5A). GABA (after 3 days of in vitro treatment) increased IP3 levels and CaMK I expression of small cholangiocytes (Fig. 5B). Knockdown of CaMK I in small cholangiocytes blocked (1) stimulatory effects of GABA on PCNA protein expression (Fig. 6A), (2) GABA-induced de novo acquisition of SR, CFTR, and Cl−/HCO3− AE2 (Fig. 6B), and (3) de novo secretin-stimulated cAMP levels (Fig. 6C). Subsequent to in vivo administration of
GABA to BDL mice, there was enhanced AC8 protein expression in small ducts, expression that was blocked by pretreatment with BAPTA/AM and W7 (Fig. 7A,B). Subsequent to in vitro treatment with GABA (3 days, 1 μM), there was increased AC8 mRNA expression in vector-transfected small cholangiocytes (Fig. 7C). GABA did not increase the expression of AC8 in small cholangiocytes transfected with CaMK I shRNA (Fig. 7C). GABA-induced de novo (1) activation of PCNA expression Selleckchem H 89 (see Fig. 3B), and (2) expression of SR, CFTR, and Cl−/HCO3− AE2 (Fig. 4A) of small cholangiocytes was blocked by the AC8 inhibitor. Our findings relate to the functional switch of small into large cholangiocytes after prolonged in vivo and in vitro: GABA treatment. We have shown that small and large cholangiocytes express the
three GABA receptor subtypes. In vivo administration of GABA: (1) induces apoptosis of large, but not small, cholangiocytes Ivacaftor in vitro and (2) decreased large IBDM, but increased de novo small IBDM, in BDL mice. GABA stimulation of small IBDM was partly blocked by BAPTA/AM and W7. The in vivo data support our recent studies11 in BDL rats, where GABA induced damage of large ducts and the de novo proliferation of
small cholangiocytes. However, our recent in vivo studies in rats11 did not (1) demonstrate the direct effects of GABA on cholangiocyte functions, effects Thiamine-diphosphate kinase that could be nonspecific and mediated by the release of unidentified growth factors, and (2) address the mechanisms by which GABA induces in vitro the differentiation of small into large cholangiocytes. Thus, we proposed to develop an in vitro model in which GABA interacts with receptors on cholangiocytes and induces differentiation of small into large functional cholangiocytes by activation of IP3/Ca2+/CaMK I-dependent AC8 signaling. The differentiation of small into large cholangiocytes (evidenced by the de novo acquisition of ultrastructural and functional phenotypes of large cholangiocytes) was associated with increased (1) IP3 levels and CaMK I phosphorylation and (2) expression of AC8 in small cholangiocytes. In small cholangiocytes, knockdown of the CaMK I gene prevented (1) GABA-induced differentiation into large cholangiocytes and (2) GABA-induced increase of AC8.