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Biomedical subjects

J B Baker

Publications and source records attributed to J B Baker.

At least 55 records · Page 3Linked to original sources
PubMed1983

Evidence that a variety of cultured cells secrete protease nexin and produce a distinct cytoplasmic serine protease-binding factor.

Four criteria were used to examine serum-free conditioned cell culture medium for protease nexin (PN):(1) formation of SDS-stable approximately 77 K Da complexes between a medium component and [125I]thrombin; (2) acceleration by heparin of the rate of formation of these complexes; (3) cellular binding of these complexes; and (4) inhibition by heparin of the cellular binding of complexes. Listed in order of decreasing PN production, PN was detected in media conditioned by the following cell types: human foreskin fibroblasts (0.18 micrograms/10(6) cells), rat embryo heart muscle cells (0.13 micrograms/10(6) cells), mouse myotubes (0.1 micrograms/10(6) cells), monkey kidney epithelial cells, human fibrosarcoma cells, human lung fibroblasts, simian virus 40 (SV-40)-transformed human fibroblasts, human epidermoid carcinoma cells, bovine aortic endothelial cells (only after phorbol ester treatment), and mouse myoblasts. No PN was found in medium conditioned by mouse 3T3 cells, SV40 virus-transformed 3T3 cells, human lymphoblasts, or mouse leukemia cells. Eleven of the cell types examined for secretion of PN were also examined for the presence of cytoplasmic thrombin-binding factors. Lysates from all of these cell types contained a factor that formed approximately 60-65 K Da sodium dodecyl sulfate (SDS)-stable complexes with [125I] thrombin. This MW is significantly lower than that of [125I] thrombin-PN complexes, indicating that the factor is distinct from PN. Nevertheless, PN and the cytoplasmic factor share similarities. Production of both PN (by HF cells and WI-26 cells) and the cytoplasmic factor (by HF cells and 3T3 cells) are stimulated by epidermal growth factor and phorbol myristate acetate. Also, both PN and the cytoplasmic factor complex trypsin, plasmin, urokinase, and thrombin, but not pancreatic elastase. Because a number of the cells that produce PN or the cytoplasmic serine protease-binding factor are known to produce plasminogen activators, both PN and the cytoplasmic factor could regulate plasminogen activator activity.

Amyloid beta-Protein Precursor↗
PubMed1983

Phorbol ester and mitogens stimulate human fibroblast secretions of plasmin-activatable plasminogen activator and protease nexin, an antiactivator/antiplasmin.

Tumor-promoting phorbol esters have been reported to greatly increase plasminogen activator (PA) activity produced in numerous cell types. Many of these studies have employed a widely used fibrinolysis assay for PA activity that involves large-scale dilution of cell lysates or conditioned medium (CM) into buffer containing plasminogen and the plasmin substrate 125I-fibrin. This assay indicates that phorbol ester and the mitogens epidermal growth factor (EGF) and thrombin all stimulate secretion of PA activity in our human foreskin fibroblast cultures. However, these effects are not observed in a modified fibrinolysis assay employing undiluted conditioned culture medium unless the medium is first treated at pH 3, which inactivates the secreted protease inhibitor, protease nexin (PN). Moreover, a direct assay for plasminogen activator activity based on cleavage of 125I-plasminogen indicates that conditioned culture medium contains little if any active plasminogen activator either before or after treatment of the cultures with phorbol ester or EGF. Phorbol ester and mitogens do stimulate secretion of (a) an inactive PA that can be activated by plasmin and (b) PN, which inhibits both the activated form of the PA and plasmin. Secretions of the inactive PA and PN are further correlated in that release of both is stimulated most by phorbol ester, somewhat less by EGF, and least by thrombin. Significantly, these effects are not accompanied by increases in total protein secretion. We propose that fibroblasts secrete PA in an inactive form in the presence of PN to confine PA activity to an as yet undefined location or event.

Amyloid beta-Protein Precursor↗
PubMed1982

Cells regulate their mitogenic response to thrombin through release of protease nexin.

We previously reported that human and mouse fibroblast-like cells release into their growth medium a protein that we termed protease nexin. Protease nexin forms a covalent acyl linkage with thrombin and certain other serine proteases via the protease active site and mediates their binding, internalization and degradation by cells. Binding of thrombin-protease nexin to cells is mediated by the protease nexin portion of the complex to a high-affinity cellular binding site. As thrombin is a potent mitogen for a variety of fibroblast-like cells in culture, we examined whether protease nexin itself regulates thrombin-stimulated cell division. Recently, we showed that heparin virtually blocked the binding of thrombin-protease nexin complexes to both mouse and human cells without affecting the ability of these cells to respond to thrombin. Thus, protease nexin does not appear to be a positive modulator in thrombin-induced cell division. Here, we show that protease nexin negatively regulates the mitogenic response of cells in culture to thrombin.

