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L Orci

Publications and source records attributed to L Orci.

At least 145 records · Page 8Linked to original sources
PubMed1988

Induction of angiogenesis in vitro by vanadate, an inhibitor of phosphotyrosine phosphatases.

We have previously shown that capillary endothelial cells grown on the surface of three-dimensional collagen gels can be induced to invade the underlying fibrillar matrix and to form capillary-like tubular structures in response to tumor-promoting phorbol esters or the angiogenic agent fibroblast growth factor (FGF). Since both phorbol esters and FGF stimulate phosphorylation of tyrosine residues, we treated endothelial cells with vanadate, an inhibitor of phosphotyrosine-specific phosphatases, to determine whether this agent could induce the expression of an angiogenic phenotype in these cells. We show here that vanadate stimulates endothelial cells to invade collagen matrices and to organize into characteristic tubules resembling those induced by FGF or phorbol esters. We have further observed that vanadate concomitantly stimulates endothelial cells to produce plasminogen activators (PAs), proteolytic enzymes which are induced by phorbol esters and FGF, and which have been implicated in the neovascular response; this stimulation can be accounted for by an increase in the levels of urokinase-type PA and tissue type PA mRNA. These results suggest a role for tyrosine phosphorylation in the regulation of the angiogenic phenotype in capillary endothelial cells.

Animals↗
PubMed1988

Junctional complexes and cell polarity in the urinary tubule.

In this review, we demonstrate how differentiated membrane domains can be detected in epithelial cells using conventional light and electron microscopy, freeze-fracture electron microscopy and the immuno- and cytochemical detection of membrane components. Using specific examples from the kidney, we show how the polarized insertion of these components into either apical or basolateral plasma membrane regions on either side of the tight junction barrier is related to specific functions of principal and intercalated cells in the collecting duct. In addition, distinct basal and lateral membrane domains have been revealed in some cells that are maintained in the absence of a tight junctional barrier in the plane of the membrane. This suggests that other factors, possibly related to cytoskeletal elements, may be involved in the functional segregation of these membrane areas. We propose that epithelial cell plasma membranes should be subdivided into apical, lateral and basal regions, and that the term "basolateral" may be an oversimplification.

Animals↗
PubMed1988

Transforming growth factor beta stimulates collagen-matrix contraction by fibroblasts: implications for wound healing.

An important event during wound healing is the contraction of newly formed connective tissue (granulation tissue) by fibroblasts. The role of polypeptide growth factors in the process of wound contraction was investigated by analyzing the influence of transforming growth factor beta (TGF-beta), platelet-derived growth factor on the ability of fibroblasts to contract a collagen matrix in an in vitro system. TGF-beta, but not the other growth factors tested, markedly enhanced the ability of BHK-21,3T3-L1, and human foreskin fibroblasts to contract collagen gels. These results suggest that TGF-beta released from platelets and inflammatory cells at sites of tissue injury stimulates fibroblasts to contract the provisional wound matrix and that this effect contributes to the ability of TGF-beta to accelerate wound healing.

Animals↗
PubMed1988

Efficient targeting to storage granules of human proinsulins with altered propeptide domain.

In neuronal and endocrine cells, peptide hormones are selectively segregated into storage granules, while other proteins are exported continuously without storage. Sorting of hormones by cellular machinery involves the recognition of specific structural domains on prohormone molecules. Since the propeptide of insulin is known to play an important role in its three-dimensional structure, it is reasonable to speculate that targeting of proinsulin to storage granules would require a functional connecting peptide. To test this hypothesis, we constructed two mutations in human proinsulin with different predicted structures. In one mutation, Ins delta C, the entire C peptide was deleted, resulting in an altered insulin in which the B and the A chains are joined contiguously. In the other mutation, Ins/IGF, the C peptide of proinsulin was replaced with the unrelated 12-amino acid connecting peptide of human insulin-like growth factor-I; this substitution should permit correct folding of the B and A chains to form a tertiary structure similar to that of proinsulin. By several biochemical and morphological criteria, we found that Ins/IGF is efficiently targeted to storage granules, suggesting that the C peptide of proinsulin does not contain necessary sorting information. Unexpectedly, Ins delta C, which presumably cannot fold properly, is also targeted to granules at a high efficiency. These results imply that either the targeting machinery can tolerate changes in the tertiary structure of transported proteins, or that the B and A chains of insulin can form a relatively intact three-dimensional structure even in the absence of C peptide.

