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

2003 ◽  
Vol 284 (5) ◽  
pp. C1205-C1214 ◽  
Author(s):  
Ai-Yu Gong ◽  
Pamela S. Tietz ◽  
Melissa A. Muff ◽  
Patrick L. Splinter ◽  
Robert C. Huebert ◽  
...  
Keyword(s):  
Bile Duct ◽  
Knockout Mice ◽  
Somatostatin Receptor ◽  
Fluid Secretion ◽  
Intracellular Camp ◽  
Fluid Absorption ◽  
Wild Type ◽  
Bile Formation ◽  
Luminal Area ◽  
Ductal Fluid

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.

Cl−-dependent secretory mechanisms in isolated rat bile duct epithelial units

2001 ◽  
Vol 281 (2) ◽  
pp. G438-G446 ◽  
Author(s):  
Satish K. Singh ◽  
Albert Mennone ◽  
Alessandro Gigliozzi ◽  
Flavia Fraioli ◽  
James L. Boyer
Keyword(s):  
Bile Duct ◽  
Intrahepatic Bile Duct ◽  
Fluid Secretion ◽  
Intracellular Camp ◽  
Camp Concentration ◽  
Uptake Pathway ◽  
Duct Epithelium ◽  
Bile Duct Epithelium ◽  
Rat Bile ◽  
Intracellular Camp Concentration

Cholangiocytes absorb and secrete fluid, modifying primary canalicular bile. In several Cl−-secreting epithelia, Na+-K+-2Cl− cotransport is a basolateral Cl− uptake pathway facilitating apical Cl− secretion. To determine if cholangiocytes possess similar mechanisms independent of CO2/HCO[Formula: see text], we assessed Cl−-dependent secretion in rat liver isolated polarized bile duct units (IBDUs) by using videomicroscopy. Without CO2/HCO[Formula: see text], forskolin (FSK) stimulated secretion entirely dependent on Na+ and Cl−and inhibited by Na+-K+-2Cl−inhibitor bumetanide. Carbonic anhydrase inhibitor ethoxyzolamide had no effect on FSK-stimulated secretion, indicating negligible endogenous CO2/HCO[Formula: see text] transport. In contrast, FSK-stimulated secretion was inhibited ∼85% by K+ channel inhibitor Ba2+ and blocked completely by bumetanide plus Ba2+. IBDU Na+-K+-2Cl− cotransport activity was assessed by recording intracellular pH during NH4Cl exposure. Bumetanide inhibited initial acidification rates due to NH[Formula: see text] entry in the presence and absence of CO2/HCO[Formula: see text]. In contrast, when stimulated by FSK, a 35% increase in Na+-K+-2Cl− cotransport activity occurred without CO2/HCO[Formula: see text]. These data suggest a cellular model of HCO[Formula: see text]-independent secretion in which Na+-K+-2Cl−cotransport maintains high intracellular Cl−concentration. Intracellular cAMP concentration increases activate basolateral K+ conductance, raises apical Cl−permeability, and causes transcellular Cl− movement into the lumen. Polarized IBDU cholangiocytes are capable of vectorial Cl−-dependent fluid secretion independent of HCO[Formula: see text]. Bumetanide-sensitive Na+-K+-2Cl− cotransport, Cl−/HCO[Formula: see text] exchange, and Ba2+-sensitive K+ channels are important components of stimulated fluid secretion in intrahepatic bile duct epithelium.

