Endomorphin-1 Research Articles

Endomorphins, endogenous opioid peptides, induce apoptosis in human leukemia HL-60 cells

Opioids play a role in the apoptosis machinery. We studied the induction of apoptosis in endomorphin 1 (EM1) and endomorphin 2 (EM2), 2 newly isolated endogenous mu-opioid receptor agonists. These endomorphins were able to reduce the viability of cultured HL-60 cells. The antiproliferative properties of endomorphins appeared to be attributable to their induction of apoptotic cell death as determined by ultrastructural change, internucleosomal DNA fragmentation, and increased proportion of the subdiploid cell population. To elucidate molecular events in the apoptosis, protein expressions of Bcl-2, Bax, Fas, and FasL were measured by western blotting using specific antibodies in HL-60 cells. The level of Bcl-2 indicated down-regulation, but the Bax, Fas, and FasL expression showed up-regulation as compared with the untreated control cells. These data support the idea that endomorphins induce apoptosis in HL-60 cells through the activation of the Bcl-2-Bax and the Fas-FasL pathway. We suggest that endomorphins may play an important role in the regulation of tumor cell death.

Internalization and down-regulation of mu opioid receptors by endomorphins and morphine in SH-SY5Y human neuroblastoma cells

The human neuroblastoma cell line, SH-SY5Y, was used to examine the effects of morphine and the endogenous opioid peptides, endomorphin-1 (EM-1) and endomorphin-2 (EM-2), on mu opioid receptor (MOR) internalization and down-regulation. Treatment for 24 h with EM-1, EM-2 or morphine at 100 nM, 1 μM and 10 μM resulted in a dose-dependent down-regulation of mu receptors. Exposure of cells to 10 μM EM-1 for 2.5, 5 and 24 h resulted in a time-dependent down-regulation of mu receptors. Down-regulation of mu receptors by morphine and EM-1 was blocked by treatment with hypertonic sucrose, consistent with an endocytosis-dependent mechanism. Sensitive cell-surface binding studies with a radiolabeled mu antagonist revealed that morphine was able to induce internalization of mu receptors naturally expressed in SH-SY5Y cells. EM-1 produced a more rapid internalization of mu receptors than morphine, but hypertonic sucrose blocked the internalization induced by each of these agonists. This study demonstrates that, like morphine, the endomorphins down-regulate mu opioid receptors in a dose- and time-dependent manner. This study also demonstrates that morphine, as well as EM-1, can induce rapid, endocytosis-dependent internalization of mu opioid receptors in SH-SY5Y cells. These results may help elucidate the ability of mu agonists to regulate the number and responsiveness of their receptors.

P.5.034 Czech university students and drugs of abuse:Preliminary results of urinalysis

In 1997, endomorphin-1 (EM-1) and -2 (EM-2) were identified as the most specific endogenous μ-opioid ligands. These two peptides have shown analgesic effects and many other opioid functions. In the present study, we attempt to investigate the possible ability of endomorphins to induce naloxone-precipitated withdrawal in comparison with that induced by morphine. Using the previously established scoring system in rats, 12 withdrawal signs (chewing, sniffing, grooming, wet-dog shakes, stretching, yawning, rearing, jumping, teeth grinding, ptosis, diarrhea, and penile erection) were observed and scored following naloxone (4 mg/kg, i.p.) challenge. Compared with the sham control, EM-1 and EM-2 (20 μg, i.c.v., b.i.d. for 5 days) both produced significant naloxone-induced withdrawal syndromes with similar severity to that induced by the same dose of morphine. There was no significant difference between EM-1, EM-2, and morphine-treated group for naloxone-induced withdrawal signs, except for grooming. EM-1 and EM-2 induced more grooming than that caused by morphine. Although EM-1 and EM-2 both led to the withdrawal, they displayed different potency for certain signs and suggest their distinct regulations. The present results indicate EM-1 and EM-2 could initiate certain mechanism involved opiate dependence.

