Analgesic activity of dipeptide Tyr-Pro

The effect of dipeptide Tyr-Pro, present in representatives of most families of opioid peptides, and two its analogs, Tyr-Pro-NH₂ and Tyr-Pro-OMe, on analgesic activity was studied in different tests (tail-flick test, tail pinch (Haffner’s) test, formalin test, and acetic acid writing test) describing different organization levels of pain sensitivity. Intraperitoneal administration of the dipeptide decreased the pain threshold in all above-mentioned tests. Coadministration of the dipeptide and naloxone or naloxone methiodide insignificantly decreased the dipeptide analgesic effect in the tail-flick and acetic acid writing tests. Its analogs Tyr-Pro-NH₂ and Tyr-Pro-OMe demonstrated a similar analgesic activity in the tail-flick test and a higher activity in the acetic acid writing test. Administration of individual amino acids (Tyr or Pro) or their mixture had no effect on the pain threshold.     

Experimental data regarding the implications of certain minimum structure enkephalin-like peptides in nociceptive processing.

The aim of this study was to investigate the importance of the amino acidic sequence at N-terminal end of certain minimum structure enkephalin-like peptides for the analgesic activity. Different groups of mice or rats were treated with 1) L-tyrosine (i.p. 200 mg/kg), 2) Tyr-Phe (i.t. 0.5 mg/rat), 3) Tyr-Pro-Phe (i.t. 0.5 mg/rat), 4) Gly-Tyr (i.t. 0.5 mg/rat), 5) Tyr-Gly-Gly (i.t. 0.5 mg/rat). Different tests were utilized to evaluate the antinociceptive effect of the substances tested: thermal nociception (hot plate test, plantar test), mechanical nociception (analgesymeter test). Tyr-Pro-Phe, Tyr-Gly-Gly, Tyr-Phe, but not Gly-Tyr, elicited analgesic activity. So, the presumption made in the case of atypical opioid peptides that opioid-like activity in case of peptides presumes a tyrosine residue at the N-terminal sequence, applies for shorter peptides. It appears also that minimal structure brain peptides with an N-terminal Tyr-Pro, rather than the Tyr-Gly-Gly-Phe sequence typical of other endogenous opioids, can provide better affinity for the opioid receptors and stronger analgesic activity. The inhibition of their analgesic effect by previous administration of naloxone proves that this effect is mediated through the endogenous opioid system.     

Analgesic effects of N-acetyl-5HTP-5HTP amide are not directly related to brain serotonin levels.

A synthetic dipeptide, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide, was shown previously to inhibit the binding of serotonin to a soluble specific serotonin binding protein as well as to alter brain serotonin levels. When injected into rats intraventricularly, the dipeptide caused an increase in pain threshold, lasting for several hours, as determined by either a flinch-jump test or a tail-flick test. This effect was reversed by naloxone. The dipeptide is a very weak inhibitor of the binding of labelled naloxone or dihydromorphine to a membranous opiate receptor preparation. The analgesic activity of the dipeptide was not diminished by p-chlorophenylalanine or the setonergic neurotoxin 5,7-dihydroxytryptamine, which depleted brain serotonin levels. This implies that the analgesic action of the dipeptide is not mediated directly by its effect on serotonin concentration.     

Kyotorphin has a novel action on rat cardiac muscle

Several endogenous peptides for G-protein-coupled receptors have been found to play physiological roles in muscle contraction in addition to their well-demonstrated actions in other tissues. To further identify such peptides, we screened over 400 peptides using an isometric tension assay of rat papillary muscle. Here, we report that kyotorphin, which is known as an analgesic dipeptide, has a cardiac effect. Although kyotorphin had no effect on the twitch tension itself, it inhibited beta-adrenergic agonist isoprenaline-induced increases in twitch tension in a dose-dependent manner. Leu-Arg, a selective antagonist of kyotorphin, reversed this inhibitory effect. The inhibitory effect was also reversed by naloxone, an opioid receptor antagonist. These results suggest that kyotorphin may release opioid peptides from rat cardiac muscle and have an indirect regulatory role in beta-adrenergic action through cross-talk with opioid receptors.     

