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Biomedical subjects

L Orci

Publications and source records attributed to L Orci.

At least 163 records · Page 9Linked to original sources
PubMed1987

Nonclathrin-coated vesicles are involved in endocytosis in kidney collecting duct intercalated cells.

Intercalated cells of the kidney collecting duct are able to modify the structure of their apical plasma membrane in response to different physiological conditions. It has been proposed that this process involves the transfer of membrane components (including a proton-pumping ATPase) to and from the apical membrane by a specialized population of tubulovesicles that are found in the apical cytoplasm of these cells. These vesicles have a prominent cytoplasmic coat of regularly arranged dense studs that we have recently shown to be immunocytochemically and morphologically distinct from clathrin. In this study, we have examined the function of these vesicles by using horseradish peroxidase as a tracer of endocytosis at the light and electron microscopic levels. Following the intravenous injection of rats with the tracer, we found a massive labeling of the tubulovesicle compartment of intercalated cells, providing direct evidence that these nonclathrin-coated vesicles are involved in endocytotic events in this cell type. This novel membrane coating material could contain the cytoplasmic domains of molecules transported to and from the plasma membrane by these vesicles (e.g., and H+ ATPase) or it could be a molecule that is involved in vesicle function, by analogy with clathrin.

Animals↗
PubMed1987

Differences in glycocalyx composition between cells of the cortical thick ascending limb of Henle and the macula densa revealed by lectin-gold cytochemistry.

The glycocalyx composition of cells from the macula densa and the cortical thick ascending limb of Henle was examined in rabbit kidney by means of the lectin-gold technique. A quantitative evaluation at the ultrastructural level showed that macula densa cells had a considerably greater affinity for Helix pomatia lectin than adjacent cells of the thick ascending limb. Wheat germ lectin and concanavalin A bound equally well to both cell types. This difference in plasma membrane glycocalyx composition may be an important aspect of the functional differentiation of cell types in this specialized nephron segment.

Animals↗
PubMed1987

Phorbol esters induce angiogenesis in vitro from large-vessel endothelial cells.

We have shown previously that the tumor promoter phorbol myristate acetate (PMA) induces capillary endothelial cells grown on the surface of three-dimensional collagen gels to invade the underlying matrix as capillary-like tubular structures, a phenomenon mimicking angiogenic processes that occur in vivo (Montesano and Orci: Cell 42:469, 1985). To establish whether the potential to invade the extracellular matrix as capillary-like sprouts is restricted to microvascular endothelial cells or is also shared by large vessel endothelium, we have examined the response to PMA of endothelial cells isolated from the human umbilical vein and the calf pulmonary artery. The results of these experiments show that both types of macrovascular endothelial cells are able to penetrate into collagen gels as vessel-like tubes following treatment with PMA. This demonstrates that endothelial cells derived from large vessels can, in response to appropriate signals, express invasive properties thought to be associated specifically with capillary endothelial cells in vivo.

Animals↗
PubMed1987

Phorbol ester induces cultured endothelial cells to invade a fibrin matrix in the presence of fibrinolytic inhibitors.

We have previously shown that the tumor promoter 4 beta-phorbol 12-myristate 13-acetate (PMA) induces capillary endothelial cells grown to confluency on the surface of three-dimensional collagen gels to invade the underlying matrix and to form capillary-like tubular structures, a phenomenon mimicking angiogenic processes that occur in vivo (Montesano and Orci: Cell, 42:469-477, 1985). Since angiogenesis frequently occurs within a fibrin-rich extracellular matrix, we have examined the ability of PMA-treated endothelial cells to invade fibrin gels. Control endothelial cells grown on fibrin gels formed a confluent monolayer on the gel surface and did not invade the underlying matrix. Treatment of the cultures with PMA resulted in a progressive lysis of the substrate without invasion of the fibrin matrix. However, if the cells were treated with PMA either in the presence of fibrinolytic inhibitors (Trasylol, epsilon-aminocaproic acid) or in the absence of detectable plasminogen, dissolution of the substrate was prevented, and the endothelial cells invaded the fibrin gel, forming vessel-like tubular structures similar to those previously observed with collagen gels. These results demonstrate that the invasive and morphogenetic events induced by PMA do not necessarily require an interaction between endothelial cells and collagen fibrils but can also occur with other biologically relevant substrata. They also suggest (1) that invasion may occur via a plasmin-independent mechanism and (2) that in vivo, neutralization of excess proteolytic activity may play an important permissive role in angiogenesis and other invasive processes by preventing uncontrolled matrix degradation.

