Regulation of fluoroquinolone uptake by human neutrophils: involvement of mitogen-activated protein kinase

Koichi Hotta1,2, Masayuki Niwa1,3,*, Masao Hirota4, Yutaka Kanamori1, Hiroyuki Matsuno1, Osamu Kozawa1, Takanobu Otsuka2, Nobuo Matsui2 and Toshihiko Uematsu1

1Department of Pharmacology and 3Medical Education Development Center, Gifu University School of Medicine, 40-Tsukasamachi, Gifu 500-8705; 2Department of Orthopedic Surgery, Nagoya City University Medical School, Nagoya 467-8601; 4Department of Drug Metabolism, Drug Safety Center, Tokushima Research Institute, Otsuka Pharmaceutical Co., Ltd, Tokushima 771-0192, Japan

Received 24 September 2001; returned 13 December 2001; revised 28 January 2002; accepted 28 February 2002.


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although human neutrophils actively internalize fluoroquinolones, the precise uptake mechanism is not fully understood. In this study, we investigated the role of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in fluoroquinolone uptake in neutrophils. Spontaneous grepafloxacin uptake was significantly enhanced by SB203580, a p38 MAPK inhibitor, in a dose-dependent manner, but not by PD98059, a specific inhibitor of the upstream kinase that activates p44/42 MAPK. Neither inhibitor affected spontaneous ciprofloxacin or ofloxacin uptake. Phorbol myristate acetate (PMA) treatment enhanced ciprofloxacin uptake, whereas it reduced grepafloxacin uptake. These effects by PMA were significantly inhibited by the pretreatment of neutrophils with GF109203X, a specific inhibitor of PKC. PMA had no effect on ofloxacin uptake. The PMA-induced enhancement of ciprofloxacin uptake was inhibited by PD98059, but not by SB203580. On the other hand, the PMA-induced reduction of grepafloxacin uptake was not inhibited by either MAPK inhibitor. Grepafloxacin, but not ciprofloxacin or ofloxacin, strongly phosphorylated p38 MAPK. This phosphorylation of p38 MAPK was not inhibited by GF109203X pretreatment. None of these three fluoroquinolones phosphorylated p44/42 MAPK. PMA phosphorylated both p38 and p44/42 MAPK. These findings indicate that grepafloxacin negatively regulates its uptake in neutrophils, and p38 MAPK activation is involved in this down-regulation of grepafloxacin uptake. Ciprofloxacin uptake is positively regulated by the activation of PKC, and p44/42 MAPK activation is involved in this up-regulation. Neither PKC, p38 nor p44/42 MAPK is involved in the regulation of ofloxacin uptake.

Keywords: fluoroquinolone, human neutrophils, MAPK, PKC


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antimicrobial agents that accumulate and remain active inside phagocytic cells are particularly useful for treating infections by intracellular bacteria. Neutrophils usually play crucial roles in host defence mechanisms, including the phagocytosis of bacteria. Many fluoroquinolones have been reported to accumulate in human neutrophils. This process enhances the killing of intracellular pathogens and could facilitate the delivery of these agents to infection sites by migrating neutrophils. The cellular-to-extracellular concentration (C/E) ratios of several fluoroquinolones have been reported previously.1,2 Grepafloxacin is a fluoroquinolone that possesses antimicrobial activity towards a broad spectrum of bacteria, and has one of the highest C/E ratios.3,4 However, the precise mechanism determining the uptake of fluoroquinolones into phagocytic cells is not yet fully elucidated.

Recently, Loo et al.5 reported that protein kinase C (PKC) activation by phorbol myristate acetate (PMA) enhances the uptake of ciprofloxacin by human neutrophils. It may be logical to hypothesize that fluoroquinolone uptake is enhanced as a consequence of human neutrophil priming or activation of host defences.6 It has also been reported that mitogen-activated protein kinase (MAPK) is involved in human neutrophil activation by various stimuli, such as tumour necrosis factor, colony simulating factor, N-formyl-methionyl-leucyl-phenylalanine and PMA. Loo et al.5 also suggested that p44/42 MAPK is involved in a PMA-induced ciprofloxacin uptake enhancement effect in human neutrophils. However, the precise mechanism of this process has not yet been fully clarified, and it is not known whether other fluoroquinolones are also regulated by PKC and/or MAPK systems.