Reversal of morphine, methadone and heroin induced effects in mice by naloxone methiodide

Opioid overdose, which is commonly associated with opioid induced respiratory depression, is a problem with both therapeutic and illicit opioid use. While the central mechanisms involved in the effects of opioids are well described, it has also been suggested that a peripheral component may contribute to the effects observed. This study aimed to further characterise the effects of the peripherally acting naloxone methiodide on the respiratory, analgesic and withdrawal effects produced by various opioid agonists. A comparison of the respiratory and analgesic effects of morphine, methadone and heroin in male Swiss-Albino mice was conducted and respiratory depressive ED(80) doses of each opioid determined. These doses (morphine 9 mg/kg i.p., methadone 7 mg/kg i.p., and heroin 17 mg/kg i.p.) were then used to show that both naloxone (3 mg/kg i.p.) and naloxone methiodide (30-100 mg/kg i.p.) could reverse the respiratory and analgesic effects of these opioid agonists, but only naloxone precipitated withdrawal. Further investigation in female C57BL/6J mice using barometric plethysmography found that both opioid antagonists could reverse methadone induced decreases in respiratory rate and increases in tidal volume. Its effects do not appear to be strain or sex dependent. It was concluded that naloxone methiodide can reverse the respiratory and analgesic actions of a variety of opioid agonists, without inducing opioid withdrawal.     

Modification of the effects of naloxone in morphine-dependent mice

Mice were rendered dependent on morphine by mixing morphine with their food (2 mg/g) for three days. Increasing doses of naloxone precipitated dose-dependent withdrawal reactions such as weight loss and jumping. These withdrawal reactions were antagonized by morphine pretreatment. Effects of morphine, such as increased locomotor activity, inhibition of intestinal transport, and analgesia were antagonized by naloxone in both non-dependent and dependent subjects. The antagonist actions of naloxone were increased in dependent subjects; lower doses of naloxone were sufficient to antagonize effects of morphine. The present results confirm earlier studies indicating that precipitation of withdrawal can be antagonized by morphine pretreatment suggesting that withdrawal reactions are due to actions of naloxone at the same receptor at which opioid agonists act. The increased antagonist potency of naloxone in dependent subjects extends earlier results obtained with analgesic effects to several other agonist effects of morphine and is consistent with the interpretation that exposure to an opioid agonist induces a change in the conformation of opioid receptors.     

Kentsin: tetrapeptide from hamster embryos produces naloxone-sensitive effects without binding to opioid receptors

The opioid nature of kentsin (Thr-Pro-Arg-Lys) and its ability to alter pain perception and intestinal transit were examined. Kentsin (30,000 nM) did not inhibit electrically stimulated contractions of the guinea pig ileum (GPI) or mouse vas deferens (MVD), nor did it cause a rightward displacement of the inhibitory concentration-response curves of the mu-selective opioid agonist PL017 in the GPI or the delta-selective agonist DPDPE in the MVD. Kentsin (10,000 nM) did not displace [3H] naloxone from rat brain homogenates. These results indicate that kentsin lacks opioid agonist and mu and delta opioid antagonist properties and does not bind to opioid receptors. In vivo, kentsin produced dose-dependent analgesia in both the hotplate and abdominal stretch tests when administered intracerebroventricularly (ICV) and intrathecally but not intravenously. The central analgesic effect of kentsin was partially antagonized by the opioid antagonist naloxone. Kentsin inhibited intestinal transit in a dose-dependent manner after ICV administration only. The intestinal antitransit effect of kentsin was not blocked by pretreatment with naloxone. These results suggest that kentsin acts centrally to produce both opioid and non-opioid effects. Further, the opioid-mediated analgesic effects of kentsin involve mechanisms other than direct interaction with opioid receptors.     

Peripheral orphanin FQ/nociceptin analgesia in the mouse.

