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M Ravazzola

Publications and source records attributed to M Ravazzola.

At least 73 records · Page 4Linked to original sources
PubMed1986

Internalization of 3H-glibenclamide in pancreatic islet cells.

As judged by autoradiographic criteria at the optical level, rat islet cells progressively accumulate 3H-glibenclamide over 1 to 30 min incubation at 24 degrees C. The labeled material associates with all endocrine cells, but is preferentially seen within B cells. The total uptake of 3H-glibenclamide (1.0 mumol/l) is little affected by the presence of unlabeled glibenclamide (0.2 mmol/l), but is significantly decreased at 4 degrees C (p less than 0.05). At the electron microscopic level, less than 15% of the autoradiographic grains are located at the B cell plasma membrane and 72-79% of the grains found over the cytoplasm are associated with insulin secretory granules. Such a pattern is observed both after short (1 min) or prolonged (30 min) incubation, and at 4 or 24 degrees C. It is proposed that the insulinotropic action of glibenclamide is not necessarily attributable to primary events located solely at the cell surface.

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

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↗
PubMed1985

Direct identification of prohormone conversion site in insulin-secreting cells.

We have localized proinsulin in B cells of human and rat pancreatic islets, using a proinsulin-specific monoclonal antibody revealed by immunocytochemistry. Proinsulin is abundant in Golgi stacks and clathrin-coated secretory granules. It rapidly disappears from these compartments when protein synthesis is inhibited. Depletion of ATP stores prevents movement of proinsulin from the Golgi stacks to the secretory granules; under these conditions, the prohormone in preformed coated granules is converted to insulin, whereas that bound to the Golgi complex is not. Non-coated granules show a low level of proinsulin reactivity under all incubation protocols. These findings provide direct evidence that coated secretory granules are the major, if not the only, cellular site of proinsulin to insulin conversion. They also suggest that the Golgi stack is not involved in conversion, and that intercisternal transport and coated granule formation are hitherto unrecognized energy-requiring steps that precede conversion.

Animals↗
PubMed1985

Clathrin-immunoreactive sites in the Golgi apparatus are concentrated at the trans pole in polypeptide hormone-secreting cells.

By analogy with receptor-mediated endocytosis, clathrin-coated membrane segments at locations other than at the plasma membrane have been implicated in intracellular membrane transport/sorting. The crucial role of the Golgi apparatus in these processes prompted us to study the distribution at this level of clathrin-immunoreactive sites in various polypeptide hormone-secreting cells. With a polyclonal antibody recognizing the polymerized form of clathrin (structural coats or cages), we found that the Golgi area had the highest degree of specific labeling of various cytoplasmic regions, including the subplasmalemmal web. Moreover, the Golgi labeling was not homogeneously distributed, most of the immunoreactive sites being associated with membrane segments at the trans pole. The labeled membranes comprised regions of trans cisternae with and without condensing secretory material, newly formed (maturing) secretory granules freshly released from trans cisternae, and typical coated vesicles. The cis Golgi cisternae, as well as the cytoplasmic region containing transfer (shuttle) vesicles and transitional cisternae of the rough endoplasmic reticulum, were only weakly labeled. Clathrin concentration, degree of assembly of triskelions into coats, duration of clathrin association with membranes, or presence of a coat protein different from clathrin could account for the low immunoreactivity observed at the cis pole as compared to the trans pole.

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

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

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

[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↗
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

[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

Pathology of endocrine cells in gastric mucosa.

Pathological changes of gastric endocrine cells are reviewed. The lesions are subdivided according to the type of mucosa in which they originate. Hyperplasias of fundic endocrine cells, probably related to the patient's concomitant hypergastrinemia, were found in Zollinger Ellison syndrome and in chronic atrophic gastritis. They may evolve into carcinoid tumors. Typical and atypical forms of fundic carcinoids have been described, usually without specific clinical syndrome. Antral gastrin G cells proliferate in achlorhydric, hypergastrinemia patients while their involvement in peptic ulcer disease is probably limited to a minority of cases. Antral gastrin secreting tumors are rare. Metaplasic intestinal epithelium also harbors endocrine cells that may present hyperplastic and neoplastic changes.

Endocrine Glands↗
PubMed1982

Gastric carcinoma with argyrophilic cells: light microscopic, electron microscopic, and immunochemical study.

An immunochemical study of a gastric adenocarcinoma with argyrophilic cells showed two areas of tumor that react differently with the usual histochemical reagents as well as with immune sera against gastrin and mucoprotein associated with antigens. Ninety per cent of the tumor cells were PAS positive and contained M2 antigen, and some also contained M1 antigen. About 30 per cent of the M2-positive cells stained strongly with an antigastrin serum as well as with the argyrophilic reagents. The remaining 10 per cent of tumor cells were signet-ring cells located in several clumps in the tumor. These cells were positive for both PAS and alcian blue and contained intestinal M3 antigen. Forty-five per cent of them also contained M1 gastric antigens. Carcinoembryonic antigen (CEA) was found in the cytoplasm of each tumor cell. The presence of CEA and M1 antigen together indicates a fetal pattern, suggesting that the cells originate from very immature gastrointestinal stem cells.

Adenocarcinoma↗
PubMed1982

Glicentin precedes glucagon in the developing human pancreas.

A quantitative evaluation of immunofluorescence elicited by anti-insulin, anti-glucagon, anti-glicentin, anti-somatostatin and anti-pancreatic polypeptide antisera has been carried out in the pancreas of 5 human fetuses from 3.0 to 9.6 cm C.R. The data obtained indicate that while insulin and somatostatin-containing cells are approximately in similar proportions with respect to the other endocrine cell types in the five fetuses studied, the glucagon and glicentin immunoreactive cells and the pancreatic polypeptide cells are not : a) pancreatic polypeptide-containing cells increase in proportion as fetuses grow older; b) the youngest fetuses (3.0 to 4.3 cm C.R.) contain a high proportion of cells reacting to anti-glicentin antiserum only (GLI-cells) and a small proportion of cells stained both with the anti-glicentin and anti-glucagon antisera (GLI/GLU-cells). However, the latter cell type which stains similarly as the postnatal and adult pancreatic A-cell (GLI-cells are not detectable in the postnatal and adult pancreas) increases iin proportion in older fetuses, while the proportion of GLI-cells decrease. The data suggest that the definitive adult-type A-cell matures from a GLI-cell type which is not able to convert glucagon precursors GLI(s) into glucagon.

Female↗