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Biomedical subjects

L Orci

Publications and source records attributed to L Orci.

At least 181 records · Page 10Linked to original sources
PubMed1986

Preproglucagon gene expression in pancreas and intestine diversifies at the level of post-translational processing.

Glucagon is a pancreatic hormone of 29 amino acids that regulates carbohydrate metabolism and glicentin is an intestinal peptide of 69 amino acids that contains the sequence of glucagon flanked by peptide extensions at the amino and carboxy termini. The glucagon gene encodes a precursor containing glucagon and two additional, structurally related, glucagon-like peptides separated by an intervening peptide. These peptides are encoded in separate exons. To determine whether the pancreatic and intestinal forms of glucagon arise by alternative RNA and/or protein processing, we used antisera to synthetic glucagon-like peptides and exon-specific, complementary oligonucleotides for analyses of proteins and mRNAs in pancreatic and intestinal extracts. Preproglucagon mRNAs are identical, but different and highly specific peptides are liberated in the two tissues. Immunocytochemistry shows colocalization of glucagon and the two glucagon-like peptides in identical cells. We conclude that diversification of preproglucagon gene expression occurs at the level of cell-specific post-translational processing.

Amino Acid Sequence↗
PubMed1986

A new type of coated vesicular carrier that appears not to contain clathrin: its possible role in protein transport within the Golgi stack.

Isolated Golgi membranes incubated in the presence of ATP and a cytosolic protein fraction form a population of coated buds or vesicles from the Golgi cisternae. The coats do not have the characteristic hexagonal-pentagonal basketwork of clathrin, and do not react with anti-clathrin polyclonal antibody. The conditions that produce these apparently nonclathrin-coated buds also reconstitute protein transport between compartments of the Golgi stack. The membrane of the buds contains the glycoprotein in transit through these Golgi stacks (VSV-encoded G protein). This suggests that protein transport through the Golgi stack is mediated by a new type of coated vesicle that does not contain clathrin. The concentration of G protein in the coated buds reflects the local concentration of G protein in the cisternae, raising the possibility that the Golgi coated vesicles may be "bulk" membrane carriers.

Animals↗
PubMed1986

Transport of horseradish peroxidase from the cell surface to the Golgi in insulin-secreting cells: preferential labelling of cisternae located in an intermediate position in the stack.

We have used serial sectioning to study the topology of Golgi cisternae in insulin-secreting cells during secretion-stimulated endocytotic uptake of exogenous horseradish peroxidase (HRP). HRP-labelled cisternae were followed on several series of consecutive sections. This revealed that labelled cisternae could always be traced to a position in the Golgi stack intermediate between the cis and the trans poles. This occurred in spite of the apparent cis or trans locations of HRP-containing cisternae on some sections. The latter images could be explained by the lack of the true cis or trans (clathrin-coated) cisternae at certain levels of the stack.

Animals↗
PubMed1986

Calcitonin and vasopressin affect epithelial properties in a renal cell line.

We have used a stable clonal variant (D + Sc), isolated from the LLC-PK1 pig kidney-derived cell line and selected for its extensive capacity to form domes, in order to study the hormonal modulation of epithelial permeability in culture. Calcitonin, vasopressin, and other agents that raise intracellular adenosine 3',5'-cyclic monophosphate levels caused a rapid and dramatic decrease in the size and number of domes. This effect was independent of RNA and protein synthesis, and thus appeared unrelated to the production of urokinase, a proteinase synthesized by the cells in response to these agents. Calcitonin caused a decrease in transepithelial electrical resistance, suggesting that the effect of the hormone on domes was due to an increase in the permeability of a paracellular pathway. Thus, in addition to the wellknown effects of vasopressin on collecting duct permeability, part of the in vivo effect(s) of calcitonin and vasopressin on the renal tubule might also involve alterations of epithelial permeability related to those described here.

Animals↗
PubMed1986

Horseradish peroxidase uptake and crinophagy in insulin-secreting cells.

Upon exposure of pancreatic B cells to exogenous horseradish peroxidase (HRP), a population of secretory granules becomes HRP-labelled. In isolated islets of Langerhans, we studied the fate of HRP-labelled secretory granules during a pulse-chase experiment with HRP in order to assess their relationship with lysosomes containing secretory granule cores. These structures (crinophagic or multigranular bodies) were previously shown to be a site of insulin degradation (Orci et al., J cell biol 98 (1984) 222) [4]. After a 15-min pulse of peroxidase, the number and volume density of HRP-labelled secretory granules decreased over an 85-min chase period, during which the number and volume density of multigranular bodies labelled with HRP was significantly increased. At both time points, the surface density of HRP-labelled Golgi elements was very small compared with that of unlabelled ones. By autoradiography after a 5-min pulse of [3H]leucine and a 55-min chase, followed by a 15-min pulse of HRP and a 85-min chase, we could show that the majority of HRP-containing secretory granules were not radioactively labelled granules. These results suggest that: The low degree of HRP labelling of the Golgi makes it unlikely that secretory granules derive their HRP by budding from HRP-labelled cisternae. HRP-labelled SGs are preferentially transferred to MGBs (which become HRP-labelled) for prospective degradation. HRP labelling does not involve newly-formed mature secretory granules.