Orphanin FQ/Nociceptin (OFQ/N) administered peripherally was an effective analgesic in the tailflick test in mice (ED50 16.3 microg). It had a peak effect at 5 min and lasted up to 30 min. The kappa3 analgesic naloxone benzoylhydrazone was also active peripherally (ED50 3.8 microg). The analgesic actions of both agents were blocked by naloxone. Neither OFQ/N(1-11) nor OFQ/N(1-7) had appreciable peripheral activity. Antisense mapping both compounds against the murine orphan opioid receptor (KOR-3) confirmed the importance of this clone in their actions. Antisense probes targeting the second and third coding exons significantly lowered the analgesic effects of both compounds. However, the antisense targeting the first coding exon blocked only the actions of OFQ/N and not kappa3 analgesia.     

Analgesic activity and selectivity of isothiocyanate derivatives of fentanyl analogs for opioid receptors

The analgesic activity and opioid receptor binding characteristics were studied for the isothiocyanate ohmefentanyl (OMFIT), and isothiocyanate carfentanil (CarFIT), isothiocyanate 4-methoxymethylfentanyl (MethoFIT), isothiocyanate 3-methylfentanyl (superFIT) and their amide analogs. Antinociceptive activity was evaluated using the mouse hot plate test; selectivity for opioid receptor was determined in bioassay and binding assay. SuperFIT, CarFIT, OMFIT and MethoFIT exhibited an analgesic ED50 lower than those of their parent compounds without isothiocyanate (SCN) group. Furthermore these compounds exhibited potent inhibitory actions on the electrically evoked contractions of mouse vas deferens, which could be antagonized by naloxone, but their actions were weaker than those of their parent compounds without SC N-group. The inhibitory actions of these compounds on binding of [3H]OMF to mouse brain membrane was weaker than those of their parent compounds without SCN-group. CarFIT and MethoFIT showed weaker inhibitory actions on the binding of [3H] DADLE than their parent compounds without SCN-group, but SuperFIT and OMFIT stronger than their parent compounds, 3-methylfentanyl and ohmefentanyl. The selectivity of these isothiocyanate derivatives for delta opioid receptors increased. In conclusion, introducing isothiocyanato-group into 1-position of phenyl ring of ohmefentanyl and other fentanyl analogs would enhance the selectivity of these compounds for delta-opioid receptors, but decrease their analgesic activity.     

The effect of opiates and opiate antagonists on heat latency response in the parasitic nematode Ascaris suum

The effects of the opiates morphine and morphine-6-glucuronide (M6G), the mu opioid receptor specific antagonist D-Phe-Cys-Tyr-D-Trp-Om-Thr-Pen-Thr-NH(2) (CTOP), and the general opiate antagonist naloxone on the latency of response to thermal stimulation were determined in the parasitic nematode Ascaris suum. Thermal detection and avoidance behaviors of the worms were evaluated with a tail flick analgesia meter using a modification of a technique employed for nociception experiments in rodents. Morphine and M6G were shown to have a dose dependent analgesic effect on A. suum's latency of response to heat with morphine being the most potent. The analgesic effect of morphine was reversed by naloxone but not CTOP. Neither naloxone nor CTOP was able to block the analgesia of M6G. CTOP but not naloxone had significant analgesic effects on its own. These findings are generally consistent with previous results on the effects of opiates and nitric oxide release from A. suum tissue. Apparently these nematodes possess opioid receptors that effect nociception.     

不同功能位点介导α干扰素的免疫调节和中枢镇痛作用

细胞因子α干扰素（ＩＦＮα）具有中枢镇痛作用。抗内源性阿片肽血清与ＩＦＮα能发生明显的交叉反应,提示ＩＦＮα与内源性阿片肽之间存在着共同的抗原决定基。采用基因定点突变技术,获得系列ＩＦＮα突变体,并分别测定其免疫学活性和镇痛能力。结果显示,ＩＦＮα突变体Ｙ１２９Ｓ－ＩＦＮα免疫学活性显著下降,但仍然保留了很强的镇痛能力,阿片受体拮抗纳洛酮能够阻断Ｙ１２９Ｓ－ＩＦＮα的镇痛作用。实验结果表明,ＩＦＮ     

