Chronic hypersensitivity for inflammatory nociceptor sensitization mediated by the epsilon isozyme of protein kinase C

Abstract

We have identified a mechanism, mediated by the epsilon isozyme of protein kinase C (PKCepsilon) in peripheral neurons, which may have a role in chronic inflammatory pain. Acute inflammation, produced by carrageenan injection in the rat hindpaw, produced mechanical hyperalgesia that resolved by 72 hr. However, for up to 3 weeks after carrageenan, injection of the inflammatory mediators prostaglandin E(2) or 5-hydroxytryptamine or of an adenosine A(2) agonist into the same site induced a markedly prolonged hyperalgesia (>24 hr compared with 5 hr or less in control rats not pretreated with carrageenan). A nonselective inhibitor of several PKC isozymes and a selective PKCepsilon inhibitor antagonized this prolonged hyperalgesic response equally. Acute carrageenan hyperalgesia could be inhibited by PKA or PKG antagonists. However, these antagonists did not inhibit development of the hypersensitivity to inflammatory mediators. Our findings indicate that different second messenger pathways underlie acute and prolonged inflammatory pain.

Fig. 1.

A, Dose (0.1–2%)–response curve of carrageenan (Carr; n = 12) induced mechanical hyperalgesia measured at 4 hr in the hindpaw of the normal rat. The Randall-Selitto paw-withdrawal test is an established method to assess heightened nociception in animals in which this subjective experience of pain cannot be directly determined. Measures using this technique have been shown to correlate with pain-like behaviors in animals. B, Time course of hyperalgesia induced by carrageenan 1% (5 μl, n = 24) in normal rats.

Fig. 2.

A, PGE₂ (100 ng,n = 24), 5-HT (1 μg, n = 6), and CGS-21680 (100 ng, n = 6)-induced mechanical hyperalgesia at 30 min, 4 hr, and 24 hr after injection in rats treated 5 d previously with carrageenan. B, Mechanical hyperalgesia induced by PGE₂ (n = 12), 5-HT (n = 6), and CGS-21680 (n= 6) at 30 min, 4 hr, and 24 hr after injection in rats treated 21 d previously with carrageenan. C, Time course of PGE₂-, 5-HT-, and CGS-21680-induced mechanical hyperalgesia in rats 5 d after vehicle used for carrageenan (n = 12 each).

Fig. 3.

A, Effect of PKA inhibitor WIPTIDE (WIP/E2; 1 μg/100 ng, n = 24), nitric oxide synthase inhibitorN^G-methyl-l-arginine (L-NMA/E2; 1 μg/100 ng, n = 12), PKC inhibitor bisindolylmalemide 1 hydrochloride (Bis/E2; 1 μg/100 ng, n = 12), PKCε inhibitor (PKCεV1–2/E2; 1 μg/100 ng,n = 12), administered 5 min before PGE₂, on PGE₂(E2)-induced mechanical hyperalgesia measured at 30 min after PGE₂ injection in control rats and in rats treated 5 d previously with carrageenan. B, Effect of PKA inhibitor WIP/E2 (n = 24), nitric oxide synthase inhibitor L-NMA/E2 (n= 12), PKC inhibitor Bis/E2 (n = 12), PKCε inhibitor PKCεV1–2/E2) (n = 12), administered 5 min before PGE₂, on PGE₂(E2)-induced mechanical hyperalgesia measured at 4 hr after PGE₂ injection in control rats and in rats treated 5 d previously with carrageenan.

Fig. 4.

A, Effect of PKA inhibitor WIPTIDE (WIP/Carr; n = 24) and PKG inhibitor (PKGI/Carr; n = 8) on carrageenan-induced mechanical hyperalgesia in the rat hindpaw. Agents were administered 5 min before carrageenan. All readings were taken 4 hr after carrageenan. B, C, Effect of PGE₂ injected at different times (30 min to 96 hr) in different groups of rats after injection of carrageenan plus WIPTIDE (B) or PKGI (C). All readings were taken 4 hr after prostaglandin E₂ injection.n = 6 each group.

Fig. 5.

A, Dose–response curve of ψεR (0.1–10,000 ng, n = 8)-induced mechanical hyperalgesia measured at 30 min in the hindpaw of the rat.B, PGE₂-induced hyperalgesia at 30 min, 4 hr, and 24 hr in rats treated 5 d previously with ψεR (1 μg,n = 6). C, Time course of ψεR (1 μg/paw, n = 12)-induced hyperalgesia (1 μg).D, Role of second messengers important in ψεR-induced hyperalgesia. PKA inhibitor WIPTIDE (WIP/ΨεR; both 1 μg, n = 24), the nitric oxide synthase inhibitor N^G-methyl-l-arginine (L-NMA/ΨεR; both 1 μg,n = 12), the PKC inhibitor bisindolylmalemide 1 hydrochloride (Bis/ΨεR; both 1 μg, n = 12), the PKCε inhibitor (PKCεV1–2/ΨεR; both 1 μg,n = 12), the PKG inhibitor (PKGI/ΨεR; both 1 μg,n = 8), the guanylyl cyclase inhibitor ODQ (ODQ/ΨεR; both 1 μg,n = 6), the calcium transport antagonist 3,4,5-trimethoxybezoic acid 8-(diethylamino) octyl ester (TMB/ΨεR; both 1 μg,n = 6), the calcium chelator (2-[(2-bis-[carboxymethyl] amino-5-methylphenoxy) methyl]-6-methoxy-8-bis[carboxymethy] aminoquinoline (Quin/ΨεR; both 1 μg, n = 6), or the NMDA receptor antagonist MK-801 (MK801/ΨεR; both 1 μg,n = 8) 5 min before injection of ψεR (1 μg). All readings were taken 30 min after injection of ψεR.E, Role of second messengers in PGE₂-induced hyperalgesia in rats pretreated with ψεR. Rats were administered the PKA inhibitor WIPTIDE (E2/WIP; 100 ng/1 μg,n = 6), the nitric oxide synthase inhibitorl-NMA (E2/L-NMA; 100 ng/1 μg,n = 6), the PKC inhibitor Bis (E2/Bis; 100 ng/1 μg, n = 10), the PKCε inhibitor (E2/PKCεV1–2; 100 ng/1 μg, n = 6), the calcium antagonists Quin-2 and TMB-8 (E2/Quin and E2/TMB; both 100 ng/1 μg, both n = 6), or the NMDA receptor antagonist MK-801 (E2/MK801; 100 ng/1 μg,n = 10) 5 min before injection of PGE₂(E2) (100 ng) on the fifth day after receiving ψεR (1 μg). All readings were taken 4 hr after injection of PGE₂.