Fig. 2.

A: dose-dependent effect of acute (15 min) pretreatment of human airway smooth muscle (HASM) cells with 1 µM to 10 mM short-chain fatty acids [SCFAs (i) acetate (n = 4–9). (ii) propionate (n = 4–19), or (iii) butyrate (n = 4–8)] before forskolin (10 µM)-stimulated (15 min) cAMP activity in HASM cells. B and C: involvement of FFAR3 (B) or either free fatty acid receptor 2 (FFAR2) or OR51E2 (C) in propionate-induced cAMP inhibition in HASM cells. HASM cells were transfected with either control nontargeting small-interfering RNA (siRNA) or FFAR3-specific siRNA (n = 7) (B) or either FFAR2- or OR51E2-specific siRNA (n = 6) (C) 2–3 days before analyses and then pretreated with propionate (10 mM) for 15 min before the addition of forskolin (10 µM) for 15 min. D–F: effect of the FFAR2 antagonist CATPB (10 µM, for 15 min pretreatment; n = 6) (D), pertussis toxin (PTX; 100 ng/mL, for 4 h pretreatment; n = 8) (E), or trichostatin A (TSA; 1 µM, for 30 min pretreatment; n = 8) (F) on propionate (10 mM)-induced attenuation of forskolin (10 µM)-stimulated cAMP activity in HASM cells. Data represent means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with forskolin alone. G: representative gel images of RT-PCR analyses of total RNA using primers specific for human FFAR2, FFAR3, or OR51E2. Total RNA extracted from nontransfected HASM cells, HASM cells that were transfected with either control nontargeting small-interfering RNA (siRNA control), FFAR2-, FFAR3-, or OR51E2-specific siRNA were analyzed. −RT, cDNA synthesis reactions performed in the absence of reverse transcriptase (RT) confirming that PCR products were not arising from contaminating gDNA. GAPDH was used as a control of relative RNA input among samples.