4B). In addition to phenylephrine, dobutamine (but not clenbuterol, and BRL 37344) increased small cholangiocyte proliferation (Fig. 4B). Because activation of β-adrenergic receptors regulates biliary functions by increased intracellular cAMP

levels in cholangiocytes,9 we focused our studies on the role of phenylephrine (an α1-AR agonist stimulating IP3/Ca2+ levels)10, 34 on Ca2+-dependent signaling in small cholangiocytes. We demonstrated that α1A (RS17053), α1B (Rec15/2615) and α1D (BMY7378) AR antagonists induced a partial yet significant reduction in phenylephrine-induced proliferation Doxorubicin of immortalized small cholangiocytes (Fig. 4C). However, levels of proliferation stimulated by phenylephrine in the presence of the antagonists remain significant in comparison to basal control proliferation, which demonstrates

that all three receptor subtypes are involved in phenylephrine-induced proliferation (Fig. 4C). Phenylephrine increased intracellular BGB324 cost IP3 (but not cAMP, not shown) levels (basal: 0.39 ± 0.03 versus phenylephrine: 0.62 ± 0.07 pmol/1 × 107 cells; P< 0.01) in immortalized small cholangiocytes. Phenylephrine-stimulated proliferation of immortalized small cholangiocytes was blocked by BAPTA/AM, CAI,4 11R-VIVIT, and MiA (Fig. 4D). To further define the role of NFAT in phenylephrine-stimulated proliferation, we performed experiments to evaluate nuclear translocation and DNA-binding

activity of NFAT2 and NFAT4 in immortalized small cholangiocytes. By immunofluorescence, phenylephrine stimulates nuclear translocation medchemexpress of both NFAT2 and NFAT4 in small cholangiocytes (Fig. 5). This translocation that was blocked by inhibitors of upstream Ca2+-dependent signaling (i.e., benoxathian [nonsubtype selective α1-AR antagonist],31 BAPTA/AM, and CAI) (Fig. 5), which confirms the results of the proliferation studies (Fig. 4D). The activation of NFAT and Sp1/3 DNA-binding activity was determined by EMSA and DNA-binding activity ELISA. We found by EMSA that phenylephrine stimulates time-dependent activation of NFAT DNA-binding in small cholangiocytes (Fig. 6). The consensus sequence used in the EMSA will bind both NFAT2 and NFAT4 (elucidation of the involvement of isoforms was determined by knockdown experiments discussed later). NFAT2 DNA-binding activity was confirmed by DNA-binding activity ELISA. The ELISA kit used recognizes the specific DNA-binding activity of NFAT2 (and not other NFAT isoforms as there are no commercially available kits). Our results demonstrate that phenylephrine stimulates NFAT2 DNA-binding activity in small cholangiocytes, which was blocked by BAPTA/AM and CAI (Fig. 7A). We also found that phenylephrine stimulates the time-dependent increase in Sp1 DNA-binding activity in small cholangiocytes as determined by EMSA (Fig. 7B).