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M Lindau

Publications and source records attributed to M Lindau.

At least 55 records · Page 3Linked to original sources
PubMed1990

GTP gamma S-induced calcium transients and exocytosis in human neutrophils.

Exocytosis and intracellular free calcium ([Ca2+]in) were simultaneously recorded in single human neutrophils using patch-clamp capacitance measurements and the fura-2 fluorescence ratio method. Intracellular application of guanosine-5'-O(3-thiotriphosphate) (GTP gamma S) stimulates both exocytosis and a calcium transient. The calcium transient starts to develop after a lag phase of approximately 40 s and normally appears to trigger the onset of exocytosis indicated by the beginning of the capacitance increase. After this delay [Ca2+]in increases from approximately 150 nM to approximately 600 nM with a sigmoidal time course. The peak concentration is reached within approximately 30 s but the main increase occurs during approximately 3 s. [Ca2+]in subsequently decays within 1-2 min to a level which is close to the resting value. This calcium transient is due to calcium release from inositoltrisphosphate-sensitive intracellular stores. Exocytosis also occurs if the calcium transient is abolished by intracellular EGTA but the lag phase is markedly prolonged. The GTP gamma S-induced calcium transient is very similar to that observed after stimulation with N-formyl-methionyl-leucyl-phenylalanine. The interplay between guanine nucleotides, [Ca2+]in and exocytosis in neutrophils closely resembles previous results obtained in mast cells suggesting a similar regulation of exocytosis in both cell types.

Calcium↗
PubMed1990

ATP-induced pore formation in the plasma membrane of rat peritoneal mast cells.

We have investigated the ATP-induced permeabilization of rat peritoneal mast cells using three different techniques: (a) by measuring uptake of fluorescent membrane and DNA marker dyes, (b) by voltage-clamp measurements using the patch-clamp technique, and (c) by measurements of exocytosis in response to entry of Ca2+ and GTP gamma S into permeabilized cells. In the absence of divalent cations cells become highly permeable at ATP concentrations as low as 3 microM. In normal saline containing 1 mM MgCl2 and 2 mM CaCl2, dye uptake and electric conductance are detectable at 100 microM ATP corresponding to 4 microM ATP4-. The permeabilization is half-maximal at an ATP4- concentration of 5-20 microM with a Hill coefficient near 2. The ATP-induced whole-cell conductance at saturating ATP concentrations was 35-70 nS, exhibiting only weak cation selectivity. The activation is very fast with a time constant less than or equal to 65 ms. Pores which are large enough to allow for permeation of substances of 300-900 D are expected to have a unit conductance of approximately 200-400 pS. However, in whole cells as well as outside-out patches, discrete openings and closings of channels could not be observed at a resolution of approximately 40 pS and the single-channel conductance obtained from noise analysis is approximately 2-10 pS. Entry of Ca2+ into cells permeabilized with ATP stimulates exocytosis at low but not at high ATP concentrations indicating loss of an essential intracellular component or components at a high degree of permeabilization. This inactivation is removed when GTP gamma S is provided in the medium and this leads to enhanced exocytosis. The enhancement only occurs at high ATP concentrations. These results strongly suggest that the ATP-induced pores are of variable size and can increase or decrease by very small units.

Adenosine Triphosphate↗
PubMed1989

Aspartic acid-96 is the internal proton donor in the reprotonation of the Schiff base of bacteriorhodopsin.

Above pH 8 the decay of the photocycle intermediate M of bacteriorhodopsin splits into two components: the usual millisecond pH-independent component and an additional slower component with a rate constant proportional to the molar concentration of H+, [H+]. In parallel, the charge translocation signal associated with the reprotonation of the Schiff base develops a similar slow component. These observations are explained by a two-step reprotonation mechanism. An internal donor first reprotonates the Schiff base in the decay of M to N and is then reprotonated from the cytoplasm in the N----O transition. The decay rate of N is proportional to [H+]. By postulating a back reaction from N to M, the M decay splits up into two components, with the slower one having the same pH dependence as the decay of N. Photocycle, photovoltage, and pH-indicator experiments with mutants in which aspartic acid-96 is replaced by asparagine or alanine, which we call D96N and D96A, suggest that Asp-96 is the internal proton donor involved in the re-uptake pathway. In both mutants the stoichiometry of proton pumping is the same as in wild type. However, the M decay is monophasic, with the logarithm of the decay time [log (tau)] linearly dependent on pH, suggesting that the internal donor is absent and that the Schiff base is directly reprotonated from the cytoplasm. Like H+, azide increases the M decay rate in D96N. The rate constant is proportional to the azide concentration and can become greater than 100 times greater than in wild type. Thus, azide functions as a mobile proton donor directly reprotonating the Schiff base in a bimolecular reaction. Both the proton and azide effects, which are absent in wild type, indicate that the internal donor is removed and that the reprotonation pathway is different from wild type in these mutants.

