Colon water transport in transgenic mice lacking aquaporin-4 water channels

Transgenic null mice were used to test the hypothesis that water channel aquaporin-4 (AQP4) is involved in colon water transport and fecal dehydration. AQP4 was immunolocalized to the basolateral membrane of colonic surface epithelium of wild-type (+/+) mice and was absent in AQP4 null (-/-) mice. The transepithelial osmotic water permeability coefficient (P(f)) of in vivo perfused colon of +/+ mice, measured using the volume marker (14)C-labeled polyethylene glycol, was 0.016 +/- 0.002 cm/s. P(f) of proximal colon was greater than that of distal colon (0.020 +/- 0.004 vs. 0. 009 +/- 0.003 cm/s, P < 0.01). P(f) was significantly lower in -/- mice when measured in full-length colon (0.009 +/- 0.002 cm/s, P < 0. 05) and proximal colon (0.013 +/- 0.002 cm/s, P < 0.05) but not in distal colon. There was no difference in water content of cecal stool from +/+ vs. -/- mice (0.80 +/- 0.01 vs. 0.81 +/- 0.01), but there was a slightly higher water content in defecated stool from -/- mice (0.68 +/- 0.01 vs. 0.65 +/- 0.01, P < 0.05). Despite the differences in water permeability with AQP4 deletion, theophylline-induced secretion was not impaired (50 +/- 9 vs. 51 +/- 8 microl. min(-1). g(-1)). These results provide evidence that transcellular water transport through AQP4 water channels in colonic epithelium facilitates transepithelial osmotic water permeability but has little or no effect on colonic fluid secretion or fecal dehydration.     

Agonist-induced coordinated trafficking of functionally related transport proteins for water and ions in cholangiocytes

We previously proposed that ductal bile formation is regulated by secretin-responsive relocation of aquaporin 1 (AQP1), a water-selective channel protein, from an intracellular vesicular compartment to the apical membrane of cholangiocytes. In this study, we immunoisolated AQP1-containing vesicles from cholangiocytes prepared from rat liver; quantitative immunoblotting revealed enrichment in these vesicles of not only AQP1 but also cystic fibrosis transmembrane regulator (CFTR) and AE2, a Cl- channel and a Cl-/HCO3- exchanger, respectively. Dual labeled immunogold electron microscopy of cultured polarized mouse cholangiocytes showed significant colocalization of AQP1, CFTR, and AE2 in an intracellular vesicular compartment; exposure of cholangiocytes to dibutyryl-cAMP (100 microm) resulted in co-redistribution of all three proteins to the apical cholangiocyte plasma membrane. After administration of secretin to rats in vivo, bile flow increased, and AQP1, CFTR, and AE2 co-redistributed to the apical cholangiocyte membrane; both events were blocked by pharmacologic disassembly of microtubules. Based on these in vitro and in vivo observations utilizing independent and complementary approaches, we propose that cholangiocytes contain an organelle that sequesters functionally related proteins that can account for ion-driven water transport, that this organelle moves to the apical cholangiocyte membrane in response to secretory agonists, and that these events account for ductal bile secretion at a molecular level.     

Cytoskeletal and motor proteins facilitate trafficking of AQP1-containing vesicles in cholangiocytes