Cell Compartmentation↗
PubMed1988

Simultaneous assessment of prohormone transport and processing in four separate islet cell types: a combined autoradiographic and biochemical study.

This study was performed to assess the relationships between prohormone transport and processing in separate cell types in pancreatic islet tissue. Anglerfish islets were subjected to pulse-chase incubation with [3H]tryptophan and/or [35S]cysteine. Tissue and media were removed at specific time points during the incubation and prepared for electron microscopic examination or biochemical analysis. Specific islet cell types were identified ultrastructurally using protein A gold immunocytochemistry. Transport of newly synthesized peptides through specific subcellular compartments was monitored using electron microscopic autoradiography. Prohormone-product ratios were established by gel filtration and high-performance liquid chromatography analyses of tissue extracts. Complete analyses were performed on A-cells (source of proglucagon-II, glucagon-II, and glucagon-like peptide-II), B-cells (proinsulin and insulin), D-cells (prosomatostatin-II and somatostatin-28), and S-cells (prosomatostatin-I and somatostatin-14). Transport of newly synthesized peptides proceeded from rough endoplasmic reticulum (RER) to Golgi complex and then to mature secretory granules in all cell types. The transport rate was most rapid in A- and B-cells, slower in S-cells, and slowest in D-cells. The T1/2 for conversion of prohormone to product(s) was shortest in S-cells (150 min), slightly longer in B-cells (155 min), much longer in D-cells (259 min), and greater than 300 min in A-cells. These results demonstrate that the transport/prohormone conversion relationships are unique in each of the islet cell types monitored.

Animals↗
PubMed1988

Lectin-gold labeling of glycoconjugates in normal and Brattleboro rat papilla: effect of vasopressin.

Some reports suggest that the plasma membrane glycocalyx of collecting duct epithelial cells, as well as interstitial glycoconjugates, may be involved in vasopressin action and urinary concentration. In view of this, we have used the lectin-gold technique to map and quantify Helix pomatia lectin (HPL)-binding sites in the inner medulla of kidneys from normal Long-Evans rats, vasopressin-deficient Brattleboro rats, and Brattleboro rats treated for up to 5 wk with exogenous vasopressin. The results show that the labeling of epithelial cell plasma membranes from collecting ducts and thin limbs of Henle is not different between normal and Brattleboro rats, and the labeling is not modified by chronic vasopressin treatment. In contrast, the heavy interstitial labeling seen in normal rats is virtually absent from Brattleboro rats, but it is progressively restored by chronic vasopressin treatment of Brattleboro rats. These results show that vasopressin does not modify HPL-binding glycoconjugates on epithelial cell plasma membranes, but that vasopressin treatment has a major effect on HPL-binding glycoconjugates in the medullary interstitium.

Animals↗
PubMed1988

Immunolocalization of betagranin: a chromogranin A-related protein of the pancreatic B-cell.

The tissue and subcellular distribution of betagranin, a chromogranin A-related, cosecreted protein produced in rat insulinoma tissue, has been investigated using a combination of density gradient centrifugation, immunoblotting, immunofluorescence, and immunoelectron microscopic techniques. Antibodies raised to insulinoma betagranin recognized antigens of the same molecular size (approximately 20,000 daltons) in insulinoma tissue and normal islets. Antigenicity was confined principally to secretory granules, and in insulinoma tissue was colocalized with insulin. Within the islet, all endocrine cells were immunoreactive, although subpopulations of beta- and alpha-cells displayed a more intense immunofluorescence. Adrenal tissue and anterior and posterior pituitaries were also highly immunoreactive, the antigen again being confined principally to the secretory granule. Higher molecular size species of 65,000, 85,000, and 100,000 daltons, which predominated in adrenal, were also present in pituitary along with equivalent amounts of the 20,000-dalton proteins. Isolated cells in the gastric antrum, small intestine, and colon were strongly immunofluorescent, but again, the molecular form differed from those of other tissues. Parallel experiments performed with antichromogranin A antisera suggested that betagranin in pancreatic B-cells is formed from chromogranin A by limited proteolysis within the secretory granule. It would appear that although chromogranin A is confined to tissues of the diffuse neuroendocrine system it can be processed differentially in tissues in this series. Potentially, the biological activity of chromogranin A resides in such derived peptides rather than in the parent molecule.

