Science and industry.

Industry is concerned with basic science as the source of its technology, as the force of its philosophy of deductive thought, as its eye to the future, and as the impetus it provides for industrial innovation. Industry's strengthened advocacy of the support of basic science is essential for its future growth.

A greater gondwanaland.

Gondwanaland at its maximum extent included Tibet, the Tarim Basin block, and parts of northern China. Tibet lay between India and Western Australia as submerged continental crust. The Himalayas are of intracontinental origin. The Tien Shatz lie along the former oceanic boundary of Gondwanaland.

Hepatic secretion of phospholipid vesicles in the mouse critically depends on mdr2 or MDR3 P-glycoprotein expression. Visualization by electron microscopy.

Hepatocellular secretion of bile salts into the biliary space induces phospholipid and cholesterol secretion, but the mechanism for integrated lipid secretion is poorly understood. Knockout mice unable to make the canalicular membrane mdr2 P-glycoprotein exhibit normal rates of bile salt secretion, yet are virtually incapable of secreting biliary phospholipid and cholesterol. As the mdr2 P-glycoprotein is thought to mediate transmembrane movement of phospholipid molecules, this mouse model was used to examine the mechanism for biliary phospholipid secretion. In wild-type mdr2 (+/+) mice, ultrarapid cryofixation of livers in situ revealed abundant unilamellar lipid vesicles within bile canalicular lumina. Although 74% of vesicles were adherent to the external aspect of the canalicular plasma membrane, bilayer exocytosis was not observed. Vesicle numbers in mdr2 (+/-) and (-/-) mice were 55 and 12% of wild-type levels, respectively. In a strain of mdr2 (-/-) mice which had been "rescued" by heterozygous genomic insertion of the MDR3 gene, the human homologue of the murine mdr2 gene, vesicle numbers returned to 95% of wild-type levels. Our findings indicate that biliary phospholipid is secreted as vesicles by a process largely dependent on the action of the murine mdr2 P-glycoprotein or human MDR3 P-glycoprotein. We conclude that mdr2-mediated phospholipid translocation from the internal to external hemileaflet of the canalicular membrane permits exovesiculation of the external hemileaflet, a vesiculation process promoted by the detergent environment of the bile canalicular lumen.

Microtubule-dependent transport of bile salts through hepatocytes: cholic vs. taurocholic acid.

Studies with taurine-conjugated bile salts have demonstrated two pathways for hepatocellular delivery of bile salts to bile: a cytosolic, microtubule-independent pathway and a membrane-based, microtubule-dependent pathway. However, a significant portion of circulating bile salts may be unconjugated. To determine whether free bile salts utilize similar pathways, we examined the effect of colchicine on the biliary excretion of intravenously administered cholic acid and taurocholate in intact rats. Basal rats were pretreated with low-dose colchicine or its inactive isomer, lumicolchicine, 1 hr before placement of intravenous and biliary cannulas and 2.75 hr before intravenous injection of [14C]cholic acid and [3H]taurocholate. Superfused rats were prepared as above but with intravenous infusion of taurocholate at 200 nmol/min.100 gm beginning 0.75 hr before [14C]cholic acid/[3H]taurocholate injection. Depleted/reinfused rats were subjected to biliary diversion for 20 hr before colchicine or lumicolchicine pretreatment, infusion of taurocholate and [14C]cholic acid/[3H]taurocholate injection. In each group, biliary excretion of [14C]taurocholate and [3H]taurocholate was inhibited equally by colchicine; for peak excretion rates the respective inhibition values were 33% and 35% in basal rats, 63% and 65% in superfused rats, and 74% and 76% in depleted/reinfused rats. Biliary excretion of [14C]taurocholate occurred consistently later than excretion of [3H]taurocholate, and maximal rates of excretion were reduced. In contrast, plasma uptake rates of [14C]cholic acid and [3H]taurocholate were essentially the same in depleted/reinfused rats. Deconvolution analysis of [14C]taurocholate vs. [3H]taurocholate biliary excretion curves revealed no significant differences among experimental groups. We conclude that conversion of [14C]cholic acid to [14C]taurocholate slightly retards its biliary excretion and diminishes its peak excretion rate compared with exogenous [3H]taurocholate.(ABSTRACT TRUNCATED AT 250 WORDS)

The normal adult human liver biopsy: a quantitative reference standard.

