Release Of Vitamin B12-binding Protein Research Articles

Kinetics of N-formyl peptide receptor up-regulation during stimulation in human neutrophils.

The kinetics of receptor up-regulation was examined in isolated neutrophils and in whole blood by flow cytometry during cell activation. Stimulation of neutrophils prepared without exposure to LPS with chemoattractants induced fast up-regulation of N-formyl peptide receptors and C receptor type 3 (CR3). Biphasic N-formyl peptide binding curves were detected for saturating concentrations of N-formyl peptide at 37 degrees C. The bulk of the rapid binding during the first 30 to 60 s is attributed to already expressed binding sites whereas the slow binding over the next 3 to 4 min represents a time course of receptor up-regulation. Support for this interpretation comes from conditions under which the number of binding sites and the progress of the binding curves were affected. Cells treated with LPS, which caused expression of internal N-formyl peptide receptors, exhibited rapid, monophasic binding curves with increased total binding. In LPS-untreated, calcium-depleted cells, N-formyl peptide receptor up-regulation was inhibited and rapid, monophasic binding to a smaller total number of expressed sites was observed. Cytochalasin B enhanced the total number of available N-formyl peptide receptors in LPS-untreated but not LPS-treated cells. In both cases binding was rapid and monophasic suggesting that receptors were either fully or rapidly up-regulated. Although not studied in real-time, C receptor type 3 up-regulation was similar to N-formyl peptide receptor up-regulation in response to LPS, or stimulation by N-formyl peptide, C product C5a, leukotriene B4, and platelet-activating factor in isolated cells and in whole blood. After stimulation with formyl peptide, LPS, or C product 5a, the release of vitamin B12-binding protein paralleled up-regulation of receptors. These data indicate that untreated cells up-regulate N-formyl peptide receptors during cell response at a rate of approximately 10,000/min in a calcium-dependent manner whereas LPS-treated cells already express the bulk of their receptors. In cytochalasin B-treated, degranulating cells 30,000 to 50,000 receptors were up-regulated within a minute.

Staurosporine inhibits the respiratory burst and induces exocytosis in human neutrophils

The protein kinase C inhibitor staurosporine influenced in different ways the functions of human neutrophils. Staurosporine prevented the enhanced protein phosphorylation in phorbol ester- and N-formylmethyionyl-leucylphenylalanine (fMLP)-stimulated cells, and was a powerful inhibitor of the respiratory burst induced by phorbol myristate acetate [IC50 (concentration causing 50% inhibition) 17 nM] and the chemotactic peptides fMLP and C5a (IC50 24 nM). It did not alter, however, the superoxide production by cell-free preparations of NADPH oxidase. Staurosporine had no effect on agonist-dependent changes in cytosolic free Ca2+ and exocytosis of specific and azurophil granules, and showed only a slight inhibition of the release of vitamin B12-binding protein induced by phorbol myristate acetate (decreased by 40% at 200 nM). On the other hand, staurosporine also exhibited neutrophil-activating properties: it induced the release of gelatinase (from secretory vesicles) and vitamin-B12-binding protein (from specific granules). These effects were protracted, concentration-dependent, insensitive to Ca2+ depletion, and strongly enhanced by cytochalasin B. Staurosporine, however, did not induce the release of beta-glucuronidase or elastase (from azurophil granules). Except for the sensitivity to cytochalasin B, these properties suggest a similarity between the exocytosis-inducing actions of staurosporine and PMA. The results obtained with staurosporine provide further evidence that different signal-transduction processes are involved in neutrophil activation, and suggest that protein phosphorylation is required for the induction of the respiratory burst, but not for exocytosis.

Botulinum C2 toxin ADP-ribosylates actin and enhances O2- production and secretion but inhibits migration of activated human neutrophils.

The binary botulinum C2 toxin ADP-ribosylated the actin of human neutrophils. Treatment of human neutrophils with botulinum C2 toxin for 45 min increased FMLP-stimulated superoxide anion (O2-) production 1.5-5-fold, whereas only a minor fraction of the cellular actin pool (approximately 20%) was ADP-ribosylated. Effects of botulinum C2 toxin depended on toxin concentrations, presence of both components of the toxin, and incubation time. Cytochalasin B similarly enhanced O2- production. The effects of botulinum C2 toxin and cytochalasin B were additive at submaximally, but not maximally effective concentrations and incubation time of either toxin. Botulinum C2 toxin also enhanced stimulation of O2- production by Con A and platelet-activating factor, but not by phorbol 12-myristate 13-acetate (PMA). Botulinum C2 toxin increased FMLP-induced release of N-acetyl-glucosaminidase by 100-250%; release of vitamin B12-binding protein induced by FMLP and PMA was enhanced by approximately 150 and 50%, respectively. Botulinum C2 toxin blocked both random migration of neutrophils and migration induced by FMLP, complement C5a, leukotriene B4, and a novel monocyte-derived chemotactic agent. The data suggest that botulinum C2 toxin-catalyzed ADP-ribosylation of a minor actin pool has a pronounced effect on the activation of human neutrophils by various stimulants.

Release of vitamin B12-binding protein from isolated rat liver perfused with a synthetic medium.

The unsaturated vitamin B12-binding capacity (UBBC) of the small molecular size vitamin B12-binding protein (SBP) release and albumin release into the medium and the fluid pressure in the portal vein were studied with respect to their ability to reflect the state of isolated perfused rat liver. The liver was perfused in vitro with a synthetic medium either with or without a gas carrier. Liver damage was induced by hypoxia or noradrenalin. Variations in experimental conditions resulted in different patterns of parameters reflecting different aspects of the liver state. The fluid pressure in the portal vein reflected sensitively the circulatory state but is not always a criterion of the general condition of the isolated liver. The albumin release is a criterion of functional state but did not always reflect liver damage. However, the amount of UBBC of SBP released into the medium is a parameter reflecting sensitively the functional capacity of the liver to produce protein and also seems to indicate liver damage. The use of this parameter provides supplementary information that cannot be obtained by either of the two more conventional parameters.