Introduction

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The opioids were among the earliest neuropeptides identified in the nervous system. The enkephalins were the first, followed soon afterward by the dynorphins and -endorphin (Evans et al., 1988; Reisine and Pasternak, 1996; Pasternak, 1993). The enkephalins are the endogenous ligands for the delta class of opioid receptors and dynorphin A is the endogenous ligand for the kappa₁ receptor. The mu receptor was the first opioid receptor identified in binding assays and its importance is further emphasized by its importance in mediating the analgesic actions of most clinically used analgesics (Reisine and Pasternak, 1996; Pasternak, 1993). Yet, the search for the endogenous ligand for the mu receptor has lagged far behind the other opioid receptor subtypes. A number of opioid peptides have high affinity for mu receptors, including dynorphin A and -endorphin. However, dynorphin A labels kappa₁ receptors far more potently than mu sites and -endorphin binds equally well to mu and delta sites. Thus, many investigators felt that these peptides were not the endogenous ligand for mu receptors based on these selectivity profiles.

The recent identification of endomorphin-1 and endomorphin-2 has opened a new area of research in the mu opioid system (Zadina et al., 1997). Although related to each other, the sequences of endomorphin-1 (Tyr-Pro-Trp-Phe-NH₂) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH₂) are quite distinct from traditional opioids in which the first four amino acids are Tyr-Gly-Gly-Phe followed by either methionine or leucine. Yet, both peptides label mu receptors with high affinity and selectivity, raising the possibility that they may represent two endogenous mu receptor ligands. To further evaluate these two peptides, we have extended these initial studies on the endomorphins.

	Materials and Methods

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Male CD-1 mice (24-30 g: Charles River Laboratories, Raleigh, NC) were housed in groups of five with food and water ad libitum. Animals were maintained on a 12-hr light/dark cycle. Fresh calf brains were obtained from a local slaughterhouse (Max Insel Cohen, NJ). Endomorphin-1 and endomorphin-2 were synthesized at our institution's Microchemistry Facility, purified by high-performance liquid chromatography, their structures verified by mass spectroscopy and the peptide content (58% for endomorphin-1 and 74% for endomorphin-2) determined by Rockefeller University's Protein Technology Center. Naloxonazine and naloxone benzoylhydrazone were synthesized in our laboratory as previously published (Luke et al., 1988; Hahn et al., 1982) and norBNI and naltrindole were purchased from Research Biochemicals International (Natick, MA). Naloxone, -FNA and the other opioids and opioid peptides were provided by the Research Technology Branch of the National Institute on Drug Abuse (Rockville, MD). Chemicals were purchased from either from Fisher Scientific (Pittsburgh, PA) or Sigma Chemical Co. (St. Louis, MO). Halothane was obtained from Halocarbon Laboratory (Hackensack, NJ).

Preparation of membranes. Brains were homogenized in 50 volumes of treated buffer (50 mM Tris, pH 7.4 at 25°C, 1 mM EDTA and 100 mM NaCl) (Clark et al., 1989; Pasternak et al., 1975; Pasternak and Snyder, 1975). The brain homogenate then was incubated with phenylmethanesulfonyl fluoride (0.1 mM) for 15 min at 25°C water bath, centrifuged at 20,000 × g for 30 min and the resulting pellet resuspended in 6 volumes of the original wet weight in sucrose (0.32 M). Membranes from CHO cells stably transfected with MOR-1 were prepared as previously described (Brown et al., 1997b) and stored in sucrose (0.32 M). Homogenates stored at 80°C retained binding for at least 4 wk. Protein content was determined by the Lowry method (Lowry et al., 1951).

