Adenoviral transfer of human aquaporin-1 gene to rat liver improves bile flow in estrogen-induced cholestasis.

Estrogens can cause liver cholestatic disease. As downregulation of hepatocyte canalicular aquaporin-8 (AQP8) water channels has been involved in estrogen-induced bile secretory failure, we tested whether the archetypal water channel AQP1 improves 17α-ethinylestradiol (EE)-induced cholestasis. EE administration to rats reduced bile flow by 50%. A recombinant adenoviral (Ad) vector encoding human AQP1 (hAQP1), AdhAQP1, or a control vector was administered by retrograde bile ductal infusion. Hepatocyte canalicular hAQP1 expression was confirmed by liver immunostaining and immunoblotting in purified membrane fractions. Accordingly, canalicular osmotic water permeability was markedly increased. Bile flow, either basal or bile salt-stimulated was significantly augmented by over 50%. The choleretic efficiency of endogenous bile salts (that is, volume of bile per µmol of excreted bile salt) was significantly increased by 45% without changes in the biliary bile salt composition. Our data suggest that the adenoviral transfer of hAQP1 gene to the livers of EE-induced cholestatic rats improves bile flow by enhancing the AQP-mediated bile salt-induced canalicular water secretion. This novel finding might have potential therapeutic implications for cholestatic diseases.

Concurrent diphtheria and infectious mononucleosis: difficulties for management, investigation and control of diphtheria in developing countries.

We report a case of concurrent diphtheria and infectious mononucleosis in an 11-year-old Brazilian child. Two days after specific treatment for diphtheria was started the patient was discharged following clinical recovery. This case highlights the difficulties in the clinical diagnosis of diphtheria in partially immunized individuals, and for the management and control of diphtheria in developing countries.

Wave and sediment dynamics along a shallow subtidal sandy beach inhabited by modern stromatolites.

To help define the habitat of modern marine stromatolites, wave-dominated flow and sediment transport were studied in the shallow subtidal region (1-2 m depth) along the slightly concave, windward face of Highborne Cay, Exuma, Bahamas - the only face of the cay that includes a population of stromatolites concentrated near the region of highest curvature of the beach. Wave energy impacting this island's most exposed beach was driven by local wind forcing which increases largely in response to the passage of atmospheric disturbances that typically affect the region for periods of a few days. Although some wave energy is almost always noted (maximum horizontal orbital speeds at the bottom are rarely <10 cm s(-1)), wave conditions remain comparatively calm until local winds increase above speeds of approximately 3-4 m s(-1) at which point maximum wave speeds rapidly increase to 50-80 cm s(-1). Stromatolites, which are largely restricted to the shoreward side of a shallow platform reef, are sheltered by the reef beyond which wave speeds are one to four times higher (depending on tidal stage). Moreover, stromatolite populations are predominantly found along a region of this wave-exposed beach that experiences comparatively reduced wave energy because of the curved morphology of the island's face. Maximum wave speeds are 1.4 to 2 times higher along more northern sections of the beach just beyond the locus of stromatolite populations. A quantitative model of sediment transport was developed that accurately predicted accumulation of suspended sediment in sediment traps deployed in the shallow subtidal zone along this beach. This model, coupled with in situ wave records, indicates that gross rates of suspended sediment deposition should be two to three times higher northward of the main stromatolite populations. Regions of the beach containing stromatolites nevertheless should experience significant rates of gross suspended sediment deposition averaging 7-10 g cm(-2) day(-1) ( approximately 4-6 cm day(-1)). Results suggest that one axis of the habitat of modern marine stromatolites may be defined by a comparatively narrow range of flow energy and sediment transport conditions.

Taurolithocholate can inhibit the biliary discharge of lysosomes in the rat.

