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Prolactin Secretion Sites Contain Syntaxin-1 and Differ from Ganglioside Monosialic Acid Rafts in Rat Lactotrophs

Paula P. Gonçalves¹, Matja Stenovec¹, Helena H. Chowdhury, Sonja Grilc, Marko Kreft and Robert Zorec

Centro de Estudos do Ambiente e do Mar (P.P.G.), Departamento de Biologia, Universidade de Aveiro, 3810-193 Aveiro, Portugal; and Celica Biomedical Center (M.S., H.H.C., S.G., M.K., R.Z.), Technology Park Ljubljana and Laboratory of Neuroendocrinology-Molecular Cell Physiology (M.S., H.H.C., S.G., M.K., R.Z.), Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia

Address all correspondence and requests for reprints to: Robert Zorec, Ph.D., Professor, Member Academia Europaea and The Slovenian Academy of Sciences and Arts, Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana; Zaloka 4, 1000 Ljubljana, Slovenia. E-mail: robert.zorec{at}mf.uni-lj.si.

In neuroendocrine cells, discharge of hormones follows the fusion of exocytotic vesicles with the plasma membrane at confined sites; however, the molecular nature of these distinct sites remains poorly understood. We studied intact pituitary lactotrophs and plasma membrane lawns by confocal microscopy in conjunction with antibodies against rat prolactin (rPRL), soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) proteins (syntaxin-1 and synaptobrevin-2,) and fluorescent cholera toxin subunit B (CT-B), a marker of ganglioside monosialic acid (GM1) lipid rafts, to examine 1) whether rPRL vesicles discharge cargo at GM1 rafts, 2) whether discharging rPRL vesicles interact with SNAREs, and 3) to examine the overlap of GM1 rafts, rPRL, and syntaxin-1 sites in plasma membrane lawns. In intact cells, immunofluorescently labeled rPRL poorly colocalized (<6%) with CT-B. In conditions favoring endocytotic trafficking, vesicle SNARE synaptobrevin-2 modestly colocalized (35%) with CT-B, whereas it highly colocalized (58%) with retrieved rPRL. Although partial mixing between rPRL and CT-B intracellular trafficking pathways is likely, our results indicated that rPRL discharge involves interactions with plasma membrane SNAREs, but not with GM1 rafts. In support of this, the plasma membrane SNARE syntaxin-1 poorly colocalized with CT-B (<5%), whereas it highly colocalized (75%) with rPRL in inside-out plasma membrane lawns. Spontaneous and stimulated rPRL discharge in live lactotrophs is thus associated with plasma membrane sites enriched with SNARE proteins, however, spatially confined to plasma membrane areas other than GM1 rafts.