Prevalence of hepatitis A, B, C and human immunodeficiency virus seropositivity among patients with acute icteric hepatitis at the Kenyatta National Hospital, Nairobi.

OBJECTIVE: To determine the prevalence of hepatitis A, B, C and HIV seropositivity among patients with acute icteric hepatitis. DESIGN: Cross-sectional descriptive survey. SETTING: Kenyatta National Hospital, Nairobi. SUBJECTS: Eighty four patients aged above six months with a history of jaundice not exceeding six months were recruited. There were 47 males and 17 females with an age range of eight months to 67 years and a median age of 25 years. METHODS: History was obtained physical examination done and blood taken for determination of bilirubin, ALT, AST and ALP levels. Sera that had disproportionately greater transaminase than ALP elevation were assayed for IgM anti-HAV, IgM anti-HBc, HbsAg, anti-HCV and anti-HIV antibodies. RESULTS: Evidence of hepatitis A, B, and C was round in 41.7%, 26.2%, and 7.1% of the patients respectively, 13.1% of the patients were HBsAg carriers while 30.1% of all patients were HIV positive. Thirty two patients did not have evidence of hepatitis A, B, or C infection and this group was significantly associated with HIV infection (p = 0.003). CONCLUSION: Hepatitis A was the commonest overall type of acute icteric hepatitis seen at the KNH, and among patients aged 15 years and below. Hepatitis B was the leading identified cause of acute hepatitis among those aged over 15 years. Hepatitis C accounted for 7.1% of acute icteric hepatitis 30.1% of all patients and 50% of those admitted with acute hepatitis were also HIV positive.

Screening for chronic hepatitis B and C virus infections in an urban sexually transmitted disease clinic: rationale for integrating services.

BACKGROUND AND OBJECTIVES: Clients attending sexually transmitted disease (STD) clinics are at risk for multiple infections (e.g., STDs, HIV, and infectious viral hepatitis). Risk assessment and serosurveys can document the need for hepatitis screening and vaccination services. GOAL: To determine hepatitis C and B virus seroprevalence, identify predictive risk factors, and provide a rationale for integrating hepatitis services in an STD clinic. METHODS: During various periods in 1998, consecutive clients completed a self-administered risk assessment and were offered screening for markers of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection (HBV core antibody and anti-HCV [enzyme-linked immunosorbent assay 3.0, confirmed by recombinant immunoblot assay 2.0]). RESULTS: Sixteen percent of 300 clients tested for an anti-HBV core were positive, with injecting-drug users (IDUs) and men who have sex with men (MSM) having higher prevalences (50% and 37%, respectively). Of 615 clients tested for anti-HCV, 21 (3.4%) were positive. Injecting-drug users (n = 34) had a 38% anti-HCV prevalence compared with 1.1% for non-IDUs. Of 66 non-IDU MSM tested, none was HCV infected. IDUs had a high prevalence of past STDs (> 50%) and unsafe sexual behavior. CONCLUSIONS: Injecting drug users and MSM are at high risk for STDs, HIV, and hepatitis infections and could benefit from a "one-stop" STD clinic that included hepatitis prevention services.

Myosin I is associated with zymogen granule membranes in the rat pancreatic acinar cell.

BACKGROUND & AIMS: The mechanisms whereby intracellular messengers mediate zymogen granule transport and exocytosis in the pancreatic acinar cell are not well defined. Electron microscopy has shown a periluminal network of actin in the acinar cell, suggesting a role for actin and myosin in the transport process. The possible involvement of two types of myosin in the secretory process was investigated, and their distribution in acinar cells was determined. METHODS: Antibodies specific to myosin I or to myosin II were used for immunocytochemistry and Western blot analysis. Ultrastructural studies were also performed. RESULTS: Western blot analysis showed that myosin I and myosin II were present in total pancreatic homogenate but that only myosin I was present on isolated zymogen granules and their membranes. By immunocytochemistry, myosin I was shown in the apical aspect of acinar cells colocalized with glycoprotein 2, a marker for zymogen granules, and actin. By immunocytochemistry, myosin I was also localized on isolated zymogen granules. CONCLUSIONS: The immunolocalization of myosin I to zymogen granule membranes and its close association with periluminal actin suggest that myosin I plays a direct role in the process of transport and exocytosis of zymogen granules in the pancreatic acinar cell.

