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. 2019 Nov 8;9:16312. doi: 10.1038/s41598-019-52540-8

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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NTS1R-D₂R bivalent ligand induced calcium release in HT22 cells. (a) The applied bivalent ligands consist of three distinct structural elements: NTS1R-agonistic pharmacophore NT(8-13) (red), spacer (blue) and D₂R-agonistic (CS142, black-orange) or D₂R-antagonistic (CS148, black-cyan) pharmacophore, respectively. (b) Treatment with 0.5–1 µM CS142 resulted in significantly reduced intracellular Ca²⁺ fluxes in co-expressing compared to NTS1R singly expressing cells (*p < 0.05). (c) The application of CS148 neutralized the diminished Ca²⁺ release in the NTS1R-D₂R co-expressing system. The observed calcium recovery is displayed by non-significant differences between mono- and co-transfected cells for all tested concentrations. Data were analyzed with one-way ANOVA and Tukey’s multiple comparisons test presented as mean ± SEM, n = 6, performed in hexaplicates. *p < 0.05, **p < 0.01, ***p < 0.001, ns = non-significant.