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Biomedical subjects

C G Cochrane

Publications and source records attributed to C G Cochrane.

At least 91 records · Page 5Linked to original sources
PubMed1985

Membrane-cytoskeleton interactions and the regulation of chemotactic peptide-induced activation of human granulocytes: the effects of dihydrocytochalasin B.

When N-formyl chemotactic peptides bind to granulocyte receptors at 37 degrees C they rapidly form a high-affinity ligand-receptor complex whose coisolation with cytoskeletal residues of Triton X-100-extracted cells is under cellular control [Jesaitis et al: J Cell Biol 98:1378, 1984]. Experiments were performed to investigate the significance of this coisolation. When the granulocytes were preincubated with dihydrocytochalasin B (dhCB) for 10 min at 37 degrees C and then stimulated with 50 nM N-formyl-Met-Leu-[3H]Phe, the rate of uptake of the radioligand by the cells was inhibited. Colocalization of the retained peptide with the Triton X-100 fraction of these cells was also reduced relative to this fraction of the untreated cells. This inhibition was apparent before the onset of FMLP endocytosis. The inhibition was 50% effective at 0.25 microgram dhCB/ml. Maximal inhibition (80-90%) occurred at doses of dhCB greater than 1 microgram/ml. The 90% retention of two plasma membrane markers by the cytoskeleton was marginally affected. These results support the hypothesis that coisolation of the high-affinity receptor-peptide complexes with granulocyte cytoskeletons occurs because of specific association of the complexes with the cytoskeleton at the cell surface. In addition, since these events precede internalization, they suggest that formation of the association between the ligand-receptor complex and cytoskeleton may be necessary for ligand-receptor endocytosis. Experiments were also performed to evaluate other functional consequences of cytoskeletal disruption on chemotactic peptide-stimulated functions. f-Met-Leu-Phe stimulation of O2- production was potentiated due to a prolongation of and an increase in the rate of O2- production. This potentiation had the same dose dependency as the inhibition of receptor modulation. The possible relationship of these various functions is discussed.

Cell Membrane↗
PubMed1985

Glutathione cycle activity and pyridine nucleotide levels in oxidant-induced injury of cells.

Exposure of target cells to a bolus of H2O2 induced cell lysis after a latent period of several hours, which was prevented only when the H2O2 was removed within the first 30 min of injury by addition of catalase. This indicated that early metabolic events take place that are important in the fate of the cell exposed to oxidants. In this study, we described two early and independent events of H2O2-induced injury in P388D1 macrophagelike tumor cells: activation of the glutathione cycle and depletion of cellular NAD. Glutathione cycle and hexose monophosphate shunt (HMPS) were activated within seconds after the addition of H2O2. High HMPS activity maintained glutathione that was largely reduced. However, when HMPS activity was inhibited--by glucose depletion or by incubation at 4 degrees C--glutathione remained in the oxidized state. Total pyridine nucleotide levels were diminished when cells were exposed to H2O2, and the breakdown product, nicotinamide, was recovered in the extracellular medium. Intracellular NAD levels fell by 80% within 20 min of exposure of cells to H2O2. The loss of NADP(H) and stimulation of the HMPS could be prevented when the glutathione cycle was inhibited by either blocking glutathione synthesis with buthionine sulfoximine (BSO) or by inhibiting glutathione reductase with (1,3-bis) 2 chlorethyl-1-nitrosourea. The loss of NAD developed independently of glutathione cycle and HMPS activity, as it also occurred in BSO-treated cells.

Animals↗
PubMed1985

Experimental pulmonary inflammatory injury in the monkey.