Phenotype analysis of aquaporin-8 null mice

2005 ◽  
Vol 288 (5) ◽  
pp. C1161-C1170 ◽  
Author(s):  
Baoxue Yang ◽  
Yuanlin Song ◽  
Dan Zhao ◽  
A. S. Verkman
Keyword(s):  
Salivary Gland ◽  
Knockout Mice ◽  
Plasma Membranes ◽  
Fluid Secretion ◽  
Intestinal Loop ◽  
Wild Type ◽  
Double Knockout ◽  
Mild Phenotype ◽  
Testicular Weight ◽  
Urinary Concentrating Ability

Aquaporin-8 (AQP8) is a water-transporting protein expressed in organs of the mammalian gastrointestinal tract (salivary gland, liver, pancreas, small intestine, and colon) and in the testes, heart, kidney, and airways. We studied the phenotype of AQP8-null mice, and mice lacking AQP8, together with AQP1 or AQP5. AQP8-knockout mice lacked detectable AQP8 transcript and protein, and had reduced water permeability in plasma membranes from testes. Breeding of AQP8 heterozygous mice yielded AQP8-null mice, whose number, survival, and growth were not different from those of wild-type mice. Organ weight and serum/urine chemistries were similar in wild-type and AQP8-null mice, except for increased testicular weight in the null mice (4.8 ± 0.7 vs. 7.3 ± 0.3 mg/g body wt). Urinary concentrating ability in AQP8-null mice was unimpaired as assessed by urine osmolality (3,590 ± 360 mosmol/kgH2O) and weight loss (22 ± 2%) after 36-h water deprivation; urinary concentrating ability was similarly impaired in AQP1-null mice vs. AQP8/AQP1 double-knockout mice. Agonist-driven fluid secretion in salivary gland was not different in AQP8 vs. wild-type mice (∼1 μl·min−1·g body wt−1) or in AQP5-null mice vs. AQP8/AQP5 double-knockout mice. Closed intestinal loop measurements in vivo indicated unimpaired osmotically driven water transport, active fluid absorption, and cholera toxin-driven fluid secretion in AQP8-null mice. After 21 days on a 50% fat diet, wild-type and AQP8-null mice had similar weight gain (∼15 g), with no evidence of steatorrhea or abnormalities in blood chemistries, except for mild hypertriglyceridemia in the null mice. The mild phenotype of AQP8-null mice was surprising in view of the multiple phenotype abnormalities found in mouse models of AQP1–5 deficiency.

Intestinal NaCl transport in NHE2 and NHE3 knockout mice

2002 ◽  
Vol 282 (5) ◽  
pp. G776-G784 ◽  
Author(s):  
Lara R. Gawenis ◽  
Xavier Stien ◽  
Gary E. Shull ◽  
Patrick J. Schultheis ◽  
Alison L. Woo ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Intestinal Tract ◽  
Apical Membrane ◽  
Intracellular Camp ◽  
Wild Type ◽  
Rt Pcr ◽  
Nacl Transport ◽  
Nacl Absorption ◽  
Small Intestinal ◽  
Absorptive Flux

Sodium/proton exchangers [Na+/H+ (NHEs)] play an important role in salt and water absorption from the intestinal tract. To investigate the contribution of the apical membrane NHEs, NHE2 and NHE3, to electroneutral NaCl absorption, we measured radioisotopic Na+ and Cl− flux across isolated jejuna from wild-type [NHE(+)], NHE2 knockout [NHE2(−)], and NHE3 knockout [NHE3(−)] mice. Under basal conditions, NHE(+) and NHE2(−) jejuna had similar rates of net Na+ (∼6 μeq/cm2 · h) and Cl− (∼3 μeq/cm2 · h) absorption. In contrast, NHE3(−) jejuna had reduced net Na+ absorption (∼2 μeq/cm2 · h) but absorbed Cl− at rates similar to NHE(+) and NHE2(−) jejuna. Treatment with 100 μM 5-( N-ethyl- N-isopropyl) amiloride (EIPA) completely inhibited net Na+ and Cl−absorption in all genotypes. Studies of the Na+ absorptive flux ( J [Formula: see text]) indicated that J [Formula: see text] in NHE(+) jejunum was not sensitive to 1 μM EIPA, whereas J [Formula: see text] in NHE3(−) jejunum was equally sensitive to 1 and 100 μM EIPA. Treatment with forskolin/IBMX to increase intracellular cAMP (cAMPi) abolished net NaCl absorption and stimulated electrogenic Cl− secretion in all three genotypes. Quantitative RT-PCR of epithelia from NHE2(−) and NHE3(−) jejuna did not reveal differences in mRNA expression of NHE3 and NHE2, respectively, when compared with jejunal epithelia from NHE(+) siblings. We conclude that 1) NHE3 is the dominant NHE involved in small intestinal Na+ absorption; 2) an amiloride-sensitive Na+ transporter partially compensates for Na+ absorption in NHE3(−) jejunum; 3) cAMPi stimulation abolishes net Na+ absorption in NHE(+), NHE2(−), and NHE3(−) jejunum; and 4) electroneutral Cl− absorption is not directly dependent on either NHE2 or NHE3.