Iodination of endomorphin-1 and endomorphin-2. The peptides were iodinated with Na¹²⁵I (Du Pont, Wilmington, DE) and chloramine T for 80 sec at room temperature with a peptide/NaI molar ratio of 10:1, after which the reaction was terminated with sodium metabisulfite, as previously described (Mathis et al., 1997). The iodinated peptides were purified by high-performance liquid chromatography over a Rainin Microsorb-MV C18 reverse-phase column (Woburn, MA) using an acetonitrile gradient (25-50%) over a 50-min period. Both ¹²⁵I endomorphin-1 and ¹²⁵I-endomorphin-2, eluting at approximately 37% acetonitrile/0.1% TFA, were readily separated from their noniodinated peptides, which eluted at about 27% acetonitrile/0.1% TFA

¹²⁵I Endomorphin-1 and endomorphin-2 binding. ¹²⁵I-Endomorphin-1 or ¹²⁵I-endomorphin-2 binding (0.2 nM) was performed in potassium phosphate buffer (50 mM, pH 7.4; 0.5 ml) with MgCl₂ (5 mM) at a tissue concentration of 10 mg wet weight/ml for brains or 0.06 mg protein/ml for MOR-1/CHO cells. Specific binding was determined in the presence and absence of either 1 µM of the corresponding unlabeled peptide. The entire mixture was then incubated at 25°C for 1 hr and filtered over no. 32 glass fiber filters (Schleicher & Schuell, Keehne, NH) which had been presoaked for 1 hr in 0.5% polyethylenimine and washed twice with ice cold Tris buffer using a Brandel cell harvester (Cambridge, MA). The filters were then counted on a Packard Cobra gamma counter. The other opioid receptor binding assays were performed as previously described (Clark et al., 1988, 1989).

Autoradiography. Brains were removed from male CD-1 mice and quickly frozen in isopentane at 50°C. The brains were cryosectioned at 10 µm and thaw-mounted onto gelatin coated slides. Tissue sections were preincubated with buffer (50 mM potassium phosphate buffer, pH 7.4 and 5 mM MgCl₂) for 15 min to remove endogenous ligands. For I-endomorphin-1 experiments the buffer also contained 0.1% bovine serum albumin to reduce binding to white matter. The sections were then incubated with ¹²⁵I-endomorphin-1 or ¹²⁵I-endomorphin-2 (1 nM) for 2 hr at 25°C. Nonspecific binding was determined in adjacent sections with the corresponding unlabeled peptide (1 µM). After the incubation, sections were rinsed in fresh buffer for 20 min at room temperature (¹²⁵I-endomorphin-1) or 0°C (¹²⁵I-endomorphin-2). Sections were then dried under a stream of cool air and exposed to Hyperfilm (Amersham Corp., Arlington Heights, IL) for 47 hr (¹²⁵I-endomorphin-1) or 17 hr (¹²⁵I-endomorphin-2).

Analgesic assays. The endomorphins were administered i.c.v. or intrathecally under light halothane anesthesia as previously reported (Rossi et al., 1995, 1997). Antinociception, termed analgesia for convenience, was assessed in the radiant heat tail-flick assay with baseline latencies between 2 to 3 sec and a maximum cutoff score of 10 sec to minimize tissue damage. Analgesia was determined quantally as a doubling or greater of baseline tailflick scores. Dose-response curves were analyzed to generate ED₅₀ values and 95% confidence limits were generated from dose-response curves using a computer program based on the Litchfield-Wilcoxin approach (Tallarida and Murray, 1987). Peak analgesia was seen at 10 min for endomorphin-1 and 15 min for endomorphin-2. Results reflect peak analgesic values unless stated otherwise.

Gastrointestinal transit. Groups of mice were treated i.c.v. with endomorphin-1 (12 µg) or endomorphin-2 (3 µg) 15 min before a 0.5-cc charcoal meal (2.5% gum tragacanth,10% activated charcoal in water). The mice were killed 30 min later and the distance the charcoal traveled was measured.

	Results

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Endomorphin binding to opioid receptors. The initial report describing the endomorphins indicated that they were mu-selective (Zadina et al., 1997). First, we examined the affinity of the two endomorphins in traditional opioid binding assays in brain homogenates (table 1). Both endomorphin-1 and endomorphin-2 competed binding to mu receptors with high affinity. As noted with most opioids, the affinity of these compounds for the mu₁ receptors was greater than for the mu₂ receptor. We also found that the endomorphins had little appreciable affinity for either delta or kappa₁ binding sites, with K_i values greater than 500 nM. They did, however, show moderate affinity for the kappa₃ site, lowering binding with K_i values between 20 and 30 nM.

