Search PubMed⌕ Search

Biomedical subjects

L Orci

Publications and source records attributed to L Orci.

At least 235 records · Page 13Linked to original sources
PubMed1984

Increase in membrane cholesterol: a possible trigger for degradation of HMG CoA reductase and crystalloid endoplasmic reticulum in UT-1 cells.

The crystalloid endoplasmic reticulum (ER) houses large amounts of HMG CoA reductase, the rate-controlling enzyme in cholesterol synthesis. The crystalloid ER appears in UT-1 cells, a line of Chinese hamster ovary cells that has been chronically starved of cholesterol as a result of growth in the presence of compactin, an inhibitor of reductase. When cholesterol was provided to UT-1 cells in the form of low density lipoprotein (LDL), the reductase and crystalloid ER were destroyed. This destruction was preceded by an increase in the cholesterol content of crystalloid ER membranes, as judged by a 4- to 8-fold increase in their ability to form complexes with filipin, a cholesterol-binding compound that can be visualized in freeze-fracture electron micrographs. Filipin binding to other membranes was unchanged. Thus insertion of cholesterol into the crystalloid ER membrane may trigger the degradation of reductase and the membrane itself.

Animals↗
PubMed1984

A clathrin-coated, Golgi-related compartment of the insulin secreting cell accumulates proinsulin in the presence of monensin.

When the intracellular transit of 3H-labeled (pro)-insulin polypeptides is perturbed by monensin in the pancreatic B-cell, proinsulin conversion is impaired and the radioactive peptides accumulate in a clathrin-coated membrane compartment related to the Golgi apparatus. Clathrin was demonstrated by immunocytochemistry using the postembedding protein A-gold technique. The coated compartment, which is dilated by monensin, comprises Golgi cisternae with condensing secretory material and newly formed secretory granules; under monensin block, the noncoated (storage) secretory granules do not become significantly labeled. These data suggest that an unperturbed passage through a Golgi-related, clathrin-coated membrane compartment which subsequently matures into noncoated secretory granules is needed for the normal processing of (pro)insulin polypeptides.

Animals↗
PubMed1984

(Pro)insulin associates with Golgi membranes of pancreatic B cells.

The immunocytochemical demonstration of (pro)insulin on intracellular membrane compartments of the pancreatic B cell reveals that the immunolabeling detected by the protein A/gold method is associated, at the level of the Golgi apparatus, with the inner aspect of the cisternal membranes; on the secretory granules, by contrast, insulin immunoreactive sites predominate over the granule core, and very little immunoreactivity is associated with the granule membrane. The localization of (pro)insulin immunoreactivity on Golgi membranes is compatible with the presence, at this level, of specific binding sites for (pro)insulin, which could be related to the proteolytic processing or sorting out (or both) of these peptides on their way from the rough endoplasmic reticulum to the storage secretory granules.

Animals↗
PubMed1984

Insulin, not C-peptide (proinsulin), is present in crinophagic bodies of the pancreatic B-cell.

We have obtained evidence by autoradiography and immunocytochemistry that mature secretory granules of the pancreatic B-cell gain access to a lysosomal compartment (multigranular or crinophagic bodies) where the secretory granule content is degraded. Whereas the mature secretory granule content shows both insulin and C-peptide (proinsulin) immunoreactivities, in crinophagic bodies only insulin, but not C-peptide, immunoreactivity was detectable. The absence of C-peptide (proinsulin) immunoreactivity in multigranular bodies, i.e., in early morphological stages of lysosomal digestion, was compatible with the ready access and breakdown of C-peptide and/or proinsulin by lysosomal degrading enzymes, while the insulin crystallized in secretory granule cores remained relatively protected. However, in the final stage of lysosomal digestion, i.e., in residual bodies where the secretory granule core material is no longer present, insulin immunoreactivity became undetectable. Lysosomal digestion thus appears to be a normal pathway for insulin degradation in the pancreatic B-cell.

Animals↗
PubMed1984

Leukocyte interleukins induce cultured endothelial cells to produce a highly organized, glycosaminoglycan-rich pericellular matrix.