Amyloid beta-Protein Precursor↗
PubMed1981

Released protease-nexin regulates cellular binding, internalization, and degradation of serine proteases.

Protease-nexin (PN), a component released by normal human fibroblasts into culture medium, forms covalent linkages with thrombin (Th) and the urinary plasminogen activator urokinase, apparently with their catalytic site serines. The present studies explored the function of PN by examining the interaction of protease-PN complexes with human fibroblasts and the consequences of this interaction. Th-PN and urokinase-PN complexes bind to cells via the PN portion of the complexes. The binding is selectively inhibited by heparin. Because PN has a heparin-binding site, this indicates that protease-PN complexes might bind to a cellular heparin-like site. After binding, the complexes are internalized. By inhibiting endocytosis with phenylarsine oxide, which does not affect cellular binding of Th-PN complexes, we showed that complexes must be internalized before they are degraded. Kinetic analysis of internalization and degradation of Th-PN showed that complexes are internalized more rapidly than they dissociate from the cell surface; by 120 min of incubation at 37 degrees C most cell-bound Th-PN complexes are degraded to amino acids. The results are summarized in a model showing how PN mediates the cellular binding, internalization, and degradation of serine proteases through formation of protease-PN complexes. This series of events may be involved in the regulation of serine protease activity at the cell surface and in the extracellular environment.

Amyloid beta-Protein Precursor↗
PubMed1981

Antifibrillatory efficacy of encainide, loracainide and ORG 6001 compared with lignocaine in isolated hearts of rabbits and guinea-pigs.

1 In Langendorff-perfused rabbit hearts, electrical stimulation threshold and ventricular fibrillation threshold (VFT) were measured by applying rectangular impulses of 3 ms duration and increasing current at frequencies of 4 and 20 Hz respectively. 2 Perfusion with lignocaine or one of three new antiarrhythmic drugs, encainide, lorcainide and ORG 6001, produced significant, dose-dependent increases in both thresholds. 3 On a dosage basis, encainide was seven times, lorcainide fourteen times and ORG 6001 twice as potent as lignocaine in raising VFT. 4 In Langendorff-perfused guinea-pig hearts, only lorcainide provided complete protection against ouabain-induced ventricular fibrillation, while 6 of 6, 3 of 6 and 2 of 6 hearts fibrillated in the presence of encainide, lignocaine and ORG 6001 respectively, but with infusion durations significantly higher than control. 5 These results indicate the potential antifibrillatory activity of these new antiarrhythmic agents.

Androstanols↗
PubMed1980

Protease-nexin: a cellular component that links thrombin and plasminogen activator and mediates their binding to cells.

This report identifies a component of normal human fibroblasts that forms a covalent linkage with thrombin and urokinase (urinary plasmingoen activator) and mediates most of the specific cellular binding of these proteases. This component, here named protease-nexin (PN), is both associated with the cell surface and released into the culture medium. In several ways PN resembles antithrombin III (AT3), a prominent inhibitor of thrombin in serum: PN links thrombin, probably via an ester bond; PN does not link thrombin blocked at its catalytic site serine; PN has a high-affinity heparin-binding site; and heparin greatly accelerates the rate of linkage between soluble PN and thrombin. Despite these similarities, PN and AT3 are distinct; they differ in size and are not immunologically cross-reactive. Whereas AT3 regulates the proteolytic activity of thrombin in serum, PN may regulates the activity of serine proteases at and near the cell surface.

Amyloid beta-Protein Precursor↗
PubMed1980

Investigation of the antifibrillatory activity of some anticonvulsant gamma-aminobutyric acid-transaminase inhibitors in the rabbit isolated heart: comparison with phenytoin and mexiletine.

1 The electrical stimulation and ventricular fibrillation thresholds were measured in the Langendorff-perfused rabbit heart by application of rectangular impulses of 3 ms duration and increasing current at frequencies of 4 and 20 Hz respectively. 2 Perfusion with either phenytoin or mexiletine produced significant dose-dependent increases in both thresholds. 3 Perfusion with four anticonvulsant inhibitors of gamma-aminobutyric acid (GABA)-transaminase, aminooxyacetic acid, gamma-acetylenic GABA, gamma-vinyl GABA and sodium valproate, up to a concentration of 20 microgram/ml, had no significant effect on either threshold. 4 The results suggest that these compounds, unlike phenytoin and mexiletine, are devoid of antifibrillatory activity.

4-Aminobutyrate Transaminase↗
PubMed1980

The antifibrillatory potency of aprindine, mexiletine, tocainide and lignocaine compared on Langendorff-perfused hearts of rabbits and guinea-pigs.

