RETRIEVED CONSTITUENTS OF LARGE DENSE-CORED VESICLES AND SYNAPTIC VESICLES INTERMIX IN STIMULATION-INDUCED EARLY ENDOSOMES OF NORADRENERGICNEURONS

Two storage compartments in cultured noradrenergic neurons derived fro m the superior cervical ganglion from fetal pig have been defined usin g sucrose density gradient centrifugation and electron microscopy: (1) large dense-cored vesicles (LDV) contain noradrenaline and dopamine-b eta-hydroxylase (D beta H); (2) small electron-lucent vesicles contain acetylcholine and p38 and represent the noradrenergic small synaptic vesicles (SSV); no small dense-cored vesicles (SDV) could be detected. Our results demonstrate that internalized LDV membrane constituents a re retrieved into early endosomes, as shown by the colocalization of r etrieved D beta H with the endosomal markers Rab5 and HRP in sucrose d ensity gradients and on confocal microscopical images. Recycling of th e SSV membranes via an endosomal intermediate is also confirmed in nor adrenergic neurons. Finally, colocalization of retrieved DPH and retri eved p38 in stimulated neurons indicates that the two sets of constitu ents intermix. These data provide the first experimental evidence for a common early endosome in which SSV and LDV membrane constituents are internalized after exocytosis and imply that endosomal sorting is an important process for the generation of different secretory vesicles i n the noradrenergic nerve terminal.