I've been where it's gone, so I know what I got ... An American Fulbright Lecturer in Albania, 1994-1995.

Spending 10 months as a Fulbright Lecturer in Medical Sciences at the University Medical Center, University of Tirana, Albania, gave me a first-hand view of academic medicine in a country merging from more than 45 years of Communist rule that impoverished the country and isolated it from the rest of the world. Even after the fall of Communism, every aspect of medicine in Albania continues to be government controlled. Early specialization is still the rule for academic physicians. Division chiefs exert absolute authority over their domain and seldom delegate this authority. Learning and teaching resources are scant, and access to current western medical literature is extremely limited because of both poverty and priority. Despite these obstacles, the medical students and postgraduate trainees I encountered were bright and receptive, which strongly reinforces the tremendous urge to help them. Fellowships abroad, however, are limited and available only to selected junior faculty; students and clinical trainees do not qualify. If we are to help, we must take the training to them. It takes time to become an effective clinical teacher in Albania: time to understand the system; time to devise the best teaching vehicles; and time to gain the trust of the students, trainees, and faculty. Given the time, the effort can be successful. The problem is, where do we find physicians with the time and interest? Might this be a role for still-energetic retired physicians? My experience in Albania permitted me only to formulate these questions; the answers must now come from this side of the Atlantic.

Prospective randomized trial of emergency portacaval shunt and emergency medical therapy in unselected cirrhotic patients with bleeding varices.

A prospective randomized trial was conducted in unselected, consecutive patients with bleeding esophageal varices resulting from cirrhosis comparing (1) emergency portacaval shunt performed within 8 hr of initial contact (21 patients) with (2) emergency medical therapy (intravenous vasopressin and esophageal balloon tamponade) followed in 9 to 30 days by elective portacaval shunt in survivors (22 patients). All patients underwent the same diagnostic workup within 3 to 6 hr of initial contact, and received identical supportive therapy initially. All patients were followed up for at least 10 yr. The protocol contained no escape or cross-over provisions. There were no statistically significant differences between the two treatment groups in the incidence of any of the clinical variables, results of laboratory tests or degree of portal hypertension. Child's risk classes in the shunt group were A-2 patients, B-8 patients and C-11 patients, whereas in the medical group they were A-10 patients, B-5 patients, and C-7 patients, a significant difference (p < 0.01) that might have favored emergency medical treatment. Bleeding was controlled initially and permanently by emergency shunt in every patient, but by medical therapy in only 45% (p < 0.001). Mean requirement for blood transfusion was 7.1 +/- 2.6 units in the shunt group and 21.4 +/- 2.6 units in the medical group (p < 0.001). Eighty-one percent of the patients in the shunt group were discharged alive compared with 45% in the medical group (p = 0.027). Five- and 10-yr observed survival rates were 67% and 57%, respectively, after emergency shunt compared with 18% and 18%, respectively, after the combination of emergency medical therapy and elective shunt (p < 0.01). These survival rates produced by emergency shunt performed within 8 hr of initial contact confirm the effectiveness of this procedure observed in our previous unrandomized studies.

Hepatocyte horseradish peroxidase uptake is saturable and inhibited by mannose-terminal glycoproteins.

In the liver, horseradish peroxidase (HRP) is thought to be taken up via mannose receptor-mediated endocytosis by non-parenchymal cells (NPC) and via fluid-phase endocytosis by hepatocytes. When we attempted to inhibit NPC uptake of HRP with mannan in the whole perfused rat liver, > 80% of HRP uptake was eliminated. Liver cell fractionation revealed that mannan not only inhibited HRP uptake by NPC (91%) but also by hepatocytes (81%). In isolated hepatocytes, HRP uptake was linear over 60 min and saturable in the range of 0 to 200 mg/l (Vmax = 4.3 ng.mg protein-1.min-1; Km = 8.3 mg/l). Mannan inhibited uptake competitively (Ki = 2.0-2.5 mg/l). At high concentrations of HRP, a nonsaturable component of HRP uptake became evident (k = 2.8 pg.mg protein-1.min-1.mg HRP-1.l-1). Hepatocyte uptake of HRP was inhibited by other glycoproteins and glycopeptides with mannose-terminal groups, as well as by mannan, but not by asialofetuin (ASF) or bovine serum albumin. Hepatocyte uptake of 125I-labeled ASF, which is taken up via the asialoglycoprotein receptor, was saturable and not inhibited by mannan. HRP binding to hepatocytes, determined at 4 degrees C, was also inhibited by mannan. Quantification of contamination of the parenchymal cell fraction by NPC by cell counting and by pronase digestibility suggested our results could not be explained by contamination of hepatocytes by NPC. At concentrations used for most morphological studies (1,000-10,000 mg/l), fluid-phase endocytosis accounts for much of HRP uptake. However, at low concentrations, a saturable low-capacity mechanism is responsible for most HRP uptake by the hepatocyte.(ABSTRACT TRUNCATED AT 250 WORDS)

