Excretion Of Horseradish Peroxidase Research Articles

Differential effects of hydroxyacetophenone analogues on the transcytotic vesicular pathway in rat liver

Insertion of transporter proteins into the apical canalicular membrane via vesicular transport is one of several choleretic mechanisms. Based on different choleretic activities of hydroxyacetophenone analogues including 4-mono; 2,6-di and 2,4,6-trihydroxy-acetophenone (MHA, DHA and THA), the present study aims to determine if these compounds stimulated vesicular transport in hepatocytes. Hydroxyacetophenone was continuously infused into the duodenum of the bile fistula rat. Bile flow rate was allowed to stabilize and then followed by an intraportal injection of horseradish peroxidase, a marker of the transcytotic vesicle pathway. MHA which stimulates bile acid independent flow, showed a dose-dependent increase in both the early (paracellular) and late (transcellular) peak of horseradish peroxidase excretion in bile. THA, which stimulates both bile acid dependent flow and bile acid independent flow, did not alter the pattern of horseradish peroxidase excretion into bile. However, DHA, which is more hydrophobic and increases only bile acid dependent flow, decreased the late peak. The stimulating effects of MHA on bile flow and horseradish peroxidase excretion were markedly inhibited by colchicine, suggesting that its choleretic action involves stimulation of exocytosis, as well as increase in paracellular permeability. In contrast, the lack of a stimulatory effect of THA and DHA on biliary horseradish peroxidase excretion suggested that their choleretic action is not associated with vesicular exocytosis. These results demonstrate a variable effect of hydroxyacetophenones on the transcytotic vesicular pathway reflecting different choleretic mechanisms and therapeutic potential.

Biliary inorganic phosphate as a tool for assessing cold preservation-reperfusion injury: a study in the isolated perfused rat liver model.

Ischemia-reperfusion injury is a major cause of early graft dysfunction after liver transplantation. The bile flow has been suggested as an index of ischemic damage, and severely impaired bile flow seems to be predictive of poor survival in experimental studies. Looking for injury markers, biliary inorganic phosphate has the potential of being a useful endogenous marker of diminished hepatobiliary function because this anion is excreted in the bile by a paracellular pathway and it can detect changes in permeability. The goal of this study was to evaluate the effects of cold preservation-reperfusion of the liver on bile flow and bile inorganic phosphate and their relationship with storage-related graft failure. The isolated and perfused rat liver was used to evaluate the injury for ischemia-reperfusion. The intrahepatic resistance, lactate dehydrogenase release, and potassium and biliary inorganic phosphate concentration were used to estimate viability and function of freshly isolated or cold-preserved livers. The intrahepatic resistance and the bile flow were consistent and significantly decreased throughout the perfusion time in relation to the increment in storage. Inorganic phosphate is more concentrated in bile from preserved livers, showing an alteration in paracellular pathway, confirmed by the biliary excretion of horseradish peroxidase. After preservation, concentration and excretion of the paracellular marker were increased during the first peak. The second peak appears earlier in preserved livers (10 minutes) with a different shape but without changes in concentration. In conclusion, inorganic phosphate in bile shows changes in paracellular permeability as occurs in livers after 48 hours of cold preservation.

Hepatocyte tight-junctional permeability is increased in rat experimental colitis

BACKGROUND & AIMS: Hepatobiliary complications occur in inflammatory bowel disease and may be caused by the translocation of intestinal toxins from portal blood into bile through leaky hepatocyte tight junctions. The role of tight junctions in the pathogenesis of hepatobiliary complications in experimental inflammatory bowel disease was investigated.METHODS: Colitis was induced in rats by intracolonic instillation of trinitrobenzene sulfonic acid. The function of hepatocellular tight junctions was evaluated in perfused livers by measuring early (paracellular) horseradish peroxidase excretion into the bile and by electron microscopy and semiquantitative analysis of lanthanum penetration through the tight junction and into bile canaliculi. Immunofluorescent localization of cingulin and ZO-1 was used to study the structure of hepatocyte junctions.RESULTS: Colitis was associated with increased serum bilirubin and bile acid concentrations, a 2.5-fold increase in paracellular biliary excretion of horseradish peroxidase, and a ninefold increase in lanthanum permeability. Liver histology and cingulin and ZO-1 localizations were similar to normal liver.CONCLUSIONS: Experimental colitis is associated with hepatobiliary complications and an increased hepatocyte tight junctional permeability to horseradish peroxidase and lanthanum. Subtle alterations in tight junction function may be involved in the pathogenesis of hepatobiliary injuries in inflammatory bowel disease.(Gastroenterology 1997 Oct;113(4):1347-54)