Effects of nociceptin/orphanin FQ and endomorphin-1 on glutamate and GABA release, intracellular [Ca 2+] and cell excitability in primary cultures of rat cortical neurons

The effects of nociceptin/orphanin FQ (N/OFQ) and endomorphin-1 (EM-1) on glutamate and GABA release, intracellular calcium, neuronal excitability and glutamate current were investigated in rat primary cortical neuronal cultures. Through their specific receptors N/OFQ and EM-1 (0.02–1 μM) inhibited the electrically evoked outflow of [ 3H]D-aspartate at most to −50% and that of [ 3H]GABA to −30%. In addition, at 1 μM, both peptides induced a decrease of the firing rate caused by electrical depolarization. N/OFQ 1–10 μM did not influence either the electrically evoked calcium influx or the glutamate-evoked currents, whereas EM-1 1 μM significantly inhibited them. Thus, in cortical neurons in culture, both N/OFQ and EM-1 inhibited the secretory process and neuronal excitability but EM-1 also affected calcium influx and cell body responsiveness to glutamate. Consequently, EM-1 appeared to dampen this excitatory signal more then N/OFQ did.

Analgesic tolerance and cross-tolerance to i.c.v. endomorphin-1, endomorphin-2, and morphine in mice

The present study examined the development of analgesic tolerance to endomorphin-1 (EM1), endomorphin-2 (EM2), and morphine, and cross-tolerance among these drugs. Male Swiss Webster mice were injected i.c.v. with EM1, EM2, morphine, or vehicle once daily for 5 days, and tested for analgesia in the tail flick test. To determine the extent of cross-tolerance, on the sixth day mice from each of the above groups received i.c.v. injections of EM1, EM2, morphine, or vehicle before analgesic testing. The development of tolerance to EM1 and EM2 closely resembled that of morphine. Complete, symmetrical cross-tolerance was observed between all drugs in the study. These results demonstrate a time-course and extent of tolerance similar to morphine, and support a common mechanism of action through the mu-opioid receptor.

Development of potent bifunctional endomorphin-2 analogues with mixed mu-/delta-opioid agonist and delta-opioid antagonist properties.

The C terminus of endomorphin-2 (EM-2) analogues (Tyr-Pro-Phe-NH-X) was modified with aromatic, heteroaromatic, or aliphatic groups (X = phenethyl,benzyl, phenyl, naphthyl, pyridyl, quinolyl, isoquinolyl, tert-butyl, cyclohexyl, or adamantyl; 3-18) to study their effect on opioid activity. Only 9 (1-naphthyl), 11 (5-quinolyl), 16 (cyclohexyl), and 18 (2-adamantyl) exhibited mu-opioid receptor affinity in the nanomolar range (K(i) = 2.41-6.59 nM), which, however, was 3- to 10-fold less than the parent peptide. Replacement of Tyr(1) by Dmt (2',6'-dimethyl-l-tyrosine) (19-32) exerted profound effects: (i) acquisition of high mu-opioid receptor affinity (K(i) = 0.11-0.52 nM) except 23 (Ph); (ii) presence of potent functional mu-opioid receptor agonism (IC(50) < 1 nM) for 19 ([Dmt(1)]EM-2), 27 (1-naphthyl), 29 (5-quinolyl), and 32 (5-isolquinolyl); (iii) association of weak delta-opioid antagonist activity (pA(2) = 5.41-7.18) except 19 ([Dmt(1)]EM-2), 20 (H), 27 (1-naphthyl), and in particular 29 (5-quinolyl) with its potent delta-agonism (IC(50) = 0.62 nM, pA(2) = 5.88); (iv) production of antinociception after ic administration of 32 (5-isoquinolyl) in mice, a bioactivity absent in the corresponding Tyr(1) analogue (14); and (v) preferential cis orientation (cis/trans = 3:2 to 7:3) at the Dmt-Pro amide bond, in contrast to the Tyr-Pro amide trans orientation (cis/trans = 1:2 to 1:3). Thus, [Dmt(1)]EM-2 analogues with hydrophobic C-terminal extensions provide model compounds with potent mu-opioid receptor bioactivity and dual functional agonism.

