Mechanisms mediating nitroglycerin-induced delayed-onset hyperalgesia in the rat

Abstract

Nitroglycerin (glycerol trinitrate, GTN) induces headache in migraineurs, an effect that has been used both diagnostically and in the study of the pathophysiology of this neurovascular pain syndrome. An important feature of this headache is a delay from the administration of GTN to headache onset that, because of GTN's very rapid metabolism, cannot be due to its pharmacokinetic profile. It has recently been suggested that activation of perivascular mast cells, which has been implicated in the pathophysiology of migraine, may contribute to this delay. We reported that hyperalgesia induced by intradermal GTN has a delay to onset of ∼ 30 min in male and ∼ 45 min in female rats. This hyperalgesia was greater in females, was prevented by pretreatment with the anti-migraine drug, sumatriptan, as well as by chronic pretreatment with the mast cell degranulator, compound 48/80. The acute administration of GTN and compound 48/80 both induced hyperalgesia that was prevented by pretreatment with octoxynol-9, which attenuates endothelial function, suggesting that GTN and mast cell-mediated hyperalgesia are endothelial cell-dependent. Furthermore, A-317491, a P2X3 antagonist, which inhibits endothelial cell-dependent hyperalgesia, also prevents GTN and mast cell-mediated hyperalgesia. We conclude that delayed-onset mechanical hyperalgesia induced by GTN is mediated by activation of mast cells, which in turn release mediators that stimulate endothelial cells to release ATP, to act on P2X3, a ligand-gated ion channel, in perivascular nociceptors. A role of the mast and endothelial cell in GTN-induced hyperalgesia suggests potential novel risk factors and targets for the treatment of migraine.

Keywords: P2X3; endothelium; mast cells; migraine; nitroglycerin; vascular pain.

Fig. 1. GTN-induced delayed onset mechanical hyperalgesia

(A). Nitroglycerin (GTN) was administered intradermally (0.1 ng, i.d.) on the dorsum of the hind paw. Mechanical nociceptive threshold was measured using the Randall-Selitto analgesymeter every 5 min for 120 min post GTN administration. GTN produced a decrease in nociceptive threshold, beginning ~30 min post-injection in males and ~45 min in females that remained undiminished for the duration of the testing period. During the plateau phase (30–120 min post administration), GTN-induced hyperalgesia was significantly greater in females (unpaired Student’s t-test, P<0.039) (B). GTN was administered intravenously (4 µg/kg/min for 20 min) and mechanical nociceptive threshold evaluated at multiple time points between 30 and 440 min after starting GTN infusion. GTN produced a decrease in nociceptive threshold, beginning ~65 min after starting infusion in males but not until ~320 min in females. There is a significant difference in the effect of GTN in males and females (two-way repeated ANOVA, P<0.0001).

Fig. 2. Effect of sumatriptan on GTN-induced delayed onset hyperalgesia

The intradermal administration of sumatriptan (1 µg i.d.), 10 min before intradermal GTN administration, markedly attenuated GTN-induced delayed onset mechanical hyperalgesia (one-tailed Student’s t-test, P<0.0005).

Fig. 3. Effect of the mast cell degranulator, compound 48/80, on GTN-induced delayed onset hyperalgesia

Three daily injection of compound 48/80 (see Methods section for details) to deplete mast cells of their pronociceptive mediators markedly attenuated GTN-induced hyperalgesia (one-tailed Student’s t-test, P<0.0001).

Fig. 4. Effect of octoxynol-9 on GTN-induced delayed onset hyperalgesia

Administration of the endothelial cell disruptor, octoxynol-9 (0.5% 1 ml/kg body weight, 30 min prior to GTN), markedly attenuated GTN-induced delayed onset mechanical hyperalgesia (one-tailed Student’s t-test, P<0.0001).

Fig. 5. Effect of octoxynol-9 on compound 48/80-induced hyperalgesia

The repeated, escalating doses of compound 48/80, which depletes mast cells (see Methods and Fig. 3), attenuated compound 48/80 induced hyperalgesia. A single injection of compound 48/80 (10 µg intradermal in hind paw) causes mast cell degranulation, releasing pronociceptive mediators (Xanthos et al., 2011), that in turn produces mechanical hyperalgesia. Disrupting endothelial cell function by pretreatment with octoxynol-9 markedly attenuated the hyperalgesia induced by a single injection of compound 48/80 (one-tailed Student’s t-test, P<0.0004).

Fig. 6. Effect of the P2X3 receptor antagonist A-317491 on hyperalgesia induced by compound 48/80 and GTN

We hypothesized that mediator(s) released from mast cells act on endothelial cells (see Fig. 5) to release ATP, which, in turn, can act at P2X3 receptors on primary afferent nociceptors, to produce mechanical hyperalgesia. Pretreatment with the selective P2X3 receptor antagonist, A-317491 markedly attenuated hyperalgesia produced by acute administration of compound 48/80 (10 µg intradermal in the hind paw; one-tailed Student’s t-test, P<0.0001, Panel A), as well as the delayed onset hyperalgesia induced by the administration of GTN (one-tailed Student’s t-test, P<0.0001, Panel B).

Fig. 7. Effect of an inhibitor of neutrophil recruitment, fucoidan, on GTN-induced hyperalgesia

Pretreatment of rats with fucoidan (25 mg/kg i.v.), to attenuate neutrophil recruited at the site of GTN administration and nociceptive threshold testing, did not significantly affect GTN-induced hyperalgesia (P=N.S.).