Adaptive regulation of hepatic bile salt transport: role of bile salt hydrophobicity and microtubule-dependent vesicular pathway

Background/Aims: The hepatic transport of bile salts can be regulated by changes in bile salt pool size and/or in the flux of bile salts through the liver. Prolonged bile salt pool depletion is associated with down-regulation of maximum taurocholate transport and decreased canalicular membrane specific bile salt binding sites. This study was undertaken to investigate; a) whether adaptive down-regulation of maximum hepatic bile salt transport occurs to the same extent for bile acids of different hydrophobicity; and b) the role of microtubule-dependent vesicular pathway in the adaptive changes of bile salt transport capacity. Methods: Male rats were subjected to 24-h or 48-h external biliary diversion to induce bile salt pool depletion. Basal bile flow, bile salt secretion and lipid secretion, maximum secretory rate of three bile salts of different hydrophobicity (tauroursodeoxycholate, taurocholate and taurochenodeoxycholate) and changes in the biliary excretion of two markers of the microtubule-dependent vesicular pathway (horseradish peroxidase and polyethyleneglycol molecular weight-900) were measured in control and bile salt-depleted rats. Taurocholate-stimulated horseradish peroxidase biliary excretion was also assessed in order to define whether the restoration of bile salt flux across the hepatocytes increased the excretion of this marker in bile salt-depleted rats. Results: The reduction in the maximum secretory rate of the three bile salts under study observed after prolonged biliary diversion was clearly related to their hydrophobicity, with greater reduction for taurochenodeoxycholate and smaller reduction for tauroursodeoxycholate, compared with taurocholate. The biliary excretion of vesicular transport markers was significantly reduced in bile salt-depleted rats. However, when stimulated by taurocholate, biliary excretion of horseradish peroxidase was similar to controls. Conclusions: The magnitude of the decrease of the hepatic bile salt maximum transport capacity seen after bile salt pool depletion is directly related to the hydrophobicity of the bile salt infused. A functionally depressed vesicular transport pathway appears to be also a contributing factor to this phenomenon.

Vasopressin reduces taurochenodeoxycholate-induced hepatotoxicity by lowering the hepatocyte taurochenodeoxycholate content

Background/Aims: Vasopressin has been reported to reduce bile flow, but its effects on bile acid secretion and bile acid-related hepatotoxicity are still unclear. We therefore investigated the influence of vasopressin on the hepatotoxicity and biliary excretion of taurochenodeoxycholic acid in primary cultured rat hepatocytes and isolated perfused rat liver models. Methods/Results: 1) Addition of vasopressin to hepatocyte culturers significantly decreased lactate dehydrogenase release as compared to cultures exposed to 1 mM taurochenodeoxycholic acid alone, and also reduced intracellular taurochenodeoxycholic acid content from 19.3±2.2 to 13.0±1.6 nmol/mg protein. After 30 min of preincubation with 1 mM taurochenodeoxycholic acid, rinsing and reculture of hepatocytes in bile acid-free medium resulted in gradual decrease in the intracellular level of the bile acid, and addition of vasopressin (10 −9 M) to the reculture medium accelerated this process. 2) Superimposition of vasopressin (330 pmol/l) for 10 min on taurochenodeoxycholic acid infusion (1.0 μmol/min: 25 μmol/l) caused a rapid increase in bile flow and biliary excretion of taurochenodeoxycholic acid (697±42 vs 584±27 nmol/10 min per g liver) from perfused rat livers, and significantly reduced lactate dehydrogenase release. 3) Superimposition of the PKC blocker H-7 (5 μmol/l) on taurochenodeoxycholic acid infusion (1.0 μmol/min: 25 μmol/l) caused a gradual increase in bile flow and biliary excretion of taurochenodeoxycholic acid. Furthermore, an additional infusion of vasopressin (100 pmol/l) for 10 min in the presence of H-7 produced a greater increase in bile flow and biliary excretion of taurochenodeoxycholic acid as compared with H-7 alone (754±71 vs 657±26 nmol/g liver). 4) Continuous infusion of vasopressin (330 pmol/l) significantly increased the late peak (10–50 min) of horseradish peroxidase excretion from perfused livers (from 8.48±1.02 to 21.7±6.02 ng/g liver). Conclusions: These findings suggest that vasopressin exerts a protective effect against taurochenodeoxycholic acid-induced hepatotoxicity by stimulating the secretion of this bile acid via intracellular vesicular transport systems.