Aminocaproic Acid↗
PubMed1987

Rapid in vitro formation of smooth endoplasmic reticulum aggregates within peptide-producing islet cells.

We report here that heptanol (3.5 mM) induces in vitro a rapid formation of smooth endoplasmic reticulum aggregates (SERA) within isolated islets of Langerhans. SERA appeared after only 15 min of exposure to the alkanol and increased in number during the first 30 min of incubation. At that time, SERA represented 2% and 6% of the volume of B- and non-B-cells, respectively. Removal of heptanol resulted in the rapid disappearance of SERA, whereas reintroduction of the alkanol rapidly induced these structures again. SERA formation was seen in different types of endocrine and nonendocrine islet cells. In the insulin-producing B-cells, SERA formation was not modified by conditions known to alter the secretory activity and the microtubular-microfilament network or to inhibit protein synthesis. By contrast, SERA formation was inhibited by low temperature and by conditions depleting the energy sources of the cells. Similar observations were made in the presence of either octanol (1 mM) or nonanol (1 mM) but not of shorter chain alkanols, alkanes, oxidative derivates of either heptanol or octanol, and of other unrelated lipid-soluble compounds. Incubations in the presence of long-chain alkanols provide, therefore, a unique model to study in vitro the formation and disposal of smooth endoplasmic reticulum, as well as a system in which rapid membrane biogenesis is amenable to direct experimental testing.

Alcohols↗
PubMed1987

Resistance of the insulin crystal to lysosomal proteases: implications for pancreatic B-cell crinophagy.

Insulin is thought to be chemically stabilized within beta-granules in the crystal form. The other major products of the beta-granule, proinsulin and C-peptide, by contrast, are not thought able to crystallize. The physico-chemical properties of peptides in soluble or crystalline form are dramatically different. The ability of insulin to crystallize in the beta-granule might thus explain why this peptide, but not proinsulin/C-peptide, remains stable even after its introduction into lysosomes as occurs during granulolysis (crinophagy). We have now studied this by exposing proinsulin or insulin to lysosomal proteases in vitro. 125I-insulin in soluble form was found to be degraded at the same rate as 125I-proinsulin. Strikingly, however, when the labelled insulin was crystallized, its rate of degradation was decreased from 1.9 to 0.2 pmol/min. We take these data as confirmation that the insulin crystal is resistant to degradation, thereby possibly accounting for (a) the presence of insulin immunoreactivity within multigranular bodies, and (b) the unusually slow rate of degradation of insulin within B cells compared with that of other hormones in their cells of origin.

Animals↗
PubMed1987

Gap junctional coupling modulates secretion of exocrine pancreas.

Dispersed pancreatic acini were studied to assess the function of junctional coupling between adult secretory cells. Nonstimulated control cells were extensively coupled to their neighbors throughout each acinus. Addition of heptanol caused their uncoupling and increased their basal amylase release. Neurotensin, secretin, and vasoactive intestinal peptide (VIP) stimulated amylase secretion without uncoupling acinar cells. Heptanol rapidly and markedly uncoupled the neurotensin-, secretin-, and VIP-stimulated acinar cells and increased their amylase secretion in an additive manner. By contrast, the secretory response to carbamoylcholine (carbachol), a secretagogue that, alone, uncoupled acinar cells, was not affected by heptanol. Basal as well as neurotensin-, secretin-, and VIP-stimulated output returned to the lower control values following removal of heptanol and recovery of normal coupling. The data provide evidence that blockage of gap junctional coupling increases the basal secretion of exocrine pancreas as well as the response of the gland to a variety of secretagogues.

