Cholangiopathies are characterized by the heterogeneous proliferation of different-sized cholangiocytes. Large cholangiocytes proliferate by a cAMP-dependent mechanism. The function of small cholangiocytes may depend on the activation of inositol trisphosphate (IP(3))/Ca(2+)-dependent signaling pathways; however, data supporting this speculation are lacking. Four histamine receptors exist (HRH1, HRH2, HRH3, and HRH4). In several cells: 1) activation of HRH1 increases intracellular Ca(2+) concentration levels; and 2) increased [Ca(2+)](i) levels are coupled with calmodulin-dependent stimulation of calmodulin-dependent protein kinase (CaMK) and activation of cAMP-response element binding protein (CREB). HRH1 agonists modulate small cholangiocyte proliferation by activation of IP(3)/Ca(2+)-dependent CaMK/CREB. We evaluated HRH1 expression in cholangiocytes. Small and large cholangiocytes were stimulated with histamine trifluoromethyl toluidide (HTMT dimaleate; HRH1 agonist) for 24-48 h with/without terfenadine, BAPTA/AM, or W7 before measuring proliferation. Expression of CaMK I, II, and IV was evaluated in small and large cholangiocytes. We measured IP(3), Ca(2+) and cAMP levels, phosphorylation of CaMK I, and activation of CREB (in the absence/presence of W7) in small cholangiocytes treated with HTMT dimaleate. CaMK I knockdown was performed in small cholangiocytes stimulated with HTMT dimaleate before measurement of proliferation and CREB activity. Small and large cholangiocytes express HRH1, CaMK I, and CaMK II. Small (but not large) cholangiocytes proliferate in response to HTMT dimaleate and are blocked by terfenadine (HRH1 antagonist), BAPTA/AM, and W7. In small cholangiocytes, HTMT dimaleate increased IP(3)/Ca(2+) levels, CaMK I phosphorylation, and CREB activity. Gene knockdown of CaMK I ablated the effects of HTMT dimaleate on small cholangiocyte proliferation and CREB activation. The IP(3)/Ca(2+)/CaMK I/CREB pathway is important in the regulation of small cholangiocyte function.

Rat and human biliary epithelium is morphologically and functionally heterogeneous. Since no information exists on the heterogeneity of the murine intrahepatic biliary epithelium, and with increased usage of transgenic mouse models to study liver disease pathogenesis, we sought to evaluate the morphological, secretory and proliferative phenotypes of small and large bile ducts and purified cholangiocytes in normal and cholestatic mouse models.MethodsFor morphometry, normal and BDL mouse livers (C57/BL6) were dissected into blocks of 2-4 μm2, embedded in paraffin, sectioned, and stained with H&E. Sizes of bile ducts and cholangiocytes were evaluated by using SigmaScan to measure the diameters of bile ducts and cholangiocytes. In small and large normal and BDL cholangiocytes, we evaluated the expression of cholangiocyte specific markers, keratin-19 (KRT19), secretin receptor (SR), cystic fibrosis transmembrane conductance regulator (CFTR), and chloride bicarbonate anion exchanger 2 (Cl-/HCO-3 AE2) by immunofluorescence and western blot; and intracellular cAMP levels and chloride efflux in response to secretin (100 nM). To evaluate cholangiocyte proliferative responses after bile duct ligation (BDL), small and large cholangiocytes were isolated from BDL mice. The proliferation status was determined by analysis of the cell cycle by FACS and bile duct mass was determined by the number of KRT19-positive bile ducts in liver sections.ResultsIn situ morphometry established that the biliary epithelium of mice is morphologically heterogeneous, which smaller cholangiocyte lining smaller bile ducts and larger cholangiocytes lining larger ducts. Both small and large cholangiocytes express KRT19 and only large cholangiocytes from normal and BDL mice express SR, CFTR, and Cl-/HCO-3 exchanger and respond to secretin with increased cAMP levels and chloride efflux. Following BDL, only large mouse cholangiocytes proliferate.ConclusionSimilar to rats, mouse intrahepatic biliary epithelium is morphologically, and functionally heterogeneous. The mouse is a suitable model for defining the heterogeneity of the biliary tree.

muscle, effects on cardiac metabolism, and vasoconstriction of blood vessels.' In addition, clinical studies demonstrating the efficiency of angiotensin converting enzyme (ACE) inhibitors in the treatment of heart failure,2 myocardial ischemia,3 4and hypertension5.6 suggest that Ang II directly promotes pathological cell growth that participates in remodeling of the failing heart. Several animal studies have implicated Ang II in cardiac hypertrophy associated with hypertension; for instance, in a rat model of "pressure-overload" cardiac hypertrophy, treatment with an ACE inhibitor prevented the increase in left ventricular mass with no effect on afterload,7 suggesting that the growth effects of Ang II are direct. Moreover, chronic infusion of Ang II into rats increased left ventricular mass, even when the pressor activity of Ang II was blocked or a subpressor dose of Ang II was used.8 Recent experiments using cultured embryonic chick cardiomyocytes lend further support to the hypothesis that Ang II can directly produce cellular hypertrophy.9"10 It has not been established whether Ang II also has a direct growth effect on nonmyocyte cells of the heart, although recent studies suggest that cardiac fibroblasts are a target for Ang II; for instance, treatment with the ACE inhibitor captopril prevented myocardial fibrosis in a rat model with renovascular hypertension" and with induced myocardial infarction.12 In the latter study,

Cardiac failure is a major cause of death in patients with type 2 diabetes, but the molecular mechanism that links diabetes to heart failure remains unclear. Insulin resistance is a hallmark of type 2 diabetes, and insulin receptor substrates 1 and 2 (IRS1 and IRS2) are the major insulin-signaling components regulating cellular metabolism and survival. To determine the role of IRS1 and IRS2 in the heart and examine whether hyperinsulinemia causes myocardial insulin resistance and cellular dysfunction via IRS1 and IRS2, we generated heart-specific IRS1 and IRS2 gene double-knockout (H-DKO) mice and liver-specific IRS1 and IRS2 double-knockout (L-DKO) mice. H-DKO mice had reduced ventricular mass; developed cardiac apoptosis, fibrosis, and failure; and showed diminished Akt→forkhead box class O-1 signaling that was accompanied by impaired cardiac metabolic gene expression and reduced ATP content. L-DKO mice had decreased cardiac IRS1 and IRS2 proteins and exhibited features of heart failure, with impaired cardiac energy metabolism gene expression and activation of p38α mitogen-activated protein kinase (p38). Using neonatal rat ventricular cardiomyocytes, we further found that chronic insulin exposure reduced IRS1 and IRS2 proteins and prevented insulin action through activation of p38, revealing a fundamental mechanism of cardiac dysfunction during insulin resistance and type 2 diabetes.