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The Endomorphin System and Its Evolving Neurophysiological Role

Laboratory of Experimental Neuropsychopharmacology, Centre National de la Recherche Scientifique-Formation de Recherche en Evolution 2735, European Institute of Peptide Research (Institut Fédératif de Recherches Multidisciplinaires sur les Peptides 23), Faculty of Medicine and Pharmacy, Institute of Biomedical Research, University of Rouen, Rouen, France (J.F., J.C., J.-C.d.R.); and Laboratory of Biomolecular Chemistry, Faculty of Medicine, Medical University of Lodz, Lodz, Poland (J.F., A.J.)

Abstract

I. Introduction

II. Structure-Activity Relationship Studies

III. Distribution

IV. Receptors

V. Enzymatic Degradation

VI. Neurophysiological Role

A. Biological Effects of Endomorphins

1. Pain.

a. Central administration of endomorphins.

b. Peripheral administration of endomorphins.

2. Tolerance.

3. Physical Dependence.

4. Effects on Locomotor Activity.

5. Behavioral Sensitization.

6. Drug Addiction, Mechanism of Reward.

7. Psychiatric disorders.

a. Stress.

b. Anxiety.

c. Depression and other psychiatric disorders.

8. Social Defeat.

9. Food Intake.

10. Sexual Behavior.

11. Learning and Memory.

12. Effects on Cardiovascular System.

13. Effects on Respiratory System.

14. Effects on Gastrointestinal Tract.

B. Endomorphins, Neurotransmitters, and Neurohormones

1. Modulation of Dopamine Transmission.

2. Modulation of Noradrenaline Transmission.

3. Modulation of Serotonin Transmission.

4. Modulation of Acetylcholine Transmission.

5. Modulation of Neurohormone Release.

VII. Conclusions

	Abstract

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	I. Introduction

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The three opioid receptors, designated µ, , and , which were found in the central and peripheral nervous systems, mediate the biological functions of opioids. After the discovery of -opioid receptor-selective enkephalins in 1975 (Hughes et al., 1975), other peptides have been characterized as endogenous ligands for the opioid receptors (Table 1) (Goldstein et al., 1979; Nakanishi et al., 1979; Bloom, 1983). Naturally occurring opioid peptides, which were shown to bind preferentially to the µ-opioid receptor, were β-casomorphin (Tyr-Pro-Phe-Pro-Gly-Pro-Ile) from the tryptic digests of β-casein (Henschen et al., 1979), hemorphin-4 (Tyr-Pro-Trp-Thr) from digests of hemoglobin (Brantl et al., 1986), Tyr-Pro-Leu-Gly-NH₂ (Tyr-MIF-1¹) and Tyr-Pro-Trp-Gly-NH₂ (Tyr-W-MIF-1), both isolated from the brain (Horvath and Kastin, 1989; Erchegyi et al., 1992). However, until 1997, no mammalian peptide was identified that would show substantial µ-opioid receptor affinity and selectivity.

In 1997, Zadina's group (Zadina et al., 1997) synthesized a number of Tyr-W-MIF-1 analogs, containing all possible natural amino acid substitutions at position 4, which were subsequently screened for the opioid receptor binding. A biologically potent sequence, Tyr-Pro-Trp-Phe-NH₂, was discovered and then identified in the bovine brain (Zadina et al., 1997) and human cortex (Hackler et al., 1997). This new peptide, which was named endomorphin-1, showed remarkable affinity for the µ-opioid receptor (360 pM) and selectivity of 4000- and 15,000-fold for the µ-opioid receptor over the - and -opioid receptors, respectively. Endomorphin-1 was extremely potent in the guinea pig ileum assay, a classic test for µ-opioid receptor agonist activity (Zadina et al., 1997). This peptide also had a potent and specific antinociceptive effect in vivo, as shown in the tail-flick test (Hackler et al., 1997; Zadina et al., 1997). A second peptide, endomorphin-2 (Tyr-Pro-Phe-Phe-NH₂), which differs by one amino acid from endomorphin-1, was also isolated. Endomorphin-2 was shown to be almost as potent as endomorphin-1 (Hackler et al., 1997; Zadina et al., 1997). Endomorphins were the first peptides isolated from brain that bind to the µ-opioid receptor with high affinity and selectivity and therefore were proposed as endogenous µ-opioid receptor ligands. However, their precursor(s) still remain(s) unidentified.

In this review we discuss the biological effects of endomorphin-1 and endomorphin-2 in relation to their distribution in the central and peripheral nervous systems. We describe the relationship between these µ-opioid receptor-selective peptides and endogenous neurohormones and neurotransmitters. We evaluate the role of endomorphins from the physiological point of view and report selectively on the most important findings in their pharmacology, rather than present all available data.

	II. Structure-Activity Relationship Studies

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Full descriptions of all the structure-activity relationships studies of endomorphins are beyond the scope of this review. For more data on endomorphin analogs, please refer to the articles by Janecka et al. (2004), Gentilucci and Tolomelli (2004), and Janecka and Kruszynski (2005).

