Introduction

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Originally proposed from studies with the benzomorphan (±)-N-allyl-normetazocine [(±)SKF-10047] (Martin et al., 1976),  receptors are now defined as nonopioid, non-phencyclidine, haloperidol-sensitive, naloxone-inaccessible, (+)benzomorphine-selective binding sites (Quirion et al., 1992). Two subtypes of  receptors have been proposed based upon their binding selectivity profiles (Bowen et al., 1993).  receptors are conserved across species (Weissman et al., 1988; Su and Wu, 1990; Walker et al., 1992) and are present in almost all tissues, with very high expression in the central nervous system, immune system, and liver (Gundlach et al., 1986; Ryan-Moro et al., 1996).

The ₁ receptor was first cloned from guinea pig liver (Hanner et al., 1996), followed by human (Kekuda et al., 1996), mouse (Pan et al., 1998), and rat clones (Seth et al., 1997, 1998; Mei and Pasternak, 2001). Structurally, ₁ receptors show no homology with any traditional receptor family. Even with the new information available from cloning, many questions regarding the functional significance of ₁ receptors remain, although recent work has reported an association with ankyrin (Hayashi and Su, 2001).

Functionally, ₁ receptors comprise an anti-opioid system and evidence suggests that some of the differences in sensitivity among strains of mice can be attributed to differing tonic levels of ₁ receptor activity (Chien and Pasternak, 1993, 1994, 1995a,b; King et al., 1997). Haloperidol, a presumed ₁ antagonist, potentiates systemic opioid analgesia, whereas the ₁ ligand (+)pentazocine diminishes it. These modulatory functions apply to µ, , ₁, and ₃ analgesics. However, haloperidol is not selective for ₁ receptors, displaying equally high affinity for D₂ dopamine receptors. Uncertainty regarding its actions is further raised by the observation that dopamine D₂ receptors also influence opioid actions (King et al., 2001). The present study compares the actions of (+)pentazocine and haloperidol with the effects of down-regulation of ₁ receptors by an antisense approach to further define the role of ₁ systems in opioid analgesia.

	Materials and Methods

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(+)Pentazocine, morphine sulfate, trans-(dl)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide methanesulfonate hydrate, (U50,488H), nalorphine, nalbuphine, and [D-Pen²,D-Pen⁵]enkephalin (DPDPE) were gifts from the National Institute on Drug Abuse Research Technology Branch (Rockville, MD). Halothane was purchased from Halocarcon Laboratories (Hackensack, NJ). Haloperidol, ketamine, and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Naloxone benzoylhydrazone (NalBzoH) was synthesized, as previously reported (Luke et al., 1988). All the drugs used in the in vivo studies were dissolved in saline, and doses were reported as the free base.

Male CD-1 (Crl:CD-1(ICR)BR) and BALB/c (BALB/cAnNCrlBR) mice (25-30 g) were purchased from Charles River Breeding Laboratories (Wilmington, MA) and maintained on a 12-h light/dark cycle with rodent chow and water available ad libitum. All animal studies were approved by the Institutional Animal Care and Use Committee of the Memorial Sloan-Kettering Cancer Center and adhere to National Institutes of Health guidelines. Spinal (i.t.) and supraspinal (i.c.v.) injections were made under halothane anesthesia as previously described (Haley and McCormick, 1957; Hylden and Wilcox, 1980). The anesthetic did not affect analgesia measurements 15 min later.

All [³H](+)pentazocine binding assays were performed as previously reported (Mei and Pasternak, 2001). In brief, membrane homogenates (100 µg of protein/ml) were incubated at 37°C for 3 h in potassium phosphate buffer (10 mM, pH 7.2) with [³H](+)- pentazocine (2 nM) and then filtered over glass fiber filters (Schleicher & Schuell, Keene, NH). All experiments were performed in triplicate and replicated at least three times. Only specific binding was reported, defined as the difference between total and nonspecific binding, as defined by that remaining in the presence of haloperidol (1 µM).

