Stimulation of sensory neuropeptide release by nociceptin/orphanin FQ leads to hyperaemia in acutely inflamed rat knees

Abstract

The peripheral effect of the 'opioid-like' peptide nociceptin/orphanin FQ (N/OFQ) on joint blood flow was investigated in acutely inflamed rats. Sensory neuropeptide release from capsaicin-sensitive nerves and the involvement of synovial mast cells and leukocytes on these vasomotor responses were also studied. Blood flow measurements of exposed knee joints were performed in urethane-anaesthetised rats (2 mg kg(-1) intraperitoneal) using laser Doppler perfusion imaging. Topical administration of N/OFQ (10(-13)-10(-8) mol) to acutely inflamed joints caused a dose-dependent increase in synovial perfusion with an ED(50) of 4.0 x 10(-10) mol. This vasodilatatory response was blocked by the selective NOP receptor antagonist [Phe(1)-(CH(2)-NH)-Gly(2)]-Nociceptin(1-13)-NH(2) (10(-9) mol) (P<0.0001).Co-administration of N/OFQ with the neurokinin-1 (NK(1)) receptor antagonist [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-Substance P (10(-12) mol), the vasoactive intestinal peptide (VIP) receptor antagonist VIP(6-28) (10(-9) mol) or the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP(8-37) (10(-9) mol) all blocked the hyperaemic effect of N/OFQ (P<0.0001). Treatment of acutely inflamed knees with capsaicin (8-methyl-N-vanillyl-6-noneamide) to destroy unmyelinated joint afferents also inhibited N/OFQ vasomotor activity. Stabilisation of synovial mast cells with disodium cromoglycate (cromolyn) ameliorated N/OFQ responses, whereas inactivation of circulating leukocytes with the pan-selectin inhibitor fucoidin completely blocked N/OFQ-induced hyperaemia in these joints. These experiments show that in acutely inflamed knee joints, N/OFQ acts on NOP receptors located on synovial mast cells and leukocytes leading to the secondary release of proinflammatory mediators into the joint. These agents subsequently stimulate sensory neuropeptide release from capsaicin-sensitive nerves culminating in vasodilatation and increased articular blood flow.

Figure 1

Time course of N/OFQ effects on synovial blood flow in acutely inflamed rat knee joints. Peak vasodilatation tended to occur 2 min after topical application of N/OFQ, although a maximal response time of 4 min was noted with the 10⁻⁸ mol dose. Data are means±s.e.m.

Figure 2

Dose–response curves of N/OFQ either alone or in the presence of the NOP receptor antagonist Noci_1–13 (10⁻⁹ mol). The 4-min time point was used for the 10⁻⁸ mol dose of N/OFQ and 2 min was used for all other doses. ^*P<0.05 Bonferroni post hoc test; n=7 and 8. Data are shown as means±s.e.m.

Figure 3

Vasomotor effect of SP on acutely inflamed knee joint blood flow and the inhibitory effect of 10⁻¹² mol [D-Arg]-SP. Means±s.e.m. are indicated.

Figure 4

Dose–response relationship to N/OFQ either alone or in the presence of the NK₁ receptor antagonist [D-Arg]-SP (10⁻¹² mol) (a), the VPAC receptor antagonist VIP_6–28 (10⁻⁹ mol) (b) or the CGRP receptor antagonist CGRP_8–37 (10⁻⁹ mol) (c). ^*P<0.05, ^**P<0.01, ^***P<0.001 Bonferroni post hoc test; n=8 and 9. Data are shown as means±s.e.m.

Figure 5

Effect of the sensory neuropeptide antagonists Noci_1–13 (10⁻⁹ mol: NOP receptor), [D-Arg]-SP (10⁻¹² mol: NK₁ receptor), CGRP_8–37 (10⁻⁹ mol: CGRP receptor) and VIP_6–28 (10⁻⁹ mol: VPAC receptor antagonist) on acutely inflamed knee joint basal blood flow. ^*P<0.05 two-tailed paired Student's t-test, n=7–10. NS: not significantly different. Data are shown as means±s.e.m.

Figure 6

Effect of capsaicin treatment on N/OFQ-induced hyperaemia in acutely inflamed knees. ^*P<0.05, ^**P<0.01, ^***P<0.001 Bonferroni post hoc test; n=6–8. Means±s.e.m. are shown for all data.

Figure 7

N/OFQ responses in control, mast cell stabilised (cromolyn treated) and leukocyte impaired (fucoidin treated) knee joints. ^*P<0.05 Bonferroni post hoc test; n=5–9. Data are shown as means±s.e.m.