Calcium-dependent subquantal peptide release from single docked lawn-resident vesicles of pituitary lactotrophs

Gonçalves, Paula P. (author), Stenovec, Matjaž (author), Grácio, Luciano (author), Kreft, Marko (author), Zorec, Robert (author)

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.

• Language: English
• Keywords: stimulus-secretion coupling, Ca$^{2+}$ signalling, confocal microscopy, lactotrophs, secretory vesicle, fusion pore, ANP.emd
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: MF - Faculty of Medicine; BF - Biotechnical Faculty
• Publication status: Published
• Publication version: Version of Record
• Year: 2023
• Number of pages: 10 str.
• Numbering: Vol. 109, art. 102687
• PID: 20.500.12556/RUL-143843
• UDC: 616-092
• ISSN on article: 1532-1991
• DOI: 10.1016/j.ceca.2022.102687
• COBISS.SI-ID: 134324739
• Publication date in RUL: 13.01.2023

Record is a part of a journal

• Title: Cell calcium
• Shortened title: Cell calcium
• Publisher: Elsevier
• ISSN: 1532-1991
• COBISS.SI-ID: 77488387

Licences

• License: CC BY-NC-ND 4.0, Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
• Link: http://creativecommons.org/licenses/by-nc-nd/4.0/
• Description: The most restrictive Creative Commons license. This only allows people to download and share the work for no commercial gain and for no other purposes.

Secondary language

• Language: Slovenian
• Keywords: stimulusno-sekrecijska sklopka, Ca$^{2+}$ signalizacija, konfokalna mikroskopija

Projects

• Funder: FCT - Fundação para a Ciência e a Tecnologia, I.P.
• Project number: UIDP/50017/2020+UIDB/50017/2020+LA/P/0094/2020

• Funder: ARRS - Slovenian Research Agency
• Project number: P3-0310
• Name: Celična fiziologija