FMLP

Previous studies indicate that osmotic gradients promote membrane fusion, while hyperosmotic conditions inhibit membrane fusion during exocytosis. Consistent with this idea is the observation that the release of lysosomal enzymes from rabbit neutrophils, induced by the chemotactic peptide J -formylmethionyl-leucyl-phenylalanine (FMLP), is inhibited almost 80% in a 700-mosmol/kg medium. Inhibition is immediate (within 10 s), increases with osmolality, and is independent of the osmoticant. Neutrophils loaded with the calcium indicator indo-1 exhibit an FMLP-induced calcium signal that is inhibited by hyperosmolality. Hyperosmolality (700 mosmol/kg) increases basal calcium levels and reduces the peak of the calcium signal elicited by FMLP at concentrations ranging from 10 ° to 10 M. 

Neutrophils represent an ideal system for studying osmotic effects on exocytosis. Stimulation of cytochalasin-B-treated neutrophils with the chemotactic peptide Jlf-formylmethionyl-leucyl-phenyl-alanine (FMLP) results in a rapid compound exocytosis up to 80% of lysosomal enzymes are released within 30 s (9-14). Secretion appears to be triggered by a rise in the level of cytosolic free calcium (15-18) promoted in part by entry of extracellular calcium through receptor-gated channels and in part by release of calcium that is sequestered or bound at some intracellular site (19-21). In this presentation, we augment our previously published data (22,23), which demonstrates that lysosomal enzyme release from neutrophils is inhibited under hyperosmotic conditions and that the rise in cytosolic calcium preceding secretion is inhibited as well. 

Figure 4. Effect of hyperosmolality on lysosomal enzyme release from rabbit neutrophils. Cells were preincubated 10 min at 37 C in either regular HEPES buffer at 320 mosmol/kg ( ) or in HEPES buffer with 0.3-M sucrose at 680 mosmol/kg ( ), 5 Mg/mL cyto-chalasin B was added, cells were stimulated with FMLP, and p-glucuronidase was released into the medium during a 6-min period measured.

Figure 5. Inhibition of lysosomal enzyme release from neutrophils by increased osmotic strength. Cells were preincubated for 10 min at 37°C in regular buffer containing no additions (o), or containing sodium sulfate ( ), sodium HEPES ( ), or sucrose ( ) to increase the osmotic strength. Cells were treated with cyto-chalasin B (5 arid FMLP (10" M) and p-glucuronidase was...

Figure 6. Effect of hyperosmolality on FMLP-induced calcium transients in indo-l-loaded neutrophils. Cells were incubated at 37°C in either regular buffer (A) regular buffer plus 0.15-ilf sodium sulfate, 662 mosmol/kg (B) 0.3-M sodium HEPES, 645 mosmol/kg (C) or 0.45-jy sucrose, 870 mosmol/kg (D). All were treated with 5 M8/ > cytochalasin B and stimulated with 10 M FMLP. Excitation at 340 nm and emission at 400 nm.

Figure 7. Sensitivity of the FMLP-induced calcium signal to removal of extracellular calcium. Indo-l-loaded neutrophils were stimulated with 10 M FMLP in a medium of normal osmolality (320 mosmol/kg) and indo-1 fluorescence was recorded as described in Figure 6. Trace 1 Cells in a medium with normal calcium (1.5 mN). Trace 2 EGTA added to chelate extracellular calcium before stimulation extracellular calcium (1.5 milf) readded 70 s after stimulation. Trace 3 Cells in a medium with normal calcium EGTA added 70 s after stimulation to chelate extracellular calcium.

Confirmation that FMLP-induced activation involves release of intracellular calcium was obtained by loading neutrophils with CTC. Addition of 20 pH CTC to a neutrophil suspension resulted in a gradual increase in CTC fluorescence as the probe entered the cells and partitioned into intracellular membranes (Figure 9, upper panel). Addition of FMLP resulted in an abrupt decrease in fluorescence, suggesting release of calcium from intracellular membranes probed by CTC. The FMLP-induced release of calcium monitored by CTC was little affected by increased medium osmolality a similar fluorescence decrease was seen in the presence of sodium HEPES (645 mosmol/kg) or sodium sulfate (662 mosmol/kg) (Figure 9, lower panel). 

