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G M Omann

Publications and source records attributed to G M Omann.

At least 19 recordsLinked to original sources
PubMed1992

Inhibition of organic anion transport in endothelial cells by hydrogen peroxide.

ATP loss is a prominent feature of cellular injury induced by oxidants or ischemia. How reduction of cellular ATP levels contributes to lethal injury is still poorly understood. In this study we examined the ability of H2O2 to inhibit in a dose-dependent manner the extrusion of fluorescent organic anions from bovine pulmonary artery endothelial cells. Extrusion of fluorescent organic anions was inhibited by probenecid, suggesting an organic anion transporter was involved. In experiments in which ATP levels in endothelial cells were varied by treatment with different degrees of metabolic inhibition, it was determined that organic anion transport was ATP-dependent. H2O2-induced inhibition of organic anion transport correlated well with the oxidant's effect on cellular ATP levels. Thus H2O2-mediated inhibition of organic anion transport appears to be via depletion of ATP, a required substrate for the transport reaction. Inhibition of organic anion transport directly by probenecid or indirectly by metabolic inhibition with reduction of cellular ATP levels was correlated with similar reductions of short term viability. This supports the hypothesis that inhibition of organic anion transport after oxidant exposure or during ischemia results from depletion of ATP and may significantly contribute to cytotoxicity.

Adenosine Triphosphate↗
PubMed1992

A threshold level of coupled G-proteins is required to transduce neutrophil responses.

Chemoattractant-induced activation of human polymorphonuclear leukocytes involves receptor coupling to guanine nucleotide binding proteins (G-proteins). Treatment of polymorphonuclear leukocytes with pertussis toxin, which ADP-ribosylates neutrophil G-proteins and uncouples G-proteins from receptors, causes a conversion of cells from responders to nonresponders rather than a gradual decrease in the ability of all cells to respond (Omann, G. M., and J. M. Harter. 1991. Cytometry 12:252; Omann, G. M., and M. M. Porasik-Lowes. 1991. J. Immunol. 146:1303). Flow-cytometric methods were used to measure N-formylpeptide-induced cytosolic Ca2+ elevation and actin polymerization over a wide range of ADP-ribosylation levels and showed that although the percentage of responding cells varied markedly, the responding cells were stimulated equivalent to controls. The conditions of pertussis toxin (PT) treatment did not interfere with non-G-protein-mediated pathways as assessed by measurement of phagocytosis, a complex process involving the cytoskeleton. We tested the explanation that the all-or-none effect may have been due to heterogeneous insertion of the catalytic subunit of PT into the cells such that responders had no ADP-ribosylation and nonresponders were completely ADP-ribosylated. Measurement of the binding of fluorescent N-formylpeptides to permeabilized cells, which allows the distinction between completely ribosylated and normal cells, showed that all cells treated with a submaximal concentration of PT had intermediate levels of receptor-coupled G-proteins. Thus, partial ADP-ribosylation had occurred in all cells and the all-or-none insertion of the catalytic subunit of PT was ruled out. Thus, there is a threshold of coupled G-proteins required to transduce responses. The ability of PT to inhibit N-formylpeptide-induced actin polymerization and cytosolic calcium elevation was compared and showed that both responses have essentially the same threshold of G-proteins required to transduce the responses. Thus, the pathways regulating actin polymerization and calcium elevation appear to be coupled with equal efficiency to the G-proteins.

Actin Cytoskeleton↗
PubMed1992

Specific G proteins mediate endothelin induced contraction.

Endothelin is a potent vasoconstrictor peptide which has recently been localized in the gastrointestinal tract. We have investigated the transmembrane signaling properties of endothelin in isolated smooth muscle cells of the rabbit rectosigmoid. Endothelin induced a dose dependent contraction of smooth muscle cells in a range of 10(-10) to 10(-6)M. In normal buffer, contraction peaked at 30 sec and was sustained for up to 8 min. Incubation in 0Ca/2mM EGTA abolished the sustained contraction induced by endothelin, but had no effect on the initial transient contraction. Preincubation of saponin treated cells with G protein antisera had no effect on control cell length. Preincubation of saponin treated isolated smooth muscle cells with specific G protein antisera (rabbit antisera) for Go alpha or Gs for 60 minutes did not inhibit contraction induced by endothelin. Preincubation with an antiserum to Gi3 alpha inhibited the initial transient contraction induced by endothelin and preincubation with an antiserum to Gi1-2 alpha inhibited the sustained phase of the endothelin induced contraction. Our data indicate that: 1) Endothelin induces a direct sustained contraction of smooth cells from the rectosigmoid; 2) The transmembrane signalling of endothelin is through two specific GTP binding components that are Gi alpha, one for the initial transient contraction, and the other for the sustained phase of the contraction.