In this report, to evaluate whether PKC activation is involved in fluoroquinolone uptake by human neutrophils, we tested the effect of PMA stimulation and/or GF109203X,7 a specific PKC inhibitor, pretreatment on the uptake of grepafloxacin, ciprofloxacin and ofloxacin. Furthermore, we also examined the contribution of MAPK systems to the uptake of these fluoroquinolones in human neutrophils.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals and reagents

Grepafloxacin and 1-cyclopropyl-6,8-difluoro-1,4-dihydro-5-ethyl-7-(4-methyl-1-piperazinyl)-4-oxo-3-quinoline carboxylic acid (OPC-17203, used as an internal standard for the measurement of fluoroquinolones) were provided by Otsuka Pharmaceutical Co., Ltd (Tokyo, Japan). Ciprofloxacin was provided by Bayer Japan Pharmaceutical Co., Ltd (Tokyo, Japan). Ofloxacin and PMA were purchased from Sigma (St Louis, MO, USA). Dextran (mol. wt 208 000) and HEPES were purchased from Nacalai (Kyoto, Japan) and DOJIN (Kumamoto, Japan), respectively. SB203580 was obtained from SmithKline Beecham Pharmaceuticals (King of Prussia, PA, USA). PD98059 was obtained from Calbiochem-Novabiochem (La Jolla, CA, USA). GF109203X was purchased from BioMol (Plymouth Meeting, PA, USA). Rabbit polyclonal antibodies against Thr-180/Tyr-182-phosphorylated p38 MAPK, p38, Thr-202/Tyr-204-phosphorylated p44/42 and p44/42 were purchased from Cell Signalling Technology (Beverly, MA, USA). The enhanced chemiluminescence (ECL) western blotting system was purchased from Amersham Pharmacia Biotech (Tokyo, Japan). SB203580, PD98059 and GF109203X were dissolved in dimethylsulphoxide (DMSO). The maximum concentration of DMSO was 0.1%, which did not affect the measurement of p38 or p44/42 MAPK activity, or the assay for fluoroquinolone uptake. All reagents used were endotoxin free, as determined by the limulus lysate assay, in which minimum detectable levels were 0.03 enzyme unit/mL.

Preparation of human neutrophils

Human neutrophils from healthy donors were isolated as previously described8 with minor changes.9 Briefly, venous blood was collected in sodium citrate solution (3.8%), centrifuged (110g, 10 min), and platelet-rich plasma was discarded. The remaining part of the blood was mixed (1:1, v/v) with a solution of 3% dextran in 0.9% sodium chloride solution in a plastic syringe and fixed vertically for 20 min at 25°C. Neutrophil-rich plasma was collected from the upper layer of the suspension and centrifuged (250g, 10 min). The pellet was subjected to hypotonic lysis to destroy the remaining erythrocytes, centrifuged and then suspended in HBSS (Hanks Balanced Salt Solution containing 10 mM HEPES, pH 7.4). The suspension was cushioned carefully on Histopaque solution (d = 1.077) and centrifuged (420g, 30 min) at 20°C. The purified neutrophil pellet was finally resuspended in HBSS. Cell number was counted by a Coulter counter model Z1 (Coulter Electronics Ltd, Bedfordshire, UK), and diluted in HBSS to the final required concentrations and kept on ice until examined. The purity of neutrophils was >95%. The viability of neutrophils used was >95%, as evaluated by the trypan blue exclusion test. Informed consent was obtained from all donors.

Determination of fluoroquinolones in human neutrophils

The previously described high performance liquid chromatography (HPLC)-fluorometric assay10 was used to measure fluoroquinolone uptake by human neutrophils. Human neutrophils (5 x 106 cells/mL) were pretreated with or without various inhibitors in HBSS for 10 min and were then incubated for 1–60 min with different concentrations of fluoroquinolones in the presence or absence of PMA (0–100 nM). After incubation, cells were separated from extracellular solution by centrifugation (10 000g, 3 min) through a water-impermeable silicon–oil barrier (SH550:SH556/1:4; Toray Dow Corning Co. Ltd, Tokyo, Japan) in a microcentrifuge tube. The human neutrophil pellet formed on the bottom of the microcentrifuge tube was obtained by cutting off this portion of the microcentrifuge tube and resuspending it with methanol by agitating vigorously on a vortex shaker. These samples were then centrifuged at 21 600g for 10 min, and the concentration of fluoroquinolones in the supernatant was determined by HPLC with a spectrofluorimeter (Shimadzu, Kyoto, Japan). The fluorescence excitation and emission maxima of fluoroquinolones in methanol were 285 and 448 nm, respectively.