Luminal hypotonicity in proximal tubules of aquaporin-1-knockout mice

2000 ◽  
Vol 278 (6) ◽  
pp. F1030-F1033 ◽  
Author(s):  
V. Vallon ◽  
A. S. Verkman ◽  
J. Schnermann
Keyword(s):  
Knockout Mice ◽  
Chloride Concentration ◽  
Plasma Osmolality ◽  
Proximal Tubules ◽  
Fluid Absorption ◽  
Wild Type ◽  
Fluid Reabsorption ◽  
Aquaporin 1 ◽  
Proximal Tubular

To examine the role of aquaporin-1 (AQP1) in near-isosmolar fluid reabsorption in the proximal tubule, we compared osmolalities in micropuncture samples of late proximal tubular fluid and plasma in wild-type (+/+) and AQP1-knockout (−/−) mice. Compared with matched wild-type mice, the −/− animals produce a relatively hypotonic urine (607 ± 42 vs. 1,856 ± 101 mosmol/kgH2O) and have a higher plasma osmolality under micropuncture conditions (346 ± 11 vs. 318 ± 5 mosmol/kgH2O; P < 0.05). Measurements of tubular fluid osmolality were done in three groups of mice, +/+, −/−, and hydrated −/− mice in which plasma osmolality was reduced to 323 ± 1 mosmol/kgH2O. Late proximal tubular fluid osmolalities were 309 ± 5 (+/+, n= 21), 309 ± 4 (−/−, n = 24), and 284 ± 3 mosmol/kgH2O (hydrated −/−, n = 19). Tubular fluid chloride concentration averaged 152 ± 1 (+/+), 154 ± 1 (−/−), and 140 ± 1 mM (hydrated −/−). Transtubular osmotic gradients in untreated and hydrated AQP1 −/− mice were 39 ± 4 ( n = 25) and 39 ± 3 mosmol/kgH2O ( n = 19), values significantly higher than in +/+ mice (12 ± 2 mosmol/kgH2O; n = 24; both P < 0.001). AQP1 deficiency in mice generates marked luminal hypotonicity in proximal tubules, resulting from the retrieval of a hypertonic absorbate and indicating that near-isosmolar fluid absorption requires functional AQP1.

scholarly journals Defective proximal tubular fluid reabsorption in transgenic aquaporin-1 null mice

1998 ◽  
Vol 95 (16) ◽  
pp. 9660-9664 ◽  
Author(s):  
Jurgen Schnermann ◽  
Chung-Lin Chou ◽  
Tonghui Ma ◽  
Timothy Traynor ◽  
Mark A. Knepper ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Water Permeability ◽  
Knockout Mice ◽  
Plasma Concentrations ◽  
Water Channels ◽  
Fluid Absorption ◽  
Wild Type ◽  
Aquaporin 1 ◽  
Tubule Fluid

