The position of mitochondria and ER in relation to that of the secretory sites in chromaffin cells.

Knowledge of the distribution of mitochondria and endoplasmic reticulum (ER) in relation to the position of exocytotic sites is relevant to understanding the influence of these organelles in tuning Ca(2+) signals and secretion. Confocal images of probes tagged to mitochondria and the F-actin cytoskeleton revealed the existence of two populations of mitochondria, one that was cortical and one that was perinuclear. This mitochondrial distribution was also confirmed by using electron microscopy. In contrast, ER was sparse in the cortex and more abundant in deep cytoplasmic regions. The mitochondrial distribution might be due to organellar transport, which experiences increasing restrictions in the cell cortex. Further study of organelle distribution in relation to the position of SNARE microdomains and the granule fusion sites revealed that a third of the cortical mitochondria colocalized with exocytotic sites and another third located at a distance closer than two vesicle diameters. ER structures were also present in the vicinity of secretory sites but at a lower density. Therefore, mitochondria and ER have a spatial distribution that suggests a specialized role in modulation of exocytosis that fits with the role of cytosolic Ca(2+) microdomains described previously.

Role of protease-activated receptor 2 in lung injury development during acute pancreatitis in rats.

OBJECTIVE: The objective of this study was to evaluate whether an uncontrolled activation of mast cells and macrophages through protease-activated receptor-2 (PAR-2) during acute pancreatitis could develop lung injury. METHODS: Pancreatitis was induced in rats by intraductal infusion of sodium taurocholate. In a group of animals, PAR-2 antagonist or trypsin (TRP) inhibitor was intravenously administered before the pancreatitis induction. In additional groups, the animals were treated with PAR-2-activating peptide or pancreatic TRP. The myeloperoxidase (MPO) activity was measured to evaluate the progression of inflammation. RESULTS: Plasma from the animals with pancreatitis and pancreatic TRP induced the secretion of mast cells and alveolar macrophages as well as increased the density of PAR-2 in the plasma membrane. The treatment of alveolar macrophages with TRP, tryptase, as well as PAR-1- and PAR-2-activating peptide led to an increase in calcium-triggered exocytosis. Similar results were obtained in acinar cells. The intravenous injection of PAR-2-activating peptide and TRP induced an increase in MPO activity in the lung. The intravenous injection of PAR-2 antagonist or TRP inhibitor before the pancreatitis induction could prevent the increase in MPO activity in the pancreas and the lung. CONCLUSIONS: The TRP generated during acute pancreatitis could be involved in the progression of lung injury through the activation of PAR-2 in alveolar macrophages.

The F-actin cortex in chromaffin granule dynamics and fusion: a minireview.

Chromaffin granules are restrained in a dense cortical cytoskeleton before releasing their complex mix of active substances in response to cell stimulation. In recent years, the complex organization and dynamics of the chromaffin cell cortex has been unveiled through its analysis with a range of techniques to visualize this structure, including confocal fluorescence, transmitted light, and evanescent field microscopy. Accordingly, it has become apparent that the cortex is a dense F-actin mesh that contains open polygonal spaces through which vesicles can access the submembrane space. In addition to its retentive role, this structure also influences vesicle motion in both the resting state and during cell stimulation with secretagogues. During secretion, the chromaffin cell cortex undergoes a complex reorganization, helping to replenish the empty fast releasable pool of vesicles. Such changes in the cortical cytoskeleton and in the vesicle motion are governed by the activity of molecular motors, such as myosins II and Va. Interestingly, the F-actin/myosin II network also affects the final stages of exocytosis, which involve the opening and expansion of the fusion pore, and the extrusion of the vesicles contents.

Vesicle motion and fusion are altered in chromaffin cells with increased SNARE cluster dynamics.

The expression of SNAP-25 fused to green fluorescent protein (GFP) has been instrumental in demonstrating SNARE role in exocytosis. The wild-type GFP-SNAP-25 and a Delta9 form, product of botulinum neurotoxin A activity, the main ingredient in the BOTOX preparation, were employed here to study SNARE implication in vesicle mobility and fusion in cultured bovine chromaffin cells, a neuroendocrine exocytotic model. Using total internal reflection fluorescent microscopy, we have identified membrane microdomains of 500-600 nm diameter that contain both SNAP-25 and syntaxin-1 and associate with synaptobrevin-2. Interestingly, while the SNAP-25 Delta9 formed similar clusters, they displayed increased mobility both laterally and in the axis perpendicular to the plasmalemma, and this correlates with the enhanced dynamics of associated chromaffin granules. SNARE cluster-enhanced motion is reversed by elevation of the intracellular calcium level. Furthermore, single vesicle fusion was unlikely in the highly mobile vesicles present in the cells expressing SNAP-25 Delta9, which, in addition, displayed in average slower fusion kinetics. Consequently, SNARE cluster dynamics is a new aspect to consider when determining the factors contributing to the mobility of the vesicles in close vicinity to the plasma membrane and also the probability of exocytosis of this granule population.

Differential participation of actin- and tubulin-based vesicle transport systems during secretion in bovine chromaffin cells.

The role of cytoskeletal elements in vesicle transport occurring during exocytosis was examined in adrenal medullary bovine chromaffin cells maintained in culture. Amperometric determination of depolarization-dependent catecholamine release from individual intact cells treated with actin or myosin inhibitors showed alterations in the fast and slow phases of secretion when compared with untreated cells. In contrast, microtubule disassemblers or stabilizers have a moderate effect on secretion, only affecting the release of slow secretory components. In experiments using confocal dynamic microscopy we have observed the drastic effect of actin and myosin inhibitors in abolishing vesicle movement throughout the cytoplasm, and the inhibition of granule mobility in deep perinuclear regions caused by the microtubule stabilizers. Following loss of mobility, vesicles were associated with filaments of F-actin or microtubules. In addition, the mobility of cortical vesicles was affected by actin-myosin inhibitors but not by microtubule inhibitors. The study of cortical cytoskeleton in living cells showed vesicles associated with dense tubular F-actin structures, with microtubules appearing as low density networks. These findings suggest that the distribution and density of both cytoskeletal elements in the cortical region may influence the recruitment of vesicle pools during secretion.

The role of myosin in vesicle transport during bovine chromaffin cell secretion.

Bovine adrenomedullary cells in culture have been used to study the role of myosin in vesicle transport during exocytosis. Amperometric determination of calcium-dependent catecholamine release from individual digitonin-permeabilized cells treated with 3 microM wortmannin or 20 mM 2,3-butanedione monoxime (BDM) and stimulated by continuous as well as repetitive calcium pulses showed alteration of slow phases of secretion when compared with control untreated cells. The specificity of these drugs for myosin inhibition was further supported by the use of peptide-18, a potent peptide affecting myosin light-chain kinase activity. These results were supported also by studying the impact of these myosin inhibitors on chromaffin granule mobility using direct visualization by dynamic confocal microscopy. Wortmannin and BDM affect drastically vesicle transport throughout the cell cytoplasm, including the region beneath the plasma membrane. Immunocytochemical studies demonstrate the presence of myosin types II and V in the cell periphery. The capability of antibodies to myosin II in abrogating the secretory response from populations of digitonin-permeabilized cells compared with the modest effect caused by anti-myosin V suggests that myosin II plays a fundamental role in the active transport of vesicles occurring in the sub-plasmalemmal area during chromaffin cell secretory activity.