ca(2+)-free solution, causes quenching of the Fura-2 emission during Fc epsilon RI clustering, indicating activation of a transmembrane pathway for the entry of extracellular calcium ions. Whole-cell current-voltage relationship of resting cells showed strong inward rectification. The inward current component at a potential of -100 mV is increased by 23 +/- 11% (n = 14) upon Fc epsilon RI clustering, whereas the outward component at +50 mV was enhanced by 45 +/- 6%. The Fc epsilon RI activated current was identified as due to K+ ions, because it reversed close to the K(+)-equilibrium potential, was blocked by Ba2+ or Cs+ containing or K(+)-free bath solutions. It was also inhibited by TEA and quinidine, while DIDS had no effect. Moreover, an inwardly rectifying K(+)-channel with a conductance of 28 pS was recorded in single channel measurements. The open probability of this channel increased by 39 +/- 16% (n = 8) upon Fc epsilon RI clustering. Superfusion of the cells with nominally K(+)-free solution also significantly inhibited both the Fc epsilon RI mediated 45Ca2+ uptake and the secretory response of the cells. We conclude that activation of K(+)-channels upon Fc epsilon RI clustering is functionally involved in the control and the maintenance of the secretory response of RBL-2H3 mast cells.

Animals↗

PubMed1995

Ca2+ release and activation of K+ and Cl- currents by extracellular ATP in distal nephron epithelial cells.

We have measured ionic currents and changes in intracellular Ca2+ concentration ([Ca2+]i) induced by extracellular ATP in single epithelial cells of the distal nephron from toad (A6 cells). ATP increased [Ca2+]i and concomitantly activated ionic currents. The ATP concentration for half-maximal increase in [Ca2+]i was approximately 10 microM. Current activation and elevation of [Ca2+]i also occurred in Ca(2+)-free bath solutions but were abolished by loading the cells via the patch pipette with 10 mM 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA) or by preincubating the cells with 10 microM BAPTA-acetoxymethyl ester for 120 min. ATP-activated currents reversed at -53.9 +/- 1.9 mV (n = 22). Tetraethylammonium (TEA, 25 mM), a K+ channel blocker, partially blocked this current but did not affect the Ca2+ transients. The TEA-insensitive component of the current reversed close to Cl- equilibrium potential. 5-Nitro-2-(3-phenylpropylamino) benzoic acid, a putative Cl- channel blocker (100 microM), abolished nearly completely the ATP-activated current. Suramin (100 microM), a P2-purinergic receptor antagonist, strongly attenuated both Ca2+ transients and currents. In cell-attached patches, single channel currents activated by ATP could be observed, i.e., an inwardly rectifying K+ channel with a slope conductance for inward currents of approximately 32 pS and an ohmic Cl- channel with a conductance of 34 pS. It is concluded that ATP activates both Cl- and K+ channels in distal nephron epithelial cells by a Ca(2+)-dependent mechanism.

Adenosine Triphosphate↗

PubMed1994

Inhibition of capacitative Ca2+ entry by a Cl- channel blocker in human endothelial cells.

We have used the patch clamp technique in combination with intracellular calcium measurements to measure simultaneously Ca2+ entry and ionic currents activated by emptying of intracellular Ca2+ stores (capacitative Ca2+ entry and Ca2+ release-activated Ca2+ currents, CRAC) in human endothelial cells from umbilical veins. Intracellular stores were depleted of Ca2+ by preincubating endothelial cells for 20 minutes with 2 microM thapsigargin in Ca(2+)-free solution. Reapplication of 10 mM [Ca2+]e evoked an increase in [Ca2+]i indicating Ca2+ influx after the thapsigargin-induced store depletion (capacitative Ca2+ entry), however no measurable CRAC could be detected. The increase in [Ca2+]i after [Ca2+]e resubmission was substantially reduced in the presence of 50 microM NPPB (5-nitro-2-(3-phenylpropylamino)-benzoic acid) from 0.77 +/- 0.25 microM to 0.2 +/- 0.06 microM (n = 6) at a holding potential of -40 mV. Estimates of the capacitative Ca2+ entry at various membrane potentials from the first time derivative of the Ca2+ transients showed a highly inwardly rectifying I-V curve with a Ca2+ inward current amplitude of 1.0 +/- 0.3 pA (membrane capacitance 59 +/- 9 pF, n = 8) at -80 mV. This current amplitude was decreased to 0.32 +/- 0.12 pA (n = 6) in the presence of 50 microM NPPB. This corresponds to a decrease in the Ca2+ permeability of the endothelial cell membrane from 0.15 x 10(-8) cm/s (control) to 0.06 x 10(-8) cm/s (50 microM NPPB).

