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Exosomes are small extracellular vesicles that are thought to participate in intercellular communication. Recent work from our laboratory suggests that, in normal and cystic liver, exosome-like vesicles accumulate in the lumen of intrahepatic bile ducts, presumably interacting with cholangiocyte cilia. However, direct evidence for exosome-ciliary interaction is limited and the physiological relevance of such interaction remains unknown. Thus, in this study, we tested the hypothesis that biliary exosomes are involved in intercellular communication by interacting with cholangiocyte cilia and inducing intracellular signaling and functional responses. Exosomes were isolated from rat bile by differential ultracentrifugation and characterized by scanning, transmission, and immunoelectron microscopy. The exosome-ciliary interaction and its effects on ERK1/2 signaling, expression of the microRNA, miR-15A, and cholangiocyte proliferation were studied on ciliated and deciliated cultured normal rat cholangiocytes. Our results show that bile contains vesicles identified as exosomes by their size, characteristic 'saucer-shaped' morphology, and specific markers, CD63 and Tsgl01. When NRCs were exposed to isolated biliary exosomes, the exosomes attached to cilia, inducing a decrease of the phosphorylated-to-total ERK1/2 ratio, an increase of miR-15A expression, and a decrease of cholangiocyte proliferation. All these effects of biliary exosomes were abolished by the pharmacological removal of cholangiocyte cilia. Our findings suggest that bile contains exosomes functioning as signaling nanovesicles and influencing intracellular regulatory mechanisms and cholangiocyte proliferation through interaction with primary cilia. bile; cholangiocytes doi: 10.1152/ajpgi.00093.2010.

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.sub.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.sub.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-[gamma]S, 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.sub.12] siRNAs, suggesting that cilia and ciliary [P2Y.sub.12] are involved in nucleotide-induced cAMP signaling. In conclusion, cholangiocyte cilia are chemosensory organelles that detect biliary nucleotides through ciliary [P2Y.sub.12] receptors and transduce corresponding signals into a cAMP response. liver; ADP; adenylyl cyclases; cAMP; protein kinase A; exchange protein directly activated by cAMP; A-kinase anchoring protein 150

Water is an essential nutrient because it must be introduced from exogenous sources to satisfy metabolic demand. Under physiologic conditions, the colon can absorb and secrete considerable amounts of water even against osmotic gradients, thus helping to maintain the body fluid balance. Here we describe studies on both aquaporin (AQP) expression and function using cells isolated from the superficial and lower crypt regions of the rat proximal colon. The expression of AQP-3, -4, and -8 in isolated colonocytes was determined by semiquantitative RT-PCR and by immunoblotting. The localization of AQP-8 in the colon was evaluated by immunohistochemistry. A stopped-flow light scattering method was used to examine osmotic water movement in isolated colonocytes. Moreover, the contribution of AQP-8 to overall water movement through isolated colonocytes was studied using RNA interference technology. Colonocytes from the proximal colon express AQP-3, -4, and -8 with increasing concentrations from the lower crypt cells toward those on the surface. Osmotic water permeability was higher in surface than in crypt colonocytes (P < 0.05); it was significantly inhibited by the water channel blocker dimethyl sulfoxide, and reversed by [beta]-mercaptoethanol (P < 0.05). Immunohistochemistry revealed a strong AQP-8 labeling in the apical membrane of the superficial colonocytes. Inhibition of aquaporin-8 expression by small interfering RNA significantly decreased osmotic water permeability (~38%; P < 0.05). Current results indicate that aquaporin-8 may play a major role in water movement through the colon by acting on the apical side of the superficial cells. KEY WORDS: * aquaporins * water channel * small interfering RNA * colon

Dynamic membrane protrusions such as lamellipodia and filopodia are driven by actin polymerization and often hijacked by intracellular microbes to enter host cells. The overall rate of membrane protrusion depends on the actin polymerization rate and the increase of localized cell volume. Although the signaling pathways involving actin polymerization are well characterized, the molecular mechanisms regulating local cell volume associated with membrane protrusion are unclear. Cryptosporidium parvum, an intracellular parasite, depends on host-cell membrane protrusion to accomplish cell entry and form the parasitophorous vacuole. Here, we report that C. parvum infection of cholangiocytes recruits host-cell SGLT1, a [Na.sup.+]/glucose cotransporter, and aquaporin 1 (AQP1), a water channel, to the attachment site. SGLT1-dependent glucose uptake occurs at the attachment site. Concordantly, the region of attachment displays localized water influx that is inhibited by either suppression of AQP1 by means of AQP1-small interfering RNA (siRNA) or inhibition of SGLT1 by a specific pharmacologic inhibitor, phlorizin. Inhibition of SGLT1 does not affect actin accumulation but decreases the membrane protrusion at the attachment site. Moreover, functional inhibition of host-cell AQP1 and SGLT1 hampers C. parvum invasion of cholangiocytes. Thus, glucose-driven, AQP-mediated localized water influx is involved in the membrane protrusion during C. parvum cellular invasion, phenomena that may also be relevant to the mechanisms of cell membrane protrusion in general. actin | aquaporin | cryptosporidiosis | [Na.sup.+]/glucosecotransporter 1 | bile ducts