Animals↗
PubMed1986

Subcellular distribution of the external and internal domains of the EGF receptor in A-431 cells.

Using specific antibodies directed against the external and internal domains of the epidermal growth factor (EGF) receptor, we have directly localized by the protein A gold technique at the electron microscopic level these receptor regions in A-431 epidermoid carcinoma cells. With all antibodies tested, 80-85% of the EGF receptors are found inside the cells, where they preferentially associate with lysosome-like structures, a tubulovesicular system, the rough endoplasmic reticulum and the nuclear envelope. The same distribution pattern is observed for antibodies directed against the external carbohydrate region of the receptor, an antibody against the protein core of the external segment of the receptor, and an antibody reacting with the internal kinase domain of the receptor, suggesting that both receptor segments are similarly distributed intracellularly.

Antibodies, Monoclonal↗
PubMed1986

Internalization and recycling of 125I-photoreactive insulin-receptor complexes in hepatocytes in primary culture.

When the insulin receptor is tagged with a 125I-photoreactive insulin analogue that can be covalently coupled to it by UV irradiation, the fate of this labeled receptor can be followed both morphologically and biochemically. In the present study we have applied this tool to trace the pathway followed by 125I-photoreactive insulin-receptor complex in hepatocytes in primary culture. As determined by quantitative electron microscopic autoradiography, the internalized labeled material first associates with clear vesicles, second is found in multivesicular bodies, third associates with dense bodies and fourth returns to the cell surface via clear vesicles. This recycling process is inhibited by lysosomotropic agents, i.e. NH4Cl or chloroquine. These data confirm, in another cell system, our previous observations carried out in freshly isolated rat hepatocytes and demonstrate the feasibility and complementarity of both freshly isolated hepatocytes and hepatocytes in primary culture to study internalization and recycling of the insulin receptor.

Ammonium Chloride↗
PubMed1986

Basic fibroblast growth factor induces angiogenesis in vitro.

Fibroblast growth factors (FGFs) are potent mitogens for vascular and capillary endothelial cells in vitro and can stimulate the formation of blood capillaries (angiogenesis) in vivo. A crucial event in this process is the invasion of the perivascular extracellular matrix by sprouting endothelial cells. Using a recently developed in vitro model of angiogenesis, we show here that highly purified basic pituitary FGF can induce capillary endothelial cells to invade a three-dimensional collagen matrix and to organize themselves to form characteristic tubules that resemble blood capillaries. We also show that basic FGF concomitantly stimulates endothelial cells to produce a urokinase-type plasminogen activator, a protease that has been implicated in the neovascular response. The results demonstrate that basic FGF can stimulate processes that are characteristic of angiogenesis in vivo, including endothelial cell migration, invasion, and production of plasminogen activator.

Animals↗
PubMed1986

Intracellular pathway followed by the insulin receptor covalently coupled to 125I-photoreactive insulin during internalization and recycling.

After it interacts with a specific receptor on the cell surface, insulin is internalized in its target cell by an adsorptive endocytotic process and eventually degraded in lysosomes. It was also recently shown that the initial surface interaction between the hormone and its receptor is followed by an internalization of the receptor, which later is recycled back to the cell surface. In the present study the insulin receptor was tagged with a 125I-photoreactive insulin analogue that can be covalently coupled to the insulin receptor by ultraviolet irradiation. Using this tool we could trace by quantitative electron microscope autoradiography the intracellular pathway followed by this labeled receptor. The quantitative analysis of the intracellular distribution of the labeled material as a function of incubation time at 37 degrees C supports the following sequence of events: association first with clear vesicles, second with multivesicular bodies, third with dense bodies, and fourth, a return to the cell surface via clear vesicles. This insulin receptor recycling process is inhibited by monensin but unaffected by cycloheximide.

Animals↗
PubMed1986

Endothelial diaphragmed fenestrae: in vitro modulation by phorbol myristate acetate.

Cultured microvascular endothelial cells isolated from fenestrated capillaries have been shown to express many properties of their in vivo differentiated phenotype, yet they contain very few diaphragmed fenestrae. We show here that treatment of capillary endothelial cells with the tumor promoter, 4 beta-phorbol 12-myristate 13-acetate, induces more than a fivefold increase in the frequency of fenestrae per micron 2 of cell surface, as determined from a quantitative evaluation on freeze-fracture replicas. In quick-frozen, deep-etched preparations, the endothelial fenestrae appeared to be bridged by a diaphragm composed of radial fibers interweaving in a central mesh, as previously observed in vivo. These results indicate that diaphragmed fenestrae are inducible structures, and provide an opportunity to study them in vitro.

Adrenal Cortex↗
PubMed1986

Blockage of cell-to-cell communication within pancreatic acini is associated with increased basal release of amylase.