Inflammatory pulmonary injury was induced in Macaca mulatta rhesus monkeys by the intrabronchial instillation of the formylated peptide norleu-leu-phe (FNLP) or phorbol myristate acetate (PMA). Indicators of pulmonary injury included an increase in mean protein content of bronchoalveolar lavage (BAL) fluid from 0.51 mg/ml in untreated animals to 3.74 mg/ml and 6.64 mg/ml in FNLP- and PMA-treated animals, respectively, the appearance of a diffuse pulmonary infiltrate in chest roentgenograms, and histologic evidence of a predominantly neutrophilic leukocytic infiltration. Concomitant with the appearance of pulmonary injury was the generation of proteases and oxidants in the BAL fluids. Neutrophil elastase, bound to alpha 1-protease inhibitor (alpha 1-PI), was found to increase from 0.47 micrograms/ml in untreated monkeys to 0.99 micrograms/ml in FNLP-treated animals and 1.23 micrograms/ml in monkeys receiving PMA. Radioiodinated human prekallikrein, instilled for 2 min into the inflammatory site and retrieved by lavaging, was found to have undergone proteolytic cleavage; this cleavage was not consistently inhibitable with the inclusion of antibody to elastase. BAL fluids were shown to contain an amidolytic activity when tested on the synthetic substrate H-D-pro-phe-arg-pNA. This activity was partially inhibitable with known inhibitors of active Hageman factor and kallikrein. beta-Glucuronidase levels in the BAL fluids increased from 0.85 U/ml to 4.36 U/ml and 8.25 U/ml in FNLP- and PMA-treated animals, respectively. Myeloperoxidase (MPO) levels also increased from 1.37 OD U/ml X min to 16.59 and 30.47 OD U/ml X min in the same groups of animals. Oxidant generation was also assessed in several different ways. The specific activity of the oxidant-sensitive inhibitor alpha 1-PI recovered in the BAL fluid decreased from 0.80 in control samples to 0.57 and 0.65 in FNLP- and PMA-treated animals. That this inactivation was due to oxidant injury of the molecule was confirmed by the return to full activity of four out of five BAL samples after their incubation with the reducing agent dithiothreitol in the presence of methionine sulfoxide peptide reductase. The specific activity of catalase in the BAL fluids of animals given 3-amino, 1,2,4 triazole (AT) 1 h before lavaging showed drops from 0.97 in untreated monkeys to 0.04 in FNLP-treated and 0.49 in PMA-treated monkeys. MPO levels also fell in the AT-treated injured animals from 16.59 to 0.85 delta OD/min X ml in FNLP animals in the absence and presence of AT, and 30.47 to 0.60 delta OD/min X ml in PMA-treated animals. Inhibition of MPO by AT was shown in vitro to be H2O2 dependent. Total glutathione levels in the BAL fluids did not change appreciably after FNLP or PMA treatment. These studies present substantial evidence of the generation of both proteases and oxidants during the establishment of acute pulmonary inflammatory injury in an experimental primate model.

Animals↗
PubMed1985

Alterations in adenosine triphosphate and energy charge in cultured endothelial and P388D1 cells after oxidant injury.

To investigate mechanisms whereby oxidant injury of cells results in cell dysfunction and death, cultured endothelial cells or P388D1 murine macrophage-like cells were exposed to oxidants including H2O2, O2-. (generated by the enzymatic oxidation of xanthine), or to stimulated polymorphonuclear leukocytes (PMN). Although Trypan Blue exclusion was not diminished before 30 min, cellular ATP was found to fall to less than 30% of control values within 3 min of exposure to 5 mM H2O2. Stimulated PMN plus P388D1 caused a 50% fall in cellular ATP levels. During the first minutes of oxidant injury, total adenylate content of cells fell by 85%. Cellular ADP increased 170%, AMP increased 900%, and an 83% loss of ATP was accompanied by a stoichiometric increase in IMP and inosine. Calculated energy charge [(ATP + 1/2 AMP)/(ATP + ADP + AMP)] fell from 0.95 to 0.66. Exposure of P388D1 to oligomycin plus 2-deoxyglucose (which inhibit oxidative and glycolytic generation of ATP, respectively) resulted in a rate of ATP fall similar to that induced by H2O2. In addition, nucleotide alterations induced by exposure to oligomycin plus 2-deoxyglucose were qualitatively similar to those induced by the oxidant. Loss of cell adenylates could not be explained by arrest of de novo purine synthesis or increased ATP consumption by the Na+-K+ ATPase or the mitochondrial F0-ATPase. These results indicate that H2O2 causes a rapid and profound fall in cellular ATP levels similar to that seen when ATP production is arrested by metabolic inhibitors.