Influence of Ethanol Dependence and Methionine Enkephalin Antisense on Serum Endomorphin‐1 and Methionine Enkephalin Levels

Opiate peptides are involved in the physical dependence on ethanol. Levels of methionine enkephalin (MEnk), for example, are affected by ethanol. No study on the effect of ethanol on endomorphin, the endogenous ligand for the mu-opiate receptor, has yet been conducted. We examined the effect of ethanol ingestion on serum endomorphin (EM)-1 and MEnk levels. We also determined the effect of antisense directed at MEnk on serum levels of EM-1 and MEnk. Serum EM-1 levels steadily decreased about 20% during 56 days of ethanol ingestion in liquid feed, whereas a similar decrease in serum MEnk levels was not statistically significant. Serum MEnk levels decreased about 20% by 48 hr after antisense injection and then returned to baseline, whereas serum EM-1 levels increased by about 80% and remained elevated for about 2 weeks. In mice not treated with antisense or alcohol, there was no correlation between the serum levels of EM-1 and MEnk. These results show that serum levels of EM-1 are decreased by physical dependence on ethanol and that this effect is not directly mediated through MEnk.

Supraspinal anti-allodynic and rewarding effects of endomorphins in rats

Two potent endogenous opioid peptides, endomorphin-1 (EM-1) and -2 (EM-2), which are selective μ-opioid agonists, have been identified from bovine and human brain. These endomorphins were demonstrated to produce a potent anti-allodynic effect at spinal level. In the present study, we further investigated their supraspinal anti-allodynic effects and rewarding effects. In a neuropathic pain model (sciatic nerve crush in rats), EM-1 and -2 (15 μg, i.c.v.) both showed significant suppressive effects in the cold-water allodynia test, but EM-1 showed a longer duration than EM-2. Naltrexone (NTX; 15 μg) and naloxonazine (NLZ; 15 μg) were both able to completely block the anti-allodynic effects of EM-1 and -2. In the tests of conditioned place preference (CPP), only EM-2 at the dose of 30 μg showed significant positive rewarding effect, whereas both endomorphins did not induce any reward at the dose of 15 μg. Due to the low solubility and the undesired effect (barrel rotation of the body trunk), EM-1 was not tested for the dose of 30 μg in the CPP tests. It was also found that acute EM-2 (30 μg) administration increased dopamine turnover in the shell of nucleus accumbens in the microdialysis experiments. From these results, it may suggest that EM-1 and -2 could be better supraspinal anti-allodynic agents compared with the other opioid drugs, although they may also induce rewarding.

Local peripheral effects of μ-opioid receptor agonists in neuropathic pain in rats

Our study was designed to demonstrate peripheral antinociception of the μ-opioid receptor agonists: morphine (MF), [ d-Ala 2, N-Me-Phe 4, Gly 5-ol]enkephalin (DAMGO), endomorphin-1 (EM-1) and endomorphin-2 (EM-2) in Bennett's rat model of neuropathic pain. All the agonists were effective in antagonizing allodynia after their intraplantar (i.pl.) but not subcutaneous (s.c.) administration. Opioid peptides: DAMGO, EM-1 and EM-2 were more effective compared with corresponding doses of morphine (opioid alkaloid) in alleviating chronic pain. Peripheral μ-opioid receptors mediated the observed effects, as was evidenced by the i.pl. treatment with naloxone methiodide (active only at the site of injection) and by cyprodime, a selective μ-opioid receptor antagonist. These results have shown that opioid peptides are effective also after local treatment, and that their peripheral use may be of therapeutic interest in long-term management of chronic pain.

Differential conditioned place preference responses to endomorphin-1 and endomorphin-2 microinjected into the posterior nucleus accumbens shell and ventral tegmental area in the rat.