The structures of endomorphin-1 and endomorphin-2 are quite distinct from those of the traditional opioid peptides (endorphins, enkephalins, and dynorphins), which all share the Tyr-Gly-Gly-Phe sequence at the N terminus. The N-terminal message sequence of endomorphin-1 is composed of two pharmacophoric amino acid residues, Tyr and Trp (in endomorphin-2 Trp is replaced by Phe), in which the amino and phenolic groups of Tyr and the aromatic ring of Trp (or Phe) are required for µ-opioid receptor recognition. The sequence of endomorphins also includes a spacer (Pro), which joins the pharmacophoric residues. Podlogar et al. (1998) presented a structural and comparative study of endomorphin-1 to identify its bioactive conformation and attributes responsible for the µ-opioid receptor selectivity. Nuclear magnetic resonance data showed that Pro² provides the necessary stereochemical requirements for activity of endomorphin-1 at the µ-opioid receptor. The bioactive conformation of endomorphin-1 is characterized by a structure, in which the Tyr¹ and Trp³ side chains have opposite orientations with respect to Pro² (Paterlini et al., 2000). Synthesizing pseudoproline-containing analogs of endomorphin-2, which are known to be quantitative inducers of the cis conformation, the group of Schiller (Keller et al., 2001) demonstrated that the Tyr-Pro amide bond in the bioactive conformation is cis.

	III. Distribution

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Radioimmunological and immunocytochemical analyses revealed that endomorphin immunoreactivities (IRs) are distributed throughout the human, bovine, and rodent central nervous systems (CNS) (Hackler et al., 1997; Martin-Schild et al., 1997, 1999; Pierce et al., 1998; Schreff et al., 1998; Pierce and Wessendorf, 2000). Both endomorphins are abundant in the areas such as the stria terminalis, the periaqueductal gray (PAG), the locus coeruleus (LC), the parabrachial nucleus, and the nucleus of the solitary tract (NTS) (Table 2). However, there are also important differences in the neuroanatomical localization of these peptides. Endomorphin-1 is widely and densely distributed throughout the brain and upper brainstem and is particularly abundant in the nucleus accumbens (Nac), the cortex, the amygdala, the thalamus, the hypothalamus, the striatum, and the dorsal root ganglia (Schreff et al., 1998; Martin-Schild et al., 1999). In contrast, endomorphin-2 is more prevalent in the spinal cord and lower brainstem (Martin-Schild et al., 1999; Pierce and Wessendorf, 2000); endomorphin-2-immunoreactive cell bodies were most prominent in the hypothalamus and the NTS, whereas endomorphin-2-immunoreactive varicose fibers were mainly observed in the substantia gelatinosa of the medulla and the spinal cord dorsal horn. More modest endomorphin-2 IR was seen in the Nac, substantia nigra, nucleus raphe magnus, ventral tegmental area (VTA), and pontine nuclei and amygdala. The differences in the distribution of endomorphins could indicate the existence of two distinct endomorphin precursors or two different processing pathways of the same precursor.

View this table: [in this window] [in a new window]   TABLE 2 Distribution of endomorphins in the selected structures of the central nervous system Data from Martin-Schild et al. (1997, 1998, 1999), Schreff et al. (1998), Barr and Zadina (1999), Pierce et al. (1998), and Pierce and Wessendorf (2000).

The distribution of endomorphin IRs in the CNS seems to be similar to that of the classic endogenous opioid peptides (Hokfelt et al., 1977; Simantov et al., 1977; Johansson et al., 1978; Sar et al., 1978; Uhl et al., 1979). However, in the case of endomorphin-2 two major differences were found. Unlike -opioid receptor-selective enkephalin (Watson et al., 1977; Sar et al., 1978) and -opioid receptor-selective dynorphin (Gramsch et al., 1982), endomorphin-2 IR was sparse in the hippocampus and striatum (Schreff et al., 1998; Martin-Schild et al., 1999). In this way the distribution of endomorphin-2 was analogous to that of β-endorphin, an endogenous µ-opioid receptor-selective ligand (Finley et al., 1981).

The reports on the presence of endomorphins outside the CNS are scarce. Jessop et al. (2000) used a combination of specific radioimmunological assays and reversed phase high-performance liquid chromatography techniques to characterize the level of endomorphin IR in rat and human peripheral tissue samples. They found that endomorphin-1 IR and endomorphin-2 IR are present in significant amounts in human spleen; relatively high levels of endomorphin IR were also detected in the rat spleen, thymus, and blood. Because very low amounts of endomorphin IR were found in anterior and posterior rat pituitaries, secretion from these glands could not account for the significant plasma level of endomorphins. The authors of that report suggested that endomorphins are secreted into the general circulation from the nerve fibers and terminals of the spinal cord. Interestingly, in the same study a number of peaks of endomorphin-1 IR and endomorphin-2 IR, which do not coelute with their respective synthetic standards, were also detected. These might represent precursor polypeptides or degradation products of endomorphin-1 and endomorphin-2, or their post-translational modifications.

Endomorphins were also detected in immune cells in inflamed subcutaneous tissue, whereas they were almost absent in noninflamed tissue (Mousa et al., 2002). The endomorphin-positive cells could only be identified in the periphery of inflammatory foci and had morphological appearances consistent with macrophages/monocytes, lymphocytes, and polymorphonuclear leukocytes.

