Streptozotocin-induced diabetes selectively enhances antinociception mediated by δ1-but not δ2-opioid receptors

We assessed the effect of diabetes on antinociception produced by intracerebroventricular injection of δ-opioid receptor agonists [D-Pen^2,5]enkephalin (DPDPE) and [D-Ala²]deltorphin II. The antinociceptive effect of DPDPE (10 nmol), administered i.c.v., was significantly greater in diabetic mice than in non-diabetic mice. The antinociceptive effect of i.c.v. DPDPE was significantly reduced in both diabetic and non-diabetic mice following pretreatment with 7-benzylidenenaltrexone (BNTX), a selective δ₁-opioid receptor antagonist, but not with naltriben (NTB), a selective δ₂- opioid receptor antagonist. There were no significant differences in the anticiceptive effect of [D-Ala²]deltorphin II (3 nmol, i.c.v.) in diabetic and non-diabetic mice. Furthermore, the antinociceptive effect of i.c.v. [D-Ala²]deltorphin II was significantly reduced in both diabetic and non-diabetic mice following pretreatment with NTB, but not with BNTX. In conclusion, mice with diabetes are selectively hyper-responsive to supraspinal δ₁-opioid receptor-mediated antinociception, but are normally responsive to activation of δ₂-opiod receptors.     

P2X3 Receptors Mediate Visceral Hypersensitivity during Acute Chemically-Induced Colitis and in the Post-Inflammatory Phase via Different Mechanisms of Sensitization

Objectives

Experiments using P2X₃ knock-out mice or more general P2X receptor antagonists suggest that P2X₃ receptors contribute to visceral hypersensitivity. We aimed to investigate the effect of the selective P2X₃ antagonist A-317491 on visceral sensitivity under physiological conditions, during acute colitis and in the post-inflammatory phase of colitis.

Methods

Trinitrobenzene sulphonic-acid colitis was monitored by colonoscopy: on day 3 to confirm the presence of colitis and then every 4 days, starting from day 10, to monitor convalescence and determine the exact timepoint of endoscopic healing in each rat. Visceral sensitivity was assessed by quantifying visceromotor responses to colorectal distension in controls, rats with acute colitis and post-colitis rats. A-317491 was administered 30 min prior to visceral sensitivity testing. Expression of P2X₃ receptors (RT-PCR and immunohistochemistry) and the intracellular signalling molecules cdk5, csk and CASK (RT-PCR) were quantified in colonic tissue and dorsal root ganglia. ATP release in response to colorectal distension was measured by luminiscence.

Results

Rats with acute TNBS-colitis displayed significant visceral hypersensitivity that was dose-dependently, but not fully, reversed by A-317491. Hypersenstivity was accompanied by an increased colonic release of ATP. Post-colitis rats also displayed visceral hypersensitivity that was dose-dependently reduced and fully normalized by A-317491 without increased release of ATP. A-317491 did not modify visceral sensitivity in controls. P2X₃ mRNA and protein expression in the colon and dorsal root ganglia were similar in control, acute colitis and post-colitis groups, while colonic mRNA expression of cdk5, csk and CASK was increased in the post-colitis group only.

Conclusions

These findings indicate that P2X₃ receptors are not involved in sensory signaling under physiological conditions whereas they modulate visceral hypersensitivity during acute TNBS-colitis and even more so in the post-inflammatory phase, albeit via different mechanisms of sensitization, validating P2X₃ receptors as potential new targets in the treatment of abdominal pain syndromes.     