Alcohols↗
PubMed1987

Use of a synthetic peptide antigen to generate antisera reactive with a proteolytic processing site in native human proinsulin: demonstration of cleavage within clathrin-coated (pro)secretory vesicles.

Polyclonal antibodies reactive with a cleavage site in human proinsulin (HPI) (C-peptide-A-chain junction) have been raised (rabbit, guinea pig) using a synthetic peptide antigen coupled with keyhole limpet hemocyanin. These antisera recognize native HPI and des-31,32-HPI equally well but react 20-50 times less well with des-64,65-HPI, the intermediate cleaved at the C-peptide-A-chain junction and lacking the Lys-Arg pair. The guinea pig antisera did not recognize insulin but reacted weakly with C peptide at high concentrations; the rabbit antisera reacted with neither insulin nor C peptide. Immunocytochemical studies with human islet tissue localized the immunoreactivity of these antisera to clathrin-coated (pro)secretory vesicles derived from the trans Golgi, indicating that cleavage of the C-peptide-A-chain junction of proinsulin occurs mainly, if not exclusively, in this compartment of the beta cell.

Antibodies↗
PubMed1987

Ligands internalized through coated or noncoated invaginations follow a common intracellular pathway.

Cholera toxin (CT) represents a class of ligands that binds preferentially to noncoated pits on the cell surface. In the present study, we have investigated the mechanism of endocytosis of this class of ligand and compared it to the classic coated pit mechanism. When either CT coupled to colloidal gold particles (CT-gold) or 125I-labeled CT were incubated with 3T3 L1 fibroblasts at 4 degrees C, both ligands bound in a preferential fashion to small noncoated pits on the cell surface. CT-gold surface-labeled cells were then incubated at 22 degrees C. The labeled ligand progressively moved into noncoated vesicles and a tubulovesicular compartment composed of a network of tubules and vesicles closely associated with multivesicular bodies but distinct from the Golgi complexes. The ligand next passed into multivesicular bodies. By contrast, alpha 2-macroglobulin (alpha 2m)-gold initially localized preferentially to coated pits and subsequently to coated vesicles and tubulovesicular structures before associating with multivesicular bodies. To directly compare the intracellular pathway followed by CT-gold to that followed by alpha 2m-gold, CT-gold (7 nm) was coincubated with alpha 2m-gold (15 nm). By 10 min of incubation at 22 degrees C, up to 66% of tubulovesicular units contained both ligands when analyzed in serial sections. Subsequently, both ligands were colocalized in multivesicular bodies. We conclude that CT-gold endocytosed via noncoated vesicles and alpha 2m-gold endocytosed through coated vesicles subsequently associate with the same tubulovesicular units, multivesicular bodies, and lysosomes.

Animals↗
PubMed1987

Urokinase-type plasminogen activator is induced in migrating capillary endothelial cells.

Cellular migration is an essential component of invasive biological processes, many of which have been correlated with an increase in plasminogen activator production. Endothelial cell migration occurs in vivo during repair of vascular lesions and angiogenesis, and can be induced in vitro by wounding a confluent monolayer of cells. By combining the wounded monolayer model with a substrate overlay technique, we show that cells migrating from the edges of an experimental wound display an increase in urokinase-type plasminogen activator (uPA) activity, and that this activity reverts to background levels upon cessation of movement, when the wound has closed. Our results demonstrate a direct temporal relationship between endothelial cell migration and uPA activity, and suggest that induction of uPA activity is a component of the migratory process.

Adrenal Cortex↗
PubMed1987

Direct visualization of the phosphorylated epidermal growth factor receptor during its internalization in A-431 cells.