An unbiased conditioned place preference (CPP) paradigm was used to evaluate the reward effects of endogenous mu-opioid receptor ligands endomorphin-1 (EM-1) and endomorphin-2 (EM-2) from the mesolimbic posterior nucleus accumbens (Acb) shell and the ventral tegmental area (VTA) in CD rats. EM-1 (1.6-8.1 nmol) microinjected into posterior Acb shell produced CPP, whereas EM-2 (8.7-17.5 nmol) given into the same Acb shell produced conditioned place aversion (CPA). EM-1 (1.6-16.3 nmol) microinjected into the VTA produced CPP, whereas EM-2 (8.7 and 17.5 nmol) given into the same VTA site did not produce any effect, but at a high dose (35 nmol) produced CPP. EM-1 (3.3 nmol) or EM-2 (17.5 nmol) microinjected into the nigrostriatal substantia nigra was not significantly different from vehicle-injected groups. D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP) at 94.13 pmol or 3-methoxynaltrexone at 0.64 pmol microinjected into the posterior Acb shell blocked EM-1-induced CPP and EM-2-induced CPA. At a higher dose, CTOP (941.3 pmol) and 3-methoxynaltrexone (6.4 pmol) produced CPA and CPP, respectively. Coadministration with antiserum against dynorphin A(1-17) (Dyn) (10 microg) microinjected into the posterior Acb shell blocked EM-2-induced CPA. However, it did not affect EM-1-induced CPP. It is concluded that EM-1 and EM-2 produce site-dependent CPP and CPA, respectively, by stimulation of different subtypes of mu-opioid-receptors; stimulation of one subtype of mu-opioid-receptor at the posterior Acb shell and VTA by EM-1 induces CPP, whereas stimulation of another subtype of mu-opioid receptor at the posterior Acb shell, but not the VTA, by EM-2 induces the release of Dyn to produce CPA.

Endomorphin 1- and endomorphin 2-like immunoreactive neurons in the hypothalamus send axons to the parabrachial nucleus in the rat

Endomorphin 1 (EM1) and endomorphin 2 (EM2) are the endogenous peptides with high affinity and selectivity for the μ-opioid receptor (MOR). We examined whether or not EM1- and EM2-expressing hypothalamic neurons might send their axons to the parabrachial nucleus (PBN), where many MOR-expressing neurons have been observed. Immunofluorescence histochemistry was combined with fluorescent retrograde tract-tracing method. In the rats injected with Fluoro-Gold (FG) into the PBN, some of EM1- and EM2-immunoreactive hypothalamic neurons were labeled retrogradely with FG. The majority of the EM1/FG and EM2/FG double-labeled neurons were distributed in the dorsomedial hypothalamus nucleus, centromedial hypothalamic region, and arcuate nucleus; a few of them were also seen in the periventricular hypothalamic nucleus and posterior hypothalamic nucleus. Endomorphins released from PBN-projecting hypothalamic neurons may modulate the gustatory, autonomic and nociceptive functions through MOR-expressing PBN neurons.

Immunohistochemical staining of endomorphin 1 and 2 in the immune cells of the spleen

Endomorphin 1 (EM-1) and EM-2 have been widely reported in the cells of the central nervous system (CNS) but limited research has been done regarding their distribution in the peripheral system. The occurrence of EM-1 and -2 in the spleen as measured by RIA and their ability to mediate immune function imply a role for EMs in this area. The current study examines the localization of EM-1 and -2 in the immune cells of the spleen of male and female rats via an immunohistochemical procedure. In both genders, EM-1 and -2 immunoreactive staining was predominantly present in macrophages and B cells with minimal EM immunoreactive staining in T cells. This is the first evidence of a differential distribution of EM-1 and -2 in cells of the immune system.