Regenerative stimulus increases hepatocyte tight junctional permeability.

Experience with young animals, animals administered certain hepatotoxins and animals with two-thirds hepatectomy suggests that tight junctional permeability is increased in states characterized by architectural remodeling in the liver. In this work we correlate changes in tight junctional morphometry induced by two-thirds hepatectomy with changes in biliary permeability assessed by sucrose and horseradish peroxidase permeation and by alterations in biliary outputs of anionic and cationic cholephilic probes. By freeze-fracture examination tight junctional strand counts, density and orientation parallel to canaliculi were all reduced 24 hr after two-thirds hepatectomy. Occasionally, strands were perpendicular to the canaliculi, creating an unobstructed communication between bile and intercellular spaces. These morphological changes correlated with increased sucrose and paracellular horseradish peroxidase access into bile, with reduced biliary outputs of low molecular weight and especially with cationic cholephilic probes. The data support an increased but still charge-selective permeability of the biliary tree, which was induced by two-thirds hepatectomy 24 hr before. Presumably, fixed intercellular connections (tight junctions and gap junctions) must be loosened or lysed to allow the architectural reorganization required by the hepatocellular regenerative process.

Breakdown of hepatic tight junctions during reoxygenation injury.

We investigated whether reoxygenation following anoxia increased biliary permeability and whether or not allopurinol had a protective effect. Isolated rat livers were perfused for 30 min in a one-pass system with buffer equilibrated with 100% nitrogen after stabilization, and then for 60 min with the oxygenated buffer. Hepatic tight junction permeability was assessed by quantifying the early appearance in the bile of horseradish peroxidase (HRP) injected with the perfusate. This early peak represents paracellular passage of HRP, whereas a later second peak results from transcellular passage. In the control livers, 7% of the total HRP passage (93 +/- 50 pg/g liver) was paracellular and 93% was transcellular. After 30 min of reoxygenation following anoxia, however, 516 +/- 20 pg/g liver of HRP passed paracellularly. Addition of allopurinol (5 micrograms/ml) to the perfusate from the start of perfusion reduced paracellular passage of HRP to 219 +/- 49 pg/g liver after anoxia and reperfusion (P less than 0.01). Allopurinol also reduced the cumulative lactate dehydrogenase (LDH) release during the first 30 min of reoxygenation from 2.1 +/- 0.3 x 10(4) to 1.4 +/- 0.4 x 10(4) units/g liver (P less than 0.01). Reduction of the anoxic period from 30 min to 25 min significantly reduced the change in tight junction permeability and the extent of cellular injury: Paracellular passage of HRP was 336 +/- 20 pg/g and LDH release was 0.7 +/- 0.1 x 10(4) units/g liver, both significantly lower than those at 30 min (P less than 0.01). No significant difference in hepatic ATP levels after 60 min of reoxygenation was noted among the experimental groups, but all had lower levels than the control group. The protective effect of allopurinol suggests that the mechanism of biliary reoxygenation injury involves free radical generation. Susceptibility of tight junctions suggests a pattern of injury similar to that involved in anoxic damage of the vascular endothelium.

Vasopressin and A23187 stimulate phosphorylation of myosin light chain-1 in isolated rat hepatocytes.

