Search PubMed⌕ Search

Biomedical subjects

C G Cochrane

Publications and source records attributed to C G Cochrane.

At least 73 records · Page 4Linked to original sources
PubMed1987

Pulmonary penetration of alpha 1-proteinase inhibitor administered parenterally to dogs.

To study the penetration of alpha 1-proteinase inhibitor (A1Pl) into the lungs of healthy dogs, 83 mg/kg of active A1Pl was administered intravenously over 30 min followed by a bolus of 131I-A1Pl. Animals were lavaged 2 to 72 h after infusion, sequential gamma camera scans were acquired, and urine was analyzed for the excretion of desmosine. After a distribution phase, infused A1Pl left the bloodstream with a half-life of 103 +/- 24 h. Analysis of plasma antiprotease activity demonstrated preservation of function of the infused A1Pl. Lavage fluid A1Pl concentration and activity were significantly increased 24 h after infusion. Gamma camera scans demonstrated that lung, liver, and spleen acquired 131I-A1Pl similarly; radioactivities per gram of tissue of these organs were similar at autopsy. Excretion of desmosine did not decrease from a baseline of 157 +/- 59 nmol/24 h after A1Pl infusion, indicating no effect of A1Pl infusion on background elastolysis. These data suggest that intravenous administration of A1Pl can raise lung antiproteinase levels within 24 h despite the absence of preferential uptake by the lung of the infused protein.

Animals↗
PubMed1987

Detection of tissue kallikrein in the bronchoalveolar lavage fluid of asthmatic subjects.

Kininogenase activity was detected by cleavage of radiolabeled substrate (125I-high molecular weight kininogen [HMWK]) in 22 of 24 bronchoalveolar lavage (BAL) fluid samples from 17 asthmatics who either responded to aerosolized allergen challenge or had symptoms of active asthma. In contrast, six of seven normal controls lacked enzymatic activity. Levels of free immunoreactive kinin found in BAL fluid correlated with the presence of kininogenase activity (P = 0.002). The cleavage pattern of 125I-HMWK by the BAL fluid kininogenase (a dominant 65,000-mol wt fragment), and synthetic inhibitor profile (phe-phe-arg-CH2Cl and phenylmethylsulfonyl fluoride) were compatible with a tissue kallikrein. Peak kininogenase activity eluted at an apparent molecular weight of 20,000-34,000 by HPLC gel filtration. Its antigenic identity was established by immunoblotting with anti-human urinary kallikrein antibody and its activity was inhibited by this antibody. Lysylbradykinin was generated during incubation of fractionated BAL fluid and purified HMWK, the characteristic cleavage product of the tissue kallikreins. We conclude that elevated amounts of tissue kallikrein and kinin are present in the bronchoalveolar spaces of asthmatic subjects. Kinin generation may contribute to the asthmatic response directly through edema formation and smooth muscle contraction and by augmenting release and/or production of preformed (histamine) and secondary mediators such as leukotrienes and platelet-activating factor.

Asthma↗
PubMed1987

Purified cytochrome b from human granulocyte plasma membrane is comprised of two polypeptides with relative molecular weights of 91,000 and 22,000.

A new method has been developed for purification of cytochrome b from stimulated human granulocytes offering the advantage of high yields from practical quantities of whole blood. Polymorphonuclear leukocytes were treated with diisopropylfluorophosphate, degranulated and disrupted by nitrogen cavitation. Membranes enriched in cytochrome b were prepared by differential centrifugation. Complete solubilization of the cytochrome from the membranes was achieved in octylglucoside after a 1-M salt wash. Wheat germ agglutinin-conjugated Sepharose 4B specifically bound the solubilized cytochrome b and afforded a threefold purification. Eluate from the immobilized wheat germ agglutinin was further enriched by chromatography on immobilized heparin. The final 260-fold purification of the b-type cytochrome with a 20-30% yield was achieved by velocity sedimentation in sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified preparation revealed two polypeptides of Mr 91,000 and Mr 22,000. Treatment of the 125I-labeled, purified preparation with peptide:N-glycosidase F, which removes N-linked sugars, decreased relative molecular weight of the larger species to approximately 50,000, whereas beta-elimination, which removes O-linked sugars, had little or no effect on the mobility of the Mr-91,000 polypeptide. Neither of the deglycosylation conditions had any effect on electrophoretic mobility of the Mr-22,000 polypeptide. Disuccinimidyl suberate cross-linked the two polypeptides to a new Mr of 120,000-135,000 by SDS-PAGE. Antibody raised to the purified preparation immunoprecipitated spectral activity and, on Western blots, bound to the Mr-22,000 polypeptide but not the Mr-91,000 polypeptide. Western blot analysis of granulocytes from patients with X-linked chronic granulomatous disease revealed a complete absence of the Mr-22,000 polypeptide. These results (a) suggest that the two polypeptides are in close association and are part of the cytochrome b, (b) provide explanation for the molecular weight discrepancies previously reported for the protein, and (c) further support the involvement of the cytochrome in superoxide production in human neutrophils.

