Figure 5. Effect of fenoldopam and methyl-β-cyclodextrin (βCD) on cAMP accumulation.

A. Effect of fenoldopam and βCD on cAMP accumulation in whole cells

HEK-hD₁R and HEK-hD₅R cells were seeded in 12-well plates at5×10⁵cells/well. The cells, pretreated for 1 h at 37°C with vehicle (control, Con), βCD (2%), or cholesterol (Chol, 100 μg/ml) plus βCD (βCD+Chol), were treated for 10 min at 37°C with vehicle (Con) or fenoldopam (Fen, 1μmol/L) or in combination (βCD+Fen, βCD+Chol+Fen). The cells were lysed in 0.1N HCL as described in “Methods”. cAMP accumulation was measured in each well using a cAMP assay kit and expressed as pmol/mg protein/min. Values are Mean ± SEM (n = 4/group). ^*P<0.001 vs. all others in HEK-hD₁R or HEK-hD₅R, respectively, ^#P<0.05 vs. Fen-D₁R, ^&P<0.05 vs. βCD, βCD+Fen, and βCD+Chol in HEK-hD₁R cells, ^$P<0.003 vs. Con-HEK-hD₅R and βCD, βCD+Fen, βCD+Chol, and βCD+Chol+Fen in HEK-hD₅R cells, ^**P<0.05, vs. βCD in HEK-hD₁R or HEK-hD₅R, n=4/group, one-way factorial ANOVA, Tukey post-hoc test.

B-C. Effect of fenoldopam and methyl- β-cyclodextrin (βCD) on cAMP accumulation in LRs and non-LRs

HEK-hD₁R (B) and HEK-hD₅R (C) cells, grown in 150-mm dishes, pre-treated with vehicle (Control) or βCD (2%) for 45 min at 37°C, were then treated with vehicle or fenoldopam (Fen, 1 μmol/L), or βCD +Fen for 15 min (total 1h). The cell lysates were then subjected to sucrose gradient centrifugation as described in “Methods” (12, 27). Cell fractions 2-6 and 7-12 were combined as LR (fractions 2-6) and non-LR (fractions 7-12) fractions and cAMP accumulation was measured using a cAMP assay kit and expressed as pmol/mg protein/min. Values are Mean ± SEM (n = 4/group). ^*P< 0.001, vs. others in HEK-hD₁R or HEK-hD₅R cells, ^#P<0.001, vs. Fen, HEK-hD₁R (B), ^$P<0.001, vs. Con, βCD, non-LRs-HEK-hD₁R and -HEK-hD₅R,^**P<0.05, vs. Con, , βCD-LRs,, non-LRs-HEK-hD₅R, one-way factorial ANOVA, Tukey post-hoc test, ^&P<0.006, vs. Fen-non-LRs HEK-hD₁R, t-test

C. Effect of fenoldopam and βCD on cAMP accumulation in hRPT cells. hRPT cells were treated with βCD and fenoldopam as described in Figure 5A. cAMP accumulation was measured in each well using a cAMP assay kit and expressed as pmol/mg protein/min. Values are Mean±SEM (n=4/group). *,^#P<0.001 vs. all others, untreated and fenoldopam-treated cells, ^$P<0.002, vs. all others, untreated and fenoldopam-treated cells, one-way factorial ANOVA, Tukey post-hoc test.