PubMed Health⌕ Search

Biomedical subjects

T Haller

Publications and source records attributed to T Haller.

33 records · Page 2Linked to original sources
PubMed1999

Exocytosis in alveolar type II cells revealed by cell capacitance and fluorescence measurements.

Measurement of lamellar body (LB) exocytosis at high spatial and temporal resolution was recently enabled by fluorescence of the dye FM 1-43 (FFM1-43). Here, the capabilities of this method were further examined and extended by simultaneous measurement of the cell membrane capacitance (Cm) and laser-scanning confocal microscopy. Step increases in Cm were evoked by extracellular ATP (20 microM) or an elevated pipette Ca2+ concentration (>/=3 microM). The delay between the first Cm step and the increase in FFM1-43 was <1 s, indicating ready access of FM 1-43 to exocytosed LB contents. A specific Cm of 0.88 microF/cm2 for the membrane of an exocytosed LB was calculated. Compound exocytosis was occasionally observed. Decreases in Cm, indicative of transient fusion or endocytosis, did not occur within 20 min of stimulation. Exocytosis was stimulated by 160 microM guanosine 5'-O-(3-thiotriphosphate) in the pipette, but compound exocytosis was unaffected. The comparison of methods revealed that FM 1-43 is ideally suited to measure the onset of exocytosis and amount of secretion. Patch clamp is superior in resolving fusion events with the plasma membrane.

Animals↗
PubMed1999

Threshold calcium levels for lamellar body exocytosis in type II pneumocytes.

Pulmonary surfactant is secreted via exocytosis of lamellar bodies (LBs) by alveolar type II cells. Here we analyzed the dependence of LB exocytosis on intracellular Ca(2+) concentration ([Ca(2+)](i)). In fura 2-loaded cells, [Ca(2+)](i) was selectively elevated by flash photolysis of a cell-permeant caged Ca(2+) compound (o-nitrophenyl EGTA-AM) or by gradually enhancing cellular Ca(2+) influx. Simultaneously, surfactant secretion by single cells was analyzed with the fluorescent dye FM 1-43, enabling detection of exocytotic events with a high temporal resolution (T. Haller, J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. Proc. Natl. Acad. Sci. USA 95: 1579-1584, 1998). Exocytosis was initiated at a threshold concentration near 320 nmol/l with both instantaneous or gradual [Ca(2+)](i) elevations. The exocytotic response to flash photolysis was highest during the first minute after the rise in [Ca(2+)](i) and thus almost identical to purinoceptor stimulation by ATP. Correspondingly, the effects of ATP on initial secretion could be sufficiently explained by its ability to mobilize Ca(2+). This was further demonstrated by the fact that exocytosis is significantly blocked by suppression of the ATP-induced Ca(2+) signal below approximately 300 nmol/l. Our results suggest a highly Ca(2+)-sensitive step in LB exocytosis.

Adenosine Triphosphate↗
PubMed1998

Dynamics of surfactant release in alveolar type II cells.

Pulmonary surfactant, secreted via exocytosis of lamellar bodies (LB) by alveolar type II (AT II) cells, maintains low alveolar surface tension and is therefore essential for normal lung function. Here we describe real-time monitoring of exocytotic activity in these cells by visualizing and quantifying LB fusion with the plasma membrane (PM). Two approaches were used. First, fluorescence of LysoTracker Green DND-26 (LTG) in LB disappeared when the dye was released after exocytosis. Second, phospholipid staining by FM 1-43 resulted in bright fluorescence when this dye entered the LB through the fusion pore. Both processes were restricted to and colocalized with LB and occurred simultaneously. In AT II cells, FM 1-43 offered the unique advantage to independently define the moment and cellular location of single exocytotic events as well as the amount of material released, and to monitor its extracellular fate. Furthermore, both dyes could be used in combination with fura-2. The results indicate considerable diversity in the dynamics of LB exocytosis. In the majority of cells stimulated with ATP and isoproterenol, the first fusion of LB coincided with the rise of [Ca2+]i, but subsequent response of other LB in the same cell considerably outlasted this signal. In other cells, however, the onset of exocytosis was delayed by several minutes. After LB fusion, release of surfactant from LB into an aqueous solution was slow. In summary, stimulated exocytosis in AT II cells occurs at a much slower rate than in most other secretory cells but is still a more dynamic process than predicted from conventional measurements of surfactant released into cell supernatants.

Adenosine Triphosphate↗
PubMed1997

Effects of bradykinin on NIH 3T3 fibroblasts pretreated with lithium. Mimicking events of Ha-ras oncogene expression.