¹²⁵I-Endomorphin binding in mouse brain and MOR-1/CHO membranes. To extend previous reports, both endomorphins were iodinated and examined in binding studies. Initial studies established that binding reached steady state levels by 60 min at 25°C in potassium phosphate buffer (pH 7.4). In addition, there was little change in specific binding between pH 7 and 7.5 (data not shown) and the physiological pH of 7.4 was used for all subsequent assays. Lowering the temperature to 0°C drastically reduced specific binding whereas increasing the temperature to 37°C had little advantage. Binding was performed with tissue at 10 mg wet weight/ml, although binding remained linear up to tissue concentrations as high as 15 mg wet weight tissue/ml. As previously observed in traditional opioid receptor assays (Pasternak et al., 1975a), magnesium enhanced binding and was included in all assays.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Saturation studies of 125I-endomorphins in brain and MOR-1/CHO cells. Saturation studies were performed using 125I-endomorphin-1 and 125I-endomorphin-2 in A, mouse brain homogenates and B, MOR-1/CHO cell membranes with concentrations of radioligands ranging from 10 pM to 3 nM. The results are from a representative experiment.

Pharmacology of endomorphin-1 and endomorphin-2. Both endomorphin-1 and endomorphin-2 were potent analgesics with peak effects seen at 10 and 15 min, respectively. All subsequent studies were performed at peak effect. Both compounds were fully active supraspinally and spinally, with no indication of ceiling effects. Endomorphin-1 was significantly more potent spinally than supraspinally and, at the spinal level, it was significantly more potent than endomorphin-2 (fig. 2; table 5). The response of both agents were readily reversed by naloxone (fig. 3). -FNA, a highly selective mu antagonist, effectively reversed the actions of both endomorphins, as previously noted (Zadina et al., 1997). Naloxonazine is another mu antagonist, but its actions are limited to mu₁ and M6G receptors (Paul et al., 1989; Ling et al., 1986; Hahn et al., 1982; Pick et al., 1991; Paul and Pasternak, 1988). Although naloxonazine significantly lowered endomorphin-1 and endomorphin-2 analgesia, its actions were not as complete as -FNA. Neither the kappa₁ antagonist norBNI nor the delta antagonist naltrindole were active against either endomorphin (fig. 4).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Supraspinal and spinal endomorphin analgesia in mice. Analgesic dose response curves for the two endomorphins were generated in groups of CD-1 mice (n  15) after A, intracerebroventricular or B, intrathecal endomorphin-1 or endomorphin-2 (n  15/group). All results are peak effects.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Effect of naloxone on endomorphin analgesia. Groups of mice received endomorphin-1 (12 µg, i.c.v., n = 20; 1 µg, intrathecally, n = 26), endomorphin-2 (3 µg, i.c.v., n = 20; 15 µg, intrathecally, n = 9) alone or with naloxone (1 mg/kg, s.c., n  10/group) 15 min before injection of each drug. Naloxone significantly decreased the analgesia produced by the endomorphins both spinally and supraspinally (P < .01).

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Sensitivity of endomorphin analgesia to opioid antagonists. The sensitivity of A, endomorphin-1 (12 µg, i.c.v.) or B, endomorphin-2 (3 µg, i.c.v.) to various opioid antagonists was assessed. The general mu antagonist -funaltrexamine (-FNA, 40 mg/kg, s.c.) and mu1 antagonist, naloxonazine (3 µg, i.c.v.) were given 24 hr before endomorphin-1 and endomorphin-2 injections although all other antagonists were administered 15 min before the endomorphin. All groups had 20 mice. -FNA and naloxonazine significantly blocked both endomorphin-1 and endomorphin-2 analgesia. No significant difference was noted in the presence of the kappa1 antagonist, norBNI (10 mg/kg, s.c.), nor the delta antagonist, naltrindole (20 mg/kg, s.c.).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Endomorphin analgesia in CD-1 and CXBK mice. Endomorphin-1 (12 µg, i.c.v.) was given to groups of CD-1 mice (n = 16) or CXBK mice (n = 19) and analgesia assessed. Endomorphin-2 (3 µg, i.c.v.) was given to groups of CD-1 mice (n = 20) or CXBK mice (n = 20). The CD-1 mice were significantly more sensitive to both endomorphins (P < .01).

