Comparison of the karyotypes of four Cercopithecoidae: Papio papio, P. anubis, Macaca mulatta, and M. fascicularis.

The karyotypes of two species of baboons, Papio papio and P. anubis, and of two species of Macaca, M. mulatta and M. fascicularis, are compared after the use of numerous banding techniques. No difference was detected between the karyotype of the two Papio species. However, a minor change in the T-staining of a short segment, probably heterochromatic, could be detected between the Papio species and M. mulatta. A paracentric inversion exists between these three and M. fascicularis. These karyotypes are briefly compared with those of the Pongidae and man. The value of the karyotypic criteria and of the methods used for taxonomy is discussed.     

Fibrinopeptides A and B of Japanese monkey (Macaca fuscata) and patas monkey (Erythrocebus patas): their amino acid sequences, restricted mutations, and a molecular phylogeny for macaques, guenons, and baboons

Amino acid sequences of fibrinopeptides A and B from the macaque, Macaca fuscata (Japanese monkey) and the guenon, Erythrocebus patas (patas monkey) were established. Fibrinopeptides A of the monkeys had a sequence identical with those of baboons: Ala-Asp-Thr-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg. Fibrinopeptides B were 9-residue, "short," peptides with the sequences Asn-Glu-Glu-Ser-Leu-Phe-Ser-Gly-Arg for M. fuscata and Asn-Glu-Glu-Val-Leu-Phe-Gly-Gly-Arg for E. patas. The sequence of the B peptide of M. fuscata differed from that of a close-related species, M. mulatta (rhesus monkey), at a single site, Leu (M.f.)----Pro (M.m.). A single replacement between the B peptides of E. patas and Cercocebus aethiops (green monkey), Val (E.p.)----Gly (C.a.), was detected. A phylogenic relationship of macaques, guenons, and baboons, named Cercopithecinae (Old World monkey), was deduced from the sequence data. A selective rather than random amino acid replacement was observed in the B peptides of these Old World monkeys, suggesting a restricted mutation of their fibrinopeptides during primate evolution.     

Effects of pancreatic polypeptide on biliary flow and bile acid secretion stimulated by secretin and cholecystokinin in the conscious pig

Fourteen castrated male Large White pigs, weighing 42.5 +/- 1.0 kg, were fitted with biliary and duodenal fistulae for biliary secretion studies. Furthermore, catheters were placed in a carotid artery for blood sampling and in a jugular vein for peptide infusion. Bile was automatically restituted to the animals and continuously sampled for analysis on experimental days. Following an 8 day recovery period, infusion studies were performed after an overnight fast. After a 30 min basal period, sustained biliary flow and bile acid output were obtained and maintained throughout the assay with secretin (36 pmol/kg/h) and CCK-8 (600 pmol/kg/h) infusion. Then, 200, 400, 600, 800 or 1200 pmol/kg/h of porcine pancreatic polypeptide (PP) were infused for 60 min. Secretin plus CCK infusion was continued for 1 h after PP infusion was stopped. Each dose of PP was given on a separate day. Biliary flow was not affected by PP except for the dose of 400 pmol/kg/h. On the contrary, bile acid concentration and output decreased with the lowest dose of PP (200 pmol/kg/h). As soon as the first dose of PP was infused, bile acid concentration and output fell to about 60% of values obtained with secretin plus CCK. Plasma levels of PP were below or similar to postprandial values for 200, 400 and 600 pmol/kg/h and they were significantly larger with 800 and 1200 pmol/kg/h. Bile acid concentration and output did not return to values obtained with secretin plus CCK infusion after cessation of PP infusion. In conclusion, porcine PP given in physiological doses to the pig decreases bile acid output whereas biliary flow remains unaffected.     