Calcium↗

PubMed1994

Amplitude modulation of Ca2+ signals induced by histamine in human endothelial cells.

We have addressed the problem of whether the agonist concentration sensed by endothelial cells is encoded by the sustained rise of the intracellular Ca2+ concentration ([Ca2+]i) or by the frequency of intracellular Ca2+ oscillations. Single or confluent endothelial cells from umbilical veins were stimulated for 15 min with histamine (0.03 to 100 mumol/l), and the concomitant changes in [Ca2+]i were measured with fura-2/AM. Application of histamine at concentrations above 0.1 mumol/l resulted always in a fast spike of [Ca2+]i, followed by a slow decline to a sustained plateau level, which depends on the presence of extracellular Ca2+. At the same time of the development of this plateau phase, quenching of the fura-2/AM signal occurred during agonist stimulation in a Ca(2+)-free, 1.5 mmol/l Mn2+ containing solution, indicating influx of divalent cations during this time. From 48 cells in 1.5 mmol/l [Ca2+]e we obtained a close relation between histamine concentration and time integral of [Ca2+]i taken over the 15 min recording of the plateau [Ca2+]i. The half-maximal increase in the integral of [Ca2+]i was at 0.7 mumol/l for solitary cells, 1.2 mumol/l for clustered cells and 1.2 mumol/l for the plateau Ca2+ level. Repetitive Ca2+ spikes or Ca2+ oscillations appeared only in 16 out of 48 cells, but their frequency was not correlated to the agonist concentration. Ca2+ oscillations were only observed in a concentration window between 0.1 and 1 mumol/l histamine, both in single and in clustered endothelial cells. Our results indicate that coding of the agonist concentration in endothelial cells is not related to the frequency of Ca2+ oscillations, but is closely correlated with the plateau level of intracellular Ca2+.

Calcium↗

PubMed1994

Cytoskeletal modulation of the response to mechanical stimulation in human vascular endothelial cells.

Possible interactions of cytoskeletal elements with mechanically induced membrane currents and Ca2+ signals were studied in human endothelial cells by using a combined patch-clamp and Fura II technique. For mechanical stimulation, cells were exposed to hypotonic solution (HTS). The concomitant cell swelling activates a Cl- current, releases Ca2+ from intracellular stores and activates Ca2+ influx. To interfere with the cytoskeleton, cells were loaded either with the F-actin-stabilizing agent phalloidin (10 mumol/l), or the F-actin-depolymerizing substance cytochalasin B (50 mumol/l). These were administered either in the bath or the pipette solutions. The tubulin structure of the endothelial cells was modulated by taxol (50 mumol/l), which supports polymerization of tubulin, or by the depolymerizing agent colcemid (10 mumol/l) both applied to the bath. Immunofluorescence experiments show that under the chosen experimental conditions the cytoskeletal modifiers employed disintegrate the F-actin and microtubuli cytoskeleton. Neither of these cytoskeletal modifiers influenced the HTS-induced Cl- current. Ca2+ release was not affected by cytochalasin B, taxol or colcemid, but was suppressed if the cells were loaded with phalloidin. Depletion of intracellular Ca2+ stores by thapsigargin renders the intracellular [Ca2+] sensitive to the extracellular [Ca2+], which is indicative of a Ca2+ entry pathway activated by store depletion. Neither cytochalasin B nor phalloidin affected this Ca2+ entry. We conclude that F-actin turnover or depolymerization is necessary for Ca2+ release by mechanical activation. The tubulin network is not involved. The Ca2+ release- activated Ca2+ entry is not modulated by the F-actin cytoskeleton.