As shown previously, expression of Ha-ras oncogene in NIH 3T3 fibroblasts (+ ras cells) increases cellular concentrations of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 and enhances bradykinin induced Ca2+ entry [1-3]. These cells respond to low concentrations of serum or bradykinin with sustained oscillations of the cell membrane potential due to pulsatile release of calcium from internal stores and subsequent activation of calcium sensitive K+ channels [1]. Furthermore Ha-ras oncogene expression leads to depolymerization of the actin filament network and delayed increase of cell volume [4-6]. Pretreatment of the same cells not expressing the oncogene (-ras cells) with Li+ similarly increases Ins(1,4,5)P3 and Ins(1,3,4,5)P4 [2]. As shown in the present study, -ras cells pretreated with Li+ similar to Ha-ras oncogene expressing cells respond to bradykinin with sustained oscillations of cell membrane potential, depolymerization of the actin filament network and increase of cell volume. The oscillations of the cell membrane potential and the depolymerization of the actin cytoskeleton can be inhibited by the calcium channel blocker lanthanum and the bradykinin induced increase of cell volume is inhibited by HOE 694, pointing to involvement of Na+/H+ exchange. The data indicate a close functional linkage of the calcium oscillations, cytoskeletal rearrangement and activation of the Na+/H+ exchanger. Thus, Li+ pretreatment mimicks crucial cellular events triggered by expression of the Ha-ras oncogene. However, unlike in cells expressing the Ha-ras oncogene, Li+ pretreatment alone does not allow for growth factor-independent proliferation of the cells.

3T3 Cells↗
PubMed1997

Activation of Na+/H(+)-exchanger by transforming Ha-ras requires stimulated cellular calcium influx and is associated with rearrangement of the actin cytoskeleton.

Expression of the Ha-ras oncogene in NIH 3T3 fibroblasts (+ras cells) results in growth factor-independent proliferation and marked alteration of cytoskeletal architecture including breakdown of actin stress fiber network. Compared to identical cells not expressing the oncogene (-ras cells), +ras cells exhibit a more alkaline intracellular pH (pHi) and a larger cell volume (CV), both of which are important mitogenic elements. They are due to a set point shift for activation of the Na+/H(+)-exchanger. Moreover +ras cells respond to stimuli like 0.5% fetal calf serum or bradykinin with sustained oscillation of the cell membrane potential (PD) due to stimulated Ca2+ entry which triggers pulsatile release of calcium from internal stores and subsequent activation of calcium-sensitive K+ channels. 10 mumol/l bepridil inhibit oscillations of PD and protect +ras cells against actin stress fiber depolymerization. It is shown that bepridil blocks both cellular calcium entry as measured by Mn2+ quenching of fura-2 fluorescence and activation of the Na+/H(+)-exchanger following expression of the Ha-ras oncogene. Inhibition of the Na+/H(+)-exchange with 10 mumol/l HOE 694, on the other hand, does not significantly alter Ha-ras stimulated calcium entry or cytoskeletal rearrangement. In -ras cells ionomycin (0.1 mumol/l) leads to a transient increase in Cai. This effect is paralleled by a transient depolymerization of actin stress fiber network which cannot be inhibited by HOE 694. Disruption of the actin cytoskeleton in -ras cells by cytochalasin D does not alter steady state cell volume or Na+/ H(+)-exchange activity. However, stimulation of cytochalasin-treated -ras cells with bradykinin leads to cell swelling which can be blunted by HOE 694. The results show that both cytoskeletal rearrangement and activation of the Na+/H(+)-exchanger following expression of the Ha-ras oncogene require stimulated calcium influx and Cai oscillations. The depolymerization of the actin cytoskeleton is permissive for the Na+/ H(+)-exchanger to cause cell swelling upon stimulation with bradykinin.

3T3 Cells↗
PubMed1996

The lysosomal Ca2+ pool in MDCK cells can be released by ins(1,4,5)P3-dependent hormones or thapsigargin but does not activate store-operated Ca2+ entry.