To investigate the role of aquaporin-1 (AQP1) water channels in proximal tubule function, in vitro proximal tubule microperfusion and in vivo micropuncture measurements were done on AQP1 knockout mice. The knockout mice were generated by targeted gene disruption and found previously to be unable to concentrate their urine in response to water deprivation. Unanesthetized knockout mice consumed 2.8-fold more fluid than wild-type mice and had lower urine osmolality (505 ± 40 vs. 1081 ± 68 milliosmolar). Transepithelial osmotic water permeability (Pf) in isolated microperfused S2 segments of proximal tubule from AQP1 knockout [−/−] mice was 0.033 ± 0.005 cm/s (SE, n = 6 mice, 37°C), much lower than that of 0.15 ± 0.03 cm/s (n = 8) in tubules from wild-type [+/+] mice (P < 0.01). In the presence of isosmolar luminal perfusate and bath solutions, spontaneous fluid absorption rates (nl/min/mm tubule length) were 0.31 ± 0.12 (−/−, n = 5) and 0.64 ± 0.15 (+/+, n = 8). As determined by free-flow micropuncture, the ratios of tubular fluid-to-plasma concentrations of an impermeant marker TF/P in end proximal tubule fluid were 1.36 ± 0.05 (−/−, n = 8 mice [53 tubules]) and 1.95 ± 0.09 (+/+, n = 7 mice [40 tubules]) (P < 0.001), corresponding to 26 ± 3% [−/−] and 48 ± 2% [+/+] absorption of the filtered fluid load. In collections of distal tubule fluid, TF/P were 2.8 ± 0.3 [−/−] and 4.4 ± 0.5 [+/+], corresponding to 62 ± 4% [−/−] and 76 ± 3% [+/+] absorption (P < 0.02). These data indicate that AQP1 deletion in mice results in decreased transepithelial proximal tubule water permeability and defective fluid absorption. Thus, the high water permeability in proximal tubule of wild-type mice is primarily transcellular, mediated by AQP1 water channels, and required for efficient near-isosmolar fluid absorption.

scholarly journals Characterization of Intracellular Signaling Mediated by Human Somatostatin Receptor 5: Role of the DRY Motif and the Third Intracellular Loop

Endocrinology ◽  
2009 ◽  
Vol 150 (7) ◽  
pp. 3169-3176 ◽  
Author(s):  
Erika Peverelli ◽  
Andrea G. Lania ◽  
Giovanna Mantovani ◽  
Paolo Beck-Peccoz ◽  
Anna Spada
Keyword(s):  
Cell Proliferation ◽  
Intracellular Calcium ◽  
Intracellular Signaling ◽  
Somatostatin Receptor ◽  
Intracellular Camp ◽  
Wild Type ◽  
Intracellular Loop ◽  
The Third ◽  
Dry Motif ◽  
Third Intracellular Loop

Somatostatin (SST) exerts inhibitory effects on hormone secretion and cell proliferation by interacting with five different receptors (SST1-SST5) linked to multiple cellular effectors. The receptor structural domains involved in these effects have been only partially elucidated. The aim of the study was to investigate the molecular determinants mediating the interaction of the human SST5 with intracellular signaling in the pituitary cell line GH3, focusing on the BBXXB domain in the third intracellular loop and the DRY motif in the second intracellular loop. We analyzed the effects of the SST5 agonist BIM23206 on cAMP accumulation, intracellular calcium, GH secretion, cell proliferation, and ERK1/2 phosphorylation in cells expressing either wild-type SST5 or mutant receptors, in particular the naturally occurring mutant R240W in the BBXXB domain and the D136A and R137A mutants in the DRY motif. We found that residues D136 and R137 were critical for SST5 signaling because their substitutions abolished all the intracellular responses. Conversely, third intracellular loop mutations resulted in receptor that inhibited intracellular cAMP levels similar to the wild-type (50 ± 9 vs. 53 ± 12% inhibition) but failed to mediate the other responses elicited by wild-type SST5, i.e. reduction of intracellular calcium levels as well as inhibition of ERK1/2. These events resulted in an absent inhibition of GH release and an impaired reduction of cell proliferation (38 ± 7 vs. 76 ± 6% inhibition in wild type, P &lt; 0.05). These data indicate that different regions of SST5 are required for the activation of different signaling pathways.

