Search PubMed⌕ Search

Biomedical subjects

L Orci

Publications and source records attributed to L Orci.

At least 271 records · Page 15Linked to original sources
PubMed1982

Orientation of rat-liver plasma membrane vesicles. A biochemical and ultrastructural study.

Using both biochemical and morphological methods, the membrane orientation of plasma membrane vesicles from rat liver which are capable of catalysing the active transport of amino acids was investigated. In intact vesicles, the plasma membrane enzyme (Na+ + K+)-ATPase displays only a minor portion of its total activity which is greatly increased upon vesicle disruption. The same intact vesicles show an almost maximal binding of ouabain, which binds only to the extracellular side of the plasma membrane. A freeze-fracture analysis of the vesicles shows that a distinct population of relatively large vesicles have predominantly the in vivo membrane orientation. These large vesicles are labelled with numerous filipin-sterol complexes following exposure to the cholesterol probe, filipin, and are therefore assumed to be plasma membrane vesicles. A population of smaller vesicles with mainly an inside-out orientation were not labelled with filipin and are probably microsomes. The data obtained with both biochemical and ultrastructural techniques indicate that the plasma membrane vesicles isolated from rat liver for transport studies are mostly (at least 70%) orientated as in vivo, i.e. inside-in.

Animals↗
PubMed1982

Circulating somatostatin acts on the islets of Langerhans by way of a somatostatin-poor compartment.

Somatostatin perfused in canine pancreases at 10 to 20 picograms per milliliter or 10 to 20 percent of the pancreatic vein somatostatin concentration inhibited insulin and glucagon secretion. This suggests that the high local concentration of endogenous somatostatin is not in contact with somatostatin receptors of the islets. The integrity of this separation may determine the sensitivity of islet cells to circulating somatostatin.

Animals↗
PubMed1982

Quantitative electron microscopic autoradiography of insulin, glucagon, and somatostatin binding sites on islets.

After monolayer cultures of rat islets were exposed to [(125)I]insulin,[(125)I]glucagon, and [(125)I]tyrosinyl somatostatin, specific autoradiographic grains associated with each radioactively labeled ligand were found on B, A, and D cells. The density of labeling of the B, A, and D cells with each labeled ligand correlated well with the known actions of the three hormones on each of the islet cells.

Animals↗
PubMed1982

Freeze-fracture cytochemistry of neuronal membranes: inhomogeneous distribution of filipin-sterol complexes in perikarya, dendrites and axons.

Vibratome slices of cerebellar cortex fixed in a glutaraldehyde solution containing filipin, a sterol-specific probe, were freeze-fractured to study the distribution of filipin-sterol complexes in neuronal plasma membranes. These complexes appear as 25-30 nm protuberances or pits in the fracture face of plasma membranes, and their density was low in dendrites of Purkinje and granule cells. In contrast, the plasma membranes of neuronal perikarya showed an abundant filipin labeling, 4-6 times greater than in dendrites. Parallel fibers, the axons of granule cells also had significantly more filipin-sterol complexes than granule cell dendrites, but fewer than granule cell perikarya. The results reveal difference in the organization of specific regions of neuronal plasma membranes, which are also characterized by a different pattern of synaptic contacts.

Animals↗
PubMed1982

Receptor mediated endocytosis of polypeptide hormones: mechanism and significance.

Polypeptide hormones bind to specific receptors on the surface of cells. Under certain conditions they localize to specific microdomains of the membrane, i.e., microvilli and coated pits. At physiologic temperature the ligand is internalized by a process of adsorptive endocytosis. This process involves several intracellular membrane bounded structures including coated vesicles, non-coated vesicles and lysosomal structure. These events provide a simple and general mechanism for removal of the ligand from the cell surface in order to terminate its signal. Linked to this process is a mechanism for surface receptor regulation. Thus, the concentration of hormone receptors on the cell surface is a function of the synthetic rate, internalization rate and the rate of recycling of surface membrane.

Animals↗
PubMed1982

Internalized insulin receptors are recycled to the cell surface in rat hepatocytes.