BACKGROUND INFORMATION: We have previously showed that: (i) cholangiocytes contain AQP1 (aquaporin 1) water channels sequestered in intracellular vesicles; and (ii) upon stimulation with choleretic agonists such as secretin or dibutyryl-cAMP (dbcAMP), the AQP1 vesicles move via microtubules to the apical cholangiocyte membrane to facilitate osmotically driven, passive water movement (i.e. ductal bile secretion). The aim of the present study was to determine which proteins and mechanisms regulate AQP1 trafficking in cholangiocytes. RESULTS: Using polarized cultured NMCs (normal mouse cholangiocytes) or NRCs (normal rat cholangiocytes) and affinity-purified antibodies, we performed immunofluorescent confocal microscopy on fixed cells or immunoblotting on cell lysates for actin, tubulin, kinesin and dynein, proteins known to regulate intracellular vesicle trafficking. By immunostaining, the appropriate orientation of the actin (i.e. sub-apical) and tubulin (i.e. generalized) cytoskeleton was apparent; kinesin and dynein displayed a homogeneous punctate distribution. Immunoblotting showed kinesin and dynein to be present in both cholangiocyte lysates and in isolated AQP1-containing vesicles. We utilized real-time fluorescence confocal microscopy of NMCs transfected with a GFP (green fluorescent protein)-AQP1 fusion construct in the presence and absence of dbcAMP. CONCLUSIONS: Our results provide additional insights into the potential molecular mechanisms of ductal bile secretion.     

Aquaporin-5 dependent fluid secretion in airway submucosal glands

Fluid and macromolecule secretion by submucosal glands in mammalian airways is believed to play an important role in airway defense and surface liquid homeostasis and in the pathogenesis of cystic fibrosis. Immunocytochemistry revealed strong expression of aquaporin water channel AQP5 at the luminal membrane of serous epithelial cells in submucosal glands throughout the mouse nasopharynx and upper airways and AQP4 at the contralateral basolateral membrane in some glands. Novel methods were applied to measure secretion rates and composition of gland fluid in wild type mice and knockout mice lacking AQP4 or AQP5. In mice breathing through a tracheotomy, total gland fluid output was measured from the dilution of a volume marker present in the fluid-filled nasopharynx and upper trachea. Pilocarpine-stimulated fluid secretion was 4.3 +/- 0.4 microl/min in wild type mice, 4.9 +/- 0.9 microl/min in AQP4 null mice, and 1.9 +/- 0.3 microl/min in AQP5 null mice (p < 0.001). Similar results were obtained when secreted fluid was collected in the oil-filled nasopharyngeal cavity. Real-time video imaging of fluid droplets secreted from individual submucosal glands near the larynx in living mice showed a 57 +/- 4% reduced fluid secretion rate in AQP5 null mice. Analysis of secreted fluid showed a 2.3 +/- 0.2-fold increase in total protein in AQP5 null mice and a smaller increase in [Cl(-)], suggesting intact protein and salt secretion across a relatively water impermeable epithelial barrier. Submucosal gland morphology and density did not differ significantly in wild type versus AQP5 null mice. These results indicate that AQP5 facilitates fluid secretion in submucosal glands and that the luminal membrane of gland epithelial cells is the rate-limiting barrier to water movement. Modulation of gland AQP5 expression or function might provide a novel approach to treat hyperviscous gland secretions in cystic fibrosis and excessive fluid secretions in infectious or allergic bronchitis/rhinitis.     

The role of aquaporin water channels in fluid secretion by the exocrine pancreas

The mammalian exocrine pancreas secretes a near-isosmotic fluid over a wide osmolarity range. The role of aquaporin (AQP) water channels in this process is now becoming clearer. AQP8 water channels, which were initially cloned from rat pancreas, are expressed at the apical membrane of pancreatic acinar cells and contribute to their osmotic permeability. However, the acinar cells secrete relatively little fluid and there is no obvious defect in pancreatic function in AQP8 knockout mice. Most of the fluid secreted by the pancreas is generated by ductal epithelial cells, which comprise only a small fraction of the gland mass. In the human pancreas, secretion occurs mainly in the intercalated ducts, where the epithelial cells express abundant AQP1 and AQP5 at the apical membrane and AQP1 alone at the basolateral membrane. In the rat and mouse, fluid secretion occurs mainly in the interlobular ducts where AQP1 and AQP5 are again co-localized at the apical membrane but appear to be expressed at relatively low levels. Nonetheless, the transepithelial osmotic permeability of rat interlobular ducts is sufficient to support near-isosmotic fluid secretion at observed rates. Furthermore, apical, but not basolateral, application of Hg²⁺ significantly reduces the transepithelial osmotic permeability, suggesting that apical AQP1 and AQP5 may contribute significantly to fluid secretion. The apparently normal fluid output of the pancreas in AQP1 knockout mice may reflect the presence of AQP5 at the apical membrane.     