The intracellular concentration of fluoroquinolones was expressed as pmol per 106 neutrophils. A previously determined intracellular volume of 3.3 x 10–13 L was used to determine the C/E ratios of fluoroquinolones.11

Western blotting

Human neutrophils (5 x 106 cells/mL) suspended in HBSS were pretreated with or without various inhibitors for 10 min at 37°C and were then stimulated with grepafloxacin (0– 200 mg/L), ciprofloxacin (0–200 mg/L), ofloxacin (0–50 mg/L) or PMA (0–100 nM) for the indicated period up to 20 min. The reactions were terminated by 1:10 dilution with cold HBSS and centrifugation at 250g for 10 min at 4°C. The cell pellets were treated as previously described.12 In short, they were resuspended in ice-cold solution containing 50 mM HEPES (pH 7.4), 1% (v/v) Triton X-100, 2 mM sodium orthovanadate, 100 mM sodium fluoride, 1 mM EDTA, 1 mM phenylmethylsulphonyl fluoride, 100 mg/L aprotinin and 10 mg/L leupeptin, and were lysed for 60 min at 4°C. After centrifugation, the supernatant was mixed 1:1 (v/v) with 2x sample buffer [4% (w/v) sodium dodecyl sulphate (SDS), 20% (v/v) glycerol, 10% (v/v) mercaptoethanol and a trace amount of bromophenol blue dye in 125 mM Tris–HCl, pH 6.8], boiled for 5 min and then frozen at –30°C until used. Samples were subjected to SDS–PAGE [10% (w/v)] at 20 mA for 90 min. After electrophoresis, proteins were electrophoretically transferred from the gel on to 0.2 µm polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA) at 10 V for 30 min. Blots were blocked with 5% (w/v) skimmed milk in Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBST) overnight at 4°C. The next day, blots were washed three times with TBST for 10 min, and were then incubated with primary antibodies against phospho-p38, p38, phospho-p44/42 and p44/42 overnight at 4°C. Antibodies were used at a dilution of 1:1000 in 5% (w/v) bovine serum albumin (fraction V) in TBST. Blots were washed with TBST three times and were incubated for 1 h with the secondary antibody goat anti-rabbit IgG-peroxidase (Chemicon International Inc., Temecula, CA, USA) at a dilution of 1:1000 in 5% (v/v) skimmed milk in TBST. Blots were subsequently washed three times with TBST and were visualized by the ECL detection system, as directed by the manufacturer.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Uptake of fluoroquinolones by human neutrophils

The time profiles of the uptake of the fluoroquinolones grepafloxacin (50 and 200 mg/L), ciprofloxacin (50 and 200 mg/L) and ofloxacin (50 mg/L) by human neutrophils were investigated. We selected these concentrations based on the results of a previous report.4 Grepafloxacin was rapidly taken up by human neutrophils, reaching a maximum at c. 5 min (Fig- ure 1a). We have previously reported4 that the Km value of grepafloxacin uptake by human neutrophils was c. 170 µM, calculated using the Eadie–Hofstee plot. Similar to grepafloxacin, ciprofloxacin (50 and 200 mg/L) was also rapidly taken up by human neutrophils, and reached a maximum at c. 20 min (Figure 1b). Ofloxacin was also rapidly taken up by human neutrophils, and reached a maximum at c. 5 min (data not shown). The absolute amounts and C/E ratios of ciprofloxacin and ofloxacin were <1/10 less than that of grepafloxacin.



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Figure 1. Time profiles of uptake of fluoroquinolones by human neutrophils. (a) Grepafloxacin (filled circles, 50 mg/L; empty circles, 200 mg/L) and (b) ciprofloxacin (filled squares, 50 mg/L; empty squares, 200 mg/L). Uptake abilities are shown as the C/E ratio of fluoroquinolones, measured as described in Materials and methods. Results are expressed as means ± S.D. of three to five experiments run in duplicate.