The antinociceptive effect of tramadol on a model of neuropathic pain in rats

The antinociceptive activity of tramadol was investigated on the vocalization threshold to paw pressure in a rat model of unilateral mononeuropathy produced by loose ligatures around the common sciatic nerve. Despite the analgesic activity of tramadol was clearly established in motor and sensory responses of the nociceptive system in rats, the effect of this atypical opioid on experimental neuropathic pain models is not investigated. The intraperitoneally injected tramadol (2.5, 5 and 10 mg/kg) produced a potent and dose-dependent antinociceptive effect on both lesioned and non-lesioned hind paws. However, the analgesic effect on the lesioned paw was significantly more potent than the non-lesioned paw. This effect was partially antagonized by intraperitoneally administered naloxone (0.1 mg/kg) suggesting an additional non-opioid mechanism. Our results suggest that tramadol may be useful for the alleviation of some symptoms in peripheral neuropathic conditions     

Augmented analgesic effects of L-tryptophan combined with low current transcranial electrostimulation

The analgesic effects of low current transcranial electrostimulation are both naloxone and pCPA-reversible, suggesting that they may be mediated in part by endogenous opioid and serotonergic activity. The present experiments indicate that pretreatment with the serotonin precursor L-tryptophan results in an increased analgesic effect of electrostimulation as measured by the 50 degrees C wet tail flick test in the rat. Rats receiving both L-tryptophan and electrostimulation displayed significantly more analgesia than rats receiving electrostimulation and injection vehicle alone, rats receiving drug and sham stimulation or rats receiving vehicle and sham stimulation.     

Effect of bone marrow opioid peptides on antibody formation in the productive phase of the immune response

The cells of intact spinal cord produce a group of biologically active peptides--myelopeptides (MP) stimulating antibody formation at peak of immune response and exerting an analgesic endorphin-like effect. The experiments on comparative studies of antibody-stimulating effect of synthetic opioid peptides and MP have shown that the mixture of opioid substances composed in aliquots corresponding to their content in MP has an antibody-stimulating effect similar to that of MP. Synthetic beta-endorphin also enhances the antibody formation during the productive phase of immune response at doses 1000-fold lower than its MP level. Leu- and met-enkephalins have no antibody-stimulating effect. An antagonist opiate, naloxone, blocks the antibody-stimulating activity of both opiates and MP. A close correlation between antibody-stimulating and analgetic endorphin-like MP activity has been established.     

Analgesia induced by intrathecal injection of dynorphin B in the rat

A dose-dependent analgesic effect of intrathecally injected dynorphin B was observed in rats using the tail flick as nociceptive test. Intrathecal injection of 20 nmol of dynorphin B increased the tail flick latency by 90 +/- 23%, an effect that lasted about 90 min. For the same degree of analgesia, dynorphin B was 50% more potent than morphine on a molar basis. The analgesic effect of this dose of dynorphin B was partially blocked by 10 mg/kg, but not by 1 mg/kg, of subcutaneous naloxone, showing a relative resistance to naloxone reversal as compared with morphine analgesia. The analgesia produced by dynorphin B was unchanged in morphine-tolerant rats but was significantly decreased in rats tolerant to ethylketazocine. These results suggest that dynorphin B produces its potent analgesic effect by activation of kappa rather than mu opioid receptors in the rat spinal cord.     

Evidence for activation of endogenous opioid systems in mice following short exposure to stable flies

Biting flies influence both physiology and behaviour of domestic and wild animals. This study demonstrates that brief (30 min) exposure of male and female mice to stable flies leads to significant increases in nociceptive responses, indicative of the induction of analgesia. The biting fly-induced analgesia was mediated by endogenous opioid systems as it was blocked by the prototypic opiate antagonist naloxone. Exposure for 30 min to the bedding of biting fly-exposed mice also induced significant opioid mediated analgesic responses in mice. Exposure to either house flies or the bedding of house fly-exposed mice had no significant effects on nociception. These results indicate that brief exposure to either stable flies, or to olfactory cues associated with mice exposed to stable flies, activates endogenous opioid systems leading to the induction of analgesia and likely other opioid mediated behavioural and physiological stress responses. These results suggest the involvement of endogenous opioid systems in the mediation of the behavioural and physiological consequences of biting fly exposure in domestic and wild animals.     