CTC, and CTC fluorescence (excitation 400 nm, emission 430 nm) was monitored versus time at 37°C. Addition of 10 H FMLP at the arrow resulted in an abrupt decrease in CTC fluorescence. Lower panel Increasing medium osmolality by addition of either sodium sulfate (middle trace) or sodium HEPES (right trace) had little effect on the FMLP induced decrease in CTC fluorescence when compared to that seen in medium of normal osmolality (left trace). ... 

Figure 10. Three phases of the FMLP-induced calcium signal in rabbit neutrophils. (Reproduced from ref. 22. Copyright 1987 Elsevier Science Publishers, BV.)...

Stenson, W.F., Mehta, J. and Spilbeig, I. (1984). Sulfiasalazine inhibition of binding of N-formyl-methionyl-leucyl-phenylalanine (FMLP) to its receptor on human neutrophils. Biochem. Pharm. 33, 407-412. 

Follin, P., Briheim, G. and Dahlgren, C. (1991). Mechanisms in neutrophil priming characterisation of the oxidative response induced by fMLP in human exudated cells. Scand. 

BMMC Bone marrow mast cell BOC-FMLP Butoxycarbonyl-FMLP bp Base pair... 

In Malaysia, a paste of the roots is applied to fingers to treat eruptions. The plant is known contain some sesquiterpene lactones including neolinderane, zeylanine, zey-lanicine and zeylanidine (59-61), the potential of which as an inhibitor of NOS would be worth investigation because pseudoneolinderane and linderalactone inhibited the production of superoxide anion generation by human neutrophils in response to fMLP/CB. The IC50 values for pseudoneolinderane and linderalactonewere 3.21 and 8.48 (xg/mL, respectively (62). 

Thus, oxygen radical production by leukocytes can be responsible for cancer development. However, the levels of leukocyte oxygen radical generation depend on the type of cancer. For example, PMNs and monocytes from peripheral blood of patients with lung cancer produced a diminished amount of superoxide [169], Timoshenko et al. [170] observed the reduction of superoxide production in bronchial carcinoma patients after the incubation of neutrophils with concanavalin A or human lectin, while neutrophils from breast cancer patients exhibited no change in their activity. Chemotherapy of lung and colorectal carcinoma patients also reduced neutrophil superoxide production. Human ALL and AML cells produced, as a rule, the diminished amounts of superoxide in response to PMA or FMLP [171], On the other hand total SOD activity was enhanced in AML cells but diminished in ALL cells, while MnSOD in AML cells was very low. It has been proposed that decreased superoxide production may be responsible for susceptibility to infections in cancer patients. 

Another pathway of the initiation of lipid peroxidation is the formation of peroxynitrite from superoxide and nitric oxide. Kausalya and Nath [228] found that the FMLP-stimulated... 

Figure 4.7. Histamine release from mast cells in response to various dilutions of HRA generated from bovine serum albumin (BSA) by medium derived from stimulated rat neutrophils [156]. Neutrophils ((50-100) x K lml) were stimulated with FMLP (10 5 M), the medium removed and incubated with BSA (10 mg I ml) at pH 4.5 for 18 h at 37° C. It was then boiled and centrifuged (11,000 x g for 30 s), the supernatant fraction was removed, its pH was adjusted to 7.2 and it was added, at various dilutions, to suspensions of mast cells. Histamine release was then measured after 10 min. As the amount of generated HRA was increased histamine release increased to a maximum at 57 4%. Mean + S.E.M., n = 5. Inset Time-course of generation of HRA as assayed by histamine release from isolated mast cells. HRA was generated as before using 50 x 106 neutrophils/ml. Aliquots were removed at the indicated times and assayed (at 50% dilution) for HRA. Note that there is a significant generation of HRA by 2 h. Mean S.E.M., n = 3.

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