Vasopressin (VP) and other hormones that elevate intracellular Ca2+ concentration also increase tight junctional permeability in the liver cell. Data derived from study of other tissues suggest that microfilaments are instrumental in regulating tight junctional permeability. By analogy to microfilament contraction in smooth muscles, it is likely that the transduction pathway for these hormones involves Ca(2+)-stimulated complex formation between calmodulin and myosin light chain (MLC) kinase with activation of this latter enzyme. MLC kinase then phosphorylates MLC, which, in the presence of actin, exerts adenosinetriphosphatase activity and produces microfilament contraction. This transduction pathway in the hepatocyte remains speculative. To demonstrate the likelihood of this pathway, we stimulated isolated hepatocytes with 10(-8) M VP and assayed MLC phosphorylation. We did this by immunoprecipitation of myosin from homogenates of liver cells prelabeled with [32P]-orthophosphate. We used a polyclonal antibody raised in rabbits against rat liver cell myosin. Our data demonstrate that VP is a potent stimulator of MLC phosphorylation. Maximal rises in intracellular Ca2+ and maximal MLC phosphorylation occur within 40 s of VP administration. The dose-response curve for MLC phosphorylation by VP is similar to that for tight junctional permeabilization in perfused liver with maximal effect at about 10(-8) M VP. The calcium ionophore A23187 also stimulated MLC phosphorylation. MLC phosphorylation, therefore, is at least coincident with, and probably responsible for, tight junctional permeabilization caused by elevation of intracellular Ca2+ in the liver cell.

The pattern of vasopressin-induced reduction in biliary output of cholephilic probes in the rat can be mimicked by dialysis.

During a recent study using isolated perfused rat liver, we concluded that the effects of vasopressin on biliary excretion of several anionic and cationic cholephilic probes could best be explained by passive diffusion of these probes through a paracellular pathway with permeability increased by vasopressin. Replication of the results of that study in an in vitro dialysis system would support the passive nature of the selective efflux of these cholephilic probes from the rat biliary tree and render metabolic events an unlikely explanation for the biliary excretory patterns of the probes. We studied biliary excretion of two bile acids-taurodehydrocholate and taurocholate-and two cholephilic cations-propidium and tributyl-methylammonium-perfused single-pass through the isolated rat liver. We collected bile and measured probe output before and during infusion of 10(-8) mol/L vasopressin. We also studied rates of diffusion of these probes in artificial solution and in whole rat bile through a 3,500 mol wt cut-off cellulose dialysis membrane. The relative order of probe efflux rates from the biliary tree with vasopressin administration paralleled their diffusion rates through the dialysis membrane. The only exception was that the biliary tree manifested charge selectivity. The data are compatible with the passive efflux of probe from the biliary tree, and metabolic events need not be invoked to explain their pattern of biliary excretion with vasopressin administration.

Specificity of the hepatocyte Na(+)-dependent taurocholate transporter: influence of side chain length and charge.

Trihydroxy bile acids with differing nonsterol chain length and charge were synthesized to define the effect of these parameters on the ability to competitively inhibit the Na(+)-dependent uptake of 14C taurocholate into isolated rat hepatocytes. Compounds with long side chains (greater than or equal to 0.8 nm) beyond carbon-17 of the sterol nucleus and carrying a negative charge or no charge were potent inhibitors. Introduction of a positive charge into the side chain weakened inhibition. When the length of the chain beyond carbon-17 fell below about 0.7 nm, charge still influenced inhibitory potency, but the effect was reversed and positively-charged chains yielded slightly greater inhibition than negatively-charged chains. From these results one may postulate a positively-charged cell surface domain extending outward from a point about 0.7 nm from the sterol nucleus receptor region. Up to about 0.7 nm from the sterol nucleus receptor region one might postulate a negative cell surface charge to account for the weaker inhibitory potency of compounds with short negatively-charged chains. Nonetheless, a short chain, regardless of charge, weakened inhibition, suggesting that a long negatively-charged side chain is necessary to orient the sterol moiety for optimal receptor fit. These data confirm that the Na(+)-dependent taurocholate transport site is sensitive to alterations of side chain charge and length and emphasize the importance of structure when designing bile acid analogs to probe taurocholate transport mechanisms.

Reoxygenation injury following anoxic perfusion preferentially impairs bile acid-independent bile flow.