δ-opioid supraspinal antinociception in mice is mediated by G13 transducer proteins

The intracerebroventricular (i.c.v.) injection to mice of antisera directed against different sequences G_i3α, impaired the antinociception produced by the selective ligands of δ opioid receptors DPDPE and [D-Ala²]-Deltorphin II, when studied 24 h later in the tail-flick test. Likewise, the potency of the μ/δ ligands DADLE, etorphine and β-endorphin-(1–31) was also reduced. Antinociception due to the μ-agonists morphine and DAMGO was slightly altered by this treatment. The selective δ antagonist ICI 174864 significantly reduced the antinociceptive activity of these opioids to the same extent observed after giving anti-G_i3α antisera. In animals treated with the antisera, ICI 174864 failed to reduce the antinociceptive effect that remained. It is concluded that G_i3 is the type of transducer protein regulated by δ opioid receptors to produce supraspinal antinociception in mice.     

The involvement of P2X3 receptors of rat sympathetic ganglia in cardiac nociceptive transmission

In this work we have examined the effects of P2X3 receptor antagonist A-317491 on P2X3 expression in superior cervical ganglion (SCG) from naive and myocardial ischemic rats to observe the effect of P2X3 receptors in cardiac nociceptive transmission. A-317491 improved nociceptive behavior. In the ganglia neurons of rats at 14 days after myocardial ischemic injury, the staining of P2X3 receptor in myocardial ischemic groups appeared to be more intense than those of naive rats detected by immunohistochemistry. After myocardial ischemic rats treated with A-317491, the intensity of the P2X3 immunoreactivity was lower than that in myocardial ischemic rats. The signals of P2X3 and its protein and mRNA in myocardial ischemic groups were higher than those in control group measured by western blotting and in situ hybridization. After myocardial ischemic rats treated with A-317491, the intensity of the P2X3 and its mRNA was lower than that in myocardial ischemic rats. These results suggest the involvement of P2X3 receptors in cardiac nociceptive transmission and A-317491 may inhibit the transmission mediated by P2X3 receptors in rat SCG after myocardial ischemia.     

α-Subunit G-protein antisense oligodeoxynucleotide effects on supraspinal (i.c.v.) α2-adrenoceptor antinociception in mice

An in vivo antisense strategy was used to examine the involvement of G-protein subunits in supraspinal (intracerebroventricular; i.c.v.) α₂-adrenoceptor-mediated antinociception. Mice that were injected with 33-mer antisense oligodeoxyribonucleotides (6 nmol) or vehicle were tested (tailflick) with an agonist (clonidine, guanfacine or BH-T 920) administered i.c.v. 18–24 h later. G_i3α antisense treatment attenuated BH-T 920 and clonidine-induced antinociception. G_i2α antisense produced differential effects on the three agonists. G_i1α and G_sα antisense treatment had no significant effect. Together with the previous demonstration that i.c.v. μ-opioid antinociception is mediated via G_i2α, the present results suggest that different receptors may mediate antinociception via different G-protein subunits and, hence, that specific subunits might offer novel targets for drug discovery.     

Antinociceptive effects of [D-Ala2]deltorphin II, a highly selective delta agonist in vivo

The present study has characterized the antinociceptive actions of [D-Ala2]deltorphin II following intracerebroventricular (i.c.v.) administration in the mouse tail-flick test. [D-Ala2]deltorphin II produced dose- and time-related antinociception, with maximal effects at +10 min and significant antinociception which lasted for 40-60 min. [D-Ala2]deltorphin II was 13-fold more potent than i.c.v. [D-Pen2, D-Pen5]enkephalin (DPDPE), a second highly selective delta agonist, and approximately equipotent with i.c.v. morphine in producing antinociception. The antinociceptive effects of i.c.v. [D-Ala2]deltorphin II and DPDPE, but not those of morphine, were antagonized by the selective delta antagonist, ICI 174,864. In contrast, pretreatment with the non-equilibrium mu antagonist, beta-funaltrexamine blocked morphine antinociception, but failed to antagonize [D-Ala2]deltorphin II and DPDPE antinociception. These data indicate that [D-Ala2]deltorphin II produced its antinociceptive effects at a supraspinal delta receptor. [D-Ala2]deltorphin II appears to be the most appropriate delta opioid agonist currently available for studies in vivo and support the involvement of delta receptors in supraspinal antinociception.     