 
Effect of PMA on fluoroquinolone uptake

The dose-dependent effect of PMA (0–100 nM) on the uptake of grepafloxacin (200 mg/L), ciprofloxacin (200 mg/L) and ofloxacin (50 mg/L) was investigated. PMA significantly reduced grepafloxacin uptake in a concentration-dependent manner for 20 min (Figure 2a). On the other hand, PMA significantly enhanced ciprofloxacin uptake (Figure 2b) and had no effect on ofloxacin uptake (data not shown).



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Figure 2. Effect of PMA and MAPK inhibitors on the uptake of (a) grepafloxacin (200 mg/L) and (b) ciprofloxacin (200 mg/L). Human neutrophils (5 x 106 cells/L) were pretreated with SB203580 (10 µM), PD98059 (50 µM) or vehicle only for 10 min. Fluoroquinolones and PMA (0–100 nM) were then concomitantly treated for 20 min. Fluoroquinolone uptake was measured as described in Materials and methods. Open columns, vehicle pretreated human neutrophils; hatched columns, SB203580 (10 µM) pretreated human neutrophils; closed columns, PD98059 (50 µM) pretreated human neutrophils. Results are expressed as means ± S.D. of three to five experiments run in duplicate. *P < 0.05 compared with no PMA-stimulated human neutrophils. **P < 0.05 compared with no inhibitor pretreated human neutrophils in each dose of PMA-stimulated ones.

 
Effect of SB203580 and PD98059 on fluoroquinolone uptake

The effect of MAPK inhibitors on the uptake of grepafloxacin (200 mg/L), ciprofloxacin (200 mg/L) and ofloxacin (50 mg/L) in the presence or absence of PMA (0–100 nM) was investigated. Human neutrophils (5 x 106 cells/L) were pretreated for 10 min with SB203580 (10 µM), a p38 MAPK inhibitor, PD98059 (50 µM), a specific inhibitor of the upstream kinase that activates p44/42 kinase or vehicle alone, and were then incubated with fluoroquinolones in the presence or absence of PMA for 20 min. SB203580 significantly enhanced spontaneous grepafloxacin uptake (Figure 2a). This enhanced effect of SB203580 reached 135% of the control value (Figure 3). PMA-induced inhibition of grepafloxacin uptake was not affected by SB203580 and PD98059 (Figure 2a).



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Figure 3. Dose-dependent effect of MAPK inhibitors on the uptake of grepafloxacin (200 mg/L). Human neutrophils were pretreated with various doses of SB203580 (filled circles) and PD98059 (empty circles) for 10 min, then grepafloxacin uptake for 20 min was measured as described in Materials and methods. Results are expressed as means ± S.D. of three to five experiments run in duplicate. *P < 0.05 compared with control.

 
On the other hand, spontaneous ciprofloxacin uptake was not affected by SB203580 and PD98059, although PMA-induced enhancement of ciprofloxacin uptake was abolished by PD98059, but not by SB203580 (Figure 2b). Neither SB203580 nor PD98059 affected ofloxacin uptake in the presence or absence of PMA (data not shown).

Effect of GF109203X on fluoroquinolone uptake

Human neutrophils (5 x 106 cells/L) were pretreated with GF109203X (1 µM) or vehicle alone for 10 min, and were then incubated with fluoroquinolones (grepafloxacin and ciprofloxacin, 200 mg/L; ofloxacin, 50 mg/L) for 20 min in the presence or absence of PMA (100 nM). Although GF109203X did not affect grepafloxacin, ciprofloxacin or ofloxacin uptake in the absence of PMA stimulation, it significantly inhibited the PMA-induced reduction of grepafloxacin uptake and abolished the PMA-induced enhancement of ciprofloxacin uptake (Figure 4).



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Figure 4. Effect of GF109203X (1 µM) on the uptake of (a) grepafloxacin (200 mg/L) and (b) ciprofloxacin (200 mg/L) in PMA- (100 nM) or vehicle-stimulated human neutrophils. Human neutrophils were pretreated with GF109203X for 10 min, and were then incubated with fluoroquinolones and PMA for 20 min. Fluoroquinolone uptake was measured as described in Materials and methods. Open columns, vehicle-treated human neutrophils; closed columns, GF109203X-pretreated human neutrophils. Results are expressed as means ± S.D. of three to five experiments run in duplicate. *P < 0.05 compared with PMA control human neutrophils. **P < 0.05 compared with vehicle-pretreated cells in each dose of PMA-stimulated human neutrophils.