In assessing adult human liver histology, questions remain concerning the normal number of portal tracts and bile ducts in a liver biopsy. We therefore reviewed liver biopsies obtained with use of a percutaneous Menghini cutting needle (14G, internal diameter 1.6 mm), from 16 patients undergoing liver biopsy for screening procedures (age 49 +/- 14 years, +/-SD) and found to be normal by histological examination. The average aggregate length of the liver tissue was 1.8 +/- 0.8 cm (area of 16.4 +/- 10.7 mm2), representing 7 +/- 3 tissue fragments. Portal triads containing at least one profile each of a portal vein, hepatic artery, and interlobular bile duct numbered 11 +/- 6 per biopsy (range 3-23). Portal dyads, which did not contain one of these profiles, usually the portal vein, numbered 8 +/- 5 (range 1-18). On a per-specimen basis, 38% of portal tracts did not contain a portal vein, 7% did not contain a bile duct, and 9% did not contain a hepatic artery. Because of multiplicity of profiles within portal tracts, however, the average number of profiles per portal tract was 6 +/- 5 (range 2-35). Notably, on average there were 2.3 +/- 2.2 interlobular bile ducts per portal tract, compared to 2.6 +/- 2.3 hepatic arteries and 0.7 +/- 0.7 portal veins. The average minimum external diameter of interlobular bile ducts was 13 +/- 4 microm, of hepatic arteries 12 +/- 5 microm, and of portal veins 35 +/- 25 microm. Bile ducts greater than 30 microm in diameter were rare, only one each in two biopsies were observed. In contrast, probable canals of Hering were occasionally evident at the periphery of portal tracts (6 +/- 6 per biopsy) and within the lobular parenchyma as strings of cuboidal cells (5 +/- 5 per biopsy). We conclude that, although multiplicity of profiles is normal, portal dyads are almost as common as portal triads in normal peripheral liver tissue. On average, there are two interlobular bile ducts, two hepatic arteries, and one portal vein per portal tract, with 6 full portal triads per linear cm of tissue obtained by external Menghini biopsy technique with use of a 14G needle, equivalent to 0.8 +/- 0.5 portal triads per mm2. By serving as a reference standard for adult human liver histology, these findings may assist in the histopathological assessment of liver biopsies, particularly those performed for disease conditions featuring loss of intrahepatic bile ducts.

Early vascular changes in the spinal grey matter following impact injury.

The ultrastructural changes of the microvasculature in the spinal grey matter were studied after impact injuries of varying severity in cats. Survival times ranged from 3 min to 6 h after injuries. The main vessels affected were capillaries and postcapillary venules. Perivascular haemorrhage and protein extravasation were present immediately after impact. Astrocytic foot processes could be swollen or shrunken and of increased electron density. The extra cellular space (ECS) was often distended with proteinaceous material. Some vessels showed an apparent increase in vesicles and small dense inclusions were seen in pericytes. By 20--30 min after impact, a number of vessels at greater impact energies showed degenerative changes in organelles and endothelial gaps. The lumina of these vessels often contained plasma, platelets, red cells and occasional neutrophils. Similar changes were seen between 3 and 6 h and at this stage neutrophils and some monocytes were present in the perivascular spaces and neuropil. Endothelial balloons and pericytic dense inclusions were also seen. It is suggested that initial events are mechanical, perhaps due to a rapid displacement of blood in venules followed by arterial hypertension (which commonly accompanies spinal cord impacts). Although initially many lumena are patent by 30 min there is a difference between the lesser and greater severities of impact.

Role of bile salt hydrophobicity in hepatic microtubule-dependent bile salt secretion.