Adenosine Triphosphate↗
PubMed1984

The dynamics of ligand-receptor interactions. Real-time analyses of association, dissociation, and internalization of an N-formyl peptide and its receptors on the human neutrophil.

Parallel cytometric and fluorimetric analyses of the interaction of a fluoresceinated N-formyl hexapeptide (N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein, Nle = norleucine) with its receptors on human neutrophils are presented. The cytometric analyses take advantage of the ability of the fluorescence flow cytometer to discriminate free and receptor-bound ligand in a homogeneous real-time assay. The spectrofluorometric analysis relies on a high affinity antibody to fluorescein to discriminate free and bound ligand. We find that the number of receptors for formyl peptides on the surface of a resting cell is 53,000 +/- 13,000 (Kd approximately 0.6 +/- 0.2 nM). We use commercially available cytometric standards to calibrate the cytometer and we obtain similar values for the number of receptors. The temperature dependence of the kinetics of ligand-receptor interactions have been examined. The association rate constant varies from approximately 3 X 10(8) M-1 min-1 at 4 degrees C to approximately 10(9) M-1 min-1 at 37 degrees C (delta H approximately 8 kcal/mol). While ligand internalization is blocked at 4 degrees C, at 37 degrees C internalization proceeds at an initial rate of approximately 24% of the occupied receptors/min following a latency period of approximately 20-30 s. Intermediate rates and longer latency periods are found at 15 and 25 degrees C. Dissociation of the ligand is heterogeneous and depends both on the length of time of association and the temperature. After short periods of association, the ligand dissociates with t1/2 approximately 1-5 min. After longer periods (30 min at 15 degrees C or 100 min at 4 degrees C), but while the ligand-receptor complex remains on the cell surface, t1/2 increases to greater than 30 min. It appears that the ligand-receptor complex undergoes an alteration in affinity, with a time course at elevated temperatures, which parallels or lags behind the time course of the participation of the occupied receptors in cell activation.

Cell Membrane↗
PubMed1984

Evidence for N-formyl chemotactic peptide-stimulated GTPase activity in human neutrophil homogenates.

Neutrophil homogenates contained a high affinity guanosine triphosphatase (GTPase) that was stimulatable (+27%) by the addition of 100 nM N-formyl chemotactic peptide (CHO-pep), but not by 1 microgram X ml-1 phorbolmyristate acetate (PMA). Kinetic analysis of the stimulation demonstrated an apparent lagtime of 14.3 +/- 6.9 s between the addition of CHO-pep and the optimal GTPase stimulation. The GTPase activity (but not CHO-pep-stimulated GTPase activity) was preserved in a highly purified plasma membrane fraction of the homogenate. From these observations we suggest that both a high affinity guanine nucleotide binding protein and GTPase are closely associated with the plasma membrane CHO-pep receptor. The possibility that GTPase activity may influence guanine nucleotide regulation of adenylate cyclase during CHO-pep stimulation of neutrophils is discussed.

Adenylyl Cyclases↗
PubMed1984

Rapid modulation of N-formyl chemotactic peptide receptors on the surface of human granulocytes: formation of high-affinity ligand-receptor complexes in transient association with cytoskeleton.

