Biomedical subjects

L Orci

Publications and source records attributed to L Orci.

At least 253 records · Page 14Linked to original sources

PubMed1983

Alterations in somatostatin and other islet cell functions in the spontaneously diabetic BB Wistar rat: biochemical and morphological characterization.

In the present study we have attempted to assess the functional status of somatostatin cells in relation to the function of the other islet cell types (B, A, and PP cells) in the BB Wistar rat. Somatostatin-like immunoreactivity (SLI), glucagon, and insulin were measured in extracted plasma obtained from the hepatic portal vein (PV) and inferior vena cava (IVC) of acutely diabetic untreated rats, insulin-treated diabetic rats and nondiabetic controls. Extracts of the pancreas were assayed for SLI, glucagon, and insulin, and the pancreatic populations of A, B, D, and PP cells were evaluated by morphometry. Extrapancreatic somatostatin changes were assessed by measurement of SLI in extracts of the whole gut, hypothalamus, and retina. Direct studies of SLI, glucagon, and insulin secretion in response to glucose, arginine, and theophylline were carried out using the isolated perfused pancreases of two separate groups of untreated diabetic and nondiabetic rats. Our results showed that in the severely insulin deficient BB Wistar rat (1) pancreatic concentrations of SLI, glucagon, and insulin were reduced; (2) the B cells are virtually eliminated and the D cells severely reduced early in diabetes; A and PP cells are resistant initially but eventually sustain major losses as observed in terminal islets; (3) retinal SLI is reduced, but SLI in gut and brain appears unchanged; (4) the secretion of SLI, glucagon, and insulin from the perfused pancreas is diminished 60%, 36%, and 99%, respectively; (5) PV and IVC blood levels of SLI and glucagon are elevated despite decreased pancreatic secretion; (6) The trans-hepatic gradient of SLI is reduced; and (7) Insulin treatment normalizes the elevated PV and IVC levels of SLI and glucagon. It is concluded that the elevated PV and IVC levels of SLI are secondary to insulin deficiency and result from increased SLI secretion most probably from the gut and from diminished hepatic metabolism. The origin of the hyperglucagonemia is less certain, but as in the case of SLI, important contributions from extra-pancreatic secretion appears likely.

Animals↗

PubMed1983

Immunocytochemical localization of prosomatostatin fragments in maturing and mature secretory granules of pancreatic and gastrointestinal D cells.

Pancreatic and gastrointestinal D cells were examined by immunocytochemistry using antisera against somatostatin-28 (SS28) and its NH2-terminal fragment SS28-(1-12), followed by the staphylococcal protein A-gold (pAg) complex. In pancreatic and gastric D cells incubated with antiserum against SS28-(1-12) the gold particles produced intense staining of the mature secretory granules but weaker staining of the immature granules associated with the Golgi area, whereas after SS28 antiserum treatment the particles accumulated selectively over the population of immature secretory granules. In intestinal D cells not only SS28-(1-12) but also SS28 antiserum produced an intense gold staining over the mature delta granules. These observations show that the relative amounts of immunoreactive sites related to SS28 and its cleavage product SS28-(1-12) in maturing and mature secretory granules are different in pancreatic, gastric, and intestinal D cells.

Humans↗

PubMed1983

Regional distribution of N-acetyl-D-galactosamine residues in the glycocalyx of glomerular podocytes.

Helix pomatia lectin (HPL) bound to colloidal gold was used as a specific cytochemical probe for the localization of terminal nonreducing N-acetyl-D-galactosamine residues in thin sections of rat kidney. In the glomerulus, lectin-binding sites were associated only with the podocyte foot process bases and were not found on the free cell surface of podocytes or on any other glomerular components. Gold-particle label was often arranged in the form of clusters which extended from the foot process base to the lamina rare externa and lamina densa of the basement membrane. In contrast, wheat germ lectin (WGL)-binding sites (beta-[1 leads to 4] linked N-acetyl-D-glucosamine residues and N-acetylneuraminic acid residues) were found in all regions of the podocyte plasma membrane and on the cell surface of all other glomerular cell types. In addition, WGL-binding sites were present in all three layers of the glomerular basement membrane (GBM) as well as in the mesangial matrix. A quantitative evaluation of the pattern of labeling for HPL-binding sites together with the sugar specificity of this lectin suggest that a component of the glycocalyx is being detected rather than a basement membrane component. This was confirmed by the absence of H. pomatia lectin-binding sites in preparations of isolated GBM which retained, however, wheat germ lectin-binding sites. These data show that the glycocalyx of the foot process base is a highly specialized cell surface domain with respect to its carbohydrate composition.

