scholarly journals Chemotactic peptide-induced changes in neutrophil actin conformation.

1984 ◽  
Vol 99 (3) ◽  
pp. 1060-1065 ◽  
Author(s):  
P J Wallace ◽  
R P Wersto ◽  
C H Packman ◽  
M A Lichtman
Keyword(s):  
Flow Cytometry ◽  
Cytochalasin B ◽  
Fluid Phase ◽  
Repetitive Stimulation ◽  
Human Neutrophils ◽  
Chemotactic Peptide ◽  
Directed Movement ◽  
Induced Changes ◽  
Dose Dependent ◽  
Stimulation Of

The effect of the chemotatic peptide, N-formylmethionylleucylphenylalanine (FMLP), on actin conformation in human neutrophils (PMN) was studied by flow cytometry using fluorescent 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin to quantitate cellular F-actin content. Uptake of NBD-phallacidin by fixed PMN was saturable and inhibited by fluid phase F-actin but not G-actin. Stimulation of PMN by greater than 1 nM FMLP resulted in a dose-dependent and reversible increase in F-actin in 70-95% of PMN by 30 s. The induced increase in F-actin was blocked by 30 microM cytochalasin B or by a t-BOC peptide that competitively inhibits FMLP binding. Under fluorescence microscopy, NBD-phallacidin stained, unstimulated PMN had faint homogeneous cytoplasmic fluorescence while cells exposed to FMLP for 30 s prior to NBD-phallacidin staining had accentuated subcortical fluorescence. In the continued presence of an initial stimulatory dose of FMLP, PMN could respond with increased F-actin content to the addition of an increased concentration of FMLP. Thus, FMLP binding to PMN induces a rapid transient conversion of unpolymerized actin to subcortical F-actin and repetitive stimulation of F-actin formation can be induced by increasing chemoattractant concentration. The directed movement of PMN in response to chemoattractant gradients may require similar rapid reversible changes in actin conformation.

scholarly journals Graded responses of human neutrophils induced by serum-treated zymosan

Blood ◽  
1985 ◽  
Vol 66 (5) ◽  
pp. 1182-1188 ◽  
Author(s):  
JC Whitin ◽  
DH Ryan ◽  
HJ Cohen
Keyword(s):  
Flow Cytometry ◽  
Membrane Potential ◽  
Fluorescent Probe ◽  
Dose Response ◽  
Dose Dependence ◽  
Human Neutrophils ◽  
Graded Responses ◽  
Dose Dependent ◽  
O2 Production ◽  
Stimulation Of

Abstract A modified zymosan preparation was used to probe the interaction of particulate stimuli with human neutrophils (PMNs). After extraction with alkali and detergent, the zymosan particles retained their ability to be opsonized in serum and to stimulate PMNs. Serum-treated zymosan (STZ) induced dose-dependent superoxide (O2-) production and membrane potential depolarization in the range of 1 to 10 mg/mL of STZ. The rate and extent of secretion of lysozyme and beta-glucuronidase were also dose-dependent in the range of 1 to 10 mg/mL of STZ. Cytochemical studies using nitroblue tetrazolium, however, showed that 92% of PMNs were stimulated to produce O2- at 0.1 mg/mL of STZ. The dose response of O2- production induced by STZ is therefore due to increasing O2- production by individual PMNs and not to the stimulation of more PMNs to produce O2-. Evidence for O2- production was found only in the area of PMN-zymosan contact, suggesting a mechanism for the graded responses of PMNs treated with particulate stimuli. In order to determine the nature of the dose dependence of depolarization (a measure of PMN activation), PMNs equilibrated with the fluorescent probe 3,3′- dipentyloxacarbocyanine were analyzed by flow cytometry. The results demonstrate that STZ induces a dose-dependent depolarization of the membrane potential of individual PMNs. These results also demonstrate that increasing concentrations of STZ can induce increasing PMN responses even when all of the PMNs have been activated. These results are consistent with the hypothesis that receptor-mediated particulate stimulation of PMNs is a phenomenon that results in graded PMN responses.

