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

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

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.

• Jezik: Angleški jezik
• Ključne besede: stimulus-secretion coupling, Ca$^{2+}$ signalling, confocal microscopy, lactotrophs, secretory vesicle, fusion pore, ANP.emd
• Vrsta gradiva: Članek v reviji
• Tipologija: 1.01 - Izvirni znanstveni članek
• Organizacija: MF - Medicinska fakulteta ; BF - Biotehniška fakulteta
• Status publikacije: Objavljeno
• Različica publikacije: Objavljena publikacija
• Leto izida: 2023
• Št. strani: 10 str.
• Številčenje: Vol. 109, art. 102687
• PID: 20.500.12556/RUL-143843
• UDK: 616-092
• ISSN pri članku: 1532-1991
• DOI: 10.1016/j.ceca.2022.102687
• COBISS.SI-ID: 134324739
• Datum objave v RUL: 13.01.2023

Gradivo je del revije

• Naslov: Cell calcium
• Skrajšan naslov: Cell calcium
• Založnik: Elsevier
• ISSN: 1532-1991
• COBISS.SI-ID: 77488387

Licence

• Licenca: CC BY-NC-ND 4.0, Creative Commons Priznanje avtorstva-Nekomercialno-Brez predelav 4.0 Mednarodna
• Povezava: http://creativecommons.org/licenses/by-nc-nd/4.0/deed.sl
• Opis: Najbolj omejujoča licenca Creative Commons. Uporabniki lahko prenesejo in delijo delo v nekomercialne namene in ga ne smejo uporabiti za nobene druge namene.

Sekundarni jezik

• Jezik: Slovenski jezik
• Ključne besede: stimulusno-sekrecijska sklopka, Ca$^{2+}$ signalizacija, konfokalna mikroskopija

Projekti

• Financer: FCT - Fundação para a Ciência e a Tecnologia, I.P.
• Številka projekta: UIDP/50017/2020+UIDB/50017/2020+LA/P/0094/2020

• Financer: ARRS - Agencija za raziskovalno dejavnost Republike Slovenije
• Številka projekta: P3-0310
• Naslov: Celična fiziologija