Gallbladder CCK receptors: species differences in glycosylation of similar protein cores.

Receptors for cholecystokinin (CCK) on gallbladder muscularis smooth muscle have different apparent sizes in man (Mr = 85,000-95,000) and cow (Mr = 70,000-85,000). In this work, these receptors were demonstrated to represent N-linked complex glycoproteins with Mr = 43,000 protein cores, based on lectin-affinity chromatography and the deglycosylation of bands affinity labeled with 125I-D-Tyr-Gly-[(Nle28,31, pNO2-Phe33)CCK-26-33] using neuraminidase, O-glycanase and endoglycosidases H and F. Similarities in the core proteins were further demonstrated by Staphylococcus aureus V8 protease peptide mapping, in which both proteins yielded similar fragment patterns. Thus, gallbladder CCK receptors present in man and cow are both N-linked complex glycoproteins, with different carbohydrate domains and similar protein cores.

Bile acid pool size and gallbladder storage capacity in gallstone disease.

Gallstone patients have a reduced total bile acid pool size. To analyze mechanisms behind this, the relation between gallbladder storage capacity and bile acid pool size was studied in 20 gallstone patients, 15 women and 5 men. At cholecystectomy bile was aspirated from the gallbladders, and the volume of bile and concentration of bile acids were recorded. The total bile acid pool size was at the same time estimated by an isotope dilution technique. A significant positive correlation between gallbladder volume and bile acid pool size was found (r = 0.45). The correlation between bile acid concentration in the gallbladders and pool size was, however, not significant (r = 0.24). It is suggested that gallbladder storage capacity may be a determinant of bile acid pool and that a diminished pool size, as seen in gallstone disease, may to some extent be caused by gallbladder fibrosis and shrinkage.

Isolation of a potent cholesterol nucleation-promoting activity from human gallbladder bile: role in the pathogenesis of gallstone disease.

Gallbladder bile contains nucleation-promoting activity that binds to concanavalin A. The activity was found in gallbladder bile from cholesterol gallstone patients but also in gallbladder bile from patients without stones and patients with pigment stones. Bile from patients with multiple cholesterol gallstones contained high concanavalin A-binding nucleation-promoting activity. The activity was much lower in bile samples from pigment stone patients, patients without stones and patients with a solitary cholesterol stone. Serum contained very little activity and no concanavalin A-binding nucleation-promoting activity could be demonstrated in gallbladder mucosa. This suggests that concanavalin A-binding nucleation promoter is produced in the liver or bile duct epithelium. The activity was fully resistant to digestion with pronase but was heat labile and could be destroyed by prolonged incubation with a mixed glycosidase preparation indicating that sugar residues are important for this activity. On a Superose 12 gel permeation column, promoting activity eluted in two major peaks at apparent molecular weights of 150 +/- 30 kD (n = 5) and less than 5 kD respectively. The mobility on the column was not influenced by pronase digestion. The factor with the higher molecular weight could be isolated further by polyacrylamide gel electrophoresis under nondenaturing conditions. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent molecular weight of the glycoprotein was 130 kD. In conclusion, gallbladder bile contains nucleation-promoting activity that binds to concanavalin A. The activity is increased in bile from patients with multiple cholesterol gallstones and could therefore play an important role in the pathogenesis of gallstone disease.

Free fatty acids stimulate mucin hypersecretion by rabbit gall-bladder epithelium in vitro.

1. Mucin, a high-molecular-weight glycoprotein secreted by the gall-bladder and biliary duct epithelium, is a well-known nucleation-prompting factor in experimental and human gall-stone disease. 2. Free fatty acids when incubated in vitro (micellar suspension with 1 mmol/l Tween 40) with rabbit gall-bladders can promote abundant mucus secretion. 3. The hexosamine content of rabbit gall-bladder walls, measured by gas-liquid chromatography, was significantly higher in gall-bladders incubated with fatty acids than in control tissues. 4. The biochemical data were supported by ultrastructural findings showing numerous droplets with a translucent content in the perinuclear cytoplasm. Exocytosis was also seen in treated gall-bladders, confirming the secretory nature of the vesicles. 5. These results suggest that free fatty acids, which appear in high amounts when bile lecithins are hydrolysed by phospholipase, play an active role in the gall-stone formation.

[A method for assessing lipid peroxidation in a biological substrate]. / Metod otsenki perekisnogo okisleniia lipidov v biologicheskom substrate.

A new method for the assessment of lipid peroxidation permits simultaneous assays of total lipids and formed malonic dialdehyde in the examined chloroform substrate; this will help define the criteria of cellular membrane destruction in various biological media of the body.

GABA-receptors in peripheral tissues.