Effect of Cytosolic Free Ca2+ on Vesicle Exocytosis in Cholestatic Cultured Hepatocytes from Bile Duct-Ligated Rats

To elucidate the effects of cytosolic free Ca2+ ( [Ca2+]i) on vesicle exocytosis in cholestatic hepatocytes, we established optimal culture conditions for experimental cholestatic hepatocytes. We also investigated the relation between vesicle exocytosis and [Ca2+] i in cholestatic hepatocytes. After the bile duct was ligated for 1 to 7 days, rats exhibited typical features of clinical and pathologic cholestasis. One day after ligation significantly greater amounts of enzymes and lipids were leaked from cultured cholestatic hepatocytes than from normal hepatocytes. These phenomena resembled those in clinical cholestasis. Uptake of horseradish peroxidase (HRP), a marker of vesicle transport, by cholestatic hepatocytes did not differ from that by normal hepatocytes, however, HRP excretion by cholestatic hepatocytes was significantly less than that by norrr.al hepatoctyes. The HRP excretion in the absence of extracellular Ca2+ was significantly less than that in the presence of Ca2+ in both normal and cholestatic hepatocytes. Treatment with nicardipine decreased HRP excretion in normal hepatocytes, but did not affect HRP excretion in cholestatic hepatocytes. Moreover, high K+-induced increases in [Ca2+] i in the presence of extracellular Ca2+ was observed in normal hepatocytes but not in cholestatic hepatocytes. These results indicate that (1) the hepatocytes isolated 1 day after ligation are a good model cholestasis, (2) cholestatic hepatocytes have impaired [Ca2+] i-dependent vesicle exocytosis, and (3) this impairment of vesicle exocytosis in cholestatic hepatocytes might be related to damage or to smaller numbers of Ca2+ channels.

Effect of Brefeldin A on transcytotic vesicular pathway and bile secretion: A study on the isolated perfused rat liver and isolated rat hepatocyte couplets

This study investigated the effect of Brefeldin A (BFA) on the transcytotic vesicular pathway labeled with horseradish peroxidase (HRP) in both isolated rat hepatocyte couplets (IRHC) and the isolated perfused rat liver (IPRL). To evaluate the role of the transcytotic vesicular pathway on bile secretion, the effect of BFA on bile secretion in the IPRL was then investigated. In the basolateral area of IRHC, BFA showed no effect on the density and percentage of area of HRP-labeled vesicles. However, HRP-labeled vesicles tended to accumulate in the juxtanuclear area of BFA-treated hepatocytes (P < .001 vs. controls). In the pericanalicular area, on the other hand, HRP-labeled vesicles were depleted compared with controls (P < .001). In keeping with these findings, although the early peak remained unchanged, BFA inhibited as much as 50% of the late peak of HRP excretion in bile, after a pulse load of HRP in the IPRL. Bile flow and the biliary secretion of bile salts (BS) and phospholipids were not modified by BFA in isolated livers perfused without BS in the perfusate or with 1 mumol/min taurocholate (TCA). In BFA-treated livers, peak bile flow and BS output decreased by 20% (P < .05 vs. controls) only when a 5 mumol TCA bolus was administered. In conclusion, this study demonstrates that BFA inhibits the transcytotic vesicular pathway in the liver. However, BFA has no significant effect on bile secretion either in basal conditions or during perfusion with physiological amounts of BS. BFA slightly decreases bile flow and BS output only after an overload of BS, providing evidence against the physiological relevance of the transcytotic vesicular pathway in the process of bile formation.