Acetylgalactosamine↗

PubMed1983

Specialization of the macrophage plasma membrane at sites of interaction with opsonized erythrocytes.

We incubated mouse peritoneal macrophages for 3-8 min at 37 degrees C with antibody-coated sheep erythrocytes and examined regions of close interaction between the two cell types by electron microscopy. At sites of focal macrophage-erythrocyte contact we observed a distinctive specialization of the macrophage plasma membrane consisting of a prominent subplasmalemmal band of electron-dense material, approximately 25-35 nm in thickness. In many instances, this band showed a periodic substructure similar to that seen in clathrin coats. Moreover, many slender erythrocyte processes penetrated into invaginations of the macrophage surface which were bristle-coated at their blind extremity. As previously shown for clathrin-coated pits, the segments of the macrophage plasma membrane beneath which the defense material was found were selectively resistant to the membrane-perturbing effect of the antibiotic, filipin. This structural specialization of the macrophage plasma membrane at sites of ligand-receptor interaction during immune phagocytosis of antibody-coated erythrocytes may represent the morphological counterpart of the zipper mechanism of phagocytosis previously demonstrated by functional studies.

Animals↗

PubMed1983

Collagen matrix promotes reorganization of pancreatic endocrine cell monolayers into islet-like organoids.

To evaluate the capacity of pancreatic endocrine cells to reassociate in vitro according to the characteristic topographical pattern observed in the islets of Langerhans in situ, we cultured cells dissociated from neonatal rat pancreas within a three-dimensional collagen matrix. Cell monolayers grown on the surface of collagen gels were covered with a second layer of collagen. This induced the monolayers of endocrine cells to reorganize into smooth-contoured, three-dimensional aggregates, in which non-B cells (identified by electron microscopy and immunofluorescence) had a preferential distribution at the periphery, whereas B cells were concentrated in a central position. These results show that cultured pancreatic endocrine cells have the capacity to reassociate into islet-like organoids in vitro, and that collagen matrices may have a permissive effect on the expression of this potential.

Animals↗

PubMed1983

In vitro rapid organization of endothelial cells into capillary-like networks is promoted by collagen matrices.

We have studied the behavior of cloned capillary endothelial cells grown inside a three dimensional collagen matrix. Cell monolayers established on the surface of collagen gels were covered with a second layer of collagen. This induced the monolayers of endothelial cells to reorganize into a network of branching and anastomosing capillary-like tubes. As seen by electron microscopy, the tubes were formed by at least two cells (in transverse sections) delimiting a narrow lumen. In addition, distinct basal lamina material was present between the abluminal face of the endothelial cells and the collagen matrix. These results showed that capillary endothelial cells have the capacity to form vessel-like structures with well-oriented cell polarity in vitro. They also suggest that an appropriate topological relationship of endothelial cells with collagen matrices, similar to that occurring in vivo, has an inducive role on the expression of this potential. This culture system provides a simple in vitro model for studying the factors involved in the formation of new blood vessels (angiogenesis).

Animals↗

PubMed1983

Immunohistochemical localization of carbonic anhydrase in postnatal and adult rat kidney.

With use of specific antibodies against human and rat erythrocyte carbonic anhydrase C and human carbonic anhydrase B, only the isozyme C could be detected by immunofluorescence in rat kidney epithelial cells. In the postnatal kidney a few cells were positive after 2 days, but the number of fluorescent cells increased during the first few weeks of life to reach the final adult levels after 3 wk in the cortex and 5 wk in the medulla. In the postnatal and adult kidney a characteristic mosaic pattern of fluorescence was seen in the late distal tubule, the connecting segment, and the collecting tubule, where the mitochondria-rich dark cells were brightly fluorescent. In addition, in later postnatal stages and in the adult, the entire epithelium of the initial portion of descending thin limbs of Henle (long loops) was labeled. Some kidney regions that had previously been shown to contain carbonic anhydrase activity by biochemical and histochemical techniques stained only weakly with the immunocytochemical method. This suggests either that the enzyme in these regions does not cross-react strongly with our antibodies or that these regions contain only low amounts of carbonic anhydrase, at the limit of the detection threshold of our techniques.