When human granulocytes were exposed to 50 nM N-formyl-Met-Leu-[3H]Phe at 37 degrees C they rapidly formed ligand-receptor complexes that dissociated 50-100 times more slowly than those on cells initially exposed to the peptide at 4 degrees C. These complexes of apparent higher affinity were stable after detergent solubilization of the cells with Triton X-100. The complexes co-isolated with the detergent insoluble cytoskeletal residues and were free of the cytosolic and Golgi markers, lactate dehydrogenase and galactosyl transferase, respectively. After 5 s of exposure to f-Met-Leu-Phe, 2,000-3,000 molecules of ligand per cell were trapped in such complexes. Continued exposure resulted in capture of a maximum of 14,000 molecules per cell by 5 min. Exposure at 15 degrees C, a temperature at which endocytosis of the receptor is prevented, resulted in complex formation at a linear rate for at least 20 min to levels twice those measured at 37 degrees C. At 4 degrees C, complex formation was approximately 10% of the maximum amount formed at 37 degrees C. Pulse-chase experiments revealed that the complex was in transient association with the cytoskeleton with a half life ranging between 30 s to 4 min depending on the length of the original incubation. Electron microscopic autoradiography indicated that after 1 min of incubation at 37 degrees C, the majority of the specific autoradiographic grains were localized to the outer circumference of the cellular cytoskeleton. After 4 min of incubation, the grains were less frequent at the cytoskeleton periphery but still threefold enriched over a random cellular distribution. We conclude that a metabolically controlled modulation of the state of the N-formyl chemotactic peptide receptor occurs in the plasma membrane which may be the result of transient association of ligand-receptor complex and the cell cytoskeleton.

Cell Membrane↗
PubMed1984

Proteases and oxidants in experimental pulmonary inflammatory injury.

We have examined various biochemical parameters of pulmonary inflammation in experimental animals. Intrabronchial instillation of glucose oxidase-glucose (GO/G) to produce oxidants or formylated norleu-leu-phe (FNLP) or phorbol myristate acetate (PMA) as leukocytic stimuli induced severe acute pulmonary injury in New Zealand white rabbits. PMA also induced inflammation when administered intravenously. Each stimulus induced transudation of protein from the vascular space into the pulmonary tissues, and an influx of leukocytes during the 4-6 h period of the experiment. Pathophysiologic changes were measured by edema formation (transudation of 125I-bovine serum albumin), and histologic examination. Biochemical analysis was performed by measuring concentrations of potentially injurious agents in bronchoalveolar lavage (BAL) fluid. Increased acid protease and myeloperoxidase levels were found in the BAL fluid after administration of either of the stimuli. Evidence of oxidant generation in vivo was obtained in two different ways. In the first, specific activities for catalase were measured in the BAL fluid in the presence or absence of 3-amino, 1,2,4 triazole (AT), injected at intervals before obtaining BAL fluid. In the presence of AT, specific activities for catalase dropped to 0.22 after a double instillation of FNLP and to 0.15 in the presence of GO/G. In neutrophil-depleted FNLP animals, catalase was not greatly inhibited by AT (sp act 0.90). In the second, intracellular levels of total glutathione (GSH + GSSG) in whole lung tissue and alveolar macrophages decreased when stimuli of neutrophils were administered. Intrabronchially instilled PMA, e.g., caused a drop of glutathione in whole lung tissue from the control value of 2.3 mumol GSH equivalent/100 mg dry wt to 0.54 mumol GSH equivalent/100 mg dry wt at 4 h. Neutrophil depletion and superoxide dismutase protected from this effect. From these results, we conclude that O-2 or its metabolites can initiate severe pulmonary injury as shown by the effect of GO/G and that, during development of pulmonary injury, stimulated neutrophils generate oxidants and release proteolytic enzymes into the surrounding tissues.

Animals↗
PubMed1984

Activation of neutrophils by N-formyl chemotactic peptides.

