Vesicular transport of horseradish peroxidase during chronic bile duct obstruction in the rat.

The vesicular transport system for biliary secretion of plasma-derived proteins was investigated in rats with chronic bile duct obstruction. Horseradish peroxidase, previously demonstrated to be a suitable tracer for vesicular transport, was employed in these studies. Both the time course of horseradish peroxidase secretion into bile and the morphological events in its uptake, transport, and biliary secretion were found to proceed in a manner essentially identical to that of sham-operated control animals. In addition, fragmentation of hepatocytes leading to sloughing into bile of large pieces of cytoplasm bearing horseradish peroxidase-containing endocytic transport vesicles frequently was observed in the cholestatic animals. These data suggest that the vesicular transport system for the secretion into bile of plasma-derived proteins remains intact and functional during chronic bile duct obstruction and that another mechanism, possibly fragmentation and solubilization of hepatocyte membranes followed by regurgitation of proteins released from endocytic vesicles, may be responsible for the elevation of biliary proteins within plasma seen during cholestasis.

Effects of cytochalasin D and colchicine on the uptake, translocation, and biliary secretion of horseradish peroxidase and [14C]sodium taurocholate in the rat.

The roles of microfilaments and microtubules in the hepatocellular uptake, translocation, and biliary excretion of horseradish peroxidase and [14C]sodium taurocholate were investigated using the microfilament inhibitor cytochalasin D and the microtubule inhibitor colchicine. In separate studies, horseradish peroxidase and [14C]taurocholate were injected separately as a bolus into rat portal veins after treatment with cytochalasin D or colchicine, and bile was collected and analyzed for the presence of horseradish peroxidase and [14C]taurocholate. Cytochalasin D treatment depressed bile flow by approximately 50% and decreased the biliary secretion of [14C]taurocholate in direct proportion to bile flow. Horseradish peroxidase secretion into bile was unaffected, and total biliary protein secretion was decreased only slightly. Because of the depression of bile secretion, concentrations in bile of horseradish peroxidase and total biliary protein increased significantly. Consistent with reported observations of cytochalasin D, decreases in microfilaments and dilated bile canaliculi were observed by electron microscopy; however, the vesicular transport of horseradish peroxidase as observed using electron microscopy cytochemistry appeared to be normal and unaffected by cytochalasin D treatment. Colchicine, in contrast, had minimal effect on bile flow and did not diminish the biliary secretion of [14C]taurocholate. Colchicine inhibited both the total amount of horseradish peroxidase secreted into bile as well as the rate of its secretion in comparison with control and cytochalasin D-treated animals. Cellular morphology was consistent with published observations for colchicine, which included a marked decrease in microtubules. In addition, after electron microscopy cytochemistry there was a paucity of horseradish peroxidase-containing vesicles within the hepatocytes, suggesting that colchicine interfered with the vesicular transport of horseradish peroxidase. Collectively, the data suggest that (a) the mechanism used by hepatocytes for the secretion of bile acids is independent of the vesicular transport of biliary proteins and is dependent upon intact microfilaments and (b) such vesicular transport of protein into bile requires an intact and functioning microtubular network.

Bile secretory apparatus: evidence for a vesicular transport mechanism for proteins in the rat, using horseradish peroxidase and [125I]insulin.

The morphologic mechanisms involved in the uptake, transport, and secretion of proteins into bile were studied in rat liver in vivo. When both horseradish peroxidase (HRP) and insulin were injected into the portal veins of anesthetized rats, these proteins were subsequently detected in bile. Utilizing the technique of combined cytochemistry and quantitative autoradiography, both HRP and [125I]insulin were coincidentally localized within endocytic vesicles within the interior of hepatocytes at various time points after simultaneous intraportal injection. The data suggest that both proteins followed two pathways involving endocytic vesicles of approximately 1000 A in diameter. In the first pathway these protein-containing vesicles were transported through the hepatocyte and subsequently fused with the bile canalicular membrane, resulting in secretion of contained proteins into the biliary space. The second pathway also involved endocytosis into 1000 A vesicles, but these vesicles were transported to the Golgi region and its associated system of lysosomes and endoplasmic reticulum (GERL). Whether the proteins in these vesicles were later secreted into bile was unclear. Measurement of HRP and [125I]insulin or its metabolites, in bile, provided direct evidence that exogenously administered proteins (or their fragments) gain entrance into the biliary space. Studies in which metabolites of [125I]insulin, [125I]monoiodotyrosine (MIT), and 125I, were injected intraportally, demonstrated that less than 10% of [125I]MIT and less than 1.5% of Na125I were retained in perfusion-fixed and processed liver tissue. This study suggests that proteins in blood plasma are taken up by hepatocytes and secreted into bile via a vesicular transport mechanism.