Aging↗

PubMed1983

The interaction of 125I-insulin with cultured 3T3-L1 adipocytes: quantitative analysis by the hypothetical grain method.

The murine 3T3-L1 fibroblast under appropriate incubation conditions differentiates into an adipocyte phenotype. This 3T3-L1 adipocyte exhibits many of the morphologic, biochemical, and insulin-responsive features of the normal rodent adipocyte. Using quantitative electron microscopic (EM) autoradiography we find that, when 125I-insulin is incubated with 3T3-L1 adipocytes, the ligand at early times of incubation localizes to the plasma membrane of the cell preferentially to microvilli and coated pits. When the incubation is continued at 37 degrees C, 125I-insulin is internalized by the cells and preferential binding to the villous surface is lost. With the internalization of the ligand, two intracellular structures become labeled, as determined by the method of hypothetical grain analysis. These include large clear, presumably endocytotic, vesicles and multivesicular bodies. Over the first hour of incubation the labeling of these structures increases in parallel, but in the second hour they diverge: the labeling of multivesicular bodies and other lysosomal forms continuing to increase and the labeling of large clear vesicles decreasing. At 3 hours limited but significant labeling occurs in small Golgi-related vesicles that have the typical distribution of GERL. The distinct morphologic features of this cell make it ideal for a quantitative morphologic analysis and allow for an unambiguous view of the sequence of events involved in receptor-mediated endocytosis of a polypeptide hormone. These events are likely to be representative of the processing of insulin by the mature rodent adipocyte.

Adipose Tissue↗

PubMed1983

Differential filipin labeling of the luminal membranes lining the pancreatic acinus.

The cytochemical labeling of cholesterol by filipin in plasma membranes of the exocrine pancreas has shown a difference in the reactivity to filipin between the acinar and centro-acinar cells. The acinar cell has a poorly labeled luminal membrane but numerous filipin-cholesterol complexes on the basolateral membrane. In contrast, the centro-acinar cell shows a comparable degree of filipin labeling on both luminal and basolateral membrane. This difference in the distribution of filipin labeling suggests underlying differences in the organization of the respective membranes, probably related to their specific functions.

Animals↗

PubMed1983

Morphological changes of the 3T3-L1 fibroblast plasma membrane upon differentiation to the adipocyte form.

By quantitative evaluation carried out on freeze-fracture replicas, we have investigated the changes in plasma membrane organization as the 3T3-L1 fibroblast phenotype differentiates into the 3T3-L1 adipocyte form. As differentiation takes place there is a dramatic change in overall appearance of the cell as it acquires large lipid-laden vacuoles. On freeze-fracture replicas we find: (1) a ninefold increase in small invaginations as the cell differentiates from the fibroblast to the adipocyte phenotype; (2) virtually no quantitative change in the larger coated invaginations upon differentiation; (3) a greater density of intramembrane particles in the large invaginations as compared to the uninvaginated membrane in both the fibroblast and adipocyte form. This remains relatively constant with differentiation. By contrast, there is a marked increase of intramembrane particles in the undifferentiated membrane as the cell changes from fibroblast to adipocyte. The functional significance of these changes in plasma membrane organization is unknown, but they significantly correlate with the onset of lipogenesis by the cell.

Adipose Tissue↗

PubMed1983

Ultrastructural analysis of crystalloid endoplasmic reticulum in UT-1 cells and its disappearance in response to cholesterol.