Antibodies↗
PubMed1987

Role of oxidants in DNA damage. Hydroxyl radical mediates the synergistic DNA damaging effects of asbestos and cigarette smoke.

The mechanism by which cigarette smoking and asbestos exposure synergistically increase the incidence of lung cancer is unknown. We hypothesized that cigarette smoke and asbestos might synergistically increase DNA damage. To test this hypothesis we exposed isolated bacteriophage PM2 DNA to cigarette smoke and/or asbestos, and assessed DNA strand breaks as an index of DNA damage. Our results supported our hypothesis. 78 +/- 12% of the DNA exposed to both cigarette smoke and asbestos developed strand breaks, while only 9.8 +/- 7.0 or 4.3 +/- 3.3% of the DNA exposed to cigarette smoke or asbestos, respectively, developed strand breaks under the conditions of the experiment. Our experimental evidence suggested that cigarette smoke and asbestos synergistically increased DNA damage by stimulating .OH formation. First, significant amounts of .OH were detected by electron paramagnetic resonance (EPR) in DNA mixtures containing both cigarette smoke and asbestos, but no .OH was detected in mixtures containing cigarette smoke alone or asbestos alone. Second, the .OH scavengers, dimethylsulfoxide (DMSO), mannitol, or Na benzoate decreased both .OH detection by EPR and strand breaks in DNA mixtures exposed to cigarette smoke and asbestos. Third, the H2O2 scavenger, catalase, and the iron chelators, 1,10-phenanthroline and desferrithiocin, decreased both .OH detection and strand breaks in DNA mixtures exposed to cigarette smoke and asbestos. These latter findings suggest that iron contained in asbestos may catalyze the formation of .OH from H2O2 generated by cigarette smoke. In summary, our study indicates that cigarette smoke and asbestos synergistically increase DNA damage and suggests that this synergism may involve .OH production.

Asbestos↗
PubMed1987

Cell-dependent chemiluminescence. Modulation of the N-formyl chemotactic peptide (FNLPNTL) mediated oxidative burst in human polymorphonuclear leukocytes (PMNL) by murine monoclonal antibody NMS-1.

Binding of purified monoclonal antibody (moAB) IgM NMS-1 to suspended initially spherical living human PMNLs is not associated with the generation of chemiluminescence but was found to enhance the chemiluminescence response to the N-formyl chemotactic peptide FNLPNTL. We investigated quantitatively the kinetics of oxygen metabolite generation by PMNLs stimulated with FNLPNTL +/- moAB NMS-1 using luminol-dependent chemiluminescence as a very sensitive detection system. Chemiluminescence detection allowed the analysis of the time sequence of onset and development of reactive oxygen metabolites following stimulation of PMNLs by FNLPNTL in the presence of moAB NMS-1. The increase of response of PMNLs stimulated with FNLPNTL in the presence of moAB NMS-1 depended on the concentration of the antibody and the sequence of stimulus addition. Stimulation of human PMNLs by 10 nM FNLPNTL induced a rapid burst of chemiluminescence which peaked approximately 5 min after stimulus addition. The subsequent addition of moAB NMS-1 (greater than or equal to 2 micrograms/ml DPBS(+) - 0.1% HSA, 37 degrees C) to FNLPNTL-stimulated PMNLs - after the FNLPNTL-mediated response had already decayed (16-18 min) - without delay induced a second burst of oxygen metabolite generation. The magnitude of this second peak of activation was dose-dependent. Treatment of PMNLs with moAB NMS-1 (greater than or equal to 1 microgram/ml DPBS(+) - 0.1% HSA, 3 min, 37 degrees C) - prior to FNLPNTL (10 nM) stimulation - increased rate and magnitude of the FNLPNTL-mediated response. This response is biphasic with the first peak at the FNLPNTL position and a second, higher peak approximately 16 min after FNLPNTL addition. The magnitude of response was dose-dependent. The latency (lag time) of the response was not changed compared to controls which received no moAB NMS-1 treatment. The observed moAB NMS-1 dependent increase in FNLPNTL-mediated chemiluminescence is transient (50-60 min), persistent activation was not detected.