Effect of brefeldin a on transcytotic vesicular pathway and bile secretion: A study on the isolated perfused rat liver and isolated rat hepatocyte couplets

This study investigated the effect of Brefeldin A (BFA) on the transcytotic vesicular pathway labeled with horseradish peroxidase (HRP) in both isolated rat hepatocyte couplets (IRHC) and the isolated perfused rat liver (IPRL). To evaluate the role of the transcytotic vesicular pathway on bile secretion, the effect of BFA on bile secretion in the IPRL was then investigated. In the basolateral area of IRHC, BFA showed no effect on the density and percentage of area of HRP-labeled vesicles. However, HRP-labeled vesicles tended to accumulate in the juxtanuclear area of BFA-treated hepatocytes ( P < .001 vs. controls). In the pericanalicular area, on the other hand, HRP-labeled vesicles were depleted compared with controls ( P < .001). In keeping with these findings, although the early peak remained unchanged, BFA inhibited as much as 50% of the late peak of HRP excretion in bile, after a pulse load of HRP in the IPRL. Bile flow and the biliary secretion of bile salts (BS) and phospholipids were not modified by BFA in isolated livers perfused without BS in the perfusate or with 1 μmol min taurocholate (TCA). In BFA-treated livers, peak bile flow and BS output decreased by 20% ( P < .05 vs. controls) only when a 5 μmol TCA bolus was administered. In conclusion, this study demonstrates that BFA inhibits the transcytotic vesicular pathway in the liver. However, BFA has no significant effect on bile secretion either in basal conditions or during perfusion with physiological amounts of BS. BFA slightly decreases bile flow and BS output only after an overload of BS, providing evidence against the physiological relevance of the transcystotic vesicular pathway in the process of bile formation.459.)

Effects of protein kinase C and cytosolic Ca2+ on exocytosis in the isolated perfused rat liver

Both protein kinase C and cytosolic Ca2+ are involved in the regulation of exocytosis in a number of cell types. However, the relative importance of each of these for apical exocytosis in the hepatocyte is unknown. To investigate this, we studied the effects of protein kinase C and Ca2+ agonists on horseradish peroxidase excretion in the isolated perfused rat liver. Vasopressin increased both horseradish peroxidase concentration and net horseradish peroxidase excretion in bile, and these effects were abolished by the protein kinase C inhibitor H-7. The protein kinase C activator phorbol dibutyrate also increased both net excretion and the concentration of biliary horseradish peroxidase. In contrast, the Ca2+ ionophore A23187 and the Ca2+ mobilizing agent 2,5'-di(tertbutyl)-1,4-benzohydroquinone both had minimal effects on horseradish peroxidase concentration and inhibited the rate of horseradish peroxidase excretion. These results suggest that protein kinase C stimulates apical exocytosis in the hepatocyte, whereas increased Cai2+ per se does not influence exocytosis and inhibits excretion only transiently by reducing bile flow.

The mutant Eisai hyperbilirubinemic rat is resistant to bile acid-induced cholestasis and cytotoxicity.

We investigated bile flow and biliary excretion of bile acids in the Eisai hyperbilirubinemic rat, a Sprague-Dawley mutant rat with conjugated hyperbilirubinemia, using both in vivo and in vitro models. In vivo bile flow was lower in Eisai hyperbilirubinemic rats than in the control rats before and after taurocholate was infused. After taurocholate was infused, bile acid output was similar in the Eisai hyperbilirubinemic rats and control rats. In the isolated perfused rat liver, biliary excretion of bile acids was higher in the Eisai hyperbilirubinemic rats than in the control rats after a high-dose infusion of taurocholate (0.33 mumol/min/gm liver). Infusion of taurochenodeoxycholate (0.22 mumol/min/gm liver) did not produce cholestasis and did not reduce the biliary excretion of bile acids in the Eisai hyperbilirubinemic rats. Taurochenodeoxycholate significantly increased the phospholipid/bile acid molar ratio and slightly reduced bile acid-induced alkaline phosphatase output into bile. The release of lactate dehydrogenase from the perfused liver 30 min after the start of the taurochenodeoxycholate infusion was 10 times lower in the Eisai hyperbilirubinemic rats than in the control rats (2.0 +/- 0.8 vs. 28.7 +/- 6.8 mU/min/gm liver). When the isolated perfused rat liver was infused with a 1-min pulse of horseradish peroxidase (25 mg), we observed an early and late peak of biliary excretion of horseradish peroxidase. The Eisai hyperbilirubinemic rats showed a significant increase in the late peak.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatobiliary function in senescent male Sprague-Dawley rats