Gamma-aminobutyric acid (GABA) and its receptors are found in a wide range of peripheral tissues, including parts of the peripheral nervous system, endocrines, and non-neural tissues such as smooth muscle and the female reproductive system. In all these, both GABAA- and GABAB-receptor types are found, with good evidence for a physiological role in the gut, pancreatic islets and the urinary bladder. In some tissues, the pharmacology of GABA-induced actions is quite atypical and should be further explored with the newer ligands and modulators for GABAA- and GABAB-receptors.

Immunohistochemical localization of proteoglycans in interstitial elements of human pancreas and biliary system.

The immunohistochemical localization of large proteoglycan and small proteoglycan was observed, using antibodies 2B1 and 6B6 (Sobue et al., 1988, 1989a), in fetal and adult pancreas and biliary system as well as in tumour tissues, obtained from 11 autopsies and 74 biopsies. The distribution of chondroitin 4- and 6-sulphate side chains, type I and IV collagen and elastin were also studied. In adult pancreas and all the biliary tracts examined, periductal fibrous tissues consisted mainly of dermatan sulphate small proteoglycan with networks of fibrous elements, which were composed of large proteoglycan, elastin, type I collagen and type IV collagen. In the interstitial components of cystadenoma of pancreas and biliary duct carcinoma, similar small proteoglycan-rich components were relatively abundant, although large proteoglycan was present in much larger amounts than that in non-neoplastic adult tissues. In some cholangiomas, the extra- and intracellular hyaline globules formed by the carcinoma cells were found to contain chondroitin sulphate large proteoglycan, laminin and fibronectin. The distribution of proteoglycans was observed to be different in the arterial walls of the interlobular tissues of the adult and the fetal pancreas. The biological significance of large and small proteoglycans in the interstitial connective tissues was discussed.

[Immunohistochemical study of cancer-associated carbohydrate antigens in carcinoma of the biliary tract].

To reveal the cell-biological character of biliary tract cancer, localization and distribution of three cancer-associated carbohydrate antigens (CA19-9, sialyl SSEA-1, NCC-ST-439) and carcinoembryonic antigen (CEA) were studied immunohistochemically in 35 cases of gallbladder carcinoma, 21 of bile duct carcinoma, 16 of chronic cholecystitis, and 3 of normal gallbladder. 1) All carbohydrate antigens and CEA were present in 70-90% of the cases of gallbladder and bile duct carcinoma. In particular, NCC-ST-439 had the highest incidence of positive staining (95.2%) in bile duct carcinoma. 2) The mode of localization was diverse and was not fixed by the kind of antigen. Antigens flowing out to the surrounding stroma were affected by the rate of positive cells. 3) No significant correlation was observed between the histological type or degree of differentiation and tissue positivity. 4) The positivity of tissue CEA was higher in the cases with serous membrane invasion, gamma INF pattern, and neuro-, vascular-, and lymphatic invasion. 5) In chronic cholecystitis, CA19-9, NCC-ST-439, and CEA were stained in mucosal cells and/or metaplastic cells, while sialyl SSEA-1 was stained only in one case in the goblet cells and the cells with pseudopyloric metaplasia. None of the antigens were stained in normal gallbladders. These results suggest that these antigens may be useful in the diagnosis and therapeutic treatments in patients with biliary tract cancer.

A method for the simultaneous analysis of unconjugated and glycine-conjugated bile acids by capillary gas-liquid chromatography.

A method for the simultaneous analysis of unconjugated and glycine-conjugated bile acids by means of capillary gas-liquid chromatography without need for prior deconjugation is described. The method involves: i) the use of an aluminum-clad fused-silica capillary column coated with a very thin film (0.1 micron) of a highly thermostable bonded and crosslinked methyl polysiloxane, and ii) the analysis of the bile acids as their methyl ester-dimethylethylsilyl ether derivatives. This method, used to separate the major free and glycine-conjugated bile acids from human gall bladder bile, should be applicable for the analysis of other biological fluids.

Gallbladder mucin and cholesterol and pigment gallstone formation in hamsters.

We measured gallbladder mucin production by hamsters fed diets lithogenic for either cholesterol or pigment gallstones. In hamsters on the cholesterol stone diet, gallbladder production of 3H-glucosamine-labeled mucin was elevated two- and seven-fold after 1 and 3 weeks, respectively. After 1 week cholesterol crystals were seen in a mucus gel on the gallbladder surface. In hamsters on the pigment stone diet, gallbladder mucin production was significantly elevated after 1 and 3 weeks. The first precipitation of pigment crystals was in mucus in bile or on the gallbladder surface. Black pigment stones grew by agglomeration of pigment crystals enmeshed in mucus. In conclusion, gallbladder mucin production is increased before cholesterol or pigment stone formation, and the earliest deposition of crystals is in mucus in bile or on the gallbladder surface.

