Electrochemical Stimulation of the Hexose Monophosphate Shunt

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Electrochemical Stimulation of the Hexose Monophosphate Shunt Proc. Nati. Acad. Sci. USA Vol. 77, No. pp. 414-417, January 1980 Cell Biology Induction of plasminogen activator secretion in macrophages by electrochemical stimulation of the hexose monophosphate shunt with methylene blue (nitro blue tetrazolium/methyl viologen/phagocytosis) JORG SCHNYDER AND MARCO BAGGIOLINI Wander Research Institute, Wander Ltd., P.O. Box 2747, CH-3001 Berne, Switzerland Communicated by Zanvil A. Cohn, October 5, 1979 ABSTRACT Resident peritoneal macrophages were ob- centrifugation, resuspended in Pi/NaCl at a concentration of tained from untreated mice and were cultured in medium 199 50 mg/ml, and autoclaved at 120°C (5). Latex beads, 1.01 ,um with or without 5% acid-treated fetal bovine serum. Three hours after harvesting, redox compounds-i.e., methylene blue, methyl (Serva, Heidelberg, W. Germany), were suspended in Pi/NaCl viologen, or nitro blue tetrazolium-were added to the cultures containing 0.01% Tween 80, washed three times by centrifu- of adherent cells. After 1 hr, the cells were washed and culturing gation, resuspended in Pi/NaCI/Tween 80, and sterilized by was continued in the absence of redox compounds. The effects UV irradiation (6). Sheep erythrocytes were washed five times of the redox compounds were tested by assaying for hexose in Pi/NaCl by centrifugation, resuspended in Pi/NaCl con- monophosphate (HMP) shunt activity and for plasminogen ac- taining 3% (wt/vol) formaldehyde, kept for 2 days at 4°C in tivator secretion, and the results were compared with the effects induced by phagocytic stimuli. Methylene blue caused a con- this medium, and then washed five more times in Pi/NaCJ (7). centration-dependent stimulation of the HMP shunt, whereas Phagocytosis experiments were performed exactly as described methyl viologen and nitro blue tetrazolium were ineffective. (1). Briefly, particles were suspended in a small volume of Shunt stimulation by methylene blue was followed, after a lag medium and were added to freshly established cultures of ad- of 2-4 days, by plasminogen activator secretion. The rate of herent cells. Phagocytosis was stopped after 1 hr by aspirating secretion was dependent on the methylene blue concentration the medium and washing three times with Culturing used. Methyl viologen and nitro blue tetrazolium were again Pi/NaCI. ineffective, whereas phagocytosis of zymosan or sheep eryth- was then continued in fresh medium for up to 10 days. rocytes, which stimulates the HMP shunt, induced plasminogen Pyridine Nucleotide Oxidation. The oxidation of NADH activator secretion at rates similar to those induced by meth- and NADPH was measured spectrophotometrically at 37°C. ylene blue. These results add further evidence to our hypothesis The reaction mixture consisted of 500 Al of 0.1 M sodium that the HMP shunt-dependent metabolic burst is involved in phosphate/HCI buffer at pH 7.0, 200 Ml of an aqueous solution macrophage activation. Because methylene blue mimics the of the redox 100 Ml of a 0.8 mM solution of NADH action of zymosan it appears that shunt stimulation by itself dye, and or initiates the activation process independently of phagocy- NADPH. Decrease in absorbance at 334 nm was recorded with tosis. an Eppendorf photometer (Eppendorf, Hamburg, W. Ger- many) equipped with a W+W linear-logarithmic recorder We have recently reported that mouse peritoneal macrophages (W+W Electronic, Munchenstein, Switzerland). become activated in culture after phagocytosis of zymosan, as HMP Shunt Assay. HMP shunt activity was always mea- indicated by the induction of plasminogen activator secretion. sured at the onset of culture by determining the amount of By testing the effects of various types of phagocytosable par- 14CO2 produced from [1-14C]glucose during 1 hr in the pres- ticles, we obtained evidence suggesting that the stimulus for ence of the agent to be tested as a possible stimulus (1). In some macrophage activation is related to the burst of hexose mono- experiments, the same technique was used on a reduced scale: phosphate (HMP) shunt activity rather than to the phagocytic 2.5 X 106 cells were plated on 33-mm dishes (Falcon Plastics, uptake per se or the intracellular fate of the particles (1). Cockeysville, MD) and nonadherent cells were washed off 3 We have now studied the effects of HMP shunt stimulation hr later. The cultures were then supplied with 1.5 ml of fresh induced by methylene blue instead of phagocytosis and have medium containing labeled glucose (1 uCi per dish; 1 Ci = 3.7 found that it equally leads to macrophage activation. As in our X 1010 becquerels) and the redox compound or other agents to earlier studies (1, 2), macrophage activation was assessed by be tested. After 1 hr, the 14CO2 released was determined (1). testing for plasminogen activator secretion (3, 4). In contrast to our former reports, results are expressed as the total amounts of CO2 formed. For this purpose, 14CO2 values MATERIALS AND METHODS were corrected for the amount escaping during incubation in Macrophage Cultures. Macrophages obtained from un- the culture dishes, which was determined to be 55.8 b 1.5 treated male OF, mice (Sandoz, Basel, Switzerland) weighing percent (mean d SEM) of the total 14CO2 that is recovered 20-24 g were cultured in medium 199 with or without 5% when the same reaction mixture is tested in gas-tight ves- acid-treated fetal bovine serum according to established tech- sels-i.e., Warburg-type flasks. The medium containing the niques (2). redox compounds was filtered (Millipore. 