Etonitazene-induced antinociception in μ1 opioid receptor deficient CXBK mice: Evidence for a role for μ2 receptors in supraspinal antinociception

The prevailing view is that supraspinal μ opioid-mediated antinociception in mice is mediated via the μ₁ subtype. The purpose of the present study was to determine if the highly μ-selective compound etonitazene could produce supraspinal (intracerebroventricular; i.c.v.) antinociception in CXBK mice, which are deficient in brain μ₁, but not μ₂, opioid receptors. CXBK or normal Crl:CD-1 ^®(ICR)BR mice were administered graded doses of etonitazene i.c.v. and 15 min later antinociception was assessed by a standard radiant-heat or 55°C water tail-flick test. Etonitazene produced dose-related antinociception that was blocked by naloxone and by β-FNA (demonstrating a μ opioid mechanism), but not by either ICI-174,864 or naltrindole (demonstrating the lack of involvement of δ opioid receptors). These findings suggest that μ₂ opioid receptors are important contributors to opioid-induced supraspinal antinociception in mice.     

In vivo characterization of the effects of ghrelin on the modulation of acute pain at the supraspinal level in mice

Ghrelin, an acylated peptide produced in the stomach, increases food intake and growth hormone secretion, inhibits pro-inflammatory cascade, etc. Ghrelin and its receptor (GHS-R1a) mRNA were found in the area related to the regions for controlling pain transmission, such as the hypothalamus, the midbrain, the spinal cord, etc. Ghrelin has been shown to have antinociceptive activity and also anti-inflammatory properties in inflammatory pain and chronic neuropathic pain. Therefore, the aim of the present study was to investigate the effects of ghrelin for the first time in the acute pain modulation at the supraspinal level, using the tail withdrawal test and hot-plate test in mice. Intracerebroventricular (i.c.v.) administration of ghrelin (mouse, 0.1–3 nmol) produced a dose- and time-related antinociceptive effect in the tail withdrawal test and hot-plate test, respectively. Antinociceptive effect elicited by ghrelin (i.c.v., 1 nmol) was significantly antagonized by opioid receptor antagonist naloxone (i.c.v., 10 nmol co-injection or i.p., 10 mg/kg, 10 min prior to ghrelin) in both tail withdrawal test and hot-plate test. At these doses, naloxone significantly antagonized the antinociceptive effect induced by morphine (i.c.v., 3 nmol). Ghrelin (i.c.v., 1 nmol)-induced antinociception was significantly antagonized by co-injection with 10 nmol [d-Lys3]-GHRP-6, the selective antagonist of GHS-R1a identified more recently, while [d-Lys3]-GHRP-6 (10 nmol) alone induced neither hyperalgesia nor antinociception. Overall this data indicate that ghrelin could produce antinociception through an interaction with GHS-R1a and with the central opioid system. Thus ghrelin may be a promising peptide for developing new analgesic drugs.     

Roles of Peripheral P2X and P2Y Receptors in the Development of Melittin-Induced Nociception and Hypersensitivity

A recent report from our laboratory shows that subcutaneous (s.c.) injection of melittin could induce persistent spontaneous nociception (PSN) and primary thermal or mechanical hyperalgesia. However, the exact peripheral mechanisms underlying melittin-induced multiple pain-related behaviors remain unclear. In this study, behavioral tests combined with pharmacological manipulations were used to explore potential roles of local P2X and P2Y receptors in melittin-induced inflammatory pain and hyperalgesia. Post-treatment of the primary injury site with s.c. injection of A-317491 (a potent P2X₃/P2X_2/3 receptor antagonist) and Reactive Blue 2 (a potent P2Y receptor antagonist) could significantly suppress the development of melittin-evoked PSN and hypersensitivity (thermal and mechanical). Our control experiments demonstrated that local administration of either antagonist into the contralateral hindpaw produced no significant effect on any kind of pain-associated behaviors. Taken together, these data indicate that activation of P2X and P2Y receptors might be essential to the maintenance of melittin-induced primary thermal and mechanical hyperalgesia as well as on-going pain. Z.-M. Lu and F. Xie are contributed equally to the work. Special issue article in honor of Dr. Ji-Sheng Han.     