Under basal conditions, bile salt secretion by the liver is not affected by microtubule disruption. However, when a bile salt load is imposed on the liver, a microtubule-dependent secretion mechanism is recruited (J. Lipid Res. 1988. 29: 144-156). We tested the hypothesis that recruitment of this microtubule-dependent mechanism is influenced by the relative hydrophobicity of the bile salts being secreted. Intact male rats were depleted of bile salts by overnight biliary diversion, pretreated with colchicine (a microtubule inhibitor) or its inactive isomer, lumicolchicine (control), and reinfused intravenously with bile salts of increasing hydrophobicity (taurodehydrocholate < tauroursodeoxycholate < taurocholate) at 200 nmol/min.100 g. After 45 min, when steady-state bile salt secretion was achieved, tracer [3H]taurocholate was administered intravenously. The colchicine-insensitive component of bulk bile salt secretion was constant at approximately 130 nmol/min.100 g, and the colchicine-sensitive component increased from approximately 0 to 35 and 60 nmol/min.100 g, respectively, with reinfusion of the more hydrophobic bile salts. Retained bile salts accumulated in the liver and serum and were detectable in urine. Peak biliary secretion of [3H]taurocholate in control animals increased linearly from 15.3 to 18.0% administered dose/min with increasing hydrophobicity of the secreted bile salts (P < 0.002). In colchicine-pretreated animals, peak secretion rates decreased linearly from 13.8 to 9.2%/min (P < 0.001), with maximal inhibition in taurocholate-reinfused animals (P < 0.01). Utilization of a microtubule-dependent secretion mechanism increases with increasing bile salt hydrophobicity. This mechanism permits more efficient hepatic secretion of bile salts, but increases the susceptibility of bile salt secretion to microtubule disruption. We postulate that microtubule-dependent insertion of bile salt transporters into the canalicular membrane underlies the enhanced bile salt secretion observed when a bile salt load is imposed upon the liver.

Enhanced Na+-dependent bile salt uptake by WIF-B cells, a rat hepatoma hybrid cell line, following growth in the presence of a physiological bile salt.

Although bile salts are toxic to the liver at high plasma concentrations, the effects of physiological concentrations of bile salts on normal hepatic function are poorly understood. We examined the effect of taurocholate (TC) on the basolateral uptake of [3H]TC in WIF-B cells, a hybrid cell line stably exhibiting in vitro the structural and functional polarity of hepatocytes. Cells were grown in the absence or presence of TC (50 micromol/L) over 12 days, and then incubated with [3H]TC concentrations ranging from 1 to 250 micromol/L. For both control and TC-grown cells, uptake of [3H]TC was linear over 2 minutes. In control cells, the Km for [3H]TC Na+-dependent uptake over 1 minute was 6 +/- 5 micromol/L, and the Vmax was 45 +/- 6 pmol TC/mg protein/min (+/- SEM). TC-grown cells exhibited no significant change in Km but showed a doubling of Vmax to 87 +/- 6 pmol TC/mg protein/min (P < .005). In both control and TC-grown cells, maximal uptake of [3H]TC occurred following 10 to 12 days in culture, with TC-grown cells consistently showing greater rates of [3H]TC uptake from 4 to 14 days in culture. Western blots immunostained for the basolateral Na+-dependent plasma membrane protein, ntcp, revealed the appropriate approximately 50-kd band in control and TC-grown cells, and confocal immunofluorescence microscopy demonstrated staining along the basolateral plasma membrane. Northern blots hybridized with a cDNA probe directed against ntcp indicated a modest TC-induced increase in mRNA levels. Reverse-transcriptase polymerase chain reaction (RT-PCR) using RNA isolated from WIF-B cells and oligonucleotide primers specific for rat ntcp or human NTCP transcripts revealed only the presence of the rat ntcp transcript. We conclude that bile salts, at concentrations normally found in mammalian portal blood, may be capable of promoting enhanced hepatocellular bile salt uptake via an increase in basolateral Na+-dependent plasma membrane transport capacity.

Imaging biliary lipid secretion in the rat: ultrastructural evidence for vesiculation of the hepatocyte canalicular membrane.

Physical-chemical and biological studies of hepatic bile suggest that biliary phospholipid molecules are secreted as unilamellar vesicles. Systematic ultrastructural studies of bile canaliculi were undertaken to visualize this event. Liver tissue was obtained from normal adult male rats (control), from bile salt-depleted rats (by overnight biliary diversion), and from depleted rats infused intravenously with a hydrophilic-hydrophobic congener series of common taurine-conjugated bile salts. Livers were fixed in situ either by modified chemical methods or by ultrarapid cryofixation. In control rats, chemical fixation revealed unilamellar vesicles 63 +/- 17 (+/- SD) nm in diameter, mostly free within canalicular lumena. Vesicles were infrequent in canaliculi of bile salt-depleted rats, but were present in canaliculi of rats infused with taurocholate. In cryofixed liver tissue, vesicles 67 +/- 13 nm in diameter were observed in canaliculi of control rats and bile-salt depleted rats infused with common bile salts. The majority of these vesicles were affixed to the luminal side of the canalicular membrane. The average number of vesicles per bile canaliculus was in agreement with that estimated on the basis of biliary phospholipid secretion rates, mean vesicle size, and area of close-packed phosphatidylcholine molecules. By immunoelectron microscopy, canalicular vesicles were free of actin and of a 100 kDa canalicular membrane protein. We conclude that biliary phospholipid molecules are secreted from hepatocytes into bile canalicular lumena as unilamellar vesicles approximately 63-67 nm in average diameter. We postulate that this secretion mechanism involves lumenal bile salt-induced vesiculation of lipid microdomains in the exoplasmic hemileaflet of the canalicular membrane.