The purpose of these studies was to investigate intrahepatic changes underlying age-related decreases in bile flow by evaluating the effects of aging on bile acid-dependent and -independent flow, canalicular versus ductular flow and hepatic tight junction permeability. The isolated perfused liver was used to assess age-related changes in intrinsic hepatobiliary function without the complications of extrahepatic factors such as circulating hormones or hemodynamics. Livers from young adults (3 to 6 mo old) or senescent (22 to 26 mo old) male Sprague-Dawley rats were isolated and perfused in a nonrecirculating, hemoglobin-free system to assess oxygen uptake, bile acid-dependent and -independent bile flow, bile acid uptake, carbon 14-labeled erythritol clearance as a measure of canalicular flow, tight junction permeability and transcellular transport into bile. Rates of oxygen uptake by livers from senescent rats were significantly lower than those of young adults (75 ± 8 μmol/gm/hr vs. 121 ± 5 μmol/gm/hr). Age-related decreases in total bile flow were observed and were associated with similar reductions in 14C-erythritol clearance suggestive of decreased canalicular bile flow. Bile acid-dependent and -independent flow was decreased by 50% and 60%, respectively, in isolated perfused livers from senescent rats. Hepatocellular uptake of taurocholate and rates of bile acid excretion also were about 50% lower in senescent than in young adult rats. Tight junction permeability and transcellular transport were assessed by monitoring appearance of tritiated inulin and horseradish peroxidase in bile after bolus injections of these compounds through the portal vein. Tritiated inulin appearance in bile was decreased slightly in senescent compared with young rats. No age-related differences in tight junction permeability to horseradish peroxidase were observed. In addition, biliary excretion of horseradish peroxidase in livers from senescent rats was not affected by colchicine, suggesting that microtubule-related transport was minimal or absent in the senescent rats. Taken together, these data indicate that decreased rates of bile flow in senescent compared with young rats are caused by age-related effects on intrinsic hepatobiliary function, specifically decreases in canalicular bile flow associated with decreased bile acid-dependent and -independent bile flow. The decreased canalicular bile flow appears to be a result in part of impairment of energy-dependent transport processes and microtubule-dependent transcellular transport across cells. These findings suggest that changes in intrinsic hepatobiliary function occur in aged populations and are not caused exclusively by extrahepatic effects such as altered blood flow or changes in hormonal status. Further, age-related decreases in bile flow may be caused at least in part by decreased active transport of osmotically active solutes into bile.

Endothelin-1 stimulates bile acid secretion and vesicular transport in the isolated perfused rat liver.

The effects of endothelin (ET) on portal pressure and bile secretion were examined using isolated perfused rat liver and rat hepatocyte preparations. ET-1 raised portal pressure dose dependently; administration at a high dose (10(-9) mol) induced a > 200% increase along with reduced bile flow and decreased secretion of bile acid and phospholipids. However, a low dose (10(-10) mol) of ET-1 brought about a < 100% portal pressure rise, enhanced both bile flow and excretion of bile acid and phospholipids, and significantly increased transfer of preadministered horseradish peroxidase (HRP) into bile. In addition, values for Ca2+ concentrations, examined by indo 1 fluorescence, were elevated in isolated hepatocytes after administration of ET-1. Papaverine suppressed the low-dose ET-1 stimulation effects on both portal pressure and bile secretion. Moreover, it also reduced the HRP excretion and suppressed intracellular Ca2+ release. This study demonstrated that ET-1 stimulates vesicular transport, probably via promotion of intracellular Ca2+ release, and, as a result, increases bile acid-dependent bile flow.

