Localization and physiological regulation of the exocytosis protein SNAP-25 in the brain and pituitary gland of Xenopus laevis

In mammals, the synaptosomal-associated protein of 25 kDa, SNAP-25, is gene rally thought to play a role in synaptic exocytosis of neuronal messengers. Using a polyclonal antiserum against rat SNAP-25, we have shown the presen ce of a SNAP-25-like protein in the brain of the South-African clawed toad Xenopus laevis by Western blotting and immunocytochemistry. Xenopus SNAP-25 is ubiquitously present throughout the brain, where its distribution in va rious identified neuronal perikarya and axon tracts is described. Western b lot analysis and immunocytochemistry also demonstrated the presence of SNAP -25 in the neural, intermediate and distal lobes of the pituitary gland. In tensity line plots of confocal laser scanning microscope images of isolated melanotropes indicated that SNAP-25 is produced and processed in the rough endoplasmatic reticulum and Golgi apparatus, and is associated with the pl asma membrane. Immunoelectron microscopy substantiated the idea that SNAP-2 5 is present in the plasma membrane but also showed a close association of SNAP-25 with the bounding membrane of peptide-containing secretory granules in both the neurohemal axon terminals in the neural lobe and the endocrine melanotropes in the intermediate lobe. Quantitative Western blotting revea led that adapting Xenopus to a dark background has a clear stimulatory effe ct on the expression of SNAP-25 in the neural lobe and in the melanotrope c ells. This background light intensity-dependent stimulation of SNAP-25 expr ession was confirmed by the demonstration of increased immunofluorescence r ecorded by confocal laser scanning microscopy of individual melanotropes of black background-adapted toads. On the basis of this study on Xenopus laev is, we conclude that SNAP-25 (i) plays a substantial role in the secretion of a wide variety of neuronal messengers; (ii) functions in the central ner vous system but also in neurohormonal and endocrine systems; (iii) acts at the plasma membrane but possibly also at the membrane of synaptic vesicles and peptide-containing secretory granules; (iv) acts not only locally (as i n synapses), but at various sites of the plasma membrane (as in the endocri ne melanotrope cell); and (v) can be upregulated in its expression by physi ological stimuli that increase the extent of the molecular machinery involv ed in exocytosis.