ctive NF ��B inhibitor Bay11 7082, which blocks activation of NF ��B signaling, attenuated ET 1 induced CO 2 protein and mRNA e pression in bEnd. 3 cells. To determine whether the involvement of NF ��B in ET 1 induced responses mediated through NF ��B trans location, as shown in Figure 5C, ET 1 time dependently stimulated translocation of NF ��B p65 from cytosol into nucleus determined by Western blot. A ma imal re sponse was obtained within 90 min and sustained over 120 min. Moreover, we also confirmed the NF ��B p65 translocation by an immunofluorescence staining. The imaging data confirmed that ET 1 stimu lated the p65 translocation at 90 min, which was inhib ited by pretreatment with Bay11 7082. We further demonstrated that ET 1 stimulated translocation of NF ��B p65 was attenuated by pretreat ment with the inhibitor of ETB receptor, MEK1 2, p38 MAPK, JNK1 2, or NF ��B.

To fur ther verify that NF ��B p65 is essential for ET 1 induced CO 2 e pression, as shown in Figure 5E, transfection with p65 siRNA significantly reduced the p65 protein e pression and the ET 1 induced CO 2 e pression. The results suggested that ET 1 stimulated NF ��B translocation mediated Brefeldin_A through ETB receptor, ERK1 2, p38 MAPK, and JNK1 2 is required for CO 2 induction in bEnd. 3 cells. Involvement of NF ��B in CO 2 gene promoter activity stimulated by ET 1 We have found that ET 1 stimulates translocation of NF ��B p65 leading to CO 2 e pression. Ne t, we e amined whether activation of NF ��B is essential for ET 1 induced CO 2 gene up regulation. The transcriptional activity of NF ��B was evaluated by a promoter luciferase ac tivity assay.

As shown in Figure 6A, ET 1 enhanced NF ��B transcriptional activity in a time dependent manner with a ma imal response within 60 min, which was sig nificantly inhibited by pretreatment with an inhibitor of NF ��B. Moreover, pretreatment with BQ 788, GPA2, GPA2A, U0126, SB202190, or SP600125 attenuated NF ��B transcriptional activity stimulated by ET 1, demonstrating that ET 1 enhances the NF ��B transcriptional activity through an ETB dependent activation of MAPKs. Subse quently, we determined that ET 1 stimulates NF ��B p65 binding activity in a time dependent manner by ChIP PCR analysis. ET 1 stimulated NF ��B p65 binding activity was inhibited by pretreatment with U0126, SB202190, SP600125, Bay11 7082, or BQ 788.

In addition, we have demon strated that ET 1 time dependently induces CO 2 pro moter activity. We further demonstrated that ET 1 increased the CO 2 promoter activity was significantly inhibited by pretreatment with BQ 788, GPA2, GPA2A, U0126, SB202190, SP600125, or Bay11 7082, suggesting that ET 1 stimulates CO 2 promoter activity via the ETB dependent activation of MAPKs and NF ��B in bEnd. 3 cells. To further ensure that NF ��B indeed mediates ET 1 induced CO 2 pro moter activity through binding to its regulatory element within the CO 2 promoter region, the wild type and mutated by a single point mutation of the NF ��B binding site CO 2 promoters