	IV. Receptors

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The µ-opioid receptors belong to the superfamily of heterotrimeric, guanine-nucleotide binding, G-protein-coupled receptors. The results of numerous in vitro studies clearly demonstrated that the µ-opioid receptors are exclusive binding sites for endomorphins. In the classic binding assays on the rat and mouse brain membrane preparations, both peptides displaced naloxone, Tyr-D-Ala-Gly-MePhe-Gly-ol (DAMGO), and other µ-opioid receptor-selective ligands in a concentration-dependent manner (for review, see Horvath, 2000). Endomorphins stimulated [³⁵S]guanosine 5'-O-(3-thio)triphosphate binding through the µ-opioid receptors in rat thalamus membrane preparations (Kakizawa et al., 1998; Sim et al., 1998; Fichna et al., 2006a), in the PAG (Narita et al., 2000), and in the pons/medulla of µ-opioid receptor-nondeficient mice (Mizoguchi et al., 2000). Both peptides were potent µ-opioid receptor agonists in the aequorin luminescence-based calcium assay performed on recombinant Chinese hamster ovary cell lines (Fichna et al., 2006b). The autoradiographic methods showed that in the CNS endomorphins labeled the same binding sites as DAMGO, a classic µ-opioid receptor-selective ligand (Goldberg et al., 1998; Kakizawa et al., 1998; Sim et al., 1998). Results of the in vivo studies also demonstrated that endomorphins are µ-opioid receptor ligands. The intracerebroventricular (i.c.v.) administration of endomorphins produced a potent antinociceptive effect in wild-type mice (for review, see Horvath, 2000) and no significant effect in µ-opioid receptor knockout mice (for reviews, see Sakurada et al., 2002; Narita et al., 1999; Zadina et al., 1999; Tseng, 2002). Intrathecal (i.t.) administration produced significant antinociception in the tail-flick, paw-withdrawal, tail pressure, and flexor-reflex tests in adult rodents (Stone et al., 1997; Zadina et al., 1997; Goldberg et al., 1998; Horvath et al., 1999; Sakurada et al., 1999, 2000, 2001; Ohsawa et al., 2001; Grass et al., 2002).

Endomorphin-1 and endomorphin-2 are regarded as partial agonists of µ-opioid receptors. The efficacy of endomorphins in many bioassays, such as guanosine 5'-O-(3-thio)triphosphate binding, is slightly lower than that of DAMGO but higher than that of morphine (Alt et al., 1998; Harrison et al., 1998; Sim et al., 1998). The extent to which these relative efficacies apply to the biological activity of endomorphins has yet to be elucidated.

The endomorphins are principal, but not exclusive, endogenous ligands of µ-opioid receptors. In the CNS, endomorphins, although anatomically positioned to activate the µ-opioid receptors, are not selectively associated with the regions expressing these binding sites. In several telencephalic and limbic structures, µ-opioid receptors and endomorphin-immunoreactive fibers are colocalized. These include septal nuclei, the bed nucleus of the stria terminalis, Nac, the amygdaloid complex, and many hypothalamic nuclei (for reviews, see Zadina et al., 1999, Zadina, 2002; Martin-Schild et al., 1999). There are also brain regions that contain low concentrations of endomorphins, in which significant numbers of µ-opioid receptors can be found, namely the amygdala (telencephalon), the thalamus, the hypothalamus (diencephalon), and the PAG (mesencephalon). Of note is the negligible amount of endomorphin IR in the striatum, a region known to express high levels of µ-opioid receptors (Pierce and Wessendorf, 2000). µ-Opioid receptors have also been detected outside the CNS, in the enteric nervous system (for review, see Olson et al., 1998) and throughout the immune tissues (Sharp et al., 1998), where they were found to be colocalized with endomorphins (Jessop et al., 2000).

Recent studies indicated that endomorphin-1 and endomorphin-2 produce their biological effects by stimulating functionally diverse subtypes of µ-opioid receptors, µ₁ and µ₂, which might be responsible for their distinct pharmacological activity (Sakurada et al., 1999, 2000; Tseng et al., 2000). The µ₁-opioid receptor antagonist naloxonazine was shown to block the antinociception induced by i.c.v. administration of endomorphin-2 more effectively than endomorphin-1, whereas β-funaltrexamine inhibited both. Spinal pretreatment with antisense oligodeoxynucleotides against different exons in the µ-opioid receptor gene differentially attenuated the antinociception induced by endomorphin-1 and endomorphin-2 (Wu et al., 2002; Garzon et al., 2004). These results showed that µ₂-opioid receptors would be stimulated by both endomorphin-1 and endomorphin-2, whereas µ₁-opioid receptors would be stimulated only by endomorphin-2. Further studies revealed that µ₁-opioid receptors mediate supraspinal analgesia and modulate acetylcholine (ACh) and prolactin release, whereas µ₂-opioid receptors mediate spinal analgesia, respiratory depression, and inhibition of gastrointestinal transit (Pasternak, 1993).

	V. Enzymatic Degradation

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The majority of opioid peptides undergo rapid enzymatic degradation (Egleton et al., 1998). Most of the extracellular peptide-degrading enzymes are membrane-bound exo- and endopeptidases, integral membrane proteins that have active sites facing the extracellular space. An intracellular cleavage of opioid peptides by cytosolic peptidases is also possible.

Endomorphins seem to be vulnerable to enzymatic cleavage, and several enzymes have been proposed as participants in endomorphin degradation (Tomboly et al., 2002) (Fig. 1). Aminopeptidase M (EC 3.4.11.2) and aminopeptidase P (EC 3.4.11.9) could degrade N-terminal Tyr-Pro peptide bonds, release an N-terminal amino acid, and generate tripeptides from endomorphins (Dua et al., 1985; Harbeck and Mentlein, 1991). The literature data also indicate that, when the N-terminal hydrophobic residue is followed by a Pro residue, the two amino acids may be released by aminopeptidase M as an intact dipeptide (Bairoch, 1996). Carboxypeptidase Y (serine peptidase, EC 3.4.16.5) and proteinase A (nonpepsin-type acid endopeptidase, EC 3.4.23.6) in the first step could convert the C-terminal amide group into a carboxyl group and then catalyze the hydrolysis of the Xaa³–Phe⁴ peptide bond, where Xaa indicates a natural amino acid) (Berne et al., 1990; Peter et al., 1999). Dipeptidyl-peptidase IV (DPP IV) (EC.3.4.14.5), a membrane-bound serine proteinase, removes dipeptides from the amino terminus of peptides containing proline as the penultimate amino acid and has also been proposed to participate in endomorphin degradation (Kato et al., 1978).