Adrenal Glands↗
PubMed1988

Localization of pancreastatin immunoreactivity in porcine endocrine cells.

Pancreastatin is a peptide isolated from the porcine pancreas and shown to inhibit insulin release. We have studied the immunocytochemical distribution of pancreastatin in three porcine endocrine tissues: pancreas, gut, and adenohypophysis. Pancreastatin-specific immunoreactivity was found in all three locations and distributed to numerous cells. In the pancreas, we performed the alternate labeling of consecutive thick (immunofluorescence) or thin (protein A-gold) sections and we observed that pancreastatin colocalizes to secretory granules of insulin and somatostatin-containing cells. The relationship of pancreastatin to chromogranin A is discussed.

APUD Cells↗
PubMed1988

Intracellular diaphragmed fenestrae in cultured capillary endothelial cells.

The endothelium of visceral capillaries is characterized by the occurrence of numerous fenestrae, which are usually bridged by a thin, single-layered diaphragm. Both in vivo and in vitro, diaphragmed fenestrae perforate the endothelial cell cytoplasm in the most attenuated regions of the cell. We report here that in capillary endothelial cells grown under experimental conditions promoting the development of intracellular lumina (for example, suspension within a three-dimensional collagen matrix), diaphragmed fenestrae can form in a unique, previously undescribed intracellular location - that is, within thin cytoplasmic septa separating contiguous luminal compartments.

Adrenal Cortex↗
PubMed1988

In vitro modulation of endothelial fenestrae: opposing effects of retinoic acid and transforming growth factor beta.

Cultured endothelial cells isolated from fenestrated capillaries express many properties characteristic of their in vivo differentiated phenotype, including the formation of a limited number of fenestrae. In this study, we have investigated whether physiological factors that control cell differentiation might regulate the surface density of fenestrae in capillary endothelial cells. We have found that treatment of the cultures with retinoic acid (10 microM) induces a more than threefold increase in the surface density of endothelial fenestrae, whereas transforming growth factor beta (TGF beta) (2 ng ml-1) causes a sevenfold decrease in the surface density of these structures. These results show that the expression of endothelial fenestrae is susceptible to bidirectional modulation by physiological signals, and suggest that retinoids and TGF beta may participate in the regulation of fenestral density of capillary endothelium in vivo.

Animals↗
PubMed1988

Vasopressin stimulates endocytosis in kidney collecting duct principal cells.

In target epithelia, a vasopressin-induced water permeability increase is accompanied by the appearance of intramembranous particle (IMP) clusters, probably representing water-permeable patches, in the apical plasma membrane of responding cells. In the collecting duct principal cell, we have previously shown that these clusters are located in clathrin-coated pits. To determine whether vasopressin induces the endocytic uptake of these membrane domains in principal cells, we have examined the uptake of horseradish peroxidase (HRP) by principal cells of normal rats, vasopressin-deficient Brattleboro rats, and vasopressin-treated Brattleboro rats, following intravenous injection of HRP. By quantitative electron microscopy, principal cells of Brattleboro homozygous rats were found to take up much less HRP into cytoplasmic vesicles than normal rats, and HRP uptake was increased to normal levels in vasopressin-treated Brattleboro rats. Many invaginating coated pits at the cell surface were loaded with HRP reaction product, indicating their participation in the observed endocytosis of HRP. We conclude that vasopressin stimulates endocytosis in collecting duct principal cells. Since we have already shown that IMP clusters are found in coated pits at the cell surface, the endocytic removal of these putative water-permeable patches from the apical membrane seems to occur via a clathrin-mediated mechanism in this tissue.

Animals↗
PubMed1987

The trans-most cisternae of the Golgi complex: a compartment for sorting of secretory and plasma membrane proteins.

The intracellular site for the sorting of proteins destined for regulated or constitutive pathways is presently unknown for any one cell. By immunoelectron microscopy, we directly followed the routes taken by a regulated hormone, insulin, and a constitutive protein, hemagglutinin. Both proteins are present in individual Golgi stacks where they appear randomly distributed throughout the cisternae. In contrast, the two proteins do not colocalize outside the Golgi area:insulin is concentrated in dense-core secretory granules, while hemagglutinin is found predominantly in clear 100-300 nm vesicles. These vesicles do not label significantly with an endocytic tracer, indicating that they are exocytic carriers for hemagglutinin. The site at which the two proteins diverge is the clathrin-coated, trans-most cisterna of the Golgi, where the packaging of proinsulin takes place.

