dependence

Brain Research 919 (2001) 20–30 www.elsevier.com / locate / bres

Research report

Cholera toxin-B subunit blocks excitatory opioid receptor-mediated hyperalgesic effects in mice, thereby unmasking potent opioid analgesia and attenuating opioid tolerance / dependence Ke-Fei Shen, Stanley M. Crain* Department of Neuroscience, Albert Einstein College of Medicine, Yeshiva University, 1300 Morris Park Ave. Bronx, NY 10461, USA Received 5 April 2001; accepted 14 August 2001

Abstract In a previous study we demonstrated that injection (i.p.) of low doses of GM1 ganglioside in mice rapidly attenuates morphine’s analgesic effects. This result is consonant with our electrophysiologic studies in nociceptive types of dorsal root ganglion (DRG) neurons in culture, which showed that exogenous GM1 rapidly increased the efficacy of excitatory (Gs-coupled) opioid receptor functions. By contrast, treatment of DRG neurons with the non-toxic B-subunit of cholera toxin (CTX-B) which binds selectively to GM1, blocked the excitatory, but not inhibitory, effects of morphine and other bimodally-acting opioid agonists, thereby resulting in a net increase in inhibitory opioid potency. The present study provides more direct evidence that endogenous GM1 plays a physiologic role in regulating excitatory opioid receptor functions in vivo by demonstrating that cotreatment with remarkably low doses of CTX-B (10 ng / kg, s.c.) selectively blocks hyperalgesic effects elicited by morphine or by a kappa opioid agonist, thereby unmasking potent opioid analgesia. These results are comparable to the effects of cotreatment of mice with morphine plus an ultra-low dose of the opioid antagonist, naltrexone (NTX) which blocks opioid-induced hyperalgesic effects, unmasking potent opioid analgesia. Low-dose NTX selectively blocks excitatory opioid receptors at their recognition site, whereas CTX-B binds to, and interferes with, a putative allosteric GM1 regulatory site on excitatory opioid receptors. Furthermore, chronic cotreatment of mice with morphine plus CTX-B attenuates development of opioid tolerance and physical dependence, as previously shown to occur during cotreatment with low-dose NTX.  2001 Elsevier Science B.V. All rights reserved. Keywords: Low-dose m and k-opioid hyperalgesia; Cholera toxin-B subunit; GM1 regulated-excitatory opioid receptor; Mouse tail-flick assay; Opioid analgesia and tolerance / dependence

1. Introduction Electrophysiologic studies of nociceptive types of mouse dorsal-root ganglion (DRG) neurons in culture showed that specific mu, delta and kappa opioid agonists evoke naloxone (NLX)-reversible prolongation of the action potential duration (APD) when applied at low nM concentrations to many of these cells [11,58]. In contrast, higher mM levels of opioids shortened the APD, as in previous studies [9,69]. This bimodal modulation of the APD of DRG neurons by exposure to either low vs. high concentrations of opioid agonists appears to be due to activation of excitatory vs. inhibitory opioid receptor-mediated func*Corresponding author. Tel.: 11-718-430-2481; fax: 11-718-4303381. E-mail address: [email protected] (S.M. Crain).

tions. Treatment with pertussis toxin (PTX), which uncouples inhibitory receptors linked to the regulatory G-proteins, Gi and Go [33,42], blocks opioid-induced shortening of the APD in DRG neurons [58] and opioid attenuation of DRG-evoked postsynaptic dorsal-horn network responses in DRG-cord explants [20]. On the other hand, opioidinduced APD prolongation in DRG neurons is blocked by treatment with the A subunit of cholera toxin (CTX-A) [59] which interferes with ligand-activation of Gs-linked excitatory receptors (see below). Cholera toxin (CTX) consists of a non-covalently assembled pentamer of the non-toxic B subunit (CTX-B) responsible for cell attachment and a toxicogenic A subunit (CTX-A) [47]. CTX-B binds selectively to GM1 ganglioside on the cell surface and facilitates penetration of CTX-A into the cell membrane [27,31,52]. CTX-A selectively catalyzes the ADP-ribosylation of Gs [32,48],

