Further characterization of membrane proteins involved in the transport of organic anions in hepatocytes Comparison of two different affinity labels: 4,4′-diisothiocyano-1,2-diphenylethane-2,2′-disulfonic acid and brominated taurodehydrocholic acid

4,4′-Diisothiocyano-1,2-diphenylethane-2,2′-disulfonic acid (H₂DIDS) known as an irreversible inhibitor of the anion transport in red blood cells (Cabantchik, Z.I. and Rothstein, A. (1972) J. Membrane Biol. 10, 311–330) blocks also the uptake of bile acids and of some foreign substrates in isolated hepatocytes (Petzinger, E. and Frimmer, M. (1980) Arch. Toxicol. 44, 127–135). [³H]H₂DIDS was used for labeling of membrane proteins probably involved in anion transport of rat liver cells. The membrane proteins modified in vitro by [³H]H₂DIDS were compared with those labeled by brominated taurodehydrocholic acid. The latter is one of a series of suitable taurocholate derivatives, all able to bind to defined membrane proteins of hepatocytes and also known to block the uptake of bile acids as well as of phallotoxins and of cholecystographic agents (Ziegler, K., Frimmer, M., Möller, W. and Fasold, H. (1982) Naunyn-Schmiedeberg's Arch. Pharmacol. 319, 254–261). The radiolabeled proteins were compared after SDS-electrophoresis with and without reducing agent present, solubilization by detergents, two-dimensional electrophoresis and after separation of integral and peripheral proteins. Our results suggest that the anion transport system of liver cells cannot distinguish between bile acids and the anionic stilbene derivative (DIDS). The labeling pattern for both kinds of affinity labels was very similar. Various combinations of separation techniques gave evidence that the radiolabeled membrane proteins are not subunits of a single native channel protein.     

500 MHz H-NMR studies of bile salt-phosphatidylcholine mixed micelles and vesicles Evidence for differential motional restraint on bile salt and phosphatidylcholine resonances

¹H nuclear magnetic resonance (NMR) spectra at 500 MHz have been obtained for taurocholate/egg phosphatidylcholine mixtures of varying composition. The excellent chemical shift dispersion permits identification of most resonances for each component. This high-resolution character of the NMR spectra is retained until the phosphatidylcholine (PC) mole fraction exceeds 60–70% (the exact limit depends on ionic strength). ¹H linewidths have been monitored as a function of solute composition in order to evaluate trends in local molecular mobility of each component as the distribution of aggregate particles is varied, and to examine the effects of added NaCl in altering micellar size and shape. Although prior light scattering studies (Mazer, N.A., Benedek, G.B. and Carey, M.C. (1980) Biochemistry 19, 601–615) and our own work indicate a 6-fold increase in particle hydrodynamic radius from pure taurocholate micelles to 1 : 1 taurocholate/PC mixtures containing 150 mM NaCl, both lipid components retain substantial motional freedom and exhibit narrow NMR signals in this compositional region. As the solubilization limit for PC is approached (approx. 2:1 PC:taurocholate), differential behavior is observed for the two components: the motion of taurocholate becomes preferentially restricted, while polar portions of the PC remain mobile until large multilayers predominate.     

Light and electron microscopy of the ducts and their subepithelial tissue in the rat ventral prostate

Summary The ducts of the rat ventral prostate have been studied by light and electron microscopy for elucidation of their role in prostatic function. The epithelium of the main duct consists of simple columnar cells and polymorphic basal cells. The columnar cells show no indication of secretory activity. The basal cells contain bundles of filaments of 5–6 nm thickness and numerous pinocytotic vesicles. The ducts are surrounded by layers of circular smooth muscle cells interspersed with nerve axons. On ultrastructural grounds the ducts do not appear to secrete material into the seminal fluid, but apparently the muscular coat actively helps drain the gland during ejaculation.     