View larger version (19K): [in this window] [in a new window]   FIG. 1. Enzymatic degradation pathways of endomorphin-1 and endomorphin-2.

Interestingly, some authors claim that endomorphin-1 is more resistant to enzymatic degradation in vivo than endomorphin-2 (Fujita and Kumamoto, 2006). This is in good agreement with the observation that the duration of spinal antinociceptive effects was significantly longer for endomorphin-1 than for endomorphin-2 (Grass et al., 2002) and that endomorphin-1 required a longer pretreatment time than endomorphin-2 before tolerance was observed (Stone et al., 1997). This might also explain a relatively shorter duration of the antidepressant-like activity of i.c.v. administered endomorphin-2 in mice (Fichna et al., 2007).

One reason the elucidation of the degradation pathways of endomorphins was necessary was to isolate their effects from these produced by the degradation products. Because of structural similarities to the parent compound, the degradation products might compete with endomorphins for the receptor binding site and could influence their biological activity. However, Szatmari et al. (2001) demonstrated that the primary degradation products of endomorphin-1, Tyr-Pro-Trp-Phe-OH and Pro-Trp-Phe-OH, possess low µ-opioid receptor binding affinity, do not activate G-proteins and have no antinociceptive activity.

The effect of peptidase inhibitors on endomorphin degradation has also been studied. Spetea et al. (1998) examined the influence of peptidase inhibitors on the binding characteristics of [³H]endomorphin-2 in rat brain membrane preparations and showed that in the absence of peptidase inhibitors 40% of the radioligand in the incubation mixture was destroyed. Actinonin, but not thiorphan, was found to be effective in inhibiting the degradation of endomorphin-1 in a 6-day-old rat spinal cord homogenate (Sugimoto-Watanabe et al., 1999). Endomorphin-2-induced antinociception was remarkably enhanced in the paw withdrawal test when diprotin A (Ile-Pro-Ile), a DPP IV inhibitor, was simultaneously injected (Sakurada et al., 2003): the peptide in combination with diprotin A was 5-fold more potent than endomorphin-2 alone in producing antinociceptive effects. Shane et al. (1999) reported that i.c.v. administered Ala-pyrrolidonyl-2-nitrile, another specific inhibitor of DPP IV, increased the magnitude, duration, and potency of endomorphin-2-induced antinociception in the rat tail-flick test.

	VI. Neurophysiological Role

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A. Biological Effects of Endomorphins

The neuroanatomical distribution of endomorphins and the µ-opioid receptors in the CNS reflects their potential endogenous role in many major biological processes. These include perception of pain, responses related to stress, and complex functions such as reward, arousal, and vigilance, as well as autonomic, cognitive, neuroendocrine, and limbic homeostasis (Fig. 2). Some of these phenomena, summarized in Tables 3 and 4, will be discussed in the following sections.

View larger version (32K): [in this window] [in a new window]   FIG. 2. Major structures in the central nervous system implicated in endomorphin-dependent effects. ARC, arcuate nucleus; DMH, dorsomedial nucleus; DTN, dorsal nucleus; HPT, hypothalamus; LC, locus coeruleus; LH, lateral nucleus; NTS, nucleus of the solitary tract (cv, caudal ventrolateral; im, intermediate, ro, rostral); PAG, periaqueductal gray; PBN, parabrachial nucleus; PVN, paraventricular nucleus; RD, caudal dorsomedial part of NTS; VMH, ventromedial nucleus

1. Pain. An important role of endomorphins in pain modulation is indicated by their presence in well-characterized nociceptive pathways (Fields and Basbaum, 1994; Guilbaud et al., 1994): endomorphin-containing neuronal elements were found in most regions of the spino(trigemino)-ponto-amygdaloid pathway (Fig. 3). These regions, which include the caudal nucleus of spinal trigeminal tract, parabrachial nucleus, NTS, PAG, nucleus ambiguous, LC, and midline thalamic nuclei, are known to be involved in the transmission of the nociceptive information by direct input from the primary afferents and/or as relay nuclei to other pain-processing circuits (for reviews, see Fields and Basbaum, 1978; Basbaum and Fields, 1984; Przewlocki et al., 1999; Przewlocki and Przewlocka, 2001). Endomorphins were also found in amygdala, which has been proposed to play an important role in nociception (Manning and Mayer, 1995), particularly with regard to the affective influences on and responses to noxious events (Bernard et al., 1992; Helmstetter and Bellgowan, 1993; Watkins et al., 1993).

View larger version (24K): [in this window] [in a new window]   FIG. 3. Neuroanatomical distribution of endomorphin-1 (EM-1) and endomorphin-2 (EM-2) in major structures involved in the sensation, transmission, and modulation of pain. ++, dense endomorphin-like immunoreactivity; +, moderate endomorphin-like immunoreactivity; -, no endomorphin-like immunoreactivity observed. Arrows represent major ascending and descending pain pathways. Adapted from Kanjhan (1995) with permission from Blackwell Publishing.

