Platelet-activating factor-mediated endosome formation causes membrane translocation of p67phox and p40phox that requires recruitment and activation of p38 MAPK, Rab5a, and phosphatidylinositol 3-kinase in human neutrophils.

Neutrophils (polymorphonuclear leukocytes, PMNs) are vital to innate immunity and receive proinflammatory signals that activate G protein-coupled receptors (GPCRs). Because GPCRs transduce signals through clathrin-mediated endocytosis (CME), we hypothesized that platelet-activating factor (PAF), an effective chemoattractant that primes the PMN oxidase, would signal through CME, specifically via dynamin-2 activation and endosomal formation resulting in membrane translocation of cytosolic phagocyte oxidase (phox) proteins. PMNs were incubated with buffer or 2 muM PAF for 1-3 min, and in some cases activated with PMA, and O(2)(-) was measured, whole-cell lysates and subcellular fractions were prepared, or the PMNs were fixed onto slides for digital or electron microscopy.

Recycling of intact dense core vesicles in neurites of NGF-treated PC12 cells.

Exocytic fusion in neuroendocrine cells does not always result in complete release of the peptide contents from dense core vesicles (DCVs). In this study, we use fluorescence imaging and immunoelectron microscopy to examine the retention, endocytosis and recycling of chromogranin B in DCVs of NGF-treated PC12 cells. Our results indicate that DCVs retained and retrieved an intact core that was available for subsequent exocytic release.

Using FM1-43 to study neuropeptide granule dynamics and exocytosis.

In the study of neuropeptide secretion and membrane trafficking, the fluorescent dye FM1-43 provides the ability to label selectively those structures that are undergoing exocytosis and endocytosis in living cells in real time. This review describes the unique properties of the FM dyes that make them ideal for studying neuropeptide granule dynamics and discusses various techniques that take advantage of FM dyes.

Retention and stimulus-dependent recycling of dense core vesicle content in neuroendocrine cells.

We have used fluorescence imaging of individual exocytic events in combination with immunogold electron microscopy and FM1-43 photoconversion to study the stimulus-dependent recycling of dense core vesicle content in isolated rat pituitary lactotrophs. Secretory stimulation with high external [K(+)] resulted in 100 exocytic sites per cell that were labeled by extracellular antibodies against the peptide hormone prolactin. Morphological analysis demonstrated that the prolactin was retained and internalized in intact dense cores.