Miniature Ag−AgCl electrode for voltage clamping of theAmbystoma collecting duct

Summary We have developed a miniature silver-silver chloride electrode. The outer diameter of the electrodes averaged 22 m and the input resistance 8.8 k. Since the core of the electrode is a glass fiber, the problem of the extreme malleability of a small diameter silver fiber is circumvented. The properties of the electrode permit us to insert it into short (600 m) fragments of the amphibian collecting duct while they are being perfusedin vitro. The passage of currents in the range of 0 to 6×10^–8 amperes allowed us to voltage clamp the nephron fragment between +20 and –20 mV. The current-voltage plots are linear over this range. Two lines of evidence suggest that the voltage clamp is homogeneous. First, the voltage measured at the perfusion end during a voltage-clamp experiment of the tubule is not significantly different from that measured at the collecting end. Secondly, the specific resistance of collecting ducts estimated from the core conductor analysis is 3.3±0.8×10⁴ cm, a value not significantly different from that computed from the current-voltage plots as determined with the Ag–AgCl electrode, 3.0±0.5×10⁴ cm. This method permits precise control of both the ionic and electrical gradients across fragments of the amphibian collecting duct.     

Permeability characteristics of muscle membrane

Unidirectional flux rates of saturated fatty acids, saturated alcohols, and bile acids were measured in an intact rat diaphragm preparation. The logarithm of the permeability coefficients for fatty acids containing from five to ten carbon atoms was a linear function of the number of carbon atoms in the fatty acid chain. Incremental free energies of solution were +336 cal · mol⁻¹ for the addition of a hydroxyl group and −258 cal · mol⁻¹ for the addition of a methylene group. These incremental free energies were similar to those obtained by other investigators in other animal tissues, and our data suggest a structural similarity between membranes in different tissues and in different species. The muscle membrane exhibited anomalously high permeabilities for fatty acids containing less than five carbon atoms. Since muscle lacks tight junctions, this result suggests that small non-electrolytes traverse polar regions or aqueous pores within the cellular membrane.     

Dictyocaulus viviparus: Migration in agar of larvae subjected to a variety of physicochemical exposures

Dictyocaulus viviparus larvae were exposed to ox bile and CO₂ at intervals during their cultivation to the infective stage. Preinfective and young infective larvae were stimulated by CO₂. Bile slightly inhibited preinfective larvae, but stimulated the infective stage. Old coiled, resting infective larvae were stimulated by bile down to a concentration of 10 ppm of bile dry matter, by vertebrate biles of pig, sheep, newborn calf, cow, guinea pig, dog, and chicken, as well as by defatted bile dry matter and by glyco-, tauro-, glycodeoxy-, and taurodeoxycholates. Continuous bile exposure appeared necessary to maintain high larval activity. A high pCO₂ as well as a low redox potential potentiated the effect of bile, but had no effect alone. Exposure to pepsin-HCl and to trypsin had only a minor stimulatory effect.     

Role of the duodenum in motilin release

In order to study the regulatory mechanism of motilin release, plasma motilin was measured in healthy dogs during the fasting state and after the ingestion of ordinary nutrient. Fasting plasma motilin levels were found to fluctuate intermittently, but ingestion of a meal completely abolished the intermittent motilin release and resulted in low motilin levels lasting for 6–8 h. To clarify the role of the duodenum in this motilin release, an operation was performed in five dogs by which we excluded from the alimentary tract the upper half of the small intestine not including the duodenum from a point 2 cm below the larger pancreatic duct. After this operation meal ingestion still caused a decrease in plasma motilin levels. However, after a modified version of the operation was performed in 5 other dogs by which the upper half of the small intestine together with the duodenum was transected at the pyloric ring, plasma motilin was not suppressed by meal ingestion. These results suggest that motilin secretion is regulated by nutrient ingestion and that the passage of nutrients through the duodenum plays a important role in its regulation.     

Light- and electron-microscopic radioautographic study of glycoprotein secretion in the granular duct of the submandibular gland of the male mouse