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02990-0

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resulting in inhibition of an associated GTPase [8], increased adenylate cyclase activity and decreased efficacy of ligand activation of Gs-coupled receptors [7,8,44,49,65]. Brief treatment of DRG neurons with very low concentrations of purified CTX-A subunit, as well as with whole toxin (1 pg / ml–1 ng / ml), selectively blocks opioidinduced prolongation of the APD, thereby providing strong evidence for mediation of this excitatory modulatory effect by opioid receptors linked to a Gs / protein kinase A / cyclic AMP second messenger system [11,59] (see also Refs. [10,21] and Section 4.6). Furthermore, pretreatment of DRG neurons with CTX-B (1–10 ng / ml) also selectively blocks opioid induced APD prolongation, but not opioidinduced APD shortening [60]. In contrast to CTX-A blockade of opioid excitatory effects by ADP-ribosylation of Gs, CTX-B blockade appears to involve interference with GM1 ganglioside regulation of opioid excitatory receptor functions. This conclusion is based on the specificity of CTX-B in binding with selective high affinity (KD 510 210 M) to GM1 ganglioside which is abundantly distributed on the external surface of neuronal cell membranes [27–29,43,72]. Treatment of DRG neurons with anti-GM1 antibodies also selectively blocks opioid induced APD prolongation, as occurs with CTX-B [59]. Subsequent studies showed that after brief treatment of DRG neurons with low (10 nM) concentrations of GM1 (but not GM2, GM3, or various other gangliosides or glycolipids) the threshold concentration of the opioid peptide dynorphin that is required to prolong the APD in many DRG neurons is markedly decreased from nM to fM–pM levels [63]. These electrophysiological studies suggest that the increased sensitivity of GM1-treated DRG neurons to the excitatory effects of opioid agonists is due to GM1 binding to an allosteric regulatory site on opioid receptors [60] which appears to enhance the efficacy of excitatory Gs-coupled opioid receptor functions [16]. Furthermore, Wu et al. [75,76] have shown that GM1 binding to opioid receptors also results in conversion of these receptors from an inhibitory Gi-coupled to an excitatory Gs-coupled mode [see also Refs. [15,16] and Section 4.1]. The increased sensitivity of GM1-treated DRG neurons to the excitatory effects of opioid agonists is consonant with recent evidence that injection of low doses of GM1 (ca. 0.1 mg / kg, i.p.) in mice rapidly attenuates morphine’s analgesic effects, resulting in ‘acute tolerance’ [17]. The present study provides more direct evidence that endogenous GM1 plays a physiologic role in regulating excitatory opioid receptor functions in vivo by demonstrating that remarkably low doses of CTX-B (10 ng / kg, s.c.) selectively block hyperalgesic effects elicited by morphine or a kappa opioid agonist, thereby unmasking potent opioid analgesia. These results are comparable to the effects of cotreatment of mice with morphine plus an ultra-low dose of naltrexone (NTX) [14,19,62] which by a different mechanism blocks opioid-induced hyperalgesic effects,

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unmasking potent opioid analgesia. Low-dose NTX selectively antagonizes excitatory opioid receptors at their recognition site, whereas CTX-B binds to, and interferes with, a putative allosteric GM1 regulatory site on excitatory opioid receptors [15,16,60]. Furthermore, chronic cotreatment of mice with morphine plus CTX-B also attenuates development of opioid tolerance and physical dependence, as previously shown to occur during cotreatment with low-dose NTX [14,62].

2. Materials and methods The protocols of this research project including the care and humane use of the mice have been approved by the Animal Institute Committee at the Albert Einstein College of Medicine.

2.1. Antinociception and hyperalgesia assays in mice Swiss–Webster (SW) male mice (20–25 g, Charles River, NY) were housed separately in groups of five, maintained on a 12 h light / dark cycle, and provided water and food ad libitum for 1–3 days prior to antinociception testing. Antinociceptive and hyperalgesic effects of opioids on these mice were measured using a hot-water-immersion tail-flick assay similar to methods previously described [14,17,37,62]. Each mouse was permitted to enter into a tapered plastic cylinder (with air holes). The size of the cylinder is a little larger than the animal body size, with the tail hanging out freely from the cylinder. The cylinder provides a secluded environment into which the animal voluntarily enters with no application of any force. During the tail-flick assay only the cylinder is handled without direct contact with the animal. One-third of the tail from the tip is immersed into a water-bath maintained at 52 or 558C (10.10) with an electronic thermoregulator (Yellow Springs). The latency to a rapid tail-flick was recorded; mice with control latencies .8 s were excluded from these tests and a 10 s cutoff was used to minimize tissue damage. Five sequential control tests were made, each with a 10 min interval. The latencies of the last four tests were averaged to provide a predrug value. Time-effect curves were plotted using tail-flick latencies as the ordinate [17,19].