Gastric acid secretion in aquaporin-4 knockout mice

The aquaporin-4 (AQP4) water channel has been proposed to play a role in gastric acid secretion. Immunocytochemistry using anti-AQP4 antibodies showed strong AQP4 protein expression at the basolateral membrane of gastric parietal cells in wild-type (+/+) mice. AQP4 involvement in gastric acid secretion was studied using transgenic null (-/-) mice deficient in AQP4 protein. -/- Mice had grossly normal growth and appearance and showed no differences in gastric morphology by light microscopy. Gastric acid secretion was measured in anesthetized mice in which the stomach was luminally perfused (0. 3 ml/min) with 0.9% NaCl containing [(14)C]polyethylene glycol ([(14)C]PEG) as a volume marker. Collected effluent was assayed for titratable acid content and [(14)C]PEG radioactivity. After 45-min baseline perfusion, acid secretion was stimulated by pentagastrin (200 microg. kg(-1). h(-1) iv) for 1 h or histamine (0.23 mg/kg iv) + intraluminal carbachol (20 mg/l). Baseline gastric acid secretion (means +/- SE, n = 25) was 0.06 +/- 0.03 and 0.03 +/- 0.02 microeq/15 min in +/+ and -/- mice, respectively. Pentagastrin-stimulated acid secretion was 0.59 +/- 0.14 and 0.70 +/- 0.15 microeq/15 min in +/+ and -/- mice, respectively. Histamine plus carbachol-stimulated acid secretion was 7.0 +/- 1.9 and 8.0 +/- 1.8 microeq/15 min in +/+ and -/- mice, respectively. In addition, AQP4 deletion did not affect gastric fluid secretion, gastric pH, or fasting serum gastrin concentrations. These results provide direct evidence against a role of AQP4 in gastric acid secretion.     

Role of aquaporin-4 in airspace-to-capillary water permeability in intact mouse lung measured by a novel gravimetric method

The mammalian peripheral lung contains at least three aquaporin (AQP) water channels: AQP1 in microvascular endothelia, AQP4 in airway epithelia, and AQP5 in alveolar epithelia. In this study, we determined the role of AQP4 in airspace-to-capillary water transport by comparing water permeability in wild-type mice and transgenic null mice lacking AQP1, AQP4, or AQP1/AQP4 together. An apparatus was constructed to measure lung weight continuously during pulmonary artery perfusion of isolated mouse lungs. Osmotically induced water flux (J(v)) between the airspace and capillary compartments was measured from the kinetics of lung weight change in saline-filled lungs in response to changes in perfusate osmolality. J(v) in wild-type mice varied linearly with osmotic gradient size (4.4 x 10(-5) cm(3) s(-1) mOsm(-1)) and was symmetric, independent of perfusate osmolyte size, weakly temperature dependent, and decreased 11-fold by AQP1 deletion. Transcapillary osmotic water permeability was greatly reduced by AQP1 deletion, as measured by the same method except that the airspace saline was replaced by an inert perfluorocarbon. Hydrostatically induced lung edema was characterized by lung weight changes in response to changes in pulmonary arterial inflow or pulmonary venous outflow pressure. At 5 cm H(2)O outflow pressure, the filtration coefficient was 4.7 cm(3) s(-1) mOsm(-1) and reduced 1.4-fold by AQP1 deletion. To study the role of AQP4 in lung water transport, AQP1/AQP4 double knockout mice were generated by crossbreeding of AQP1 and AQP4 null mice. J(v) were (cm(3) s(-1) mOsm(-1) x 10(-5), SEM, n = 7-12 mice): 3.8 +/- 0. 4 (wild type), 0.35 +/- 0.02 (AQP1 null), 3.7 +/- 0.4 (AQP4 null), and 0.25 +/- 0.01 (AQP1/AQP4 null). The significant reduction in P(f) in AQP1 vs. AQP1/AQP4 null mice was confirmed by an independent pleural surface fluorescence method showing a 1.6 +/- 0.2-fold (SEM, five mice) reduced P(f) in the AQP1/AQP4 double knockout mice vs. AQP1 null mice. These results establish a simple gravimetric method to quantify osmosis and filtration in intact mouse lung and provide direct evidence for a contribution of the distal airways to airspace-to-capillary water transport.     