The crystalloid endoplasmic reticulum (ER) consists of hexagonally packed membrane tubules that contain 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase), an intrinsic membrane protein that catalyses the rate-limiting step in cholesterol synthesis. The crystalloid ER appears in a clone of Chinese hamster ovary cells, designated UT-1, that contain high levels of HMG CoA reductase as a result of growth in the presence of compactin, a competitive inhibitor of the reductase. In the present studies, we have used ultrastructural morphometry to estimate that the crystalloid ER: (1) occupies about 15% of the volume of UT-1 cells; (2) contains 3.4-fold more membrane area than the plasma membrane; and (3) contains less than 700 subunits of HMG CoA reductase per micrometer2 of membrane surface. The crystalloid ER tubules contain 2000 intramembrane particles per micrometer2 with a mean diameter of 10.4 nm, as determined by freeze-fracture. The crystalloid ER membranes are low in cholesterol, as indicated by the small number of filipin-cholesterol complexes in freeze-fracture images after treatment with filipin. The addition of cholesterol or related sterols to UT-1 cells promoted a rapid and stepwise disappearance of the crystalloid ER. Initially, the crystalloid ER fragmented into randomly arranged vesicles and tubules. Subsequently, membrane-bound structures disappeared from the cell so that after incubation with cholesterol for 24-72 h, the cells appeared completely normal. We found no morphological evidence that autophagic vacuoles participate in the degradation. We conclude: (1) that the crystalloid ER is more extensive than necessary merely to support HMG CoA reductase; and (2) that upon exposure to cholesterol the crystalloid ER is degraded by a process that does not involve autophagy.

Animals↗

PubMed1983

Functional and morphologic characterization of human insulinomas.

Circulating levels of insulin, proinsulin-like component, glucagon, growth hormone, and pancreatic polypeptide were measured in 12 patients with functioning insulinomas, and the suppressibility of serum insulin by somatostatin and diazoxide was assessed before surgical removal of the tumors. The hormone content of the tumors was evaluated by radioimmunoassay and by immunofluorescence and the structure of the tumor cells by electron microscopy. Based on these findings, we propose a new classification of insulinomas in two groups: group A is characterized morphologically by abundant well-granulated typical B-cells, trabecular arrangement of tumor cells, and uniform insulin immunofluorescence; functionally, these tumors are associated with a moderate elevation of proinsulin-like component and with an almost complete suppressibility of serum insulin by somatostatin and diazoxide. In contrast, tumors of group B are characterized by scarce well-granulated typical B-cells, a medullary-type histologic structure, and irregular insulin immunofluorescence; functionally these tumors show elevated circulating levels of proinsulin-like component and a marked resistance of insulin secretion to somatostatin and diazoxide inhibition. This way of separating human insulinomas in groups A and B represents a simplification of existing classifications and emphasizes the quantitative ultrastructure in relationship to suppressibility of insulin secretion. The proposed classification of human insulinomas in groups A and B, however, does not allow the assessment of the clinical or histopathologic malignancy of the tumors.

Adenoma, Islet Cell↗

PubMed1983

A quantitative immunofluorescent study of the endocrine cell populations in the developing human pancreas.

The immunofluorescent cell content of the pancreas of 8--40-wk fetuses and of 1.5--5-mo Caucasian infants was quantitatively evaluated using anti-insulin, anti-glicentin, anti-glucagon, anti-somatostatin, and anti-pancreatic polypeptide antisera. The most significant findings are: (1) the pancreas of 8--10 wk fetuses contains a sizable population of endocrine cells reacting exclusively to anti-glicentin antiserum. This cell population decreases and disappears in later stages and is replaced by the adult type glucagon/glicentin immunoreactive cell; (2) the pancreatic polypeptide-rich region shows a lower relative endocrine cell content as compared with the glucagon-rich region and its islets appear smaller; (3) in the total pancreas, the relative (volume density) and absolute (microliter) insulin cell content increases regularly with age, while the relative volume of glucagon cells peaks in fetal life (wk 17--20) to decrease in infants, although remaining at higher levels than in adults; the relative and absolute volumes of somatostatin cells are elevated in fetal and infant stages studied where they represent the second most abundant cell type, while pancreatic polypeptide cells appear to least abundant cells during prenatal and infant life. These data show several differences with the pattern of the respective endocrine cell populations in the adult pancreas.

Female↗

PubMed1983

In vivo modulation of gap junctions and dye coupling between B-cells of the intact pancreatic islet.