View larger version (40K): [in this window] [in a new window]   Fig. 6.   Effect of endomorphin on gastrointestinal transit. Groups of mice (n  10) received saline, endomorphin-1 (12 µg, i.c.v.) or endomorphin-2 (3 µg) 15 min before a charcoal meal bolus given by gavage. Mice were then killed 30 min after the bolus and the distance traveled by the meal measured as an indication of gastrointestinal (GI) transit. Endomorphin-1 decreased GI transit by 46% (P < .001) and endomorphin-2 lowered GI transit by 41% (P < .001).

Autoradiography of ¹²⁵I-endomorphin binding in mice. Finally, we examined the regional distribution of ¹²⁵I-endomorphin binding in mouse brain (fig. 7). ¹²⁵I-Endomorphin-1 and ¹²⁵I-endomorphin-2 binding patterns were established in sections from the striatum through the brain stem. Overall, the pattern of labeling was quite similar to that previously reported for mu receptors (Goodman and Pasternak, 1985; Atweh and Kuhar, 1977a, b, c; Moskowitz and Goodman, 1985; Kuhar et al., 1973). In the striatum (fig. 7a and b), both peptides exhibited higher labeling in the patches as compared to the surrounding matrix. High binding levels were also seen in the nucleus accumbens core and shell, as well as the anterior and medial thalamic and the amygdaloid nuclei (fig. 7c-h). At the level of the brainstem, both peptides labeled the periaqueductal gray, superior colliculus and interpeduncular nucleus (fig. 7I and h). Finally, a layered distribution of binding was observed with both peptides throughout the cortex and hippocampus.

View larger version (86K): [in this window] [in a new window]   Fig. 7.   Autoradiographic distribution of 125I-endomorphin binding sites in mouse brain. Tissue sections taken through the nucleus accumbens (A, B), posterior striatum (C, D), thalamus (E, F, G, H), and brainstem (I, J) were incubated with either 125I-endomorphin-1 or 125I-endomorphin-2 as described in "Materials and Methods."

	Discussion

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The original description of the two endomorphins revealed that both compounds had a profound mu selectivity (Zadina et al., 1997). In this initial study both endomorphins competed mu binding over 1000-fold more potently than either delta or kappa₁ receptors (Zadina et al., 1997). In the current studies, the two endomorphins also displayed very poor affinities for delta and kappa₁ receptors and high affinity for both mu receptor subtypes. Most opioids label mu₁ receptors more potently than mu₂ sites and the same trend was seen with the endomorphins, which displayed a 5- to 10-fold greater affinity in the mu₁ binding assay. The high affinity of the two endomorphins for mu receptors was confirmed in competition studies against the cloned mu receptor MOR-1.

The initial study exploring the selectivity of the compounds did not examine kappa₃ binding. We found that the endomorphins also competed kappa₃ receptors moderately well, lowering binding with K_i values between 20 and 30 nM. Although the endomorphin-1 and endomorphin-2 remain selective for mu receptors, their selectivity is not as great as initially proposed.

Radiolabeling the endomorphins did not appreciably affect their affinity in assays using either mouse brain homogenates or MOR-1/CHO cell membranes. ¹²⁵I-Endomorphin-1 displayed an affinity in the MOR-1/CHO cells which was slightly better than endomorphin-1 itself although ¹²⁵I-endomorphin-2 labeled sites with an affinity similar to that seen with the noniodinated endomorphin-2. The maintenance of affinity after iodination contrasts with the typical 10-fold decrease in affinity seen when traditional opioid peptides are iodinated. The iodine is located on the N-terminal tyrosine in all the compounds, suggesting significant differences in the way the traditional opioid peptides and the endomorphins sit in the binding pocket.