Aspartic Acid↗
PubMed1988

Patch-clamp techniques for time-resolved capacitance measurements in single cells.

Two methods are described for estimation of passive cell parameters such as membrane capacitance, membrane conductance and access resistance in tight-seal whole cell recording. Both methods are restricted in their application to cases where the cell under study can be approximated by a simple three-component network with linear properties over some voltage range. One method, referred to as the time domain technique, requires only standard electrophysiological equipment and a computer. Parameters are derived from an analysis of capacitive transients during square wave stimulation. It is readily adaptable to wide variations in experimental parameters. Particularly, it is equally applicable to the "slow whole-cell" configuration (access resistance in the range 100 M omega to 1 G omega) and to normal whole-cell measurements (access resistance typically 10 M omega). The other method applies a sine wave command signal to the cell and employs a lock-in amplifier to analyse the resulting current signal. Two modes of operating the lock-in amplifier are described. One mode provides an output signal directly proportional to small changes in capacitance at maximum resolution (1-10 fF). The other mode, in conjunction with a digital computer, supplies estimates of all passive cell parameters, as does the time domain technique, but with a large amount of data reduction performed by the lock-in amplifier itself. Due to the special hardware, however, this method is not as flexible as the time domain technique.

Animals↗
PubMed1988

The dynamics of exocytosis in human neutrophils.

We have investigated the dynamics of exocytosis in single human neutrophils. The increase of membrane area associated with granule fusion was followed by time-resolved patch-clamp capacitance measurements. Intracellular application of 20 microM guanosine-5'-O(3-thiotriphosphate) (GTP gamma S) in the presence of 2.5 mM ATP stimulated exocytosis and led to an increase of membrane capacitance from 3.0 to integral of 8.4 pF corresponding to a 540 micron 2 increase of membrane area. This capacitance change is very close to the value expected from morphological data if all primary and secondary granules fuse with the plasma membrane. High resolution measurements revealed stepwise capacitance changes corresponding to the fusion of individual granules. GTP gamma S-stimulated exocytosis did not require pretreatment with cytochalasin B and the amplitude was independent of the intracellular-free calcium concentration between 10 nM and integral of 2.5 microM. In the absence of GTP gamma S elevation of intracellular-free calcium concentration to the micromolar range led to the fusion of only a limited number of granules. Degranulation stimulated with GTP gamma S started after a lag phase of 2-7 min and was usually complete within 5-20 min. The time course was affected by the intracellular ATP and calcium concentration. Exocytosis was markedly accelerated by pretreatment with cytochalasin B. Our results demonstrate that the final steps leading to primary and secondary granule fusion are controlled by a guanine nucleotide-binding protein and do not require an elevation of intracellular calcium. Calcium and other factors are, however, involved in the regulation having pronounced effects on the dynamics of exocytosis.

Calcium↗
PubMed1987

Pertussis toxin does not affect the time course of exocytosis in mast cells stimulated by intracellular application of GTP-gamma-S.

Exocytosis was studied in single rat peritoneal mast cells. Granule fusion was monitored by time-resolved capacitance measurements using the patch-clamp technique. Intracellular stimulation of mast cells with 20 microM GTP-gamma-S stimulates exocytosis with a calcium-dependent time course. Secretion in response to receptor-mediated stimulation with compound 48/80 was completely abolished by treatment with pertussis toxin (IAP) at 180 ng/ml for 4 h. The time course of exocytosis in response to GTP-gamma-S remained unaffected in IAP-treated cells supporting the involvement of a second GTP-binding protein in stimulus-secretion coupling.

Animals↗
PubMed1987

Intracellular stimulation of mast cells with guanine nucleotides mimic antigenic stimulation.

Exocytosis was followed in single rat peritoneal mast cells, by measuring the cell membrane capacitance using circuit analysis and patch-clamp techniques. After antigenic stimulation or intracellular perfusion with guanine nucleotides, exocytosis followed a time course characterized by a lag period d, area expansion factor A, and a time constant tau. We suggest that A depends entirely on the cell's morphology, d reflects the properties of a GTP-binding regulatory protein that appears to rate limit the response and tau is due to an independent and yet unknown process. In contrast, cells stimulated by compound 48/80 can respond without a measurable delay and degranulate within 2 s, suggesting that this compound acts at a site after the GTP-binding regulatory protein.