Zadina (2002) suggested that endogenous endomorphin-2, due to its specific neuroanatomical localization, might be involved in the earliest stages of nociceptive information processing. To begin with, endomorphin-2 IR was found to be predominantly present in the spinal cord, in the superficial layers of the dorsal horn, and in the primary afferent fibers (including small diameter primary afferent neurons, i.e., most likely nociceptors), whose cell bodies are localized within the dorsal root ganglia (Mousa et al., 2002). These are the regions with the highest densities of µ-receptors in the nervous system (Martin-Schild et al., 1997) and are thought to play an important role in the modulation of nociceptive transmission by various endogenous substances, including opioids (for review, see Fürst, 1999). Moreover, the electrical stimulation of the dorsal roots was shown to promote the release of endomorphin-2 from dense-cored vesicles, situated in dorsal horn axons (Williams et al., 1999; Wang et al., 2002). Thus, it was hypothesized that endomorphin-2 could serve both a regulatory function, by hyperpolarizing the membranes on intrinsic neurons of the dorsal horn and decreasing the excitability of postsynaptic µ-opioid receptors (Wu et al., 1999), and an autoregulatory function, by limiting the release of excitatory transmitters, glutamate (Glu), substance P, -aminobutyric acid, glycine, and calcitonin gene-related peptide through the activation of presynaptic µ-opioid autoreceptors on primary afferent fibers in the spinal cord (Yajiri and Huang, 2000; Wu et al., 2003) (Fig. 4). Endomorphin-2 might also modify pain sensations from the visceral organs and alter the efferent components of the autonomic nervous system by interacting with their preganglionic neurons (Martin-Schild et al., 1997). This latter hypothesis was recently confirmed by Silverman et al. (2005).

View larger version (28K): [in this window] [in a new window]   FIG. 4. Hypothetical involvement of endogenous endomorphin-2 (EM-2) in the earliest stage of pain sensation, transmission, and modulation. 1, regulatory function. Decrease of excitability of postsynaptic µ-opioid receptors. 2, autoregulatory function. Inhibition of the release of excitatory transmitters [calcitonin gene-related peptide (CGRP), glutamate (Glu), substance P (SP)]. DYN A, dynorphin A.

View larger version (31K): [in this window] [in a new window]   FIG. 5. Simplified representation of pain modulatory pathways activated by supraspinally and spinally administered endomorphin-1 and endomorphin-2. DYN A, dynorphin A; MET-ENK, Met-enkephalin. Adapted from Tseng (2002).

Hung et al. (2003) determined the effects of i.t. pretreatment with 6-OHDA and 5,7-DHT to deplete NA and 5-HT, respectively, on the antinociception induced by supraspinally administered endomorphins. They found that 3-day i.t. pretreatment with 6-OHDA, which depleted spinal NA (>90%), completely abolished the antinociception induced by i.c.v. administration of endomorphins. These results strongly indicate that the release of NA in the spinal cord plays an essential role in the endomorphin-induced antinociception. The 3-day i.t. pretreatment with 5,7-DHT, which depleted both 5-HT (>90%) and NA (up to 25%) attenuated, but did not block, the endomorphin-induced antinociception. These data demonstrate that the adrenergic pathway is more important than the serotonergic pathway in mediating the antinociception activated by supraspinally administered endomorphins.

In the same study the antinociception induced by spinally administered endomorphins was blocked by i.t. pretreatment with the µ-receptor antagonists, but not with 6-OHDA or 5,7-DHT (Ohsawa et al., 2001; Hung et al., 2003). These observations clearly indicated that direct stimulation of the µ-opioid receptors, located in the dorsal horn of the spinal cord, mediates the antinociception induced by spinally administered endomorphins, without the involvement of NA or 5-HT transmission.

Supraspinal endomorphin-2- but not endomorphin-1-induced tail-flick inhibition was blocked by i.c.v. or i.t. pretreatment with an antiserum against dynorphin A(1-17) or norbinaltorphimine. Similarly, it was blocked by i.t. pretreatment with an antiserum against Met-enkephalin or naltriben (Tseng et al., 2000). These findings indicated that the supraspinal endomorphin-2-induced antinociception also involved additional components, corresponding to the release of dynorphin A(1-17), as well as Met-enkephalin, acting on the - and ₂-opioid receptors, respectively. This accounts for the differences in the antinociceptive effects between endomorphin-1 and endomorphin-2 (Fig. 4).

b. Peripheral administration of endomorphins. Several studies have shown that peripherally administered opioids produce analgesic effects mediated through peripheral µ- and -opioid receptors located on primary afferent neurons (Craft et al., 1995; Kolesnikov and Pasternak, 1999; Nozaki-Taguchi and Yaksh, 1999). However, detailed pathways and exact mechanisms, through which peripherally administered opioids produce antinociception, have not been elucidated.

As for endomorphins, there is only one report on their analgesic effect after peripheral administration. Li et al. (2001) used various nociceptive tests to demonstrate that endomorphin-1 produced a dose-dependent, naloxone-reversible analgesia after i.p. administration in rats. The peak analgesic effect appeared later in the time course and was less pronounced than that induced by centrally (i.c.v. and i.t.) administered peptide. Because it is generally believed that peripherally administered endomorphins, because of their rapid enzymatic degradation in peripheral tissues and low permeation through the brain-blood barrier, cannot reach the CNS in an amount sufficient to elicit analgesia (Hau et al., 2002; Spampinato et al., 2003), these observations need to be further investigated. The question whether the analgesic effect resulting from this route of administration has a peripheral or a central origin needs to be answered as well.

2. Tolerance. The development of tolerance, as well as physical dependence limits the clinical use of the opioids as pharmacological agents. Numerous studies have shown that the µ-opioid receptor ligands are responsible for the emergence of both phenomena (Schiller et al., 1999; Shen et al., 2000; Spreekmeester and Rochford, 2000). Recently, much emphasis has been put on the µ-opioid receptor-mediated intracellular signal transduction mechanisms and long-lasting molecular and cellular adaptations after chronic treatment with the µ-opioid receptor ligands (Defer et al., 2000; Heyne et al., 2000; Law et al., 2000). The ability of endomorphins, being potent µ-opioid receptor-selective agonists, to develop tolerance and dependence has also been investigated.