Analgesia was determined using the radiant heat tail-flick technique (D'Amour and Smith, 1941; Gistrak et al., 1990; Paul et al., 1990, 1991; Chien and Pasternak, 1994). Baseline latencies were determined before each experimental treatment for all animals as the mean of two trials and generally ranged between 2 and 3 s. Tail-flick latencies were measured again 15 min after either i.c.v. and i.t. injections and 30 min following s.c. injections. These times correspond to peak effects of the drugs (Paul et al., 1989; D. Paul and G. W. Pasternak, unpublished observations). A maximal latency of 10 s was used to minimize tissue damage. Analgesia was defined quantally as a doubling or greater of the baseline latency for each mouse. All studies employed groups of at least 10 mice. Quantal data were compared using Fisher's exact test. When evaluating the effects of an agent upon a single agonist dose, it is important to use a dose that is on the linear portion of the dose-response curve. We used agonist doses giving approximately a 75% response when we anticipated seeing decreased responses and a dose of approximately 15 to 20% when looking for increased responses.

Antisense oligodeoxynucleotides were designed based upon the mouse ₁ receptor cDNA sequences (GenBank accession no. AF004927) and were purchased from Midland Certified Reagent Co. (Midland, TX). Oligodeoxynucleotides were repurified using sodium acetate precipitation and dissolved in saline for a final concentration of 5 µg/µl. The three antisense oligodeoxynucleotides corresponding to the cloned mouse ₁ receptor (S2-1a) are: AS1, 5'-GAGTGCCCAGCCACAACCAGG-3' (bp 97-77); AS2, 5'-GGCCGTACTCCACCATCCACG-3' (bp 523-503); and AS3, 5'-CTGGCTGGTCAGGAGTCTTGGC-3' (bp 680-659). The mismatch oligo was generated with the same base composition as AS1, in which three pairs of bases were switched: 5'-GAGGTCCCGACCACACACAGG-3'. Groups of mice were treated with antisense oligodeoxynucleotides (10 µg in 2 µl) or vehicle (saline) under light halothane anesthesia (Standifer et al., 1994) on days 1, 2, and 4 and tested for analgesia on day 5 with the indicated drug.

	Results

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Anti-Opioid Properties of ₁ Receptors in the Central Nervous System. When administrated systemically (s.c.), (+)pentazocine antagonized both systemic and central morphine analgesia (Chien and Pasternak, 1994, 1995a,b). To determine the site of action of (+)pentazocine, we examined its effects when given centrally. (+)Pentazocine alone had no effect on tail-flick latencies when given intracerebroventricularly (data not shown). Supraspinal (+)pentazocine significantly reduced the analgesic actions of systemic and supraspinal morphine (Figs. 1 and 2). Spinal morphine analgesia was unaffected by (+)pentazocine given either spinally or supraspinally (Fig. 2).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Effects of supraspinal (+)pentazocine on systemic opioid analgesia. Groups of CD-1 mice (n  20) received vehicle or (+)pentazocine (13 µg, i.c.v.) immediately prior to testing with either morphine (5 mg/kg, s.c.), U50,488 (5 mg/kg, s.c.), NalBzoH (75 mg/kg, s.c.), nalbuphine (50 mg/kg, s.c.), or nalorphine (50 mg/kg, s.c.). Agonist doses were chosen to give approximately 75% responses to enable the assessment of either decreased or increased responses. (+)Pentazocine significantly decreased the number of analgesic animals following systemic morphine (p < 0.05), U50,488H (p < 0.01), NalBzoH (p < 0.005), nalbuphine (p < 0.01), and nalorphine (p < 0.005).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Effects of (+)pentazocine on central opioid analgesia. Groups of CD-1 mice (n  20) received either vehicle or (+)pentazocine (13 µg, i.t. or i.c.v.) immediately prior to testing with morphine (700 ng, i.c.v. or i.t.), nalbuphine (20 µg, i.c.v.), or DPDPE (8 µg, i.c.v.). (+)Pentazocine significantly decreased supraspinal morphine (p < 0.001), DPDPE (p < 0.005), and nalbuphine (p < 0.01) analgesia. However, spinal morphine analgesia was unaffected by (+)pentazocine given either i.c.v. or i.t.