Antibodies, Monoclonal↗
PubMed1987

Oxidant injury of cultured cells: biochemical consequences.

The experimental results summarized here suggest a mechanism of oxidant induced cell injury which begins with the generation of DNA strand breaks. The mechanism of the generation of these breaks is currently under investigation. The presence of DNA strand breaks activates pADPr polymerase which causes the conversion of cellular NAD to pADPr and free nicotinamide. It is likely that low levels of NAD are associated with inhibition of glycolysis and thereby contribute to the oxidant-induced fall in cell ATP. Additional factors are likely to contribute to altered cell metabolism following oxidant injury. Hyslop has shown that the Vmax of a critical enzyme of the glycolytic pathway, glyceraldehyde-3-phosphate dehydrogenase, is decreased following exposure to oxidant. Finally, it is likely that the oxidative phosphorylation functions of the mitochondrial membrane may also be perturbed by oxidant injury. The extent to which these alterations in cell metabolism occur in vivo following exposure to oxidants may be of great importance to our understanding of acute inflammatory tissue injury.

Adenosine Triphosphate↗
PubMed1986

Physicochemical properties of the N-formyl peptide receptor on human neutrophils.

In order to investigate the physicochemical properties of the N-formyl peptide receptor of human neutrophils, the receptor was specifically and covalently labeled with an iodinated, photoactivatable derivative of the chemotactic hexapeptide, N-formyl-norleucylleucyl- phenylalanyl-norleucyl-[125I]iodotyrosyl-N epsilon-(6- (4'-azido-2'-nitrophenylamino) hexanoyl)-lysine. After labeling isolated neutrophil membranes, the receptor was extracted with Triton X-100, digitonin, or octyl glucoside and subjected to gel filtration on a calibrated Ultrogel AcA 34 column. The Triton X-100- and digitonin-extracted receptor eluted as single molecular species, with Stokes radii of 40 and 33 A, respectively. This material was subjected to further physicochemical analysis. When octyl glucoside-extracted material was gel-filtered, a second peak containing specifically labeled material eluted in the void volume. Subsequent sodium dodecyl sulfate-polyacryl-amide amide gel electrophoresis analysis indicated that this species was the result of disulfide bonded aggregates containing the monomeric species. Sedimentation equilibrium analysis was carried out in H2O and D2O/H2O mixtures, yielding an apparent molecular mass of 63,000 daltons for both Triton X-100- and digitonin-extracted receptor. This agrees closely with the reduced sodium dodecyl sulfate-polyacrylamide gel electrophoretic value of 50,000-60,000 daltons, indicating that the receptor extracted from unstimulated membranes is monomeric in these detergents. From the sedimentation equilibrium data, the partial specific volume (v) and frictional ratio (f/f0) were calculated. The v is high in both Triton X-100 (0.880) and digitonin (0.829), indicating that the receptor may be associated with tightly bound endogenous lipid or that it is a hydrophobic membrane protein. This latter likelihood is further supported by the quantitative extraction of receptor into Triton X-114 by a phase-separation method. The frictional ratio of 1.1-1.3 is consistent with an elongated globular protein having an axial ratio of approximately 3:1. This in conjunction with the Stokes radius of 40 A would indicate that the receptor is capable of spanning the 35-40-A nonpolar center of the lipid bilayer. The state of the receptor in situ is discussed.

