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E K Hoffmann

Publications and source records attributed to E K Hoffmann.

At least 37 records · Page 2Linked to original sources
PubMed1999

Role of the F-actin cytoskeleton in the RVD and RVI processes in Ehrlich ascites tumor cells.

The role of the F-actin cytoskeleton in cell volume regulation was studied in Ehrlich ascites tumor cells, using a quantitative rhodamine-phalloidin assay, confocal laser scanning microscopy, and electronic cell sizing. A hypotonic challenge (160 mOsm) was associated with a decrease in cellular F-actin content at 1 and 3 min and a hypertonic challenge (600 mOsm) with an increase in cellular F-actin content at 1, 3, and 5 min, respectively, compared to isotonic (310 mOsm) control cells. Confocal visualization of F-actin in fixed, intact Ehrlich cells demonstrated that osmotic challenges mainly affect the F-actin in the cortical region of the cells, with no visible changes in F-actin in other cell regions. The possible role of the F-actin cytoskeleton in RVD was studied using 0. 5 microM cytochalasin B (CB), cytochalasin D (CD), or chaetoglobosin C (ChtC), a cytochalasin analog with little or no affinity for F-actin. Recovery of cell volume after hypotonic swelling was slower in cells pretreated for 3 min with 0.5 microM CB, but not in CD- and ChtC-treated cells, compared to osmotically swollen control cells. Moreover, the maximal cell volume after swelling was decreased in CB-treated, but not in CD- or Chtc-treated cells. Following a hypertonic challenge imposed using the RVD/RVI protocol, recovery from cell shrinkage was slower in CB-treated, but not in CD- or Chtc-treated cells, whereas the minimal cell volume after shrinkage was unaltered by either of these treatments. It is concluded that osmotic cell swelling and shrinkage elicit a decrease and an increase in the F-actin content in Ehrlich cells, respectively. The RVD and RVI processes are inhibited by 0.5 microM CB, but not by 0.5 microM CD, which is more specific for actin.

Actins↗
PubMed1999

Increases in [Ca2+]i and changes in intracellular pH during chemical anoxia in mouse neocortical neurons in primary culture.

The effect of chemical anoxia (azide) in the presence of glucose on the free intracellular Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) in mouse neocortical neurons was investigated using Fura-2 and BCECF. Anoxia induced a reversible increase in [Ca2+]i which was significantly inhibited in nominally Ca2+-free medium. A change in pHo (8.2 or 6.6), or addition of NMDA and non-NMDA receptor antagonists (D-AP5 and CNQX) in combination, significantly reduced the increase in [Ca2+]i, pointing to a protective effect of extracellular alkalosis or acidosis, and involvement of excitatory amino acids. An initial anoxia-induced acidification was observed under all experimental conditions. In the control situation, this acidification was followed by a recovery/alkalinization of pHi in about 50% of the cells, a few cells showed no recovery, and some showed further acidification. EIPA, an inhibitor of Na+/H+ exchangers, prevented alkalinization, pointing towards anoxia-induced activation of a Na+/H+ exchanger. In a nominally Ca2+-free medium, the initial acidification was followed by a significant alkalinization. At pHo 8.2, the alkalinization was significantly increased, while at pHo 6.2, the initial acidification was followed by further acidification in about 50% of the cells, and by no further change in the remaining cells.

2-Amino-5-phosphonovalerate↗
PubMed1999

Mechanical stress induces release of ATP from Ehrlich ascites tumor cells.