Tauroursodeoxycholic acid stimulates hepatocellular exocytosis and mobilizes extracellular Ca++ mechanisms defective in cholestasis.

To assess the effects of tauroursodeoxycholic acid (TUDCA) on bile excretory function, we examined whether TUDCA modulates vesicular exocytosis in the isolated perfused liver of normal rats in the presence of high (1.9 mM) or low (0.19 mM) extracellular Ca++ and in cholestatic rats 24 h after bile duct ligation. In addition, the effects of TUDCA on Ca++ homeostasis were compared in normal and in cholestatic hepatocytes. In the isolated perfused rat liver, TUDCA (25 microM) stimulated a sustained increase in the biliary excretion of horseradish peroxidase, a marker of the vesicular pathway, in the presence of high, but not low extracellular Ca++ or in the cholestatic liver. In contrast, TUDCA stimulated bile flow to the same extent regardless of the concentration of extracellular Ca++ or the presence of cholestasis. In indo-1-loaded hepatocytes, basal cytosolic free Ca++ ([Ca++]i) levels were not different between normal and cholestatic cells. However, in cholestatic cells [Ca++]i increases induced by TUDCA (10 microM) and its 7 alpha-OH epimer taurochenodeoxycholic acid (50 microM) were reduced to 22% and 26%, respectively, compared to normal cells. The impairment of TUDCA-induced [Ca++]i increase in cholestatic cells could be mimicked by exposing normal cells to low extracellular Ca++ (21%) or to the Ca++ channel blocker NiCl2 (23%). These data indicate that (a) dihydroxy bile acid-induced Ca++ entry may be of functional importance in the regulation of hepatocellular vesicular exocytosis, and (b) this Ca++ entry mechanism across the plasma membrane is impaired in cholestatic hepatocytes. We speculate that the beneficial effect of ursodeoxycholic acid in cholestatic liver diseases may be related to the Ca+(+)-dependent stimulation of vesicular exocytosis by its conjugate.

Intracellular alkalinization stimulates bile flow and vesicular-mediated exocytosis in IPRL.

Intracellular pH recovery from an acute alkaline load in rat hepatocytes is mediated by a Cl(-)-HCO3- exchanger, which is electroneutral, Na+ independent, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) sensitive. Stimulation of this Cl(-)-HCO3- exchanger requires intact microtubules, suggesting that vesicular transport may be required to activate this exchanger. To determine if intracellular alkalinization stimulates biliary HCO3- excretion and bile flow in the intact liver by vesicle-mediated exocytosis, isolated perfused rat livers (IPRL) were alkalinized by two protocols. Isohydric changes in CO2 and HCO3- concentrations induced transient increases in bile flow by 36% (P < 0.01), which were abolished by DIDS (0.01 mM), inhibited by pretreatment with colchicine (P = 0.01), but not affected by membrane depolarization with the K(+)-channel blocker BaCl2 (1 mM). Similarly, perfusion with 20 mM NH4Cl produced a 42% increase in bile flow (P < 0.01) and a 26% increase in biliary HCO3- excretion. Both the increases in bile flow and HCO3- excretion were almost completely blocked by DIDS and inhibited by pretreatment with colchicine (P < 0.01). Biliary excretion of horseradish peroxidase was also increased during intracellular alkalinization with either protocol (P < 0.01). These findings suggest that intracellular alkalinization stimulates bile flow and biliary HCO3- excretion. Microtubule-dependent vesicular-mediated exocytosis is involved in this response.

Hepatic accumulation of lysosomes and defective transcytotic vesicular pathways in cirrhotic rat liver.