Endomorphins exit the brain by a saturable efflux system at the basolateral surface of cerebral endothelial cells

Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) are two highly selective mu-opiate receptor agonists. We recently demonstrated that EM-1 and EM-2 have a saturable transport system from brain-to-blood in vivo. Since the endothelial cells are the main component of the non-fenestrated microvessels of the blood-brain barrier (BBB), we examined whether these endogenous tetrapeptides have a saturable transport system in cultured cerebral endothelial cells. EM-1 and EM-2 binding and transport were studied in a transwell system in which primary mouse endothelial cells were co-cultured with rat glioma cells. We found that binding of both endomorphins was greater on the basolateral than the apical cell surface. Flux of EM-1 and EM-2 occurred predominantly in the basolateral to apical direction, each showing self-inhibition with an excess of the respective endomorphin. Transport was not influenced by the addition of the P-glycoprotein inhibitor, cyclosporin A. Neither the mu-opiate receptor agonist DAMGO nor the delta-opiate receptor agonist DPDPE had any effect on the transport. Thus, the results show that a saturable transport system for EM-1 and EM-2 occurs at the level of endothelial cells of the BBB, and unlike beta-endorphin and morphine, P-glycoprotein is not needed for the brain-to-blood transport. Cross-inhibition of the transport of each endomorphin by the other suggests a shared transport system that is different from mu- or delta-opiate receptors. As endormorphins are mainly produced in the CNS, the presence of the efflux system at the BBB could play an important role in pain modulation and neuroendocrine control.

Endomorphins, endogenous opioid peptides, provide antioxidant defense in the brain against free radical-induced damage

Oxidative stress has been considered to be a major cause of cellular injuries in a variety of chronic health problems, such as carcinogenesis and neurodegenerative disorders. The brain appears to be more susceptible to oxidative damage than other organs. Therefore, the existence of antioxidants may be essential in brain protective systems. The antioxidative and free radical scavenging effects of endomorphin 1 (EM1) and endomorphin 2 (EM2), endogenous opioid peptides in the brain, have been investigated in vitro. The oxidative damage was initiated by a water-soluble initiator 2,2′-azobis(2-amidinopropane hydrocholoride) (AAPH) and hydrogen peroxide (H2O2). The linoleic acid peroxidation, DNA and protein damage were monitored by formation of hydroperoxides, by plasmid pBR 322 DNA nicking assay and single-cell alkaline electrophoresis, and by SDS-polyacrylamide gel electrophoresis. Endomorphins can inhibit lipid peroxidation, DNA strand breakage, and protein fragmentation induced by free radical. Endomorphins also reacted with galvinoxyl radicals in homogeneous solution, and the pseudo-first-order rate constants were determined spectrophotometrically by following the disappearance of galvinoxyl radicals. In all assay systems, EM1 was more potent than EM2 and GSH, a major intracellular water-soluble antioxidant. We propose that endomorphins are one of the protective systems against free radical-induced damage in the brain.

Dynorphinergic mechanism mediating endomorphin-2-induced antianalgesia in the mouse spinal cord.

We have previously demonstrated that both endomorphin-1 (EM-1) and endomorphin-2 (EM-2) at high doses (1.75-35 nmol) given intrathecally (i.t.) or intracerebroventricularly produce antinociception by stimulation of mu-opioid receptors. Now, we report that EM-2 at small doses (0.05-1.75 nmol), which injected alone did not produce antinociception, produces anti-analgesia against opioid agonist-induced antinociception. The tail-flick (TF) response was used to test the antinociception in male CD-1 mice. Intrathecal pretreatment with EM-2 (0.02-1.75 nmol) 45 min before i.t. morphine (3.0 nmol) injection dose dependently attenuated morphine-induced TF inhibition. On the other hand, a similar dose of EM-1 (1.64 nmol) failed to produce any antianalgesic effect. The EM-2 (1.75 nmol)-produced anti-analgesia against morphine-induced TF inhibition was blocked by i.t. pretreatment with the mu-opioid antagonist naloxone or 3-methoxynaltrexone, but not delta-opioid receptor antagonist naltrindole, kappa-opioid receptor antagonist nor-binaltorphimine, or N-methyl-d-aspartate (NMDA) receptor antagonist MK-801. The EM-2-induced antianalgesic effect against morphine-induced TF inhibition was blocked by i.t. pretreatment with antiserum against dynorphin A(1-17), but not beta-endorphin, [Met]-enkephalin, [Leu]-enkephalin, or cholecystokinin antiserum (200 microg each). The i.t. EM-2 pretreatment also attenuated the TF inhibition induced by other mu-opioid agonists, [d-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin, EM-1 and EM-2, delta-opioid agonist deltorphin II, and kappa-opioid agonist (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methane-sulfonate hydrate (U50,488H). It is concluded that EM-2 at subanalgesic doses presumably stimulates a subtype of mu-opioid receptor and subsequently induces the release of dynorphin A(1-17) to produce antianalgesic effects against mu-, delta-, or kappa-agonists-induced antinociception. The EM-2-induced antianalgesia is not mediated by the release of [Met]-enkephalin, [Leu]-enkephalin, beta-endorphin, or cholecystokinin, nor does it involve kappa- or delta-opioid or NMDA receptors in the spinal cord.