Actins↗

PubMed1994

Responses of endothelial cells to hypotonic solutions: lack of regulatory volume decrease.

Hypotonic stress (HTS) activates a Cl- current and releases intracellular Ca2+ in vascular endothelial cells, but there is no co-activation of K(+)-channels. The concomitant increase in cell volume, as assessed from the changes in endothelial cell thickness, is not followed by a regulatory volume decrease (RVD). This lack compensation of the volume increase may be explained by the absence of a concomitant activation of a K+ outward current, resulting in an insufficient efflux of osmolytes during HTS.

Cell Size↗

PubMed1994

The volume-activated chloride current in human endothelial cells depends on intracellular ATP.

We have studied the effect of intracellular ATP on volume-activated CI(-)-currents in endothelial cells from human umbilical veins by means of the whole-cell patch-clamp technique. The run-down of this current in ruptured patches during repetitive applications of hypotonic solutions (HTS) could be significantly reduced if the cells were internally perfused with a pipette solution that contained 4 mmol/l ATP. This run-down was much less pronounced if currents were recorded using nystatin-perforated patches. The amplitude of the current was drastically reduced and its activation became slower if the cells were superfused with a glucose-free medium with 1 mmol/l KCN. Adding 4 mmol/l ATP gamma S, a poorly hydrolyzable ATP-analogue, to the patch pipette prevented run-down of the current during repetitive activations by HTS, even if the cells were superfused with glucose-free solution with 1 mmol/l KCN. It is concluded that activation of the mechanosensitive Cl- conductance in human endothelial cells requires the presence of intracellular ATP, but not its hydrolysis.

Adenosine Triphosphate↗

PubMed1994

Volume-activated Cl- currents in different mammalian non-excitable cell types.

The existence and properties of volume-activated Cl- currents were studied in 15 different cell types (endothelium: human umbilical vein, human aorta, bovine pulmonary artery; fibroblasts: Swiss 3T3, L, C3H 10T1/2 and COS-1; epithelium: KB3, HeLa and A6; blood cells: RBL-2H3 and Jurkat; endothelioma cells derived from both subcutaneous and thymic hemangiomas; skin: IGR1 melanoma). Volume-activated Cl- currents with common characteristics, i.e. small conductance, outward rectification, higher permeability for iodide than for chloride and sensitivity to block by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) could be elicited in all cells. The block of this current by tamoxifen and dideoxyforskolin is different for the various cell types, as well as the time course and the amplitude of the responses induced by repetitive applications of hypotonicity. Volume-activated Cl- channels with similar biophysical properties are therefore wide-spread among mammalian cells. This may reflect either a single Cl- channel that is ubiquitously expressed or a family of functionally related Cl- channels with cell specific expression patterns.

Animals↗

PubMed1994

Calcium entry activated by store depletion in human umbilical vein endothelial cells.