2.2. Animal test groups Comparative tests were generally carried out on the same day with two or more groups of eight mice receiving a specific cotreatment plus an appropriate control group of eight mice treated with morphine (or other test opioid agonist) alone. All animal test groups were used for only one assay, except in chronic drug treatment tests.

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2.3. Statistical analyses Differences between treatment groups were examined for statistical significance by means of ANOVA with Neuman–Keuls tests or by means of Student t-test [66].

with the same or even lower doses of rCTX-B (10 ng / kg) also blocked low-dose morphine-induced hyperalgesia, unmasking potent opioid analgesia (Fig. 1C; cf. Fig. 1B).

2.4. Materials

3.2. Cotreatment with CTX-B also blocks acute low-dose kappa opioid-induced hyperalgesia, unmasking potent analgesia

The following drugs were used: NTX, NLX, morphine, U-50, 488H, norbinaltorphimine (Sigma); CTX-B (‘choleragenoid’) and recombinant CTX-B (List). Stock solutions of CTX-B (commercially purified from whole CTX) were heated to 568C for 20 min to eliminate possible traces of CTX-A in the commercial product. This protocol is based upon our previous study showing that the potent blocking effects of both CTX-A (tested up to 1 mg / ml) and whole CTX (tested up to l ng / ml) on opioid excitatory modulation of the APD were completely eliminated after this heat treatment [59]. In contrast to the thermolabile, enzymatic properties of the A subunit, the B subunit preparations showed relatively little loss in potency after heating to 568C [60]. These results demonstrate that pretreatment of DRG neurons with CTX-B can block opioid induced APD prolongation even under conditions which preclude effects mediated by the A subunit. Control tests were also carried out on some groups of mice injected with recombinant CTX-B (rCTX-B) (produced and purified from a recombinant strain of Vibro cholerae lacking the CTX-A gene [54]).

Acute hyperalgesia elicited by the kappa opioid agonist, U-50, 488H (10 ng / kg) (Fig. 2A: d) was also blocked by CTX-B (0.1 mg / kg), unmasking potent analgesia (Fig. 2A: ,), comparable to the effects of cotreatment with pg / kg doses of either NTX (Fig. 2A: s) or the specific kappa receptor antagonist, nor-binaltorphimine (Fig. 2A: .). When the group of mice cotreated with U-50, 488H plus CTX-B (Fig. 2A: ,) was retested 1 day later with the kappa opioid alone, the time-effect curve (Fig. 2B: s) showed that the blocking effect of CTX-B had disappeared, unmasking typical hyperalgesic responses (cf. Fig. 2A: d). Furthermore, when the group of mice showing low-dose U-50, 488H-induced hyperalgesia (Fig. 2A: d) was retested 1 day later together with a low dose of CTX-B, 10 ng / kg, the hyperalgesia was blocked, unmasking prominent kappa opioid analgesia (Fig. 2B: d). Cotreatment of another group of mice with 10 ng / kg rCTX-B plus 10 ng / kg U-50, 488H also blocked acute kappa opioid-induced hyperalgesia, unmasking potent analgesia, as occurred with regular CTX-B (Fig. 2C: s vs. d; cf. Fig. 2B).

3. Results

3.3. Chronic cotreatment of mice with morphine plus CTX-B attenuates the development of opioid tolerance

3.1. Cotreatment of mice with CTX-B blocks acute lowdose morphine-induced thermal hyperalgesia, thereby unmasking potent opioid analgesia Hot-water (528C)-immersion nociceptive assays in normal, naive SW mice result in control tail-flick latencies of about 4 s [19]. Administration of very low doses of morphine (ca. 1 mg / kg, s.c.), to these mice resulted in rapid onset of decreases in tail-flick latencies (ca. 1–2 s) which lasted for 4–5 h after drug injection (Fig. 1A: d). Cotreatment of other groups of mice with 1 mg / kg CTX-B (s.c.) blocked this low-dose morphine-induced thermal hyperalgesia and unmasked potent opioid analgesia lasting for .6 h (Fig. 1A: .), similar to the effect of cotreatment with ultra-low-dose NTX (Fig. 1A: s; Ref. [19]). Cotreatment with a still lower dose of CTX-B, 0.1 mg / kg, was also effective in blocking low-dose morphine-induced hyperalgesia and unmasking potent analgesia (Fig. 1B: .). Control tests showed that introduction of CTX-B alone was ineffective (Fig. 1B: ,). In order to confirm that our heat treatment of regular CTX-B did, in fact, eliminate trace amounts of CTX-A (Section 2.4), several groups of mice were tested with recombinant CTX-B. Cotreatment