Role of sodium/hydrogen exchanger isoform NHE3 in fluid secretion and absorption in mouse and rat cholangiocytes

Na+/H+ exchanger (NHE) isoforms play important roles in intracellular pH regulation and in fluid absorption. The isoform NHE3 has been localized to apical surfaces of epithelia and in some tissues may facilitate the absorption of NaCl. To determine whether the apical isoform NHE3 is present in cholangiocytes and to examine whether it has a functional role in cholangiocyte fluid secretion and absorption, immunocytochemical studies were performed in rat liver with NHE3 antibodies and functional studies were obtained in isolated bile duct units from wild-type and NHE3-/- mice after stimulation with forskolin, using videomicroscopic techniques. Our results indicate that NHE3 protein is present on the apical membranes of rat cholangiocytes and on the canalicular membrane of hepatocytes. Western blots also detect NHE3 protein in rat cholangiocytes and isolated canalicular membranes. After stimulation with forskolin, duct units from NHE3-/- mice fail to absorb the secreted fluid from the cholangiocyte lumen compared with control animals. Similar findings were observed in isolated bile duct units from wild-type mice and rats in the presence of the Na+/H+ exchanger inhibitor 5-(N-ethyl-N-isopropyl)-amiloride. In contrast, we could not demonstrate absorption of fluid from the canalicular lumen of mouse or rat hepatocyte couplets after stimulation of secretion with forskolin. These findings indicate that NHE3 is located on the apical membrane of rat cholangiocytes and that this NHE isoform can function to absorb fluid from the lumens of isolated rat and mouse cholangiocyte preparations.     

Salivary acinar cells from aquaporin 5-deficient mice have decreased membrane water permeability and altered cell volume regulation

Aquaporins (AQPs) are channel proteins that regulate the movement of water through the plasma membrane of secretory and absorptive cells in response to osmotic gradients. In the salivary gland, AQP5 is the major aquaporin expressed on the apical membrane of acinar cells. Previous studies have shown that the volume of saliva secreted by AQP5-deficient mice is decreased, indicating a role for AQP5 in saliva secretion; however, the mechanism by which AQP5 regulates water transport in salivary acinar cells remains to be determined. Here we show that the decreased salivary flow rate and increased tonicity of the saliva secreted by Aqp5(-)/- mice in response to pilocarpine stimulation are not caused by changes in whole body fluid homeostasis, indicated by similar blood gas and electrolyte concentrations in urine and blood in wild-type and AQP5-deficient mice. In contrast, the water permeability in parotid and sublingual acinar cells isolated from Aqp5(-)/- mice is decreased significantly. Water permeability decreased by 65% in parotid and 77% in sublingual acinar cells from Aqp5(-)/- mice in response to hypertonicity-induced cell shrinkage and hypotonicity-induced cell swelling. These data show that AQP5 is the major pathway for regulating the water permeability in acinar cells, a critical property of the plasma membrane which determines the flow rate and ionic composition of secreted saliva.     

Role of the neuropeptide,bombesin, in bile secretion.