Antibodies raised against the N-terminal sequence of δ opioid receptors blocjed δ-mediated supraspinal antinociception in mice

A polyclonal antiserum directed against the first 16 aminoacids of the N-terminal sequence of the murine δ opioid receptor was raised in rabbits. The intracerebroventricular (i.c.v.) injection to mice of the anti δ receptor IgGs impaired the antinociception produced by DPDPE, [D-Ala²]- Deltorphin II, DADLE and β-endorphin-(1–31) when studied 24 h later in the tail-flick test. Antinociception produced by morphine and DAMGO was fully expressed in mice undergoing this treatment. The selective δ antagonist ICI 174864 (0.8 nmols/mouse, i.c.v.) significantly reduced the antinociceptive activity of opioids to the extent observed after giving the antibodies. ICI 174864 did not decrease further the antinociception that remained after the anti δ receptor serum. The specific binding displayed by 3 nM [³H]-DPDPE was reduced in membranes pre-incubated with the antiserum, whereas no change could be detected for 0.6 nM [³H]-DAMGO labelling μ receptors. This experimental approach revealed the δ component of opioid-evoked supraspinal antinociception in mice.     

Antinociceptive profile of LP1, a non-peptide multitarget opioid ligand

AimsOpioid drugs are the principal treatment option for moderate to severe pain and exert their biological effects through interactions with opioid receptors that are widely distributed throughout the CNS and peripheral tissues. Ligands capable of simultaneously targeting different receptors could be successful candidates for the treatment of chronic pain. Enhanced antinociception coupled with a low incidence of side effects has been demonstrated for ligands possessing mixed mu-opioid receptor (MOR) and delta-opioid receptor (DOR) activity. We previously reported that 3-[(2R,6R,11R)-8-hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2 H)-yl]-N-phenylpropanamide (LP1) acted as a MOR-DOR ligand in in vitro functional assays and moreover this drug produced a valid antinociception that was longer lasting than that of morphine. The aim of this work was to determine whether the antinociceptive effect produced by LP1 was central or peripheral and to assess which opioid receptor subtypes are involved in its effects.Main methodsWe explored the effects of naloxone methiodide (NX-M), a quaternary opioid antagonist, administered either intracerebroventricularly (i.c.v.) or subcutaneously (s.c.), on LP1-mediated antinociception in male Sprague–Dawley rats. In addition, we administered s.c. selective antagonists for MOR, DOR and kappa-opioid receptor (KOR) to investigate the effects of LP1. To characterise this drug's DOR profile better, we also investigated the effects of LP1 on DPDPE, a selective DOR agonist.Key findingsData obtained by tail flick test showed that LP1 induced predominantly MOR-mediated supraspinal antinociception and was able to counteract DPDPE analgesia.SignificanceLP1, a multitarget opioid ligand, is a supraspinal acting antinociceptive agent that is useful for the treatment of chronic pain.     

Possible involvement of mu1-opioid receptors in the fentanyl- or morphine-induced antinociception at supraspinal and spinal sites