Affinity Labels↗
PubMed1986

Intracellular calcium homeostasis during hydrogen peroxide injury to cultured P388D1 cells.

The effects of exposure of cultured P388D1 cells to H2O2 on intracellular free calcium ([Ca++]i) was investigated utilizing the intracellular fluorescent calcium chelator "Quin 2." [Ca++]i rose from approximately 150 nM to greater than 2 microM over a time course that was strongly dependent on the concentration of H2O2 used (5 X 10(-5) to 5 X 10(-3) M). After exposure of P388D1 cells to 5 X 10(-3) M H2O2, Quin 2 was fully saturated between 15 and 30 min exposure. During this time, no apparent change in the rate of equilibration of 45Ca++ from the extracellular medium could be detected, whereas in cells preloaded with 45Ca, net 45Ca was lost from the cells at a greater rate than controls. Measurements of total cellular calcium by atomic absorption spectroscopy confirmed that there was a net loss of calcium from the cells during the first 30 min. At time points greater than 45 min after exposure to H2O2 the influx of extracellular 45Ca and net intracellular Ca++, Na+ and K+ rapidly increased. Half times for H2O2 catabolism by the cells varied from about 8 min at 5.0 X 10(-4) M H2O2 to 14.0 min at 5.0 X 10(-3) M. When the total [Ca++]i-buffering capacity of the Quin 2 pool was varied by increasing the loading of intracellular Quin 2 by 68-fold (1.1 X 10(2) - 7.6 X 10(3) amol per cell), the rate of rise of [Ca++]i was depressed by only 1.6-fold following exposure to 5 mM H2O2. During the rise of intracellular [Ca++]i, cell morphology was observed by both light and scanning electron microscopy and revealed that "surface blebs" appeared during this phase of injury. Both the rise in [Ca++]i and "blebbing" were observable before any loss in cell viability was detected by either loss of Trypan blue exclusion or loss of preloaded 51Cr from the cells. From these results we conclude the following, H2O2 exposure induces a dose-dependent disturbance of intracellular calcium homeostatis; the rise in [Ca++]i is mediated by exposure to H2O2 in the early phase of the injury, and is not dependent on the continuing presence of the oxidant; the rate of rise of [Ca++]i is largely independent of the quantity of calcium mobilized to the Quin 2 pool; during the early phase (less than 30 min) of rise of [Ca++]i, only intracellular calcium is involved in the response; these events occur concomitantly with gross morphological changes to the plasma membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1986

Hageman factor dependent pathway in local vascular permeability enhancement.

The possibility of an involvement of the kallikrein-kinin system in increasing vascular permeability induced by intradermal injection of the guinea pig activated Hageman factor (beta HFa) was examined. In vitro system, the kinin generation was observed in guinea pig plasma when the plasma was incubated with beta HFa. This kinin generation was dependent upon the dose of beta HFa added and upon the presence of plasma prekallikrein, since 97 percent of the kinin release was diminished by removing the prekallikrein in plasma by treatment with anti-prekallikrein antibody. These results suggested that the Hageman factor-kallikrein-kinin cascade in guinea pig was similar to those in human and bovine plasma. In the permeability experiment in vivo, a simultaneous injection of soybean trypsin inhibitor (10(-6) M), which is the inhibitor of guinea pig plasma kallikrein, inhibited the permeability response to beta HFa by more than 90 percent. The permeability response to beta HFa was attenuated 5 fold in animals depleted of the circulating plasma prekallikrein by intraarterial antibody administration. These results indicated the participation of plasma prekallikrein in the permeability reaction to beta HFa. A simultaneous injection of a kinin destructive enzyme, carboxypeptidase B, diminished the permeability reaction to beta HFa, without any inhibition of the amidolytic activity of beta HFa or plasma kallikrein. A simultaneous injection of an inhibitor of a kinin destructive enzyme (kininase II), SQ 20,881(Glu-Tyr-Pro-Arg-Pro-Gln-Ile-Pro-Pro-OH), augmented the permeability reaction to beta HFa 10 fold, without any effect on the amidolytic activity of beta HFa or plasma kallikrein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Human surfactant treatment of severe respiratory distress syndrome: pulmonary effluent indicators of lung inflammation.