Since ancient times, bile secretion has been considered vital for maintaining health. One of the main functions of bile secretion is gastric acid neutralization with biliary bicarbonate during a meal or Pavlovian response. Although the liver has many extrinsic and intrinsic nerve innervations, the functional role of these nerves in biliary physiology is poorly understood. To understand the role of neural regulation in bile secretion, our recent studies on the effect of bombesin, a neuropeptide, on bile secretion and its underlying mechanisms will be reviewed. Using isolated perfused rat livers (IPRL) from both normal and 2 week bile duct ligated rats, as well as hepatocyte couplets and isolated bile duct units (IBDU) from normal rat livers, bombesin was shown to stimulate biliary bicarbonate and fluid secretion from bile ducts. Detailed pH studies indicated that bombesin stimulated the activity of Cl-/HCO3- exchanger, which was counterbalanced by a secondary activation of electrogenic Na+/HCO3- symport. Quantitative videomicroscopic studies showed that bombesin-stimulated fluid secretion in IBDU was dependent on Cl- and HCO3- in the media, anion exchanger(s), Cl- and K+ channels, and carbonic anhydrase, but not on the microtubular system. Furthermore, this bombesin response is inhibited by somatostatin but not substance P. Finally, studies of secondary messengers in isolated cholangiocytes and IBDU indicated that bombesin had no effect on intracellular cAMP, cGMP, or Ca++ levels in cholangiocytes. These results provide evidence that neuropeptides such as bombesin can directly stimulate fluid and bicarbonate secretion from cholangiocytes by activating luminal Cl-/HCO3- exchange, but by different mechanisms from those established for secretin. These findings, in turn, suggest that neuropeptides may play an important regulatory role in biliary transport and secretion. Thus, this neuropeptidergic regulation of bile secretion may provide a plausible mechanism for the bicarbonate-rich choleresis seen with meals or Pavlovian response.     

Carbon dioxide permeability of aquaporin-1 measured in erythrocytes and lung of aquaporin-1 null mice and in reconstituted proteoliposomes

Measurements of CO(2) permeability in oocytes and liposomes containing water channel aquaporin-1 (AQP1) have suggested that AQP1 is able to transport both water and CO(2). We studied the physiological consequences of CO(2) transport by AQP1 by comparing CO(2) permeabilities in erythrocytes and intact lung of wild-type and AQP1 null mice. Erythrocytes from wild-type mice strongly expressed AQP1 protein and had 7-fold greater osmotic water permeability than did erythrocytes from null mice. CO(2) permeability was measured from the rate of intracellular acidification in response to addition of CO(2)/HCO(3)(-) in a stopped-flow fluorometer using 2',7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF) as a cytoplasmic pH indicator. In erythrocytes from wild-type mice, acidification was rapid (t((1)/(2)), 7.3 +/- 0.4 ms, S.E., n = 11 mice) and blocked by acetazolamide and increasing external pH (to decrease CO(2)/HCO(3)(-) ratio). Apparent CO(2) permeability (P(CO(2))) was not different in erythrocytes from wild-type (0.012 +/- 0.0008 cm/s) versus null (0.011 +/- 0.001 cm/s) mice. Lung CO(2) transport was measured in anesthetized, ventilated mice subjected to a decrease in inspired CO(2) content from 5% to 0%, producing an average decrease in arterial blood pCO(2) from 77 +/- 4 to 39 +/- 3 mm Hg (14 mice) with a t((1)/(2)) of 1.4 min. The pCO(2) values and kinetics of decreasing pCO(2) were not different in wild-type versus null mice. Because AQP1 deletion did not affect CO(2) transport in erythrocytes and lung, we re-examined CO(2) permeability in AQP1-reconstituted liposomes containing carbonic anhydrase (CA) and a fluorescent pH indicator. Whereas osmotic water permeability in AQP1-reconstituted liposomes was >100-fold greater than that in control liposomes, apparent P(CO(2)) (approximately 10(-3) cm/s) did not differ. Measurements using different CA concentrations and HgCl(2) indicated that liposome P(CO(2)) is unstirred layer-limited and that HgCl(2) slows acidification because of inhibition of CA rather than AQP1. These results provide direct evidence against physiologically significant AQP1-mediated CO(2) transport and establish an upper limit to the CO(2) permeability through single AQP1 water channels.     