As it was shown in the in vitro assays, acute treatment with or chronic administration of endomorphins may stimulate the development of tolerance (Higashida et al., 1998; McConalogue et al., 1999). Nevo et al. (2000) demonstrated that forskolin-stimulated adenylyl cyclase activity was elevated above control levels after naloxone in µ-opioid receptor-expressing Chinese hamster ovary cells chronically treated with endomorphin-1 and endomorphin-2. Similarly, chronic exposure to endomorphins stimulated adenylyl cyclase activity in µ-opioid receptor-expressing African green monkey kidney cells cotransfected with type I and V isozymes (Nevo et al., 2000).

In vivo investigations also showed that pretreatment with endomorphins may develop tolerance. First, it was demonstrated that i.t. pretreatment with endomorphin-1 and endomorphin-2 attenuated the antinociceptive response in mice induced by i.t. administration of endomorphin-1 and endomorphin-2, respectively, and developed acute tolerance to endomorphins (Stone et al., 1997; Higashida et al., 1998; Horvath et al., 1999). Further studies showed that i.c.v. or i.t. administration in mice and rats of a single high dose of endomorphin-1 or endomorphin-2 induced an acute antinociceptive tolerance to the subsequent challenging dose of i.c.v. or i.t. administered endomorphin-1 or endomorphin-2, respectively (Wu et al., 2001, 2003; Hung et al., 2002; Labuz et al., 2002). Interestingly, endomorphins induced the development of tolerance much faster than morphine, although endomorphin-1 required a longer pretreatment time than endomorphin-2 before the acute antinociceptive tolerance was observed. It was proposed that these diverse effects might result from different peptide half-lives or differences in the µ-opioid receptor selectivity or divergent neuronal mechanisms and not from the degree of receptor stimulation by endomorphins or differences in the opioid receptor desensitization (Wu et al., 2001).

Labuz et al. (2002) reported on results obtained in a cross-tolerance study, in which the antinociceptive effects of endomorphins and morphine were compared in tail-flick and paw pressure tests in rats. The animals made tolerant to endomorphin-2 exhibited a partial antinociceptive cross-tolerance to endomorphin-1, whereas rats tolerant to endomorphin-1 showed no cross-tolerance to endomorphin-2. The study also described the cross-tolerance between morphine and endomorphin-1, suggesting a common target, which was probably the µ₂-opioid receptor subtype. Because no cross-tolerance was observed between morphine and endomorphin-2, it was suggested that endomorphin-2 acts via another µ-opioid receptor subtype, apparently µ₁, which could also be a different splice variant or a physical state of the µ-opioid receptor.

As discussed above, so far only animal models of pain have been used to characterize the influence of endomorphins on the development of tolerance. However, tolerance to other endomorphin effects needs to be elucidated. This study might be particularly interesting, as tolerance did not develop to all of the effects of morphine: for example, it did not occur to morphine-induced locomotor activity, which is linked to the physical dependence (Spanagel et al., 1998).

3. Physical Dependence. Chronic administration of opioids usually results in physical dependence, as measured in terms of the appearance of withdrawal symptoms after cessation of the drug or when an opioid antagonist is administered. In animals, the opioid withdrawal symptoms include abnormal posture (Pineda et al., 1998), diarrhea (Miranda and Pinardi, 1998; Pineda et al., 1998), hypothermia (Thornton and Smith, 1998), changes in blood pressure (Zhang and Buccafusco, 1998), and several others (for reviews, see Vaccarino et al., 1999; Vaccarino and Kastin, 2000, 2001). In some cases single, but not chronic, administration of opioids is necessary for the development of dependence (Kest et al., 1998). The µ-opioid receptor ligands are regarded as being mainly responsible for the development of physical dependence and drug addiction (Reisine and Pasternak, 1996; Rockhold et al., 2000).

McConalogue et al. (1999) demonstrated that endomorphins specifically activated the µ-opioid receptor and induced its endocytosis in cells transfected with the µ-opioid receptor cDNA, as well as in the enteric neurons that naturally express the µ-opioid binding sites. Endomorphin-induced endocytosis and trafficking of the µ-opioid receptor may mediate receptor desensitization, resensitization and down-regulation, mechanisms that regulate cellular responsiveness to ligand stimulation and that might be important in the development of opioid tolerance and addiction (Bohm et al., 1997).

To examine the possible effects of endomorphins on physical dependence in vivo, Chen et al. (2003) investigated the ability of both peptides to induce naloxone-precipitated withdrawal in rats. Using a previously established scoring system, 12 withdrawal signs (chewing, sniffing, grooming, wet-dog shakes, stretching, yawning, rearing, jumping, teeth grinding, ptosis, diarrhea, and penile erection) were observed and scored after a naloxone (4 mg/kg i.p.) challenge. Endomorphins (20 µg i.c.v., b.i.d. for 5 days) were shown to induce physical dependence, but they displayed different potency for certain signs. The severity of the endomorphin-induced withdrawal was similar to that induced by the same dose of morphine.

Unfortunately, no data on the interactions between endomorphins and the nonopioid systems are available, although dopamine (DA) (El-Kadi and Sharif, 1998; Samini et al., 2000; Tokuyama et al., 2000), NA (Milanes et al., 1998; Fuentealba et al., 2000; Fuertes et al., 2000; Laorden et al., 2000), ACh (Zhang and Buccafusco, 1998; Buccafusco et al., 2000), benzodiazepine (Tejwani et al., 1998), N-methyl-D-aspartate (Popik et al., 1998), imidazoline (Su et al., 2000), Glu (Rockhold et al., 2000), and GABAergic (Sayin et al., 1998) pathways were suggested to play an important role in the development of physical dependence. Nitric oxide (NO) may also be involved in endomorphin-induced dependence, both directly, as systemic injections of NO synthase inhibitors attenuated some signs of naloxone-precipitated withdrawal and hyperactivity of the LC (Pineda et al., 1998), and indirectly, as the i.t. infusion of morphine increased N-methyl-D-aspartate binding activity and up-regulated neuronal NO synthase expression (Wong et al., 2000).