Acute cotreatment of mice with CTX-B markedly enhanced the analgesic effects of higher doses of morphine by blocking opioid hyperalgesic ‘side-effects’, as observed in mice cotreated with morphine plus ultra-low-dose NTX [14,62]. Cotreatment with 3 mg / kg morphine plus 0.1 mg / kg CTX-B greatly increased the magnitude as well as the duration of morphine’s antinociceptive effects (Fig. 3A: s vs. d). The cotreated mice showed prominent analgesia for .6 h after dosing, whereas analgesic effects in mice treated with morphine alone were sharply attenuated by 2–3 h after dosing. This series of assays were carried out using a slightly higher water-immersion temperature (558C) (as utilized in our previous studies of morphine6low-dose NTX [14,62]). In assays at 558C control tail-flick latencies were about 2 s so that although possible hyperalgesic effects were more difficult to quantitate, opioid antinociceptive effects were quite reliable and comparable with assays at 528C (cf. Fig. 3 vs. Figs. 1 and 2). After daily injections of 3 mg / kg morphine for 5 days (b.i.d.), the magnitude and duration of morphine’s antinociceptive effects were sharply decreased (Fig. 3B: d). By contrast, chronic cotreatment with 3 mg / kg morphine plus 0.1 mg / kg CTX-B for 5 days (b.i.d.) resulted in

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Fig. 1. Cotreatment of mice with CTX-B blocks acute low-dose morphine-induced thermal hyperalgesia, unmasking potent opioid analgesia. (Time-effect curves of hot-water (528C)-immersion tail flick tests). (A): Administration of 1 mg / kg morphine (s.c.) results in onset of decreases in tail-flick latencies, which lasts for 4–5 h after drug injection (d). Cotreatment with 1 mg / kg morphine plus 1 mg / kg CTX-B (s.c.) blocks this low-dose morphine-induced hyperalgesia and unmasks potent opioid analgesia lasting for .6 h (.), similar to the effect of cotreatment with 1 pg / kg NTX (s). (B): Cotreatment with a still lower dose of CTX-B, 0.1 mg / kg is also effective (.; cf. 1 mg / kg morphine alone). Control tests showed that 10 mg / kg CTX-B alone is ineffective (,; cf. 1 mg / kg Mor110 mg / kg CTX-B: s). (C): Cotreatment with recombinant CTX-B at an even lower dose, 10 ng / kg, also blocks low-dose (100 ng / kg) morphine-induced hyperalgesia, unmasking potent opioid analgesia (s; cf. d). Note: in this and all subsequent figures, n58 for each curve; error bars indicate S.E.M. Dashed line has been inserted in Fig. 1A and some of the subsequent figures to facilitate visualization of the hyperalgesic effects.

maintenance of prominent antinociception, which was maintained for .6 h after the test dose (Fig. 3B: s; cf. Fig. 3A). The ability of CTX-B to attenuate the development of morphine tolerance is quite comparable to the results obtained by cotreatment of morphine with ultralow-dose NTX, utilizing similar tail-flick assays at 558C [62].

3.4. Chronic cotreatment of mice with morphine plus CTX-B prevents development of NLX-precipitated withdrawal hyperalgesia Injection of naive mice with low doses of NLX or NTX

does not alter the baseline tail-flick latency in our antinociceptive assays [14,62]. On the other hand, in chronic morphine-treated mice (e.g. 10 mg / kg for 5 days, b.i.d.) which had developed a moderate degree of tolerance (Fig. 4A), withdrawal of morphine and injection of 10 mg / kg NLX evoked a prominent hyperalgesic response lasting for .5 h (Fig. 4B: d). By contrast, no significant NLXprecipitated withdrawal hyperalgesia occurred in mice that had been chronically cotreated with CTX-B plus the same morphine dosage (Fig. 4B: s). It is of interest that NLXprecipitated withdrawal hyperalgesia could be elicited in chronic morphine-treated mice well before the development of a marked degree of antinociceptive tolerance [61]; see also Ref. [6].