Pulmonary effluent from infants who received exogenous human surfactant for severe respiratory distress syndrome was evaluated for inflammatory changes previously identified with lung injury during the first 2 weeks after birth. The number of pulmonary effluent inflammatory cells was higher only on day 1 in infants given surfactant. No other evidence of enhanced inflammation was detected in cytologic assessment of tracheal secretions. The classical pathway of complement was not activated in infants given surfactant or in control infants 2 weeks after birth. Albumin content of airway secretions was higher on the first day but not significantly altered on subsequent days. Human surfactant treatment was not associated with increased proteolytic activity, measured as neutrophilic elastase per milligram of albumin in lung effluent, but was associated with significantly higher alpha 1-proteinase inhibitor levels than in control infants from days 2 to 7 after birth. These findings provide evidence that exogenous human surfactant instilled into the lungs of preterm infants with severe respiratory distress syndrome is not associated with enhanced lung inflammation, compared with conventional mechanical ventilation alone. These data support additional clinical trials using human surfactant.

Albumins↗
PubMed1986

Hydrogen peroxide-induced injury of cells and its prevention by inhibitors of poly(ADP-ribose) polymerase.

H2O2, in concentrations achieved in the proximity of stimulated leukocytes, induces injury and lysis of target cells. This may be an important aspect of inflammatory injury of tissues. Cell lysis in two target cells, the murine macrophage-like tumor cell line P388D1 and human peripheral lymphocytes, was found to be associated with activation of poly(ADP-ribose) polymerase (EC 2.4.2.30), a nuclear enzyme. This enzyme is activated under various conditions of DNA damage. Poly(ADP-ribose) polymerase utilizes nicotinamide adenine dinucleotide (NAD) as substrate and has been previously shown to consume NAD during exposure of cells to oxidants that was associated with inhibition of glycolysis, a decrease in cellular ATP, and cell death. In the current studies, inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide, nicotinamide, or theophylline in cells exposed to lethal concentrations of H2O2 prevented the sequence of events that eventually led to cell lysis--i.e., the decrease in NAD, followed by depletion of ATP, influx of extracellular Ca2+, actin polymerization and, finally, cell death. DNA damage, the initial stimulus for poly(ADP-ribose) polymerase activation, occurred despite the inhibition of this enzyme. Cells exposed to oxidant in the presence of the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide failed to demonstrate repair of DNA strand breaks.

Actin Cytoskeleton↗
PubMed1986

Reconstitution of surfactant activity using purified human apoprotein and phospholipids measured in vitro and in vivo.

The major apoprotein of human lung surfactant was isolated from amniotic fluid obtained at term gestation. It was found to be a disulfide-linked oligomer composed of polypeptide chains of 35,000 daltons. The monomeric unit was shown to be a glycoprotein, and treatment with peptide: N-glycosidase F resulted in a decrease in molecular weight to 31,000 daltons. The isolated apoprotein could be recombined in the presence of Ca++ with the phospholipids dipalmitoylphosphatidylcholine and phosphatidylglycerol (3:1) at a weight ratio of 1:100. The surface tension (gamma min) measured on a pulsating bubble formed in 4 mg/ml of phospholipids was reduced from 32.3 +/- 2.0 dyn X cm-1 to 18.0 +/- 0.6 dyn X cm-1 after 15 s when 1% apoprotein was present. Reduction of disulfide bonds and deglycosylation of the apoprotein did not alter its ability to lower gamma min. Fetal rabbits of 27 days gestation had instilled intratracheally at delivery, saline, phospholipids, phospholipids plus apoprotein, or natural human surfactant. The latter 2 resulted in increased lung compliance and striking improvement in homogeneous alveolar expansion when the lungs were expanded to 10 cm H2O pressure, fixed, and viewed histologically. This effect was also shown to be independent of the disulfide-dependent oligomeric structure of the apoprotein or its state of glycosylation. The surfactant produced by recombination of the phospholipids with the isolated apoprotein was, therefore, shown to be biophysically active both in vitro and in vivo. These data suggest that apoprotein can be recombined with phospholipids to produce a biologically active surfactant for use in clinical trials of human surfactant replacement.