 
Phosphorylation of p38 MAPK

When human neutrophils (5 x 106 cells/L) were stimulated with 100 nM PMA for 3 min or 10 nM PMA for 5 min, significant increases in phosphorylation of p38 MAPK were detected (Figure 5a). Grepafloxacin-induced phosphorylation of p38 MAPK was rapidly detected 1 min after stimulation and was dependent on the concentration used as the stimulus (0–200 mg/L). Maximal stimulation was observed at 100 mg/L (Figure 5b). p38 MAPK was not phosphorylated by ciprofloxacin (200 mg/L) or ofloxacin (50 mg/L) (data not shown).



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Figure 5. Phosphorylation of p38 MAPK in human neutrophils stimulated by (a) PMA (0–100 nM) for indicated periods and (b) grepafloxacin (0–200 mg/L) for 1 min at 37°C. Phosphorylation of p38 MAPK was analysed by immunoblotting analysis using antibody against the phosphorylated form and the total of each protein as described in Materials and methods. The results shown are representative of three independent experiments.

 
Phosphorylation of p44/42 MAPK

When human neutrophils (5 x 106 cells/L) were stimulated with 100 nM PMA, weak phosphorylation of p44/42 MAPK was detected as early as 1 min and phosphorylation increased up to 5 min. PMA (10 nM) caused weak phosphorylation of p44/42 MAPK at 3 min and maximal phosphorylation was observed at 5 min (data not shown). Grepafloxacin (200 mg/L), ciprofloxacin (200 mg/L) and ofloxacin (50 mg/L) did not phosphorylate p44/42 MAPK up to 20 min (data not shown).

Effect of GF109203X on phosphorylation of p38 MAPK

Human neutrophils (5 x 106 cells/L) were pretreated for 10 min with GF109203X (1 µM) or vehicle only, and were then stimulated with PMA (10 nM) or grepafloxacin (100 mg/L) for 5 or 1 min, respectively. GF109203X inhibited PMA-induced phosphorylation of p38 MAPK (Figure 6a). On the other hand, GF109203X did not show any effect on grepafloxacin-induced p38 MAPK phosphorylation (Figure 6b).



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Figure 6. Effects of GF109203X (1 µM) on the phosphorylation of p38 MAPK in human neutrophils. (a) PMA (10 nM)-induced and (b) grepafloxacin (100 mg/L)-induced p38 MAPK phosphorylation. Human neutrophils were pretreated with GF109203X or vehicle only for 10 min, and were then stimulated by PMA or grepafloxacin for 5 or 1 min, respectively. Phosphorylation of p38 MAPK was analysed by immunoblotting using antibody against the phosphorylated form and the total of each protein as described in Materials and methods. The results shown are representative of three independent experiments.

 

    Discussion
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 Abstract
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 Materials and methods
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 Discussion
 References
 
The uptake of fluoroquinolones by neutrophils is a recently defined pharmacokinetic parameter that is increasing in importance in clinical practice, especially for immunocompromised patients whose neutrophils have their bactericidal systems impaired, or in infections due to bacteria that are able to survive in the phagocytes. As a consequence of active internalization, human neutrophils can accumulate intracellular fluoroquinolone to levels much higher than the extracellular concentration. Taira et al.3 have shown C/E ratios of 66.2 for grepafloxacin, 9.8 for levofloxacin and 7.6 for ofloxacin, and suggested that some active transport system is involved in the mechanism for these high cellular uptakes. However, the specific mechanism by which human neutrophils accumulate fluoroquinolones has not yet been clearly defined.

It has been reported that chelerythrine, a selective inhibitor of the catalytic domain of PKC,13 and H7, a widely used PKC antagonist, both inhibit the activation of ciprofloxacin uptake by PMA.5 These results indicate that PKC activation might be involved in the uptake not only of ciprofloxacin but also of other fluoroquinolones. Therefore, we compared the effects of PMA on the uptake of grepafloxacin, ciprofloxacin and ofloxacin in human neutrophils. While PMA increased the uptake of ciprofloxacin dose dependently, it decreased grepafloxacin uptake. Uptake of ofloxacin was not affected by PMA stimulation. Furthermore, GF109203X inhibited both the activation of ciprofloxacin uptake by PMA and the reduction of grepafloxacin uptake by PMA. These results indicate that uptake mechanisms of fluoroquinolones are varied, and that activation of PKC positively regulates ciprofloxacin uptake and negatively regulates grepafloxacin uptake. It seems likely that PKC activation is not involved in ofloxacin uptake.