In several cell types, Ca2+ release from intracellular Ca2+ stores by Ins(1,4,5)P3 elicits Ca2+ influx from the extracellular space into the cytoplasm, termed store-operated Ca2+ entry (SOCE). In MDCK cells, the Ins(1,4,5)P3-sensitive Ca2+ store giving rise to SOCE essentially overlaps with the thapsigargin (TG)-sensitive store. Recent evidence suggests that in MDCK cells lysosomes form a Ca2+ pool that is functionally coupled with the Ins(1,4,5)P3-sensitive Ca2+ store: Ca2+ can be selectively released from lysosomes by glycyl-L-phenylalanine naphthylamide, an agent inducing lysosomal swelling with subsequent and reversible permeabilization of the vesicular membranes. This compartment is also depleted by Ins(1,4,5)P3-dependent agonists or TG, indicating that it is part of a larger, Ins(1,4,5)P3-sensitive Ca2+ pool. Here we show that whereas SOCE is triggered by Ca2+ release from the entire Ins(1,4,5)P3-sensitive Ca2+ pool, selective Ca2+ release from lysosomes alone is unable to trigger SOCE. This finding is consistent with measurements of the store-operated cation current, a direct parameter for store-operated Ca2+ and Na+ entry into MDCK cells. Hence it is proposed that the Ins(1,4,5)P3-sensitive Ca2+ pool is composed of different intracellular compartments that do not uniformly stimulate Ca2+ entry into the cell.

Animals↗
PubMed1996

Two different store-operated Ca2+ entry pathways in MDCK cells.

Whole cell patch clamp experiments in conjunction with Fura-2 fluorescence microscopy were performed to study the mechanisms of 'store-operated' (capacitative) Ca2+ entry. In MDCK cells, depletion of inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores activates a store-operated cation current (SOCC) predominantly selective for Ca2+ than for Na+ or Mn2+ [Delles C., Haller T., Dietl P. A highly calcium-selective cation current activated by intracellular calcium release in MDCK cells. J Physiol 1995, 486: 557-569]. In the presence of extracellular Ca2+, thapsigargin (TG) stimulated both SOCC and a Ca(2+)-dependent K+ current (IK(Ca)), reflecting stimulation of store-operated Ca2+ entry. The Ca2+ entry blocker 1-[3-(4-methoxyphenyl) propoxyl]-1-(4-methoxyphenyl)-ethyl-1H-imidazole HCI (SK&F96365; 30 microM) did not inhibit SOCC. At the same concentration, it exerted a transient partial inhibition on IK(Ca) activated by TG-induced Ca2+ entry. It did, however, not directly inhibit IK(Ca). This was demonstrated by an unchanged relationship between the cytosolic Ca2+ concentration ([Ca2+]i) and IK(Ca) in experiments where [Ca2+]i was measured under whole cell patch clamp conditions and by a lacking effect of SK&F96365 on IK(Ca) prestimulated by a high 'clamped' [Ca2+]i. La3+ partially, but not directly, inhibited the TG-induced IK(Ca) at a concentration (10 microM) sufficient to entirely block SOCC. La3+ and SK&F96365 in combination exerted an additive reduction on the TG-induced whole cell conductance (G) and completely blocked IK(Ca) stimulated by TG. We conclude that two Ca2+ entry pathways with different pharmacological and biophysical properties are involved in 'store-operated' Ca2+ entry in MDCK cells.

Animals↗
PubMed1996

The lysosomal compartment as intracellular calcium store in MDCK cells: a possible involvement in InsP3-mediated Ca2+ release.

To test for a possible role of lysosomes in intracellular Ca2+ homeostasis, the effects of glycyl-L-phenylalanine-beta-naphthylamide (GPN), known to permeabilize these organelles by osmotic swelling, were studied in single MDCK cells. Fluorescence of acridine orange, rhodol green dextran, lysotracker green and FITC-dextran indicated that GPN (0.2 mmol/l) elicited a reversible permeabilization of lysosomes. Cytosolic Ca2+ ([Ca2+]i) as determined by Fura-2 fluorescence increased from 60 +/- 11 to 534 +/- 66 nmol/l (n = 41) in the presence of GPN. Whereas only a single intracellular Ca2+ release could be induced by GPN in a Ca(2+)-free perfusate, repetitive release could be evoked in Ca2+ containing solutions suggesting reuptake of Ca2+ into lysosomal stores. GPN-induced Ca2+ release was blunted after pretreatment with thapsigargin (TG), an inhibitor of Ca(2+)-ATPase, or repeated applications of ATP inducing Ca2+ release from inositol trisphosphate (InsP3) sensitive Ca2+ stores. The effect of ATP on Ca2+ release was, however, not abolished by preceding GPN treatment. GPN-induced Ca2+ release from lysosomes was independent of InsP3 formation or Ca(2+)-induced Ca2+ release, since it was unaffected by the phospholipase C inhibitor U-73, 122 or by caffeine and ruthenium red. These results suggest that Ca2+ largely accumulates in lysosomal vesicles. Moreover, these organelles seem to be part or functionally coupled with InsP3-sensitive Ca2+ stores.