Animals↗
PubMed1991

Dual effects of guanosine 5'-[gamma-thio]triphosphate on secretion by electroporated human neutrophils.

It is generally believed that G-proteins play stimulatory roles on cell activation. In contrast, we found that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) was a potent inhibitor of Ca(2+)-induced secretion from specific granules (as monitored by vitamin B-12-binding protein). GTP[S] inhibition of specific-granule release occurred in the presence or absence of adenine nucleotides, required Mg2+ (1-3 mM), and was half-maximal at 30 microM-GTP[S]. The dual stimulatory and inhibitory effects of GTP[S] could be readily observed and differentiated when degranulation was monitored over a range of Ca2+ concentrations. Inhibition of specific-granule release by GTP[S] was observed at low Ca2+ concentrations and resulted from shifting the Ca2+ dose-response curves to the right. In contrast, GTP[S] promoted azurophil-granule secretion at relatively high concentrations of Ca2+ and appeared to be due to a general enhancement at all Ca2+ concentrations. A series of hydrolysable and non-hydrolysable nucleotides did not mimic GTP[S] or block its action. Inhibition by GTP[S] occurred in cells which were sensitized with a protein kinase C agonist, suggesting that inhibition of secretion took place distal to this enzyme. However, the inhibitory effects of GTP[S] on specific-granule secretion were reversed by cytochalasin D, which prevents new microfilament formation; this compound also enhanced the stimulation of azurophil-granule release by GTP[S]. We also found that GTP[S] greatly increased the F-actin content of permeabilized neutrophils, whereas Ca2+ (to a lesser extent) decreased F-actin. These data are consistent with the hypothesis that at least two G-proteins are involved in regulating secretion: one which has been previously described as stimulating Ca(2+)-induced secretion (particularly from azurophil granules) and a second, possibly involved in promoting microfilament assembly, which inhibits the discharge of specific granules.

Actins↗
PubMed1991

Graded G-protein uncoupling by pertussis toxin treatment of human polymorphonuclear leukocytes.

Pertussis toxin (PT) inhibits polymorphonuclear leukocyte (PMN) function by ADP-ribosylating and inactivating guanine nucleotide binding proteins (G-proteins) that transduce activation by chemoattractants such as N-formyl peptides (FP). Studies of PMN activation during the time course of PT treatment yielded these results. 1) Responses were differentiated based on their sensitivity to PT treatment. Suboptimal PT treatment that resulted in 50% inhibition of the FP-induced actin-associated right angle light scatter response resulted in greater than 90% inhibition of oxidant production. Exhaustive PT treatment was required to completely inhibit the right angle light scatter response. This is consistent with previous observations that, relative to oxidant production, actin polymerization requires 100-fold fewer active N-formylpeptide receptors to elicit the response. This differential sensitivity to PT treatment has important implications for studies that use pertussis toxin to determine if the neutrophil G-protein is involved in the signaling of responses. If inhibition of oxidant production is used as the only indicator of the effectiveness of PT treatment, significant cytoskeletal changes may still be activated in these cells. Inhibition of actin polymerization is a much more rigorous indicator of complete G-protein inhibition by PT. 2) Analysis of FP-induced actin polymerization and cytosolic calcium elevation using flow cytometry, which measures individual cell responses, revealed that PT treatment resulted in the conversion of PMN from a responding to a non-responding population. In contrast, in control PMN, submaximal doses of FP caused submaximal stimulation of all the cells. The all-or-none effect of PT may result from heterogeneous insertion of the A-promoter of PT into the cell or it may result from a sharp threshold of coupled G-proteins required to transduce the responses.

Actins↗
PubMed1991

Intracellular calcium changes associated with in vitro lymphokine-activated killer and natural killer cell cytotoxicity.