We have used the patch clamp technique combined with simultaneous measurement of intracellular Ca2+ to record ionic currents activated by depletion of intracellular Ca(2+)-stores in endothelial cells from human umbilical veins. Two protocols were used to release Ca2+ from intracellular stores, i.e. loading of the cells via the patch pipette with Ins(1,4,5)P3, and extracellular application of thapsigargin. Ins(1,4,5)P3 (10 microM) evoked a transient increase in [Ca2+]i in cells exposed to Ca(2+)-free extracellular solutions. A subsequent reapplication of extracellular Ca2+ induced an elevation of [Ca2+]i. These changes in [Ca2+]i were very reproducible. The concomitant membrane currents were neither correlated in time nor in size with the changes in [Ca2+]i. Similar changes in [Ca2+]i and membrane currents were observed if the Ca(2+)-stores were depleted with thapsigargin. Activation of these currents was prevented and holding currents at -40 mV were small if store depletion was induced in the presence of 50 microM NPPB. This identifies the large currents, which are activated as a consequence of store-depletion, as mechanically activated Cl- currents, which have been described previously [1,2]. Loading the cells with Ins(1,4,5)P3 together with 10 mM BAPTA induced only a very short lasting Ca2+ transient, which was not accompanied by activation of a detectable current, even in a 10 mM Ca(2+)-containing extracellular solution. Also thapsigargin does not activate any membrane current if the pipette solution contains 10 mM BAPTA (ruptured patches). The contribution of Ca(2+)-influx to the membrane current during reapplication of 10 mM extracellular calcium to thapsigargin-pretreated cells was estimated from the first time derivative of the corresponding Ca2+ transients at different holding potentials. These current values showed strong inward rectification, with a maximal amplitude of 1.0 +/- 0.3 pA at -80 mV (n = 8; membrane capacitance 59 +/- 9 pF).(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗

PubMed1994

Multiple effects of SK&F 96365 on ionic currents and intracellular calcium in human endothelial cells.

1. Multiple effects of the imidazole compound SK&F 96365 have been evaluated on endothelial cells from human umbilical vein using a combined patch clamp and Ca(2+)-microfluorimetric technique (Fura-2). 2. At concentrations of 100 mumol/l or higher of SK&F 96365, the block of the receptor-mediated Ca2+ entry overlaps with the activation of another Ca(2+)-entry mechanism, which is associated with a non selective cationic current. 3. This rise in [Ca2+]i depends on the extracellular Ca(2+)-concentration, and the entry pathway is in contrast with the receptor-mediated Ca(2+)-entry pathway permeable to Ni2+, as shown by quenching of the Fura-2 fluorescence signal. 4. The concentration of SK&F 96365 for half maximal increase in [Ca2+]i was 141 +/- 19 mumol/l (n = 16). 5. SK&F 96365 activated a current that reversed at +11.8 +/- 2.1 mV (n = 21) when measured using nystatin-perforated patches with either Cs+ or K+ in the pipette and 140 Na+, 1.5 Ca2+ in the bath (chloride equilibrium potential ECl = -36 mV). 6. SK&F 96365 (200 mumol/l) blocked an inwardly rectifying K+ current in endothelial cells independently of [Ca2+]i. This block caused depolarization of the endothelial cells from -55.3 +/- 2.57 mV (n = 33) to -10 +/- 5.5 mV (n = 6). This block was concentration-dependent, half maximal block occurred at a concentration of about 40 mumol/l SK&F 96365. 7. In cells which showed an outwardly rectifying current, this outward component was also completely blocked by 200 mumol/l SK&F 96365. 8. It is concluded that SK&F 96365 reversibly activates a non-selective cation channel at concentrations higher than 100 mumol/l, but also blocks K+ currents in endothelial cells independently of [Ca2+]i. These multiple effects overlap with the proposed block of receptor-mediated Ca2+ entry. The block of K(+)-channels may in unclamped cells reduce the driving force for Ca2+, and thereby interfere with the Ca(2+)-influx.

Calcium↗

PubMed1994

Activation of a Cl- current by hypotonic volume increase in human endothelial cells.