We report here that interleukins have a dramatic effect on extracellular matrix production by cultured endothelial cells. Human umbilical vein endothelial cells incubated with growth media conditioned by lectin-activated human peripheral blood mononuclear leukocytes undergo marked changes in cell shape and elaborate a highly organized extracellular material that is not detectable in untreated cultures. This material has the following characteristics: (a) it is not recognizable by electron microscopy unless the cationic dye, Alcian blue, is added to the fixative; (b) it is visualized as a network of branching and anastomosing fibrils of various thickness that can be resolved into bundles of fine filaments; (c) it is associated with the cell surface, extends between contiguous cells, and coats the culture substrate; (d) it is removed by digestion with glycosaminoglycan-degrading enzymes, such as crude heparinase and chondroitinase ABC. These results demonstrate that soluble factors released by activated peripheral blood mononuclear leukocytes (interleukins) stimulate cultured human umbilical vein endothelial cells to produce a highly structured pericellular matrix containing glycosaminoglycans (probably chondroitin sulfate and/or hyaluronic acid) as a major constituent. We speculate that this phenomenon corresponds to an early step of angiogenesis as observed in vivo as a consequence of interleukin release.

Alcian Blue↗
PubMed1984

Nonconverted, amino acid analog-modified proinsulin stays in a Golgi-derived clathrin-coated membrane compartment.

The secretion of insulin by the pancreatic B-cell involves a passage of the newly synthetized (pro)insulin polypeptides across the Golgi apparatus, at the trans pole of which secretory proteins are released as a population of secretory granules characterized by a clathrinlike coat on segments of their limiting membrane. When the conversion of radiolabeled proinsulin to insulin was inhibited by replacing arginine and lysine with the aminoacid analogs, canavanine and thialysine, the nonconverted radioactive material remained associated with Golgi-derived, coated secretory granules. The coat was characterized as clathrin-containing by immunocytochemistry. Under analog treatment, the noncoated, storage secretory granules did not become markedly labeled during the pulse-chase experiment. These data are compatible with the hypothesis that in normal conditions, the maturation of the coated compartment into noncoated granules is linked to the effective conversion of the prohormone.

Animals↗
PubMed1984

Insulin within islets is a physiologic glucagon release inhibitor.

To determine if glucagon secretion is under physiological control of intra-islet insulin, pancreata from normal rats were perfused at a 100 mg/dl glucose concentration with either guinea pig antiinsulin serum or normal guinea pig serum in a nonrecirculating system. Perfusion of antiserum was followed within 3 min by a significant rise in glucagon that reached peak levels three times the base-line values and assumed a hectic pattern that returned rapidly to base-line levels upon termination of the antiserum perfusion. Nonimmune guinea pig serum had no effect. To gain insight into the probable site of insulin neutralization, 125I-labeled human gamma-globulin was added to antiserum or nonimmune serum and perfused for 3 min. More than 83% of the radioactivity was recovered in the effluent within 3 min after termination of the infusion, and only 0.05 +/- 0.015% of the radioactivity injected was present in the pancreas 10 min after the perfusion. The maximal amount of insulin that could be completely bound to insulin antibody at a dilution and under conditions simulating those of the perfusion experiments was 20 mU/min. It is concluded that insulin maintains an ongoing restraint upon alpha cell secretion and in its absence causes hectic hypersecretion of glucagon. This restraint probably occurs largely in the intravascular compartment. Loss of this release-inhibiting action of insulin may account for initiation of hyperglucagonemia in insulin-deficient states.

Animals↗
PubMed1984

The insulin receptor of a human monocyte-like cell line: characterization and function.