Potentiation of Opioid Analgesia by ₁ Receptor Antisense Treatment. We next examined ₁-opioid interactions at the molecular level by targeting the ₁ receptors using an antisense approach. To validate the technique, we first confirmed the down-regulation of ₁ receptor binding following antisense treatment. Mice were treated with saline, antisense, or a mismatch control (20 µg, i.c.v.) on days 1, 2, 3, and 4. On day 5, the periaqueductal gray region, an area important in µ opioid analgesia, was dissected from the animal and used in homogenate binding assays (Fig. 3). Antisense treatment (AS1) significantly decreased [³H](+)pentazocine binding by almost 80% compared with controls (p < 0.05). There were no significant differences in binding levels between the control and mismatch groups. Thus, antisense treatment effectively down-regulated ₁ receptor binding. The inactivity of the mismatch control supported the specificity of the approach.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Effect of 1 receptor antisense treatment on [3H](+)pentazocine binding. CD-1 mice received antisense AS1 (20 µg, i.c.v.) on days 1, 2, 3, and 4. The periaqueductal gray region was dissected on day 5 and membranes prepared. Binding assays were performed as described under Materials and Methods, and only specific binding was shown. Results are the means ± S.E.M. values of three independent determinations.

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Effect of 1 antisense on opioid analgesia in CD-1 mice. A, groups of CD-1 mice (n  20) received saline or AS1 (10 µg, i.c.v.) on days 1, 2, and 4. On day 5 all mice were tested with morphine (2 mg/kg, s.c.), U50,488H (2 mg/kg, s.c.), or NalBzoH (30 mg/kg, s.c.), and analgesia was assessed quantally. Antisense AS1 significantly potentiated systemic morphine (p < 0.005), U50,488H (p < 0.001), and NalBzoH (p < 0.001) analgesia. B, groups of CD-1 mice (n  20) received saline or AS1 (10 µg, i.c.v.) on days 1, 2, and 4. On day 5 all mice were tested with morphine (175 ng, i.c.v.) or DPDPE (2 µg, i.c.v.), and analgesia was assessed quantally. Antisense AS1 significantly potentiated morphine supraspinal (p < 0.001) and DPDPE supraspinal (p < 0.005) analgesia.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Effect of different 1 receptor antisense probes on systemic  opioid analgesia. Groups of mice (n  20) received saline or the indicated oligodeoxynucleotides (10 µg, i.c.v.) on days 1, 2, and 4. On day 5 all mice were tested with U50,488H (2 mg/kg, s.c.) or NalBzoH (30 mg/kg, s.c.), and analgesia was assessed quantally. Analgesic testing of mice was performed as described under Materials and Methods. All the antisense probes tested significantly potentiated U50,488H 1 analgesia (p < 0.01) and potentiated NalBzoH analgesia (p < 0.02).

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Effect of 1 antisense on opioid analgesia in BALB-c mice. Groups of mice (n  20) received saline or AS1 (10 µg, i.c.v.) on days 1, 2, and 4. A, on day 5 all mice were tested with morphine (1 mg/kg, s.c.), U50,488H (5 mg/kg, s.c.), or NalBzoH (50 mg/kg, s.c.), and analgesia was assessed quantally. Antisense AS1 significantly potentiated systemic morphine (*, p < 0.02) and systemic U50,488H, and NalBzoH (**, p < 0.001) analgesia. B, on day 5 all mice were tested with morphine (125 ng, i.c.v.) or DPDPE (3 µg, i.c.v.), and analgesia was assessed quantally. Antisense AS1 significantly potentiated supraspinal morphine and DPDPE analgesia (*, p < 0.001).

	Discussion

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Previously, we observed strong interactions between the ₁ receptor system and opioid analgesia (Chien and Pasternak, 1994, 1995a,b). In these studies, systemic (+)pentazocine, a selective ₁ receptor ligand diminished opioid analgesia, whereas systemic haloperidol potentiated it (Chien and Pasternak, 1994). The anti-opioid actions of the ₁ receptor system were restricted to opioid analgesia, with (+)pentazocine having no appreciable effect upon morphine-induced inhibition of gastrointestinal transit (Chien and Pasternak, 1994).