The response of neutrophils to N-formyl peptides is mediated via a specific 50,000- to 60,000-dalton (Mr) receptor. Real-time kinetic analysis indicated that most of the cellular responses elicited by this ligand began within 5-10 s of addition to the cells at 37 C. Of three possible biochemical changes measured that could serve as transducers or second messengers, two, i.e., increases in cyclic AMP (cAMP) and intracellular free Ca2+, occurred within 5 s of stimulus addition. In contrast, internalization of the ligand by cells showed a latency time of 20-30 s, which indicates that it probably plays no role in triggering later responses. Using pulse binding techniques that allow the level of a given response to be measured as a function of the measured level of surface receptor occupancy, we found that O2- production required up to 30% receptor occupancy to elicit 50% of maximal response. In contrast, secretion, cAMP changes, Ca2+ changes, and membrane potential changes required less than 5% occupancy. Within 5 s, occupied receptors were converted at the cell surface to a slowly dissociating form. The receptors, exhibiting apparent higher affinity, were transiently associated with the cell cytoskeleton as defined by their conversion to a Triton X-100-insoluble form. Internalized receptor-ligand complexes were transported, in large part, to the Golgi apparatus. Further analyses of the mechanism of stimulation of leukocytes have been performed with monoclonal antibodies directed against the neutrophil surface. Data with these antibodies, which are not directed to the N-formyl peptide receptor, reveal that some modulated the N-formyl peptide-mediated responses and other antibodies initiated responses of the cells.

Antibodies, Monoclonal↗
PubMed1984

In vitro activation of the contact (Hageman factor) system of plasma by heparin and chondroitin sulfate E.

A large number of negatively charged macromolecules, including DNA, glycosaminoglycans, and proteoglycans, were tested as possible activators of the contact (Hageman factor) system in vitro. Activation was assessed by conversion of prekallikrein to kallikrein, as determined by amidolytic assay and by cleavage of 125I-Hageman factor into 52,000- and 28,000-dalton fragments. Of particular interest to these studies, heparin proteoglycan and glycosaminoglycan from rat peritoneal mast cells, and squid chondroitin sulfate E, which is representative of the glycosaminoglycan from cultured mouse bone marrow derived mast cells, induced the reciprocal activation between Hageman factor and prekallikrein. In addition, naturally occurring heparin glycosaminoglycans from pig mucosa, bovine lung, and rat mast cells also induced activation. In contrast, native connective tissue matrix glycosaminoglycans and proteoglycans from several sources were inactive, although when one such chondroitin sulfate was further sulfated in vitro, it gained activity. When the negative charge of the activating agents was blocked by the addition of hexadimethrine bromide, the cleavage of 125I-Hageman factor in the presence of prekallikrein was prevented. The active negatively charged macromolecules induced cleavage of 125I-high molecular weight kininogen in normal plasma but not in Hageman factor-deficient or prekallikrein-deficient plasmas. Reconstitution of prekallikrein-deficient plasma with purified prekallikrein restored the kininogen cleavage upon addition of the active proteoglycans. These results suggest that both heparin from connective tissue mast cells and highly sulfated chondroitin sulfate E from cultured mouse bone marrow derived mast cells (which are considered synonomous with mucosal mast cells) could activate the contact system of plasma subsequent to an activation secretion response.

Animals↗
PubMed1984

Biochemical factors in pulmonary inflammatory disease.

Various biochemical events taking place during pulmonary inflammation were examined in the bronchoalveolar lavage (BAL) fluids from patients with acute respiratory distress syndrome (ARDS) and in experimental animal models. In patients with ARDS, active neutrophil elastase was found in the BAL fluids. In these fluids, inactivation of the major elastase inhibitor alpha 1-protease inhibitor (alpha 1-PI) occurred. This was caused by oxidation of a methionine residue at the active site of the alpha 1-PI, and offered indirect evidence of oxidation occurring in the inflamed pulmonary tissues. Studies with experimental animals have been initiated to gain understanding of the relative roles of proteases, oxidants, arachidonate metabolites, complement and contact system components, and other mediators in the pathogenesis of pulmonary inflammation. Intrabronchial instillation of glucose oxidase/glucose to produce oxidants or formylated norleucylleucylphenylalanine or phorbol myristate acetate as leukocytic stimuli induced severe acute pulmonary injury in New Zealand white rabbits and rhesus monkeys. The injury was accompanied by leukocytic protease (acid cathepsins) release in rabbit lungs and oxidant formation, and could be inhibited by neutrophil depletion. Oxidant formation was demonstrated by the inactivation of catalase by 3-amino-1,2,4-triazole in the presence of H2O2, a drop in intracellular glutathione levels, and in the rhesus monkey by inactivation of alpha 1-PI.