Interleukin 2-activated peripheral blood lymphocytes (lymphokine-activated killer [LAK] cells) have been shown to lyse tumor cells in a nonspecific fashion. Although the mechanisms involved in this cell-mediated lysis are unknown, previous work has shown that T-lymphocyte-mediated cytolysis is associated with massive fluctuations in the intracellular calcium concentration within target cells. Studies were undertaken to determine whether intracellular calcium concentration changes were associated with LAK-mediated cytotoxicity and to determine the effects of calcium channel blockade on in vitro cell-mediated cytotoxicity. Natural killer (NK) and LAK cell cytotoxicity in vitro were measured against head and neck squamous cell carcinoma (UMSCC-11a and UMSCC-38) and Daudi cell lines. Assays were performed in parallel with flow cytometry to measure changes in intracellular calcium concentration within the target cells. Compared with NK cells, LAK cells showed enhanced cytotoxicity against the UMSCC-11a and Daudi lines but not the UMSCC-38 cell line. Both NK and LAK cell cytotoxicity against all the target cell lines directly paralleled significant increases in calcium concentration in the target cells. The addition of verapamil hydrochloride inhibited the rise in intracellular calcium concentration in the sensitive target cells and significantly inhibited both NK and LAK cell cytotoxicity in all the cell lines. The NK cell activity was more sensitive than LAK cell activity to verapamil inhibition. These data suggest that changes in the target cell calcium concentration are early and important events in both NK and LAK cell cytotoxicity in vitro.

Calcium↗
PubMed1991

Pertussis toxin effects on chemoattractant-induced response heterogeneity in human PMNs utilizing Fluo-3 and flow cytometry.

Flow cytometric methods were utilized to determine N-formylpeptide-induced cytosolic calcium levels in human polymorphonuclear leukocytes (PMNs) detected with the calcium indicator Fluo-3. Fluo-3 was readily loaded into PMNs as the acetoxymethyl ester. At room temperature Fluo-3 extrusion was minimal (less than 10%) over a 2 h time period. Flow cytometric histograms yielded symmetric distributions indicating homogeneous labelling of the cells. Stimulation of the cells with N-formyl-met-leu-phe (FMLP) caused homogeneous activation of all cells as indicated by a shift of the fluorescence distribution to higher fluorescence levels while still maintaining a symmetrical distribution. Resting values or FMLP-induced cytosolic calcium levels were similar in cells loaded over a 20-fold range of Fluo-3-acetoxymethyl ester. The effect of graded pertussis toxin (PT) treatment on the calcium response was determined by incubating cells with different concentrations of pertussis toxin for a time period that yielded a range of ADP ribosolation levels inside the cells. When these cells were activated with FMLP, the fluorescence histograms showed that pertussis toxin treatment resulted in a conversion of cells from responders to nonresponders. The responding cells responded with maximum calcium elevations similar to controls. This behavior may reflect heterogeneous insertion of the A-protomer of PT or a very sharp threshold of coupled G-proteins required to transduce the responses.

Aniline Compounds↗
PubMed1991

Flow cytometric analysis of human erythrocytes: II. Possible identification of senescent RBC with fluorescently labelled wheat germ agglutinin.

In the first paper of a series (Gutowski, et al., 1991) we discussed the use of flow cytometry to follow at the cellular level the aging of red blood cells (RBC) in circulation, using fluorescently labelled lectins and goat anti-human-IgG and -IgM. The Coulter Epics 541 was used for those studies. In this report we describe more extensive experiments using the Becton-Dickinson FACScan flow cytometer, and compare the results with those obtained with the Coulter Epics 541. By changing sample conditions from isotonic to hypotonic, compensation for differences of the two instruments was accomplished. We confirmed our previous observations that RBC react very strongly with fluorescein isothiocyanate labelled wheat germ agglutinin (FITC-WGA) and that there is little change in the intensity of fluorescence given by RBC of all sizes with the exception of the smallest. Reactivity with FITC-WGA is markedly decreased in the presence of competitive inhibitors of sialic acid or upon enzymatic removal of sialic acid from RBC. Removal of sialic acid is accompanied by increased reaction with peanut agglutinin (FITC-PNA). Flow cytometry was also used to monitor the enrichment of a population of smallest RBC (less than 0.05%), isolated from both counterflow centrifugation and the interface obtained from Histopaque separation. These smallest RBC showed low reactivity with FITC-WGA and higher binding of FITC-goat-anti-human-IgG, and -IgM, and therefore represent the most senescent RBC, just prior to their clearance from circulation by the reticuloendothelial system. These observations are in compliance with the hypothesis that physiological desialylation of glycophorin is responsible for clearance of senescent RBC from circulation (Aminoff, 1988).