Regulation of biliary protein secretion in dogs

The biliary secretion of protein in response to bile acids and other agents known to increase bile flow was examined in a chronic bile fistula dog model. Infusion of 25, 50, or 75 mumole/kg/hr sodium taurocholate after 3 hr of bile fistulization increased biliary protein output significantly by 52, 86, and 108% respectively compared to preinfusion values. A proportionate increase in biliary albumin output during taurocholate choleresis was demonstrated. Protein outputs during bile fistulization without taurocholate replacement were unchanged. The non-micelle-forming bile acid dehydrocholate markedly increased bile flow but did not change protein output. Similarly, the hormonal choleretics glucagon and secretin caused significant decreases in biliary protein concentration but no change in protein output. These data indicate a correlation between biliary protein secretion and bile acid-dependent bile flow. It is likely that regulation of certain proteins is dependent on the micelle-forming properties of bile acids.     

Profiling circulating and urinary bile acids in patients with biliary obstruction before and after biliary stenting

Bile acids are considered as extremely toxic at the high concentrations reached during bile duct obstruction, but each acid displays variable cytotoxic properties. This study investigates how biliary obstruction and restoration of bile flow interferes with urinary and circulating levels of 17 common bile acids. Bile acids (conjugated and unconjugated) were quantified by liquid chromatography coupled with tandem mass spectrometry in serum and urine samples from 17 patients (8 men and 9 women) with biliary obstruction, before and after biliary stenting. Results were compared with serum concentrations measured in 40 age- and sex-paired control donors (20 men and 20 women). The total circulating bile acid concentration increases from 2.7 μM in control donors to 156.9 μM in untreated patients with biliary stenosis. Serum taurocholic and glycocholic acids exhibit 304- and 241-fold accumulations in patients with biliary obstruction compared to controls. The enrichment in chenodeoxycholic acid species reached a maximum of only 39-fold, while all secondary and 6α-hydroxylated species--except taurolithocholic acids--were either unchanged or significantly reduced. Stenting was efficient in restoring an almost normal circulating profile and in reducing urinary bile acids. Conclusion: These results demonstrate that biliary obstruction affects differentially the circulating and/or urinary levels of the various bile acids. The observation that the most drastically affected acids correspond to the less toxic species supports the activation of self-protecting mechanisms aimed at limiting the inherent toxicity of bile acids in face of biliary obstruction.     

Role of microtubules in estradiol-17beta-D-glucuronide-induced alteration of canalicular Mrp2 localization and activity

Estradiol-17beta-D-glucuronide (E2-17G) induces a marked but reversible inhibition of bile flow in the rat together with endocytic retrieval of multidrug resistance-associated protein 2 (Mrp2) from the canalicular membrane to intracellular structures. We analyzed the effect of pretreatment (100 min) with the microtubule inhibitor colchicine or lumicholchicine, its inactive isomer (1 micromol/kg iv), on changes in bile flow and localization and function of Mrp2 induced by E2-17G (15 micromol/kg iv). Bile flow and biliary excretion of bilirubin, an endogenous Mrp2 substrate, were measured throughout, whereas Mrp2 localization was examined at 20 and 120 min after E2-17G by confocal immunofluorescence microscopy and Western analysis. Colchicine pretreatment alone did not affect bile flow or Mrp2 localization and activity over the short time scale examined (3-4 h). Administration of E2-17G to colchicine-pretreated rats induced a marked decrease (85%) in bile flow and biliary excretion of bilirubin as well as internalization of Mrp2 at 20 min. These alterations were of a similar magnitude as in rats pretreated with lumicolchicine followed by E2-17G. Bile flow and Mrp2 localization and activity were restored to control levels within 120 min of E2-17G in animals pretreated with lumicolchicine. In contrast, in colchicine-pretreated rats followed by E2-17G, bile flow and Mrp2 activity remained significantly inhibited by 60%, and confocal and Western studies revealed sustained internalization of Mrp2 120 min after E2-17G. We conclude that recovery from E2-17G cholestasis, associated with exocytic insertion of Mrp2 in the canalicular membrane, but not its initial E2-17G-induced endocytosis, is a microtubule-dependent process.     

