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Biomedical subjects

N Savion

Publications and source records attributed to N Savion.

At least 91 records · Page 5Linked to original sources
PubMed1989

Cholesterol efflux from and high-density-lipoproteins binding to cultured bovine vascular endothelial cells are higher than with vascular smooth muscle cells.

Cholesterol metabolism was studied and compared in confluent cultures of adult bovine aortic endothelial and bovine aortic smooth muscle cells which were grown under similar conditions. The total cholesterol content/mg protein was only slightly higher in smooth muscle cells than in endothelial cells and upon exposure to [3H]cholesterol the maximal specific activity/mg protein obtained was similar in both cell types. Most (98%) of the incorporated [3H]cholesterol remained in the form of free cholesterol in both cell types, and provided a system for the study of cholesterol efflux. The role of high-density lipoproteins (HDL) and human serum in cholesterol influx and efflux, in both endothelial and smooth muscle cells, was studied. Net cholesterol transport in the cultures was calculated and net efflux was observed in both cell types. This was higher in endothelial than in smooth muscle cells and HDL was more efficient than human serum in promoting net cholesterol efflux. During the influx experiments, no conversion of [3H]cholesterol to [3H]cholesteryl ester was observed either in the cell layer or in the incubation medium. On the other hand, during efflux experiments when HDL but not human serum was the acceptor, some (about 6%) conversion of [3H]cholesterol to [3H]cholesteryl ester occurred in the incubation medium. 125I-HDL3 binding to endothelial and smooth muscle cells was studied and demonstrated saturation at a concentration of about 100 micrograms protein/ml for both cell types. However, endothelial cells bound about six times more 125I-HDL3 than smooth muscle cells. These studies indicate that vascular endothelial cells are more protected against cholesterol accumulation than vascular smooth muscle cells. The greater ability of endothelial cells to bind HDL complexes when compared with smooth muscle cells, and thereby to be more susceptible to HDL induced cholesterol efflux, may add a new mechanism through which endothelial cells protect the blood vessel from cholesterol accumulation.

Animals↗
PubMed1989

Basic fibroblast growth factor enhances the capacity of bone marrow cells to form bone-like nodules in vitro.

The role of basic fibroblast growth factor (bFGF) in the proliferation and differentiation of rat bone marrow cells in culture was studied. bFGF stimulated [3H]thymidine incorporation into these cells by 4-fold at a concentration of 0.3 ng/ml and half-maximal effect was observed at a concentration of 15 pg/ml. In addition to its mitogenic effect, bFGF stimulated alkaline phosphatase activity by 3.6-fold. Continuous treatment with bFGF (for 21 days) resulted in a 6.3-fold increase in the culture dish surface area covered by bone-like mineralized tissue. Maximal bone-like tissue formation was observed in the presence of 3 ng/ml bFGF with half-maximal effect at a concentration of 0.3 ng/ml. These results indicate the possible role of bFGF in the proliferation of osteogenic rat bone marrow cells and their differentiation into cells of osteoblast-like phenotype.

Alkaline Phosphatase↗
PubMed1989

Monoclonal antibodies against antithrombin III. Identification of their epitopes and effects on antithrombin III activities.