To investigate the potential role of lysosomes in cirrhosis, we analyzed the activity of lysosomal enzymes in rats exposed long-term to phenobarbital and carbon tetrachloride. The activity of lysosomal enzymes was markedly increased in the homogenate of cirrhotic livers (e.g., arylsulfatase 9 +/- S.D.2 vs. 16 +/- 6 nmoles.min-1.mg-1 in control rats and cirrhotic rats, respectively; p less than 0.001). The corresponding plasma levels were also increased (7 +/- 1 vs. 12 +/- 3 nmoles.min-1.mg-1; p less than 0.01), whereas biliary excretion was diminished (16 +/- 7 vs. 7 +/- 2 pmol.min-1.gm liver-1; p less than 0.05) in cirrhotic rats. Stereological quantification of lysosomes visualized cytochemically revealed an increase of pericanalicular lysosomes averaging 1.5 +/- 0.4 around a canaliculus in controls and 3.7 +/- 1.0 in cirrhotic rats (p less than 0.01). Because this suggested a defect in the transcellular vesicular pathway, we investigated the biliary excretion of horseradish peroxidase and epidermal growth factor in perfused livers. Bile flow and total horseradish peroxidase excretion were similar in control rats and cirrhotic rats. However, the early peak of biliary horseradish peroxidase excretion--usually taken as evidence of paracellular transport--was increased in cirrhotic rats (13 +/- 7 vs. 57 +/- 22%; p less than 0.01), whereas the second peak--reflecting the transcellular vesicular pathway(s)--was markedly reduced (87 +/- 7 vs. 43 +/- 22%; p less than 0.001). A similar reduction in the biliary excretion of intact epidermal growth factor and of its degradation products was found. These results demonstrate an increased number of lysosomes in hepatocytes of cirrhotic livers; this appears to be the result of accumulation rather than proliferation, in view of the reduced transcellular vesicular movement of different markers into bile.

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

The effects of the lysosomotropic agents chloroquine and leupeptin on the taurocholatestimulated 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 μmol/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.

Regulatory volume decrease stimulates bile flow, bile acid excretion, and exocytosis in isolated perfused rat liver.

To study the effect of volume regulation on bile secretory function, isolated perfused rat livers (IPRL) were exposed to hypotonic stress (45 mM NaCl) while bile flow and the biliary excretion of bile acids and horseradish peroxidase (HRP) were assessed. Hypotonic stress induced a biphasic increase in bile flow, which rose in the first minute from 1.1 +/- 0.2 to 1.7 +/- 0.1 microliter.min-1.g liver-1 (P less than 0.01), an effect attributed to rapid osmotic equilibration of water, then increased further between 3 and 5 min to 1.6 +/- 0.1 microliter.min-1.g liver-1 (P less than 0.01, followed by a subsequent return to baseline. HRP excretion in bile increased during the second peak of bile flow from 0.9 +/- 0.2 to 1.1 +/- 0.2 ng.min-1.g liver-1, P less than 0.01. Pretreatment with colchicine but not lumicolchicine completely abolished the latter increase in bile flow and HRP excretion as did BaCl2 (1 mM), an inhibitor of both K+ channels and regulatory volume decrease (RVD) in hepatocytes. When sodium taurocholate was infused (1 mumol/min), hypotonic stress induced an even larger increase in the second peak of bile flow (5.1 +/- 0.7 microliters/g liver, P less than 0.01) and higher rates of bile acid excretion than in control perfusions with bile acid (126.2 +/- 21.0 vs. 99.0 +/- 17.1 nmol.min-1.g liver-1, P less than 0.05). These data suggest that both bile flow and bile acid excretion are stimulated during RVD by mechanisms that involve both K+ channels and microtubule-dependent exocytosis at the canalicular (apical) membrane domain.

DBcAMP stimulates vesicle transport and HRP excretion in isolated perfused rat liver