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Fig. 2. Cotreatment with CTX-B blocks acute low-dose kappa opioid-induced hyperalgesia, unmasking potent analgesia. (A): Administration of a low dose of the kappa opioid agonist, U-50, 488H (10 ng / kg, s.c.) results in similar hyperalgesia as elicited by low doses of the mu agonist morphine (d; cf. Fig. 1A: d). Cotreatment with U-50, 488H plus 0.1 mg / kg CTX-B blocks this low-dose kappa opioid hyperalgesia, unmasking potent analgesia (,), similar to the effects of cotreatment with either ultra-low doses of NTX (s) or the specific kappa antagonist, nor-binaltorphimine (.). (B): When the same group of mice that showed prominent analgesia following cotreatment with U-50, 488H plus CTX-B (A: ,) was retested after 24 h with U-50, 488H alone, typical hyperalgesia occurs (s) indicating disappearance of the CTX-B blocking effect. Furthermore, when the same group of mice that showed hyperalgesia following U-50, 488H alone (A: d) was retested after 24 h by cotreatment with a remarkably low dose of CTX-B, 10 ng / kg, the hyperalgesia is blocked, unmasking prominent analgesia (d). (C): Cotreatment of another group of mice with 10 ng / kg recombinant CTX-B (rCTX-B) plus 10 ng / kg U-50, 488H also blocks acute kappa opioid-induced hyperalgesia (d) unmasking potent analgesia (s), as occurs with regular CTX-B (B: d). Treatment with rCTX-B alone was ineffective (.).

3.5. Oral cotreatment of mice with CTX-B blocks hyperalgesic effects of morphine injections, unmasking potent analgesia

than the control group given morphine alone (Fig. 5C: s vs. d).

CTX-B was added to the drinking-water bottles of mice for 1 day prior to antinociception assays with low-dose of morphine (0.1 mg / kg, s.c.). Mice pretreated with CTX-B showed prominent, long-lasting antinociception, in sharp contrast to the marked hyperalgesia elicited by this extremely low dose of morphine in the control group (Fig. 5A: s vs. d). After a second day on oral CTX-B these mice were retested with a 10 000-fold higher dose of morphine (1 mg / kg, s.c.). Mice treated with CTX-B showed much larger and longer-lasting analgesic effects

4. Discussion The results of the present study provide strong evidence suggesting that GM1 ganglioside-binding to excitatory Gscoupled opioid receptors plays a physiological role in regulating these receptors on neurons in nociceptive networks in vivo. We previously showed that injection of low doses of exogenous GM1 (0.1–1 mg / kg, i.p.) in mice rapidly attenuates morphine’s analgesic effects (‘acute tolerance’) [17], presumably by enhancing the efficacy of

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Fig. 3. Chronic cotreatment of mice with CTX-B plus morphine blocks the development of opioid tolerance. (A): Acute cotreatment of another group of mice with 0.1 mg / kg CTX-B plus a high dose of morphine (3 mg / kg) (s.c.) markedly increases the magnitude as well as the duration of morphine’s antinociceptive effects (d). (B): After daily injections of 3 mg / kg morphine plus CTX-B for 5 days (b.i.d.), the magnitude and duration of morphine’s antinociceptive effects are still quite large (s), whereas the analgesic effects of 3 mg / kg morphine alone (b.i.d.) are sharply decreased (d). Note: The tail-flick assays in this figure were carried out using a slightly higher water-immersion temperature (558C) (see text: Section 3.3); note shorter control tail-flick latencies, ca. 2 s (vs. 4 s in assays at 528C: Figs. 1 and 2).

excitatory opioid receptor functions of nociceptive neurons. This interpretation is supported by the present evidence that injection of remarkably low doses of CTX-B (s.c.) rapidly blocks acute opioid-induced hyperalgesic effects, thereby unmasking potent opioid analgesia. Similar effects occur after systemic cotreatment of mice with morphine plus ultra-low-dose naltrexone [14,62] and intrathecal, as well as systemic, cotreatment of rats with morphine plus ultra-low-dose NTX [1]. The prolonged duration of prominent antinociceptive effects for .6 h after dosing in mice cotreated with CTX-B plus low mg / kg morphine (Fig. 1A) as well as high 3 mg / kg morphine (Fig. 3A) is quite remarkable. The