Four monoclonal antibodies with distinct epitopes were prepared against antithrombin III. None of them is directed against the heparin-binding region nor the active site, yet two mAb namely A36 and B108, interfere with antithrombin III inhibition of thrombin. The epitope of monoclonal antibody A36 is located within amino acid residues 1-393, at a site different from the active site since it recognizes antithrombin III and antithrombin-III-thrombin complexes with the same affinity. A36 partially prevents the intrinsic antithrombin III activity and has no effect on the heparin-enhanced antithrombin III activity when added to the antithrombin-III--heparin complex. If A36 is first reacted with antithrombin III and then heparin is added to the reaction mixture, A36 fixes the conformation of antithrombin III so that heparin binds to antithrombin III, but is not able to induce the conformational change in the antithrombin III molecule required for the enhanced activity. The epitope for monoclonal antibody B108 is located within residues 282-393, close to the active site. It does not recognize antithrombin-III-thrombin complexes by solid-phase radioimmunoassay. Its binding to antithrombin III induces a conformational change that enhances antithrombin III activity in a manner that resembles the heparin effect, but its effect is additive to the heparin effect, since when it was added to a reaction mixture which contained a saturating amount of heparin, inhibition of thrombin was enhanced. The epitope for monoclonal antibody A5 is located within residues 1-393, and its recognition of antithrombin III or antithrombin-III-thrombin is strongly dependent on the integrity of the disulfide bonds. A5 has no effect on antithrombin III activities. The epitope for monoclonal antibody A10 is well defined within a narrow range of 55 amino acid residues, 339-393, on the antithrombin III molecule, close to the active site, yet it has no effect on antithrombin III inhibitory activity. These monoclonal antibodies may be developed for various diagnostic or clinical purposes and offer a powerful tool for studying the conformational changes and structure/activity relationships in the antithrombin III molecule.

Animals↗
PubMed1989

Characterization of bicarbonate-dependent potassium uptake in cultured corneal endothelial cells.

Bovine corneal endothelial (BCE) cells in culture demonstrated 86Rb+ uptake which was mostly ouabain-sensitive with some (15 to 50%) ouabain-insensitive uptake that was dependent on the presence of bicarbonate in the incubation medium. Bovine smooth muscle (SM) cells demonstrated ouabain-sensitive 86Rb+ uptake but the ouabain-insensitive 86Rb+ uptake was not bicarbonate-dependent. Although omission of bicarbonate from the incubation buffer resulted in some reduction in the pH, this change was not responsible for the reduction in the ouabain-insensitive 86Rb+ uptake. Furthermore, the removal of bicarbonate decreased the 86Rb+ influx but not its efflux. This ouabain-insensitive and bicarbonate-dependent 86Rb+ influx in BCE cells proceeded at a linear rate for at least 60 min and increased as a function of bicarbonate concentration such that almost maximal uptake was observed at a concentration of about 10 to 15 mM. Saturation of the bicarbonate-dependent 86Rb+ pump in BCE cells occurred at a concentration of 2 mM Rb+ in the incubation buffer, similar to the previously observed value for the Na+, K+-ATPase. Competition experiments with both unlabeled Rb+ and K+ demonstrated that likewise in the Na+, K+-ATPase the 86Rb+ influx represented physiological influx of K+. Furthermore, the energy requirements of the bicarbonate-dependent 86Rb+ uptake were similar to those of the 86Rb+ uptake via the Na+, K+-ATPase. The results described in this work demonstrated a novel bicarbonate-dependent K+ pump in addition to the Na+, K+-ATPase pump.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Anti-thrombin activity in cultured aortic and capillary endothelial cells: binding, internalization and degradation of thrombin.

Thrombin (Th) binds specifically to confluent cultures of adult bovine aortic (ABAE) and bovine brain capillary (BBC) endothelial cells. Saturation of 125I-Th binding is observed after 1 h exposure to the ligand and at an extracellular concentration of 0.5 and 1.0 microgram/ml for ABAE and BBC cells, respectively. Under optimal conditions both ABAE and BBC cultures bind about 2 to 5 ng/10(6) cells, which represents about 20% of Th binding to bovine corneal endothelial (BCE) cells. Under optimal conditions less than 30% of the total cell associated 125I-Th is internalized in ABAE and BBC cells, while in BCE cells the extent of internalization is more than 50%. The internalized 125I-Th is degraded both in ABAE and BBC cells as previously demonstrated in BCE cells. As analyzed by SDS-PAGE, 17%, 22% and 77% of the bound 125I-Th is in complex with anti-thrombins (anti-Ths) in BBC, ABAE and BCE cultures, respectively. ABAE cells possess 3 types of complexes, one which appears only on the cell surface with a molecular weight of 78 kDa, and two others which appear only in the conditioned medium (CM) with molecular weights of 84 and 85 kDa. BBC and BCE cells demonstrate only one type of complex with a molecular weight of 77 kDa which appears both on the cell surface and in the CM. The 125I-Th 77 kDa complex formed in the CM of BCE cells is recognized and bound by BBC cells and ABAE cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

The effect of transfusion of fresh whole blood versus platelet concentrates after cardiac operations. A scanning electron microscope study of platelet aggregation on extracellular matrix.