Summary L-³H-fucose was injected intravenously into adult male mice, after which, at different time intervals, the submandibular glands were removed and processed for light-and electron-microscopic radioautography. This radio active hexose was taken up by newly synthesized glycoproteins in the cells lining the granular ducts which were maximally labeled at 4 h after injection. Between 4 and 72 h the amount of labeled glycoproteins decreased moderately indicating that these macromolecules undergo a slow renewal. The main subcellular site of incorporation of 3 H-fucose into glycoproteins was the Golgi apparatus. From this organelle labeled glycoproteins were transferred to small secretory granules (diameter up to 1.0 m) located not only near the Golgi region but also throughout the apical cytoplasm. At 1 h after injection the concentration of label reached a maximum in the small secretory granules and labeling of medium (diameter between 1.1 and 2.0 m) and large (diameter over 2.0 m) granules was very low. At this postinjection interval the secretion product inside the lumen of the duct was already labeled. Between 1 and 72 h after injection the concentration of radioactivity in the small secretory granules decreased intensely while increasing in the medium and in the large ones. The concentration of fucose label reached a maximum in the medium secretory granules at 24 h and in the large ones at 72 h after injection. Additional experiments using mice previously injected with 4 intraperitoneal doses of ³H-fucose given 3 h apart demonstrated that the large granules undergo a very slow renewal. Some were found to be labeled as long as 28 days after administration of ³H-fucose. Recorded in this latter series of experiments was the labeling pattern of dense bodies that were regularly visualized in the cells lining the granular ducts. Their significance in the secretory process is discussed. In conclusion, newly synthesized glycoproteins are transferred from the Golgi apparatus to small secretory granules which carry a readily releasible pool of these macromolecules to the lumen of the duct. The small secretory granules also transfer newly synthesized glycoproteins to medium and large secretion granules which store a pool that is released very slowly. This characterizes the large secretory granules as the intracellular sites of storage of secretion products. The results of this investigation were correlated with the knowledge about the chemical composition of the different macromolecules that are known to be synthesized by the secretory cells of the granular ducts of the submandibular gland of the mouse.     

A scanning electron microscopic study of the liver of the monkey Macaca speciosa

Summary The bile canalicular network of the monkey was studied by fracturing fixed liver tissue and examination by scanning electron microscopy. Bile canaliculi do not differ remarkably from those described in other species. Their course and luminal diameter vary, depending on their position in the liver lobule. In one specimen the continuity of a canaliculus with a terminal bile ductule (canal of Hering) is presented. Several constrictions occur in this part of the ductular lumen. The interlobular bile duct wall shows two kinds of niches. A single cilium arises from a primary niche. The walls of secondary niches contain numerous primary niches. Simple columnar epithelium lines the common bile duct, the main pancreatic duct and the gallbladder. A common feature is the presence of microplicae on their lateral cell surfaces.

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Zusammenfassung Das Netzwerk der Gallekanälchen beim Affen wird durch Brechen von fixiertem Lebergewebe sichtbar. Strukturen der Portalfelder und der extrahepatischen Gänge werden durch Schneiden von Gewebe dargestellt. DieGallekanälchenunterscheidensichnichtwesentlich von den bei anderen Spezies beschriebenen. Ihr unterschiedlicher Verlauf und Lumendurchmesser hängen von ihrer intralobulären Lage ab. Die Kontinuität eines Gallekanälchens mit einem Ductulus (Heringscher Kanal) wird in einem Fall dargestellt. Im ductulären Lumen kommen mehrere Konstriktionen vor. Die Wand der interlobulären Gallengänge weist zwei Arten von Nischen auf. Eine Einzelzilie kommt aus den primären Nischen. Sekundäre Nischen bestehen aus mehreren primären Nischen. Einschichtiges hochprismatisches Epithel kleidet den Ductus choledochus, den Ductus pancreaticus und die Gallenblase aus. Ein gemeinsames Merkmal ihrer lateralen Zelloberflächen sind Mikroplicae.

     

Ultrastructural and histochemical observations on the tegument of Gastrodiscoides hominis (Paramphistoma: Digenea).

The tegument of the paramphistome, Gastrodiscoides hominis, is basically similar to that of other digeneans. It is folded into concentrically arranged furrows and ridges bearing numerous tightly packed tubercules, and extends into the oral cavity. An area of specialized tegument is present on the ventral surface, anterior to the disc region. Mitochondria are absent from the tegumental syncytium and underlying tegumental cells, suggesting that the tegument may serve principally as a protective layer rather than in active uptake phenomena. However, extensions of the lymph and parenchyma systems are closely associated with the base of the tegumental syncytium and may provide ATP for active processes. Ciliated and non-ciliated sensory papillae are present, particularly around the oral opening. Numerous lymph channels are present in the sub-tegument and may be involved in osmoregulation.