Acute antinociceptive tolerance and partial cross-tolerance to endomorphin-1 and endomorphin-2 given intrathecally in the mouse

The effect of pretreatment with endomorphin-1 (EM-1) or endomorphin-2 (EM-2) given intrathecally (i.t.) on the tail-flick inhibition induced by subsequent i.t. injection of EM-1 or EM-2 was studied in CD-1 mice. Pretreatment with EM-1 (32.7 nmol) for 1–3 h, but not 4 h, attenuated the tail-flick inhibition induced by i.t. EM-1 (16.3 nmol), while pretreatment with EM-2 (70 nmol) for 0.5–1.5 h, but not 2 h, attenuated tail-flick inhibition induced by i.t. EM-2 (35 nmol). EM-1 (32.7 nmol) pretreatment for 1.5 h produced 5.3- and only 2.4-folds shift to the right of the dose-response curves for EM-1- and EM-2-induced tail-flick inhibition, respectively, while EM-2 (70 nmol) pretreatment for 1 h caused 4.3- and 4.5-folds shift to the right for EM-2- and EM-1-induced tail-flick inhibition, respectively. Thus, mice made antinociceptive tolerant to EM-1 were partially cross-tolerant to EM-2 and mice made antinociceptive tolerant to EM-2 were completely cross-tolerant to EM-1. It is proposed that EM-1- and EM-2-induced antinociception are mediated by the stimulation of two different subtypes of μ-opioid receptors in mouse spinal cord; one subtype is stimulated by both EM-1 and EM-2, and another subtype is stimulated by EM-2.

The involvement of dopamine and nitric oxide in the endocrine and behavioural action of endomorphin-1

Previous publications have demonstrated a prominent central and corticotropin releasing hormone-mediated action of the endomorphins (EMs) on both open-field behaviour and the hypothalamo–pituitary–adrenal (HPA) axis. In the present experiments, the direct action of endomorphin-1 (EM1) on pituitary adrenocorticotropic hormone (ACTH) release, adrenal corticosterone secretion and the roles of nitric oxide (NO) and dopamine (DA) in the HPA and behavioural responses elicited by EM1 were investigated in mice. In vitro perifusion studies indicated that the action of EM1 on the HPA system appears to be confined to the hypothalamus, as EM1 did not influence the corticosterone secretion from adrenal slices and moderately attenuated the ACTH release from anterior pituitary slices. In in vivo experiments, N G-nitro- l-arginine (L-NNArg) pretreatment brought about a profound inhibition of both the endocrine and the behavioural responses. On the other hand, haloperidol completely abolished the increases in square crossing and rearing, without affecting corticosterone release. The direct action of EM1 on striatal DA release was therefore also investigated in an in vitro superfusion system. Although EM1 did not influence the basal release of tritiated DA, it significantly enhanced the transmitter release evoked by electric impulses and pretreatment with L-NNArg resulted in a considerable inhibition of the release elicited by EM1. In conclusion, our endocrine studies suggest an important role of NO in the mediation of the EM1-evoked corticosterone secretion. They also indicate that EM1 activates the HPA axis at a hypothalamic level and dopamine is not involved in this process. In contrast, the behavioural experiments reflect that the locomotor activation induced by EM1 is mediated by NO and dopamine, and the superfusion studies demonstrate that NO transmits the dopamine release enhancing effect of EM1.