We have used whole-cell and perforated patches to study ionic currents induced by hypotonic extracellular solutions (HTS, 185 mOsm instead of 290 mOsm) in endothelial cells from human umbilical veins. These currents activated within 30-50 s after application of HTS, reached a maximum value after approximately 50-150 s and recovered completely after re-exposing the cells to normal osmolarity. They slowly inactivated at potentials positive to +50 mV. The same current was also activated by breaking into endothelial cells with a hypertonic pipette solution (377 mOsm instead of 290 mOsm). The reversal potential of these volume-induced currents using different extracellular and intracellular Cl- concentrations was always close to the Cl(-)-equilibrium potential. These currents are therefore mainly carried by Cl-. DIDS only weakly blocked the current (KI = 120 microM), while another Cl(-)-channel blocker, DCDPC (20 microM) was ineffective. We were unable to record single channel activity in cell-attached patches but we always observed an increased current variance during HTS. From the mean current-variance relation of the whole-cell current records, we determined a single channel conductance of 1.1 pS. The size and kinetics of the current were not correlated with the concomitant changes in intracellular calcium. Furthermore, the currents could still be activated in the presence of 10 mmol/liter intracellular EGTA and are thus Ca2+ independent. A similar current was also activated with iso-osmotic pipette solutions containing 300 mumol/liter GTP gamma S. Neomycin (1 mmol/liter), a blocker of PLC, did not prevent activation of this current. TPA (4 mumol/liter) was also ineffective in modulation of this current. The HTS-induced current was completely blocked by 10 mumol/liter pBPB, a PLA2 inhibitor. NDGA (4 mumol/liter) and indomethacin (5 mumol/liter), blockers of lipoxygenase and cyclo-oxygenase respectively, did however not affect the current induced by hypotonic solutions. The effects of arachidonic acid (10 mumol/liter) were variable. In 12 out of 40 cells it either directly activated a Cl- current or potentiated the current activated by HTS. The membrane current was decreased at all potentials in 18 cells, and was not affected in 10 cells. The HTS-induced currents may therefore be modulated by cleavage products of PLA2, but not by messengers downstream of arachidonic acid. Loading the cells with a segment of the heat stable protein kinase A inhibitor PKI (5-24) did not prevent activation of the HTS-induced current.(ABSTRACT TRUNCATED AT 400 WORDS)

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Permeation properties and modulation of volume-activated Cl(-)-currents in human endothelial cells.

1. We have studied the permeation and pharmacological properties of a recently described volume-activated, calcium-insensitive, small-conductance Cl(-)-channel in endothelial cells from human umbilical vein. 2. The relative permeability for various anions was I- > Cl- approximately Br- > F- > gluconate- (1.63 +/- 0.36: 1:0.95 +/- 0.16:0.46 +/- 0.04:0.19 +/- 0.07, n = 10). 3. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) induced a fast and reversible block of the current (Ki = 29 mumol l-1). 4. Extracellular ATP induced a low-affinity block of the current, that showed a small voltage-dependence (K1 = 4.9 mmol l-1 at +80 mV and K1 = 8.2 mmol l-1 at -80 mV). 5. Extracellularly applied arachidonic acid (10 mumol l-1) irreversibly blocked the current in 5 out of 9 cells. This block seems to be non-specific, because other ionic currents, e.g. inwardly rectifying K+ currents, were blocked as well. 6. Tamoxifen induced a high affinity block of the current (K1 = 2.9 mumol l-1). Block and reversal of block were however much slower than with NPPB. 7. Cytotoxic compounds, which are substrates of the P-glycoprotein multidrug transporter, loaded into endothelial cells via the patch pipette, exerted only minor effects on the volume-activated current. Vinblastine and colcemid did not affect the volume-activated current, whereas daunomycin and vincristine induced a slow 'run-down' of the current. 8. The similarity between permeation and pharmacological properties of volume-activated Cl--currents in endothelial cells and those in many other cell types may suggest that they all belong to the same family of volume-activated small-conductance Cl--channels. Evidence that they belong to the class of P-glycoprotein associated Cl--channels is however only marginal, whereas their biophysical characteristics differ significantly from those of the CIC-2 volume-activated Cl--channels.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Nonselective ion pathways in human endothelial cells.

