Purinergic regulation of cholangiocyte secretion: Identification of a novel role for P2X receptors

The P2X family of ligand-gated cation channels is comprised of seven distinct isoforms activated by binding of extracellular purines. Although originally identified in neurons, there is increasing evidence for expression of P2X receptors in epithelia as well. Because ATP is released by both hepatocytes and cholangiocytes, these studies were performed to evaluate whether P2X receptors are present in cholangiocytes and contribute to local regulation of biliary secretion and bile formation. RT-PCR of cDNA from cultured normal rat cholangiocytes detected transcripts for P2X receptors 2, 3, 4, and 6; products from P2X3 and P2X4 were robust and always detectable. In cholangiocyte lysates, P2X4 protein was readily detected, and immunohistochemical staining of intact rat liver revealed P2X4 protein concentrated in intrahepatic bile ducts. To assess the functional significance of P2X4, isolated Mz-ChA-1 cells were exposed to the P2X4-preferring agonist 2′,3′-O-(4-benzoyl-benzoyl)-ATP (BzATP), which activated inward currents of -18.2 + 3.0 pA/pF. In cholangiocyte monolayers, BzATP but not P2X3 agonists elicited robust Cl^- secretory responses (short-circuit current) when applied to either the apical (ΔI_sc 22.1 ± 3.3 μA) or basolateral (18.5 ± 1.6 μA) chamber, with half-maximal stimulation at ∼10 μM and ∼1 μM, respectively. The response to BzATP was unaffected by suramin (not significant) and was inhibited by Cu²⁺ (P < 0.01). These studies provide molecular and biochemical evidence for the presence of P2X receptors in cholangiocytes. Functional studies indicate that P2X4 is likely the primary isoform involved, representing a novel and functionally important component of the purinergic signaling complex modulating biliary secretion.