Most clinical studies of the insulin receptor in man have been carried out in circulating cells, mainly monocytes. It is important to establish whether the function of the insulin receptor in blood cells reflects that of the major target tissues. Since peripheral monocytes do not divide, they cannot be studied under continuous cell culture conditions. We, therefore, studied the insulin receptor of a monocyte-like human cell line (U-937) to determine whether it could serve as a model for further investigation of the function of the insulin receptor. We found that the U-937 cells bind [125I]insulin specifically and in a time-, temperature-, concentration-, and pH-dependent fashion, similar to circulating monocytes. In addition, they internalize the hormone-receptor complex rapidly and extensively at 37 C in a fashion analogous to that of blood monocytes and hepatocytes. U-937 cells show a similar affinity for insulin as monocytes. Unlike hepatocytes, these cells do not appear to release extensive insulin-degrading activity and do not degrade cell surface-associated ligand during initial incubations. [125I] Insulin extracted or dissociated spontaneously from the cell surface shows full rebindability to fresh cells. The insulin receptor of the U-937 cells can be down-regulated as in the major target cells. As in cultured human lymphocytes, preincubation of U-937 cells with prednisolone results in significantly increased insulin binding. Incubation with a phorbol ester tumor promoter, in contrast, inhibits the extent of specific [125I]insulin binding to U-937 cells by altering the receptor affinity. The insulin receptor of the U-937 monocyte-like cell line mirrors the insulin receptor of blood monocytes and target cells; it is a useful tool for further in vitro studies of the insulin receptor.

Cell Line↗
PubMed1984

Immunocytochemical characterization of secretory granule maturation in pancreatic A-cells.

Antigenic sites related to glucagon and glucagon precursors were characterized by ultrastructural immunocytochemistry in secretory compartments of the pancreatic A-cell, namely Golgi cisternae, condensing granules in Golgi cisternae, coated immature secretory granules, and noncoated mature secretory granules. The C-terminal glucagon immunoreactivity was low in all these compartments except in the noncoated mature secretory granules. By contrast, N-terminal glucagon and glicentin immunoreactivities were high at the condensing granule stage and, respectively, increased (N-terminal) or decreased (glicentin) until the mature secretory granule stage. These data suggest that: 1) glucagon precursors and glucagon coexist at different concentrations in secretory compartments of the A-cell; 2) the condensing granules in Golgi cisternae and immature granules consist predominantly of glucagon precursors; and 3) the removal of the peptide masking the C-terminal of the glucagon molecule occurs between the coated and the mature granule stages.

Animals↗
PubMed1984

Immunohistochemically detectable vitamin D-dependent calcium-binding protein is reduced in cerebellum of diabetic subjects.

The immunohistochemical distribution of the vitamin D-dependent calcium-binding protein (CaBP) was studied in the cerebellar vermis of 14 adult type (NIDDM) diabetic and 13 nondiabetic human subjects. In both groups, CaBP-immunoreactive material was found in Purkinje cells and in axons of the cortical white matter, but, when compared with controls, the positive structures had a significantly (P less than 0.001) reduced volume density in 12 of the 14 diabetic subjects. The reduction of the volume density of CaBP-positive structures in diabetic subjects was not due to an overall reduced volume of the molecular layer or of the cortical white matter. These results complement previous reports of a reduction of CaBP levels in duodenum and kidney of diabetic subjects.

Aged↗
PubMed1984

The topography of electrical synchrony among beta-cells in the mouse islet of Langerhans.

beta-Cells in microdissected islets of Langerhans produce rhythmical bursts of electrical activity. This was monitored with two micro-electrodes simultaneously and the frequency and phase (collectively referred to as synchrony) of the two signals was investigated. At any instant two impaled cells produced bursts of the same frequency even when separated by up to 400 micron. When the electrode tips were separated by less than about 20 micron and current injection showed the cells to be ionically coupled the two signals were in phase and had almost identical shape. The phase relations between cells further apart were variable, the leading cell usually being located deeper within the islet than the other impaled cell. Increasing the glucose concentration increased electrical activity, reduced any phase lags and made the shape of the bursts more similar. There was less lag between the responses from two cells when the glucose concentration was suddenly reduced, than when it was suddenly increased. Qualitatively similar observations were made in glibenclamide-treated mice, a treatment previously shown to increase dye coupling between islet cells. However, the response to increasing glucose concentrations showed less phase lag; likewise the phase lag between bursts was reduced. Furthermore the response to current injected into one cell could be detected at much larger distances (up to 80 micron) than in control islets. This suggests that electrical coupling of beta-cells was improved in sulphonylurea-treated mice. Electron microscopy of both control and glibenclamide-treated mouse islets fixed at the end of each electrophysiological experiment showed the region impaled by the electrodes to be well preserved and, whenever the electrodes penetrated at least 20 micron into the islet, to contain a large proportion of beta-cells. The data support the view that, within an islet, most but not necessarily all cells are electrically synchronized, and that the coupling can be modulated by natural and pharmacological secretagogues.