Aquaporin-1 in the peritoneal membrane: implications for peritoneal dialysis and endothelial cell function

PD (peritoneal dialysis) is an established mode of renal replacement therapy, based on the exchange of fluid and solutes between blood in peritoneal capillaries and a dialysate that has been introduced into the peritoneal cavity. The dialysis process involves diffusive and convective transports and osmosis through the PM (peritoneal membrane). Computer simulations predicted that the PM contains ultrasmall pores (radius <3 A, 1 A=10(-10) m), responsible for up to 50% of UF (ultrafiltration), i.e. the osmotically driven water movement during PD. Several lines of evidence suggest that AQP1 (aquaporin-1) is the ultrasmall pore responsible for transcellular water permeability during PD. Treatment with corticosteroids induces the expression of AQP1 in the PM and improves water permeability and UF in rats without affecting the osmotic gradient and permeability for small solutes. Studies in knockout mice provided further evidence that osmotically driven water transport across the PM is mediated by AQP1. AQP1 and eNOS (endothelial nitric oxide synthase) show a distinct regulation within the endothelium lining the peritoneal capillaries. In acute peritonitis, the up-regulation of eNOS and increased release of nitric oxide dissipate the osmotic gradient and prevent UF, whereas AQP1 expression is unchanged. These results illustrate the usefulness of the PM to investigate the role and regulation of AQP1 in the endothelium. The results also emphasize the critical role of AQP1 during PD and suggest that manipulation of AQP1 expression may be used to increase water permeability across the PM.     

Evidence against functionally significant aquaporin expression in mitochondria

Recent reports suggest the expression of aquaporin (AQP)-type water channels in mitochondria from liver (AQP8) (Calamita, G., Ferri, D., Gena, P., Liquori, G. E., Cavalier, A., Thomas, D., and Svelto, M. (2005) J. Biol. Chem. 280, 17149-17153) and brain (AQP9) (Amiry-Moghaddam, M., Lindland, H., Zelenin, S., Roberg, B. A., Gundersen, B. B., Petersen, P., Rinvik, E., Torgner, I. A., and Ottersen, O. P. (2005) FASEB J. 19, 1459-1467), where they were speculated to be involved in metabolism, apoptosis, and Parkinson disease. Here, we systematically examined the functional consequence of AQP expression in mitochondria by measurement of water and glycerol permeabilities in mitochondrial membrane preparations from rat brain, liver, and kidney and from wild-type versus knock-out mice deficient in AQPs -1, -4, or -8. Osmotic water permeability, measured by stopped-flow light scattering, was similar in all mitochondrial preparations, with a permeability coefficient P(f) approximately 0.009 cm/s. Glycerol permeability was also similar ( approximately 5 x 10(-6) cm/s) in the various preparations. HgCl(2) slowed osmotic equilibration comparably in mitochondria from wild-type and AQP-deficient mice, although the slowing was explained by altered mitochondrial size rather than reduced P(f). Immunoblot analysis of mouse liver mitochondria failed to detect AQP8 expression, with liver homogenates from wild-type/AQP8 null mice as positive/negative controls. Our results provide evidence against functionally significant AQP expression in mitochondria, which is consistent with the high mitochondrial surface-to-volume ratio producing millisecond osmotic equilibration, even when intrinsic membrane water permeability is not high.     