Cellular Senescence↗
PubMed1990

Kinetics and amplification in neutrophil activation and adaptation.

Neutrophil activation and adaptation are beginning to be understood in quantitative and mechanistic detail. Neutrophil responses to chemoattractants involve cell surface receptors, guanine nucleotide binding (G) proteins and intracellular second messengers which are generated within a few seconds of the presentation of the ligand. Novel real-time fluorescent methods have made it possible to examine the dynamics of assembly and disassembly of receptors and G proteins during cell activation and to model, by computer, the complex sequence of ligand-receptor events. Rapid amplification cascades of signals are observed in both stimulatory and inhibitory receptor pathways. The adaptation of neutrophils involves uncoupling between receptors and G proteins, both by sequestering receptors from the transduction sequence and by reducing the activatibility of the G proteins.

Adaptation, Physiological↗
PubMed1989

Oscillating actin polymerization/depolymerization responses in human polymorphonuclear leukocytes.

Leukotriene B4 and platelet-activating factor induced a rapidly oscillating actin polymerization/depolymerization response in human polymorphonuclear leukocytes. N-Formylpeptides were deficient in the ability to induce these oscillations. Flow cytometric analysis of filamentous actin verified that all cells were synchronously responding in this cyclic manner. The hypothesis was tested that these oscillations were analogous to chemical oscillations, i.e. oscillations of intermediate species in chemical systems that are far from equilibrium (Epstein, I. R., Kustin, K., DeKepper, P., and Orban, M. (1983) Sci. Am. 248, 112). Actin polymerization/depolymerization cycles were terminated by adding receptor antagonist a few seconds after initiation of the response by agonists. Thus the oscillations did not represent chemical oscillations that hypothetically could result from a rapid jump of the intracellular milieu to a state far from equilibrium. Rather, continued occupancy of receptors and/or occupancy of new receptors was required to sustain the oscillations. This suggested that the oscillations resulted from regulated polymerization and depolymerization pathways. In simultaneous measurements of actin-associated right angle light scatter and intracellular calcium, no calcium oscillations were detected. Thus, cycles of actin polymerization/depolymerization were not regulated by calcium oscillations.

Actins↗
PubMed1989

Transient increase in phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol trisphosphate during activation of human neutrophils.

We recently showed that phosphatidylinositol trisphosphate (PIP3) was present in a unique lipid fraction generated in neutrophils during activation. Here, we demonstrate that the band containing this fraction isolated from thin layer chromatography consists primarily of PIP3 and that only small amounts of radiolabeled PIP3 exist prior to activation. In addition, high performance liquid chromatography of deacylated phospholipids from stimulated cells reveals an increase in a fraction eluting ahead of glycerophosphoinositol 4,5-P2. After removal of the glycerol we found that it coeluted with inositol 1,3,4-P3 when resubjected to high performance liquid chromatography. Thus, we have detected a second, novel form of phosphatidylinositol bisphosphate in activated neutrophils, PI-(3,4)P2. The elevation of PIP3 through the formyl peptide receptor is blocked by pretreatment with pertussis toxin, implicating mediation of the increase in PIP3 by a guanosine triphosphate-binding (G) protein. The rise in PIP3 is not secondary to calcium elevation. Buffering the rise in intracellular calcium did not diminish the increase in PIP3. The elevation of PIP3 appears to occur during activation with physiological agonists, its level varying with the degree of activation. Leukotriene B4, which elicits many of the same responses as stimulation of the formyl peptide receptor but with minimal oxidant production, stimulates a much attenuated rise in PIP3. Isoproterenol, which inhibits oxidant production also reduces the rise in PIP3. Hence formation of PI(3,4)P2 and PIP3 (presumed to be PI(3,4,5)P3) correlates closely with the early events of neutrophil activation.

