File S1 - Dextromethorphan Mediated Bitter Taste Receptor Activation in the Pulmonary Circuit Causes Vasoconstriction

Table S1, A list of bitter taste receptors activated by the compounds used in the study. Table S2, DXM mediated effects on T2Rs expressed in PASMCs, ASMCs, pulmonary artery and airway rings. Figure S1, Representative calcium traces for primary cultures of hPASMCs stimulated with different concentrations of DXM or assay buffer (bottom trace). The calcium mobilized (Relative Fluorescence Units or RFUs) was detected using the calcium sensitive dye Fluo-4 NW (Invitrogen), and fluorescence measured using the automated Flex Station 3 microplate reader as described before [15], [34], [38]. In brief, the basal calcium mobilized was measured for the first 20 sec in all the 8 wells (one column) of a 96 well plate, followed by the simultaneous addition of different concentrations of the test compound, shown by arrows in the figure, to all the 8 wells by the in-built automated dispenser in Flex Station 3. Then calcium traces were recorded for the next 180 sec. Figure S2, Comparison of intracellular calcium release in hPASMCs and hASMCs in response to different concentrations of DXM. A. Concentration-dependent changes in [Ca2+]i of hPASMCs and hASMCs induced by different concentrations of bitter agonist DXM (log M). Data were collected from 3–5 independent experiments carried out in triplicate. Dose response curves were generated using Graph Pad Prism software. B. Bar graph showing difference in intracellular calcium release in hPASMCs and hASMCs in response to 2 mM DXM (Emax concentration). Significant calcium release was observed in hPASMCs in comparison to hASMCs with significance level of *p<0.05. Figure S3, Quantification of T2R1 expression in human and porcine cells. A. Relative expression level of T2R1 in porcine PASMCs and ASMCs as determined by quantitative (q)-PCR. T2R1 expression in porcine PASMCs was considered as 100% and relative expression of T2R1 in ASMCs was normalized to it. B. Relative expression of T2R1 in human and porcine PASMCs. The relative expression of T2R1 in porcine PASMCs was normalized to that of hPASMCs, which was considered as 100%. Data presented are from five independent experiments done in triplicates. Results are normalized to the expression of GAPDH. Values are plotted as mean ± SEM. Relative expressions were computed using 2−ΔCT method. Student's t-test was used to check the significance. Figure S4, Myograph analysis of the effects of DXM on U46619 precontracted porcine pulmonary arterial rings. A. Raw traces showing effect of DXM (10−5 to 6.5×10−5 M) stimulation on resting tension of U46619 (30 nM) precontracted pulmonary artery rings. Force generation started from 100 µM and reaching a plateau after 650 µM DXM. B. Effects of DXM and chloroquine on precontracted porcine arterial rings. Data are representative of n = 6 rings and presented as means ± S.E.M. The levels of precontraction to U46619 (30 nM) in pulmonary arteries was ∼140% of KCl (50 mM)-induced contractions. Figure S5, Myograph analysis of the effects of DXM on endothelium-denuded porcine pulmonary arterial rings. Dose response curve of DXM normalized to maximal KCl stimulation in pulmonary arterial rings. The figure represents a cumulative dose response curve of DXM with highest concentration being 10−3 M and lowest 10−5 M on isometric tension of pulmonary artery rings. The DXM responses were normalized to maximal KCl stimulation and the EC50 was calculated to be 238±1 µM. The results are presented as mean ± SEM and are from a minimum of n = 15 rings from 5 piglets. Figure S6, Effects of bitter compound chloroquine on porcine airway rings. Piglet airway rings were contracted with 10 µM of acetylcholine (ACh) and then chloroquine (10 µM–300 µM) was added to the bath as shown, resulting in relaxation in a dose dependent manner. Results are representative of five independent experiments and are from a minimum of n = 15 rings from 5 piglets. Figure S7, IP3 produced in human PASMCs and ASMCs. Bar plot representation of total IP3 produced (nanomoles) in hPASMCs and hASMCs upon treatment with DXM. hASMCs and hPASMCs were stimulated with T2R1 agonist DXM (500 µM) and the vasoconstrictor U46619 (TP agonist, 1 µM),which was used as a positive control. Shown are the agonist-independent or basal activity (-), and activity after stimulation (+) with agonist. Results are from a minimum of four independent experiments performed in triplicates. A one way ANOVA with Tukey's post hoc test was used to check the significance level of the amount of IP3 produced. DXM treatment caused no change in IP3 level in hASMCs, whereas, a significantly increased IP3 was observed in DXM-treated hPASMCs, as compared to hASMCs, at significance level of ***p<0.001. Error bars represent mean ± SEM. Figure S8, Effect of DXM on superoxide production in hPASMCs. hPASMCs were treated with 0.5 mM DXM or media alone for 4 hours. DHE fluorescence assay was done to measure the superoxide production as described in methods. Superoxide production was expressed as fluorescence intensity and as the mean ± SEM of three independent experiments performed in triplicates.
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