To assess whether junctional coupling is involved in the secretory activity of pancreatic acinar cells, dispersed rat acini were incubated for 30 min in the presence of either heptanol (3.5 mM) or octanol (1.0 mM). Exposure to either alkanol caused a marked uncoupling of the acinar cells which, in control acini, were extensively coupled. Uncoupling was associated with an increased basal release of amylase that was at least twice that of controls. By contrast, carbamylcholine (10(-5) M)-induced maximal amylase secretion, cytosolic pH, and free Ca2+, as well as the structure of gap junctions joining the acinar cells, were unaffected. Both uncoupling and the alteration of basal secretion were already observed after only 5 min of exposure to heptanol, they both persisted throughout the 30-min exposure to the alkanols, and were reversible after removal of either heptanol or octanol. Since neither of the two uncouplers appeared to alter unspecifically the secretory machinery and the nonjunctional membrane of acinar cells, the data are consistent with the view that junctional coupling participates in the control of the basal secretion of acinar cells.

Alcohols↗
PubMed1986

Role of intracellular calcium and protein kinase C in the endocytosis of transferrin and insulin by HL60 cells.

The role of the cytosolic free calcium concentration ([Ca2+]i) and of protein kinase C on the internalization of transferrin and insulin in the human promyelocytic cell line HL60 was investigated. [Ca2+]i was selectively monitored and manipulated by the use of the fluorescent Ca2+ indicator and buffer quin2, while receptor-ligand internalization was studied directly by quantitative electron microscope autoradiography. Decreasing the [Ca2+]i up to 10-fold below resting level had no effect on the internalization of transferrin or insulin. Similarly, a 10-fold elevation of the [Ca2+]i using the calcium ionophore ionomycin caused little or no change in the endocytosis of the two ligands. In contrast, activation of protein kinase C by phorbol myristate acetate markedly stimulated the internalization of both occupied and unoccupied transferrin receptors, even in cells with very low [Ca2+]i. The insulin receptor was found to behave differently in response to phorbol myristate acetate, however, in that only the occupied receptors were stimulated to internalize. We conclude that the [Ca2+]i plays only a minor role in regulating receptor-mediated endocytosis, whereas protein kinase C can selectively modulate receptor internalization depending on receptor type and occupancy.

Calcium↗
PubMed1986

Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles.

Proinsulin is a single polypeptide chain composed of the B and A subunits of insulin joined by the C-peptide region. Proinsulin is converted to insulin during the maturation of secretory vesicles by the action of two proteases and conversion is inhibited by ionophores that disrupted intracellular H+ gradients. To determine if conversion of prohormone to hormone actually occurs in an acidic secretory vesicle, cultured rat islet cells were incubated in the presence of 3-(2,4-dinitroanilino)-3' amino-N-methyldipropylamine (DAMP), a basic congener of dinitrophenol that concentrates in acidic compartments and is retained there after aldehyde fixation. The cells were processed for indirect protein A-gold colocalization of DAMP, using a monoclonal antibody to dinitrophenol, and proinsulin, using a monoclonal antibody that exclusively reacts with the prohormone. The average density of DAMP-specific gold particles in immature secretory vesicles that contained proinsulin was 71/micron 2 (18 times cytoplasmic background), which indicated that this compartment was acidic. However, the density of DAMP-specific gold particles in the insulin-rich mature secretory vesicle averaged 433/micron 2. This suggests that although proinsulin conversion occurs in an acidic compartment, the secretory vesicles become more acidic as they mature. Since the concentration of anti-proinsulin IgG binding in secretory vesicles is inversely proportional to the conversion of proinsulin to insulin, we were able to determine that maturing secretory vesicles had to reach a critical pH before proinsulin conversion occurred.

Animals↗
PubMed1986

Somatostatin analog-induced remission of necrolytic migratory erythema without changes in plasma glucagon concentration.

A 41-year-old woman with metastatic glucagonoma and the characteristic disabling rash, necrolytic migratory erythema, was treated with a synthetic somatostatin analog while waiting to undergo curative surgical resection. Plasma glucagon concentration (1,500-3,300 pg/ml, normal less than 200) remained elevated during analog therapy as the rash cleared. Only with surgical resection (partial pancreatectomy and partial hepatectomy) did glucagon levels return to normal. The therapeutic benefit caused by the analog in this syndrome differs from that in other endocrine tumor syndromes such as pancreatic cholera, carcinoid, or gastrinoma where circulating levels of tumor-produced agents are suppressed in conjunction with control of symptoms.

Adult↗
PubMed1986

The "coat" of kidney intercalated cell tubulovesicles does not contain clathrin.

Intercalated cells of kidney collecting ducts contain a population of tubulovesicles in their apical cytoplasm, whose limiting membranes are decorated by arrays of dense, club-shaped projections oriented toward the cytoplasm. These tubulovesicles have been implicated in endo-exocytotic events in these cells. To determine a possible relationship between this "coating" material and clathrin, the coat protein associated with endocytotic coated pits and coated vesicles in other cell types, we applied a monospecific, affinity-purified anti-clathrin antibody to thin sections of rat kidney embedded at low temperature in Lowicryl K4M. We found that no specific labeling was associated with the studlike material of intercalated cell tubulovesicles.

Animals↗