Preparation and culture of adrenal chromaffin cells.

Cultured chromaffin cells have been used for almost 40 years in the study of different cell functions using biochemical, electrophysiological, pharmacological, and toxicological approaches. Chromaffin cells are essentially secretory cells that are used to model sympathetic neurons or neuroendocrine cells. In this chapter, we describe the most common methods currently used to isolate and culture chromaffin cells from the animals used most commonly: cows, rats, and mice. We also provide some advice on the use of these cells in the laboratory.

The quantal secretion of catecholamines is impaired by the accumulation of beta-adrenoceptor antagonists into chromaffin cell vesicles.

BACKGROUND AND PURPOSE: The delayed onset of certain effects of antagonists of beta-adrenoceptors (beta-blockers), such as lowering arterial blood pressure (several days), cannot be explained solely by their effects on beta-adrenoceptors, an action that occurs within minutes. Although several mechanisms have been proposed, none of them explain this temporal delay. This work aimed at providing a new explanation based on the interference of these drugs with the functional accumulation of catecholamines within neurosecretory vesicles. EXPERIMENTAL APPROACH: We used the simultaneous on-line monitoring of catecholamine and labetalol release from bovine isolated chromaffin cells and from rat perfused adrenal glands, as well as single cell amperometry, intracellular electrochemistry, patch amperometry and HPLC. KEY RESULTS: Using amperometry, three beta-blockers, labetalol, atenolol and propranolol, reduced the quantal size of secretory events in chromaffin cells, accompanied by a slowing down of exocytosis. By patch amperometry, we found that treatment with beta-blockers also increases the chromaffin vesicle volume, thereby creating a functional dilution of catecholamines. Experiments with intracellular electrochemistry show that vesicles cannot uptake new catecholamines. There was progressive accumulation of labetalol in secretory vesicles of bovine adrenal chromaffin cells, and this beta-blocker was co-released with catecholamines from rat and bovine chromaffin tissues. CONCLUSIONS AND IMPLICATIONS: We propose that beta-blockers are progressively concentrated into sympathetic secretory vesicles, and interfere with the storage of catecholamines and are co-released with the natural transmitters, resulting in a decrease in the sympathetic tone. This could explain the delayed onset of the hypotensive effects of beta-blockers.

The crucial role of chromogranins in storage and exocytosis revealed using chromaffin cells from chromogranin A null mouse.

Chromogranins (Cgs) are the major soluble proteins of dense-core secretory vesicles. Chromaffin cells from Chga null mice [chromogranin A knock-out (CgA-KO)] exhibited approximately 30% reduction in the content and in the release of catecholamines compared with wild type. This was because of a lower secretion per single exocytotic event, rather than to a lower frequency of exocytotic events. Cell incubation with L-DOPA produced an increase in the vesicular amine content of wild-type, but not CgA-KO vesicles. In contrast, intracellular electrochemistry showed that L-DOPA produced a significantly larger increase in cytosolic amines in CgA-KO cells than in the wild type. These data indicate that the mechanisms for vesicular accumulation in CgA-KO cells were fully saturated. Patch-amperometry recordings showed a delayed initiation of the amperometric signal after vesicle fusion, whereas no changes were observed in vesicle size or fusion pore kinetics despite the smaller amine content. We conclude that intravesicular proteins are highly efficient systems directly implicated in transmitter accumulation and in the control of neurosecretion.

Pharmacological regulation of the late steps of exocytosis.

We used amperometry to analyze the role of several second messengers and drugs in the exocytotic kinetics of bovine chromaffin cells. Activation of PKG produces a slowing down of exocytosis, which is not generally accompanied by changes in the net granule content of catecholamines. These effects are also observed after mild PKA activation. However, strong PKA stimulation also causes an increase in the apparent granule content of catecholamines, suggesting the presence of composed fusion. Conversely, PKC activation promotes acceleration of the exocytotic process. We also analyzed the contribution of different Ca(2+) channel subtypes to the exocytotic kinetics at the single event level. Although N-subtype channels do not contribute to total catecholamine release, their blockade produces a slowing down of exocytosis without changes in granule content. However, L or P/Q blockade causes, in addition, a reduction in the apparent granule content. The L-type agonist BAY-K-8644 produces giant secretory amperometric spikes, indicating that Ca(2+) favors composed fusion prior to exocytosis. Our data suggest that second messengers continuously regulate exocytotic kinetics and granule content. In addition, several well-known antihypertensive agents, such as sodium nitroprusside, organic nitrates, hydralazine, or Ca(2+) antagonists, could be acting through these novel mechanisms on sympathetic synapses by changing the synaptic performance, thereby producing additional vasodilatory effects.

New approaches for analysis of amperometrical recordings.

Amperometry is a powerful tool for studying the exocytotic process. Catecholamines released from a single secretory vesicle are oxidized by a carbon fiber microelectrode placed on a chromaffin cell membrane. This phenomenon can be recorded online as an amperometric signal. Each event of exocytosis is called a secretory spike. Several kinetic parameters can be extracted from spikes to get important information about catecholamine storage and to follow the time course of exocytosis. The large amount of data requires the use of computer programs. We describe software, written for Igor Pro (Wavemetrics, Lake Oswego, OR, USA), that allows the offline analysis of amperometric signals. It includes (1) the automatic analysis of a large collection of experiments without user intervention; (2) the visual check of located spikes; (3) data pooling from several experiments to create galleries with hundreds of thousands of spikes. In addition, we have designed a new filtering method for amperometric data. It provides an excellent tool to enhance the signal/noise ratio with minimal artifacts. This filter allows one to obtain more reliable spike parameters.