Biliary lipid composition and gallstone formation in rabbits fed on soy protein, cholesterol, casein and modified casein.

In four experimental groups, rabbits were fed on diets containing soy beans, soy beans plus cholesterol (1%, w/w), casein and modified casein for 8 weeks. Biliary lipid levels, lithogenic-index values and the rate of gallstone formation were determined. The highest mean relative concentrations (mol%) of cholesterol and phospholipid were found in the soy bean + cholesterol group, and the highest mean relative bile acid concentration was in the soy bean group. The lowest mean relative cholesterol and phospholipid values were found in the soy bean and modified casein groups respectively. The lowest mean relative bile acid level was in the soy bean + cholesterol group. The highest lithogenic index and rate of gallstone formation were in the soy bean + cholesterol group, and the lowest values were in the soy bean group. The modification of casein used was effective in decreasing the lithogenic effect of casein on gallstone formation.

Identification of 3 alpha,4 beta,7 alpha-trihydroxy-5 beta-cholanoic acid in human bile: reflection of a new pathway in bile acid metabolism in humans.

The chemical synthesis, nuclear magnetic resonance, and mass spectrometric characteristics of the first C-4 hydroxylated bile acid analogues are described. The data definitively confirm, for the first time, the identity of 3 alpha,4 beta,7 alpha-trihydroxy-5 beta-cholanoic acid in human fetal gallbladder bile. In addition, 3 alpha,4 beta,7 alpha-12 alpha-tetrahydroxy-5 beta-cholanoic was identified in the feces from healthy newborn infants many days after birth, indicating a hepatic origin for C-4 hydroxylation of bile acids. To our knowledge bile acids hydroxylated at the C-4 position of the steroid nucleus have never been previously recognized in any mammalian species. The finding of this novel bile acid which accounts for 5-15% of the total biliary bile acids in early gestation indicates that C-4 hydroxylation is a unique and important metabolic pathway in early human development.

Functional and biochemical characterization of the human gallbladder muscularis cholecystokinin receptor.

Gallbladders removed at cholecystectomy are a potentially useful source of human receptor for the gastrointestinal peptide hormone cholecystokinin (CCK). Seven healthy gallbladders (removed incidentally at time of resection of hepatic metastases) and 50 diseased gallbladders were studied. Cholecystokinin radioligand binding to an enriched plasma membrane preparation from these tissues was shown to be rapid, reversible, temperature-dependent, saturable, specific, and high-affinity. Computer analysis of equilibrium binding data using the Ligand program best fit a single class of binding sites with Kd = 1.0 +/- 0.1 nM (mean +/- SEM). This was similar in health and disease, with no apparent differences related to age, gender, or body habitus. The structural specificity for binding to this site correlated well with relative potencies for CCK-gastrin peptides to stimulate gallbladder contraction. To biochemically characterize this receptor, we used a battery of reagents, including "long" (125I-Bolton Hunter-CCK-33) and "short" 125I-D-Try-Gly-[(Nle28,31)CCK-26-33] probes that were cross-linkable through their amino terminus and a monofunctional probe with a photolabile group at its carboxyl terminus 125I-D-Tyr-Gly[(Nle28,31,pNO2-Phe33)CCK-26-33]. All probes specifically labeled a human gallbladder muscularis protein of Mr = 85,000-95,000, which was also independent of diagnosis. Labeling of this band was inhibited in a concentration-dependent manner by CCK-8 and by L-364,718. Thus, the CCK receptor present on the very common surgically removed human gallbladder is functionally and biochemically intact and is useful for further characterization.

Down-regulation of pancreatic growth and gallbladder contractility by bile salts.

Luminal sequestration of bile salts with cholestyramine and the oral administration of bile salts represent current forms of therapy for some diseases. We have recently reported that secretion of these salts exerts negative feedback control on the release of cholecystokinin (CCK). The purpose of this study was to examine the effects of long-term alterations of luminal concentrations of bile salts on CCK target organs, the pancreas and gallbladder. The bile salt pool in adult guinea pigs was either enriched by feeding 0.5 percent sodium taurocholate or depleted by feeding 2 percent cholestyramine. Pancreatic growth, gallbladder contractility, the concentration of cholecystokinin receptors in the gallbladder muscle, and meal-stimulated plasma levels of cholecystokinin were significantly stimulated by feeding the bile salt sequestrant cholestyramine to guinea pigs. Administration of the bile salt taurocholate produced the opposite effects. Inhibition of CCK release by bile salts and up-regulation of CCK receptors by CCK may be the mechanisms responsible for the actions of bile salts on CCK target organs.

[Determination of bile acids in bear gall drainage by thin layer chromatographic scanning].