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

2001 ◽  
Vol 280 (2) ◽  
pp. G247-G254 ◽  
Author(s):  
Albert Mennone ◽  
Daniel Biemesderfer ◽  
Daniel Negoianu ◽  
Chao-Ling Yang ◽  
Thecla Abbiati ◽  
...  
Keyword(s):  
Bile Duct ◽  
Ph Regulation ◽  
Fluid Secretion ◽  
Wild Type ◽  
Western Blots ◽  
Functional Studies ◽  
Sodium Hydrogen Exchanger ◽  
Apical Membranes ◽  
Nhe Isoforms

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.

Defective fluid and HCO3 − absorption in proximal tubule of neuronal nitric oxide synthase-knockout mice

2000 ◽  
Vol 279 (3) ◽  
pp. F518-F524 ◽  
Author(s):  
Tong Wang ◽  
Fiona M. Inglis ◽  
Robert G. Kalb
Keyword(s):  
Nitric Oxide ◽  
Blood Pressure ◽  
Metabolic Acidosis ◽  
Nitric Oxide Synthase ◽  
Proximal Tubule ◽  
Knockout Mice ◽  
Neuronal Nitric Oxide Synthase ◽  
Fluid Absorption ◽  
Wild Type ◽  
Oxide Synthase

Using renal clearance techniques and in situ microperfusion of proximal tubules, we examined the effects of N G-monomethyl-l-arginine methyl ester (l-NAME) on fluid and HCO3 −transport in wild-type mice and also investigated proximal tubule transport in neuronal nitric oxide synthase (nNOS)-knockout mice. In wild-type mice, administration of l-NAME (3 mg/kg bolus iv) significantly increased mean blood pressure, urine volume, and urinary Na+ excretion. l-NAME, given by intravenous bolus and added to the luminal perfusion solution, decreased absorption of fluid (60%) and HCO3 − (49%) in the proximal tubule. In nNOS-knockout mice, the urinary excretion of HCO3 − was significantly higher than in the wild-type mice (3.12 ± 0.52 vs. 1.40 ± 0.33 mM) and the rates of HCO3 − and fluid absorption were 62 and 72% lower, respectively. Both arterial blood HCO3 − concentration (20.7 vs. 25.7 mM) and blood pH (7.27 vs. 7.34) were lower, indicating a significant metabolic acidosis in nNOS-knockout mice. Blood pressure was lower in nNOS-knockout mice (76.2 ± 4.6 mmHg) than in wild-type control animals (102.9 ± 8.4 mmHg); however, it increased in response to l-NAME (125.5 ± 5.07 mmHg). Plasma Na+ and K+ were not significantly different from control values. Our data show that a large component of HCO3 − and fluid absorption in the proximal tubule is controlled by nNOS. Mice without this isozyme are defective in absorption of fluid and HCO3 − in the proximal tubule and develop metabolic acidosis, suggesting that nNOS plays an important role in the regulation of acid-base balance.

Channel-mediated water movement across enclosed or perfused mouse intrahepatic bile duct units

2002 ◽  
Vol 283 (1) ◽  
pp. C338-C346 ◽  
Author(s):  
Ai-Yu Gong ◽  
Anatoly I. Masyuk ◽  
Patrick L. Splinter ◽  
Robert C. Huebert ◽  
Pamela S. Tietz ◽  
...  
Keyword(s):  
Bile Duct ◽  
Water Transport ◽  
Water Movement ◽  
Water Channel ◽  
Intrahepatic Bile Duct ◽  
Enzymatic Digestion ◽  
Bile Formation ◽  
Initial Application ◽  
Luminal Area ◽  
Water Secretion

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 HgCl2, a water channel inhibitor. With the use of a quantitative epifluorescence technique in perfused mouse IBDUs, a high osmotic water permeability ( P f = 2.5–5.6 × 10−2 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.

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