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© Copyright 2004 The British Pharmacological Society

139P GKT, University of London
Winter Meeting December 2003

Histamine-induced superoxide production by equine eosinophils is map kinase-dependent


C.F. Titterton, M. Andrews, N.T. Goode & F.M. Cunningham, Veterinary Basic Sciences, The Royal Veterinary College, London, UK.

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Nasra J
Winny C
Nichols A
Butler K

Equine eosinophils produce superoxide anions when stimulated with histamine (Foster & Cunningham, 1997) and protein kinase C (PKC) has been implicated in this response (Greenaway et al., 2002). In the present study the role of mitogen-activated protein (MAP) kinases (specifically p44/42 extra-cellular regulated kinases (ERK-1/2) and p38 MAP kinases) in histamine-induced superoxide production was investigated. Phorbol 12-myristate 13-acetate (PMA), the potent PKC activator, was used as a positive control. Activation of MAP kinases was assessed by Western blotting using antibodies that detect the phosphorylated (activated) form of the enzymes. Superoxide production was measured colorimetrically (Foster & Cunningham, 1997).

PMA (10-9M) strongly induced superoxide production, and also activated ERK-1/2, but not p38 MAP kinase (n=7; Table 1). Histamine (10-4M) activated ERK-1/2 and p38 MAP kinases, although the increases were not significant (n=4; Table 1). However, in the bioassay, inhibitors of ERK-1/2 activation (PD98059, an inhibitor of the upstream activator of ERKs, MEK1/2) and p38 activity (SB203580) significantly reduced histamine-induced superoxide production (n=3; Table 2). In contrast, neither compound inhibited PMA-induced superoxide production (PMA alone 145±12, PMA + 10µM PD98059 144±13; PMA alone 144±18, PMA + 10µM SB203580 142±19. Results are expressed as means±SEM nmol reduced cytochrome (Cyt) C/106 cells; n=4). Neither inhibitor had any effect when used alone.

Table 1: Effects of histamine and PMA on superoxide production (nmol reduced cyt C/106 cells) and MAP kinase phosphorylation (arbitrary absorbance units).

superoxide
MAP kinase
control
treated
control
treated
PMA (10-9M)
1.6±1.4
#169±44
ERK-1
0.5±0.1
*2.0±0.4
 
ERK-2
0.5±0.2
*2.3±0.4
p38
0.6±0.1
0.5±0.2
histamine (10-4M)
3.9±1.7
11.2±2.9
ERK-1
0.5±0.2
1.2±0.4
 
ERK-2
0.4±0.1
0.9±0.4
p38
0.7±0.1
0.9±0.2

# p<0.01; * p<0.05 versus control; unpaired t test.

Table 2: Effect of MAP kinase inhibitors on histamine-induced superoxide production (nmol reduced cyt C/106 cells)

histamine
Inhibitor
Inhibitor concentration (µM)
1
5
10
5.3±0.2
PD98059
5.8±0.1
#4.2±0.2
*2.4±0.2
SB203580
*1.9±0.2
*1.5±0.2
*-1.3±0.2

#p<0.05; *p<0.001 versus histamine alone; repeat measures ANOVA and Bonferroni's post hoc test.

This study suggests that ERK-1/2 and p38 MAP kinases are involved in transduction of the histamine signal that induces superoxide production in equine eosinophils but do not play a significant role in the PMA-induced response.

Foster AP & Cunningham FM (1997). Vet. Immunol. Immunopathol. 59, 225-237.
Greenaway EC, Goode NT & Cunningham FM (2002). Brit. J. Pharmacol. 136, 34P.