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Biomedical subjects

W G Hardison

Publications and source records attributed to W G Hardison.

At least 37 records · Page 2Linked to original sources
PubMed1988

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.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1987

Modulation of hepatic biotransformation and biliary excretion of bile acid by age and sinusoidal bile acid load.

Pericentral hepatocytes excrete bile acids more slowly and biotransform them more than periportal cells. This may reflect adaptation to low pericentral bile acid concentration or may be intrinsic. We studied two models in which pericentral bile acid concentrations are high: the 72-h choledocho-caval shunt (CCS) rat and the 3- to 4-wk-old rat. Livers were perfused forward or backward to assess periportal or pericentral hepatocyte function. Taurodeoxycholate (TDC) was infused at 32 nmol X min-1 X g liver-1, and a bolus of [3H]TDC was given to assess metabolism and excretion of bile acids. In CCS livers perfused backward, pericentral cells resembled periportal cells of controls in that time to excrete 50% of administered [3H]TDC (t50) was reduced by two-thirds and [3H]TDC biotransformation was reduced by about half. In young livers t50 was half that of adult livers when perfused backward. Biotransformation, however, was not reduced. Young livers biotransformed more than adult controls for any given residence time of bile acid in the liver. We conclude that the difference between pericentral and periportal cells as regards bile acid processing is adaptive. Livers from young rats biotransform more bile acid than those from controls under similar conditions.

Aging↗
PubMed1987

Bile acid inhibition of taurocholate uptake by rat hepatocytes: role of OH groups.

To define further the structural specificity of the taurocholate uptake site, we studied the ability of a variety of taurine-conjugated bile acids with differing hydroxyl substituents on the sterol moiety to inhibit [14C]taurocholate uptake. Rat hepatocytes isolated by collagenase perfusion were incubated in a tris(hydroxymethyl)aminomethane-phosphate buffer containing [14C]taurocholate (2.5-100 microM) in the presence or absence of inhibitor bile acid. Stronger inhibitors were studied at a fixed concentration of 5 microM, weaker ones at 25 microM. Initial uptake velocity was measured by sedimenting an aliquot of cells through silicone oil into 3 N KOH every 15 s for 1 min. Uptake velocity (nmol X mg protein-1 X min-1) could then be related to taurocholate concentration and a Vmax and Km could be determined by applying a nonlinear least squares fit to the data obtained with or without inhibitor. The kinetic parameters allowed the determination of the type of inhibition and of inhibition constants (Ki) of the various test bile acids. The data indicate that bile acids containing a 6- or 7-OH group exhibit competitive inhibition, whereas bile acids with no 6- or 7-OH group exhibit noncompetitive inhibition. Of the compounds exhibiting competitive inhibition, Ki varied with the number of hydroxyl groups on the sterol moiety. We conclude that the presence or absence of a 6- or 7-OH group dictates the mechanism of inhibition; the number of hydroxyl substituents determines the potency of competitive inhibition.

Animals↗
PubMed1987

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.

Animals↗
PubMed1986

Postobstructive subcellular organelle and biliary lipid composition in the rat. A selective increase in biliary lecithin output is not reflected by changes in organelle composition.

Rats infused intravenously with taurocholate shortly after relief of 48 h of biliary obstruction excrete in bile twice as much phospholipid in relation to cholesterol and bile acid as controls. In an effort to identify a subcellular compartment as the source of this biliary lipid, we examined several hepatic subcellular organelles for an increase in phospholipid to cholesterol ratio or an increase in biliary-type lecithins (16:0-18:2; 16:0-20:4) either absolute or relative to non-biliary-type lecithins (18:0-20:4; 18:0-22:6) as analyzed by high-performance liquid chromatography. Subcellular fractions studied were: three microsomal subfractions, Golgi heavy and intermediate fractions, and plasma membrane light (canaliculus-enriched) fraction. No organelle fraction from rats with biliary obstruction displayed either a significant increase in phospholipid to cholesterol ratio or a relative or absolute increase in biliary type lecithin. This suggests that biliary lipid changes are not attributable to measurable alterations in lipid composition of any anatomical compartment in the liver cells; changes are probably more related to changes in lipid turnover than to organelle total lipid pool sizes.