The supernatant from a suspension of Ehrlich cells exposed to centrifugation at 700xg for 45 s induced a transient increase in the intracellular concentration of free, cytosolic Ca2+, [Ca2+]i, as well as activation of an outwardly rectifying whole-cell current when added to a suspension of non-stimulated cells. These effects were inhibited by suramin, a non-specific P2 receptor antagonist, and mimicked by ATP. Reversed phase HPLC analysis revealed that the supernatant from Ehrlich cells exposed to centrifugation contained 2. 6+/-0.2 microM ATP, and that the mechanical stress-induced release of ATP was inhibited by glibenclamide and verapamil, non-specific inhibitors of the cystic fibrosis transmembrane conductance regulator and P-glycoprotein, respectively. After trypan blue staining, less than 0.5% of the cells were unable to extrude the dye. Addition of extracellular ATP induced a suramin-sensitive, transient, concentration-dependent increase in [Ca2+]i, activation of an outwardly rectifying whole-cell current and a hyperpolarization of the plasma membrane. The ATP-induced hyperpolarization of the plasma membrane was strongly inhibited in the presence of charybdotoxin (ChTX), an inhibitor of several Ca2+-activated K+ channels, suggesting that stimulation of P2 receptors in Ehrlich cells evokes a Ca2+-activated K+ current. The relative potencies of several nucleotides (ATP, UTP, ADP, 2-MeSATP, alpha,beta-MeATP, bzATP) in eliciting an increase in [Ca2+]i, as well as the effect of repetitive addition of nucleotides were investigated. The results lead us to conclude that mechanical stimulation of Ehrlich cells leads to release of ATP, which in turn stimulates both P2Y1 and P2Y2 receptors, resulting in Ca2+ influx as well as release and activation of an outwardly rectifying whole-cell current.

Adenosine Triphosphate↗
PubMed1999

The Na+2Cl-K+ cotransporter in the rectal gland of Squalus acanthias is activated by cell shrinkage.

Effects of cAMP on Cl- secretion, intracellular Cl- activity and cell volume were studied in isolated perfused rectal gland tubules (RGT) of Squalus acanthias with electrophysiological and fluorescence methods. Recording of equivalent short-circuit current (Isc) showed that cAMP stimulates Na+Cl- secretion in a biphasic manner. The first and rapid phase corresponds to Cl- exit via the respective protein-kinase-A- (PKA-) phosphorylated Cl- conductance. The inhibitory effect of the loop diuretic furosemide (0.5 mmol/l, n=12) indicates that second phase reflects the delayed (1-2 min) activation of the Na+2Cl-K+ cotransporter. During the first phase cytosolic Cl- activity, as monitored by 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) fluorescence, fell to 78% (n=23) of the control value. Concomitantly, a transient fall in cell volume was recorded by calcein fluorescence to 92% (n=5) of the control value. Preincubation of the RGT with phalloidin (0.1 mmol/l, n=6) or cytochalasin D (0.1 mmol/l, n=4) almost completely prevented the development of the second phase of Isc activation. When cytosolic Cl- activity was increased by exposing the RGT to a high K+ concentration (25 mmol/l), in the presence of mannitol to prevent volume increases, stimulation was unaffected and biphasic. In contrast, when cell volume was clamped to an increased value (115%, n=8) by removing extracellular NaCl, the second phase was abolished completely (n=11). These data suggest that the primary and key process for triggering the Na+2Cl-K+ cotransport is transient cell shrinkage.

Animals↗
PubMed1999

Leukotriene D4 (LTD4) activates charybdotoxin-sensitive and -insensitive K+ channels in ehrlich ascites tumor cells.

The putative role for ATP, UTP, bradykinin and leukotriene D4 (LTD4) in the activation of the charybdotoxin-insensitive, volume-activated K+ leak pathway has been assessed in Ehrlich cells. K+ channel activity is evaluated from bumetanide-insensitive 86Rb+ efflux using Rb+ as a tracer for K+. Addition of the Ca2+-mobilizing agonists bradykinin, ATP, UTP or LTD4 accelerates the regulatory volume decrease (RVD) response and activates a fast bumetanide-insensitive, charybdotoxin-sensitive efflux of K+. In addition LTD4 activates a charybdotoxin-insensitive K+ efflux, whereas bradykinin, ATP and UTP do not. The charybdotoxin-insensitive K+ efflux dominates after addition of LTD4 at concentrations too low to elicit an increase in [Ca2+]i but still high enough to be effective in accelerating the RVD response. The EC50 values for LTD4-induced K+ effluxes are estimated at 2 nM and 15 nM for the charybdotoxin-insensitive and charybdotoxin-sensitive components, respectively. The LTD4 (cysLT1) receptor antagonist L660,711(MK-571) blocks the activation of the charybdotoxin-sensitive but not the charybdotoxin-insensitive K+ efflux. Thus, LTD4 activates two different K+ leak pathways in Ehrlich cells, one pathway activated by an increase in [Ca2+]i and the other via an alternative signalling pathway. LTD4 is thus a potential candidate for an autocrine messenger activating the Ca2+-independent, charybdotoxin-insensitive K+ channel during the RVD response in Ehrlich cells.