The natural bile salt taurolithocholate (TLC) impairs the biliary excretion of lipids and proteins, which are known to reach the canaliculus via vesicles. In this study we examined whether these observations could be extended to the exocytic discharge of lysosomal contents into bile. The single intravenous injection of a cholestatic dose of TLC, 3 micromol/100 g body wt., markedly inhibited the biliary excretion of the lysosomal enzymes acid phosphatase and beta-glucuronidase, despite the excretion of bile salts being normalized after a transient diminution. Under such a condition, TLC did not affect the normal transport to and the processing in lysosomes of the exogenously administered [14C]sucrose-labeled horseradish peroxidase. However, the biliary excretion of the radioactive lysosomal metabolites of the protein was significantly reduced. The results indicate that TLC can inhibit the biliary discharge of lysosomes in the rat without altering the functional integrity of these organelles. Possible explanations for these findings are discussed.

Assessment of the in vivo hepatic lysosomal processing of horseradish peroxidase.

The lysosomal processing of horseradish peroxidase (HRP) was assessed in this study, i.e., its lysosomal proteolysis and the biliary output of its possible lysosomal metabolites by rat liver in vivo. HRP was covalently linked to [14C]sucrose to provide a label that remains trapped within lysosomes after proteolysis. The [14C]sucrose-labelled HRP was injected into the portal vein of rat, and after 30 min about 34% of the injected radiolabel was present in the liver. Subcellular fractionation by differential centrifugation and further purification of lysosomes in a Percoll gradient showed that radiolabel was concentrated in lysosomes and indicated that about 91% of the total proteolysis of HRP in liver could be attributed to these organelles. The in vivo lysosomal degradation rate of HRP at 30 min was about 40%/h, decreasing over time. The lysosomal inhibitors chloroquine and leupeptin suppressed proteolysis of HRP by about 30 and 60%, respectively. Analysis of the 14C excreted in bile by trichloroacetic acid precipitation and by SDS-polyacrylamide gel electrophoresis showed a minor fraction, which was intact HRP (40 kDa), and a major fraction, which was associated with material smaller than 3 kDa. The biliary output of these low molecular mass products, in contrast to that of intact HRP, did not gradually decline with time and represented about 3% of the corresponding amounts in liver. Chloroquine and leupeptin specifically decreased their biliary excretion (about 60%), giving additional support to their lysosomal origin. In addition, the overall hepatic processing of [14C]sucrose-labelled HRP did not differ from that of the native HRP measured by enzyme assay, indicating no significant alteration caused by the labelling procedure.

Effect of cholephilic dyes on hepatic tight junctional permeability in the rat.

Changes in biliary permeability during cholephilic dye-induced choleresis, as assessed by measuring the movement into bile of two permeability probes, [14C]sucrose and horseradish peroxidase, were analyzed following an i.v. infusion (60 nmol/min per 100 g body wt) of the model cholephilic organic anion sulfobromophthalein in rats. Dye infusion led to a progressive increase of the [14C]sucrose bile-to-plasma ratio, which reached a maximum value after 100 min of dye infusion (+97%). Paracellular entry of horseradish peroxidase, as evaluated by the early peak of its biliary appearance curve, was also selectively increased (+69%), without changes in the later (transcytotic) access of the protein. Additional dose-response studies of biliary permeability to [14C]sucrose, using sulfobromophthalein and rose bengal, showed that this effect was dose-dependent and rapidly reversed by interruption of dye administration. The influence of hydrophobic/hydrophilic balance on this effect was also studied by infusing four dyes covering a broad range of hydrophobicity (phenol red, bromocresol green, sulfobromophthalein, and rose bengal), so as to attain a similar value of dye hepatic content at the end of the experiment (approximately 150 nmol/g liver wt). Under these conditions, a strong positive correlation was found between the increase in biliary permeability to [14C]sucrose and dye hydrophobicity. These results suggest that cholephilic dyes increase tight junctional permeability in a reversible and dose-dependent manner, and that this effect depends on the hydrophobic/hydrophilic balance of the dye.

Effect of lysosomotropic agents on the taurocholate-stimulated biliary excretion of horseradish peroxidase.