黑叶猴和灰叶猴的线粒体DNA限制性片段长度多态研究

本文以15种限制性内切酶分析黑叶猴和灰叶猴种内及种间mtDNA多态。从各个样品中分别检出了41—50个酶切位点。综合15种限制内酶的酶切类型,在2只黑叶猴和2只灰叶猴中分别检出了两种限制性类型,并与其4个地理来源相对应。结合恒河猴和红面猴的资料,构建了4种猴科动物的分子系统树。结果表明,黑叶猴和灰叶猴种内的分歧分别始于30和35万年以前,两种叶猴的分离始于190万年以前,猴亚科和疣猴亚科的分离应早于1100万年。叶猴属在中国的扩散不是很晚才发生的。     

Role of the hepatocyte microtubular system in the excretion of bile salts and biliary lipid: implications for intracellular vesicular transport

The role of the hepatocyte microtubular system in the transport and excretion of bile salts and biliary lipid has not been defined. In this study the effects of microtubule inhibition on biliary excretion of micelle- and non-micelle-forming bile salts and associated lipid were examined in rats. Low-dose colchicine pretreatment had no effect on the baseline excretion of biliary bile salts and phospholipid in animals studied 1 hr after surgery (basal animals), but slightly retarded the excretion of tracer [14C]taurocholate relative to that of lumicolchicine-pretreated (control) rats. However, colchicine pretreatment resulted in a marked reduction in the excretion of 2 mumol/100 g doses of a series of four micelle-forming bile salts of differing hydrophilicity, but had no significant effect on the excretion of the non-micelle-forming bile salt, taurodehydrocholate. Continuous infusion of 0.2 mumol of taurocholate/(100 g.min) following 24 hr of biliary drainage (depleted/reinfused animals) resulted in physiologic bile flow with biliary excretion rates of bile salts, phospholipid, and cholesterol that were markedly inhibited (mean 33, 39, and 42%, respectively) by colchicine or vinblastine pretreatment. Excretion of tracer [14C]taurocholate also was markedly delayed by colchicine in these bile salt-depleted/reinfused animals. In contrast, colchicine did not inhibit bile salt excretion in response to reinfusion of taurodehydrocholate. Thus, under basal conditions, the microtubular system appears to play a minor role in hepatic transport and excretion of bile salts and biliary lipid. However, biliary excretion of micelle-forming bile salts and associated phospholipid and cholesterol becomes increasingly dependent on microtubular integrity as the transcellular flux and biliary excretion of bile salts increases, in both bile salt-depleted and basal animals. We postulate that cotransport of micelle-forming bile salts and lipids destined for biliary excretion, via an intracellular vesicular pathway, forms the basis for this microtubule dependence.     

Age dependence of hepatic transport in control and phenobarbital-pretreated rats.

Eosine is excreted in rat bile unchanged, which makes it suitable for the study of age dependent changes in hepatic uptake and excretion. Bile flow was approximately 40 μl/kg/min in 20-day-old rats and twice as high in 30-day-old animals. In 60- and 120-day-old rats the bile volume was decreased, moreover in 220-day-old ones it fell to the level of 20-day-old rats. The biliary excretion of eosine (150 μmol/kg i.v.) was highest in 60-day-old rats, however, the biliary flow reached its peak in 30-day-old rats. After phenobarbital (PB) pretreatment (75 mg/kg i.p. daily for five days) each age group showed enhancement in liver weight and bile volume. On the other hand, the hepatic concentration of eosine did not change after PB pretreatment caused an increase in the biliary excretion of eosine in 30-, 60-, 120- and 220-day-old rats but no significant change in 20-day-old animals. Our results indicate that the hepatic transport in young rats was immature and was not induced by PB. However, PB can increase the low excretion rate in old rats.     

