Cyclo-oxygenase and lipoxygenase pathways in mast cell dependent-neurogenic inflammation induced by electrical stimulation of the rat saphenous nerve

Abstract

1. We investigated the role of arachidonic acid metabolism and assessed the participation of mast cells and leukocytes in neurogenic inflammation in rat paw skin. We compared the effect of lipoxygenase (LOX) and cyclo-oxygenase (COX) inhibitors on oedema induced by saphenous nerve stimulation, substance P (SP), and compound 48/80. 2. Intravenous (i.v.) pre-treatment with a dual COX/LOX inhibitor (RWJ 63556), a dual LOX inhibitor/cysteinyl-leukotriene (CysLt) receptor antagonist (Rev 5901), a LOX inhibitor (AA 861), a five-lipoxygenase activating factor (FLAP) inhibitor (MK 886), or a glutathione S-transferase inhibitor (ethacrynic acid) significantly inhibited (40 to 60%) the development of neurogenic oedema, but did not affect cutaneous blood flow. Intradermal (i.d.) injection of LOX inhibitors reduced SP-induced oedema (up to 50% for RWJ 63556 and MK 886), whereas ethacrynic acid had a potentiating effect. 3. Indomethacin and rofecoxib, a highly selective COX-2 inhibitor, did not affect neurogenic and SP-induced oedema. Surprisingly, the structurally related COX-2 inhibitors, NS 398 and nimesulide, significantly reduced both neurogenic and SP-induced oedema (70% and 42% for neurogenic oedema, respectively; 49% and 46% for SP-induced oedema, respectively). 4. COX-2 mRNA was undetectable in saphenous nerves and paw skin biopsy samples, before and after saphenous nerve stimulation. 5. A mast cell stabilizer, cromolyn, and a H(1) receptor antagonist, mepyramine, significantly inhibited neurogenic (51% and 43%, respectively) and SP-induced oedema (67% and 63%, respectively). 6. The co-injection of LOX inhibitors and compound 48/80 did not alter the effects of compound 48/80. Conversely, ethacrynic acid had a significant potentiating effect. The pharmacological profile of the effect of COX inhibitors on compound 48/80-induced oedema was similar to that of neurogenic and SP-induced oedema. 7. The polysaccharide, fucoidan (an inhibitor of leukocyte rolling) did not affect neurogenic or SP-induced oedema. 8. Thus, (i) SP-induced leukotriene synthesis is involved in the development of neurogenic oedema in rat paw skin; (ii) this leukotriene-mediated plasma extravasation might be independent of mast cell activation and/or of the adhesion of leukocytes to the endothelium; (iii) COX did not appear to play a significant role in this process.

Figure 1

The effect of LOX pathway inhibitors on neurogenic and SP-induced oedema. RWJ 63556 (1 mg kg⁻¹ i.v.), Rev 5901 (1 mg kg⁻¹ i.v.), AA 861 (0.1 mg kg⁻¹ i.v.), MK 886 (10 μg kg⁻¹ i.v.), or ethacrynic acid (1 mg kg⁻¹ i.v.) were injected 15 min before a 5-min stimulation (neurogenic oedema). RWJ 63556 (1 μg site⁻¹ i.d.), Rev 5901 (1 μg site⁻¹ i.d.), AA 861 (1 μg site⁻¹ i.d.), MK 886 (1 μg site⁻¹ i.d.), or ethacrynic acid (1 μg site⁻¹ i.d.) were co-injected with SP (100 pmol site⁻¹; SP-induced oedema). The anti-inflammatory effects are expressed as per cent inhibition of the oedema volume compared to untreated animals (see Methods). Asterisks indicate significant differences in volumes of neurogenic or SP-induced oedema between treated and untreated animals. (^*P<0.05, ^***P<0.001 according to ANOVA, followed by Bonferroni's test for multiple comparisons).