Epidermal growth factor (EGF) rapidly stimulates receptor autophosphorylation in A-431 cells. After 1 min the phosphorylated receptor can be identified at the plasma membrane using an anti-phosphotyrosine antibody. With further incubation at 37 degrees C, approximately 50% of the phosphorylated EGF receptor was internalized (t1/2 = 5 min) and associated with the tubulovesicular system and later with multivesicular bodies, but not the nucleus. During this period, there was no change in the extent or sites of phosphorylation. At all times the phosphotyrosine remained on the cytoplasmic side of the membrane, opposite to the EGF ligand identified by anti-EGF antibody. These data indicate that (a) the tyrosine-phosphorylated EGF receptor is internalized in its activated form providing a mechanism for translocation of the receptor kinase to substrates in the cell interior; (b) the internalized receptor remains intact for at least 60 min, does not associate with the nucleus, and does not generate any tyrosine-phosphorylated fragments; and (c) tyrosine phosphorylation alone is not the signal for receptor internalization.

Amino Acids↗
PubMed1987

Stimulation of insulin secretion reveals heterogeneity of pancreatic B cells in vivo.

We examined the immunofluorescence and ultrastructural changes of insulin-producing B cells in the center and at the periphery of islets of Langerhans during in vivo stimulation by glucose and glibenclamide. A decreased insulin immunostaining was detected in islets from the splenic rat pancreas after 1.5 h of glucose stimulation. By contrast, immunofluorescence changes became apparent in islets from the duodenal pancreas only after greater than 3 h of hyperglycemia. In both cases, the immunolabeling of central B cells decreased before that of peripheral B cells. Similar changes were seen following in vivo stimulation of insulin secretion by glibenclamide. At the ultrastructural level, hyperglycemia decreased the volume density of B cell secretory granules and increased that of rough endoplasmic reticulum and Golgi apparatus. These changes were also detected earlier in central than in peripheral B cells and earlier in splenic than in duodenal islets. The data show that B cells form a heterogeneous population in vivo.

Animals↗
PubMed1987

Selective binding of somatostatin-14 and somatostatin-28 to islet cells revealed by quantitative electron microscopic autoradiography.

Quantitative electron microscopic autoradiography was used for comparing the binding of labeled somatostatin-14 (S-14) and somatostatin-28 (S-28 section) to islet cells. Monolayer cultures of rat islet cells were incubated with [125I-Tyr11]S-14 (S-14 section) or [125I-Leu8, D-Trp22, Tyr25]S-28 (S-28 section) in the presence or absence of excess unlabeled peptides. Autoradiographic grains (ARG) associated with individual islet cells were identified and expressed as the mean number per B, A, and D cells. Specific ARG associated with S-14 were found over B and A cells. S-28 section-related specific ARG were concentrated over B, A, as well as D cells. The highest density of S-14 section labeling occurred over A cells, which under conditions of maximum labeling (37 degrees C for 60 min) contained five times as many ARG as did B cells. By contrast, under the same incubation conditions, the labeling density with S-28 section was maximal over B cells, which contained four and five times as many grains as A and D cells, respectively. These observations show preferential association of S-14 section with the A cell and S-28 section with the B cel provide strong evidence for the existence of separate binding sites for S-14 section and S-28 section on A and B cells, respectively, which presumably mediate the previously reported glucagon selective inhibitory effect of S-14 and the insulin-selective action of S-28.

Animals↗
PubMed1987

Somatostatin-14-like antigenic sites in fixed islet D-cells are unaltered by cysteamine: a quantitative electron microscopic immunocytochemical evaluation.