Somatostatin release induced by gastrin-releasing peptide in man. Effect of proximal gastric vagotomy and cholinergic blockade

The influence of intragastric pH on the basal release of somatostatin has been studied in healthy controls and in duodenal ulcer patients. In addition the somatostatin response to gastrin-releasing peptide infusion has been evaluated both regarding the effect of intragastric pH and the influence of vagal innervation and muscarinic blockade. No difference was found in basal blood levels, when changing the intraluminal pH, although a slightly higher basal somatostatin concentration was noticed in patients with duodenal ulcer disease. Neither proximal gastric vagotomy nor cholinergic blockade had any effect on basal somatostatin concentrations. GRP infused in stepwise increasing doses from 20 pmol/kg/h to 400 pmol/kg/h induced a small but significant response. This effect of GRP was most evident, when the stomach was perfused with 0.1 M HCl. The small, somatostatin response to GRP infusion was not influenced by vagal denervation of the parietal cell area, neither by cholinergic blockade. Despite the previously observed effects of vagotomy and cholinergic blockade on gastrin release induced by GRP, a corresponding inverse effect on somatostatin is not apparent.     

Substance P in the control of extrahepatic biliary motility in the cat

The effects of regional intra-arterial injections of substance P (SP) or efferent electrical stimulation of the vagal nerves on feline extrahepatic biliary motility were studied in anesthetized cats using a constant perfusion model. Each of these procedures elicited contractile motor responses of the gallbladder and the sphincter of Oddi. Since SP is present in feline vagal axons, these findings may indicate a role of SP in the vagal motor control of biliary motility. Immunocytochemically neurons with SP-like immunoreactivity were found in the smooth muscle layers of the biliary tree as well as adjacent to acetylcholinesterase-positive ganglion cells indicating either direct activation of smooth muscle cells and/or indirect activation via cholinergic neurons. Depending on the type of stimulation different SP mechanisms were demonstrated; exogenous SP induced contraction of both the sphincter and the gallbladder which were probably direct (resistant to atropine but sensitive to a SP analogue), while vagal stimulation elicited contraction of both regions via a mechanism sensitive to atropine and to a SP analogue.     

Effect of taurochenodeoxycholate or tauroursodeoxycholate upon biliary output of phospholipids and plasma-membrane enzymes, and the extent of cell damage, in isolated perfused rat livers.

Isolated perfused rat livers were used to study the effects of taurochenodeoxycholate (TCDC) and tauroursodeoxycholate (TUDC) upon some aspects of biliary composition. After depletion of the endogenous bile salt pool of the liver, introduction of either bile salt brought about increases in bile flow, bile salt output and biliary phospholipid output. Taurochenodeoxycholate needed a lower biliary concentration to produce phospholipid output than did tauroursodeoxycholate. TCDC perfusion caused a substantial output of plasma-membrane enzymes (5'-nucleotidase and alkaline phosphodiesterase) into the bile, whereas TUDC caused little output of either enzyme; this may represent a characteristic difference between the effects of the two bile salts on the hepatobiliary system. The results from TUDC perfusion indicate also that much of the output of biliary phospholipid promoted by bile salts, may be independent of the output of plasma-membrane enzymes promoted by bile salts.     

The biliary excretion of sulfbromophthalein in the pig

The characteristics of the hepatic metabolism of Sulfbromophthalein (BSP) have not been described previously for the pig. This is an important deficiency, since the pig is particularly suitable for studies of hepatic physiology and pharmacology which might apply to man. The aim of these experiments was to establish the pattern of serum clearance and biliary excretion of BSP and to determine that dose which would produce a maximal concentration in bile. A dose response and pattern of biliary excretion of BSP was studied at three dose levels administered either as a single bolus of a continuous infusion. All experiments were performed in conscious, conditioned pigs. The patterns of serum clearance and biliary excretion were found to be similar to other laboratory animals and to man. Maximary biliary concentration of BSP was achieved by a single bolus of 5-9 mumol/kg or a constant infusion of 0-59 mumol/kg/min. At these dose levels no significant alteration in bile flow was demonstrated nor was there any correlation between bile flow and BSP excretion. Supra-maximal doses produced a significant increase in bile flow and with these doses there was a significant positive correlation between bile flow and BSP excretion.     