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Fig. 4. Chronic cotreatment of mice with CTX-B plus morphine blocks the development of naloxone-precipitated withdrawal hyperalgesia. (A): After daily injections of 10 mg / kg morphine for 5 days (b.i.d.), morphine’s antinociceptive effects are still quite prominent (d), in comparison with the marked tolerance observed in the group of mice chronically treated with a three-fold lower dose of morphine (cf. Fig. 3B: d). Chronic cotreatment with daily injections (b.i.d.) of 10 mg / kg morphine plus a 1000-fold lower dose of CTX-B (0.1 mg / kg) results in enhanced antinociception (s), but not as dramatic as in the group treated with 0.1 mg / kg CTX-B (cf. Fig. 3B: s). (B) Nevertheless, after withdrawal of morphine on day 6, injection of a low dose of NLX (10 mg / kg) evokes a prominent long-lasting hyperalgesia in the mice that had been chronically treated with morphine alone (d), whereas no significant NLX-precipitated withdrawal hyperalgesia occurs in mice that had been cotreated with CTX-B plus morphine (s) (nor in control tests on naive mice injected with 10 mg / kg NLX; not shown – see text).

results of the present study provide further support for our hypothesis that the ‘much longer duration of antinociceptive action of morphine plus [ultra-low-dose] NTX vs. morphine alone may be due to blockade of the antianalgesic effects . . . initiated by morphine activation of putative excitatory opioid receptors on nociceptive neurons. These long-lasting higher-efficacy excitatory opioid receptor-mediated [hyperalgesic] activities would normally mask inhibitory opioid receptor-mediated analgesic effects elicited by the low levels of morphine remaining in the

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Fig. 5. Cotreatment of mice with orally administered CTX-B blocks acute morphine-induced hyperalgesic effects, unmasking potent opioid analgesia. (Tail-flick tests at 528C as in Figs. 1 and 2). (A): Administration of 0.1 mg / kg morphine (s.c.) results in characteristic hyperalgesia: d (as in Fig. 1: d). By contrast, after oral pretreatment of another group of mice with CTX-B (added a day earlier to the drinking-water bottles at 1 mg / ml), this low-dose morphine-induced hyperalgesia is blocked, unmasking prominent opioid analgesia: s. (B): Assays of same groups of mice after a 2nd day of oral CTX-B treatment by testing effect of a 10 000-fold increase in acute morphine dose (1 mg / kg, s.c.). Note much larger increase in magnitude and duration of morphine’s antinociceptive effects in CTX-B-treated mice (s).

central nervous system at .1 h after injection’ [14]; see also Ref. [62].

4.1. Consonance of CTX-B blockade of opioid-induced hyperalgesia in mice and CTX-B blockade of excitatory opioid receptor functions in DRG neurons in culture A possible role for GM1 ganglioside in regulating excitatory opioid receptor functions was initially proposed following evidence that acute application of CTX-B to DRG neurons blocks excitatory, but not inhibitory, opioid

receptor functions [60]. Because CTX-B binds selectively to GM1 on neuronal cell membranes, the results suggested that CTX-B might thereby interfere with a putative allosteric GM1 regulatory site on Gs-coupled excitatory opioid receptors [60]. These studies on DRG neurons also showed that CTX-blockade of opioid-induced excitatory, APDprolonging effects in DRG neurons does not appear to be due to direct interference with ion channel functions. Firstly, exposure to CTX-B alone did not result in significant alterations in the APD of DRG neurons. Secondly, exposure to forskolin resulted in characteristic prolongation of the APD of CTX-B treated neurons [60], demonstrating that CTX-B did not interfere with the responsiveness of cyclic AMP-dependent ion channels, which mediate the excitatory effects of forskolin [34,58,71] as well as opioids [10,11,16,58]. Furthermore, in recent studies of non-opioid GM1-deficient CHO cells transfected with cloned opioid receptors Wu et al. [75,76] showed that acute application of GM1 results in rapid (ca. 30 min) conversion of these receptors from an inhibitory Gi-coupled to an excitatory Gs-coupled mode. Elevation of GM1 levels in nociceptive neurons may therefore have two major effects: (1) an increase in the number of opioid receptors in the excitatory Gscoupled mode; and (2) an increase in efficacy of coupling of these receptors to the adenylate cyclase / cyclic AMP transducer system [15,16] (see Sections 1, 4.5 and 4.6). The present evidence that CTX-B blocks acute low-dose opioid-induced hyperalgesia and unmasks potent opioid analgesia in mice is remarkably consonant with our previous study of CTX-B effects on DRG neurons in culture. Cotreatment of these cells with 1–10 ng / ml CTXB blocked low-dose (1–10 nM) mu, delta and kappa opioid induced excitatory, APD-prolonging (‘hyperalgesic’) effects in |15 min, unmasking potent opioid inhibitory APD-shortening (‘analgesic’) effects which required 100- to 1000-fold higher opioid concentrations when applied alone [60].