To clarify the effect of adenosine 3',5'-cyclic monophosphate (cAMP) on the transcytotic vesicle pathway, we measured the biliary excretion of bile acid, phospholipid, and horseradish peroxidase (HRP) in the isolated perfused rat liver (IPRL) with or without infusion of N6,2'-O-dibutyryl-cAMP (DBcAMP). A linear relationship between bile flow and bile acid excretion was observed in both control and DBcAMP-infused livers. DBcAMP increased the y-axis intercept from 1.10 +/- 0.16 to 1.48 +/- 0.19 microliters.min-1.g liver-1 (P less than 0.01) and the slope from 6.5 +/- 1.99 to 10.77 +/- 1.71 microliters/mumol bile acid (P less than 0.01). DBcAMP also increased the biliary excretion of bile acid and phospholipid during a 1.0 mumol/min infusion of taurocholate. When HRP was pulse loaded for 1 min, HRP appeared in bile in early (4-6 min) and late (20-25 min) peaks. DBcAMP markedly increased the late peak of HRP from 0.33 +/- 0.08 to 1.15 +/- 0.32 ng.min-1.g liver-1 (P less than 0.01), a phenomenon blocked by colchicine. An electron-microscopic morphometric analysis indicated that DBcAMP increased both the density and %area of HRP-containing vesicles in the pericanalicular area, compared with controls, 18 min after a 1-min pulse of HRP. DBcAMP had no effect on the uptake rate of HRP in 4-h primary hepatocyte cultures but stimulated biliary excretion of HRP when preloaded in the IPRL. These findings indicate that cAMP regulates excretory function in part by stimulating the microtubule-dependent transcytotic vesicle transport system.

Taurocholate stimulates transcytotic vesicular pathways labeled by horseradish peroxidase in the isolated perfused rat liver

The effect of taurocholate on transcytotic vesicular pathways labeled with horseradish peroxidase was assessed in isolated perfused rat liver preparations. Forty-five minutes after a horseradish peroxidase load in a recirculating system, continuous infusion of taurocholate but not taurodehydrocholate significantly increased horseradish peroxidase excretion in bile by 50% compared with controls. When horseradish peroxidase (25 mg) was pulse loaded for 1 minute in control perfusions, it appeared in bile in early (4–6 minutes) and late (20–25 minutes) peaks, the latter accounting for 90% of total horseradish peroxidase output. Taurocholate infusion significantly increased horseradish peroxidase output in both early and late peaks, whereas only a small increase in the early peak was observed with taurodehydrocholate. Colchicine pretreatment increased the early peak in bile but abolished the second peak. Electron micrographs from control livers revealed the accumulation of horseradish peroxidase-containing vesicles in pericanalicular regions at early (2 minutes) as well as late (18 minutes) periods. When a morphometric analysis of electron micrographs was performed from pericanalicular regions 2 minutes after a 1-minute pulse of horseradish peroxidase (500 mg), taurocholate but not taurodehydrocholate increased both the density and percent area of horseradish peroxidase-containing vesicles compared with controls. In contrast, colchicine pretreatment had no effect on the density of the early-appearing vesicles, although their individual sizes were reduced. Taurocholate but not taurodehydrocholate also increased the percent of tubular structures in the pericanalicular region. These findings indicate that taurocholate stimulates both early and late transcytotic vesicle pathways and that a rapid transcellular-colchicine-insensitive and therefore probably microtubule-independent vesicle pathway is present in hepatocytes that must be distinguished from paracellular routes.

The fate of electron opaque tracers (horseradish peroxidase and lanthanum chloride) during valproic acid-induced choleresis.

Horseradish peroxidase (HRP) and lanthanum chloride (LaCl3) are useful tools for, respectively, the study of vesicular transport through the hepatocyte and the study of the permeability of junctional complexes. These tracers have been used to detect the changes associated with the choleresis independent of bile acids induced by valproic acid (VPA) in rats. The animals were given a single dose of VPA (600 mg/kg, ip). HRP (100 mg/kg) or 5 mM LaCl3 were given intraportally after 1 h, when bile flow had increased twofold. The excretion of HRP in bile was measured colorimetrically up to 2 h after HRP. Ultrastructural morphometry was conducted on liver of intact rats taken from 1 to 40 min after HRP. The volume density (VD) of HRP-containing vesicles and of HRP-containing multivesicular bodies (MVB) was counted. In VPA-treated rats, HRP appeared in bile with a peak showing at 5 min against 20 min in controls, but the total amount of HRP excreted was less than in controls. The intrahepatocytic vesicular transport of HRP was also modified, showing a peak at 3 min in VPA-treated rats compared to 10 min in controls, together with a decreased VD of pericanalicular vesicles. This was accompanied by an increase of HRP-containing MVB, already evident at 5 min.(ABSTRACT TRUNCATED AT 250 WORDS)