Fentanyl has been shown to be a potent analgesic with a lower propensity to produce tolerance and physical dependence in the clinical setting. The present study was designed to investigate the mechanisms of fentanyl- or morphine-induced antinociception at both supraspinal and spinal sites. In the mouse tail-flick test, the antinociceptive effects induced by both fentanyl and morphine were blocked by either the mu1-opioid receptor antagonist naloxonazine or the mu1/mu2-opioid receptor antagonist beta-funaltrexamine (beta-FNA) after s.c., i.c.v. or i.t. injection. In contrast, both fentanyl and morphine given i.c.v. or i.t. failed to produce antinociception in mu1-deficient CXBK mice. These findings indicate that like morphine, the antinociception induced by fentanyl may be mediated predominantly through mu1-opioid receptors at both supraspinal and spinal sites in mice. We also determined the ED50 values for s.c.-, i.c.v.- and i.t.-administered fentanyl- or morphine-induced antinociception in mice. The ED50 values for s.c.-, i.c.v.- and i.t.-administered fentanyl-induced antinociception were 73.7, 18.5 and 1.2-fold lower than that of morphine, respectively. The present data clearly suggest the usefulness of peripheral treatment with fentanyl for the control of pain.     

Endomorphin 1[psi] and endomorphin 2[psi], endomorphins analogues containing a reduced (CH2NH) amide bond between Tyr1 and Pro2, display partial agonist potency but significant antinociception

Endomorphin 1 (EM1) and endomorphin 2 (EM2) are highly potent and selective mu-opioid receptor agonists and have significant antinociceptive action. In the mu-selective pocket of endomorphins (EMs), Pro2 residue is a spacer and directs the Tyr1 and Trp3/Phe3 side chains into the required orientation. The present work was designed to substitute the peptide bond between Tyr1 and Pro2 of EMs with a reduced (CH2NH) bond and study the agonist potency and antinociception of EM1[psi] (Tyr[psi(CH2NH)]Pro-Trp-Phe-NH2) and EM2[psi] (Tyr[psi(CH2NH)]Pro-Phe-Phe-NH2). Both EM1[psi] and EM2[psi] are partial mu opioid receptor agonists showing significant loss of agonist potency in GPI assay. However, EMs[psi] exhibited potent supraspinal antinociceptive action in vivo. In the mice tail-flick test, EMs[psi] (1, 5, 10 nmol/mouse, i.c.v.) produced potent and short-lasting antinociception in a dose-dependent and naloxone (1 mg/kg) reversed manner. At the highest dose of 10 nmol, the effect of EM2[psi] was prolonged and more significant than that of EM2. In the rat model of formalin injection induced inflammatory pain, EMs[psi] (0.1, 1, 10 nmol/rat, i.c.v.), like EMs, exerted transient but not dose-dependent antinociception. These results suggested that in the mu-selective pocket of EMs, the rigid conformation induced by the peptide bond between Tyr1 and Pro2 is essential to regulate their agonist properties at the mu opioid receptors. However, the increased conformational flexibility induced by the reduced (CH2NH) bond made less influence on their antinociception.     

Study on the molecular mechanism of antinociception induced by ghrelin in acute pain in mice

Ghrelin has been identified as the endogenous ligand for the GHS-R1α (growth hormone secretagogue receptor 1 alpha). Our previous experiments have indicated that ghrelin (i.c.v.) induces antinociceptive effects in acute pain in mice, and the effects were mediated through the central opioid receptors and GHS-R1α. However, which opioid receptor (OR) mediates the antinociceptive effects and the molecular mechanisms are also needed to be further explored. In the present study, the antinociceptive effects of ghrelin (i.c.v.) could be fully antagonized by δ-opioid receptor antagonist NTI. Furthermore, the mRNA and protein levels of δ-opioid peptide PENK and δ-opioid receptor OPRD were increased after i.c.v injection of ghrelin. Thus, it showed that the antinociception of ghrelin was correlated with the GHS-R1α and δ-opioid receptors. To explore which receptor was firstly activated by ghrelin, GHS-R1α antagonist [D-Lys³]-GHRP-6 was co-injection (i.c.v.) with deltorphin II (selective δ-opioid receptor agonist). Finally, the antinociception induced by deltorphin II wasn’t blocked by the co-injection (i.c.v.) of [D-Lys³]-GHRP-6, indicating that the GHS-R1α isn’t on the backward position of δ-opioid receptor. The results suggested that i.c.v. injection of ghrelin initially activated the GHS-R1α, which in turn increased the release of endogenous PENK to activation of OPRD to produce antinociception.     