To evaluate the effect of fresh whole blood transfusion versus platelet concentrates transfusion on platelet aggregation after cardiac operations, 24 patients were randomized to receive either one unit of fresh whole blood (12 patients) or 10 platelet units (12 patients) after cardiopulmonary bypass. Platelet aggregation on extracellular matrix, platelet count, and mean platelet volume were studied preoperatively, at termination of cardiopulmonary bypass, after protamine administration, and after the transfusion of fresh whole blood or after transfusion of each two platelet units. Extracellular matrix produced by cultured bovine corneal cells closely resembles the vascular subendothelial basal lamina, and is an ideal in vitro model in the study of platelet interaction with the subendothelium. Platelet aggregation on extracellular matrix, studied by a scanning electron microscope, was graded from 1 to 4, wherein grade 1 represents nonactivated platelets and grade 4 a mature platelet aggregate. With this grading system, the two groups were similar in preoperative values (3.3 +/- 0.9 versus 3.7 +/- 0.4) and values after cardiopulmonary bypass (1.5 +/- 1.0 in both groups). One unit of fresh whole blood restored platelet aggregation on extracellular matrix to preoperative status (3.0 +/- 1.0), whereas eight platelet units were needed for the same result (3.2 +/- 0.8). One unit of fresh whole blood increased platelet count in a manner similar to that achieved by six platelet units and increased mean platelet volume to a level higher than that achieved by 10 platelet units. These results suggest that the effect of one unit of fresh whole blood on platelet aggregation after cardiopulmonary bypass is at least equal, if not superior, to the effect of 8 to 10 platelet units.

Adult↗
PubMed1988

The effects of partial demineralization and fibronectin on migration and growth of gingival epithelial cells on cementum in vitro.

The capacity of mineralized cementum to support epithelial cell migration and growth and the effect that fibronectin and partial demineralization of cementum have on these processes were assessed in vitro. Dog gingival explants, 1 X 2 mm, were cultured on the cementum surfaces of pig root pieces in a defined medium consisting of DMEM and F12 (1V/1V), transferrin, insulin, epidermal growth factor, cortisone, selenium, and high-density lipoprotein. Sixty root pieces were divided into four equal groups according to the treatment: (1) untreated mineralized cementum; (2) treated with 5 micrograms of fibronectin; (3) partially demineralized in 18% EDTA for 30 min; and (4) both partially demineralized and fibronectin-treated as above. Epithelial cell migration and growth on each of the four differently treated cementum surfaces were assessed histomorphometrically by means of scanning electron microscopy. The defined culture medium supported the selective migration and growth of epithelial cells from the gingival explants onto the mineralized cementum. This was confirmed by the positive immunostaining of these cells with antikeratin antibodies. Partial demineralization of cementum inhibited epithelial migration and growth by 83% and 91%, respectively. Fibronectin treatment did not affect epithelial cell migration and growth on mineralized cementum, but it decreased the degree of epithelial cell migration and growth inhibition on partially demineralized cementum to 57% and 43%, respectively. The results indicate that: (i) mineralized cementum may consist of components that are recognized by gingival epithelial cells and support their migration and growth in vitro; (ii) these components can be removed by demineralization; and (iii) fibronectin partially restores epithelial cell migration and growth on partial demineralized cementum in vitro.

Animals↗
PubMed1988

Growth and migration of gingival epithelial cells on mineralized and partially demineralized root surfaces in an in vitro system.

