Tracing G-Protein-Mediated Contraction and Relaxation in Vascular Smooth Muscle Cell Spheroids
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-dimen...
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MDPI AG
2022-12-01
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| Series: | Cells |
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| Online Access: | https://www.mdpi.com/2073-4409/12/1/128 |
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| author | Jaspal Garg Alexandra Sporkova Markus Hecker Thomas Korff |
| author_facet | Jaspal Garg Alexandra Sporkova Markus Hecker Thomas Korff |
| author_sort | Jaspal Garg |
| collection | DOAJ |
| description | 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 (<i>Ednra</i>). 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 (<i>Rgs5</i>) 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. |
| first_indexed | 2024-03-11T10:05:28Z |
| format | Article |
| id | doaj.art-fe1f1b01670f4c0db9ad920726194961 |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-11T10:05:28Z |
| publishDate | 2022-12-01 |
| publisher | MDPI AG |
| record_format | Article |
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| spelling | doaj.art-fe1f1b01670f4c0db9ad9207261949612023-11-16T15:06:30ZengMDPI AGCells2073-44092022-12-0112112810.3390/cells12010128Tracing G-Protein-Mediated Contraction and Relaxation in Vascular Smooth Muscle Cell SpheroidsJaspal Garg0Alexandra Sporkova1Markus Hecker2Thomas Korff3Institute of Physiology and Pathophysiology, Department of Cardiovascular Physiology, Heidelberg University, 69120 Heidelberg, GermanyInstitute of Physiology and Pathophysiology, Department of Cardiovascular Physiology, Heidelberg University, 69120 Heidelberg, GermanyInstitute of Physiology and Pathophysiology, Department of Cardiovascular Physiology, Heidelberg University, 69120 Heidelberg, GermanyInstitute of Physiology and Pathophysiology, Department of Cardiovascular Physiology, Heidelberg University, 69120 Heidelberg, GermanyAnalyses 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 (<i>Ednra</i>). 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 (<i>Rgs5</i>) 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.https://www.mdpi.com/2073-4409/12/1/128G-protein signalingRGSVSMC phenotypeMAPKcontractionrelaxation |
| spellingShingle | Jaspal Garg Alexandra Sporkova Markus Hecker Thomas Korff Tracing G-Protein-Mediated Contraction and Relaxation in Vascular Smooth Muscle Cell Spheroids Cells G-protein signaling RGS VSMC phenotype MAPK contraction relaxation |
| title | Tracing G-Protein-Mediated Contraction and Relaxation in Vascular Smooth Muscle Cell Spheroids |
| title_full | Tracing G-Protein-Mediated Contraction and Relaxation in Vascular Smooth Muscle Cell Spheroids |
| title_fullStr | Tracing G-Protein-Mediated Contraction and Relaxation in Vascular Smooth Muscle Cell Spheroids |
| title_full_unstemmed | Tracing G-Protein-Mediated Contraction and Relaxation in Vascular Smooth Muscle Cell Spheroids |
| title_short | Tracing G-Protein-Mediated Contraction and Relaxation in Vascular Smooth Muscle Cell Spheroids |
| title_sort | tracing g protein mediated contraction and relaxation in vascular smooth muscle cell spheroids |
| topic | G-protein signaling RGS VSMC phenotype MAPK contraction relaxation |
| url | https://www.mdpi.com/2073-4409/12/1/128 |
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