Somatostatin stimulates ductal bile absorption and inhibits ductal bile secretion in mice via SSTR2 on cholangiocytes

With an in vitro model using enclosedintrahepatic bile duct units (IBDUs) isolated from wild-type andsomatostatin receptor (SSTR) subtype 2 knockout mice, we tested theeffects of somatostatin, secretin, and a selective SSTR2 agonist(L-779976) on fluid movement across the bile duct epithelial celllayer. By RT-PCR, four of five known subtypes of SSTRs (SSTR1,SSTR2A/2B, SSTR3, and SSTR4, but not SSTR5) were detected incholangiocytes in wild-type mice. In contrast, SSTR2A/2B werecompletely depleted in the SSTR2 knockout mice whereas SSTR1, SSTR3 andSSTR4 were expressed in these cholangiocytes. Somatostatin induced adecrease of luminal area of IBDUs isolated from wild-type mice,reflecting net fluid absorption; L-779976 also induced a comparabledecrease of luminal area. No significant decrease of luminal area byeither somatostatin or L-779976 was observed in IBDUs from SSTR2knockout mice. Secretin, a choleretic hormone, induced a significantincrease of luminal area of IBDUs of wild-type mice, reflecting netfluid secretion; somatostatin and L-779976 inhibited (P < 0.01) secretin-induced fluid secretion. The inhibitory effect ofboth somatostatin and L-779976 on secretin-induced IBDUsecretion was absent in IBDUs of SSTR2 knockout mice. Somatostatin induced an increase of intracellular cGMP and inhibitedsecretin-stimulated cAMP synthesis in cholangiocytes; depletion ofSSTR2 blocked these effects of somatostatin. These data suggest thatsomatostatin regulates ductal bile formation in mice not only byinhibition of ductal fluid secretion but also by stimulation of ductalfluid absorption via interacting with SSTR2 on cholangiocytes, aprocess involving the intracellular cAMP/cGMP second messengers.

     

Secretin stimulates exocytosis in isolated bile duct epithelial cells by a cyclic AMP-mediated mechanism.

Intrahepatic bile duct epithelial cells, or cholangiocytes, contribute to bile secretion in response to hormones, including secretin. However, the mechanism by which secretin stimulates ductular bile flow is unknown. Since recent data in nonhepatic epithelia have suggested a role for exocytosis in fluid secretion, we tested the hypothesis that secretin stimulates exocytosis by isolated cholangiocytes. Cholangiocytes were isolated from normal rat liver by a newly described method employing enzymatic digestion and mechanical disruption followed by immunomagnetic separation using specific monoclonal antibodies, and exocytosis was measured using a fluorescence unquenching assay employing acridine orange. Secretin caused a dose-dependent (10(-12)-10(-7) M) increase in acridine orange fluorescence by acridine orange-loaded cholangiocytes with a peak response at 10 min; the half-maximal concentration of secretin was 7 x 10(-9) M. The secretin effect was inhibited by preincubation of cholangiocytes with colchicine (30% inhibition, p less than 0.05) or trypsin (90% inhibition, p less than 0.001); no inhibition was seen with lumicolchicine and heat-inactivated trypsin. Cholecystokinin, insulin, and somatostatin had no effect on fluorescence of acridine orange-loaded cholangiocytes; secretin had no effect on fluorescence of acridine orange-loaded hepatocytes or hepatic endothelial cells. Exposure of isolated cholangiocytes to secretin at doses that stimulated exocytosis caused a dose-dependent increase in cyclic AMP levels (218% maximal increase, p less than 0.05); moreover, an analogue of cyclic AMP stimulated exocytosis by cholangiocytes. Secretin had no effect on intracellular calcium concentration using Fura-2-loaded cholangiocytes assessed by digitized video microscopy. Our results demonstrate, for the first time, that secretin stimulates exocytosis by rat cholangiocytes. The effect is cell- and hormone-specific, dependent on intact microtubules, on a protein(s) on the external surface of cholangiocytes, and on changes in cellular levels of cyclic AMP. The results are consistent with the hypothesis that secretin-induced changes in bile flow may involve an exocytic process.     

