Fig. 6. GTP--s and dominant negative dyn-2 K44A reduce BK-dependent, L-NAME-inhibited NO production from cells. (A) Digitonin (1 µM)-permeabilized BAEC were coincubated with GTP--S (10 µM) or vehicle for 5 minutes. After washing, cells were loaded with the NO fluorescent probe DAF-2DA and stimulated with 10 µM BK in the presence or absence of L-NAME. Time series photographs, taken to observe the temporal effects of BK on NO production in each experimental group, demonstrated an increase in intracellular DAF-2DA fluorescence intensity in cells treated with BK (top panel). The increase in fluorescence intensity in response to BK was markedly abrogated in cells treated with GTP--S (middle panel), or with both GTP--S and L-NAME (lower panel). The graph below, depicts the quantified fluorescence intensity over time within each group and shows a significant abrogation in NO production in BK-stimulated cells incubated with GTP--S or both GTP--S and L-NAME as compared to BK stimulation alone. Data are representative of three independent experiments with similar results. (B) eNOS-GFP ECV 304 cells were transfected with vectors encoding V5-K44A, a dominant negative mutant of dynamin-2, or empty pcDNA3.1 vector, then serum starved for 24 hours prior to experiments. Cells were stimulated with BK or vehicle in the presence of L-arginine or L-NAME and NO release was assessed from the media using the NO probe DAF-2. After collection of media for fluorimetry, cell lysates were prepared for western blot analysis using V5 mAb, dyn-2 pAb, and eNOS mAb, as well as protein assay. In cells transfected with empty vector, BK stimulated a 2-fold increase in NO production, which was entirely inhibited in the presence of the NOS inhibitor L-NAME. Overexpression of dyn-2 K44A markedly abrogated BK stimulated NO production in the presence and absence of L-NAME. (Closed bars: 0 µM BK; open bars: 10 µM BK; n=5 independent experiments each measured in duplicate). The control immunoblot panels demonstrate overexpression of V5 epitope-tagged dyn-2 using antibodies for V5 as well as dyn-2, and similar levels of eNOS protein between the experimental groups. (C) eNOS-GFP ECV 304 cells were transfected as described above for DAF-2 fluorimetry experiments. Samples of media were collected 20 minutes after BK stimulation and analyzed for nitrite levels using NO-specific chemiluminescence. In cells transfected with empty vector, L-NAME-inhibited NO production was increased by approximately twofold in cells stimulated with BK as compared to control cells (n=3 independent experiments each analyzed with duplicate samples; ^*P<0.05 compared to other groups). In cells transfected with dyn-2 K44A, no prominent increase in L-NAME inhibited, BK-mediated NO production was detected.

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