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E Neher

Publications and source records attributed to E Neher.

At least 37 records · Page 2Linked to original sources
PubMed1993

A Ca-dependent early step in the release of catecholamines from adrenal chromaffin cells.

Intense stimuli, such as trains of depolarizing pulses or the caffeine-induced release of calcium from intracellular stores, readily depress the secretory response in neuroendocrine cells. Secretory responses are restored by rest periods of minutes in duration. This recovery was accelerated when the concentration of cytosolic calcium was moderately increased and probably resulted from calcium-dependent replenishment of a pool of release-ready granules. Continuously increased concentrations of calcium led the over-filling of such a pool. Subsequently, secretory responses to stronger calcium stimuli were augmented. Hormone-induced calcium transients with a plateau phase of increased concentration of calcium may enhance the secretory response in this way.

Adrenal Medulla↗
PubMed1993

A two-step model of secretion control in neuroendocrine cells.

Recent experiments on a variety of neuroendocrine cells indicate that intense stimuli readily depress the secretory response. The most likely explanation for this depression is that a pool of release-ready granules is depleted. We present a two-step model of secretion that allows one to simulate the dynamics of such a pool for different time courses of free intracellular Ca concentration [Ca2+]i. We derive rate constants of the model from two types of experiment and find that, for the simplest type of model, not only the rate of consumption (exocytosis) but also the rate of vesicle supply to the pool of release-ready granules must be made Ca-dependent. Given these functional dependences a variety of results from the literature can be simulated. In particular, the model predicts the occurrence of secretory depression and augmentation under appropriate conditions.

Animals↗
PubMed1993

Calcium permeability of nicotinic acetylcholine receptor channels in bovine adrenal chromaffin cells.

The fractional contribution of Ca to current flow through neuronal-type nicotinic acetylcholine receptor channels was determined by quantitative fluorescence microfluorimetry using fura-2. The method, which has been applied already to several types of cells and channels is described in detail here. At -70 mV and 2 mM external Ca concentration it was found that Ca contributes 2.5% to the net current. The fractional contribution was found to be voltage dependent, increasing at negative potentials e-fold for a 110 mV potential difference. Total non-specific cation current was found to have a bell-shaped dependence on external Ca concentration peaking at 2 mM.

Adrenal Glands↗
PubMed1993

Small-conductance Ca(2+)-activated K+ channels in bovine chromaffin cells.

Simultaneous whole-cell patch-clamp and fura-2 fluorescence [Ca2+]i measurements were used to characterize Ca(2+)-activated K+ currents in cultured bovine chromaffin cells. Extracellular application of histamine (10 microM) induced a rise of [Ca2+]i concomitantly with an outward current at holding potentials positive to -80 mV. The activation of the current reflected an increase in conductance, which did not depend on membrane potential in the range -80 mV to -40 mV. Increasing the extracellular K+ concentration to 20 mM at the holding potential of -78 mV was associated with inwardly directed currents during the [Ca2+]i elevations induced either by histamine (10 microM) or short voltage-clamp depolarizations. The current reversal potential was close to the K+ equilibrium potential, being a function of external K+ concentration. Current fluctuation analysis suggested a unit conductance of 3-5 pS for the channel that underlies this K+ current. The current could be blocked by apamin (1 microM). Whole-cell current-clamp recordings showed that histamine (10 microM) application caused a transient hyperpolarization, which evolved in parallel with the [Ca2+]i changes. It is proposed that a small-conductance Ca(2+)-activated K+ channel is present in the membrane of bovine chromaffin cells and may be involved in regulating catecholamine secretion by the adrenal glands of various species.

Adrenal Glands↗
PubMed1993

Multiple calcium-dependent processes related to secretion in bovine chromaffin cells.

We have used the caged calcium compound DM-nitrophen to investigate the kinetics of calcium-dependent secretion in bovine chromaffin cells. Perfusion with partially calcium-loaded nitrophen often caused a loading transient--slow secretion for up to 1 min due to displacement of Ca2+ by cytoplasmic Mg2+. Flash photolysis elicited 100 microM [Ca2+]i steps that evoked intense secretion, lasting a few seconds. In cells experiencing a loading transient, [Ca2+]i steps evoked an especially fast secretion. A persistent, slow secretion often followed these fast phases. Distinct kinetic components may reflect secretion from pools that are differentially capable of release. Both secretion and movement of vesicles between pools appear to be [Ca2+]i sensitive. Later [Ca2+]i steps sometimes evoked a rapid capacitance decrease, indicating a fast, [Ca2+]i-dependent phase of endocytosis.

Acetates↗
PubMed1993

Fractional contribution of calcium to the cation current through glutamate receptor channels.

The Ca2+ fraction of the ion current flowing through glutamatergic NMDA and AMPA/kainate receptor channels was determined in forebrain neurons of the medial septum. The neurons were overloaded with the Ca2+ indicator dye fura-2 (1 mM) via the recording patch pipettes. This approach allowed the direct determination of the Ca2+ influx from changes in the Ca(2+)-sensitive fura-2 fluorescence. We found that, at negative membrane potentials and at an extracellular free Ca2+ concentration of 1.6 mM, the Ca2+ fraction of the current through the NMDA receptor channels is only 6.8%, about 2-fold lower than previously estimated from reversal potential measurements. Interestingly, a quite high fractional Ca2+ current of 1.4% was determined for the linearly conducting AMPA/kainate receptor channels found in these neurons.