Autoradiography↗
PubMed1988

Response of neutrophils to stimulus infusion: differential sensitivity of cytoskeletal activation and oxidant production.

The response of human neutrophils to N-formyl peptides were studied under conditions where ligand binding was controlled by infusing a cell suspension with the peptide over a time period comparable to the normal half-time for binding. Receptor occupancy was measured in real time with a fluorescently labeled peptide using flow cytometry. This binding was approximated by a simple reversible model using typical on (7 X 10(8) M- min-1) and off (0.35/min) rate constants and the infusion rates (0.02-0.2 nM/min). Under conditions of stimulus infusion intracellular calcium elevation, superoxide generation, and right angle light scatter and F-actin formation were measured. As the infusion rate was decreased into the range of 10 pM/min, lowering the rate of increase of receptor occupancy to approximately 0.5% per min, the calcium and right angle light scatter responses elongated in time and decreased in magnitude. Superoxide generation decreased below infusion rates of approximately 100 pM/min (occupancy increasing at a rate in the range of 5% per min). This behavior could contribute to differences between chemotactic responses, which appear to require low rates of receptor occupancy over long periods, and bactericidal or inflammatory responses (free radical generation and degranulation), which require bursts of occupancy of several percent of the receptors.

Actins↗
PubMed1988

The chemotactic peptide receptor. A model for future understanding of chemotactic disorders.

Chemotaxis by human phagocytes enables these cells to find sites of injury or inflammation in the host. This function is a complex orchestration of biochemical and morphologic events that begins with the occupancy of specific surface receptors for chemotactic agents. The response is propagated and amplified by a biochemical cascade of transduction reactions. This cascade is mediated by receptor interactions with guanyl nucleotide binding proteins, subsequent activation of phospholipase C, and a resulting increase in phospholipid turnover. These events are spatially and temporarily integrated as elevations in the concentrations of intracellular Ca++ and diacylglycerol combine to activate protein kinase C. At the macromolecular and subcellular level, integration of these functions with as yet undiscovered pathways results in stimulation of actin polymerization, shape change, degranulation, and membrane flow. Final integration of these events results in the morphologic process of directed migration of the cell along concentration gradients of chemotaxins. During this process, chemotactic receptors are continuously regulated and feed back information to the response system that adjusts the biochemistry and morphology accordingly. This article has briefly considered some of the relationships between these transduction events and the control of a specific chemotactic receptor system employing the N-formylated chemotactic peptides. It is hoped that knowledge of this regulation will aid the physician in understanding the origin of chemotactic disorders derived from defects in chemotactic receptor systems of the phagocytic cells.

Calcium↗
PubMed1987

N-formylpeptide-receptor dynamics, cytoskeletal activation, and intracellular calcium response in human neutrophil cytoplasts.

Cytoplasts (enucleated neutrophils which are depleted of dense granules) were prepared from human neutrophils with a modified procedure which employed dihydrocytochalasin B instead of cytochalasin B. These cytoplasts retained an activatable cytoskeletal network similar to cells in that filamentous actin polymerization in response to an N-formylpeptide (fluoresceinated N-formyl-nle-leu-phe-nle-tyr-lys, FLPEP) occurred with similar dose-response characteristics and was inhibitable by cytochalasin B and dihydrocytochalasin B. Cytoplasts had the same number of receptors per surface area as cells and binding constants and dissociation kinetics were the same for cells and cytoplasts. The conversion of receptors from a rapidly dissociating form to a slowly dissociating form was comparable in cells and cytoplasts. This conversion was not inhibited by cytochalasins and thus did not require actin polymerization. Cytoplasts were capable of internalizing 30% of bound FLPEP after 3 min of binding. Cytochalasins did not block this internalization which thus did not appear to require actin polymerization. After 5 min of binding, [3H]-N-formyl-met-leu-phe cosedimented with the Golgi marker enzymes when cytoplasts were fractionated on sucrose density gradients after N2 cavitation. These results indicate that the internalization mechanism is functional in cytoplasts. The Indo-1-detectable calcium response in cytoplasts had a ED50 similar to cells, though the maximum increase in Ca2+ concentration was about one-half that of cells. The response recovered with time after stimulation and the calcium detected was primarily from intracellular stores. The decay of responses after addition of formylpeptide antagonists was parallel for cells and cytoplasts, and leukotriene B4-induced responses in both cells and cytoplasts. Thus the regulation of the responses in cells and cytoplasts was analogous.