The effects of the lysosomotropic agents chloroquine and leupeptin on the taurocholate-stimulated biliary excretion of horseradish peroxidase (HRP) was studied in bile fistula rats. HRP (0.5 mg/100 g body wt) was injected into the portal vein during taurocholate (0.4 mumol/min/100 g body wt) or saline infusion. HRP appeared in bile showing both an early (approx. 5 min) and a late (approx. 25 min) excretion peak. The late peak, which represented about 95% of the total HRP excreted, is due to transcellular vesicular transport. The early peak is mainly due to paracellular leakage although a rapid vesicular transport also contributes. Taurocholate infusion significantly increased the biliary output of HRP (both peaks) and of the endogenous lysosomal enzyme acid phosphatase. Pretreatment with chloroquine or leupeptin inhibited the taurocholate-stimulated late excretion of HRP into bile, without affecting its early excretion. The lysosomotropic agents did not affect the biliary excretion of bile salts but significantly inhibited the taurocholate-stimulated biliary excretion of acid phosphatase. The results are consistent with a role of lysosomes in the taurocholate-stimulated major transcellular vesicular transport of HRP into bile.

Taurolithocholate-induced inhibition of biliary lipid and protein excretion in the rat.

Taurolithocholate (TLC), a natural bile salt, induces selective impairment on canalicular membrane of the hepatocyte, which seems to be a major determinant of its cholestatic effect in experimental animals. In order to extend existing studies about the effects of TLC on bile secretion, we examined in TLC-treated rats the biliary excretion of compounds that are transported to canalicular membrane via vesicles, such as lipids and proteins. The single intravenous injection of TLC (3 mumol/100 g body wt.) inhibited transiently the biliary bile salt excretion, while the biliary excretion of lipids (i.e., cholesterol and phospholipids) and proteins remained inhibited even though the biliary excretion and composition of bile salts were normalized. Under such a condition, TLC also inhibited the transcellular vesicular pathway to the exogenous protein horseradish peroxidase entry into bile, without altering the paracellular biliary access of the protein. The hepatic uptake of horseradish peroxidase was unaffected by TLC-treatment. The results indicate that TLC can inhibit the biliary excretion of compounds that reach the canaliculus via a vesicular pathway, such as lipids and proteins, by a mechanism not related to a defective bile salt excretion. Possible explanations for these findings are discussed.

Biliary excretion of polyethylene glycol molecular weight 900. Evidence for a bile salt-stimulated vesicular transport mechanism.

Polyethylene glycol molecular weight 900 (PEG-900) has been used as a marker of vectorial water transport into bile canaliculus. However, the mechanisms by which this compound is excreted have not been clarified. To gain more information on this process, we studied the biliary excretion of [3H]PEG-900 in rats during choleresis induced by canalicular choleretics. In addition, the effects of the microtubule inhibitors colchicine and vinblastine, and of the acidotropic agent chloroquine, on PEG-900 excretion were studied to determine whether a vesicular pathway is involved. Continuous i.v. infusion of either dehydrocholate (DHC, a non-micelle forming bile salt choleretic) or 4-methylumbelliferone (4-MU, a non-bile salt canalicular choleretic) at stepwise-increasing rates [0.7, 1.0 and 1.2 mumol.min-1.(100 g body wt)-1] induced a gradual increment in bile flow, whereas a transient increment of [3H]PEG-900 excretion was observed only during DHC-induced choleresis. Furthermore, studies in which two consecutive i.v. injections of DHC (10 mumol/100 g body wt) were administered showed that [3H]PEG-900 excretion induced by a second administration of DHC was 54% lower than that induced by the first one, despite a similar excretion in bile flow. Finally, colchicine (0.5 mumol/100 g body wt), vinblastine (0.5 mumol/100 g body wt) and chloroquine (50 mg/kg body wt) pretreatments inhibited the DHC-induced increment in biliary [3H]PEG-900 output, while DHC-induced choleresis was almost unaffected. Conversely, excretion of [14C]sucrose, when coadministered with [3H]PEG-900, was not impaired by the treatments. These results suggest that, unlike sucrose, PEG-900 excretion is not associated with canalicular water movements. Instead, it may be related to a vesicular transport process followed by a bile acid-stimulated discharge of secretory vesicles into bile through the lysosomal compartment.