Figure 2

Comparison of the effects of the FLAP inhibitor, MK 886 (10 μg kg⁻¹ i.v.), CGRP_8 – 37 (0.3 mg kg⁻¹ i.v.,) and the μ-opioid agonist, DAMGO (1 mg kg⁻¹ i.v.), on neurogenic vasodilatation. (a, b) Representative blood flow traces obtained from individual experiments showing the effects of MK 886 and CGRP_8 – 37 on neurogenic vasodilatation. Arrows (up) delineate the 10-s stimulation period. Arrows (down) indicate the time at which the test agents were injected. (c) Summarized results obtained with MK 886, CGRP_8 – 37, and DAMGO. The amplitude of the increase in blood flow (in arbitrary units) induced by the third stimulation was compared with that induced by the second stimulation. The effects of the test agent and vehicle are expressed as per cent change±s.e.mean in neurogenic response between the second and third electrical stimulations. Asterisks indicate significant differences between the neurogenic increase in blood flow measured during the second and third stimulations (^***P<0.001 according to ANOVA, followed by Bonferroni's test for multiple comparisons).

Figure 3

(a) Effect of COX pathway inhibitors on neurogenic oedema and SP-induced oedema. Indomethacin (5 mg kg⁻¹ i.v.), rofecoxib (10 μg kg⁻¹ i.v.), NS 398 (10 μkg⁻¹ i.v.), or nimesulide (10 μg kg⁻¹ i.v.) were injected 15 min before a 5-min stimulation (neurogenic oedema). Indomethacin (1 μg site⁻¹ i.d.), rofecoxib (1 μg site⁻¹ i.d.), NS 398 (1 μg site⁻¹ i.d.), or nimesulide (3 μg site⁻¹ i.d.) were co-injected with SP (100 pmol site⁻¹; SP-induced oedema). The anti-inflammatory effects are expressed as per cent inhibition of the oedema volume measured in untreated animals (see Methods). Asterisks indicate significant differences in the volume of neurogenic or SP-induced oedema between treated and untreated animals. (^*P<0.05, ^***P<0.001 according to ANOVA, followed by Bonferroni's test for multiple comparisons). (b) A representative trace from an individual experiment showing the lack of effect of NS 398 (10 μg kg⁻¹ i.v.) on neurogenic vasodilatation. Arrows (up) delineate the 10-s stimulation periods. Arrows (down) indicate the time at which the test agent was injected. (c) Analysis of COX-1 and COX-2 mRNA levels in rat paw skin and saphenous nerve by RT – PCR. Total RNA was extracted from the saphenous nerve and skin biopsy samples of ipsilateral (I) and contralateral (C) paws before and after a 5-min stimulation. COX-1 and COX-2 mRNA levels were studied by RT – PCR with specific rat COX-1 and COX-2 mRNA primers. Rat brain RNA was used as a positive control for COX-1 and COX-2.

Figure 4

(a) Involvement of NK₁ receptors, mast cells and PMN adhesion in neurogenic and SP-induced oedema. The NK₁ receptor antagonist, L-703,606 (0.5 mg kg⁻¹ i.v.), the mast cell stabilizer, cromolyn (5 mg kg⁻¹ i.v.), the H₁ receptor antagonist, mepyramine (6 mg kg⁻¹ i.v.), or the polysaccharide fucoidan (5 mg kg⁻¹ i.v.) were injected 15 min before a 5-min stimulation (neurogenic oedema). L-703,606 (1 μg site⁻¹ i.d.), cromolyn (10 μg site⁻¹ i.d.), mepyramine (1 μg site⁻¹ i.d.), or fucoidan (10 μg site⁻¹ i.d.) were co-injected with SP (100 pmol site⁻¹ i.d., SP-induced oedema) or compound 48/80 (1 μg site⁻¹ i.d.; compound 48/80-induced oedema). The anti-inflammatory effects are expressed as per cent inhibition of the oedema volume in untreated animals (see Methods). Asterisks indicate significant differences in volume of neurogenic or SP-induced oedema between treated and untreated animals. (^*P<0.05, ^**P<0.01 according to ANOVA, followed by Bonferroni's test for multiple comparisons). (b) Effect of LOX and COX pathway inhibitors on compound 48/80-induced oedema. Drugs were co-injected with compound 48/80 (1 μg site⁻¹ i.d.), at the same doses used in Figures 1 and 3 for SP-induced oedema. The anti-inflammatory effects are expressed as per cent inhibition of the oedema volume in untreated animals (see Methods). Asterisks indicate significant differences in the volume of compound 48/80-induced oedema between treated and untreated animals. (^*P<0.05 according to ANOVA, followed by Bonferroni's test for multiple comparisons).