With an in vitro model using enclosed intrahepatic bile duct units (IBDUs) isolated from wild-type and somatostatin receptor (SSTR) subtype 2 knockout mice, we tested the effects of somatostatin, secretin, and a selective SSTR2 agonist (L-779976) on fluid movement across the bile duct epithelial cell layer. By RT-PCR, four of five known subtypes of SSTRs (SSTR1, SSTR2A/2B, SSTR3, and SSTR4, but not SSTR5) were detected in cholangiocytes in wild-type mice. In contrast, SSTR2A/2B were completely depleted in the SSTR2 knockout mice whereas SSTR1, SSTR3 and SSTR4 were expressed in these cholangiocytes. Somatostatin induced a decrease of luminal area of IBDUs isolated from wild-type mice, reflecting net fluid absorption; L-779976 also induced a comparable decrease of luminal area. No significant decrease of luminal area by either somatostatin or L-779976 was observed in IBDUs from SSTR2 knockout mice. Secretin, a choleretic hormone, induced a significant increase of luminal area of IBDUs of wild-type mice, reflecting net fluid secretion; somatostatin and L-779976 inhibited (P < 0.01) secretin-induced fluid secretion. The inhibitory effect of both somatostatin and L-779976 on secretin-induced IBDU secretion was absent in IBDUs of SSTR2 knockout mice. Somatostatin induced an increase of intracellular cGMP and inhibited secretin-stimulated cAMP synthesis in cholangiocytes; depletion of SSTR2 blocked these effects of somatostatin. These data suggest that somatostatin regulates ductal bile formation in mice not only by inhibition of ductal fluid secretion but also by stimulation of ductal fluid absorption via interacting with SSTR2 on cholangiocytes, a process involving the intracellular cAMP/cGMP second messengers. bile flow; biliary secretion; cholestasis; secretin; receptors

The physiological relevance of the absorption of glucose from bile by cholangiocytes remains unclear. The aim of this study was to test the hypothesis that absorbed glucose drives aquaporin (AQP)-mediated water transport by biliary epithelia and is thus involved in ductal bile formation. Glucose absorption and water transport by biliary epithelia were studied in vitro by microperfusing intrahepatic bile duct units (IBDUs) isolated from rat liver. In a separate set of in vivo experiments, bile flow and absorption of biliary glucose were measured after intraportal infusion of D-glucose or phlorizin. IBDUs absorbed D-glucose in a dose- and phlorizin-dependent manner with an absorption maximum of 92.8 [+ or -] 6.2 pmol*[min.sup.-1]*[mm.sup.-1]. Absorption of D-glucose by microperfused IBDUs resulted in an increase of water absorption ([J.sub.v] = 3-10 nl*[min.sup.-1]*[mm.sup.-1], [P.sub.f] = 40 x [10.sup.-3] cm/sec). Glucose-driven water absorption by IBDUs was inhibited by Hg[Cl.sub.2], suggesting that water passively follows absorbed D-glucose mainly transcellularly via mercury-sensitive AQPs. In vivo studies showed that as the amount of absorbed biliary glucose increased after intraportal infusion of D-glucose, bile flow decreased. In contrast, as the absorption of biliary glucose decreased after phlorizin, bile flow increased. Results support the hypothesis that the physiological significance of the absorption of biliary glucose by cholangiocytes is likely related to regulation of ductal bile formation. liver; cholangiocytes; absorption; secretion; aquaporins; phlorizin; microperfusion; intrahepatic bile duct units

Channel-mediated water movement across enclosed or perfused mouse intrahepatic bile duct units. Am J Physiol Cell Physiol 283: C338-C346, 2002; 10.1152/ajpcell. 00162.2001.--We previously reported the development of reproducible techniques for isolating and perfusing intact intrahepatic bile duct units (IBDUs) from rats. Given the advantages of transgenic and knockout mice for exploring ductal bile formation, we report here the adaptation of those techniques to mice and their initial application to the study of water transport across mouse intrahepatic biliary epithelia. IBDUs were isolated from livers of normal mice by microdissection combined with enzymatic digestion. After culture, isolated IBDUs sealed to form intact, polarized compartments, and a microperfusion system employing those isolated IBDUs developed. A quantitative image analysis technique was used to observe a rapid increase of luminal area when sealed IBDUs were exposed to a series of inward osmotic gradients reflecting net water secretion; the choleretic agonists secretin and forskolin also induced water secretion into IBDUs. The increase of IBDU luminal area induced by inward osmotic gradients and choleretic agonists was reversibly inhibited by Hg[Cl.sub.2], a water channel inhibitor. With the use of a quantitative epifiuorescence technique in perfused mouse IBDUs, a high osmotic water permeability ([P.sub.f] = 2.55.6 x [10.sup.-2]cm/s) was found in response to osmotic gradients, further supporting the presence of water channels. These findings suggest that, as in the rat, water transport across intrahepatic biliary epithelia in mice is water channel mediated. epithelial cells; aquaporins; perfusion