Biliary excretion of proteins in the rat during dehydrocholate choleresis.

Choleresis induced by dehydrocholate (DHC) stimulates the discharge into bile of lysosomes, which are implicated in the biliary excretion of proteins. Contrary to taurocholate-induced choleresis, DHC choleresis is not affected by microtubule (mt) inhibition. Therefore, the role of mt's in the biliary protein excretion during bile salt choleresis was analyzed in this study. Normal rats and rats treated with the mt poisons colchicine or vinblastine or with the acidotropic agent chloroquine (Cq) were used. The analysis of the protein component in bile was made on SDS-polyacrylamide gel, and the individual polypeptides were quantitated by densitometry. The excretion of bile polypeptides were compared with that of lysosomal acid phosphatase. Bile flow and bile salt output did not show changes on account of treatments. The biliary excretion of acid phosphatase was stimulated by DHC, and it was not affected by mt inhibitors but was markedly diminished by Cq. DHC choleresis produced different effects on the bile polypeptides. The biliary excretion of polypeptide of high molecular mass (84-140 kDa) was stimulated by DHC. Cq treatment increased their basal biliary excretions, whereas DHC-induced secretion was qualitatively and quantitatively similar to that of controls. The 69-kDa polypeptide (albumin) also increased during DHC-induced choleresis, but it showed a different excretory pattern. Cq treatment inhibited such an increase but no correlation with the excretory pattern of the lysosomal marker was found. The biliary excretion of polypeptides of low molecular mass (down to 14 kDa) suffered a transitory decrease and then a subsequent increase over basal values during the DHC choleresis.(ABSTRACT TRUNCATED AT 250 WORDS)

A simple method for the correction of biliary excretion curves distorted by the biliary dead space.

Biliary solute concentrations measured at the tip of the cannula suffer a delay with respect to bile flow due to the transit time through the biliary tree volume. This study proposes a simple method, which is valid under variable bile flow conditions, to correct the distortion introduced by the biliary tree volume on the kinetic curves of the biliary excretion rate. The biliary transit time (tt) was calculated as the time needed to excrete a bile volume equal to the biliary tree volume by means of the interpolation of biliary cumulative volume versus time curves. Such tt permits one to estimate the canalicular concentration at time t, interpolating the biliary concentration curves at time t-tt. The product between the estimated canalicular concentration and the bile flow allows the calculation of the corrected biliary excretion rate. This method was evaluated by a comparison between biliary excretion rate curves of [14C]taurocholate [( 14C]TC) injected as a bolus under basal and sodium dehydrocholate (DHC)-induced choleresis conditions. Since the canalicular excretion rate of [14C]TC is considered independent of bile flow, the significant differences observed in its excretion kinetics under both conditions were attributed to distortion due to the biliary tree volume. After the correction, both curves showed a significant overlapping. This result indicates that the method improves the time-course representation of canalicular events in biliary excretion kinetic studies.

Bile protein secretion in the rat stimulated by taurocholate: effect of chloroquine.

The biliary protein excretion during sodium taurocholate induced choleresis was studied in normal rats and in rats treated with the lysosomotropic agent, chloroquine. The analysis of the protein component in bile was made on SDS-polyacrilamide gel, and the individual polypeptides were quantitated by densitometry. The excretion of bile polypeptides was compared with that of lysosomal acid phosphatase. The biliary excretion of polypeptides of molecular mass lower than and equal to 54 kDa was markedly stimulated by taurocholate-induced choleresis. Chloroquine treatment of rats diminished the biliary excretion of such polypeptides and also inhibited their excretion induced by taurocholate. The behaviour of these polypeptides was well correlated to that of the lysosomal marker. The biliary excretion of polypeptide bands of a higher molecular mass (up to 140 kDa) did not show major changes during taurocholate-induced choleresis in any of the groups. The results indicate that biliary excretion of proteins in the rat may be either stimulated by taurocholate or may be independent of the bile salt. The former requires the functional integrity of chloroquine-sensitive hepatocyte compartments, which may involve the lysosomes.

