Figure 3
Effects of L-NAME and aminoguanidine (AG) on the release of sPLA 2 -IIA from BMVECs. (A) Representative bands showing the sPLA2-IIA (upper panel) and BSA (middle panel) from the medium from BMVECs pretreated with L-NAME. Brain endothelial cells were treated for 16 h with 1 or 5 μg/ml of LPS in the presence or absence of L-NAME as indicated, and Western blots were performed on the culture medium using an antibody against sPLA2-IIA. Quantitative data on the release of sPLA2-IIA from BMVECs are shown in the lower panel. The data are presented as the means ± SE of the three separate experiments. *P < 0.05 versus the normal group, ^ P < 0.05 versus the group without L-NAME. (B) Representative bands showing the sPLA2-IIA (upper panel) and BSA (middle panel) from the medium from BMVECs pretreated with AG. The cells were treated for 16 h with 5 μg/ml of LPS in the presence or absence of 1 mM AG, as indicated, and Western blots were performed on the culture medium using an antibody against sPLA2-IIA. Quantitative data on the release of sPLA2-IIA from BMVECs are shown in the lower panel. The data are presented as the means ± SE of the three separate experiments. *P < 0.05 versus the normal group and AG-alone group, # P < 0.05 versus the group without AG treatment. (C) A histogram showing the representative results of sPLA2 enzyme activity in the culture medium after 5 μg/ml LPS treatment for 16 h and pretreatment with L-NAME and AG. The data shown here are the averages of four separate experiments from four individual platings (means ± SE). *P < 0.05 versus the normal group, ^ P < 0.05 versus LPS treatment alone. (D) Time course of nitrite production in BMVECs after LPS treatment with or without L-NAME and AG. Upper line: Nitrite concentration from the medium from BMVECs treated with LPS alone for different times (0, 8, 16, 24 h). The culture medium from BMVECs collected at the indicated times. Middle line: BMVECs were treated with LPS in the presence of L-NAME for 15 min prior to LPS stimulus, the culture medium was collected at the indicated times, and nitrite concentrations were calculated. Bottom line: BMVECs were treated with LPS in the presence of AG 15 min prior to LPS stimulus, the culture medium was collected at the indicated times, and nitrite concentrations were calculated. The data shown are the averages of five separate experiments from six separate platings; *P < 0.05 versus their respective normal groups (time 0 h) and 8-h treatment groups, ^ P < 0.05 versus their respective 16-h LPS treatment groups, $ P < 0.05 versus treatment with LPS alone.