Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP3/Ca2+ and cAMP-dependent signaling mechanisms

Abstract

Although large cholangiocytes exert their functions by activation of cyclic adenosine 3′,5′-monophosphate (cAMP), Ca2+-dependent signaling regulates the function of small cholangiocytes. Histamine interacts with four receptors, H1–H4HRs. H1HR acts by Gαq activating IP3/Ca2+, whereas H2HR activates Gαs stimulating cAMP. We hypothesize that histamine increases biliary growth by activating H1HR on small and H2HR on large cholangiocytes. The expression of H1–H4HRs was evaluated in liver sections, isolated and cultured (normal rat intrahepatic cholangiocyte culture (NRIC)) cholangiocytes. In vivo, normal rats were treated with histamine or H1–H4HR agonists for 1 week. We evaluated: (1) intrahepatic bile duct mass (IBDM); (2) the effects of histamine, H1HR or H2HR agonists on NRIC proliferation, IP3 and cAMP levels and PKCα and protein kinase A (PKA) phosphorylation; and (3) PKCα silencing on H1HR-stimulated NRIC proliferation. Small and large cholangiocytes express H1–H4HRs. Histamine and the H1HR agonist increased small IBDM, whereas histamine and the H2HR agonist increased large IBDM. H1HR agonists stimulated IP3 levels, as well as PKCα phosphorylation and NRIC proliferation, whereas H2HR agonists increased cAMP levels, as well as PKA phosphorylation and NRIC proliferation. The H1HR agonist did not increase proliferation in PKCα siRNA-transfected NRICs. The activation of differential signaling mechanisms targeting small and large cholangiocytes is important for repopulation of the biliary epithelium during pathologies affecting different-sized bile ducts.

Inhibition of mast cell-derived histamine secretion by cromolyn sodium treatment decreases biliary hyperplasia in cholestatic rodents

“…26,34,44,45 After BDL, the biliary epithelium proliferates and cholangiocytes secrete abundant amounts of these neuroendocrine factors to support the increased demands of the proliferating biliary tree. 26,34,44 We have previously shown that (i) histamine treatment increases normal rat cholangiocyte proliferation; 28 and (ii) after BDL, histamine levels are increased in both serum and cholangiocyte supernatant. 7 Autocrine regulation by histamine has been demonstrated in both biliary hyperplasia and neoplasia, as evidenced by our finding that inhibition of HDC decreases biliary growth.…”

“…25,28 Virtually pure cholangiocytes (verified by g-glutamyltranspetidase histochemistry 29 ) were isolated as described by us 25,26 using a monoclonal antibody (a mouse IgG 2a , a gift from Dr R Faris, Brown University, Providence, RI, USA) against an unidentified antigen expressed by all rat cholangiocytes. Cell viability was evaluated using trypan blue.…”

“…In sham-operated and BDL rats treated with saline or cromolyn sodium, we measured intrahepatic bile duct mass (IBDM) and cholangiocyte proliferation by (i) immunohistochemistry for CK-19 (a specific marker of cholangiocytes) and PCNA, respectively; 7,28 (ii) real-time PCR for CK-19 and PCNA expression in total liver and isolated cholangiocytes; and (iii) PCNA expression by immunoblots in isolated cholangiocytes. Apoptosis was evaluated by realtime PCR for the apoptotic markers, BAX and pro-caspase 3, and the anti-apoptotic marker, Bcl-2, in RNA obtained from isolated cholangiocytes.…”

“…33 Western blotting was performed in isolated cholangiocytes for PCNA expression with 20 mg of protein; b-actin was used as the housekeeping gene. 28,34 Band intensity was determined on the Odyssey system from LI-COR Biosciences (Lincoln, NE, USA), Model no. 9120.…”

“…We also evaluated the expression of CK-19 (the cholangiocyte-specific marker 25,28 ) by real-time PCR. Our cultured mast cell line expressed high levels of HDC, MAO-B, chymase, tryptase and c-kit but had virtually no expression of CK-19 ( Figure 7).…”