The extent of gap junctions and dye coupling between insulin-producing B-cells was analyzed on islets of Langerhans isolated from adult rats treated for one day with glibenclamide, an insulin secretagogue, or diazoxide, a blocker of insulin release, or a combination of the two drugs. Glibenclamide treatment was associated with a marked depletion of the islet insulin content, an effect which was blocked by pretreatment of the rats with diazoxide. Diazoxide alone caused a marked increase in the plasma glucose level, and a decrease in the level of circulating insulin and in the hormone content of the B-cells. Quantitative analysis showed that (1) under control conditions, B-cells are connected by minute gap junctions (as evaluated on freeze-fracture replicas) and show a nonuniform and apparently restricted dye coupling (as determined by microinjection of the low-molecular-weight fluorescent probe Lucifer Yellow CH); (2) each of the three treatments tested significantly increased the relative and absolute gap junction area of the B-cells and the number of detectable, dye coupled B-cells per microinjection. After treatment with glibenclamide alone or with diazoxide plus glibenclamide, a 1.5-1.8-fold increase in gap junction area and a 2.7-3.7-fold increase in the number of dye-coupled B-cells were observed. In contrast, following treatment with diazoxide alone, gap junctions and dye coupling were found increased 1.8 and 8.7 times, respectively, as compared with control values.

Animals↗

PubMed1983

Surface interactions and intracellular localization of cationic ferritin: similarity to 125I-insulin in 3T3-L1 adipocytes.

We previously reported that in 3T3-L1 adipocytes 125I-insulin associates preferentially with microvilli and coated pits at low temperatures and early times of incubation. At higher temperatures it is internalized through a series of membrane limited intracellular compartments. In the present study, we used a high resolution probe, cationic ferritin (CF), to track adsorptive endocytosis in the 3T3-L1 adipocyte. We find that CF initially associates with coated pits at 2 min of incubation at 37 degrees C. With further incubation at 37 degrees C CF is internalized and after 2 to 10 min of incubation is predominantly localized to coated and non-coated clear vesicles. Approximately 50% of the apparent coated vesicles seen near the plasma membrane on single thin sections are shown by serial sectioning to be true vesicles (i.e., without a surface connection). At later time points CF is localized predominantly to lysosomal structures and, to a much smaller extent, Golgi-related structures. The remarkable similarity between 125I-insulin and CF with respect to post-binding processing suggests that while the membrane receptor confers the initial specificity, post-binding events are common for different types of ligands after they bind to cell surfaces and are subject to adsorptive endocytosis.

Adipose Tissue↗

PubMed1983

[Ultrastructural detection of the heterogeneity of glycocalyx in convoluted and straight proximal tubules of rat kidney by the lectin-gold complex technic].

A new histochemical technique in which lectin-gold complexes are applied directly to thin and semi-thin sections of tissue was used to determine the distribution of Helix pomatia lectin binding sites in the proximal tubule of Rat kidney. Whereas a strong labeling was seen on the brush border and baso-lateral plasma membranes of convoluted tubules, the plasma membrane of cells from the medullary straight segment was unlabeled. Some profiles at the frontier between cortex and medulla had a mosaic of positive and negative cells. These results show, therefore, considerable segmental variation in the glycocalyx composition of Rat proximal tubules.

Animals↗

PubMed1983

[Membrane binding of insulin antigenic sites in the Golgi apparatus of pancreatic B cells].

Ultrastructural immunocytochemistry with anti-insulin antiserum allowed the detection of insulin antigenic sites localized at the inner aspect of the cisternae of the Golgi apparatus in pancreatic B-cells. By contrast, in secretory granules released from the Golgi apparatus, insulin antigenic sites were localized over the granule content but not on the membrane. The demonstration of insulin antigenic sites at the level of the Golgi membranes is compatible with the presence of specific insulin receptors and/or charge interactions between the insulin and the membrane. Binding at this level could in turn be involved in the sorting out of insulin polypeptides destined to be concentrated in secretory granules.

Cell Membrane↗

PubMed1983

Distribution of filipin-sterol complexes in plasma membranes of the kidney. II. The thin limbs of Henle's loop.

Thin limbs of Henle from the rat kidney were investigated by freeze fracture electron microscopy after incubation of the tissue with the polyene antibiotic, filipin, a morphologic probe for membrane cholesterol. Distinct patterns of distribution of filipin-sterol complexes were found in plasma membranes of epithelial cells from each of the four thin limb regions examined. In particular, adjacent cells in the thin ascending limb had either heavily or poorly labeled apical membranes, whereas in the lower part of long descending thin limbs, all apical membranes were poorly labeled. In contrast, both apical and basolateral membranes of short descending limbs were heavily labeled with filipin-sterol complexes. These results demonstrate the existence of plasma membranes with different properties both within specific segments of Henle's loop (ascending thin limb) and between different thin limb segments (short and long descending limbs).

Animals↗