Four probably different transmembrane pathways are described in human endothelial (EN) cells that are all nonselective for cations. i) A nonselective cation channel that is more permeable for Na+ and K+ than for Ca2+ can be gated by agonists such as histamine. This channel provides an agonist-gated entry route for Ca2+ into EN cells with a single-channel conductance of 25 pS for Na+, K+, and approximately 4 pS for Ca2+ (110 mM). ii) Another Ca(2+)-permeable pathway can be activated by shear stress. This supposedly mechanically activated channel is more permeable for divalent than for monovalent cations and provides mechano-sensing properties to EN cells. iii) A third ionic current, activated by the selective Ca(2+)-ATPase blocker thapsigargin, seems to be related to Ca(2+)-release from Ca(2+)-stores in the endoplasmic reticulum. In EN cells, this Ca(2+)-entry route is cation selective, but cannot differentiate between Na+ and K+. Activation of this nonselective current is associated with an increase in intracellular Ca2+. We therefore assume a Ca(2+)-entry through this thapsigargin-activated pathway. iv) A nickel-blockable, Ca(2+)-permeable, nonselective leak is described that is present in nonstimulated EN cells. It will be discussed whether agonist-gated channels and leak channels might be related to the Ca(2+)-release activated Ca(2+)-entry mechanism.

Animals↗

PubMed1993

Histamine-activated, non-selective cation currents and Ca2+ transients in endothelial cells from human umbilical vein.

Permeation properties and modulation of an ionic current gated by histamine were measured in single endothelial cells from human umbilical cord veins by use of the patch-clamp technique in the ruptured-whole-cell mode or using perforated patches. We combined these current measurements with a microfluorimetric method to measure concomitantly free intracellular calcium concentration ([Ca2+]i). Application of histamine induced an intracellular calcium transient and an ionic current that reversed near 0 mV. The amplitude of the current ranged from -0.2 to -2 nA at -100 mV. The tonic rise in [Ca2+]i and the ionic current are partly due to Ca2+ influx. This Ca2+ entry pathway is also permeable for Ba2+ and Mn2+. The amplitude of the histamine-activated current was also closely correlated with the amplitude of the concomitant Ca2+ transient, suggesting that the latter is at least partially due to Ca2+ influx through histamine-activated channels. The reversal potential of the histamine-induced current was 7.6 +/- 4.1 mV (n = 14) when the calcium concentration in the bath solution ([Ca2+]o) was 1.5 mmol/l. With 10 mmol/l [Ca2+]o it was -13.7 +/- 4.7 mV and shifted to + 13.0 +/- 1.5 mV in nominally Ca(2+)-free solution (n = 3 cells). The amplitude of the current in Ca(2+)-free solution was enhanced compared to that in 10 mmol/l [Ca2+]o. The shift of the reversal potential and the concomitant change of the current amplitude suggest that the channel is permeable for calcium but has a smaller permeability for calcium than for monovalent cations.(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1992

Shear stress induced membrane currents and calcium transients in human vascular endothelial cells.

We have measured membrane currents induced by shear stress together with intracellular calcium signals in endothelial cells from human umbilical cord veins. In the presence of extracellular calcium (Ca2+]o), shear stress induced an inward current at a holding potential of 0 mV which is accompanied by a rise in intracellular Ca2+ ([Ca2+]i). In the absence of extracellular calcium shear stress was unable to evoke a calcium signal but still induced a membrane current. The voltage dependence of the shear stress induced current was obtained from difference currents evoked by linear voltage ramps before and during application of shear stress. Its reversal potential Erev shifted from -2.3 +/- 0.8 mV (n = 4) in a nominally Ca2+ free solution to +1.5 +/- 1.6 mV at 1.5 mM [Ca2+]o (n = 4) and to +21.9 +/- 4.4 mV (n = 7) at 10 mM [Ca2+]o. From our data we conclude that shear stress opens an ion channel that is 12.5 +/- 2.9 (n = 7) times more permeable for calcium than for sodium or cesium.

Calcium↗