The alpha2-adrenergic receptor agonist UK 14,304 inhibits secretin-stimulated ductal secretion by downregulation of the cAMP system in bile duct-ligated rats

Secretin stimulates ductal secretion by activation of cAMP PKA CFTR Cl^–/HCO₃^– exchanger in cholangiocytes. We evaluated the expression of _2A-, _2B-, and _2C-adrenergic receptors in cholangiocytes and the effects of the selective ₂-adrenergic agonist UK 14,304, on basal and secretin-stimulated ductal secretion. In normal rats, we evaluated the effect of UK 14,304 on bile and bicarbonate secretion. In bile duct-ligated (BDL) rats, we evaluated the effect of UK 14,304 on basal and secretin-stimulated 1) bile and bicarbonate secretion; 2) duct secretion in intrahepatic bile duct units (IBDU) in the absence or presence of 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na⁺/H⁺ exchanger isoform NHE3; and 3) cAMP levels, PKA activity, Cl^– efflux, and Cl^–/HCO₃^– exchanger activity in purified cholangiocytes. ₂-Adrenergic receptors were expressed by all cholangiocytes in normal and BDL liver sections. UK 14,304 did not change bile and bicarbonate secretion of normal rats. In BDL rats, UK 14,304 inhibited secretin-stimulated 1) bile and bicarbonate secretion, 2) expansion of IBDU luminal spaces, and 3) cAMP levels, PKA activity, Cl^– efflux, and Cl^–/HCO₃^– exchanger activity in cholangiocytes. There was decreased lumen size after removal of secretin in IBDU pretreated with UK 14,304. In IBDU pretreated with EIPA, there was no significant decrease in luminal space after removal of secretin in either the absence or presence of UK 14,304. The inhibitory effect of UK 14,304 on ductal secretion is not mediated by the apical cholangiocyte NHE3. ₂-Adrenergic receptors play a role in counterregulating enhanced ductal secretion associated with cholangiocyte proliferation in chronic cholestatic liver diseases. bicarbonate secretion; chloride efflux; gastrointestinal hormones; intrahepatic biliary epithelium; protein kinase A     

Analysis of double knockout mice lacking aquaporin-1 and urea transporter UT-B. Evidence for UT-B-facilitated water transport in erythrocytes

We reported increased water permeability and a low urea reflection coefficient in Xenopus oocytes expressing urea transporter UT-B (former name UT3), suggesting that water and urea share a common aqueous pathway (Yang, B., and Verkman, A. S. (1998) J. Biol. Chem. 273, 9369-9372). Although increased water permeability was confirmed in the Xenopus oocyte expression system, it has been argued (Sidoux-Walter, F., Lucien, N., Olives, B., Gobin, R., Rousselet, G., Kamsteeg, E. J., Ripoche, P., Deen, P. M., Cartron, J. P., and Bailly, P. (1999) J. Biol. Chem. 274, 30228-30235) that UT-B does not transport water when expressed at normal levels in mammalian cells such as erythrocytes. To quantify UT-B-mediated water transport, we generated double knockout mice lacking UT-B and the major erythrocyte water channel, aquaporin-1 (AQP1). The mice had reduced survival, retarded growth, and defective urinary concentrating ability. However, erythrocyte size and morphology were not affected. Stopped-flow light scattering measurements indicated erythrocyte osmotic water permeabilities (in cm/s x 0.01, 10 degrees C): 2.1 +/- 0.2 (wild-type mice), 2.1 +/- 0.05 (UT-B null), 0.19 +/- 0.02 (AQP1 null), and 0.045 +/- 0.009 (AQP1/UT-B null). The low water permeability found in AQP1/UT-B null erythrocytes was also seen after HgCl(2) treatment of UT-B null erythrocytes or phloretin treatment of AQP1 null erythrocytes. The apparent activation energy for UT-B-mediated water transport was low, <2 kcal/mol. Estimating 14,000 UT-B molecules per mouse erythrocyte, the UT-B-dependent P(f) of 0.15 x 10(-4) cm/s indicated a substantial single channel water permeability of UT-B of 7.5 x 10(-14) cm(3)/s, similar to that of AQP1. These results provide direct functional evidence for UT-B-facilitated water transport in erythrocytes and suggest that urea traverses an aqueous pore in the UT-B protein.