Animals↗
PubMed1986

Greater taurodeoxycholate biotransformation during backward perfusion of rat liver.

Differing patterns of taurodeoxycholate (TDC) metabolism and biliary excretion were studied with a forward-and-backward perfusion model of the isolated rat liver. Livers were perfused with 8 microM TDC via either the portal vein (forward) or the hepatic vein (backward). Bile was collected, total bile acids measured enzymatically, and bile composition determined by high-pressure liquid chromatography and thin-layer chromatography. During backward perfusion 48% of infused TDC was metabolized to taurocholate (TC) and 22.0% to other minor TDC metabolites. During forward perfusion, however, only 16% of administered TDC was metabolized to TC and 6.5% to minor metabolites. Total bile acid output was similar for both forward and backward perfusions. The kinetics of biliary bile acid excretion also differed between forward and backward perfusion. The time necessary for 50% excretion of labeled TDC and its metabolites was 3.5 +/- 0.45 min during forward and 18 +/- 3.50 min during backward perfusion, a time difference of 14.5 min. The greater biotransformation of TDC during backward perfusion could be explained by its longer intracellular residence. The reason for the delayed excretion of TDC during backward perfusion is unknown.

Animals↗
PubMed1985

Phospholipid, cholesterol, polypeptide and glycoprotein composition of hepatic endosome subfractions.

The composition of hepatic endosome subfractions was compared directly with that of plasma membranes and Golgi-apparatus fractions. The neutral lipid and phospholipid composition of two endosome subfractions separated on Nycodenz gradients from a parent endosome fraction was similar to that of plasma membranes. The phospholipid/cholesterol ratios and the sphingomyelin contents were high, as in plasma membranes. However, the phosphatidylserine content was low. Endosomal subfractions contained a simpler polypeptide profile than did plasma membranes. However, a large number of glycoproteins were common to both fractions. Two endosome-specific glycoproteins, of Mr 59 000 and 38 000, were identified. Sialic acid was present at concentrations higher than in plasma membranes. The results indicate that endosomal membranes have a similar composition to plasma membranes that probably reflects their functional interaction during endocytosis and receptor recycling.

Animals↗
PubMed1985

Mechanisms of liver adaptation to prolonged selective biliary obstruction (SBO) in the rat.

To determine changes which occur in selective biliary obstruction (SBO), we studied male adult Fischer rats after one month of either SBO or selective biliary cannulation (SBC) of the median lobe duct (MLD). In rats with SBC the ML was obstructed with a sealed PE-10 catheter placed in the MLD. One month later at laparotomy the ML was either drained or not drained for 30 min before the injection of 200 microCi [99Tc]DIDA (2,6-diethylacetoanilido-imino-diacetic acid). Bile was collected and biopsies of the obstructed ML and non-obstructed right lobe (RL) were taken at 1, 3, 10 and 30 min. Serum bile acid concentrations were higher in SBC not drained rats than in control as were hepatic bile acid concentrations. The latter, however, did not achieve statistical significance. In SBC-drained rats biliary bile acid secretion from the obstructed lobe was lower than that from the non-obstructed lobe for 30 min after the release of obstruction but was thereafter the same. Hepatic DIDA levels in both the obstructed and non-obstructed portions of liver from SBO animals were higher than in liver from controls, despite normal DIDA biliary excretion. This is in part explained by increased cytosolic binding of DIDA. In rats with SBO the MLD was simply ligated and transected. After one month uptake kinetics of [14C]taurocholate in freshly isolated hepatocytes from obstructed and non-obstructed lobes were similar suggesting that no major impairment of BA uptake occurs. We conclude that cholestasis is still present after 30 days of SBO in spite of the presence of interlobular biliary connections. The observed increased hepatic storage capacity for DIDA is probably an adaptive mechanism in mild chronic cholestasis.