The capacity of epithelial cells to migrate and grow from gingival explants cultured on mineralized and partially demineralized root surfaces in an in vitro system was assessed. Explants of attached gingiva were obtained from mongrel dogs, cut into rectangular pieces (1 x 2 mm) and cultured either on mineralized or partially demineralized cementum in a defined culture medium containing transferrin, insulin, epidermal growth factor, cortisone, high density lipoprotein and selenium. After seven days of culture, the specimens were prepared for scanning electron microscopic examination. The amount of epithelial outgrowth from each explant was assessed by measuring the distances between the four aspects of the rectangular explant and the furthest epithelial cell located opposite to each of these aspects. The mean value obtained for epithelial outgrowth of explants grown on mineralized cementum was six times higher than that for explants cultured on partially demineralized cementum. These results indicate that partially demineralized cementum does not support epithelial growth and migration in vitro.

Animals↗
PubMed1987

Identification of a factor IX/IXa binding protein on the endothelial cell surface.

Endothelium provides a specific binding site for Factor IX/IXa which can propagate activation of coagulation by promoting Factor IXa-VIII-mediated activation of Factor X. In this report the endothelial cell Factor IX/IXa binding site has been identified and the coagulant function of the receptor blocked. Studies using [3H]Factor IX derivatized with the photoaffinity labeling agent N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH) and cultured bovine endothelial cells demonstrated cross-linking to a trypsin-sensitive cell surface protein of Mr approximately equal to 140,000. Immunoprecipitation of metabolically labeled endothelium with Factor IX derivatized with the cleavable cross-linking agent N-succinimidyl(4-azidophenyl)-1,3'-dithiopropionate and antibody to Factor IX demonstrated the endothelial cell origin of the Mr 140,000 cell surface protein. Blockade of the Factor IX/IXa binding protein by covalently linking SANPAH-5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg-Factor IXa or SANPAH-Factor IX prevented both specific Factor IXa binding and effective Factor IXa-VIII-mediated activation of Factor X on endothelium. Following extraction of endothelium with detergents, Factor IX/IXa binding activity was solubilized and could be assayed using a polyvinyl chloride plate binding assay. Western blots of cell extracts demonstrated binding of 125I-Factor IX at Mr approximately equal to 140,000 which was blocked by excess Factor IX, but not antisera to Factor VIII, von Willebrand factor, alpha 2-macroglobulin, or epidermal growth factor receptor. These data indicate that endothelium provides a distinct binding site for Factor IX/IXa consisting, at least in part, of a membrane protein which can modulate the coagulant activity of Factor IXa on the cell surface.

Animals↗
PubMed1987

Free apolipoproteins A-I and A-IV present in human plasma displace high-density lipoprotein on cultured bovine aortic endothelial cells.