Exposure of somatostatin cells to cysteamine (CSH) produces a marked reduction in somatostatin-14-like immunoreactivity (S-14 LI) in cell extracts. In the present study we have evaluated the effects of CSH on S-14-like sites in fixed islet D-cells using immunofluorescence and quantitative electron microscopic immunocytochemistry. Monolayer cultures of rat islet cells exposed to CSH (10 mM) for 1 h and subsequently extracted in 1 M acetic acid exhibited a severe reduction in S-14 LI from 6.6 +/- 0.48 to 0.7 +/- 0.06 ng/dish. CSH-induced reduction in S-14 LI persisted when cells were fixed in Zamboni's solution for 16 h and subsequently extracted and assayed. By immunofluorescence, however, the relative numbers of somatostatin-positive cells as well as the fluorescent intensity were identical in control and CSH-treated cells. CSH did not produce any identifiable abnormality in the ultrastructural appearance of D-cells. Protein A-gold labeling of the islet cells showed a uniform distribution of gold particles in both control and CSH-treated cultures. The density of gold particles over D-cell secretory granules from CSH-exposed cultures (36.6 +/- 3.5 particles/micron2) was not different from that in control D-cell granules (42.2 +/- 5.9 particles/micron2). These data clearly indicate that despite a profound reduction by CSH of S-14 LI in tissue extracts, there is no detectable decrease in the same antigenic sites in tissue sections when assessed immunocytochemically.

Animals↗
PubMed1987

Morphologic and functional changes in sympathetic nerve relationships with pancreatic alpha-cells after destruction of beta-cells in rats.

Insulin-dependent diabetes mellitus (IDDM) in humans is accompanied by an attenuation of the response of glucagon to hypoglycemia. To identify an animal model of IDDM with alpha-cell unresponsiveness to glucopenia in which to pursue morphologic and in vitro functional investigation of the lesion, pancreases isolated from rats with IDDM induced by streptozocin (STZ) or occurring spontaneously in BB/W rats were perfused with buffer containing 150, 25, and 150 mg/dl of glucose. In both forms of IDDM the normal glucagon rise during glucopenia was markedly impaired, suggesting an abnormality comparable to that of human IDDM. Studies of the insular sympathetic apparatus were conducted in these rat models. Electron-microscopic examination of peri-insular nerve endings disclosed no discernible abnormality in either form of rat IDDM. However, morphometric analysis of contacts between [3H]norepinephrine-labeled sympathetic nerve terminals and alpha-cells in pancreases from STZ-induced diabetic (STZ-D) rats revealed a 65-70% reduction in direct contacts. An 80% reduction in the number of nerve endings (not labeled) in direct contact with alpha-cells was also noted in the BB/W diabetic rats. Norepinephrine reuptake, studied only in the STZ-D group, was not impaired. The availability of local endogenous norepinephrine to alpha-cells and their sensitivity to exogenous norepinephrine was determined by perfusing 2, 5, or 10 micrograms/ml of tyramine, a releaser of endogenous norepinephrine, and norepinephrine at a concentration that in pancreases from nondiabetic rats gave a quantitatively similar glucagon response.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Phorbol ester induces diaphragmed fenestrae in large vessel endothelium in vitro.

The endothelium of visceral capillaries is fenestrated, whereas that of large vessels is not. We show here that the tumor promoter, 4 beta-phorbol 12-myristate 13-acetate (PMA), triggers formation of diaphragmed fenestrae in endothelial cells cultured from the calf pulmonary artery and the human umbilical vein. This demonstrates that large vessel endothelial cells have the potential to express, in response to exogenous signals, a structural specialization uniquely associated with the endothelium of visceral capillaries in vivo.

Animals↗
PubMed1986

Selective localization of calcium-binding protein in human brainstem, cerebellum and spinal cord.

The 28,000-Da vitamin D-dependent calcium-binding protein, CaBP, which is induced by one hormonally active form of vitamin D3, 1,25-dihydroxyvitamin D3, was localized by immunocytochemistry in the human brainstem, cerebellum and cervical segment of the spinal cord. Positive structures (neurons and their processes) were restricted to some well-defined motor and sensory pathways. In motor regions, the highest density of immunoreactive sites was found in the Purkinje cell layer of the cerebellar cortex, and CaBP-positive neurons were also found in the reticular formation and the inferior olivary nucleus. In sensory pathways, positive neurons were mainly localized in structures associated with protopathic thermalgesia (pain and temperature), as well as in the solitary nucleus and parabrachial nucleus of the taste pathway.

Aged↗