Recent evidence indicates that MAPK is downstream of PKC activation in several cells, including human neutrophils. So, we determined whether the MAPK system is involved in fluoroquinolone uptake. PMA-induced increase of ciprofloxacin uptake was completely blocked by PD98059, but not by SB203580. PD98059 had no effect on spontaneous ciprofloxacin uptake in human neutrophils. Furthermore, PMA phosphorylated p38 and p44/42 MAPK, which were inhibited by pretreatment with GF109203X, although ciprofloxacin did not phosphorylate both kinases. These results strongly suggest that p44/42 MAPK positively regulates ciprofloxacin uptake when p44/42 MAPK is activated by PKC, and also suggest that p44/42 MAPK is downstream of PKC in the regulation of ciprofloxacin uptake (Figure 7b).



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Figure 7. Schematic diagram of MAPK-regulated (a) grepafloxacin uptake and (b) ciprofloxacin uptake in human neutrophils.

 
In the case of grepafloxacin uptake, SB203580, but not PD98059, significantly and dose dependently enhanced spontaneous grepafloxacin uptake, without PMA stimulation. Furthermore, grepafloxacin treatment produced phosphorylation of p38 MAPK, but not that of p44/42 MAPK. Of note, PMA reduced grepafloxacin uptake in a concentration-dependent manner. This PMA effect was not inhibited by treatment with either SB203580 or PD98059, but it was significantly inhibited by GF109203X. These results taken together indicate that activation of p38 MAPK negatively regulates spontaneous grepafloxacin uptake, and that grepafloxacin by itself activates a negative feedback system to affect grepafloxacin uptake through p38 MAPK activation. These data, however, do not conclusively prove that phosphorylation of p38 MAPK is the cause of the decreased uptake of grepafloxacin. We have recently reported that grepafloxacin uptake is mediated, at least in part, by an active transport system.4 So, it is likely that this transport system is regulated by the activation of the p38 MAPK cascade. Furthermore, PKC activation may also negatively regulate grepafloxacin uptake, independently of the p38 MAPK system (Figure 7a). In addition, PKC activation is not involved in grepafloxacin-induced p38 MAPK activation, since GF109203X did not show a significant effect on the spontaneous grepafloxacin uptake. These data are supported by the finding that grepafloxacin-induced phosphorylation of p38 MAPK was not inhibited by the pretreatment with GF109203X.

Neither PMA nor SB203580 or PD98059 affected ofloxacin uptake. Phosphorylation of neither p38 MAPK nor p44/42 MAPK was observed in ofloxacin-stimulated human neutrophils. This suggests that neither MAPK nor PKC is involved in the regulation of ofloxacin uptake by human neutrophils.

Our results indicate that human neutrophils activated by PKC or p38 MAPK reduce their grepafloxacin uptake. Although a physiological role is not clear, it seems likely that p38 MAPK-activated human neutrophils down-regulate grepafloxacin uptake, resulting in a decrease in intracellular concentrations of grepafloxacin. Furthermore, grepafloxacin uptake is negatively self-regulated via activation of p38 MAPK. These interesting observations may explain the extremely high penetrating ability of grepafloxacin into human neutrophils.

This is the first report showing that p38 MAPK is involved in the down-regulation of grepafloxacin uptake and that this is the key difference between other fluoroquinolones such as ciprofloxacin, whose uptake involves p44/42 MAPK, and ofloxacin, whose uptake does not involve either p38 MAPK or p44/42 MAPK. The mechanisms underlying the cellular uptake of fluoroquinolones are not yet fully understood, but appear to involve both PKC and MAPK, and it is likely that more than one mechanism is involved. While further studies will be needed to understand the basis for the differences in cellular uptake of fluoroquinolones (e.g. because of differences in chemical structure), the present study provides a better understanding of how fluoroquinolone uptake is influenced by the PKC and MAPK cascades.


    Acknowledgements
 
This work was supported in part by a grant-in-aid for Scientific Research (No. 12470015 and No. 13670084) from the Ministry of Education, Science, Sports and Culture of Japan.


    Footnotes
 
* Corresponding author. Tel: +81-58-267-2233; Fax: +81-58-267-2959; E-mail: mniwa{at}cc.gifu-u.ac.jp Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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