We have followed the fate of cell surface insulin receptors in isolated rat hepatocytes by both a biochemical and a morphological approach. Hepatocytes were labeled with the photoreactive and biologically active 125I-labeled insulin analogue, [2-nitro-4-azidophenylacetylB2]des-PheB1-insulin, under conditions that allow for minimal internalization (2 hr at 15 degrees C). Analysis of the cell-associated radioactivity by NaDodSO4/polyacrylamide gel electrophoresis under reducing conditions followed by autoradiography revealed the specific labeling of a major insulin receptor subunit with Mr 130,000 and a minor degradation product with Mr 125,000. When the cells were exposed at 15 degrees C to trypsin at the end of the association period, these two bands were no longer observed, indicating that the labeled receptors were at the cell surface. This trypsin sensitivity of the receptor disappeared within 30-60 min of incubation of the cells at 37 degrees C, reflecting the internalization of the hormone-receptor complexes. Over the subsequent 4 hr of incubation, this was followed by a progressive reappearance of the receptor complexes at the cell surface, as indicated by the recovery of trypsin sensitivity of the labeled insulin receptors. An identical (both chronologically and quantitatively) journey of the insulin receptors was observed when the labeled material was studied by quantitative electron microscopic autoradiography. Thus, when the cells were incubated at 37 degrees C there was a rapid decrease (30-60 min) in the percentage of autoradiographic grains associated with the plasma membrane, followed by a progressive increase in this percentage over the subsequent 4 hr of incubation. In conclusion, using a biochemical and morphological approach to trace the photoaffinity-labeled insulin receptor, we have shown that the internalized hormone-receptor complex is recycled back to the cell surface.

Affinity Labels↗
PubMed1982

Receptor-mediated endocytosis of insulin: role of microvilli, coated pits, and coated vesicles.

When 125I-labeled insulin (125I-insulin) is incubated with 3T3-L1 adipocytes and cells processed for electron microscopic autoradiography, the ligand initially localizes preferentially to microvilli and coated pits. As a function of time and temperature, this initial preferential localization to microvilli is lost, and the ligand is internalized by the cell. Serial sections of apparent coated vesicles near the cell surface indicate that about half of these structures are true vesicles and, therefore, intermediates in this receptor-mediated endocytotic process. With time, 125I-insulin localizes to larger intracellular membrane-bounded structures. When cells are incubated with another ligand, cationic ferritin, that is taken up by adsorptive endocytosis, essentially the same structures are involved as for the endocytosis of 125I-insulin. The data suggest that specificity for receptor-mediated endocytosis is conferred by the specific ligand receptor and possibly by ligand-induced receptor mobility in the plane of the plasma membrane. Other structures such as coated pits, coated vesicles, larger vesicles, and secondary lysosomes are common for different ligands.

Animals↗
PubMed1982

Direct communication of homologous and heterologous endocrine islet cells in culture.

The transfer of 6-carboxyfluorescein between islet cells in monolayer culture was observed by fluorescence microscopy, and the endocrine cells involved in this transfer were identified by immunohistochemistry and electron microscopy. The results show that carboxyfluorescein was directly exchanged between homologous B-cells and also between B- and A- or D-cells. Successive microinjections of the probe into different cells of the same cluster showed the existence of separate territories, each formed by 2-8 communicating cells. Intercellular communication was not observed after every dye microinjection, and communicating and noncommunicating islet cells were found to coexist within the same cluster. The data indicate that the exchange of exogenous cytoplasmic molecules occurs between different types of endocrine islet cells. However, within a single cluster, all islet cells are not metabolically coupled to one another, at a given time.

Animals↗
PubMed1982

Biochemical and morphological evidence that the insulin receptor is internalized with insulin in hepatocytes.

There is morphological and biochemical evidence that insulin is internalized in hepatocytes. The present study was designed to investigate the fate of the insulin receptor itself, subsequently to the initial binding step of the hormone to the hepatocyte plasma membrane. The insulin receptor was labeled with a 125I-photoreactive insulin analogue (B2[2-nitro,4-azidophenylacetyl]des-PheB1-insulin). This photoprobe was covalently coupled to the receptor by UV irradiation of hepatocytes after an initial binding step of 2-4 h at 15 degrees C. At this temperature, only limited (approximately 20%) internalization of the ligand occurred. In a second step, hepatocytes were resuspended in insulin-free buffer and further incubated for 2-4 h at 37 degrees C. After h at 37 degrees C, no significant radioactivity could be detected in non-UV-irradiated cells, whereas 12-15 % of the radioactivity initially bound remained associated to UV-irradiated cells. Morphological analysis after electron microscopy revealed that approximately 70% of this radioactivity was internalized and preferentially associated with lysosomal structures. SDS PAGE analysis under reducing conditions revealed that most of the radioactivity was associated with a 130,000-dalton band, previously identified as the major subunit of the insulin receptor in a variety of tissues. Internalization of the labeled insulin-receptor complex at the end of the 37 degrees C incubation was further demonstrated by its inaccessibility to trypsin. Conversely, at the end of the association step, the receptor (also characterized as a predominant 130,000-dalton species) was localized on the cell surface since it was cleaved by trypsin. We conclude that in hepatocytes the insulin receptor is internalized with insulin.

