Comparing analgesia and μ-opioid receptor internalization produced by intrathecal enkephalin: Requirement for peptidase inhibition

Abstract

Opioid receptors in the spinal cord produce strong analgesia, but the mechanisms controlling their activation by endogenous opioids remain unclear. We have previously shown in spinal cord slices that peptidases preclude mu-opioid receptor (MOR) internalization by opioids. Our present goals were to investigate whether enkephalin-induced analgesia is also precluded by peptidases, and whether it is mediated by MORs or delta-opioid receptors (DORs). Tail-flick analgesia and MOR internalization were measured in rats injected intrathecally with Leu-enkephalin and peptidase inhibitors. Without peptidase inhibitors, Leu-enkephalin produced neither analgesia nor MOR internalization at doses up to 100 nmol, whereas with peptidase inhibitors it produced analgesia at 0.3 nmol and MOR internalization at 1 nmol. Leu-enkephalin was 10 times more potent to produce analgesia than to produce MOR internalization, suggesting that DORs were involved. Selective MOR or DOR antagonists completely blocked the analgesia elicited by 0.3 nmol Leu-enkephalin (a dose that produced little MOR internalization), indicating that it involved these two receptors, possibly by an additive or synergistic interaction. The selective MOR agonist endomorphin-2 produced analgesia even in the presence of a DOR antagonist, but at doses substantially higher than Leu-enkephalin. Unlike Leu-enkephalin, endomorphin-2 had the same potencies to induce analgesia and MOR internalization. We concluded that low doses of enkephalins produce analgesia by activating both MORs and DORs. Analgesia can also be produced exclusively by MORs at higher agonist doses. Since peptidases prevent the activation of spinal opioid receptors by enkephalins, the coincident release of opioids and endogenous peptidase inhibitors may be required for analgesia.

Noxious mechanical stimulation evokes the segmental release of opioid peptides that induce μ-opioid receptor internalization in the presence of peptidase inhibitors

“…In them the percentage of MOR neurons with internalization in lumbar spinal segments was negligible: L1, 0.4±0.4 %; L2, 1.8±1.1 %; L3, 1.0±0.5 %; L4, 2.3±1.8 %; L5, 3.5±2.2 %; L6, 1.5±1.5 % (n=3 rats). We have previously shown that peptidase inhibitors by themselves do not produce MOR internalization (Chen et al, 2007). In other experiments we also measured the internalization of NK1Rs in lamina I neurons in order to assess the intensity of the noxious stimuli.…”

“…Importantly, the inability of opioids to produce MOR internalization in the absence of peptidase inhibitors does reflect their inability to activate them. Thus, intrathecal Leu-enkephalin failed to produce analgesia in the tail-flick test, even at high doses (100 nmol), but it produced analgesia at low very doses (0.3 nmol) when it was co-injected with peptidase inhibitors (Chen et al, 2007). Another study (Kishioka et al, 1994) found that peptidase inhibitors substantially increased the analgesia produced by Metenkephalin, dynorphin or electroacupuncture.…”

“…Indeed, we found that inhibitors of three peptidases (aminopeptidase, dipeptidyl carboxypeptidase and neutral endopeptidase) increased a hundred times the apparent potency of Leu-enkephalin to induce MOR internalization in spinal cord slices (Song and Marvizon, 2003a). In vivo, intrathecal Leu-enkephalin was unable to produce not only MOR internalization, but also analgesia, unless it was co-injected with inhibitors of these three peptidases (Chen et al, 2007).…”

Sustained Suppression of Hyperalgesia during Latent Sensitization by μ-, δ-, and κ-opioid receptors and α2AAdrenergic Receptors: Role of Constitutive Activity

“…Spinal cord sections were labeled for MORs as described previously (Song and Marvizó n, 2003b;Chen et al, 2007;Chen et al, 2008;Chen and Marvizó n, 2009). Rats were killed with pentobarbital (100 mg/ kg) and fixed immediately by aortic perfusion of 100 ml phosphate buffer (0.1 M sodium phosphate, pH 7.4) containing 0.01% heparin, followed by 400 ml of ice-cold fixative (4% paraformaldehyde, 0.18% picric acid in phosphate buffer).…”

“…1A) suggests that receptors other that MORs contribute to the suppression of hyperalgesia during the remissionphaseoflatentsensitization.Descendingnoradrenergicpathwaysproduceanalgesia by acting on ␣ 2 receptors in the dorsal horn (Yaksh, 1985;Pertovaara, 2006), which are mainly ␣ 2A Rs located in primary afferent terminals (Stone et al, 1998;Chen et al, 2008). Therefore, we hypothesized that ␣ 2A Rs contribute to suppressing hyperalgesia in latent sensitization and predicted that an ␣ 2A R antagonist would produce reinstatement during the remission phase.…”