Animals↗
PubMed1986

Oxidant injury of cells. DNA strand-breaks activate polyadenosine diphosphate-ribose polymerase and lead to depletion of nicotinamide adenine dinucleotide.

To determine the biochemical basis of the oxidant-induced injury of cells, we have studied early changes after exposure of P388D1 murine macrophages to hydrogen peroxide. Total intracellular NAD+ levels in P388D1 cells decreased with H2O2 concentrations of 40 microM or higher. Doses of H2O2 between 0.1 and 2.5 mM led to an 80% depletion of NAD within 20 min. With doses of H2O2 of 250 microM or lower, the fall in NAD and, as shown previously, ATP, was reversible. Higher doses of H2O2 that cause ultimate lysis of the cells, induced an irreversible depletion of NAD and ATP. Poly-ADP-ribose polymerase, a nuclear enzyme associated with DNA damage and repair, which catalyzes conversion of NAD to nicotinamide and protein-bound poly-ADP-ribose, was activated by exposure of the cells to concentrations of 40 microM H2O2 or higher. Activation of poly-ADP-ribose polymerase was also observed in peripheral lymphocytes incubated in the presence of phorbol myristate acetate-stimulated polymorphonuclear neutrophils. Examination of the possibility that DNA alteration was involved was performed by measurement of thymidine incorporation and determination of DNA single-strand breaks (SSB) in cells exposed to H2O2. H2O2 at 40 microM or higher inhibited DNA synthesis, and induced SSB within less than 30 s. These results suggest that DNA damage induced within seconds after addition of oxidant may lead to stimulation of poly-ADP-ribose polymerase, and a consequent fall in NAD. Excessive stimulation of poly-ADP-ribose polymerase leads to a fall in NAD sufficient to interfere with ATP synthesis.

Adenosine Triphosphate↗
PubMed1986

Intravascular release of intact cellular fibronectin during oxidant-induced injury of the in vitro perfused rabbit lung.

Fibronectin (Fn) is produced by cells in blood vessels at inflammatory sites in vivo. Fn release into the circulation thus may be a marker for vascular injury. In support of this, we found that oxidant-induced vascular injury of isolated perfused rabbit lungs caused elevated circulating Fn levels. Western blot analysis indicated that Fn released from the injured blood vessels was intact, dimeric, and possessed electrophoretic mobility identical with Fn produced by fibroblasts. Unlike Fn isolated from rabbit plasma, Fn derived from lung perfusate or produced by fibroblasts reacted with antibodies raised to a synthetic peptide containing sequences from the extra type III Fn domain that is transcribed in fibroblasts but not hepatocytes. Vascular injury by protease was also associated with intravascular release of Fn, but with cleavage. Oxidant-induced vascular injury causes release of tissue-derived Fn, which can be distinguished from plasma Fn by its size and content of antigenic determinants of the extra type III domain.

Animals↗
PubMed1986

Cytoskeletal and morphologic impact of cellular oxidant injury.

The relationship between changes in cell morphology and the cytoskeleton in oxidant injury was examined in the P388D1 cell line. Flow cytometry of cells stained with NBD-phallacidin, a fluorescent probe specific for filamentous (F) actin, revealed a substantial increase in F actin content in H2O2-injured cells over 3-4 hours. Doses of H2O2 as low as 500 microM produced sustained increases in F actin content. Experiments where catalase was used to interrupt H2O2 exposure over a long time course revealed 15-30 minutes to be the critical period of exposure to 5 mM H2O2 necessary for a sustained increase in F actin as well as large increases in membrane blebbing and later cell death. The increase in F actin with H2O2 injury was confirmed with the use of electrophoresis in acrylamide gels of 1% Triton X-100 cytoskeletal extracts from P388D1 cells. Scanning electron microscopy revealed major loss of surface convolutions in addition to the formation of blebs. Fluorescence microscopy of adherent cells using rhodamine phalloidin showed considerable cell rounding and rearrangement of cellular F actin by 30 minutes of exposure to H2O2. Transmission electron microscopy revealed side to side aggregation of F actin bundles (microfilaments) developing during this time. Considerable swelling of mitochondria and other subcellular organelles was seen after 2 hours of injury. The apparent area of attachment to the substrate was markedly diminished in injured cells. H2O2 injury produced a marked increase in F actin with an associated rearrangement of the microfilaments and simultaneous changes in the plasma membrane prior to cell death in the P388D1 cell line.