Adenosine Triphosphate↗
PubMed1999

Thrombin-, bradykinin-, and arachidonic acid-induced Ca2+ signaling in Ehrlich ascites tumor cells.

Stimulation of single Ehrlich ascites tumor cells with agonists (bradykinin, thrombin) and with arachidonic acid (AA) induces increases in the free intracellular Ca2+ concentration ([Ca2+]i) in the presence and absence of extracellular Ca2+, measured using the Ca2+-sensitive probe fura 2. Sequential stimulation with two agonists elicits sequential increases in [Ca2+]i, unlike addition of the same agonist twice. Bradykinin and thrombin have additive effects on [Ca2+]i in Ca2+-free medium. The phosphoinositidase C inhibitor U-73122 inhibits the agonist-induced increases in [Ca2+]i, whereas ryanodine has no effect. Pretreatment of cells in Ca2+-free medium with thapsigargin abolishes the bradykinin-induced increase in [Ca2+]i but not the response to thrombin. The AA-induced response is not inhibited by U-73122 and cannot be mimicked by the inactive structural analog trifluoromethylarachidonyl ketone. Pretreatment of the cells with 50 microM AA (but not with 10 microM AA) abolishes the agonist-induced increase in [Ca2+]i. Thus bradykinin, thrombin, and AA induce increases in [Ca2+]i in Ehrlich cells due to Ca2+ entry and release from intracellular stores. Thrombin causes release of Ca2+ from an intracellular store that is insensitive to bradykinin and is not depleted by thapsigargin but is depleted by AA.

Animals↗
PubMed1998

P2 receptor-mediated signal transduction in Ehrlich ascites tumor cells.

The mechanisms, by which the P2 receptor agonists adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) evoke an increase in the free cytosolic calcium concentration ([Ca2+]i) and in intracellular pH (pHi), have been investigated in Ehrlich ascites tumor cells. The increase in [Ca2+]i evoked by ATP or UTP is abolished after depletion of intracellular Ca2+ stores with thapsigargin in Ca2+-free medium, and is inhibited by U73122, an inhibitor of phospholipase C (PLC), indicating that the increase in [Ca2+]i is primarily due to release from intracellular, Ins(1,4,5)P3-sensitive Ca2+ stores. ATP also activates a capacitative Ca2+-entry pathway. ATP as well as UTP evokes a biphasic change in pHi, consisting of an initial acidification followed by alkalinization. Suramin and 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS) inhibit the biphasic change in pHi, apparently by acting as antagonists at P2 receptors. The alkalinization evoked by the P2 receptor agonists is found to be due to activation of a 5'-(N-ethyl-N-isopropyl)amiloride (EIPA)-sensitive Na+/H+ exchanger. ATP and UTP elicit rapid cell shrinkage, presumably due to activation of Ca2+ sensitive K+ and Cl- efflux pathways. Preventing cell shrinkage, either by incubating the cells at high extracellular K+ concentration, or by adding the K+-channel blocker, charybdotoxin, does not affect the increase in [Ca2+]i, but abolishes the activation of the Na+/H+ exchanger, indicating that activation of the Na+/H+ exchanger is secondary to the Ca2+-induced cell shrinkage.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1998

Separate swelling- and Ca2+-activated anion currents in Ehrlich ascites tumor cells.