We perfused isolated rat livers with Krebs-Ringer buffer, with no recirculation. Bile flow virtually stopped during 30 min of anoxia and resumed following reoxygenation to reach a plateau of 44% of the control level. When taurodehydrocholic acid (TDHC, 50 nmol/min/g liver) was administered during reoxygenation, bile flow increased three-fold (16.1 +/- 1.3 to 45.3 +/- 6.3 microliters/g liver). The increase in bile output with TDHC was 27.8 microliters/g liver, which was 89% of the control output. Bile acid output during this period was 1.4 mumol/g liver, which was 93% of the control level. Addition of allopurinol (50 nmol/min/g liver) without TDHC increased bile flow significantly (16.1 +/- 1.3 to 21.3 +/- 1.2 microliters/g liver), but the change was not significant when allopurinol and TDHC were given. The addition of allopurinol also reduced the cumulative release of lactate dehydrogenase from the liver during the reoxygenation period, but had no effect on hepatic adenosine triphosphate levels. Our data suggest that the bile acid-independent bile flow is sensitive to reoxygenation injury following anoxia whereas bile acid output and bile acid-dependent bile flow are resistant.

Function of rat hepatocyte tight junctions: studies with bile acid infusions.

To determine the effects of alteration of biliary paracellular permeability on bile flow and composition, we measured the biliary outputs of compounds highly concentrated in bile, all infused at a constant rate in the isolated rat liver perfused with Krebs-Henseleit buffer in a one-pass fashion. Paracellular permeability was increased by infusing 10(-8) M vasopressin (VP). The cholephilic compounds were three cations of various molecular weights, tributylmethylammonium (TBuMA), N-acetylprocainamide ethobromide (APAEB), and propidium iodide, and two anions, taurocholate (TC), a micelle-forming bile acid, and taurodehydrocholate (TDHC), an nonmicelle former. When TC was infused and paracellular permeability increased with VP, neither bile flow nor TC output changed, whereas outputs of cations fell. When TDHC was infused, TDHC output fell, as did outputs of all cations. The decrements in cation outputs exceeded that of TDHC and were inversely related to the molecular weight of the cation. To document that these changes were not related to reduced uptake of these compounds, we tested the uptakes of TBuMA, APAEB, and TDHC into isolated hepatocytes. In no case did 10(-8) M VP significantly reduce uptake. The data demonstrate that micelle-forming bile acids, with their high effective molecular weights, do not efflux from the biliary tree when permeability is increased with VP, whereas nonmicelle-forming bile acids do. Cations efflux more readily than anions, and within this group efflux rate is inversely related to molecular weight. The data confirm the size and charge selectivity of biliary tree permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline phosphatase activity in hepatic tissue and serum correlates with amount and type of bile acid load.

Bile acids stimulate synthesis of alkaline phosphatase (ALPase) in the liver. We studied how alterations in bile acid type and load affect ATPase activity in hepatic tissue and in the serum. We increased the load of natural bile acids in rats by bile duct obstruction (BDO) or by creating a shunt between the common bile duct and superior vena cava (choledocho-caval shunt or CCS). Concentration of bile acids and ALPase activity in hepatic tissue rose more rapidly in the BDO model than in CCS. ALPase activity on the hepatocellular surface, normally confined to the canaliculus, spread outward to involve basolateral membrane in livers with high total hepatic ALPase activity. When the bile acid pool was reduced by a 12-hour biliary drainage in the CCS model, surface distribution of ALPase reverted to a nearly normal pattern. We substituted the endogenous bile acid pool with an equimolar amount of the single bile acid, taurocholic (TCA), tauroursocholic, taurohyocholic, or tauroursodeoxycholic acid in the CCS. The first two bile acids have a 12 alpha-hydroxyl group, whereas the latter two do not. After 12 hours, hepatic ALPase activity was increased with TCA or tauroursocholic substitution, but not with taurohyocholic or tauroursodeoxycholic. Again, surface distribution of ALPase activity correlated with the tissue ALPase activity. However, the serum activity increased significantly only with TCA, the most detergent of the bile acids. In bile fistula rats only infusion of TCA accelerated biliary secretion of ALPase. The above results suggest that hepatic synthesis and serum activity of ALPase are influenced by two different features of bile acids: the former by structure (the 12 alpha-hydroxyl group) and the latter by a physical property (detergency).

Effect of molecular charge on para- and transcellular access of horseradish peroxidase into rat bile.

