Spinal substance P receptor expression and internalization in acute, short-term, and long-term inflammatory pain states.

Inflammatory pain involves the sensitization of both primary afferent and spinal cord neurons. To explore the neurochemical changes that contribute to inflammatory pain, we have examined the expression and ligand-induced internalization of the substance P receptor (SPR) in the spinal cord in acute, short-term, and long-term inflammatory pain states. These inflammatory models included unilateral injection of formalin (8-60 min), carrageenan (3 hr), and complete Freund's adjuvant (CFA; 3 d) into the rat hindpaw as well as adjuvant-induced polyarthritis (21 d). In acute inflammatory pain there is ongoing release of substance P (SP) as measured by SPR internalization in lamina I neurons at both 8 and 60 min after formalin injection. Although there is no tonic release of SP in short-term inflammatory pain, at 3 hr after carrageenan injection, SP is released in response to both noxious and non-noxious somatosensory stimulation with SPR internalization being observed in neurons located in both laminae I and III-IV. In long-term inflammatory pain models (CFA and polyarthritis) the same pattern of SP release and SPR activation occurs as is observed in short-term inflammation with the addition that there is a significant upregulation of the SPR in lamina I neurons. These results suggest that SPR internalization might serve as a marker of the contribution of ongoing primary afferent input in acute and persistent pain states. These stereotypical neurochemical changes suggest that there are unique neurochemical signatures for acute, short-term, and long-term inflammatory pain.

Primary afferent fibers that contribute to increased substance P receptor internalization in the spinal cord after injury.

Upon noxious stimulation, substance P (SP) is released from primary afferent fibers into the spinal cord where it interacts with the SP receptor (SPR). The SPR is located throughout the dorsal horn and undergoes endocytosis after agonist binding, which provides a spatial image of SPR-containing neurons that undergo agonist interaction. Under normal conditions, SPR internalization occurs only in SPR+ cell bodies and dendrites in the superficial dorsal horn after noxious stimulation. After nerve transection and inflammation, SPR immunoreactivity increases, and both noxious as well as nonnoxious stimulation produces SPR internalization in the superficial and deep dorsal horn. We investigated the primary afferent fibers that contribute to enhanced SPR internalization in the spinal cord after nerve transection and inflammation. Internalization evoked by electrical stimulation of the sciatic nerve was examined in untreated animals, at 14 days after sciatic nerve transection or sham surgery and at 3 days after hindpaw inflammation. Electrical stimulation was delivered at intensities to excite Abeta fibers only, Abeta and Adelta fibers or A and C fibers as determined by the compound action potential recorded from the tibial nerve. Electrical stimuli were delivered at a constant rate of 10 Hz for a duration of 5 min. Transection of the sciatic nerve and inflammation produced a 33.7 and 32.5% increase in SPR and immunoreactivity in lamina I, respectively. Under normal conditions, stimulation of Adelta or C fibers evoked internalization that was confined to the superficial dorsal horn. After transection or inflammation, there was a 20-24% increase in the proportion of SPR+ lamina I neurons that exhibited internalization evoked by stimulation of Adelta fibers. The proportion of lamina I SPR+ neurons that exhibited internalization after stimulation of C-fibers was not altered by transection or inflammation because this was nearly maximal under normal conditions. Moreover, electrical stimulation sufficient to excite C fibers evoked SPR internalization in 22% of SPR+ lamina III neurons after nerve transection and in 32-36% of SPR+ neurons in lamina III and IV after inflammation. Stimulation of Abeta fibers alone never evoked internalization in the superficial or deep dorsal horn. These results indicate that activation of small-caliber afferent fibers contributes to the enhanced SPR internalization in the spinal cord after nerve transection and inflammation and suggest that recruitment of neurons that possess the SPR contributes to hyperalgesia.

Noxious cutaneous thermal stimuli induce a graded release of endogenous substance P in the spinal cord: imaging peptide action in vivo.

Dorsal root ganglia (DRG) neurons synthesize and transport substance P (SP) to the spinal cord where it is released in response to intense noxious somatosensory stimuli. We have shown previously that SP release in vivo causes a rapid and reversible internalization of SP receptors (SPRs) in dorsal horn neurons, which may provide a pharmacologically specific image of neurons activated by SP. Here, we report that noxious heat (43 degrees, 48 degrees, and 55 degrees C) and cold (10 degrees, 0 degrees, -10 degrees, and -20 degrees C) stimuli, but not innocuous warm (38 degrees C) and cold (20 degrees C) stimuli, applied to the hindpaw of anesthetized rats induce SPR internalization in spinal cord neurons that is graded with respect to the intensity of the thermal stimulus. Thus, with increasing stimulus intensities, both the total number of SPR+ lamina I neurons showing SPR internalization and the number of internalized SPR+ endosomes within each SPR immunoreactive neuron showed a significant increase. These data suggest that thermal stimuli induce a graded release of SP from primary afferent terminals and that agonist dependent receptor endocytosis provides evidence of a spatially and pharmacologically unique "neurochemical signature" after specific somatosensory stimuli.

Endogenous Alzheimer's brain factor and oxidized glutathione inhibit antagonist binding to the muscarinic receptor.

An endogenous inhibitor (< 3,500 Da) of antagonist binding to the muscarinic acetylcholine receptor has been extracted from Alzheimer's disease (AD) brain with trifluoracetic acid. Oxidized glutathione, (GSSG) has also been found to inhibit antagonist binding to the receptor. However, in its reduced form, glutathione (GSH) like other reducing agents, markedly enhances the inhibitory effect of both GSSG and the endogenous AD inhibitor. EDTA and the free radical scavengers Mn(2+) and Trolox, a vitamin E analog, block the action of the endogenous AD inhibitor but not of GSSG in the presence of GSH. Further, while GSSG inhibition is reversible, the action of the endogenous AD inhibitor is irreversible, consistent with a free radical mechanism. The enhancement of endogenous AD inhibitor activity by GSH suggested that GSH may be involved in formation of the free radical generated by the inhibitor. The glutathione thiyl radical is shown to inhibit antagonist binding to the receptor and is, therefore, a good candidate for the free radical formed by the endogenous AD inhibitor. The ability of Trolox to block the reduction in muscarinic receptor binding caused by the endogenous AD inhibitor is encouraging and suggests that free radical scavengers, such as vitamin E, may have a potential therapeutic role in AD by protecting the integrity of the muscarinic receptor.