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The density of the immunolabeled ternary soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) complexes that bind complexin was approximately 10 times lower than the PRL-positive, lawn-resident vesicles; this indicates that some but not all vesicles are associated with ternary SNARE complexes. However, lawn-resident PRL vesicles colocalized relatively well with particular SNARE proteins: synaptobrevin 2 (35%), syntaxin 1 (22%), and 25-kDa synaptosome associated protein (6%). To study vesicle discharge, we prepared lawn-resident vesicles, derived from atrial natriuretic peptide tagged with emerald fluorescent protein (ANP.emd)-transfected cells, which label vesicles. These maintained the structural passage to the exterior because approximately 40% of ANP.emd-loaded vesicles were labeled by extracellular PRL antibodies. Cargo release from the lawn-resident vesicles, monitored by the decline in the ANP.emd fluorescence intensity, was similar to that in intact cells. It is likely that SNARE proteins are required for calcium-dependent release from these vesicles. This is because the expression of the dominant-negative SNARE peptide, which interferes with SNARE complex formation, reduced the number of PRL-positive spots per cell (PRL antibodies placed extracellularly) significantly, from 58 \u00b1 9 to 4 \u00b1 2. In dominant-negative SNARE-treated cells, the PRL-positive area was reduced from 0.259 \u00b1 0.013 to 0.123 \u00b1 0.014 \u03bcm2, which is consistent with a hindered vesicle luminal access for extracellular PRL antibodies. These results indicate that vesicle discharge is regulated by SNARE-mediated fusion pore widening.<\/jats:p>","DOI":"10.1210\/en.2012-2022","type":"journal-article","created":{"date-parts":[[2013,2,1]],"date-time":"2013-02-01T13:52:41Z","timestamp":1359726761000},"page":"1235-1246","source":"Crossref","is-referenced-by-count":6,"title":["Peptide Hormone Release Monitored From Single Vesicles in \u201cMembrane Lawns\u201d of Differentiated Male Pituitary Cells: SNAREs and Fusion Pore Widening"],"prefix":"10.1210","volume":"154","author":[{"given":"Matja\u017e","family":"Stenovec","sequence":"first","affiliation":[{"name":"Celica Biomedical Center (M.S., R.Z.), University of Ljubljana, 1000 Ljubljana, Slovenia"},{"name":"Laboratory of Neuroendocrinology-Molecular Cell Physiology (M.S., R.Z.), Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia"}]},{"given":"Paula P.","family":"Gon\u00e7alves","sequence":"first","affiliation":[{"name":"Department of Biology and Centro 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