A Ca2+-activated (ICl,Ca) and a swelling-activated anion current (ICl,vol) were investigated in Ehrlich ascites tumor cells using the whole cell patch clamp technique. Large, outwardly rectifying currents were activated by an increase in the free intracellular calcium concentration ([Ca2+]i), or by hypotonic exposure of the cells, respectively. The reversal potential of both currents was dependent on the extracellular Cl- concentration. ICl,Ca current density increased with increasing [Ca2+]i, and this current was abolished by lowering [Ca2+]i to <1 nm using 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA). In contrast, activation of ICl,vol did not require an increase in [Ca2+]i. The kinetics of ICl,Ca and ICl,vol were different: at depolarized potentials, ICl,Ca as activated in a [Ca2+]i- and voltage-dependent manner, while at hyperpolarized potentials, the current was deactivated. In contrast, ICl,vol exhibited time- and voltage-dependent deactivation at depolarized potentials and reactivation at hyperpolarized potentials. The deactivation of ICl, vol was dependent on the extracellular Mg2+ concentration. The anion permeability sequence for both currents was I- > Cl- > gluconate. ICl,Ca was inhibited by niflumic acid (100 micron), 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 micron) and 4, 4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS, 100 micron), niflumic acid being the most potent inhibitor. In contrast, ICl,vol was unaffected by niflumic acid (100 micron), but abolished by tamoxifen (10 micron). Thus, in Ehrlich cells, separate chloride currents, ICl,Ca and ICl,vol, are activated by an increase in [Ca2+]i and by cell swelling, respectively.

Animals↗
PubMed1998

Swelling-activated potassium currents of Ehrlich ascites tumour cells.

The K+ and Cl- currents activated by Ca2+-ionophore treatment or by hypotonic cell swelling have been studied in Ehrlich ascites tumour cells by the patch-clamp technique. A charybdotoxin-inhibitable K+ current was activated by increasing intracellular Ca2+ concentration. In contrast, the K+ current activated by cell swelling was insensitive to charybdotoxin as well as to apamin, suggesting that channels different from those sensitive to Ca2+ are responsible for regulatory volume adjustments in these cells. The magnitude of the K+ and Cl- currents activated by hypotonic challenge was markedly temperature-dependent, possibly reflecting the temperature-dependence of enzymes involved in the intracellular signalling of cell volume regulation.

Animals↗
PubMed1998

Mechanisms of pHi regulation studied in individual neurons cultured from mouse cerebral cortex.

Maintenance and regulation of intracellular pH (pHi) was studied in single cultured mouse neocortical neurons using the fluorescent probe 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). Reversal of the Na+ gradient by reduction of the extracellular Na+ concentration ([Na+]o) resulted in rapid intracellular acidification, inhibited by 5'-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of Na+/H+ exchange. In the presence of EIPA and/or 4',4'-diisothiocyano-stilbene-2',2'-sulfonic acid (DIDS), an inhibitor of Na+-coupled anion exchangers and Na+-HCO3- cotransport, a slow decline of pHi was seen. Following intracellular acidification imposed by an NH4Cl prepulse, pHi recovered at a rapid rate, which was reduced by reduction of [Na+]o and was virtually abolished by EIPA and DIDS in combination. Creating an outward Cl- gradient by removal of extracellular Cl- significantly increased the rate of pHi recovery. In HCO3(-)-free media, the pHi recovery rate was reduced in control cells and was abolished at zero [Na+]o and by EIPA. After intracellular alkalinization imposed by an acetate prepulse, pHi recovery was unaffected by DIDS but was significantly reduced in the absence of extracellular Cl-, as well as in the presence of Zn2+, which is a blocker of proton channels. Together, this points toward a combined role of DIDS-insensitive Cl-/HCO3- and passive H+ influx in the recovery of pHi after alkalinization.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1998

Dynamics of Ca2+i and pHi in Ehrlich ascites tumor cells after Ca2+-mobilizing agonists or exposure to hypertonic solution.