0.22 um pore size) Phagocytosis. Zymosan (Koch-Light Laboratories, Coln- before use. All these experiments were performed in the dark brook, Buckinghamshire, England) was boiled in phosphate- (Forma CO2 incubator, model 3156, Forma Scientific, Marietta, buffered saline (Pi/NaCI) for 1 hr, washed three times by OH). Chemicals. The following reagents were used: f3-NADPH The publication costs of this article were defrayed in part by page tetrasodium salt and f3-NADH disodium salt (Boehringer charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviations: HMP, hexose monophosphate; Pi/NaCl, phosphate- this fact. buffered saline. 414 Downloaded by guest on September 26, 2021 Cell Biology: Schnyder and Baggiolini Proc. Natl. Acad. Sci. USA 77 (1980) 415 Table 1. Oxidation of pyridine nucleotides by redox compounds Table 3. HMP shunt stimulation in nonelicited mouse peritoneal Concentration (MM) macrophages by methylene blue and by phagocytosis at which pyridine CO2 liberation from glucoset nucleotides are oxidized at Stimulus* [1-14C]- [6-14C]- E0* 1 Mmol/min at pH 7 Compound V NADH NADPH None 1.30 + 0.10 (23) 0.47 ± 0.02 (9) Methylene blue (0.1 mM) 12.75 + 0.51 (11) 1.64 + 0.19 (9) Methyl viologen -0.45 >250 >250 Zymosan 17.99 i 1.23 (12) 0.94 ± 0.01 (6) NADPH -0.32 Sheep erythrocytes 12.82 ± 1.48 (6) 0.77 + 0.02 (6) >25 Nitro blue tetrazolium 0 >25 * Methylene blue or particles were added to the cultures for 1 hr and Methylene blue 0.01 6.3 7.0 the 14CO2 formed during this time from labeled glucose was deter- * E' is the standard redox potential at pH 7. mined. Particle-to-cell ratios were 8:1 for zymosan and 5:1 for sheep erythrocytes. t Nanomoles of CO2 per 106 cells; mean values + SEM; in parentheses, number of assays. Mannheim); methyl viologen, pure analytical reagent and nitro blue tetrazolium chloride, pure analytical reagent (Koch-Light after phagocytosis of particles that stimulate the HMP shunt Laboratories); methylene blue B and H202 (E. Merck, Darm- (1). We therefore tested the effect of methylene blue under stadt, W. Germany); D-[1-14C]glucose (New England Nuclear); similar experimental conditions. Nonelicited mouse peritoneal D-[6-14C]glucose (The Radiochemical Centre, Amersham, macrophages were put in culture and the adherent cells were England); catalase from bovine liver, type V glucose oxidase challenged 3 hr later with either zymosan or 10MM methylene from Aspergillus niger, and type I superoxide dismutase from blue for 1 hr. Both treatments resulted in a marked increase in bovine blood (Sigma). Reagents used for culturing macrophages plasminogen activator secretion as compared to the control (Fig. were described in a previous paper (2). 1). As in earlier experiments, secretion occurred after a lag period (which in this case was somewhat shorter than usual). RESULTS Fig. 2 shows that the rate of plasminogen activator secretion Pyridine nucleotide oxidation and stimulation of the depended on the methylene blue concentration to which the HMP shunt by methylene blue cells were exposed. At the two higher concentrations (100 and Pyridine nucleotides were readily oxidized by methylene blue, 300 ,uM), the methylene blue pulse induced levels of plasmin- but not by two other redox compounds that we have studied, ogen activator secretion similar to those for zymosan phago- methyl viologen and nitro blue tetrazolium (Table 1). In ac- cytosis (see Fig. 1 and Table 5). The experiments presented in cordance with these results, methylene blue also stimulated the Fig. 1 were performed in the absence and those presented in HMP shunt of macrophages in culture in a concentration- Fig. 2 in the presence of serum. Methylene blue was ictive dependent manner, presumably by reoxidizing the NADPH under both conditions. When serum was present, however, formed, whereas both methyl viologen and nitro blue tetrazo- higher concentrations were needed, presumably because the lium were ineffective (Table 2). As shown in Table 3, the dye is partially trapped by binding to serum proteins. Plas- stimulatory effect of methylene blue compared well with that minogen activator secretion was also induced when methylene of phagocytosis. An approximately 10-fold enhancement of blue was supplied to the media during the entire culture period 4CO2 formation from 1-14C-labeled glucose was obtained with (Fig.
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