Analgesic activity of morphiceptin, beta-casomorphin-4, and deltakephalin in normotensive Wistar-Glaxo and spontaneously hypertensive rats

The effects of intraventricular injection of beta-casomorphin-4, morphiceptin and deltakephalin (DTLET) on hot water tail flick and tail compression responses were investigated in Wistar Albino Glaxo (WAG) and spontaneously hypertensive rats (SHR). The effects of the mu agonist morphiceptin (20 nmol/rat), as assessed by the tail compression test, were significantly greater in SHR rats but did not differ between both strains when measured by tail flick latency. Opioid agonist deltakephalin (2 nmol/rat) in both tests elicited stronger analgesic effects in SHR as compared to WAG and these effects were blocked by naloxone in both tests used. beta-Casomorphin-4 exhibits moderate activity for mu receptors. In the tail flick test peptide (60 nmol/rat) produced an increase in latencies in SHR rats that was significantly greater than was observed in WAG rats. Naloxone pretreatment abolished the analgesic activity of beta-casomorphin-4 solely in the tail compression test in SHR. Analysis of the slopes of the dose-response curves seems to suggest that differences between the activity of these opioid peptides in SHR and WAG rats are based on a difference in the density and affinity of the subpopulation of the opioid receptors in these strains of rats.     

Suppression of appetitive behavior in the rat by naloxone: lack of effect of prior morphine dependence

Naloxone (0.3–10 mg/kg) produced a dose-related suppression of eating and drinking in rats that had been deprived of food for 48 hr or water for 24 hr. The suppression of water intake by naloxone was unaltered in rats that had been physically dependent upon morphine one week earlier and which were tolerant to the analgesic effect of morphine at the time naloxone was tested. These results confirm the ability of naloxone to suppress appetitive behavior in the rat but do not resolve the issue of whether or not this effect of naloxone is the consequence of an interaction with an endogenous opioid system.     

Analgesic effect of metoclopramide and its mechanism

Metoclopramide produced a significant analgesic effect when tested by both acetic acid induced writhing and hot plate test. This effect was reduced by naloxone suggesting opioid involvement. Further, bromocriptine which inhibits the release of PRL attenuated the effect of metoclopramide indicating that this drug could act by releasing PRL. The unaltered analgesic effect of metoclopramide by yohimbine reveals that alpha-2 adrenoceptors may not be involved in this action.     

High-affinity naloxone binding to filamin a prevents mu opioid receptor-Gs coupling underlying opioid tolerance and dependence

Ultra-low-dose opioid antagonists enhance opioid analgesia and reduce analgesic tolerance and dependence by preventing a G protein coupling switch (Gi/o to Gs) by the mu opioid receptor (MOR), although the binding site of such ultra-low-dose opioid antagonists was previously unknown. Here we show that with approximately 200-fold higher affinity than for the mu opioid receptor, naloxone binds a pentapeptide segment of the scaffolding protein filamin A, known to interact with the mu opioid receptor, to disrupt its chronic opioid-induced Gs coupling. Naloxone binding to filamin A is demonstrated by the absence of [(3)H]-and FITC-naloxone binding in the melanoma M2 cell line that does not contain filamin or MOR, contrasting with strong [(3)H]naloxone binding to its filamin A-transfected subclone A7 or to immunopurified filamin A. Naloxone binding to A7 cells was displaced by naltrexone but not by morphine, indicating a target distinct from opioid receptors and perhaps unique to naloxone and its analogs. The intracellular location of this binding site was confirmed by FITC-NLX binding in intact A7 cells. Overlapping peptide fragments from c-terminal filamin A revealed filamin A(2561-2565) as the binding site, and an alanine scan of this pentapeptide revealed an essential mid-point lysine. Finally, in organotypic striatal slice cultures, peptide fragments containing filamin A(2561-2565) abolished the prevention by 10 pM naloxone of both the chronic morphine-induced mu opioid receptor-Gs coupling and the downstream cAMP excitatory signal. These results establish filamin A as the target for ultra-low-dose opioid antagonists previously shown to enhance opioid analgesia and to prevent opioid tolerance and dependence.