Animals↗
PubMed1982

Immunocytochemical localization of the vitamin D-dependent calcium binding protein in chick duodenum.

The vitamin D-dependent calcium binding protein (CaBP) of chick duodenum has been localized by immunocytochemistry and by radioimmunoassay. Light microscopically, CaBP was seen to be present in the absorptive cells of the villi while in other cell types of the villi and the crypts, including goblet cells and endocrine cells, no CaBP was seen. At the electron microscopic level, CaBP was shown to be localized in the cytosol and the euchromatin of the nucleus but not in membrane-bounded cytoplasmic compartments. Quantitative evaluation of the immunocytochemical protein A-gold label showed that the terminal web and the cytosol of basal cellular regions were most highly labeled while the brush border was weakly labeled. The radioimmunoassay evaluation of intestinal subcellular fractions indicated that 96% of the homogenate CaBP is in the cytosol high-speed supernatant fraction. Collectively, these results support the hypothesis that the vitamin D-dependent intestinal CaBP may play a role in either regulation of intracellular calcium concentration or movement of calcium across the brush border membrane from the gut lumen.

Animals↗
PubMed1982

Co-localization of 125I-epidermal growth factor and ferritin-low density lipoprotein in coated pits: a quantitative electron microscopic study in normal and mutant human fibroblasts.

Low density lipoprotein (LDL) and epidermal growth factor (EGF) bind to receptors on the surface of human fibroblasts and are internalized in coated vesicles. Each of the ligands has been studied separately by electron microscopy in human fibroblasts using ferritin-LDL as one visual probe and 125I-EGF as a second visual probe. A mutant strain of human fibroblasts (J.D.) has been described in which LDL does not localize to coated pits and hence is not internalized. Because LDL and EGF do not compete with each other for binding, in the current studies we coincubated the two ligands with normal and mutant cells to visualize their cellular fates. In normal fibroblasts ferritin-LDL and 125I-EGF both bound preferentially to coated pits at 4 degrees C and both ligands were internalized into endocytotic vesicles and lysosomes. Quantitative studies in normal cells showed that 75% of the coated pits and vesicles that contained 125I-EGF also contained ferritin-LDL, indicating that both ligands enter the cell through the same endocytotic vesicles. In the LDL internalization-mutant J.D. cells, ferritin-LDL did not localize in coated pits and was not internalized, but 125I-EGF bound to coated pits and was internalized just as in normal fibroblasts.

Cell Line↗
PubMed1982

Localization of the vitamin D-dependent calcium-binding protein in mammalian kidney.

The vitamin D-dependent calcium-binding protein (CaBP) was localized by immunocytochemistry in the rat and human kidney. In both species 80% of the cells lining the distal convoluted tubules contained CaBP. In the connecting segment and the initial collecting tubule of rat kidney, 50% of the cells was positive; in the outer medullary collecting duct only 15% was positive. In the human kidney, collecting ducts in medullary rays contained 50% positive cells, whereas in the rest of the medulla no positive cells were found. The CaBP-positive cells were identified as principal or clear cells by immunoelectron-microscopy, using the protein A-gold technique. Mitochondria-rich dark cells were negative. In principal cells, CaBP immunoreactive sites were found throughout the cytosol and the nuclear euchromatin. No preferential labeling of cellular membranes was found. The data show that CaBP-positive cells are present in tubular regions that are important in regulating the final excretion of calcium. However, the subcellular distribution of CaBP does not suggest a role in the initial transmembrane transport of Ca2+ but rather indicates an involvement in processes regulating intracellular calcium.

Adult↗
PubMed1982

Patterns of filipin-sterol complex distribution in intact erythrocytes and intramembrane particle-aggregated ghost membranes.

When fixed erythrocytes are exposed to the cholesterol probe, filipin, and freeze-fractured, their membrane is labeled with only a limited number of typical, 25 nm diameter filipin-sterol complexes. In addition, the membrane contains many less distinct filipin-induced perturbations that give it an overall rippled appearance. In contrast, red cell ghosts subjected to conditions that result in an aggregation of their intramembrane particles have large particle-free membrane domains which are completely filled with 25 nm filipin-sterol complexes. These results suggest that some constraint exists in the intact erythrocyte membrane which restricts the formation of typical filipin-sterol complexes.

Animals↗