The permeability pathway into the biliary tree for small inert molecules exhibits a charge selectivity. Using a method which distinguishes trans- from paracellular access, we have examined the charge selectivity of biliary access pathways for the 40-kD protein horseradish peroxidase (pI 7.5), which was derivatized to strongly anionic (pI less than 3.5) and strongly cationic (pI greater than 9.5) isoenzymes. Each isoenzyme was injected as a bolus into the perfusate of an isolated rat liver perfused in situ with a nonrecirculating Krebs-Ringer buffer. Bile was collected at intervals and horseradish peroxidase activity was measured. Its appearance allowed differentiation of paracellular from transcellular access, and the amount entering via each pathway was quantified. The species of enzyme entering bile was the same as that injected as determined by cation-exchange high-performance liquid chromatography of biliary horseradish peroxidase. Paracellular biliary access of anionic horseradish peroxidase was less than 50% that of neutral and cationic horseradish peroxidase both in the control state and when paracellular entry was augmented with 10(-10) M vasopressin. Transcellular access of anionic horseradish peroxidase was similarly restricted. To determine whether this restriction of anionic transcellular access was brought about by diminished hepatocellular uptake or augmented catabolism, we studied these parameters in 4-hr primary hepatocyte cultures. The uptake rates of all species were similar. Little or no degradation or efflux of any horseradish peroxidase species occurred over 30 min in the cultured cells. We conclude that access is charge selective for macromolecules and that this selectivity holds for trans- as well as for paracellular pathways.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of bicarbonate in biliary excretion of diisothiocyanostilbene disulfonate.

Hepatic transport of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) was studied in isolated perfused rat livers and in isolated rat hepatocytes to determine if DIDS-induced decrease in biliary HCO3- excretion is due to a DIDS-HCO3- exchange and/or due to inhibition of Cl(-)-HCO3- exchange. In isolated perfused rat livers, DIDS reversibly decreased biliary HCO3- concentration and excretion. The changes in biliary HCO3- concentration were inversely related to biliary DIDS concentration. DIDS was concentrated in bile, indicating active hepatic transport. Replacement of perfusate HCO3- with equimolar dimethyloxazolidinedione (DMO) or tricine decreased biliary excretion, but not hepatic uptake, of DIDS. Biliary excretion of DIDS was also associated with a decrease in bile pH, and this decrease in pH was greater in the presence of HCO3-. HCO3-, but not DMO or tricine, stimulated DIDS efflux from preloaded hepatocytes. DIDS efflux was also temperature dependent and increased with increasing extracellular pH. Collectively, these results are consistent with the presence of a DIDS-HCO3- (OH-) exchange mechanism at the canalicular membrane. HCO3(-)-dependent Cl- uptake in hepatocytes was competitively inhibited by DIDS (Ki = 0.24 mM), confirming the presence of DIDS-inhibitable Cl(-)-HCO3- exchange. However, the ability of DIDS to decrease biliary HCO3- excretion persisted when perfusate Cl- was replaced by isethionate. Moreover, biliary HCO3- concentration returned to base line despite the presence of 2-6 mM DIDS in bile. Thus it seems unlikely that the inhibition of Cl(-)-HCO3- exchange by DIDS is a major mechanism of inhibition of HCO3- excretion. We, therefore, conclude that a DIDS-HCO3- (OH-) exchange at the canalicular membrane is the most likely explanation for the observed decrease in biliary HCO3- excretion.

Hormonal regulation of hepatocyte tight junctional permeability.

We have investigated the effects of hormones on the permeability of the hepatocyte tight junction to two probes, [14C]sucrose and horseradish peroxidase, using one-pass perfused rat livers. Using a single injection of horseradish peroxidase we have demonstrated that this probe can enter bile by two pathways that are kinetically distinct, a fast pathway, which corresponds to the passage of the probe through the hepatocyte tight junctions, and a slow pathway, which corresponds to the transcytotic entry into bile. The passage of horseradish peroxidase through the hepatocyte tight junctions was confirmed by electron microscopic histochemistry. Vasopressin, epinephrine, and angiotensin II, hormones that act in the hepatocyte through the intracellular mediators calcium, the inositol polyphosphates, and diacylglycerol, increased the bile-to-perfusion fluid ratio of [14C]sucrose and the rapid entry of horseradish peroxidase into bile, indicating that the permeability of the tight junctions to these probes was increased. The effect of these hormones was dose dependent and in the cases of angiotensin II and epinephrine was inhibited by the specific inhibitors [Sar1, Thr8]angiotensin II and prazosin, respectively. Dibutyryl adenosine 3',5'-cyclic monophosphate did not affect the [14C]sucrose bile-to-perfusion fluid ratio or the fast entry of horseradish peroxidase into bile. These results suggest that the hepatocyte tight junction can no longer be considered a static system of pores separating blood from bile. It is rather a dynamic barrier potentially capable of influencing the composition of the bile.