The response to thrombin of human neutrophils: evidence for two novel receptors

1995 ◽  
Vol 108 (9) ◽  
pp. 3059-3066 ◽  
Author(s):  
A.L. Jenkins ◽  
G.L. Howells ◽  
E. Scott ◽  
B.F. Le Bonniec ◽  
M.A. Curtis ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Human Neutrophils ◽  
Maximal Response ◽  
Thrombin Receptor ◽  
Neutrophil Chemotaxis ◽  
Similar Magnitude ◽  
Wild Type ◽  
Dose Dependent ◽  
Made In ◽  
Stimulation Of

Human alpha-thrombin was a chemoattractant for human neutrophils yielding a maximal response of similar magnitude to that observed with formyl-Met-Leu-Phe. The observed chemotaxis was not due to stimulation of the proteolytically activated thrombin receptor since: (1) this receptor was not detected by flow cytometry; (2) the inactive thrombin mutant Ser195-->Ala elicited a chemotactic response indistinguishable from that caused by wild-type thrombin; (3) antibodies to the cleavage site of the proteolytically activated receptor did not affect thrombin-induced chemotaxis; (4) a thrombin receptor activating peptide (TRAP) failed to stimulate chemotaxis. These data indicate the existence of a thrombin receptor for neutrophil chemotaxis which is not activated by proteolysis. In addition, although wild-type and ser195-->Ala thrombin did not cause an increase in intracellular Ca2+, a Ca2+ response to TRAP was observed with neutrophils from some donors. The TRAP-induced increase in Ca2+ was reproducible, dose dependent and specific. The use of alanine-substituted peptides demonstrated that the Ca2+ response was due to TRAP stimulation of a receptor other than the proteolytically activated thrombin receptor. Thus, it is necessary to re-evaluate the assumption made in previous studies that responses to TRAP are mediated by the proteolytically activated thrombin receptor.

scholarly journals Graded responses of human neutrophils induced by serum-treated zymosan

Blood ◽  
1985 ◽  
Vol 66 (5) ◽  
pp. 1182-1188
Author(s):  
JC Whitin ◽  
DH Ryan ◽  
HJ Cohen
Keyword(s):  
Flow Cytometry ◽  
Membrane Potential ◽  
Fluorescent Probe ◽  
Dose Response ◽  
Dose Dependence ◽  
Human Neutrophils ◽  
Graded Responses ◽  
Dose Dependent ◽  
O2 Production ◽  
Stimulation Of

A modified zymosan preparation was used to probe the interaction of particulate stimuli with human neutrophils (PMNs). After extraction with alkali and detergent, the zymosan particles retained their ability to be opsonized in serum and to stimulate PMNs. Serum-treated zymosan (STZ) induced dose-dependent superoxide (O2-) production and membrane potential depolarization in the range of 1 to 10 mg/mL of STZ. The rate and extent of secretion of lysozyme and beta-glucuronidase were also dose-dependent in the range of 1 to 10 mg/mL of STZ. Cytochemical studies using nitroblue tetrazolium, however, showed that 92% of PMNs were stimulated to produce O2- at 0.1 mg/mL of STZ. The dose response of O2- production induced by STZ is therefore due to increasing O2- production by individual PMNs and not to the stimulation of more PMNs to produce O2-. Evidence for O2- production was found only in the area of PMN-zymosan contact, suggesting a mechanism for the graded responses of PMNs treated with particulate stimuli. In order to determine the nature of the dose dependence of depolarization (a measure of PMN activation), PMNs equilibrated with the fluorescent probe 3,3′- dipentyloxacarbocyanine were analyzed by flow cytometry. The results demonstrate that STZ induces a dose-dependent depolarization of the membrane potential of individual PMNs. These results also demonstrate that increasing concentrations of STZ can induce increasing PMN responses even when all of the PMNs have been activated. These results are consistent with the hypothesis that receptor-mediated particulate stimulation of PMNs is a phenomenon that results in graded PMN responses.

scholarly journals Oxidative inactivation of myeloperoxidase released from human neutrophils

1987 ◽  
Vol 245 (3) ◽  
pp. 925-928 ◽  
Author(s):  
S W Edwards ◽  
H L Nurcombe ◽  
C A Hart
Keyword(s):  
Respiratory Burst ◽  
Cytochalasin B ◽  
The Other ◽  
Human Neutrophils ◽  
Granulomatous Disease ◽  
Cellular Activity ◽  
Chemotactic Peptide ◽  
Oxidative Inactivation ◽  
Stimulation Of

Within 1 min of stimulation of human neutrophils by the chemotactic peptide (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) plus cytochalasin B, myeloperoxidase (together with other granule enzymes) was secreted and detected extracellularly. In contrast with the other granule constituents assayed (vitamin B12-binding protein and beta-glucuronidase), the activity of released myeloperoxidase rapidly decreased, so that, by 10 min after stimulation, only about 5% of the total cellular activity was detected. This inactivation was shown to be dependent on oxidant generation during the respiratory burst, since inactivation was not observed (a) after stimulation of anaerobic suspensions or (b) after release from neutrophils from a patient with chronic granulomatous disease; purified myeloperoxidase was rapidly inactivated after incubation with H2O2, presumably owing to the formation of an inactive enzyme-H2O2 complex. These results show that experiments designed to assess the role of myeloperoxidase in neutrophil functions which utilize assays based on peroxidase activity will grossly underestimate this enzyme if oxidant generation during the respiratory burst has also been activated.