Adenosine Triphosphate↗
PubMed1996

Calcium entry stimulated by swelling of Madin-Darby canine kidney cells.

Cell swelling in Madin-Darby canine kidney (MDCK) cells by reduction of extracellular osmolarity (omission of 70 ad 150 mmol/l mannitol, respectively) leads to the activation of anion of channels and Ca2+ sensitive K+ channels. The K+ channel activation leads to an initial transient hyperpolarization of the cell membrane potential (PD) followed by a sustained depolarization due to activation of anion channels. The present study elucidates the role of intracellular calcium (Ca2+i) in regulatory cell volume decrease (RVD) of MDCK cells. While reduction of extracellular osmolarity by omitting 70 mmol/l mannitol did not lead to a detectable change in Ca2+i, severe cell swelling by omitting 150 mmol/l mannitol led to a transient rise in Ca2+i. PD changes, on the other hand, were not different under either condition. In addition, the response of PD to cell swelling was not altered by treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate diester, pertussis toxin or cholera toxin. In the nominal absence of extracellular Ca2+, reduction of extracellular osmolarity did not lead to an increase in Ca2+i and no initial transient hyperpolarization was observed, whereas addition of 10 mumol/l ATP still led to a significant hyperpolarization. Omission of extracellular Ca2+ was followed by a strong decrease in cell membrane resistance (Rm) due to activation of a depolarizing cation conductance. Subsequent readdition of Ca2+ caused a marked increase in Ca2+i due to Ca2+ influx. This Ca2+ entry was further stimulated by cell swelling. RVD was significantly blunted in the absence of extracellular Ca2+. The results suggest that cell swelling stimulates a Ca2+-permeable pathway in the cell membrane favoring Ca2+ entry into the cell with subsequent activation of Ca2+-sensitive K+ channels.

Animals↗
PubMed1995

A highly calcium-selective cation current activated by intracellular calcium release in MDCK cells.

1. The whole-cell patch clamp technique and fluorescence microscopy with the Ca2+ indicators fura-2 and fluo-3 were used to measure the whole-cell current and the free intracellular Ca2+ concentration ([Ca2+]i) in Madin-Darby canine kidney (MDCK) cells. 2. In a Ca(2+)-free bath solution, thapsigargin (TG) caused a transient increase of [Ca2+]i. Subsequent addition of Ca2+ caused a long lasting elevation of [Ca2+]i. 3. In a Ca(2+)-free bath solution, extracellular application of TG, ATP or ionomycin, or intracellular application of inositol 1,4,5-trisphosphate (IP3), caused a small but significant inward current (Iin) and a transient outward Ca(2+)-dependent K+ current (IK(Ca)), consistent with intracellular Ca2+ release. Subsequent addition of Ca2+ induced a prominent Iin with a current density of -4.2 +/- 0.7 pA pF-1. This Iin was unaffected by inositol 1,3,4,5-tetrakisphosphate (IP4). 4. Na+ replacement by mannitol, N-methyl-D-glucamine+ (NMG+), aminomethylidin-trimethanol+ (Tris+) or choline+ reduced Iin by 54, 65, 52 and 56%, respectively. This indicates an apparent Ca2+ selectivity over Na+ of 26:1. Iin was, however, unaffected by replacing Cl- with gluconate- or by the K+ channel blocker charybdotoxin (CTX). 5. Iin was completely blocked by La3+ (IC50 = 0.77 microM). Consistently, La3+ completely reversed the TG-induced elevation of [Ca2+]i. SK&F 96365 (1-[3-(4-methoxyphenyl)-propoxyl]-1-(4-methoxy-phenyl)-ethyl-1H-im idazole) HCl did not inhibit the TG-induced Iin. It did, however, exhibit a biphasic effect on [Ca2+]i, consisting of an initial Ca2+ decay and a subsequent Ca2+ elevation. La3+ completely reversed the SK&F 96365-induced elevation of [Ca2+]i. 6. In the absence of Na+, Iin was dependent on the bath Ca2+ concentration (EC50 = 1.02 mM). Ca2+ replacement by Ba2+ or Mn2+ resulted in a reduction of Iin by 95 and 94%, respectively. 7. From these experiments we conclude that Ca2+ release from intracellular Ca2+ stores, induced by different independent methods, stimulates La(3+)-inhibitable Ca2+ entry in MDCK cells. Ca2+ entry is at least, in part, mediated by a cation current, which is highly, but not exclusively, selective for Ca2+ over Na+ and insensitive to SK&F 96365.