Nature of taurodehydrocholic acid uptake in rat hepatocytes.

We studied uptake into isolated rat hepatocytes of the bile acid analogue taurodehydrocholate (TDHC) over a concentration range of 2.5-4,000 microM. Uptake was mainly by a saturable sodium-dependent process with a Km of approximately 50 microM and a Vmax of 0.036 nmol.s-1.mg protein-1. A lesser sodium-independent process was evident but was linear in the range studied. Both processes were inhibited by incubation at 37 degrees C under nitrogen in the presence of 3 mM sodium cyanide or by incubation at 0 degrees C. A single transport site was suggested by the Eadie-Hofstee plot of TDHC uptake from 2.5 to 750 microM. TDHC was a weak competitive inhibitor of taurocholic acid (TCA) uptake (Ki = 236 microM) but was not itself taken up by the TCA transport site. TCA exhibited moderately potent mixed inhibition of TDHC uptake. Uptake of both compounds was strongly inhibited by bromosulfophthalein (BSP) and Rose Bengal, whereas 0.5 mM alanine uptake was not affected. BSP exhibited a complex pattern of inhibition of TDHC uptake: mixed partial inhibition. Degree of inhibition of both TDHC and TCA uptake did not increase as BSP concentrations were increased from 50 to 100 microM. BSP did not exert its inhibitory effects by alteration of membrane potential or sodium gradients; 50 microM BSP changed membrane potential less than 10% and sodium gradient not at all. The data indicate that despite close structural analogy between TDHC and TCA, the two compounds are taken up by different sodium-dependent mechanisms. Nonetheless, the similar qualitative and quantitative effects of BSP on their uptakes suggests the mechanisms are related.

Reduced cholestatic potency of taurolithocholate during backward perfusion of the rat liver.

Rat livers perfused backward are less susceptible to taurolithocholate (TLC) cholestasis than livers perfused forward. To understand this phenomenon, the uptake, metabolism, and excretion of TLC were studied in rat livers perfused forward and backward with TLC. Bolus injections of [3H]TLC were administered 20 minutes after the onset of unlabeled TLC infusion. Biliary bile acids were measured enzymatically and bile acid species were separated and quantified by thin-layer chromatography. Determination of radioactivity in each spot yielded the percentage distribution of various biliary bile acid metabolites. After perfusion, livers were examined by light and transmission electron microscopy. TLC infusion at 32 nmoles/minute/gm of liver reduced bile flow by 80% or more within 30 minutes of forward perfusion but not at all during backward perfusion. TLC uptake was over 95% regardless of perfusion direction. Although the distribution of biliary metabolites of TLC was the same during forward and backward perfusion, maximal rate of total biliary bile acid excretion was four-fold higher and total recovery of radioactivity in bile was six-fold higher during backward perfusion. Less than 6% of excreted radioactivity was in the form of unmetabolized TLC. Severe cholestasis induced by forward TLC perfusion was associated with extensive structural distortion of bile canalicular membranes almost exclusively in the periportal zone but with little hepatocellular necrosis. Livers perfused backward manifested no cholestasis. They showed cell necrosis in the pericentral zone which became extensive (10%) by 60 minutes, but the canalicular changes occurred only in a small proportion of canaliculi in the pericentral region. Bile canalicular membrane changes developed extensively only when very high doses of TLC were perfused backward. Even then, bile flow fell only 60%. We conclude: the lesser susceptibility to TLC cholestasis of livers perfused backward is in part related to the greater biotransformation and consequent excretion of TLC by pericentral hepatocytes. TLC-induced cholestasis is associated more closely with bile canalicular membrane changes than with extent of hepatocellular necrosis. The greater reduction of bile flow with periportal than with pericentral canalicular change is compatible with current concepts of biliary microanatomy which postulate a flow of bile from pericentral through periportal regions of the lobule into the bile ductules.