The effectiveness of three new antiarrhythmic drugs, aprindine, mexiletine and tocainide in elevating ventricular fibrillation threshold measured in the Langendorff-perfused rabbit heart and in protecting against ouabain-induced arrhythmias in the Langendorff-perfused guinea-pig heart has been compared with that of lignocaine. All these drugs have produced a significant rise in the threshold but quantitatively mexiletine was about equal to, tocainide five times less and aprindine thirty-eight times more potent than lignocaine in this effect. Aprindine differed from the other three drugs in that it had a slow onset of action and its effect was not entirely removed upon reperfusion with the drug-free solution. Only aprindine and mexiletine provided complete protection against ouabain-induced ventricular fibrillation while 3 of 6 and 4 of 6 hearts fibrillated in the presence of lignocaine or tocainide respectively.

Anilides↗
PubMed1979

Direct linkage of thrombin to its cell surface receptors in different cell types.

When 125I-thrombin was incubated with foreskin fibroblasts, cervical carcinoma cells or fibrosarcoma cells of human origin, or with secondary chick embryo cells or Chinese hamster lung cells, it became directly linked to its cell surface receptors. The thrombin-receptor complex (TH-R) was derived exclusively from a pool of 125I-thrombin that had become specifically bound to the cell surface. The linkage was probably covalent, since the complex was resistant to boiling in sodium dodecyl sulfate and 2-mercaptoethanol. Raising the pH to 12 disrupted TH-R, but did not affect a similar complex between epidermal growth factor and its receptor, suggesting that the linkage of these mitogens to their receptors was different. Mild trypsin treatment removed the ability of cells to form TH-R; however, after a 24-h incubation in serum-free medium, trypsin-treated cells recovered the capacity to form TH-R, suggesting that TH-R resulted from interaction of 125I-thrombin with a cellular rather than a serum component. The mitogenic response of cells to thrombin was inversely related to the fraction of specifically bound 125I-thrombin represented by TH-R. The role of TH-R in mitogenesis may be clarified in future studies by obtaining clones of Chinese hamster lung cells that vary in their capacities to form TH-R and to respond to the mitogenic action of thrombin.

Animals↗
PubMed1978

Glucocorticoid-mediated alteration in growth factor binding and action: analysis of the binding change.

The addition of the glucocorticoid analog dexamethasone (DX) to serum-free cultures of human fibroblasts caused a twofold enhancement of the mitogenic response to epidermal growth factor (EGF), although DX by itself was not mitogenic. A basis for this effect was suggested by studies showing that DX also increased the cellular binding of 125I-EGF. DX increased the ability of the cells to bind 125I-EGF only at low physiological concentrations of this polypeptide. Thus, data from 125 I-EGF binding to cells incubated without DX produced a linear Scatchard plot, whereas the data from 125I-EGF binding to DX-treated cells led to an upwardly curvilinear Scatchard plot. Measurements of 125I-EGF association with the cell surface and cytoplasm indicated that this binding change involved an alteration of cell surface EGF receptors. The binding change appeared not to involve negatively cooperative interactions between EGF receptors, nor a change in the number of receptors. The binding alteration could be explained by a model in which DX converted 25--30% of the cell surface EGF receptors to a form having a fourfold increased affinity.

Cell Division↗
PubMed1978

Dexamethasone modulates binding and action of epidermal growth factor in serum-free cell culture.

Experiments probing the mechanism by which glucocorticoids modulate cell proliferation were carried out on serum-free cell cultures of quiescent human diploid foreskin (HF) cells. Added alone, the synthetic glucocorticoid dexamethasone had no effect on cell number. However, dexamethasone enhanced the mitogenic response of HF cells to epidermal growth factor (EGF) by 50% at all EGF concentrations. The mitogenic action of EGF was maximally promoted by a dexamethasone concentration of 100 ng/ml (0.25 muM). Binding studies with (125)I-labeled EGF ((125)I-EGF) suggested that dexamethasone caused this "permissive" effect by modulating cell surface receptors for EGF. Paralleling their increased responsiveness to EGF growth stimulation, dexamethasone-treated cells exhibited a 50-100% increased ability to bind physiological concentrations of (125)I-EGF. A binding increase was apparent after a 4-hr dexamethasone treatment. The dexamethasone-treated cells maintained an increased ability to bind (125)I-EGF during the prolonged exposure to EGF that was required to stimulate cell division. Moreover, the increase in (125)I-EGF binding exhibited a dexamethasone dose-dependence similar to that for the enhancement of EGF mitogenesis, suggesting a relationship between the dexamethasone effects on binding and growth. An investigation of the binding increase showed that it was specific for glucocorticoids, and required protein synthesis. The enhancement of (125)I-EGF binding diminished with increasing concentrations of (125)I-EGF, indicating that dexamethasone caused a qualitative change in the EGF receptors (possibly a change in receptor affinity or cooperativity). The alteration in (125)I-EGF binding may occur as part of a far-reaching dexamethasone-mediated change in the cell surface, because dexamethasone treatment slightly increased the ability of HF cells to bind (125)I-insulin, and decreased by half their ability to bind (125)I-thrombin.

Cell Division↗