Animals↗
PubMed1993

Mobile and immobile calcium buffers in bovine adrenal chromaffin cells.

1. The calcium binding capacity (kappa S) of bovine chromaffin cells preloaded with fura-2 was measured during nystatin-perforated-patch recordings. 2. Subsequently, the perforated patch was ruptured to obtain a whole-cell recording situation, and the time course of kappa S was monitored during periods of up to one hour. 3. No rapid change (within 10-20 s) of kappa S was observed upon transition to whole-cell recording, as would be expected, if highly mobile organic anions contributed significantly to calcium buffering. However, approximately half of the cells investigated displayed a drop in kappa S within 2-5 min, indicative of the loss of soluble Ca2+ binding proteins in the range of 7-20 kDa. 4. The average Ca2+ binding capacity (differential ratio of bound calcium over free calcium) was 9 +/- 7 (mean +/- S.E.M.) for the poorly mobile component and 31 +/- 10 for the fixed component. It was concluded that a contribution of 7 from highly mobile buffer would have been detected, if present. Thus, this value can be considered as an upper bound to highly mobile Ca2+ buffer. 5. Both mobile and fixed calcium binding capacity appeared to have relatively low Ca2+ affinity, since kappa S did not change in the range of Ca2+ concentrations between 0.1 and 3 microM. 6. It was found that cellular autofluorescence and contributions to fluorescence of non-hydrolysed or compartmentalized dye contribute a serious error in estimation of kappa S. 'Balanced loading', a degree of fura-2 loading such that the calcium binding capacity of fura-2 equals cellular calcium binding capacity, minimizes these errors. Also, changes in kappa S at the transition from perforated-patch to whole-cell recording can be most faithfully recorded for similar degrees of loading in both situations. 7. Nystatin was found unable to make pores from inside of the plasma membrane of chromaffin cells. With careful preparation and storage the diluted nystatin solution maintained its high activity of membrane perforation for more than one week. 8. An equation for the effective diffusion constant for total cytoplasmic calcium, D'Ca, was derived, which takes into account fixed buffer and poorly mobile buffer as determined, as well as calcium bound to fura-2 and some highly mobile buffers.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin cells.

In synapses, a rise in presynaptic intracellular calcium leads to secretory vesicle fusion in less than a millisecond, as indicated by the short delay from excitation to postsynaptic signal. In nonsynaptic secretory cells, studies at high time resolution have been limited by the lack of a detector as fast and sensitive as the postsynaptic membrane. Electrochemical methods may be sensitive enough to detect catecholamines released from single vesicles. Here, we show that under voltage-clamp conditions, stochastically occurring signals can be recorded from adrenal chromaffin cells using a carbon-fibre electrode as an electrochemical detector. These signals obey statistics characteristic for quantal release; however, in contrast to neuronal transmitter release, secretion occurs with a significant delay after short step depolarizations. Furthermore, we identify a pedestal or 'foot' at the onset of unitary events which may represent the slow leak of catecholamine molecules out of a narrow 'fusion pore' before the pore dilates for complete exocytosis.

Adrenal Medulla↗
PubMed1992

Correction for liquid junction potentials in patch clamp experiments.

This chapter describes corrections that have to be applied to measured membrane potentials in patch clamp experiments. Some of them [Eqs. (1)-(3)] are required regardless of the nature of the reference electrode (in the Ringer's solution bath) whenever the pipette-filling solution is different from the bath solution. They represent the liquid junction potentials that are present at the pipette tip before patch formation. In addition, corrections have to be applied when the bath solution is being changed during a measurement (i.e., after seal formation). In that case the following rules apply. (1) The new solution should never get into contact with the bare silver/silver chloride wire of the reference electrode. This requirement is best met by using a salt bridge. (2) The "best" salt bridge is a 3 M KCl bridge with an abrupt KCl-bath fluid boundary at its tip (see above). This bridge does not require any additional potential corrections, but it may lead to KCl poisoning of the bath or become contaminated by solutions used previously. (3) Local solution changes (microperfusion by puffer pipette, U tool or sewer pipe arrangements) as well as recessed KCl bridges require additional corrections, which (together with the simple liquid junction potential correction) are approximately given by Eqs. (6)-(8). It should be stressed that all equations given here represent approximate corrections, since liquid junction potentials are thermodynamically ill-defined. This is particularly relevant for Eqs. (6) and (7) where the sum of two liquid junction potentials appears.

Amplifiers, Electronic↗
PubMed1992

Neuronal Ca2+ signalling takes the local route.

The past year has seen several sets of experimental results demonstrate that fast, large and highly localized rises in intracellular Ca2+ concentration can occur in neurons. These results confirm previous theoretical predictions of acute spatial compartmentalization of Ca2+ signalling, and document a form of signalling that may occur whenever rapid and local signal processing is the goal. The dimensions involved present severe challenges for attempts to directly measure these signalling events.

Animals↗