Excreción biliar de enzimas en la rata / Biliary excretion of enzymes in rats

En el presente trabajo, se estudió en ratas con fístula biliar, la variación de la excreción biliar de enzimas procedentes de distintas regiones del hepatocito y de proteínas totales así como la relación existente con la excreción de sales biliares. Las enzimas estudiadas incluyeron láctico deshidrogenasa (LDH) como enzima del citosol, fosfatasa ácida (FAc) lisosomal, fosfatasa alcalina (FAlc) de origen canalicular y gama glutamil transpeptidasa (gama-GT), enzima asociada con membrana canalicular y de las células epiteliales de los conductilos biliares. Se utilizaron ratas Wistar machos, adultas. Se cateterizó el calédoco y la vena femoral, mediante catéteres de polietileno y bajo anestesia con pentobarbital sódico. La bilis fue recogida durante tres períodos consecutivos de 2h, efectuándose además la infusión continua i.v. de solución glucosada isotónica, a fin de compensar la pérdida de agua. Durante el transcurso del experimento, se mantuvo la temperatura rectal a 38 + ou - 0,5-C, mediante una lámpara de calentamiento. En todas las muestras de bilis se determinó flujo biliar, concentración de sales biliares y proteínas totales y las actividades de LDH, FAc, FAlc y gama-GT. Para los distintos componentes estudiados, se calcularon las respectivas velocidades de excreción biliar. Se observó una progresiva disminución del flujo biliar de las velocidades de excreción de sales biliares y FAc. La velocidad de excreción biliar de proteínas totales disminuyó inicialmente, para luego estabilizarse. La excreción biliar de FAlc disminuyó en el segundo perídodo de recolección, aunque luego fue seguida de un aumento de su actividad. Contrariamente, la excreción biliar de gama-GT aumentó durante la recolección biliar, mientras que la de LDH, se mantuvo constante durante el experimento. La velocidad de excreción biliar de FAc estuvo positivamente correlacionada con la de sales biliares y proteínas totales. El cálculo de los coeficientes de ...

Excreción biliar de enzimas en la rata / Biliary excretion of enzymes in rats

En el presente trabajo, se estudió en ratas con fístula biliar, la variación de la excreción biliar de enzimas procedentes de distintas regiones del hepatocito y de proteínas totales así como la relación existente con la excreción de sales biliares. Las enzimas estudiadas incluyeron láctico deshidrogenasa (LDH) como enzima del citosol, fosfatasa ácida (FAc) lisosomal, fosfatasa alcalina (FAlc) de origen canalicular y gama glutamil transpeptidasa (gama-GT), enzima asociada con membrana canalicular y de las células epiteliales de los conductilos biliares. Se utilizaron ratas Wistar machos, adultas. Se cateterizó el calédoco y la vena femoral, mediante catéteres de polietileno y bajo anestesia con pentobarbital sódico. La bilis fue recogida durante tres períodos consecutivos de 2h, efectuándose además la infusión continua i.v. de solución glucosada isotónica, a fin de compensar la pérdida de agua. Durante el transcurso del experimento, se mantuvo la temperatura rectal a 38 + ou - 0,5-C, mediante una lámpara de calentamiento. En todas las muestras de bilis se determinó flujo biliar, concentración de sales biliares y proteínas totales y las actividades de LDH, FAc, FAlc y gama-GT. Para los distintos componentes estudiados, se calcularon las respectivas velocidades de excreción biliar. Se observó una progresiva disminución del flujo biliar de las velocidades de excreción de sales biliares y FAc. La velocidad de excreción biliar de proteínas totales disminuyó inicialmente, para luego estabilizarse. La excreción biliar de FAlc disminuyó en el segundo perídodo de recolección, aunque luego fue seguida de un aumento de su actividad. Contrariamente, la excreción biliar de gama-GT aumentó durante la recolección biliar, mientras que la de LDH, se mantuvo constante durante el experimento. La velocidad de excreción biliar de FAc estuvo positivamente correlacionada con la de sales biliares y proteínas totales. El cálculo de los coeficientes de ... (AU)