Synthesis and antinociceptive effects of endomorphin-1 analogs with C-terminal linked by oligoarginine

Endomorphins (EMs) cannot be delivered into the central nervous system (CNS) in sufficient quantity to elicit antinociception when given systemically because they are severely restricted by the blood-brain barrier (BBB). In the present study, we investigated herein a series of EM-1 analogs with C-terminal linked by oligoarginine in order to improve the brain delivery and antinociception after systemic administration. Indeed, all these analogs decreased the opioid receptor affinity and in vitro pharmacological activity. Moreover, analogs 4, 7-9 produced a less potent antinociceptive activity after intracerebroventricular (i.c.v.) administration, with the ED₅₀ values about 11- to 13-fold lower potencies than that of EM-1. Nevertheless, our results revealed that EM-1 failed to induce any significant antinociception at a dose of 50 μmol/kg after subcutaneous (s.c.) administration, whereas equimolar dose of these four analogs produced a little low but significant antinociceptive effects. Naloxone (10 nmol/kg, i.c.v.) significantly blocked the antinociceptive effects, indicating an opioid and central mechanism. These results demonstrated that C-terminal of EM-1 linked to oligoarginine improved the brain delivery, eliciting potent antinociception following peripheral administration.     

An examination of the relationship between μ-opioid antinociceptive efficacy and G-protein coupling using pertussis and cholera toxins

The hypothesis that μ-opioid agonists having low antinociceptive efficacy might be more susceptible to interference with G-protein coupling than μ-opioid agonists having higher antinocicep-tive efficacy was tested. Supraspinal antinociceptive efficacy for the three μ-opioid agonists morphine, [D-Ala², NMePhe⁴, Gly⁵-ol]-enkephalin (DAMGO) and sufentanil in the mouse 55 °C warm-water tail-flick test was evaluated 18–24 h after intracerebroventricular (i.c.v.) administration of β-funaltrexamine (β-FNA). The β-FNA pretreatment (0.2–2.0 nmol) attenuated antinociception in the order morphine > DAMGO > sufentanil, consistent with previous reports of their relative antinociceptive efficacy. The association of efficacy with G-protein coupling was then assessed by determining sensitivity to i.c.v. (0.1–3.0 μg) pertussis toxin (PTX) or cholera toxin (CTX). The effect of PTX on equiantinociceptive doses was in the inverse order of agonist efficacy. CTX augmented sufentanil-induced antinociception. Morphine- and DAMGO-induced antinociception were unaffected by CTX. These data suggest that: (i) highly efficacious μ agonists (viz., sufentanil) couple more efficiently to PTX-sensitive inhibitory G_i-proteins than do agonists of lower efficacy (viz., morphine, DAMGO) and (ii) highly efficacious μ agonists have greater capacity to utilize CTX-sensitive stimulatory G_s-proteins than do μ-agonists with lower efficacy.     