Evidence for a vesicular transport mechanism in hepatocytes for biliary secretion of immunoglobulin A.

Quantitative electron microscopic autoradiography and diaminobenzidine cytochemistry provide evidence for an uptake and vesicular transport mechanism for iodine-125-labeled immunoglobulin A from plasma to bile by hepatocytes in vivo. The data confirm the existence of a hepatobiliary pathway for secretion of immunoglobulin A into the intestine and are consistent with a vesicular transport mechanism for biliary proteins within liver parenchymal cells.

Autoradiographic evidence for hepatic lobular concentration gradient of bile acid derivative.

Using an iodinated bile-acid analog with hepatic uptake and transport characteristics similar to conventional bile acids, the hepatic lobular gradient concept of Goresky was studied utilizing autoradiography. 125I-labeled cholylglycylhistamine (125I-CGH) was infused into the portal veins of male Sprague-Dawley rats and the livers were fixed for light microscopic autoradiography at 1 and 5 min after infusion. In two animals, sequential samples of bile were collected to assess the transport characteristics of 125I-CGH. By 1 min, virtually all (98%) of the injected 125I-CGH was taken up and retained by hepatocytes after perfusion fixation. Less than 15% of the label was lost during subsequent tissue processing. 125I-CGH appeared in bile within minutes, reaching maximum levels at 7 min. Quantitative autoradiography demonstrated that the first six to nine periportal hepatocytes were, by far, the most active (P less than 0.0005) in sequestering the bile-acid analog than were the remaining cells in the lobule. This study, therefore, provides the first autoradiographic evidence of a hepatic lobular concentration gradient for the uptake of a bile-acid analog.

Application of electron microscopy in the definitive diagnosis of effusions.

Electron microscopy was performed on a series of body cavity fluid specimens to evaluate the usefulness of this technique in the diagnosis of effusions. During a six-month period, 42 specimens were examined, 19 benign and 23 malignant. In two cases, the electron microscopic findings were crucial to the diagnosis. In a third case, electron microscopy helped to confirm the diagnosis and avoid unnecessary surgery. In a number of other cases, ultrastructural evaluation demonstrated special cell characteristics not evident in routine light microscopic studies. Based on these findings, we conclude that electron microscopy has a definite place in the diagnosis of effusions and should be increasingly utilized on a selective basis in the evaluation of body cavity fluid specimens. In addition, electron microscopic studies on cells in effusions provide an excellent source of material for educational and research purposes.

Cholesterol ester hydrolysis by homogenates of whole testis, seminiferous tubules and interstitial cells of mature rats.

The characteristics and localization of a cholesterol ester hydrolase enzyme in homogenates of whole testis and in isolated seminiferous tubules and interstitial cells of mature rats have been investigated. Hydrolysis of cholesteryl [1(-14)C]oleate occurred at an optimum pH of 7.0, was linearly related to time up to 5--6 h of incubation and increased linearly up to 0.25 mg protein/incubation. Hydrolytic activity was inhibited by increasing the incubation temperature from 29 to 41 degrees C and by sonication. Cholesterol ester hydrolase activity/mg protein was three times greater in homogenates of seminiferous tubules than in interstitial cells. Cholesterol ester hydrolase may function to provide precursors for use in seminiferous tubular steroid hormone biosynthesis or germ cell maturation.

Postnatal differentiation of Leydig cells in the rabbit testis.

After the gradual disappearance of fetal Leydig cells in the first week after birth, the interstitial tissue of the rabbit testis is composed principally of undifferentiated mesenchymal cells between one and five weeks of age. Also present during this time are scattered partially differentiated cells with oval-shaped nuclei, prominent nucleoli and abundant cytoplasm. These cells exhibit some of the cytoplasmic features of steroid-secreting cells, but extensive development of smooth endoplasmic reticulum and the grouped perivascular arrangement of characteristic of fully differentiated Leydig cells are not present. The latter features appear at five weeks of age, indicating the formation of mature Leydig cells at that time. By seven weeks, the bulk of the interstitial tissue consists of Leydig cell aggregates, typical of the appearance in adult testis. Since spermatogonial mitoses first appear at seven to eight weeks of age, the findings indicate that Leydig cell differentiation precedes the onset of spermatogenesis.

Concentrations of free and esterified cholesterol in the testes of maturing rabbits.

Levels of cholesterol and cholesterol esters were established in the testes of maturing New Zealand white rabbits. Free cholesterol levels remained relatively constant throughout pre- and post-pubertal development. The total cholesterol, cholesterol esters and percentage esterified cholesterol values were highest during the prepubertal period and decreased steadily thereafter, reaching their lowest after 90 days of age. Correlations of these findings with hormonal and morphological changes during testicular development are discussed.