Animals↗
PubMed1986

A patch-clamp study of histamine-secreting cells.

The ionic conductances in rat basophilic leukemia cells (RBL-2H3) and rat peritoneal mast cells were investigated using the patch-clamp technique. These two cell types were found to have different electrophysiological properties in the resting state. The only significant conductance of RBL-2H3 cells was a K+-selective inward rectifier. The single channel conductance at room temperature increased from 2-3 pS at 2.8 mM external K+ to 26 pS at 130 mM K+. This conductance, which appeared to determine the resting potential, could be blocked by Na+ and Ba2+ in a voltage-dependent manner. Rat peritoneal mast cells had a whole-cell conductance of only 10-30 pS, and the resting potential was close to zero. Sometimes discrete openings of channels were observed in the whole-cell configuration. When the Ca2+ concentration on the cytoplasmic side of the membrane was elevated, two types of channels with poor ion specificity appeared. A cation channel, observed at a Ca2+ concentration of approximately 1 microM, had a unit conductance of 30 pS. The other channel, activated at several hundred micromolar Ca2+, was anion selective and had a unit conductance of approximately 380 pS in normal Ringer solution and a bell-shaped voltage dependence. Antigenic stimulation did not cause significant changes in the ionic conductances in either cell type, which suggests that these cells use a mechanism different from ionic currents in stimulus-secretion coupling.

Animals↗
PubMed1986

Testing a model of intonation in a tone language.

Schematic fundamental frequency curves of simple statements and questions are generated for Hausa, a two-tone language of Nigeria, using a modified version of an intonational model developed by Gårding and Bruce [Nordic Prosody II, edited by T. Fretheim (Tapir, Trondheim, 1981), pp. 33-39]. In this model, rules for intonation and tones are separated. Intonation is represented as sloping grids of (near) parallel lines, inside which tones are placed. The tones are associated with turning points of the fundamental frequency contour. Local rules may also modify the exact placement of a tone within the grid. The continuous fundamental frequency contour is modeled by concatenating the tonal points using polynomial equations. Thus the final pitch contour is modeled as an interaction between global and local factors. The slope of the intonational grid lines depends at least on sentence type (statement or question), sentence length, and tone pattern. The model is tested by reference to data from nine speakers of Kano Hausa.

Adult↗
PubMed1982

On the origin and the signal-shaping mechanism of the fast photosignal in the vertebrate retina.

Fast photosignals (FPS) with R(1) and R(2) components were measured in retinas of cattle, rat, and frog within a temperature range of 0 degrees to 60 degrees C. Except for temperatures near 0 degrees C the signal rise of the R(1) component was determined by the duration of the exciting flash. The kinetics of the R(2) component and the meta transition of rhodopsin in the cattle and rat retina were compared. For the analysis of the FPS it is presupposed that the signal is produced by light-induced charges on the outer segment envelope membrane that spread onto the whole plasma membrane of the photoreceptor cell. To a good approximation, this mechanism can be described by a model circuit with two distinct capacitors. In this model, the charging capacitance of the pigmented outer segment envelope membrane and the capacitance of the receptor's nonpigmented plasma membrane are connected via the extra- and intracellular electrolyte resistances. The active charging is explained by two independent processes, both with exponential rise (R(1) and R(2)), that are due to charge displacements within the pigmented envelope membrane. The time constant tau(2) of the R(2) membrane charging process shows a strong temperature dependence that of the charge redistribution, tau(r), a weak one. In frog and cattle retinas the active charging is much slower within a large temperature range than the passive charge redistribution. From the two-capacitor model it follows for tau(r) << tau(2) that the rise of the R(2) component is determined by tau(r), whereas the decay is given by tau(2). For the rat retina, however, tau(2) approaches tau(r) at physiological temperatures and becomes <tau(r) above 45 degrees C. In this temperature range where tau(2) approximately tau(r), both processes affect rise and decay of the photosignal. The absolute values of tau(r) are in good accordance with the known electric parameters of the photoreceptors. At least in the cattle retina, the time constant tau(2) is identical with that of the slow component of the meta II formation. The strong temperature dependence of the meta transition time gives rise to the marked decrease of the R(2) amplitude with falling temperature. As the R(1) rise could not be fully time resolved the signal analysis does not yield the time constant tau(1) of the R(1) generating process. It could be established, however, within the whole temperature range that the decay of the R(1) component is determined by tau(r). Using an extended model that allows for membrane leakage, we show that in normal ringer solution the membrane time constant does not influence the signal time-course and amplitude.

Animals↗