Adult bovine aortic endothelial (ABAE) cells, exposed to serum-free medium, specifically bind 125I-labeled human high-density lipoprotein (125I-HDL). Addition of human lipoprotein-deficient serum (LPDS) reduces the specific binding of 125I-HDL in a concentration-dependent manner, such that LPDS at a concentration of 6 mg protein/ml almost completely inhibits the specific binding of 125I-HDL. ABAE cultures exposed to 125I-labeled LPDS (125I-LPDS) specifically bind two peptides, which appear as minor iodinated components in 125I-LPDS. The binding of these two components is abolished in the presence of excess amounts of unlabeled LPDS or HDL. Preincubation of ABAE cells with 25-hydroxycholesterol (25-HC) results in an increase in the binding of the two 125I-LPDS components, similar to the increase observed in 125I-HDL binding in the presence of 25-HC. These two LPDS components comigrate on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) with apolipoproteins A-I and A-IV of molecular masses 28 kDa and 43 kDa respectively. Furthermore, these two proteins were transferred from the SDS gel to nitrocellulose paper and interacted specifically with anti-(A-I) and anti-(A-IV) sera respectively. When ABAE cultures, pretreated with 25-HC in the presence of LPDS, are subjected to cell-surface iodination, the A-IV appears as one of the major proteins on the cell surface accessible to iodination. The interaction of A-IV with the cell surface of 25-HC-treated cells is not specific to ABAE cells and appears also in human skin fibroblasts. Analysis of the relative amounts of various apolipoproteins in the 125I-HDL bound to ABAE cells demonstrates a decrease in the relative amount of iodinated A-II concomitant with increase in the relative amounts of the other iodinated apolipoproteins, when compared to the composition of the native 125I-HDL. These changes are similar whether the binding is done in the presence or absence of LPDS. It indicates that the decrease in 125I-HDL binding in the presence of LPDS is not due to displacement of the iodinated apolipoproteins A-I and A-IV in the 125I-HDL by unlabeled A-I and A-IV present in LPDS. The results indicate that free apolipoproteins A-I and A-IV, present in LPDS, can displace HDL on the cell surface of ABAE cells. Thus, free A-I and A-IV, present in plasma, control the binding of HDL to endothelial cells and may regulate the process of cholesterol removal from the cells performed by HDL.

Animals↗
PubMed1987

Murine macrophage heparanase: inhibition and comparison with metastatic tumor cells.

Circulating macrophages and metastatic tumor cells can penetrate the vascular endothelium and migrate from the circulatory system to extravascular compartments. Both activated murine macrophages and different metastatic tumor cells (B16-BL6 melanoma; ESb T-lymphoma) attach, invade, and penetrate confluent vascular endothelial cell monlayer in vitro, by degrading heparan sulfate proteoglycans in the subendothelial extracellular matrix. The sensitivity of the enzymes from the various sources degrading the heparan sulfate proteoglycan was challenged and compared by a series of inhibitors. Activated macrophages demonstrate a heparanase with an endoglycosidase activity that cleaves from the [35S]O4 = -labeled heparan sulfate proteoglycans of the extracellular matrix 10 kDa glycosaminoglycan fragments. The macrophages do not store the heparanase intracellularly but it is instead found pericellularly and requires a continuous cell-matrix contact at the optimal pH for maintaining cell growth. The degradation of [35S]O4 = -labeled extracellular matrix proteoglycans by the macrophages' heparanase is significantly inhibited in the presence of heparan sulfate (10 micrograms/ml), arteparon (10 micrograms/ml), and heparin at a concentration of 3 micrograms/ml. In contrast, other glycosaminoglycans such as hyaluronic acid, dermatan sulfate, and chondroitin sulfate as well as the specific inhibitor of exo-beta-glucuronidase D-saccharic acid 1,4-lactone failed to inhibit the degradation of sulfated proteoglycans in the subendothelial extracellular matrix. Degradation of this heparan sulfate proteoglycan is a two-step sequential process involving protease activity followed by heparanase activity. However, the following antiproteases--alpha 2-macroglobulin, antithrombin III, leupeptin, and phenylmethylsulfony fluoride (PMSF)--failed to inhibit this degradation process, and only alpha 1-antitrypsin inhibited the heparanase activity. B16-BL6 metastatic melanoma cell heparanase, which is also a cell-associated enzyme, was inhibited by heparin to the same extent as the macrophage heparanase. On the other hand, heparanase of the highly metastatic variant (ESb) of a methylcholanthrene-induced T lymphoma, which is an extracellular enzyme released by the cells to the incubation medium, was more sensitive to heparin and arteparon than the macrophages' heparanase, inhibited at concentrations of 1 and 3 micrograms/ml, respectively. These results may indicate the potential use of heparin or other glycosaminoglycans as specific and differential inhibitors for the formation in certain cases of blood-borne tumor metastasis.

Animals↗
PubMed1987

Regeneration of cat corneal endothelium induced in vivo by fibroblast growth factor.

