Fig 1.
Synergistic ERK1/2 activation in (BK 10−11 M plus IBOP) co-stimulated RASMC.
(a): Concentration-response curves representing “fold/basal” ERK1/2 phosphorylation in RASMC treated with IBOP alone or (BK 10−11 M plus IBOP) with or without prior incubation with the TP antagonist, SQ29548. Results are mean ± SEM of at least three independent experiments. * p < 0.05; ** p < 0.01; and *** p < 0.001 as compared to unstimulated basal, One Way ANOVA. (b): Western blots corresponding to curves in (a). (c, d and, e): Analysis of synergy in (BK 10−11 M and IBOP) binary combinations in RASMCs based on the method of Chou and colleagues at five different fractional effects (Fa). (c) The normalized isobologram analysis: The x-coordinate for each data point at a given Fa was calculated by dividing the concentration (DA) of BK in the (BK+IBOP) combination from its corresponding single-agent (Dx,A) value. The y-coordinate was calculated by dividing the concentration (DB) of IBOP in the (BK+IBOP) combination from its corresponding single-agent (Dx,B) value. Data points below the line of additivity indicate synergy, whereas points above the line of additivity indicate antagonism. (d) Combination index (CI) analysis: CI values at any given Fa were derived from actual data points in the dose-response non-linear regression curves. Combinations are additive at CI = 1, synergistic at CI < 1, and antagonistic at CI > 1. (e) Dose Reduction Index (DRI) was calculated at the above Fa levels and plotted as Log(DRI)-Fa plot, where Log(DRI) > 0 would be favorable in case of synergy.
Fig 2.
Differential mechanism of ERK1/2 activation in single versus double stimulation of B2R and TP in RASMC.
Cells were pretreated with the PKC inhibitor, Gö6983 (a), or the EGFR Tyr kinase inhibitor, AG1478 (b), prior to stimulation with BK, IBOP, or combination. Results are plotted as “fold/basal” ERK1/2 phosphorylation. Representative western blots are seen for pERK1/2 and tERK2. Results are mean ± SEM of at least three independent experiments. *: p < 0.05; as compared to unstimulated basal. #: p < 0.05; for pairwise comparisons, One Way ANOVA.
Fig 3.
Quantitative analysis of single GPCR (B2R or TP) occupancy in VSMC using PLA.
(a): Representative 3D maximum intensity projection of z-stack images obtained for RASMC analyzed by PLA for single B2R or TP expression at basal conditions (red). Nuclei appear in blue. (b): Scatter plot representing single cell analysis of the total number of PLA blobs per cell for B2R (n = 801 cells) or TP (n = 779 cells). The mean total blobs per cell is also plotted for B2R versus TP. Bar graphs representing mean ± SEM of nuclear blobs (c) or extra-nuclear blobs (d) per cell were plotted for B2R or TP. Statistical analysis was conducted using Mann-Whitney Rank Sum t-test. N = 3 independent experiments. (NS): not statistically significant; (****): p ≤ 0.0001.
Fig 4.
Quantitative analysis of B2R-TP proximity in VSMC using dual receptor PLA recognition workflow.
(a): Representative 3D images of B2R-TP PLA blobs (red) obtained for RASMC that were either kept untreated, or stimulated with BK, IBOP, or combinations for 10 min. (b): Scattergram of the total number of PLA blobs per cell for B2R-TP heteromer. The total number of cells analyzed is shown at the top of the scattergram for each treatment group. Bar graphs of the mean± SEM total B2R-TP PLA blobs per cell were also plotted. Statistical analyses were performed using Kruskal-Wallis One Way ANOVA (p ≤ 0.0001) followed by Dunn’s multiple comparison analysis. N = 3 independent experiments. (*) p < 0.05, versus basal; (#): p < 0.05, all pairwise comparisons.
Fig 5.
B2R-TP interactions in RASMC as revealed by co-IP followed by SDS PAGE.
RASMC lysates were immunoprecipitated with anti-B2R followed by immunoblotting with anti-TP (upper panels) and anti-B2R (lower panels) antibodies, successively. E and L represent eluates and matching lysates of unstimulated RASMC (E1, L1), or RASMC that were stimulated with BK 10−11 M (E2, L2), IBOP 10−7 M (E3, L3), or [BK 10−11 M + IBOP 10−7 M] (E4, L4) for 10 min. EM and LM represent eluates and matching lysates of mock co-IP condition. (EB) represents eluates of a control co-IP condition, whereby Dynabeads-protein-A-anti-B2R were incubated with PBS instead of RASMC lysates. Images are representative of three qualitatively similar independent experiments. A denatured broad molecular weight protein ladder was loaded in parallel (upper and lower left-hand lanes).
Fig 6.
The unique aspects of B2R-TP crosstalk in VSMC.
(a): Constitutive B2R-TP interaction exists at the plasma membrane and nuclei implying possible B2R-TP association early during ontogenesis. (b): BK provokes positive allosteric modulation on IBOP resulting in synergistic ERK1/2 signaling possibly mediated via a biased signaling pathway in rat VSMC.