Animals↗
PubMed1983

Survey of plant inhibitors of polymorphonuclear leukocyte elastase, pancreatic elastase, cathepsin G, cathepsin B, Hageman factor fragments, and other serine proteinases.

Various flower bulbs and vegetable and legume seeds were tested for inhibitors of polymorphonuclear leukocyte elastase, pancreatic elastase, cathepsin G, cathepsin B, trypsin, alpha-chymotrypsin, Hageman factor fragments, plasma kallikrein, and plasmin. Calla bulbs contained a 33,000 dalton polymorphonuclear leukocyte elastase inhibitor and a 4,000 dalton cathepsin G inhibitor. Seeds of some members in the Cruciferae family, such as radish and broccoli, were found to contain one or more 2,500-4,000 dalton inhibitors which inhibited cathepsin G, trypsin, Hageman factor fragments, and plasmin, but not plasma kallikrein. These seeds also contained a 1,000 dalton cathepsin B inhibitor. The above inhibitors were probably polypeptides which inhibited proteinases by making an enzyme-inhibitor complex, with the exception of the cathepsin B inhibitor. These newly found inhibitors with their characteristic profiles of inhibition should be useful in biochemical and pathophysiological studies on granulocyte proteinases and enzymes of the coagulation and fibrinolytic pathways.

Cathepsin B↗
PubMed1983

The fate of an N-formylated chemotactic peptide in stimulated human granulocytes. Subcellular fractionation studies.

Experiments were performed to examine how human granulocytes process the chemotactic peptide N-formyl-Met-Leu-Phe after stimulation by the same peptide. Purified human granulocytes were stimulated with 50 nM N-formyl-Met-Leu-[3H]Phe at 37 degrees C for various times, washed, lysed by N2 cavitation, and fractionated by isopycnic sucrose density gradient sedimentation. The major subcellular fractions identified were plasma membrane, Golgi, granules, endoplasmic reticulum, and mitochondria. After 1 min of stimulation, radioactivity was found only in the plasma membrane (sedimentable) and cytosol (soluble) fraction. At 5, 10, and 25 min, radioactivity also appeared in a sedimentable, low density fraction (25-28% sucrose) enriched in galactosyl transferase activity and containing Golgi structures. The accumulation in the sedimentable fractions was maximal after 5 min but continued to increase linearly in the cytosol fraction. Incorporation of radioactivity into cells or membrane and soluble fractions was 60 to 85% specific and was inhibited if incubation with N-formyl-Met-Leu-[3H]Phe was performed at 4 degrees C. 80-90% of the radiolabel in the plasma membrane or Golgi-containing fractions remained sedimentable despite freeze thawing or sonication. Solubilization of these fractions in Triton X-100 followed by Sepharose 4B column chromatography revealed that the radiolabel eluted in the void volume. Our results are consistent with internalization which proceeds by passage of an occupied receptor in a high affinity, supramolecular complex from the plasma membrane to the Golgi followed by accumulation of peptide in the cytosol.

Biological Transport↗
PubMed1983

The plasma contact system in atopic asthma.

An in vitro test for the rate of appearance of kallikrein in plasma due to contact system activation by dextran sulfate at 0 degree C was applied to plasmas of 19 atopic asthma patients and 19 age- and sex-matched controls without atopy. The average prekallikrein activation rate was markedly higher in the plasmas of the atopic patients. Mean endogenous heparin levels were also elevated.

Adolescent↗