Figure 1.
Response of B2K-CC FRET sensor and PM-CC (control FRET sensor) to stimulation by polyunsaturated fatty acids.
B2K-CC transfected HEK-293 cells treated with (A) EPA, EPA and B2R inhibitor (HOE-140), oleic acid or (B) DDA, DGLA. Experiments were performed 24 h after transfection of B2K-CC FRET sensor or PM-CC in HEK293 cells on chambered cover glass at 37°C. FRET ratio was defined as ratio of Citrine emission intensity at 525 nm to Cerulean emission intensity at 475 nm.
Figure 2.
Activation of B2R by fatty acids in HEK293 cells.
BK concentration in (A) was 100 nM (positive control) and (B) was 10 nM. Fatty acid concentrations were 10 µM in HEK293 cells transfected with B2R as described under Materials & Methods and treated for (A) 5 minutes or (B) 15 seconds in PBS at 37°C. Western blots were used to determine ERK1/2 phosphorylation. Data represent mean of n ≥ 6 experiments. Error bars indicate standard error of the mean (SEM).
Figure 3.
B2R antagonist inhibits ERK1/2 response to bradykinin and fatty acids.
(A) Effect of 1µM of B2R antagonist HOE-140 on pERK stimulated with: 100 nM bradykinin, 1 µM EPA and 10 µM DHA in DPBS for 5 minutes at 37°C. Experiments were done 24 h after transfection of HEK293 cells with B2R in 12-well plates. (B) Effect of 1 µM of B2R antagonist HOE-140, on pERK stimulated with 10 nM BK, 1 µM EPA and 1 µM DHA in BAECs expressing endogenous B2R. Ratio of pERK to ERK was measured using western blots. (C) Effect of 1 µM of B2R antagonist HOE-140, on pERK stimulated with 100 nM NA in BAECs expressing endogenous adrenergic receptors. Data represent the mean of at least four independent experiments.
Figure 4.
B2R antagonist shifts the EPA EC50 by inhibiting ERK1/2 response to fatty acids.
(A) Dose response to EPA at different concentrations of antagonist HOE-140 in HEK293 cells. (B) Schild plot of HOE-140 antagonism on EPA-stimulated B2R. Experiments were done 24 h after transfection of HEK293 cells with B2R in 12-well plates. Ratio of pERK to ERK was measured using western blots. Data represent the mean of at least four independent experiments.
Figure 5.
Fatty acids cause eNOS activation and alters NOS3 and KLF2 expression.
(A) Effect of 1 µM of B2R antagonist HOE-140 on peNOS stimulated with: 10 nM bradykinin or 1 µM EPA on BAECs in DPBS for 5 minutes at 37°C. Effect of 1 µM of B2R antagonist HOE-140 on NOS3 expression with 10 nM BK or 1 µM EPA (B) BAECs and (C) HCAECs DPBS for 1 hour at 37°C. (D) Effect of 1 µM of B2R antagonist HOE-140 on KLF2 expression with 10 nM bradykinin or 1 µM EPA. Data represent the mean of at least four independent experiments.
Figure 6.
Membrane viscosity changes with fatty acids.
Measured signal intensity of HEK293 cells stained with FCVJ as a response to 100 µM EPA or control treatment in chambered cover glass at 37°C. Data represents the mean of at least 12 independent experiments.