Overall, the competition studies were consistent with a high affinity of the ¹²⁵I-endomorphins for mu receptors. Typical mu drugs were quite potent in these studies although kappa and delta selective agents were not. In the MOR-1/CHO cell assay that contains only a single site, the Hill coefficients were close to unity, contrasting with the shallow competition studies in brain. For example, morphine has a Hill coefficient close to unity in the MOR-1/CHO membrane binding assay, but only about 0.5 against ¹²⁵I-endomorphin-1 in brain. The meaning of the low Hill slopes in the brain is not clear, but they might represent evidence that the labeling in brain may not reflect a single site. These issues will require further study.

In vivo, the endomorphins are potent analgesics, both spinally and supraspinally. In the first description of endomorphin-1, Zadina et al. (1997) reported significant analgesic activity after both spinal and supraspinal administration, with a 3-fold greater potency after spinal administration (Zadina et al., 1997). We also observed this difference between the two sites. Indeed, our ED₅₀ values are remarkably similar to the earlier ones.

Previous work from our laboratory has suggested that different mu receptor subtypes are responsible for spinal and supraspinal morphine analgesia (Pick et al., 1991, 1993; Ling and Pasternak, 1983). Mu₁ receptors mediate supraspinal morphine analgesia although mu₂ receptors are responsible for spinal analgesia. The affinity of endomorphin-1 for the mu receptor subtypes in binding assays does not explain its greater spinal activity because it competed mu₁ binding approximately 5-fold more potently than mu₂ sites. Thus, the reasons for the enhanced spinal activity remain unclear. However, both the earlier results and the current ones find significant spinal/supraspinal differences in the potency of endomorphin-1.

Endomorphin-2 was active at both levels of the neuroaxis, with no significant difference in the sensitivity of the two sites. However, spinal endomorphin-2 was significantly less active than endomorphin-1. An earlier study exploring the analgesic activity of the endomorphins at the spinal level found that the two compounds were not significantly different in a thermal assay (Stone et al., 1997). Although our results with endomorphin-1 were quite similar to the earlier report, endomorphin-2 was far less active in our studies, perhaps due to different assays. Whereas we used a quantal radiant tailflick assay, the earlier report used a warm water immersion tailflick assay with graded responses. The question of peptide stability also must be considered.

The mu characteristics of the endomorphins were further supported by a variety of studies. Pharmacologically, endomorphin analgesia was reversed by the mu-selective antagonist -FNA, implying that they were acting through mu receptors. Although the actions of both endomorphins were significantly reduced by the mu₁ antagonist naloxonazine, the blockade was not as great as with -FNA, particularly for endomorphin-1. However, the significance of this difference is not clear. Neither the kappa nor the delta-selective antagonists were effective, but the general opioid antagonist naloxone potently blocked the response, confirming its opioid nature. The inactivity of the two endomorphins in CXBK mice, a strain that is insensitive to morphine (Moskowitz and Goodman, 1985a; Reith et al., 1981a; Baron et al., 1975a; Pick et al., 1993a), also supported their mu selectivity. Mu drugs typically inhibit gastrointestinal transit and both endomorphins had similar actions. Finally, the regional distribution of ¹²⁵I-labeled endomorphins resembled that seen with traditional mu radioligands (Goodman and Pasternak, 1985; Kuhar et al., 1973).

In conclusion, the endomorphins are highly selective endogenous mu ligands. The binding selectivities and pharmacology of the peptides in the current studies support the possibility that they may represent the endogenous mu receptor ligands, as originally proposed (Zadina et al., 1997). However, several features of these peptides raise the possibility that their actions may involve more than just traditional mu receptors. A number of opioids competed binding with shallow hill slopes and preliminary studies suggest that the endomorphins retain analgesic activity in an MOR-1 knockout mouse model (King M, Schiller A, Pintar J and Pasternak GW, in preparation). The significance of these observations is not yet clear, but they do suggest that additional studies are needed to more fully define these potential endomorphin systems.