G. Alvarez de Toledo

High calcium concentrations shift the mode of exocytosis to the kiss-and-run mechanism.

Exocytosis, the fusion of secretory vesicles with the plasma membrane to allow release of the contents of the vesicles into the extracellular environment, and endocytosis, the internalization of these vesicles to allow another round of secretion, are coupled. It is, however, uncertain whether exocytosis and endocytosis are tightly coupled, such that secretory vesicles fuse only transiently with the plasma membrane before being internalized (the ‘kiss-and-run’ mechanism), or whether endocytosis occurs by an independent process following complete incorporation of the secretory vesicle into the plasma membrane. Here we investigate the fate of single secretory vesicles after fusion with the plasma membrane by measuring capacitance changes and transmitter release in rat chromaffin cells using the cell-attached patch-amperometry technique. We show that raised concentrations of extracellular calcium ions shift the preferred mode of exocytosis to the kiss-and-run mechanism in a calcium-concentration-dependent manner. We propose that, during secretion of neurotransmitters at synapses, the mode of exocytosis is modulated by calcium to attain optimal conditions for coupled exocytosis and endocytosis according to synaptic activity.

Is swelling of the secretory granule matrix the force that dilates the exocytotic fusion pore

The swelling of the secretory granule matrix which follows fusion has been proposed as the driving force for the rapid expansion of the fusion pore necessary for exocytosis. To test this hypothesis, we have combined simultaneous measurements of secretory granule swelling using videomicroscopy with patch clamp measurements of the time course of the exocytotic fusion pore in mast cells from the beige mouse. We show that isotonic acidic histamine solutions are able to inhibit swelling of the secretory granule matrix both in purified secretory granules lysed by electroporation and in intact cells stimulated to exocytose by guanine nucleotides. In contrast to the inhibitory effects on granule swelling, the rate of expansion of the exocytotic fusion pore is unaffected. Therefore, as the rate of granule swelling was more than 20 times slower under these conditions, swelling of the secretory granule matrix due to water entry through the fusion pore cannot be the force responsible for the characteristic rapid expansion of the exocytotic fusion pore. We suggest that tension in the secretory granule membrane, which has recently been demonstrated in fused secretory granules, might be the force that drives the irreversible expansion of the fusion pore.

Ionic basis of the differential neuronal activity of guinea‐pig septal nucleus studied in vitro.

1. The electrical properties and ionic conductances of septal neurones were studied by intracellular recording in an in vitro slice preparation. Within the total number of cells recorded (n = 150) we identified three electrophysiological cell types, each one of them located in a separate septal region. Dorsolateral septal neurones comprised 60% of the cells, intermediate septal neurons 10%, and medical septal neurones 30%. 2. Passive electrical constants of dorsolateral, intermediate and medial septal neurones were, respectively:resting potential (‐60.2 +/‐ 4.8, ‐59.8 +/‐ 3.3 and ‐56 +/‐ 4.3 mV); input resistance (82.5 +/‐ 17, 63 +/‐ 16 and 83 +/‐ 18 M omega) and membrane time constant (18.5 +/‐ 7.3, 14.2 +/‐ 6.8 and 10.7 +/‐ 3.4 ms). 3. Direct activation of dorsolateral septal neurones by current injection below 0.2 nA triggered repetitive firing of fast action potentials. Larger current pulses elicited a characteristic response consisting of an initial fast action potential followed by a train of slow spikes. An after‐hyperpolarization followed termination of the pulse and the characteristic response. 4. In dorsolateral septal neurons tetrodotoxin (TTX) abolished the fast action potentials. The slow spikes and the after‐hyperpolarization disappeared in presence of Co2+ or after brief removal of external Ca2+. This suggests that the characteristic response is mediated by Ca2+ and the after‐hyperpolarization by a Ca2+‐dependent K+ conductance. 5. The firing pattern of intermediate septal neurones activated from the resting potential spontaneously measured in the cells was similar to that of dorsolateral septal neurones; but direct activation from a hyperpolarized membrane potential evoked in intermediate septal cells a bursting response due to the generation of a low‐threshold spike. The low‐threshold spike was TTX‐resistant but abolished by Co2+ and reached a maximal amplitude after hyperpolarization to ‐75 mV lasting for 100‐150 ms. These results suggest the existence in intermediate septal neurons of a low‐threshold Ca2+ conductance inactivated at the resting potential and deinactivated by hyperpolarization. 6. Depolarization of medial septal neurons by current pulses of amplitude greater than 0.2‐0.3 nA elicited a typical burst of two to six action potentials. The bursts lasted for 20‐50 ms and were followed by a marked after‐hyperpolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrophysiological properties of guinea pig septal neurons in vitro

The electrophysiological properties of septal neurons have been examined in vitro in guinea pig brain slices. These cells display different firing modes when stimulated by transmembrane current pulses depending on the amplitude of the depolarization. With small pulses septal neurons fire repetitive Na spikes but on larger depolarizations they respond with a single full-Na action potential which is followed by a number of spikes of smaller amplitude. A further increase in the amplitude of the pulse evokes powerful Ca spikes possibly generated in the dendrites. These Ca spikes appear with larger amplitude in presumptive intradendritic recordings. In many cells stimulation of the fimbria evoked postsynaptic responses consisting of either a depolarization, a hyperpolarization or a depolarization-hyperpolarization sequence.

On line quantification of mast cell degranulation with electrophysiological techniques

Mast cells play a crucial role in inflammation by releasing mediators that orchestrate the action of neutrophils, macrophages and lymphocytes [1]. This action may lead to the effective arrest of a bacterial infection [2, 3] or, by contrast, evoked the undesirable effects of anaphylaxis [4]. These two dramatically opposite actions are elicited by two different mechanisms but mediated by the same secretory products stored and released by mast cells. Therefore, the rate and the extent of mast cells degranulation may be critical for determining the quality of the inflammatory response. In this paper we have combined cell membrane capacitance measurements with fast cyclic voltammetry in single isolated peritoneal mast cells to monitor the time course of degranulation. This in vitro method may be useful to estimate the concentration in the extracellular space of mast cell mediators and to determine factors that regulate this process.