Cholangiocyte primary cilia are chemosensory organelles that detect biliary nucleotides via P2Y12 purinergic receptors

Abstract

Cholangiocytes, the epithelial cells lining intrahepatic bile ducts, contain primary cilia, which are mechano- and osmosensory organelles detecting changes in bile flow and osmolality and transducing them into intracellular signals. Here, we asked whether cholangiocyte cilia are chemosensory organelles by testing the expression of P2Y purinergic receptors and components of the cAMP signaling cascade in cilia and their involvement in nucleotide-induced cAMP signaling in the cells. We found that P2Y(12) purinergic receptor, adenylyl cyclases (i.e., AC4, AC6, and AC8), and protein kinase A (i.e., PKA RI-beta and PKA RII-alpha regulatory subunits), exchange protein directly activated by cAMP (EPAC) isoform 2, and A-kinase anchoring proteins (i.e., AKAP150) are expressed in cholangiocyte cilia. ADP, an endogenous agonist of P2Y(12) receptors, perfused through the lumen of isolated rat intrahepatic bile ducts or applied to the ciliated apical surface of normal rat cholangiocytes (NRCs) in culture induced a 1.9- and 1.5-fold decrease of forskolin-induced cAMP levels, respectively. In NRCs, the forskolin-induced cAMP increase was also lowered by 1.3-fold in response to ATP-gammaS, a nonhydrolyzed analog of ATP but was not affected by UTP. The ADP-induced changes in cAMP levels in cholangiocytes were abolished by chloral hydrate (a reagent that removes cilia) and by P2Y(12) siRNAs, suggesting that cilia and ciliary P2Y(12) are involved in nucleotide-induced cAMP signaling. In conclusion, cholangiocyte cilia are chemosensory organelles that detect biliary nucleotides through ciliary P2Y(12) receptors and transduce corresponding signals into a cAMP response.

Fig. 1.

P2Y₁₂ and P2Y₁₃ are expressed in rat cholangiocytes; P2Y₁₂ is localized to primary cilia. A: RT-PCR. Positive bands for both P2Y₁₂ and P2Y₁₃ were detected in freshly isolated intrahepatic bile ducts (IBD), normal rat cholangiocytes (NRCs), hepatocytes, and brain (a positive control). No bands were detected in the negative control (i.e., no template cDNA). First line represents 100 bp ladder. B: by Western blotting, positive bands for both P2Y₁₂ and P2Y₁₃ were detected in cholangiocytes, hepatocytes, and brain. C: paraffin sections of rat liver were stained with antibodies to a ciliary marker, acetylated α-tubulin (shown in red), and to P2Y₁₂ (shown in green). Cholangiocyte nuclei were visualized by staining with 4′,6-diamidino-2-phenylindole (DAPI, shown in blue). On overlay of the images, colocalization of acetylated α-tubulin and P2Y₁₂ (yellow) was seen, indicating that P2Y₁₂ is present in cholangiocyte cilia. D: localization of P2Y₁₂ to cholangiocyte cilia was confirmed by immunogold scanning electron microscopy (SEM). Conventional SEM image of an isolated split-open IBD showing a single primary cilium (a) and the image generated by backscattered electrons (b) reveals the presence of the gold-labeled secondary antibodies to P2Y₁₂ on cilia.

Fig. 2.

Adenylyl cyclases (ACs) are expressed in cholangiocyte primary cilia. Isolated split-open rat IBDs were stained with antibodies to a ciliary marker, acetylated α-tubulin (shown in red), and antibodies to AC4, AC5, AC6, AC7, and AC8 (shown in green). Overlay images in yellow show that AC4, AC6, and AC8 but not AC5 and AC7 are expressed in cholangiocyte cilia.

Fig. 3.

PKA is expressed in cholangiocyte primary cilia. Paraffin sections of rat liver were stained with an antibody to a ciliary marker acetylated α-tubulin (shown in red) and antibodies to PKA regulatory subunits (shown in green). Cholangiocyte nuclei were visualized by staining with DAPI (shown in blue). On overlay of the images, colocalization of acetylated α-tubulin and PKAI-β and PKAII-α (shown in yellow) was seen, indicating that 2 of 4 PKA subtypes are expressed in cholangiocyte cilia.

Fig. 4.

Exchange protein directly activated by cAMP (EPAC)2 and A-kinase anchoring protein (AKAP)150 are expressed in cholangiocyte primary cilia. Paraffin sections of rat liver were stained with an antibody to a ciliary marker, acetylated α-tubulin (shown in red), and antibodies to EPAC1, EPAC2, and AKAP150 (shown in green). Cholangiocyte nuclei were visualized by staining with DAPI (shown in blue). On overlay of the images, colocalization of acetylated α-tubulin and EPAC2 (but not EPAC1) and AKAP150 (shown in yellow) was seen, indicating that 1 of 2 EPAC isoforms (i.e., EPAC2) and AKAP150 are expressed in cholangiocyte cilia.

Fig. 5.

Apically applied nucleotides affect cAMP levels in rat cholangiocytes. In microperfused IBDs (A) and NRCs (B and C), the basal levels of cAMP were increased by forskolin (FSK). ADP, ATP-γS, and UTP alone did not affect basal cAMP levels either in IBDs or NRCs (A, B, and C). However, ADP perfused through the lumen of IBDs (A) or apically applied to ciliated NRCs (B) significantly lowered the FSK-induced cAMP increase. ATP-γS also decreased FSK-induced cAMP levels in NRCs (C), whereas UTP did not show any affect on cAMP levels (C); n = 4–6 IBDs or wells containing NRCs in each group; *P < 0.05.

Fig. 6.

The effects of apically applied nucleotides on the FSK-induced cAMP increase in rat cholangiocytes depend on P2Y receptors. A nonspecific inhibitor of P2Y purinergic receptors, suramin, completely abolished effects of ADP on FSK-induced cAMP levels in NRCs (n = 4–6 wells containing NRCs in each group; *P < 0.05).

Fig. 7.

Apically applied nucleotides affect the FSK-induced cAMP increase in rat cholangiocytes in a P2Y₁₂-dependent manner. Incubation of IBDs for 24 h with siRNAs to P2Y₁₂ resulted in the suppression of P2Y₁₂ expression on both mRNA and protein levels (A and B). Suppression of P2Y₁₂ by siRNAs abolished effects of ADP on FSK-induced cAMP levels in microperfused IBDs (C); scr, scrambled siRNA. n = 4–6 IBDs in each group; *P < 0.05.

Fig. 8.

Apically applied nucleotides affect the FSK-induced cAMP increase in rat cholangiocytes in a ciliary-dependent manner. Pretreatment of IBDs (A) or NRCs (B) for 24 h with chloral hydrate completely abolished effects of ADP on FSK-induced cAMP levels in cholangiocytes; n = 4–6 IBDs or wells containing NRCs in each group; *P < 0.05.

Fig. 9.

A working model of coordinated sensory functions of cholangiocyte cilia. (See discussion for details).