Tracing G-protein-mediated contraction and relaxation in vascular smooth muscle cell spheroids

• Verfasst von: Garg, Jaspal [VerfasserIn]
• Verfasst von: Sporkova, Alexandra [VerfasserIn]
• Verfasst von: Hecker, Markus [VerfasserIn]
• Verfasst von: Korff, Thomas [VerfasserIn]
• Titel: Tracing G-protein-mediated contraction and relaxation in vascular smooth muscle cell spheroids
• Verf.angabe: Jaspal Garg, Alexandra Sporkova, Markus Hecker and Thomas Korff
• Jahr: 2023
• Umfang: 15 S.
• Fussnoten: Online veröffentlicht: 28 December 2022 ; Gesehen am 01.02.2023
• Titel Quelle: Enthalten in: Cells
• Ort Quelle: Basel : MDPI, 2012
• Jahr Quelle: 2023
• Band/Heft Quelle: 12(2023), 1, Artikel-ID 128, Seite 1-15
• ISSN Quelle: 2073-4409
• DOI: doi:10.3390/cells12010128
• Datenträger: Online-Ressource
• Sprache: eng
• Sach-SW: contraction
• Sach-SW: G-protein signaling
• Sach-SW: MAPK
• Sach-SW: relaxation
• Sach-SW: RGS
• Sach-SW: VSMC phenotype
• K10plus-PPN: 183290959X

Abstract

Analyses of G-protein-mediated contraction and relaxation of vascular smooth muscle cells (VSMCs) are usually hampered by a rigid growth surface and culture conditions promoting cell proliferation and a less contractile phenotype. Our studies indicated that mouse aortic VSMCs cultured in three-dimensional spheroids acquire a quiescent contractile status while decreasing the baseline G-protein-dependent inositolphosphate formation and increasing the expression of endothelin receptor type A (Ednra). Endothelin-1 (ET-1) promoted inositolphosphate formation in VSMC spheroids, but not in VSMCs cultured under standard conditions. To trace ET-1-mediated contraction of VSMC spheroids, we developed an assay by adhering them to collagen hydrogels and recording structural changes by time-lapse microscopy. Under these conditions, mouse and human VSMC spheroids contracted upon treatment with ET-1 and potassium chloride or relaxed in response to caffeine and the prostacyclin analogue Iloprost. ET-1 activated AKT-, MKK1-, and MKK3/6-dependent signaling cascades, which were inhibited by an overexpressing regulator of G-protein signaling 5 (Rgs5) to terminate the activity of Gα subunits. In summary, culture of VSMCs in three-dimensional spheroids lowers baseline G-protein activity and enables analyses of both contraction and relaxation of mouse and human VSMCs. This model serves as a simple and versatile tool for drug testing and investigating G-protein-depending signaling.