Actins↗
PubMed1987

LTB4 induced activation signals and responses in neutrophils are short-lived compared to formylpeptide.

Leukotriene B4 (LTB4) was shown to be a potent stimulator of neutrophil actin polymerization as detected by right-angle light scatter and rhodamine-phalloidin staining of F-actin. When we compared the kinetics of this neutrophil cytoskeletal response to the chemoattractants formylpeptide and LTB4, we observed that the response to LTB4 was markedly shorter-lived. To understand the basis of this result, we re-examined the kinetics of superoxide generation, elastase release, intracellular calcium elevation, and phosphoinositide metabolism in neutrophils stimulated with LTB4 and N-formylhexapeptide. LTB4 was relatively inefficient in producing superoxide and in releasing elastase. Although both responses were initiated with similar rapidity, they turned off sooner with LTB4 as compared with N-formylhexapeptide stimulation. Intracellular calcium elevation, a signal that is necessary for superoxide generation and degranulation, was of similar magnitude but of shorter duration in response to LTB4 as compared with the N-formylhexapeptide. The LTB4-induced rise of phosphatidic acid also was not sustained as long as the N-formylhexapeptide-induced increase. Prior exposure of neutrophils to LTB4 did not inhibit subsequent stimulation of superoxide generation by N-formylhexapeptide. Thus, the inability of LTB4 to stimulate superoxide generation was not due to LTB4-induced global inhibitory signals. The deficiency in LTB4-induced superoxide and elastase responses may be related to short-lived LTB4-induced activation signals and/or the number of receptors contributing to these responses.

Actins↗
PubMed1987

A high-yield, high-purity elutriation method for preparing human granulocytes demonstrating enhanced experimental lifetimes.

A counterflow centrifugal elutriation procedure for preparing human granulocytes is described. This method permits isolation of 1-2 X 10(9) granulocytes with a yield of 70% +/- 7% from a unit of whole blood in 3 h. These cells are 99% +/- 1% pure and 95-99% viable. When compared to cells prepared by a conventional procedure employing hypotonic lysis to remove red cells, the elutriated cells show enhanced oxidative responses resulting from enhanced longevity. Other responses including receptor-mediated uptake of chemoattractant were not significantly different from those of conventionally prepared cells after incubation at 37 degrees C prior to stimulation. The population of elutriated granulocytes, however, was significantly more homogeneous and pure than cells prepared conventionally.

Cell Separation↗
PubMed1986

Transient catecholamine modulation of neutrophil activation: kinetic and intracellular aspects of isoproterenol action.

Modulation of neutrophil activation by catecholamines may reflect regulatory mechanisms that couple beta-adrenergic and N-formyl peptide receptors to antagonistic biochemical pathways. We examined kinetic and mechanistic aspects of the inhibition by catecholamines of neutrophil activation by formyl peptides. Inhibition of oxidant production by isoproterenol (ISO) was detected as low as 3 nM, had an ID50 of 10(-7) M, and could be blocked and reversed by propranolol. Recovery of cell function occurred over a period of minutes when the concentration of ISO was less than 10(-6) M. These observations are discussed in terms of the interaction of ISO with the adrenergic receptors. The site of catecholamine action is addressed. ISO neither influences formyl peptide-receptor interaction nor does it inhibit oxidant production by phorbol ester. These results suggest an impairment of intracellular signalling processes that couple the formyl peptide-receptor binding to cell activation. We observed inhibition of intracellular Ca++ elevation by ISO only at low formyl peptide concentrations. This inhibition is consistent with a partial inhibition of phosphoinositide metabolism, which was observed. Several other cell responses, including actin polymerization and right angle light scatter, are minimally inhibited by 10(-6) M ISO indicating that the cell activation process is not entirely obliterated. The presence of catecholamine and formyl peptide results in a synergistic elevation of cAMP. The intracellular targets of ISO action may be regulated by cAMP dependent kinases and could follow a branchpoint in the activation sequence that leads distinctly to oxidase activation and cytoskeletal activation.

Actins↗