Adrenocorticotropic Hormone↗
PubMed1987

Involvement of GTP-binding "G" proteins in transport through the Golgi stack.

GTP gamma S irreversibly inhibits protein transport between successive compartments of the Golgi stack in a cell-free system. Fluoride, potentiated by the addition of aluminum ion, also causes a strong inhibition. These are hallmarks of the involvement of a guanine nucleotide-binding or regulatory "G" protein. Inhibition by GTP gamma S requires a cytosolic inhibitory factor that binds to Golgi membranes during inhibition. Preincubation experiments reveal that GTP gamma S blocks the function of acceptor Golgi but not donor Golgi membranes. More specifically, a processing step in between vesicle attachment and the actual fusion event seems to be affected. Electron microscopy demonstrates a corresponding 5-fold accumulation of non-clathrin-coated buds and vesicles associated with the Golgi cisternae during inhibition by GTP gamma S.

Aluminum↗
PubMed1987

Stimulation by ATP of proinsulin to insulin conversion in isolated rat pancreatic islet secretory granules. Association with the ATP-dependent proton pump.

Isolated rat pancreatic islets were pulse-labeled for 5 min with [3H]leucine then chased for 25 min, during which time endogenously labeled [3H]proinsulin becomes predominantly compartmented in immature secretory granules. The islets were then homogenized in isotonic sucrose (pH 7.4) and a beta-granule preparation obtained by differential centrifugation and discontinuous sucrose gradient ultracentrifugation. This preparation was enriched 8-fold in beta-granules. Aside from contamination with mitochondria and a limited number of lysosomes, the beta-granule preparation was essentially free of any other organelles involved in proinsulin synthesis and packaging (i.e. microsomal elements and, more particularly, Golgi complex). Conversion of endogenously labeled [3H]proinsulin was followed in this beta-granule fraction for up to 2 h at 37 degrees C in a buffer (pH 7.3) that mimicked the cationic constituents of B-cell cytosol, during which time 92% of the beta-granules remained intact. Proinsulin conversion was analyzed by high performance liquid chromatography. The rate of proinsulin conversion to insulin was stimulated by 2.2 +/- 0.1-fold (n = 6) (at a 60-min incubation) in the presence of ATP (2 mM) and an ATP regenerating system compared to beta-granule preparations incubated without ATP. This ATP stimulation was abolished in the presence of beta-granule proton pump ATPase inhibitors (tributyltin, 2.5 microM, or 1,3-dicyclohexylcarbodiimide, 50 microM). Inhibitors of mitochondrial proton pump ATPases (sodium azide, 20 mM, or oligomycin, 10 micrograms/ml) had no effect on the ATP stimulation of proinsulin conversion. When granules were incubated in a more acidic buffer (pH 5.5), proinsulin conversion was increased relative to that at pH 7.3. At pH 5.5, ATP no longer stimulated conversion, and tributyltin and 1,3-dicyclohexylcarbodiimide had no effect. Disrupted granules only converted proinsulin to a limited extent, and neither ATP nor the inhibitors affected conversion. It is therefore suggested that ATP stimulation of proinsulin conversion in isolated, intact, beta-granules is secondary to intragranular acidification by an ATP-dependent proton pump (reflecting the low pH optimum for proinsulin conversion), rather than ATP dependence of converting activity per se.

Adenosine Triphosphatases↗
PubMed1987

Proteolytic maturation of insulin is a post-Golgi event which occurs in acidifying clathrin-coated secretory vesicles.

The direct identification of the intracellular site where proinsulin is proteolytically processed into insulin has been achieved by immunocytochemistry using an insulin-specific monoclonal antibody. Insulin immunoreactivity is absent from the Golgi stack of pancreatic B-cells and first becomes detectable in clathrin-coated secretory vesicles released from the trans Golgi pole. Clathrin-coated secretory vesicles transform into mature noncoated secretory granules which contain the highest concentration of insulin immunoreactive sites. Maturation of clathrin-coated secretory vesicles is accompanied by a progressive acidification of the vesicular milieu, as evidenced by a cytochemical probe that accumulates in acidic compartments whereupon it can be revealed by immunocytochemistry. Thus packaging of the prohormone in secretory vesicles, and acidification of this compartment, are critical steps in the proper proteolytic maturation of insulin.

Antibodies, Monoclonal↗