Calcium-dependent subquantal peptide release from single docked lawn-resident vesicles of pituitary lactotrophs. (January 2023)

• Record Type: Journal Article
• Authors: Gonçalves, Paula P.; Stenovec, Matjaž; Grácio, Luciano; Kreft, Marko; Zorec, Robert
• Is Part Of: Cell calcium. Volume 109(2023)
• Publication Date: 2023-01
• Subjects: Stimulus-secretion coupling -- Ca2+ signalling -- Lactotroph -- Secretory vesicle -- Fusion pore -- ANP.emd -- Confocal microscopy; Calcium -- Metabolism -- Periodicals; Vertebrates -- Physiology -- Periodicals; Calcium -- Physiological effect -- Periodicals; Cell physiology -- Periodicals; Calcium in the body -- Periodicals; 572.516
• DOI: 10.1016/j.ceca.2022.102687
• Languages: English
• ISSNs: 0143-4160

Abstract

Highlights: A release-competent exocytotic apparatus is preserved in lactotroph lawns. Stimulated peptide discharge from single vesicles is subquantal, and unitary or sequential in membrane lawns and intact lactotrophs. Micromolar Ca 2+ triggers predominantly unitary peptide discharge from single vesicles in lawns. Abstract: Regulated exocytosis consists of the fusion between vesicles and the plasma membranes, leading to the formation of a narrow fusion pore through which secretions exit the vesicle lumen into the extracellular space. An increase in the cytosolic concentration of free Ca 2+ ([Ca 2+ ]i ) is considered the stimulus of this process. However, whether this mechanism can be preserved in a simplified system of membrane lawns with docked secretory vesicles, devoid of cellular components, is poorly understood. Here, we studied peptide discharge from individual secretory vesicles docked at the plasma membrane, prepared from primary endocrine pituitary cells (the lactotrophs), releasing hormone prolactin. To label secretory vesicles, we transfected lactotrophs to express the fluorescent atrial natriuretic peptide (ANP.emd), previously shown to be expressed in and released from prolactin-containing vesicles. We used stimulating solutions containing different [Ca 2+ ] to evoke vesicle peptide discharge, which appeared similar in membrane lawns and in intact stimulated lactotrophs. All vesicles examined discharged peptides in a subquantal manner, either exhibiting a unitary or sequential time course. In the membrane lawns, the unitary vesicle peptide discharge was predominant and slightly slower than that recorded in intact cells, but with a shorter delay with respect to the stimulation onset. This study revealed directly that Ca 2+ triggers peptide discharge from docked single vesicles in the membrane lawns with a half-maximal response of ∼8 µM [Ca 2+ ], consistent with previous whole-cell patch-clamp studies in endocrine cells where the rapid component of exocytosis, interpreted to represent docked vesicles, was fully activated at <10 µM [Ca 2+ ]. Interestingly, the sequential subquantal peptide vesicle discharge indicates that fluctuations between constricted and dilated fusion pore states are preserved in membrane lawns and that fusion pore regulation appears to be an autonomously controlled process. Graphical abstract: Calcium-dependent subquantal peptide release from single secretory vesicles Image, graphical abstract