The current studies further defined the actions of (+)pentazocine. Alone, (+)pentazocine had no effect when given spinally or supraspinally. When given supraspinally, (+)pentazocine had anti-opioid actions against µ, , ₁, and ₃ analgesia. In contrast, spinal morphine analgesia was not influenced by (+)pentazocine given either supraspinally or spinally. This observation was not anticipated since systemic (+)pentazocine reverses spinal morphine analgesia (Chien and Pasternak, 1994). The reasons underlying the difference in sensitivity of spinal morphine analgesia to systemic as opposed to either supraspinal or spinal (+)pentazocine are not clear. It may require simultaneous administration of (+)pentazocine to both sites for activity or it may also involve other sites of action, perhaps even peripheral ones.

The cloning of the ₁ receptor (Kekuda et al., 1996; Pan et al., 1998; Seth et al., 1998) has not resolved the question of its function at the molecular level. A recent report has found a physical association of the  receptor with ankyrin (Hayashi and Su, 2001), but even here the functional significance of this association is unclear. However, cloning the protein opens a number of possibilities to explore its pharmacology at the molecular level using antisense approaches (Standifer et al., 1994, 1995; Pasternak and Standifer, 1995). In the current studies, antisense treatments produced a down-regulation of the ₁ receptor at the protein level, as shown by approximately an 80% decrease in [³H](+)pentazocine binding compared controls, with no change in binding in mismatch-treated animals. This decreased receptor binding corresponded well to the decrease in mRNA that we reported earlier (Pan et al., 1998), confirming that the antisense treatment down-regulates both mRNA and protein. Functionally, the supraspinal antisense treatment potentiated µ, , ₁, and ₃ opioid analgesia. Furthermore, multiple antisense probes targeting the ₁ receptor gave similar results against the  analgesics. Thus, the actions of the antisense were not dependent upon a single sequence, further supporting the specificity of the response.

Haloperidol potentiates opioid analgesia both in patients (Maltbie et al., 1979) and rodents (Chien and Pasternak, 1993, 1994, 1995a,b). However, haloperidol is not selective. In addition to its high affinity for the ₁ receptor, haloperidol also labels dopamine D₂ receptors equally well. Thus, the actions of haloperidol might be due to activity at either, or both, of these receptors. The possibility of a combined mechanism of action is more intriguing in view of studies on a D₂ receptor knockout mouse (King et al., 2001). In these studies, opioid analgesia was enhanced in D₂ receptor knockout mice. A similar potentiation was produced by the D₂ receptor antagonist sulpiride in wild-type, but not knockout, mice. It is not clear why an earlier study failed to detect an effect of sulpiride on opioid analgesia, but it may be due in part to using different strains of mouse. These D₂ knockout animals also confirmed the presence of an independent ₁ receptor system. (+)Pentazocine retained its ability to reduce opioid analgesia in these knockout mice. Haloperidol still potentiated opioid analgesia in the D₂ knockout mice when given alone and reversed the actions of (+)pentazocine in the knockout mice, further establishing its role as a ₁ receptor antagonist. Together with the current studies, these observations argue strongly for independent ₁ and D₂ receptor modulation of opioid analgesia.

The activity of the ₁ anti-opioid system varies among strains, possibly explaining some of their sensitivity differences to opioids (Chien and Pasternak, 1994). For example,  drugs are far less effective analgesics in BALB-c mice than CD-1 mice. The ₁ drug U50,488H is 6-fold more potent in CD-1 mice, and the ₃ analgesic NalBzoH shows a similar difference. Yet, coadministration of haloperidol with the opioids potentiates the analgesic responses in both strains and virtually eliminates the strain differences among the opioids (Chien and Pasternak, 1994). As in the CD-1 mice, antisense treatment significantly enhanced the opioid responses in the BALB-c mice, with responses similar to those in CD-1 animals. The similar activity of the antisense to our earlier studies with haloperidol is consistent with a ₁ receptor mechanism in both paradigms.

In conclusion, the present study confirms and extends prior observations on the functional interactions between ₁ receptors and opioid analgesia action and establishes their interactions supraspinally. It is still unclear whether the receptors are physically associated or influence each other at the level of their circuitry, but supraspinal interactions appear to be important. Our studies also support the concept that haloperidol is a ₁ antagonist based on the similarity of its actions compared with those seen following down-regulation of the receptor by antisense. Conversely, these observations support the classification of (+)pentazocine as an agonist. Although the molecular actions of the ₁ receptor are unclear despite having been cloned, ₁ receptors clearly are functionally important in modulating opioid analgesic actions.