Endomorphin-1 causes synovial hypoaemia in rat knee joints via a capsaicin-sensitive neural pathway

In joints, synthetic μ-opioids reduce inflammatory changes such as protein extravasation and associated oedema formation. However, the effect of endogenous opioid peptides on other inflammatory processes such as altered tissue blood flow has not been investigated. The present study examined the peripheral effects of the endogenous μ-opioid ligand endomorphin-1 (EM-1) on rat knee joint blood flow using laser Doppler perfusion imaging. Topical application of EM-1 (10 −16–10 −9 mol) to exposed rat knee joints resulted in a dose-dependent increase in synovial vascular resistance with a maximum rise of 56% occurring with the 10 −9 mol dose. Destruction of unmyelinated articular afferents by capsaicin treatment completely abolished the hypoaemic effects of EM-1. These findings suggest that EM-1 acts peripherally in knee joints to decrease synovial blood flow, and this hypoaemic response is dependent on the presence of capsaicin-sensitive nerves.

Endomorphin-1 and -2 induce naloxone-precipitated withdrawal syndromes in rats

In 1997, endomorphin-1 (EM-1) and -2 (EM-2) were identified as the most specific endogenous μ-opioid ligands. These two peptides have shown analgesic effects and many other opioid functions. In the present study, we attempt to investigate the possible ability of endomorphins to induce naloxone-precipitated withdrawal in comparison with that induced by morphine. Using the previously established scoring system in rats, 12 withdrawal signs (chewing, sniffing, grooming, wet-dog shakes, stretching, yawning, rearing, jumping, teeth grinding, ptosis, diarrhea, and penile erection) were observed and scored following naloxone (4 mg/kg, i.p.) challenge. Compared with the sham control, EM-1 and EM-2 (20 μg, i.c.v., b.i.d. for 5 days) both produced significant naloxone-induced withdrawal syndromes with similar severity to that induced by the same dose of morphine. There was no significant difference between EM-1, EM-2, and morphine-treated group for naloxone-induced withdrawal signs, except for grooming. EM-1 and EM-2 induced more grooming than that caused by morphine. Although EM-1 and EM-2 both led to the withdrawal, they displayed different potency for certain signs and suggest their distinct regulations. The present results indicate EM-1 and EM-2 could initiate certain mechanism involved opiate dependence.

Anatomical and functional correlation of the endomorphins with mu opioid receptor splice variants.

The present study characterizes the relationship between the endogenous mu opioid peptides endomorphin-1 (EM-1) and endomorphin-2 (EM-2) and several splice variants of the cloned mu opioid receptor (MOR-1) encoded by the mu opioid receptor gene (Oprm). Confocal laser microscopy revealed that fibers containing EM-2-like immunoreactivity (-LI) were distributed in close apposition to fibers showing MOR-1-LI (exon 4-LI) and to MOR-1C-LI (exons 7/8/9-LI) in the superficial laminae of the lumbar spinal cord. We also observed colocalization of EM-2-LI and MOR-1-LI in a few fibers of lamina II, and colocalization of EM-2-LI and MOR-1C-LI in laminae I-II, and V-VI. To assess the functional relevance of the MOR-1 variants in endomorphin analgesia, we examined the effects of antisense treatments that targeted individual exons within the Oprm1 gene on EM-1 and EM-2 analgesia in the tail flick test. This antisense mapping study implied mu opioid receptor mechanisms for the endomorphins are distinct from those of morphine or morphine-6beta-glucuronide (M6G).