4. Effects on Locomotor Activity. The effects of opioid receptor ligands on locomotor activity are influenced by a number of variables, including the dose and the paradigm used in the experiment. However, in most studies the µ- and -opioid receptor ligands stimulated locomotor activity by increasing the synthesis and the release of DA from dopaminergic neurons in the nigrostriatal and the mesolimbic dopaminergic system (Chesselet et al., 1981; Urwyler and Tabakoff, 1981; Broderick, 1985; Locke and Holtzman, 1986; Cunningham and Kelley, 1992). In contrast, -opioid receptor agonists were shown to decrease both vertical and horizontal locomotion (Kuzmin et al., 2000).

The opioid alkaloid morphine, a µ-opioid receptor ligand with low affinities to - and -opioid receptors, was shown to increase locomotion under most conditions (Latimer et al., 1987; Austin and Kalivas, 1990; Calenco-Choukroun et al., 1991; Aguilar et al., 1998; Kimmel et al., 1998; Schildein et al., 1998; Stinus et al., 1998), but in some cases had no effect (Waddell and Holtzman, 1998). Acute injections of morphine stimulated horizontal locomotion through the µ-opioid receptors and grooming through the -opioid binding sites, localized in the VTA, Nac, and PAG (Babbini and Davis, 1972; Joyce and Iversen, 1979; Katz, 1979; Havemann et al., 1983; Morgan et al., 1998; Schildein et al., 1998). A single administration of morphine was shown to increase behavioral sensitivity to the DA agonist, apomorphine (de la Baume et al., 1979), whereas chronic treatment resulted in the development of supersensitive DA receptors, as determined by induction of the stereotypic behaviors by the DA agonists (Ritzmann et al., 1979). To elucidate the relationship between morphine and DA, Jang et al. (2001) compared the effect of morphine on the modulation of the apomorphine-induced climbing behavior in wild-type and µ-receptor knockout mice. Treatment with morphine potentiated apomorphine-induced climbing behavior in wild-type mice, whereas it did not produce any significant effect in the µ-receptor knockout mice. These results indicated that µ-receptors play an important role in potentiation of the climbing behavior induced by DA receptor agonists (Jang et al., 2000) and proved that µ-receptor ligands influence the dopaminergic system.

Effects of centrally administered endomorphin-1 and endomorphin-2 on locomotor activity were evaluated and seem confusing. In some studies endomorphins, like morphine, were shown to increase horizontal and vertical activity (i.e., hyperlocomotion) and not to alter grooming activity (Bujdoso et al., 2001, 2003). Interestingly, the concentration-response curves for endomorphin-1 and endomorphin-2 in mice were of a bell-shaped type (Bujdoso et al., 2001). However, relatively lower concentrations of endomorphin-2 than of endomorphin-1 were used to obtain the same response (Bujdoso et al., 2001), suggesting differences in activation of the µ-opioid receptor subtypes (Sakurada et al., 1999, 2000) or the involvement of the enkephalinergic or dynorphinergic system (Sanchez-Blazquez et al., 1999; Tseng et al., 2000).

In contrast, some studies showed that endomorphins did not influence locomotor activity. Our group (Fichna et al., 2007) investigated the effect of i.c.v. administration of endomorphin-1 and endomorphin-2 on locomotor activity in mice. The peptides did not modify horizontal locomotor activity in any of the time periods of the test. Furthermore, endomorphin-1, at the highest dose (30 µg/mouse), and endomorphin-2, at the lowest doses (0.3 and 1 µg/animal), produced weak inhibition of vertical locomotor activity.

Our results are consistent with those obtained by Mehta et al. (2001), who examined the influence of endomorphins on the activity of basal ganglia, specific subcortical brain structures that play an important role in the control of movement (Delfs et al., 1994; Peckys and Landwehrmeyer, 1999). Dysfunction of the basal ganglia, resulting from specific degeneration of neurons, as in Parkinson's and Huntington's diseases, or from the administration of pharmacological agents, leads to severe motor disorders (Albin et al., 1991; Chesselet and Delfs, 1996). Within the basal ganglia, a subpopulation of neurons of the globus pallidus expresses particularly high levels of µ-opioid receptor mRNA (Delfs et al., 1994; Obeso et al., 2000). Morphine injections into the globus pallidus produced a robust increase in locomotor activity (Anagnostakis et al., 1992), whereas endomorphin-1 induced orofacial dyskinesia (Mehta et al., 2001). The authors of that report suggested that stimulation of the locomotor activity by morphine could be mediated by - and -opioid receptors and the inhibitory activity of endomorphin-1 might involve µ-opioid receptors. Previous studies showed that stimulation of GABA receptors induced catalepsy in rats (Egan et al., 1995). Endomorphin-1 and GABA could therefore induce opposite behavioral effects in the globus pallidus and alterations in their equilibrium could play a crucial role in control of movement and development of dyskinesia.