Intracellular free calcium concentration ([Ca2+]i) and intracellular pH (pHi) were monitored in Ehrlich ascites tumor cells using Fura-2 or 2',7',-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), or both probes in combination. An increase in [Ca2+]i induced by thrombin or bradykinin, agonists known to elicit transient cell shrinkage in these cells, evoked a transient intracellular acidification, followed by an alkalinization. The latter was due to activation of a Na+/H+ exchanger and was inhibited under conditions preventing agonist-induced cell shrinkage without preventing the increase in [Ca2+]i. In contrast, a smaller, slower increase in [Ca2+]i elicited by thapsigargin did not cause cell shrinkage, and did not activate the Na+/H+ exchanger. Exposure to hypertonic solution was not associated with an increase in [Ca2+]i, but elicited an intracellular alkalinization similar to that induced by thrombin or bradykinin, via activation of the Na+/H+ exchanger. Thus, activation of the exchanger by the Ca2+-mobilizing agonists is suggested to be secondary to the cell shrinkage induced by these compounds. NH4Cl-induced intracellular alkalinization resulted in an increase in [Ca2+]i, apparently via stimulation of Ca2+ influx, whereas shrinkage-induced intracellular alkalinization did not stimulate Ca2+ influx. Thus, cell shrinkage appears to inhibit the Ca2+ influx otherwise resulting from alkalosis. In agreement with that notion, thapsigargin-induced Ca2+ influx was inhibited by cell shrinkage.

Ammonium Chloride↗
PubMed1998

Na+-K+-2Cl- cotransport in Ehrlich cells: regulation by protein phosphatases and kinases.

To identify protein kinases (PK) and phosphatases (PP) involved in regulation of the Na+-K+-2Cl- cotransporter in Ehrlich cells, the effect of various PK and PP inhibitors was examined. The PP-1, PP-2A, and PP-3 inhibitor calyculin A (Cal-A) was a potent activator of Na+-K+-2Cl- cotransport (EC50 = 35 nM). Activation by Cal-A was rapid (<1 min) but transient. Inactivation is probably due to a 10% cell swelling and/or the concurrent increase in intracellular Cl- concentration. Cell shrinkage also activates the Na+-K+-2Cl- cotransport system. Combining cell shrinkage with Cal-A treatment prolonged the cotransport activation compared with stimulation with Cal-A alone, suggesting PK stimulation by cell shrinkage. Shrinkage-induced cotransport activation was pH and Ca2+/calmodulin dependent. Inhibition of myosin light chain kinase by ML-7 and ML-9 or of PKA by H-89 and KT-5720 inhibited cotransport activity induced by Cal-A and by cell shrinkage, with IC50 values similar to reported inhibition constants of the respective kinases in vitro. Cell shrinkage increased the ML-7-sensitive cotransport activity, whereas the H-89-sensitive activity was unchanged, suggesting that myosin light chain kinase is a modulator of the Na+-K+-2Cl- cotransport activity during regulatory volume increase.

Animals↗
PubMed1998

Swelling-induced arachidonic acid release via the 85-kDa cPLA2 in human neuroblastoma cells.

Arachidonic acid or its metabolites have been implicated in the regulatory volume decrease (RVD) response after hypotonic cell swelling in some mammalian cells. The present study investigated the role of arachidonic acid (AA) during RVD in the human neuroblastoma cell line CHP-100. During the first nine minutes of hypo-osmotic exposure the rate of 3H-arachidonic acid (3H-AA) release increased to 250 +/- 19% (mean +/- SE, n = 22) as compared with cells under iso-osmotic conditions. This release was significantly inhibited after preincubation with AACOCF3, an inhibitor of the 85-kDa cytosolic phospholipase A2 (cPLA2). This indicates that a PLA2, most likely the 85-kDa cPLA2 is activated during cell swelling. In contrast, preincubation with U73122, an inhibitor of phospholipase C, did not affect the swelling-induced release of 3H-AA. Swelling-activated efflux of 36Cl and 3H-taurine were inhibited after preincubation with AACOCF3. Thus the swelling-induced activation of cPLA2 may be essential for stimulation of both 36Cl and 3H-taurine efflux during RVD. As the above observation could result from a direct effect of AA or its metabolite leukotriene D4 (LTD4), the effects of these agents were investigated on swelling-induced 36Cl and 3H-taurine effluxes. In the presence of high concentrations of extracellular AA, the swelling-induced efflux of 36Cl and 3H-taurine were inhibited significantly. In contrast, addition of exogenous LTD4 had no significant effect on the swelling-activated 36Cl efflux. Furthermore, exogenous AA increased cytosolic calcium levels as measured in single cells loaded with the calcium sensitive dye Fura-2. On the basis of these results we propose that cell swelling activates phospholipase A2 and that this activation via an increased production of AA or some AA metabolite(s) other than LTD4 is essential for RVD.