The corneal endothelium of man, primates and carnivores has limited regenerative ability. Fibroblast growth factor (FGF) stimulates the proliferation of various mesoderm-derived cells, including corneal endothelial cells, in vitro. In the present work the effect of FGF on the regeneration of injured cat endothelium in vivo was studied. Scraping of cat corneal endothelium off Descemet's membrane was carried out by a specially designed instrument. FGF from crude preparations (25 micrograms in 50 microliter saline) or affinity purified FGF (0.5 micrograms in 25 microliter saline) were injected immediately after the scraping of the endothelium, and in both cases significant stimulation of endothelium regeneration was observed. During the first 2 weeks after scraping, the endothelial cell density (ECD) in FGF-treated eyes was higher by a factor of 1.7-2.3 than that in the control eyes. This marked difference decreased with time, but even after 12 weeks the ECD in FGF-treated eyes was higher by a factor of 1.15-1.30 than in the control eyes. Furthermore, FGF improved the polygonal shape of the cells and decreased the corneal thickness. These results clearly demonstrate the efficacy of FGF in inducing the proliferation of cat corneal endothelial cells in vivo and indicate its potential application in clinical practice.

Animals↗
PubMed1987

Molecular and cellular aspects of axon-glia interaction in CNS regeneration.

The relationships of neurons and non-neuronal cells are vital for the maintenance and function of neurons. Trauma alters these relationships causing proliferation of non-neuronal cells and, in adult mammalian CNS, presumably disturbs the environmental support needed for regeneration. A supportive environment can be restored by introducing a regenerating nerve to injured mammalian CNS. This response is probably due, at least in part, to diffusible substances secreted by the non-neuronal cells. We have obtained diffusible substances from either regenerating fish optic nerves or neonatal rabbit optic nerves and applied them around crushed adult rabbit optic nerves. This manipulation caused the adult nerve to show regenerative changes: a general increase of protein synthesis in the retinas; selective increase in synthesis of a few polypeptides in the retinas; sprouting from the retinas in vitro; increased viability of nerve fibers as shown by HRP staining; and the appearance of growth cones adjacent to glial limitans in the injured nerves. We termed these diffusible, active substances "Growth Associated Triggering Factors" (GATFs). In addition to the phenomena described above, the active substances (obtained in the form of media conditioned by regenerating fish optic nerve or neonatal rabbit optic nerve) caused various other changes in the injured nerve itself: acceleration of non-neuronal cell proliferation; changes in the protein pattern, e.g. an increase in a 12 kDa polypeptide which might be a second mediator in the cascade of events leading to regeneration; increased laminin immunoreactive sites in the nerve; and the acquisition of growth supportive activity in media conditioned by the implanted injured nerves.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Comparative study of antithrombin III.protease complex metabolism by fibroblasts and vascular endothelial cells.

125I-labeled human antithrombin III (125I-AT III).protease complexes are specifically bound to both cultured human skin fibroblast (HSF) cells and adult bovine aortic endothelial (ABAE) cells; however, there is a significant difference in the rate and degree of metabolism of the complexes by these two cell types. HSF cells appear to internalize the complexes at a rate of about 2.5 pmole/1 X 10(6) cells/h and subsequently degrade them at a rate of 0.6 pmole/1 X 10(6) cells/h. ABAE cells internalize and degrade the complexes at rates approximately 100 and 30 times lower, respectively. Neither cell type interacts with free 125I-AT III but only with its combined form with either thrombin or trypsin. These data indicate the major role of HSF cells in the removal of AT III.protease complexes from extravascular spaces in the body, in contrast to the inert vascular surface with regard to AT III.protease complexes provided by the vascular endothelium.

Animals↗
PubMed1986

Characterization of the Na, K-ATPase pump in cultured bovine corneal endothelial cells.

