Nitric oxide signaling in pain and nociceptor sensitization in the rat

Abstract

We investigated the role of nitric oxide (NO) in inflammatory hyperalgesia. Coinjection of prostaglandin E2 (PGE2) with the nitric oxide synthase (NOS) inhibitor NG-methyl-L-arginine (L-NMA) inhibited PGE2-induced hyperalgesia. L-NMA was also able to reverse that hyperalgesia. This suggests that NO contributes to the maintenance of, as well as to the induction of, PGE2-induced hyperalgesia. Consistent with the hypothesis that the NO that contributes to PGE2-induced sensitization of primary afferents is generated in the dorsal root ganglion (DRG) neurons themselves, L-NMA also inhibited the PGE2-induced increase in tetrodotoxin-resistant sodium current in patch-clamp electrophysiological studies of small diameter DRG neurons in vitro. Although NO, the product of NOS, often activates guanylyl cyclase, we found that PGE2-induced hyperalgesia was not inhibited by coinjection of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylyl cyclase inhibitor. We then tested whether the effect of NO depended on interaction with the adenylyl cyclase-protein kinase A (PKA) pathway, which is known to mediate PGE2-induced hyperalgesia. L-NMA inhibited hyperalgesia produced by 8-bromo-cAMP (a stable membrane permeable analog of cAMP) or by forskolin (an adenylyl cyclase activator). However, L-NMA did not inhibit hyperalgesia produced by injection of the catalytic subunit of PKA. Therefore, the contribution of NO to PGE2-induced hyperalgesia may occur in the cAMP second messenger pathway at a point before the action of PKA. We next performed experiments to test whether administration of exogenous NO precursor or donor could mimic the hyperalgesic effect of endogenous NO. Intradermal injection of either the NOS substrate L-arginine or the NO donor 3-(4-morphinolinyl)-sydnonimine hydrochloride (SIN-1) produced hyperalgesia. However, this hyperalgesia differed from PGE2-induced hyperalgesia, because it was independent of the cAMP second messenger system and blocked by the guanylyl cyclase inhibitor ODQ. Therefore, although exogenous NO induces hyperalgesia, it acts by a mechanism different from that by which endogenous NO facilitates PGE2-induced hyperalgesia. Consistent with the hypothesis that these mechanisms are distinct, we found that inhibition of PGE2-induced hyperalgesia caused by L-NMA could be reversed by a low dose of the NO donor SIN-1. The following facts suggest that this dose of SIN-1 mimics a permissive effect of basal levels of NO with regard to PGE2-induced hyperalgesia: (1) this dose of SIN-1 does not produce hyperalgesia when administered alone, and (2) the effect was not blocked by ODQ. In conclusion, we have shown that low levels of NO facilitate cAMP-dependent PGE2-induced hyperalgesia, whereas higher levels of NO produce a cGMP-dependent hyperalgesia.

Fig. 1.

Reduction of mechanical nociceptive threshold (hyperalgesia) produced by PGE₂ 15 min after injection (PGE2) (100 ng; n = 12),l-NMA (1 μg; n = 6) plus PGE₂ (l-NMA/PGE2) (n = 12; p < 0.05), PGE₂ 5 min after injection [PGE2(5′)] (n = 6), l-NMA 5 min after PGE₂ (PGE2/l-NMA5′post) (n = 6; p < 0.05),d-NMA (10 μg) plus PGE₂(d-NMA/PGE2) (n = 6), ODQ (1 μg) plus PGE₂ (ODQ/PGE2) (n = 6), and l-NMA(n = 6) on mechanical paw-withdrawal threshold in rats. In this and subsequent figures, *p < 0.05. Higher values indicate greater hyperalgesia. The data for one behavioral experimental group, PGE₂, is repeated in more than one figure for ease of comparison.

Fig. 2.

l-NMA reduces the PGE₂-induced potentiation of TTX-R I_Na. TTX-R I_Na was monitored by test pulses given every 20 sec. Voltage of the test pulse (−20 to −5 mV) was selected for each neuron to give an approximately half-maximal current amplitude. One hundred micromolar d-NMA or l-NMA was included in the bath for 15 min before exposure to 1 μm PGE₂. PGE₂ typically causes a potentiation of TTX-R I_Na in approximately half of cells tested (Gold et al., 1996a,b). Experiments were alternated between using the active and inactive enantiomers of the NOS inhibitor, and data from all neurons were used, including those in which the current was not affected by PGE₂. Peak current amplitudes were normalized to mean of the baseline measurements.

Fig. 3.

Reduction of the paw-withdrawal threshold by the A₂ adenosine agonist CGS21680 after injection (1 μg; n = 6), l-NMA plus CGS21680 (l-NMA/CGS) (n= 6; p < 0.05), 5HT_1A agonist8-OH-DPAT after injection (1 μg; n= 6), and l-NMA plus 8-OH-DPAT (l-NMA/8-OH) (n = 6; p < 0.05) on mechanical paw-withdrawal threshold in rats.

Fig. 4.

Reduction of the paw-withdrawal threshold by forskolin (10 μg) 5 min after injection (Forsk) (n = 8), l-NMA plus forskolin (l-NMA/Forsk) (n = 6;p < 0.05), WIPTIDE plus forskolin (WIPTIDE/Forsk) (n = 10;p < 0.05), 8-bromo-cAMP after injection (8brcAMP) (1 μg; n = 12),l-NMA plus 8-bromo-cAMP (l-NMA/8brcAMP) (n = 8;p < 0.05), WIPTIDE plus 8-bromo-cAMP (WIPTIDE/8brcAMP) (n = 6;p < 0.05), PKACS (15 U;n = 12), WIPTIDE plus PKACS (WIPTIDE/PKACS) (n = 6;p < 0.05), and l-NMA plus PKACS (l-NMA/PKACS) (n = 6; not statistically significant) on mechanical paw-withdrawal threshold in rats.

Fig. 5.

A, Dose–response curve forl-Arg-induced hyperalgesia (n = 6).B, Latency to onset of l-Arg-induced (10 μg) mechanical hyperalgesia (n = 6).C, Time course of l-Arg-induced mechanical hyperalgesia (n = 6). D, Dose–response curve of SIN-1-induced hyperalgesia (n = 6). E, Latency to onset of SIN-1-induced hyperalgesia (10 μg; n = 6).F, Time course of SIN-1-induced hyperalgesia (n = 8).

Fig. 6.

A, Change in mechanical paw withdrawal after injection of l-Arg (l-Arg) (10 μg; n = 12), ODQ plus l-Arg (ODQ/l-Arg) (n = 6), WIPTIDE plus l-Arg (WIPTIDE/l-Arg) (n = 6),l-NMA plus l-Arg (l-NMA/l-Arg) (n = 6), d-NMA/l-Arg (n = 6), d-Arg (d-Arg) (10 μg; n = 6), PGE₂ (PGE2) (n = 12), WIPTIDE plus PGE₂ (WIPTIDE/PGE2) (n = 12), ODQ plus PGE₂(ODQ/PGE2) (n = 8),SIN-1 (10 μg; n = 6), ODQ plus SIN-1 (ODQ/SIN) (n = 8), and WIPTIDE plus SIN-1 (WIPTIDE/SIN) (n = 6). B, Change in mechanical paw-withdrawal threshold after injection of PGE₂(n = 12), l-NMA plus PGE₂(l-NMA/PGE2) (n = 6), SIN-1 (SIN) (100 ng; n = 6), and l-NMA plus ODQ plus PGE₂ plus SIN-1 (l-NMA/ODQ/PGE2/SIN) (100 ng;n = 12).