scholarly journals Regulation of superoxide generation by myeloperoxidase during the respiratory burst of human neutrophils

1986 ◽  
Vol 237 (2) ◽  
pp. 601-604 ◽  
Author(s):  
S W Edwards ◽  
T F Swan
Keyword(s):  
Respiratory Burst ◽  
Cytochalasin B ◽  
Superoxide Radical ◽  
Human Neutrophils ◽  
Second Phase ◽  
Chemotactic Peptide ◽  
O2 Uptake ◽  
Superoxide Generation ◽  
Lower Magnitude

The role of myeloperoxidase in the regulation of the respiratory burst of human neutrophils activated by the chemotactic peptide (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) plus cytochalasin B was determined by using anti-(human myeloperoxidase) antibody. The respiratory burst activated under these conditions consisted of an initial (1-2 min) phase with high rates of O2 uptake, luminol-dependent chemiluminescence and superoxide radical (O2-.) generation and a second, more sustained, phase of lower magnitude of chemiluminescence and O2 uptake: O2-. generation did not occur during this second phase. In cell suspensions stimulated in the presence of anti-(human myeloperoxidase) antibody, the magnitude of the initial phase of both O2 uptake and O2-. generation was unaffected, but these high rates were maintained over much longer periods than in control suspensions. It is therefore proposed that a product of myeloperoxidase normally regulates the duration of O2-. generation during the respiratory burst, possibly by inhibition of NADPH oxidase.

LUTEINIZING HORMONE RELEASING FACTOR IN THE QUAIL HYPOTHALAMUS

1972 ◽  
Vol 53 (1) ◽  
pp. 131-138 ◽  
Author(s):  
P. M. SMITH ◽  
B. K. FOLLETT
Keyword(s):  
Luteinizing Hormone ◽  
Japanese Quail ◽  
Release Rate ◽  
Initial Period ◽  
Repetitive Stimulation ◽  
Pituitary Glands ◽  
Lh Release ◽  
A Minor ◽  
Dose Dependent ◽  
Stimulation Of

SUMMARY Pituitaries from Japanese quail were superfused continuously for up to 12 h and the luteinizing hormone (LH) in the superfusate was measured by radioimmunoassay. After an initial period the release rate remained low and relatively constant. The introduction of hypothalamic extracts prepared from quail substantially increased immunoreactive LH release. The responses were dose-dependent. Cortical extracts caused a minor but significant response. Dopamine was inactive in the system. The technique is attractive because it allows for repetitive stimulation of the same pituitary glands with treatments being administered every 30–45 min.

Protein synthesis is activated in primed neutrophils: a possible role in inflammation

1987 ◽  
Vol 7 (11) ◽  
pp. 881-890 ◽  
Author(s):  
Valerie Hughes ◽  
John M. Humphreys ◽  
Steven W. Edwards
Keyword(s):  
Protein Synthesis ◽  
Actinomycin D ◽  
Protein Biosynthesis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Neutrophils ◽  
Chemotactic Peptide ◽  
Inflammatory Agent ◽  
Maximal Stimulation ◽  
Stimulation Of ◽  
Primed Neutrophil

Circulating human neutrophils exhibited low rates of protein biosynthesis, as determined by their ability to incorporate [35S]methionine into TCA-precipitable material. Exposure of cells to the chemotactic peptide (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) increased their rate of protein synthesis, and the maximal stimulation of biosynthesis by this inflammatory agent was observed at 0.1 μM: this concentration of chemotactic peptide “primed” neutrophil activity and only activated the oxidase of these cells by 8% of maximum. The newly-synthesized proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis and compared with those synthesized in control cells. Two classes of proteins were observed in “primed” cells. The first of these comprised proteins whose rate of biosynthesis changed very little upon “priming” whereas the second class comprised proteins whose rate of synthesis increased greatly after exposure to chemotactic peptide. The fMet-Leu-Phe stimulated protein synthesis was inhibited by actinomycin D and cycloheximide showing that this phenomenon required both transcription and translation. We propose that these fMet-Leu-Phe regulated proteins play an important role in the function of neutrophils during an inflammatory response.