Endotoxin-stimulated macrophages decrease bile acid uptake in WIF-B cells, a rat hepatoma hybrid cell line.

Endotoxemia leads to cytokine-mediated alterations of the hepatocellular sodium-taurocholate-cotransporting polypeptide (ntcp). We hypothesized that stimulated macrophages are essential transducers for down-regulating hepatocellular bile salt uptake in response to endotoxin (lipopolysaccharide [LPS]) exposure. Using an in vitro model, we exposed mouse macrophages (IC-21 cell line) to LPS for 24 hours. Concentrations of cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6 increased 10.6-fold, 12.5-fold, and 444-fold, respectively, in LPS-conditioned IC-21 medium (CM) versus unconditioned IC-21 medium (UM). WIF-B rat hepatoma hybrid cells were incubated with either CM or UM or treated directly with medium containing recombinant TNF-alpha, IL-1beta, and IL-6. [(3)H]Taurocholate ([(3)H]TC) uptake decreased in WIF-B cells exposed to either TNF-alpha (54% of control), IL-1beta (78%), IL-6 (55%) as single additives, or in triple combination (TCC) (43%). A virtually identical decrease was observed after exposing WIF-B cells to CM (52%, P <.001). LPS had no direct effect on [(3)H]TC uptake. CM treatment did not decrease L-alanine transport in WIF-B cells. Blocking antibodies against TNF-alpha, IL-1beta, and IL-6 restored the diminished [(3)H]TC uptake in cells exposed to TCC and CM to 87% and 107% of controls, respectively. Northern blotting revealed that ntcp messenger RNA (mRNA) expression was significantly reduced in WIF-B cells after exposure to CM, and in primary rat hepatocytes exposed to CM or TNF-alpha (68%, 14%, and 29% of control, respectively). We conclude that macrophages and their ability to secrete the cytokines TNF-alpha, IL-1beta, and IL-6 may be essential in mediating the endotoxin-induced cholestatic effect of decreased hepatocellular bile salt uptake.

Short-term regulation of bile acid uptake by microfilament-dependent translocation of rat ntcp to the plasma membrane.

The Na+-taurocholate cotransport polypeptide (ntcp) is the primary transporter for the uptake of bile acids in the liver. The second messenger adenosine 3':5'-cyclic monophosphate (cAMP) rapidly increases ntcp protein concentration in the plasma membrane, yet the mechanism is unknown. To investigate this, HepG2 cells were transiently transfected with a carboxy-terminal-tagged green fluorescence protein (GFP) conjugate of ntcp, and then examined by confocal video microscopy. Transporter activity was directly assayed with 3H-taurocholic acid (TC) scintigraphy. ntcp-GFP targeted to the plasma membrane in transfected cells, and the conjugate protein transported 3H-TC as effectively as unmodified rat ntcp. Stimulation of ntcp-GFP cells with cAMP increased GFP fluorescence in the plasma membrane by 40% (P <.0001) within 2.5 minutes and by 55% within 10 minutes. Similarly, cAMP increased transport of bile acids by 30%. Cytochalasin D, an inhibitor of microfilaments, did not prevent ntcp-GFP from targeting to the plasma membrane, but completely abolished the increase in GFP fluorescence seen in response to cAMP. In contrast, the microtubule inhibitor, nocodazole, prevented development of membrane fluorescence in 48 (96%) of 50 cells. Cells regained plasma membrane fluorescence within 2 hours after nocodazole removal. These findings suggest that targeting of ntcp to the plasma membrane consists of 2 steps: 1) delivery of ntcp to the region of the plasma membrane via microtubules; and 2) insertion of ntcp into the plasma membrane, in a microfilament- and cAMP-sensitive fashion.