Animals↗
PubMed1984

Specificity of an Na+ -dependent taurocholate transport site in isolated rat hepatocytes.

The structural specificity of the taurocholate uptake mechanism was studied by determining the ability of 100 microM structural analogues of taurocholate to inhibit the uptake of 5 microM [14C]-taurocholate into freshly isolated rat hepatocytes. Taurocholate was a more potent inhibitor than cholate. The fewer the number of hydroxyl groups, the more potent the inhibitors. Neither hydroxyl group orientation (alpha or beta) nor esterification with formic acid strongly influenced inhibitory potency. Replacement of hydroxyl groups with keto groups, however, diminished inhibitory potency. These facts suggested that distortion of the sterol backbone may be an important factor in diminishing inhibitory potency. Charge on the nonsteroid moiety affected potency. Both neutral and negatively charged compounds inhibited more strongly than positively charged compounds. As important as charge, however, was length of the nonsteroidal moiety. Inhibitory potency increased progressively as length beyond C-17 increased up to 11 A. Inhibitory potency was reduced again, however, with an extremely long end group of 18 A. The inhibitors do not act as cytotoxins since cellular oxygen consumption was not diminished by any of them. Furthermore, inhibition was specific for the taurocholate uptake mechanism. The three most potent inhibitory compounds inhibited L-alanine uptake little or not at all. The kinetics of inhibition, determined for three selected compounds, were consistent with competitive inhibition. The structural specificity of the rat hepatocyte sodium-dependent taurocholate uptake site is similar to that of the guinea pig ileum uptake site.

Animals↗
PubMed1984

Bile-acid binding to isolated rat liver plasma membranes. Failure to find a specific binding site.

Bile-acid transport across the hepatocyte is an active Na-dependent process and specific putative binding sites on liver plasma membrane have been described. We studied cholic-acid binding to isolated liver plasma membranes over a wide concentration range. We found no saturability of binding over a concentration range of 0.001-2000 microM. We did demonstrate saturability of binding over a concentration range (1-30 nM) at pH 6.0 and 4 degrees C, but the saturability was an artifact caused by the increasing concentration of ethanol necessary to avoid precipitation of cholic acid at pH 6.0, 4 degrees C. We were unable to eliminate specific binding by heating the plasma membrane for 3 h at 37 degrees C, as other authors have. Although we were able to show a structural specificity of the bile-acid uptake site on the isolated liver cells, we were unable to demonstrate a specific saturable bile-acid binding site on isolated liver plasma membranes.

Animals↗
PubMed1984

Effect of biliary pressure versus high bile acid flux on the permeability of hepatocellular tight junction.

Creation of a shunt between the common bile duct and vena cava, choledocho-caval shunt (CCS), causes biliary retention without obstruction. This in turn induces a marked choleresis (up to three times the normal bile flow) and increases bile acid output, both of which reach a plateau by 6 hours and remain elevated thereafter. In contrast, bile duct obstruction produces high biliary pressure and stoppage of bile flow and bile acid output. To compare the effect of biliary pressure and bile acid flux on permeability of the hepatocellular tight junction, we examined lanthanum permeation through tight junctions and bile to plasma concentration ratios of 3H-sucrose in these two models. In addition, we examined the effect of biliary pressure further by adjusting biliary pressure to 15 cm of H2O in modified CCS animals. At 6 hours after establishing these experimental models in male Sprague-Dawley rats, we infused lanthanum chloride (5 mM) in physiologic saline through the aorta (120 mm Hg) for 3 minutes, followed by perfusion fixation of the liver. Transmission electron microscopy on 144 bile canaliculi in each group (three rats each) revealed penetration of lanthanum in descending order of pressure rather than bile acid output: bile duct obstruction (50.7%), modified CCS (19.4%), CCS (13.2%), and control (5.6%). Bile to plasma concentration ratios of 3H-sucrose measured at 0, 2, and 6 hours in CCS and modified CCS animals revealed a marked increase of bile to plasma ratios only in the modified CCS group. These data lead us to conclude that biliary pressure is the significant determinant of biliary permeability and that bile acid output is important only insofar as the resultant choleresis elevates pressure.