Enzymes and proteins in bile. Variations in output in rat cannula bile during and after depletion of the bile-salt pool.

The protein concentration in bile from several species is reported. The changes in output of protein, bile salts and several enzymes have been followed in rat bile over a 48 h cannulation period. Bile-salt concentration dropped rapidly owing to interruption of the enterohepatic circulation but the output of protein, lysosomal enzymes [acid phosphatase (EC 3.1.3.2) and beta-D-glucuronidase (EC 3.2.1.31)] and plasma-membrane enzymes [5'-nucleotidase (EC 3.1.3.5) and phosphodiesterase I (EC 3.1.4.1)] was maintained. Liver cell damage, monitored by output of lactate dehydrogenase, was very low throughout. Protein, lysosomal enzymes and plasma-membrane enzymes showed different patterns of output with time, but all showed a net increase between 12 and 24 h. The output of lysosomal and plasma-membrane enzymes was between 1 and 5% of the total liver complement over the first 24 h; if inhibition by biliary components is taken into account the output of some of these enzymes, particularly acid phosphatase, may be greater. Ultracentrifugation of bile showed that as the concentration of bile salts decreases the proportion of plasma-membrane enzymes in a sedimentable form increases. The results are discussed in relation to other studies of biliary proteins and to studies of the perturbation of membranes and cells with bile salts.     

Interleukin-6 (IL-6) and acute-phase proteins in rats with biliary sepsis.

We measured serum interleukin-6 (IL-6) and acute-phase proteins, alpha 1-acid glycoprotein (AGP) and alpha 2-macroglobulin (alpha 2M), after a retrograde intrabiliary bacterial infection in rats with biliary obstruction. Maximum serum IL-6 was obtained at 6 h in rats following inoculation of bacteria (10(6) CFU/ml E. Coli) in the bile duct and it was higher than that observed in rats undergoing a bile duct ligation or a laparotomy. There was a strict relationship between the level of IL-6 at 6 h and the modified levels of AGP and alpha 2M at 48 h. AGP and alpha 2M levels were the highest in sera of rats with bile duct infection as compared with those found in sera of rats with bile duct ligation or laparotomy. After inoculation of E. Coli or E. Fecalis, blood IL-6 level was always higher at 6 h in inferior vena cava as compared with that found in the supra hepatic vein. These results indicate that IL-6 is synthesized after a biliary sepsis and that its blood level is higher in the systemic circulation than in the local circulation.     

Biliary excretion of [C]succinylsulphathiazole in the rat and rabbit

1. After intravenous injection about 30% of the dose (20mg./kg.) of succinylsulphathiazole is excreted unchanged in the bile in 3hr. by the rat, whereas only about 1% is excreted by the rabbit. When the renal pedicles are ligated the biliary excretion of succinylsulphathiazole in the rat increases to about 80% of the dose, but in the rabbit under these conditions the biliary excretion is only 2% of the dose. 2. In the rat, the sulphonamide readily enters the liver and biliary excretion occurs against a concentration gradient from liver to bile; further, the excretory process can be saturated, and can be depressed by the simultaneous administration of phenolphthalein glucuronide or bile salts. 3. In the rabbit, these conditions have not been found; succinylsulphathiazole does not readily enter the liver from the plasma, there is no transfer of the drug from the liver cells to the bile against a concentration gradient, and no saturation or depression of the biliary excretion of succinylsulphathiazole is found. 4. It is suggested that two factors responsible, at least partly, for the low biliary excretion of succinylsulphathiazole in the rabbit are the poor entry of the sulphonamide into the liver in this species and a deficiency of the concentrative mechanism for its excretion in the bile.     