5. Behavioral Sensitization. Repeated administration of psychoactive drugs can lead either to a decrease (tolerance) or an increase (sensitization) in their behavioral effects. The term "behavioral sensitization" was first used to describe the augmented motoric stimulant effect produced by a given dose of a psychomotor-stimulant drug, such as amphetamine or cocaine, after repeated intermittent injections (Segal and Kuczenski, 1997). This phenomenon could persist for a long period after drug abstinence (Robinson and Becker, 1986). The term is now more readily used for the phenomenon of increased behavioral and neurochemical responsiveness of any type to the administration of the same or lower doses of drug after repeated drug injections (Spyraki et al., 1983).

Behavioral sensitization plays an important role in the development and maintenance of drug addiction (Gaiardi et al., 1991; Hunt and Lands, 1992; Robinson and Berridge, 1993); simple "preference" for a drug becomes "sensitized" (i.e., increases) up to the point where the urge to take the drug becomes overwhelming (i.e., loss of behavioral control or drug craving) (Wise and Bozarth, 1987; Robinson and Berridge, 1993). Virtually all human drugs of abuse that show positive results in animal models of reward and addiction produce behavioral sensitization (Stewart and Badiani, 1993).

In the search for neuroanatomical and neurochemical bases of drug-induced sensitization, research has focused on the mesolimbocortical dopaminergic pathway, which arises in the VTA and projects mainly to the Nac (Spanagel, 1995). There are several transmitter systems in the VTA known to evoke behavioral sensitization, including DA (Hooks et al., 1993, Hooks and Kalivas, 1994'02; Vezina, 1996), GABA (Johnson and North, 1992), Glu/aspartate (Karler et al., 1990), and tonically active endogenous opioid systems (Spanagel et al., 1992).

In vivo DA release measurements (microdialysis) clearly demonstrated that systemic administration of µ-opioid receptor agonists increases DA release in the Nac (Di Chiara and Imperato, 1988a,b; Spanagel et al., 1990; Pentney and Gratton, 1991). DAMGO and morphine increased DA release in the Nac after injection into the VTA (Leone et al., 1991; Longoni et al., 1991; Spanagel et al., 1992; Devine et al., 1993), whereas D-Phe-c(Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH₂) (CTOP)], a highly specific µ-opioid receptor antagonist, produced a significant decrease of DA release in the Nac (Spanagel et al., 1992). In contrast, infusion of either ligand into the Nac was without any effect.

Chen et al. (2001) reported that repeated administration of endomorphins in the VTA has a significant effect on the development of sensitization to amphetamine in rats. To understand the neural basis of the behavioral outcome of the chronic endomorphin treatment, the tissue contents of several neurotransmitters and their metabolites in the limbic forebrain (ventral striatum and medial prefrontal cortex) and extrapyramidal area (dorsal striatum) of rats treated with amphetamine or endomorphin were analyzed. It has been demonstrated that Glu concentrations increased significantly in all tested brain areas in both groups of animals. It was suggested that µ-opioid receptor agonists could activate dopaminergic neurons through the activation of GABA or Glu neurons in the VTA (Karler et al., 1990, Wolf and Xue, 1998; Johnson and North, 1992).

6. Drug Addiction, Mechanism of Reward. Numerous studies, in which µ-selective agonists and antagonists were used, showed that the µ-opioid receptor is the primary factor in the development of drug addiction because of its central role in the mediation of reward (Negus et al., 1993, Devine and Wise, 1994; Piepponen et al., 1997). µ-Opioid receptor ligands, including DAMGO, morphine, codeine, and sufentanyl, elicited strong rewarding effects in several brain structures, such as the VTA or Nac (for review, see Wise, 1989; Suzuki, 1996; Martin-Schild et al., 1999), whereas selective -opioid receptor agonists produced significant aversion (Becker et al., 2000). The µ-opioid receptor agonists were also shown to mediate the reward effects induced by stress (Zacharko et al., 1998).

It is generally assumed that the critical brain region for µ-opioid effects on motivation is the VTA. The rewarding effects of µ-opioid injections into the VTA are mediated by the µ-opioid receptors expressed on the GABA-containing cells. µ-Opioid receptor agonists inhibit GABAergic inputs to the dopaminergic VTA principal cells, projecting to the Nac, and disinhibit release of DA in the Nac, a major component of the endogenous reward circuitry of the brain (Johnson and North, 1992; Margolis et al., 2003).

Similarly to morphine and DAMGO, endomorphins injected into the posterior VTA established a conditioned place preference and produced significant psychomotor stimulating effects (Zangen et al., 2002). Endomorphin-1 injected into the posterior VTA gave also strong rewarding effects in the self-administration paradigm. Injection of endomorphin-1 and DAMGO into the Nac gave no rewarding effect (Zangen et al., 2002). The effect of DAMGO was not only weak but also generally delayed, which suggests the lack of sites of action for µ-opioid agonists in the Nac (Churchill and Kalivas, 1992; Johnson et al., 1996; Churchill et al., 1998).

The possible rewarding effect of i.c.v.-administered endomorphins has also been investigated in different species; however, the results obtained were inconsistent. Narita et al. (2001a,b) reported that endomorphin-1 produced a significant place preference and endomorphin-2 produced a significant place aversion in mice, which were inhibited by pretreatment with either an antiserum against the endogenous -opioid receptor agonist dynorphin A(1-17) or coadministration with a -opioid receptor antagonist (Wu et al., 2004). The authors suggested that endomorphins stimulate different subtypes of the µ-opioid receptor and that endomorphin-2 could additionally stimulate the release of dynorphins, which are responsible for its aversive effect (Fig. 6).

View larger version (25K): [in this window] [in a new window]   FIG. 6. Different motivational effects of endomorphin-1 (EM-1) and endomorphin-2 (EM-2). DYN A, dynorphin A. Adapted from Narita et al. (2002).