Actins↗
PubMed1985

Signal transduction and ligand-receptor dynamics in the human neutrophil. Transient responses and occupancy-response relations at the formyl peptide receptor.

The responses of neutrophils to formyl peptides are initiated and in many cases achieve a maximal level prior to equilibrium receptor occupancy. In order to begin to understand the linkage between receptor occupancy and cell response we have used a pulsed binding procedure to analyze: 1) the number of receptors contributing to three potential signalling events and six functional responses and 2) the evolution of these responses once ligand binding is interrupted. We find that the half-optimal elevations of the potential signals are produced by less than 1% occupancy (Ca2+) or 1-3% occupancy (cAMP, membrane depolarization). In contrast, actin polymerization and a rapid light scatter response are elicited by less than 0.1% occupancy. Half-optimal elastase release and degranulation require approximately 3% occupancy. While half-optimal O2- production and aggregation require approximately 30% occupancy, the half-optimal rate of O2- production requires less than 10% occupancy. To resolve the apparent lack of correlation between the responses and the signals we examined their time courses following the pulse of stimulation. At least four responses and one signal are transient and decay while occupied receptors remain on the membrane surface. These include the Quin 2-Ca2+ signal, actin polymerization, the light scatter response, O2- generation, and aggregation. Ca2+ elevation is correlated with the responses in that: 1) each of these responses is transient unless new receptors are occupied; 2) occupancy of nearly all of the receptors contributes to the time course of these responses; 3) when binding is interrupted, the responses decay with a half-time of 15 s, following a latency of approximately 10 s or less (except for disaggregation where latency is 30-40 s). We discuss evidence in support of the hypothesis that transient cell responses arise from transient receptor activation.

Actins↗
PubMed1985

Regulation of the oxidative response of human granulocytes to chemoattractants. No evidence for stimulated traffic of redox enzymes between endo and plasma membranes.

Experiments were performed to probe the role of exocytotic and endocytotic processes in the regulation of the human granulocyte O-2-generating system. Analytical subcellular fractionation studies indicated that 25-30% of the total cellular b-cytochrome and 8-10% of the flavin co-sedimented with plasma membrane markers, irrespective of stimulation of the cells by the chemoattractants N-formyl-Met-Leu-Phe (FMLP) or C5a. Phorbol myristate acetate stimulation resulted in significant translocation of b-cytochrome but not flavin from the specific granule/Golgi to the plasma membrane-enriched fractions. These results indicated that approximately 3.1 X 10(5) flavin and 0.8-1 X 10(6) b-cytochrome molecules are present in the plasma membrane of an isolated unstimulated human granulocyte and that these levels are invariant upon stimulation with chemoattractants. Maximal instantaneous rates of O-2 generation by cells in these preparations, however, were equivalent for all the stimuli. Since stimulation of granulocytes by phorbol myristate acetate, FMLP, or C5a results in exocytosis and/or endocytosis, then the role of these processes in regulating stimulated O-2 production by controlling the content of plasma membrane redox enzymes is questionable. This conclusion was supported by observations made with cytoplasts, which do not have an intracellular reserve of granules. Cytoplasts prepared from granulocytes produced O-2 at equivalent rates as their parent cells on a per unit surface area basis. These results suggest: 1) that stimulation of granulocytes with chemotactic peptides leads to full generation of O-2 at the cell surface without exocytotic recruitment of additional b-cytochrome and flavoprotein from the cytoplasmic compartment; 2) that these redox enzymes are not internalized along with chemoattractant receptors; and 3) that traffic of these redox enzymes between endo- and plasma membranes is not involved in the regulation of O-2 production in suspensions of human granulocytes stimulated by chemoattractants.

Cell Fractionation↗