Adenosine Triphosphate↗
PubMed1995

Activation of L-type Ca2+ channels after purinoceptor stimulation by ATP in an alveolar epithelial cell (L2).

In the alveolar epithelium, ATP increases the intracellular Ca2+ concentration ([Ca2+]i) and stimulates the secretion of surfactant. We investigated the effects of extracellular ATP on the membrane potential (Vm), the whole cell current, and [Ca2+]i in a cloned rat alveolar epithelial cell line (L2). In microelectrode experiments, ATP caused a sustained depolarization of Vm, resulting from the activation of cation and Cl- conductances, as revealed by ion replacements. The depolarizing phase of the Vm shift was superimposed by Ca(2+)-dependent depolarizing spikes. Spikes were also induced by depolarizing Vm with charybdotoxin or maitotoxin. Replacement of bath Ca2+ with Ba2+ or Sr2+ also evoked repetitive spikes. Ca2+ (Ba2+, Sr2+)-induced spikes were unaffected by pretreatment with ionomycin or thapsigargin. They were, however, completely abolished by (+)-isradipine (100 nM) and stimulated by BAY K 8644 (100 nM). Whole cell L-type Ca2+ (Ba2+, Sr2+) currents were similarly abolished by (+)-isradipine and enhanced by BAY K 8644. L-type Ca2+ channels were further confirmed by demonstrating high-affinity dihydropyridine receptors stereoselectively labeled by (+)-[3H]-isradipine, apparent dissociation constant < 1 nM. In fura 2 experiments, ATP evoked a transient elevation of [Ca2+]i in the absence of Ca2+ and a biphasic sustained elevation in the presence of Ca2+, indicating intracellular Ca2+ release and Ca2+ entry. The ATP-induced fura 2 signals were unaffected by (+)-isradipine. We conclude that in L2 cells, L-type Ca2+ channels are activated after purinoceptor stimulation by ATP. The overall [Ca2+]i response is, however, mediated by Ca2+ entry through and (+)-isradipine-insensitive mechanism and by intracellular Ca2+ release.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1994

A respirometer for investigating oxidative cell metabolism: toward optimization of respiratory studies.

Oxygen flux measurements are critical at low rates of. respiration and at oxygen tensions below air saturation. Difficulties are primarily due to increased oxygen diffusion and consumption processes which may occur in conventional closed respirometric chambers. To avoid measurement errors encountered with standard equipment, a new type of oxygraph was developed. This instrument provides reliable quantification of oxygen flux in small organisms, tissue samples, cells, isolated mitochondria, and chloroplasts. The instrument is available commercially or can be rebuilt in a well-equipped laboratory workshop.

Cells↗
PubMed1993

[Discordant growth in twin pregnancy--value of Doppler ultrasound].

A 4 MHz continuous-wave Doppler device was used to study uterine and umbilical arterial wave forms in 91 pairs of twins between 18th and 40th week of gestation. Biometry and cord localisation were effected by real-time ultrasound. The results of 182 Doppler flow examinations showed that umbilical flow velocimetry may prove relevant for early identification of twin pregnancies with discordant growth. Depending on the interval between examination and delivery, sensitivity and specificity values between 44% and 66%, and 66% and 73%, respectively, were obtained. A high resistance index in umbilical arteries was indicative of intrauterine growth retardation, at a specificity of 69% and a sensitivity of 44%. For uteroplacental as well as foetoplacental flow velocity waveform assessment, singleton reference values may be used, whereas, by reason of its low sensitivity, Doppler flow velocimetry does not lend itself as a primary diagnostic tool for intrauterine growth retardation. It can signal pathologic blood flow profiles, which are often associated with added risks, such as pregnancy-induced hypertension, foetal acidosis and stillbirth and can contribute to early detection of twin pregnancies that require close clinical and cardiotocographic surveillance.

Blood Flow Velocity↗
PubMed1990

Effects of taurine on Ca2(+)-dependent force development of skinned muscle fibre preparations.

The effects of the naturally occurring amino acid taurine (2-aminoethanesulphonic acid) on isometric force development were investigated using skinned muscle fibre preparations. In atrial and ventricular pig heart muscles, as well as in fibres of slow abdominal extensor muscle of crayfish, an increase of submaximal isometric force was observed in Ca2(+)-activated skinned fibre preparations at physiological concentrations of taurine. The maximal isometric force remained unaffected in all preparations. It is assumed that taurine increases the Ca2+ sensitivity of the force-generating myofilaments in mammalian hearts and crustacean slow skeletal muscle fibres.

Animals↗