Propofol inhibits FMLP-stimulated phosphorylation of p42 mitogen-activated protein kinase and chemotaxis in human neutrophils  T. Nagata, M. Kansha, K. Irita, S. Takahashi  British Journal of Anaesthesia  Volume 86, Issue 6, Pages 853-858 (June 2001) DOI: 10.1093/bja/86.6.853 Copyright © 2001 British Journal of Anaesthesia Terms and Conditions

Fig 1 Possible signal transduction pathway via MAPK in neutrophils. The binding of FMLP to its receptor on the plasma membrane activates the Ras–Raf–MEK–p44/42 MAPK pathway through heterotrimeric GTP binding proteins. MEK is a specific activator of p44/42 MAPK. PD98059 selectively inhibits MEK. This signalling cascade is considered to participate in the activation of various functions of neutrophils. The participation of other MAPK families, such as p38, has been reported recently. In various cells, the activated p44/42 MAPK activates transcription factors by protein phosphorylation, and thus leads to gene expression. British Journal of Anaesthesia 2001 86, 853-858DOI: (10.1093/bja/86.6.853) Copyright © 2001 British Journal of Anaesthesia Terms and Conditions

Fig 2 (a) Schematic representation of the under-agarose method. Four series of three wells, measuring 3 mm in diameter and spaced 6 mm apart, were cut into 60×15 mm agarose plates. The centre well received 10 µl of the prepared cell suspension. The outer wells received 10 µl FMLP dissolved in MEM and the inner wells received 10 µl MEM. (b) Chemotaxis pattern of neutrophils. The number of neutrophils was counted in 1×1 mm square areas, which were 2 mm from the edges of the neutrophil wells. British Journal of Anaesthesia 2001 86, 853-858DOI: (10.1093/bja/86.6.853) Copyright © 2001 British Journal of Anaesthesia Terms and Conditions

Fig 3 Time course of phosphorylation of p44/42 MAPK of human neutrophils. The cells were stimulated with 5 µg ml–1 cytochalasin B and 100 nM FMLP, as described in Materials and methods. The arrow indicates the bands of phosphorylated p42 MAPK. The band of phosphorylated p44 MAPK is not shown in this blot. Phosphorylation of p42 MAPK reached a maximum 1 min after FMLP stimulation. British Journal of Anaesthesia 2001 86, 853-858DOI: (10.1093/bja/86.6.853) Copyright © 2001 British Journal of Anaesthesia Terms and Conditions

Fig 4 (a) Effect of propofol on p42 MAPK phosphorylation. A typical blot is shown. Cells were stimulated with 5 µg ml–1 cytochalasin B and 100 nM FMLP, as described in Materials and methods. The protein blots were probed with phospho-p44/42 MAP kinase antibody or anti-ERK1 antibody on the same samples. The arrows indicate the bands of phosphorylated p42 MAPK and p44/42 MAPK. nc=negative control sample that was stimulated only with cytochalasin B. (b) Densitometric analysis of p42 MAPK phosphorylation. Phosphorylation is expressed relative to the phosphorylation produced by 5 µg ml–1 cytochalasin B and 100 nM FMLP without pretreatment (positive control). Means (sem) *P<0.001 vs positive control; P<0.01 was considered to indicate significance. Negative control, FMLP 0 µM. British Journal of Anaesthesia 2001 86, 853-858DOI: (10.1093/bja/86.6.853) Copyright © 2001 British Journal of Anaesthesia Terms and Conditions

Fig 4 (a) Effect of propofol on p42 MAPK phosphorylation. A typical blot is shown. Cells were stimulated with 5 µg ml–1 cytochalasin B and 100 nM FMLP, as described in Materials and methods. The protein blots were probed with phospho-p44/42 MAP kinase antibody or anti-ERK1 antibody on the same samples. The arrows indicate the bands of phosphorylated p42 MAPK and p44/42 MAPK. nc=negative control sample that was stimulated only with cytochalasin B. (b) Densitometric analysis of p42 MAPK phosphorylation. Phosphorylation is expressed relative to the phosphorylation produced by 5 µg ml–1 cytochalasin B and 100 nM FMLP without pretreatment (positive control). Means (sem) *P<0.001 vs positive control; P<0.01 was considered to indicate significance. Negative control, FMLP 0 µM. British Journal of Anaesthesia 2001 86, 853-858DOI: (10.1093/bja/86.6.853) Copyright © 2001 British Journal of Anaesthesia Terms and Conditions

Fig 5 Effect of propofol and PD98059 on neutrophil chemotaxis. The under-agarose method was used to study chemotaxis as described in Materials and methods. Chemotaxis is expressed as the percentage of chemotaxis caused by 1 µM FMLP without pretreatment (positive control). Mean (sem); *P<0.0001 vs positive control; P<0.01 was considered to indicate significance. British Journal of Anaesthesia 2001 86, 853-858DOI: (10.1093/bja/86.6.853) Copyright © 2001 British Journal of Anaesthesia Terms and Conditions