Bovine corneal endothelial cells in culture possess Na, K-ATPase pump sites, as measured by [3H]ouabain binding, and demonstrated an active ouabain-sensitive 86Rb+ uptake. The binding of [3H]ouabain, a specific inhibitor of Na, K-ATPase, was used to quantitative the density of Na, K-ATPase pump sites in bovine corneal endothelial cell cultures. [3H]ouabain binding was time-dependent and reached saturation after 1-2 hr. The specific binding represented more than 99% of the total cell-associated [3H]ouabain, and about 85% of this binding was abolished in the presence of K+ ions. The binding was concentration-dependent and saturated at a ouabain concentration of 2 X 10(-8)M with a dissociation constant (Kd) of 1.0 X 10(-8)M. The number of [3H]ouabain binding sites was maximal in sparse, activity growing cultures and decreased accompanying the development of a confluent monolayer. A pump density of 2.2 X 10(6) pump sites cell-1 was estimated for sparse cultures, declining to 0.8 X 10(6) pump site cell-1 at confluence. The activity of the Na, K-ATPase pump in bovine corneal endothelial cell cultures was evaluated by measuring 86Rb+ influx. Sparse and confluent cultures demonstrated 86Rb+ ouabain-sensitive uptake at a rate of 4.2 nmol (10(6) cells)-1 min-1 and 1.5 nmol (10(6) cells)-1 min-1, respectively. The ouabain-sensitive 86Rb+ uptake was linear for at least 30 min, while the ouabain-insensitive 86Rb+ uptake was slower and declined during the 30-min time period.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Chloroquine and primary amines inhibit the internalization of antithrombin III.trypsin complex in cultured cells.

Bovine corneal endothelial (BCE) cells have been shown to specifically bind, internalize and degrade antithrombin III (AT III).protease complexes as well as thrombin. Previous studies have indicated that chloroquine has no effect on the internalization of thrombin or other cell surface-bound ligands, but it inhibits their subsequent degradation. In contrast, the present study demonstrates the unique inhibitory effect of chloroquine on the internalization of 125I-AT III.trypsin complex by BCE cultures. Similarly, the primary amines, monodansylcadaverine and methylamine, inhibit the internalization of 125I-AT III.trypsin complex, but not the internalization of 125I-thrombin. The various amines used in this study revealed: (1) differences in the process of cellular binding and internalization between AT III.protease complex and thrombin, although the degradation of both internalized ligands proceed in an analogous manner; and (2) the unique sensitivity to chloroquine of 125I-AT III.trypsin complex internalization by cultured cells. These results might indicate that AT III.protease complexes are internalized via a distinct receptor and/or a different mechanism from thrombin.

Amines↗
PubMed1985

Correlation between trypsin binding to a specific receptor and prostacyclin production in cultured vascular endothelial cells.

The correlation between the binding and processing of trypsin and its effect on prostacyclin (PGI2) production in cultured adult bovine aortic endothelial (ABAE) cells was studied. ABAE cells demonstrated an ability to produce PGI2 in a dose-response manner to trypsin at the range of 0.1-2.0 micrograms/ml with a saturation at a concentration of 1 microgram/ml. Likewise, 125I-trypsin binding to the cultured cells increased in a dose-response way and reached saturation at a concentration of about 1 microgram/ml; 125I-trypsin was bound to a specific high-affinity cell-surface receptor with a dissociation constant (Kd) of 1.5 X 10(-8) M and each of the confluent ABAE cells has about 1.2 X 10(5) such receptors sites. The cell-surface receptor for trypsin displayed specific characteristics and an excess amount of unlabeled trypsin successfully abolished 125I-trypsin binding while thrombin in excess failed to compete for 125I-trypsin binding. Only a small fraction of the cell-surface-bound 125I-trypsin was internalized and subsequently degraded by ABAE cells as compared to the process of 125I-trypsin internalization by human skin fibroblasts (HSF). This study demonstrated that the stimulatory effect of trypsin on prostacyclin production and release by ABAE cells might be mediated by a specific cell-surface receptor for trypsin on these cells distinct from the thrombin receptor.

6-Ketoprostaglandin F1 alpha↗