Role of motilin in the control of intestinal absorption, and gastric and biliary secretions in the rat

The effects of motilin on proline absorption and gastric and biliary secretions were examined in the rat. Prolonged intravenous administration of motilin (50 pmol/kg/min) significantly inhibited (P < 0.05) proline transport across the jejunum and reduced basal acid secretion to 40% of control value. The same concentration of motilin induced choleresis and increased bile output by 32%. Incubation of intestinal strips with different concentrations of motilin produced a dose-dependent inhibitory pattern of proline accumulation in the intestinal cells.     

Bile acid secretion and pool size during phenobarbital induced hypercholeresis

An increase in bile flow after phenobarbital administration occurs in the rat and other species; however, the mechanism(s) of the choleretic effect is incompletely understood and the role of the increase in liver weight is controversial. We therefore measured bile flow, bile acid secretion and pool size in male Sprague-Dawley rats pretreated with phenobarbital (75 mg/kg/day) for 6 days; liver weight, liver cell volume and DNA content were also evaluated. Phenobarbital treatment increased liver weight and mean hepatocyte volume by 39 and 26%, respectively, while total DNA content did not change, thus indicating that the hepatomegaly results principally from hypertrophy rather than hyperplasia. Bile flow was significantly higher in treated rats when expressed per unit of body weight (64.6 +/- 2.4 (S.E.) vs 53.3 +/- 1.6 microliter/min/kg; P less than 0.05) but was unchanged when expressed per gram of liver (1.40 +/- 0.04 vs 1.37 +/- 0.06 microliter/min/g; P greater than 0.5). The initial bile acid secretion rate and pool size were both significantly reduced in the phenobarbital group compared to controls (1224.2 +/- 110.4 vs 1656.6 +/- 163.2 nmol/kg/min and 562.8 +/- 41.5 vs 814.3 +/- 78.3 mumol/kg; both P less than 0.05), whereas the basal synthetic rate was unchanged. These findings suggest that the enlarged, phenobarbital-treated hepatocyte produces more bile than the normal cell, despite the decreased secretion of bile acids. Therefore, the drug-induced choleresis involves a selective increase in the bile acid-independent fraction of bile flow.     

Biliary lipid secretion in hypercholesterolemia.

A report on the effects of primary bile acid ingestion alone or in combination with plant sterols on serum cholesterol levels, biliary lipid secretion, and bile acid metabolism. Biliary bile acid and cholesterol secretion were measured in four patients with type IIa hypercholesterolemia before and after randomized treatment periods. During these periods either a bile acid mixture (cholic-chenodeoxycholic 2:1, a proportion similar to that endogenously synthesized in health), at a level of 20 mg/kg, or the same mixture plus sitosterols, 200 mg/kg, was fed. Serum cholesterol and the cholesterol saturation of fasting-state bile was also measured. Pretreatment biliary lipid secretion was within normal limits. Bile acid kinetic measurements were also recorded before treatment and showed that cholic acid synthesis was disproportionately decreased relative to that of chenodeoxycholic acid, a finding previously reported by others. Administration of the bile acid mixture increased biliary bile acid secretion in 3 of 4 patients, but did not influence biliary cholesterol secretion. The combination of sitosterol-bile acid, however, caused a relative decrease in cholesterol secretion in bile, and fasting-state bile became unsaturated in all patients. No change in fecal neutral sterol excretion occurred during the beta-sitosterol-bile acid regimen, suggesting that simultaneous bile acid feeding blocks the compensatory increase in cholesterol synthesis known to be induced by beta-sitosterol feeding in hypercholesterolemic patients. Serum cholesterol levels also fell modestly during the sitosterol-bile acid regimen, the decrease averaging 15%. We conclude that the abnormally low rate of bile acid synthesis in patients with type IIa hyperlipoproteinemia does not influence biliary lipid secretion; that increasing the input of the two primary bile acids into the enterohepatic circulation does not increase biliary cholesterol secretion or lower serum cholesterol levels in such patients; and that the usual increase in cholesterol synthesis induced by beta-sitosterol feeding does not occur if bile acids are administered simultaneously.