Depolarization evokes different patterns of calcium signals and exocytosis in bovine and mouse chromaffin cells: the role of mitochondria.

This study was planned on the assumptions that different high-voltage activated calcium channels and/or the ability of mitochondria to take up Ca(2+) could be responsible for different cytosolic Ca(2+) concentrations ([Ca(2+)](c)) and catecholamine release responses in adrenal chromaffin cells of bovine and mouse species. Short K(+) pulses (2-5 s, 70 mM K(+)) increased [Ca(2+)](c) to a peak of about 1 microM; however, in bovine cells the decline was slower than in mouse cells. Secretory responses were faster in mouse but were otherwise quantitatively similar. Upon longer K(+) applications (1 min), elevations of [Ca(2+)](c) and secretion were prolonged in bovine cells; in contrast [Ca(2+)](c) in mouse cells declined three-fold faster and failed to sustain a continued secretion. Confocal [Ca(2+)](c) imaging following a 50-ms depolarizing pulse showed a similar Ca(2+) entry, but a rate of [Ca(2+)](c) increase and a maximum peak significantly higher in bovine cells; the rate of dissipation of the Ca(2+) wave was faster in the mouse. The mitochondrial protonophore CCCP (2 microm) halved the K(+)-evoked [Ca(2+)](c) and secretory signals in mouse cells, but had little affect on bovine responses. We conclude that the relative densities of L (15% in bovine and 50% in mouse) and P/Q Ca(2+) channels (50% in bovine and 15% in mouse) do not contribute to the observed differences; rather, the different intracellular distribution of Ca(2+), which is strongly influenced by mitochondria, is responsible for a more sustained secretory response in bovine, and for a faster and more transient secretory response in mouse chromaffin cells. It seems that mitochondria near the plasmalemma sequester Ca(2+) more rapidly and efficiently in the mouse than in the bovine chromaffin cell.

Exocytosis of catecholamine (CA)-containing and CA-free granules in chromaffin cells.

Recent evidence suggests that endocytosis in neuroendocrine cells and neurons can be tightly coupled to exocytosis, allowing rapid retrieval from the plasma membrane of fused vesicles for future use. This can be a much faster mechanism for membrane recycling than classical clathrin-mediated endocytosis. During a fast exo-endocytotic cycle, the vesicle membrane does not fully collapse into the plasma membrane; nevertheless, it releases the vesicular contents through the fusion pore. Once the vesicle is depleted of transmitter, its membrane is recovered without renouncing its identity. In this report, we show that chromaffin cells contain catecholamine-free granules that retain their ability to fuse with the plasma membrane. These catecholamine-free granules represent 7% of the total population of fused vesicles, but they contributed to 47% of the fusion events when the cells were treated with reserpine for several hours. We propose that rat chromaffin granules that transiently fuse with the plasma membrane preserve their exocytotic machinery, allowing another round of exocytosis.

The sea anemone toxin Bc2 induces continuous or transient exocytosis, in the presence of sustained levels of high cytosolic Ca2+ in chromaffin cells.

We have isolated and characterized a new excitatory toxin from the venom of the sea anemone Bunodosoma caissarum, named Bc2. We investigated the mechanism of action of the toxin on Ca(2+)-regulated exocytosis in single bovine adrenal chromaffin cells, monitoring simultaneously fura-2 fluorescence measurements and electrochemical recordings using a carbon fiber microelectrode. Bc2 induced quantal release of catecholamines in a calcium-dependent manner. This release was associated with a sustained rise in cytosolic Ca(2+) and displayed two different patterns of response: a continuous discharge of prolonged duration that changed to a transient burst as the toxin concentration (or incubation time) increased. Continuous secretion was dependent on the activity of native voltage-dependent Ca(2+) channels and showed a pattern similar to that of alpha-latrotoxin; however, its kinetics adjusted better to that of continuous cell depolarization with high K(+) concentration. In contrast, transient secretion was independent of Ca(2+) entry through native voltage-dependent Ca(2+) channels and showed inhibition of late vesicle fusion that was accompanied by "freezing" of F-actin disassembly. These new features make Bc2 a promising new tool for studying the machinery of neurotransmitter release.

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.

[Neurotransmitter release: a process of membrane fusion occurring in fractions of milliseconds]. / Liberación de neurotransmisores: un proceso de fusión de membranas que transcurre en fracciones de milisegundos.

INTRODUCTION: The physiological mechanisms involved in neurotransmitter release were established thanks to the pioneer work of Katz, Del Castillo and Miledi at the neuromuscular junction. They termed their work as the quantal hypothesis of synaptic transmission. This hypothesis was further established morphologically by the work of Heuser and Reese. However, the molecular events underlying this process are poorly understood. We are starting to know which proteins interact between the vesicle and plasma membrane to promote fusion and to identify which molecules participate in the sensing of cytosolic calcium. DEVELOPMENT: Thanks to the combination of molecular biology, electrophysiology and microfluorometry, a huge amount of new information is been obtained on the mechanisms participating in synaptic transmission. This data deals with processes concerning to different pools of synaptic vesicles and their availability to reach the presynaptic membrane; the molecular events responsible for the targeting of these vesicles with the plasma membrane; the sensitivity to calcium at the presynaptic membrane; the fusion of membranes required to release the vesicle contents, and the mechanisms responsible for membrane retrieval needed for presynaptic homeostasis. CONCLUSIONS: In this review we discuss new data regarding synaptic function. However, some key points are still a matter of controversy. Meanwhile the quantal hypothesis is valid, the precise processes by which channels and vesicles interact, membrane is recycled and vesicles reused are still controversial. New techniques should be developed to address these points.