Animals↗
PubMed1984

Effect of ursodeoxycholate and its taurine conjugate on bile acid synthesis and cholesterol absorption.

Six male subjects with normal gastroenterologic function were studied to determine the effects of ursodeoxycholic (15 mg/kg X day) and tauroursodeoxycholic (20 mg/kg X day) acid feeding on bile acid synthesis and cholesterol absorption. Each bile acid was fed for 1 mo and withheld for the next month. Subjects remained on a metabolic ward and consumed a constant diet of 500 mg of cholesterol mixed with solid and liquid formulas. Before the study started, each subject received 50 muCi of [4-14C]cholesterol intravenously. During the study, stools were collected for the determination of 24-h fecal acidic and neutral sterol excretion, and blood was drawn twice weekly for determination of serum cholesterol specific activity. At the end of each month an intestinal perfusion study was performed to measure total bile acid pool size and hourly biliary secretion rates of cholesterol, phospholipid, and bile acid. From these data, the percentage of cholesterol absorption and total endogenous bile acid synthesis could be calculated. Neither ursodeoxycholic nor tauroursodeoxycholic acid feeding decreased endogenous bile acid synthesis. During bile acid feeding periods, the percentage of cholesterol absorption was decreased.

Bile Acids and Salts↗
PubMed1983

Uptake of iopanoic acid by isolated rat hepatocytes in primary culture.

Uptake of iopanoic acid (IOP) was studied in 3-day primary cultures of rat hepatocytes isolated by the collagenase perfusion method. 125I activity of cells after incubation with 125I-IOP (1.0-100 microM) was used as a measure of uptake. At each IOP concentration uptake was linear for the first 45 s. The initial uptake velocity was directly proportional to IOP concentration and was nonsaturable up to 100 microM. The calculated uptake rate constant was 0.67 nmol . mg prot-1 . min-1 . microM-1. Uptake was only slightly reduced when the incubation was performed at 4 degrees C and was independent of sodium concentration. Albumin in the medium reduced IOP uptake. Uptake, however, was always greater than that predicted from the unbound IOP concentration alone. The data indicate that the hepatocyte uptake of IOP occurs by both a passive process and a saturable process. The saturable uptake component depends on an albumin-IOP-hepatocyte interaction. The influence of albumin on uptake occurs possibly by an undefined specific cell surface phenomenon of albumin that promotes uptake of IOP or by enhancement of the diffusibility of IOP across the unstirred layer.

Animals↗
PubMed1983

Effect of bile acid conjugation pattern on bile acid metabolism in normal humans.

Six male subjects were fed taurine 0.5 g six times daily for 2 wk to determine the effect of a shift in bile acid conjugation pattern upon bile acid metabolism. Duodenal bile acids were analyzed, and bile acid pool size, daily fecal excretion, and biliary excretion rate were quantified. In addition, daily biliary excretion rate of cholesterol and phospholipid were quantified, and biliary saturation with cholesterol was estimated. The dose of taurine caused reversal of the bile acid glycine-to-taurine conjugation ratio. Total bile acid pool size decreased, as did the pool size of chenodeoxycholic acid. Pool sizes of cholic and deoxycholic acids did not change. Daily fecal bile acid excretion decreased slightly. Biliary secretion rates of cholesterol, phospholipid, and bile acids did not change, nor did biliary cholesterol saturation. Pool size can decrease because of increased bile acid catabolism or decreased synthesis. The fact that bile acid excretion failed to increase, and actually decreased slightly, suggests that the effect is upon bile acid synthesis. In normal humans, the effect is small and probably physiologically unimportant. In special cases, however, such as during ursodeoxycholic acid therapy, the effect of shifting conjugation pattern may become important.

Adult↗