Action Potentials↗
PubMed1984

[Asymmetrical distribution of clathrin in the Golgi apparatus of polypeptide-secreting cells].

Using an antibody revealed by the protein A-gold technique, we have studied the distribution of clathrin antigenic sites in the Golgi area of pancreatic B-cells. Golgi compartments showing an immunolabelling comprised extensive segments of cisternae, typical coated vesicles, dilated extremities of cisternae with condensing secretory material, and newly formed secretory granules. Most of the labelled membranes were observed at the trans Golgi pole while little immunoreactivity was found on the cis pole.

Clathrin↗
PubMed1984

Appearance of Helix pomatia lectin-binding sites on podocyte plasma membrane during glomerular differentiation. A quantitative analysis using the lectin-gold technique.

The visceral glomerular epithelial cells (podocytes) of newborn rat kidneys were examined by the lectin-gold technique to determine the distribution of Helix pomatia lectin-binding sites during glomerular development. Previous work has shown that this lectin, which primarily recognizes terminal N-acetyl-D-galactosamine residues of glycoconjugates (but also N-acetyl-D-glucosamine to a lesser extent), specifically labels the plasma membrane glycocalyx of podocyte foot process bases, whereas the plasma membrane of the podocyte cell body that is in contact with the urinary space is unlabeled. In neonatal glomeruli, the labeling was most intense in those podocytes whose pedicel development corresponded to that seen in adult rat kidneys, whereas immature podocytes were poorly labeled. A quantitative evaluation of the labeling showed a highly significant correlation between the degree of podocyte development (as assessed by counting the number of pedicels per micrometer of basal lamina) and the intensity of H. pomatia lectin-gold particle labeling. These results show that the appearance of pedicels on developing podocytes is accompanied by the appearance of a specific cell coat component(s), detectable using H. pomatia lectin-gold complexes.

Acetylgalactosamine↗
PubMed1984

Perturbation of podocyte plasma membrane domains in experimental nephrosis. A lectin-binding and freeze-fracture study.

Alterations in the ultrastructural organization of podocyte plasma membrane domains were quantitatively assessed in puromycin aminonucleoside-treated rats by the use of 1) Helix pomatia lectin-gold complexes for detection of a specific glycocalyx component(s) normally associated with foot process bases and 2) freeze-fracture for detection of intramembrane particles and endocytotic invaginations on the plasma membrane. Lectin-binding sites were significantly reduced on podocyte foot process bases during the 7-day treatment period; and in freeze-fracture, the plasma membrane of the foot process base showed an increase in intramembrane particle number and size and an increased number of endocytotic invaginations, compared with the numbers in control animals. The cell body of nephrotic animals also had a significantly increased intramembrane particle density, compared with the control animals. These results provide direct evidence that the normal structure of specific plasma membrane regions is perturbed in podocytes that have lost their characteristic array of foot processes and support a role for these domains in the maintenance of normal podocyte architecture.

Animals↗
PubMed1983

Short and reversible uncoupling evokes little change in the gap junctions of pancreatic acinar cells.

Three different preparations of mouse pancreatic fragments where all the cells tested electrophysiologically showed (a) complete electrical coupling (control), (b) complete uncoupling (after 1-to 2-min exposure to 100% CO2), or (c) complete recoupling (1-2 min after removal of 100% CO2) were fixed, with the electrodes in situ, with 0.2% glutaraldehyde and freeze-fractured for quantitative analysis of acinar cell gap junctions. No obvious difference was observed between gap junctions of coupled and uncoupled acinar cells. However, quantitation revealed a small (2.3-5.6%) increase in particle diameter and spacing within junctions of uncoupled cells. Such increase was rapidly reversed upon cell recoupling. In all preparations, most of the gap junctions were made up of disordered arrays of particles but a few of them showed a more tight packing of their particles of which most had lost the usual globular appearance. These "amorphous" gap junctions had larger particle diameter but smaller particle spacing than the other gap junctions and these parameters were not modified during cell uncoupling. However, "amorphous" gap junctions were more frequent in the latter condition.

Animals↗