P2X3 receptors contribute to transition from acute to chronic muscle pain

This study aimed to evaluate whether the development and/or maintenance of chronic-latent muscle hyperalgesia is modulated by P2X3 receptors. We also evaluate the expression of P2X3 receptors and PKCε of dorsal root ganglions during these processes. A mouse model of chronic-latent muscle hyperalgesia, induced by carrageenan and evidenced by PGE₂, was used. Mechanical muscle hyperalgesia was measured by Randall-Selitto analgesimeter. The involvement of P2X3 receptors was analyzed by using the selective P2X3 receptors antagonist A-317491 by intramuscular or intrathecal injections. Expression of P2X3 and PKCε in dorsal root ganglion (L4-S1) were evaluated by Western blotting. Intrathecal blockade of P2X3 receptors previously to carrageenan prevented the development and maintenance of acute and chronic-latent muscle hyperalgesia, while intramuscular blockade of P2X3 receptors previously to carrageenan only reduced the acute muscle hyperalgesia and had no effect on chronic-latent muscle hyperalgesia. Intrathecal, but not intramuscular, blockade of P2X3 receptors immediately before PGE₂, in animals previously sensitized by carrageenan, reversed the chronic-latent muscle hyperalgesia. There was an increase in total and phosphorylated PKCε 48 h after the beginning of acute muscle hyperalgesia, and in P2X3 receptors at the period of chronic muscle hyperalgesia. P2X3 receptors expressed on spinal cord dorsal horn contribute to transition from acute to chronic muscle pain. We also suggest an interaction of PKCε and P2X3 receptors in this process. Therefore, we point out P2X3 receptors of the spinal cord dorsal horn as a pharmacological target to prevent the development or reverse the chronic muscle pain conditions.

     

Involvement of spinal release of α-neo-endorphin on the antinociceptive effect of TAPA

The antinociceptive effect of i.t.-administered Tyr-d-Arg-Phe-β-Ala (TAPA), an N-terminal tetrapeptide analog of dermorphin, was characterized in ddY mice. In the mouse tail-flick test, TAPA administered i.t. produced a potent antinociception. The antinociception induced by TAPA was significantly attenuated by i.t. pretreatment with the κ-opioid receptor antagonist nor-binaltorphimine, as well as by the μ-opioid receptor antagonist β-funaltrexamine and the μ₁-opioid receptor antagonist naloxonazine. TAPA-induced antinociception was also significantly suppressed by co-administration of the μ₁-opioid receptor antagonist Tyr-d-Pro-Phe-Phe-NH₂ (d-Pro²-endomorphin-2) but not by co-administration of the μ₂-opioid receptor antagonists Tyr-d-Pro-Trp-Phe-NH₂ (d-Pro²-endomorphin-1) and Tyr-d-Pro-Trp-Gly-NH₂ (d-Pro²-Tyr-W-MIF-1). In CXBK mice whose μ₁-opioid receptors were naturally reduced, the antinociceptive effect of TAPA was markedly suppressed compared to the parental strain C57BL/6ByJ mice. Moreover, the antinociception induced by TAPA was significantly attenuated by i.t. pretreatment with antiserum against the endogenous κ-opioid peptide α-neo-endorphin but not antisera against other endogenous opioid peptides. In prodynorphin-deficient mice, the antinociceptive effect of TAPA was significantly reduced compared to wild-type mice. These results suggest that the spinal antinociception induced by TAPA is mediated in part through the release of α-neo-endorphin in the spinal cord via activation of spinal μ₁-opioid receptors.     

Antinociception induced by beta-lactotensin,a neurotensin agonist peptide derived from beta-lactoglobulin,is mediated by NT2 and D1 receptors

In this study, we examined the antinociceptive effect of beta-lactotensin, a neurotensin agonist that has been isolated from the chymotrypsin digest of beta-lactoglobulin as an ileum-contracting peptide. Beta-lactotensin showed naloxone-insensitive antinociceptive activity by the tail-pinch test after i.c.v. (200 nmol/mouse) or s.c. (300 mg/kg) administration in ddY mice. Tolerance was not developed to antinociception induced by beta-lactotensin after repeated s.c. administration for 5 days. The antinociceptive activity of beta-lactotensin was blocked by treatment with the neurotensin NT2 receptor antisense ODN, while treatment with the NT1 receptor antisense ODN had no effect. The antinociceptive activity was also blocked by a dopamine D1 receptor antagonist, SCH23390 (1 microg/mouse, i.c.v.), while a D2 receptor antagonist, raclopride (0.5 microg/mouse, i.c.v.), did not block the activity. These results indicate that the antinociceptive activity of beta-lactotensin is mediated by NT2 and D1 receptors.