A method for the quantitative determination of three main bile acids, cholic acid (CA), ursodesoxycholic acid (UDCA) and chenodesoxycholic acid (CDCA), in bear gall, drainage from bear gall and bear gallstone is described. Experimental conditions: TLC Scanner CS-910, fluorescence scanning, lambda ex 470 nm and lambda em 550 nm for CA; lambda ex 380 nm and lambda em 450 nm for UDCA and CDCA. The results showed that the contents of UDCA and CDCA in bear gall drainage were higher than those in bear gall. The method is simple, rapid and sensitive. The reproducibility is good. The average recovery is 98.4%, CV is 1.4%.

[Crystal morphology of cholesterol monohydrate in cholesterol gallstones and gallbladder bile].

To understand the pathogenesis of cholesterol gallstones from a crystallographic point of view, we investigated gallstones and precipitates in gallbladder bile by the optical method used in crystallography. First, ground thin sections of cholesterol gallstones in 28 cases were examined by a polarizing microscope. The crystalline fragments which were peeled off from gallstones by sonication were also examined in the same manner. Secondly, the cholesterol monohydrate crystals in gallbladder bile were examined by a polarizing and phase contrast microscope. The bile samples were obtained from 15 patients with cholesterol gallstones by the needle puncture of gallbladder during surgery. The results of observation were as follows. 1) The cholesterol gallstones were composed of many small plated cholesterol monohydrate crystals aggregated radially. 2) Each small plated crystal was grown by the lateral growth mechanism in bile. Spiral growth patterns were often observed on the (001) faces. 3) The cholesterol gallstones were formed by two successive mechanisms: The aggregation of cholesterol monohydrate crystals and the lateral growth of each single crystal.

Culture behavior of healthy bovine gallbladder muscularis smooth muscle cells.

In contrast to the extensive experience culturing vascular smooth muscle cells, little is known about the culture behavior of gastrointestinal smooth muscle cells. In this work, we have studied smooth muscle cells from bovine gallbladder muscularis in culture. Properties reflecting their state of differentiation, including cellular morphology, multicellular arrangements, intermediate filament protein expression, and content of contractile proteins, were studied after dispersed cell preparations were placed into short- and long-term culture on a variety of substrates. Immunocytochemical analysis of intact healthy gallbladder wall demonstrated that the muscularis smooth muscle cells express actin and the intermediate filament protein desmin, while being vimentin-negative. In this tissue they are, however, surrounded by sheets of vimentin-positive fibroblasts. Optimal microdissection of mucosa and serosa from the muscularis therefore still produced a combination of smooth muscle cells and fibroblasts; however, multiple strategies for enriching the yield and purity of muscularis smooth muscle cells for culture were partially successful. Like vascular smooth muscle cells in culture, these visceral smooth muscle cells rapidly underwent morphological dedifferentiation, losing their contactile phenotype. By 5 days in culture, the desmin-positive muscle cells took on a spread, fibroblast-like morphology, likely representing modulation to a proliferative, dedifferentiated state. After long-term culture, the muscle cells were observed to regain some markers of differentiation, but they were never observed to attain complete morphological and functional redifferentiation.

Affinity labeling the bovine gallbladder cholecystokinin receptor using a battery of probes.

Although the gallbladder was the first recognized target of the peptide hormone cholecystokinin (CCK) and is a physiologically important target, only one preliminary report of the biochemical characterization of this receptor exists. Recently, a series of molecular probes for the affinity labeling of different domains of the pancreatic CCK receptor have been developed. In this work we report the application of several of those probes toward the biochemical characterization of the bovine gallbladder muscularis receptor. These include "long" (125I-Bolton-Hunter-CCK-33) and "short" (125I-D-Tyr-Gly-[Nle28,31)CCK-(26-33)]) probes chemically cross-linkable through their amino-terminal amino groups and monofunctional probes with their photolabile moieties at their amino terminus (2-diazo-3,3,3-trifluoropropionyl-125I-D-Tyr-Gly-[(Nle28,31) CCK-(26-33)]) and carboxyl terminus (125I-D-Tyr-Gly-[(Nle28,31,pNO2-Phe33)CCK-(26-33)]), that span the receptor-binding region. Each of these bound specifically and saturably to a preparation enriched in plasma membranes from bovine gallbladder muscularis (mean inhibitor constants: 5.2, 1.1, 0.8, and 1.8 nM, respectively). A major relative molecular weight (Mr) 70,000-85,000 band was specifically and reproducibly labeled with the appropriate apparent affinity by each of the probes, whereas labeling of minor bands of Mr 40,000-50,000, Mr 92,000, Mr 120,000, and Mr 200,000 was dependent on cross-linker type or concentration. These observations support the identification of the Mr 70,000-85,000 protein as the bovine gallbladder CCK-binding subunit and, since this is a different size from the pancreatic CCK-binding subunit, provide biochemical evidence for molecular heterogeneity of peripheral CCK receptors.