Calcium stimulates glucose transport in skeletal muscle by a pathway independent of contraction

1991 ◽  
Vol 260 (3) ◽  
pp. C555-C561 ◽  
Author(s):  
J. H. Youn ◽  
E. A. Gulve ◽  
J. O. Holloszy
Keyword(s):  
Skeletal Muscle ◽  
Glucose Transport ◽  
Cytochalasin B ◽  
Muscle Tension ◽  
Creatine Phosphate ◽  
Transport Activity ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
Glucose Analogue ◽  
Stimulation Of

In this study we investigated the possibility that an increase in cytoplasmic Ca2+ concentration that is too low to cause muscle contraction can induce an increase in glucose transport activity in skeletal muscle. The compound N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which induces Ca2+ release from the sarcoplasmic reticulum (SR), caused a dose-dependent increase in tension in rat epitrochlearis muscles at concentrations more than approximately 200 microM. Although 100 microM W-7 did not increase muscle tension, it accelerated loss of preloaded 45Ca2+. Glucose transport activity, measured with the nonmetabolizable glucose analogue 3-O-methylglucose, increased sixfold in muscles treated for 100 min with 50 microM W-7 (P less than 0.001) and eightfold in response to 100 microM W-7 (P less than 0.001). The increase in glucose transport activity was completely blocked with 25 microM cytochalasin B. There was no decrease in ATP or creatine phosphate concentrations ([approximately P]) in muscles incubated with 50 microM W-7. Dantrolene (25 microM), which blocks Ca2+ release from the SR, blocked the effects of W-7 both on 45Ca2+ release and on glucose transport activity. 9-Aminoacridine, another inhibitor of Ca2+ release from the SR, also blocked the stimulation of hexose transport by W-7. Caffeine, a compound structurally unrelated to W-7 that also releases Ca2+ from the SR, also increased glucose transport activity. Incubation of muscles with 3 mM caffeine for 30 min, which did not cause contraction or lower [approximately P], induced a threefold increase in 3-O-methylglucose transport (P less than 0.001). These results provide evidence suggesting that an increase in cytoplasmic Ca2+ too low to cause contraction or [approximately P] depletion can bring about an increase in glucose transport activity in skeletal muscle.

scholarly journals Two distinct mechanisms for stimulation of oxygen-radical production by polymorphonuclear leucocytes

1983 ◽  
Vol 216 (2) ◽  
pp. 459-465 ◽  
Author(s):  
M B Hallett ◽  
A K Campbell
Keyword(s):  
Peroxidase Activity ◽  
Cytochalasin B ◽  
Oxygen Radical ◽  
Polymorphonuclear Leucocytes ◽  
Chemotactic Peptide ◽  
Latex Particles ◽  
Radical Production ◽  
Latex Beads ◽  
Oxygen Radical Production ◽  
Stimulation Of

Oxygen-radical production stimulated from rat polymorphonuclear leucocytes by either unopsonized latex particles (diameter = 1.01 microM) or chemotactic peptide (N-formyl-Met-Leu-Phe) was monitored by using luminol-dependent chemiluminescence. Azide inhibited by more than 80% the luminescence response induced by chemotactic peptide whether added before or after stimulation. However, the luminescence response to latex particles was progressively less susceptible to azide inhibition if the azide was added after the stimulus. Cytochalasin B, which was shown to abolish phagocytosis of the latex beads, also abolished the chemiluminescence response. However, the same cells showed a greatly enhanced response to chemotactic peptide. Cytochalasin B-treated cells secreted approx. 45% of total cellular myeloperoxidase in response to chemotactic peptide, but there was no detectable secretion in response to unopsonized latex particles. Microperoxidase equivalent to 20% of cellular peroxidase activity added to the cells before addition of the stimulus had no effect on the response to latex particles but increased approx. 2-fold the peak rate of chemiluminescence induced by chemotactic peptide. It was concluded that the unopsonized latex particles stimulated oxygen-radical production by the mechanism that involved endocytosis, whereas chemotactic peptide stimulated production by a mechanism that involved exocytosis of myeloperoxidase, the latter mechanism requiring an increase in intracellular free [Ca2+].

Light-scattering changes during chemotactic stimulation of human neutrophils: Kinetics followed by flow cytometry

Cytometry ◽  
1985 ◽  
Vol 6 (1) ◽  
pp. 7-12 ◽  
Author(s):  
Paul L. McNeil ◽  
Amy L. Kennedy ◽  
Alan S. Waggoner ◽  
D. Lansing Taylor ◽  
Robert F. Murphy
Keyword(s):  
Flow Cytometry ◽  
Light Scattering ◽  
Human Neutrophils ◽  
Stimulation Of
Sign in / Sign up

Export Citation Format

Share Document