Arachidonic Acid↗
PubMed1998

pH regulation in sensitive and multidrug resistant Ehrlich ascites tumor cells.

Maintenance and regulation of intracellular pH (pHi) was studied in wild-type Ehrlich ascites tumor cells (EHR2) and five progressively daunorubicin-resistant, P-glycoprotein (P-gp)-expressing strains, the maximally resistant of which is EHR2/1.3. Steady-state pHi was similar in cells expressing different amounts of P-gp, in the absence and presence of glucose. In EHR2/1.3, glucose-induced acidification was reduced, and proton efflux was increased, compared to the wild-type EHR2, differences which were not caused by increased activity of a Na+/H+ exchanger in the resistant cells. Comparing all six cell lines, no evidence was found for a correlation between the amount of P-gp in the membrane and pHi regulation, which was also unaffected by P-gp modulators. However, a correlation was seen between relative resistance/daunorubicin accumulation and acid extrusion rate, which is likely to be due to aspects of development of drug resistance other than P-gp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Cell swelling activates phospholipase A2 in Ehrlich ascites tumor cells.

Ehrlich ascites tumor cells, loaded with 3H-labeled arachidonic acid and 14C-labeled stearic acid for two hours, were washed and transferred to either isotonic or hypotonic media containing BSA to scavenge the labeled fatty acids released from the cells. During the first two minutes of hypo-osmotic exposure the rate of 3H-labeled arachidonic acid release is 3.3 times higher than that observed at normal osmolality. Cell swelling also causes an increase in the production of 14C-stearic acid-labeled lysophosphatidylcholine. This indicates that a phospholipase A2 is activated by cell swelling in the Ehrlich cells. Within the same time frame there is no swelling-induced increase in 14C-labeled stearic acid release nor in the synthesis of phosphatidyl 14C-butanol in the presence of 14C-butanol. Furthermore, U7312, an inhibitor of phospholipase C, does not affect the swelling induced release of 14C-labeled arachidonic acid. Taken together these results exclude involvement of phospholipase A1, C and D in the swelling-induced liberation of arachidonic acid. The swelling-induced release of 3H-labeled arachidonic acid from Ehrlich cells as well as the volume regulatory response are inhibited after preincubation with GDP beta S or with AACOCF3, an inhibitor of the 85 kDa, cytosolic phospholipase A2. Based on these results we propose that cell swelling activates a phospholipase A2--perhaps the cytosolic 85 kDa type--by a partly G-protein coupled process, and that this activation is essential for the subsequent volume regulatory response.

Arachidonic Acid↗
PubMed1997

Hypertonicity enhances expression of functional Na+/K+/2Cl- cotransporters in Ehrlich ascites tumour cells.

Ehrlich cells exposed to a hypertonic medium for five hours respond by an increased expression of Na+/K+/2Cl- cotransport proteins as estimated from immunoprecipitations using polyclonal anti-cotransporter antibodies. The 3.4-fold increase in cotransport expression is followed by a concomitant 2.6-fold increase in the maximal bumetanide-sensitive K+ influx during regulatory volume increase, indicating a 2.6-fold increase in the number of functional cotransporters in the plasma membrane.

Animals↗