4.2. Attenuation of opioid tolerance by chronic cotreatment with CTX-B Attenuation of tolerance in chronic opioid-exposed mice by cotreatment with CTX-B is consonant with our previous study on DRG neurons in culture which showed that chronic cotreatment with nM concentrations of CTX-B attenuated development of tolerance to the inhibitory APDshortening effects of mM concentrations of the stable delta / mu opioid peptide, D-ala 2 – D-leu 5 –enkephalin [61]. Attenuation of the development of tolerance in chronic opioid-treated DRG neurons in culture and mice in vivo by cotreatment with CTX-B can be accounted for by selective blockade of excitatory Gs-coupled opioid receptor functions (which would otherwise be upregulated and supersensitized during chronic exposure to a bimodally-acting opioid agonist), thereby unmasking inhibitory opioid re-

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ceptor functions [12,14,16,61]. These effects of CTX-B are in good agreement with the attenuation of tolerance during chronic cotreatment with morphine plus ultra-low-dose NTX in mice [14,62] and in rats [1]. It should be noted, however, that in the present in vivo study (Fig. 3B vs. A) as well as in our in vitro study [61] a moderate degree of tolerance developed in the chronic CTX-B-treated test groups, probably due to homologous desensitization of inhibitory opioid receptor functions [6].

4.3. Prevention of opioid physical dependence by chronic cotreatment with CTX-B Previous studies have demonstrated that enhanced responsiveness to noxious stimuli, i.e. hyperalgesia, is a reliable index of the magnitude of physical dependence that occurs after withdrawal of acute, as well as chronic, opioid agonists in animals and in humans [39,40,45,46,67,70]. The low dose of NLX (10 mg / kg) used to evoke hyperalgesia in chronic morphine-treated mice in the present study (Fig. 4B: d) is comparable to doses used clinically to elicit characteristic autonomic withdrawal symptoms in opiate-dependent humans (e.g. Refs. [51,70]). Much higher doses of NLX (ca. 10 mg / kg) were required to precipitate withdrawal-jumping activity in our previous assays of dependence in chronic morphinetreated mice [14,62]. Prevention of NLX-precipitated withdrawal hyperalgesia in chronic morphine-treated mice by cotreatment with CTX-B (Fig. 4B: s) is remarkably consonant with our study of chronic opioid-treated DRG neurons in culture where the development of NLX-precipitated excitatory APD-prolonging effects in opioid-supersensitized neurons [12,14] was prevented by chronic cotreatment with CTX-B [61]. We had previously shown that NLX did not alter the APD of any DRG neurons when tested alone at concentrations of 3 nM to 3 mM, although higher concentrations (30 mM) prolonged the APD in two out of 10 cells tested [13]. In contrast, after acute treatment of DRG neurons with GM1 ganglioside (1 mM for .10 min), application of 3 to 30 nM NLX prolonged the APD in most cells [13]. These results suggest that acute elevation of GM1 in naive DRG neurons greatly enhances the efficacy of opioid excitatory receptor functions so that even the extremely weak partial agonist properties of NLX become effective in prolonging the APD of these treated neurons [13]. On the other hand, the antagonist properties of NLX at inhibitory opioid receptors did not appear to be altered by GM1 treatment. The increased partial agonist potency of NLX at excitatory opioid receptors on acute GM1-treated DRG neurons, while maintaining its characteristic antagonist properties at inhibitory opioid receptors, is quite similar to the NLX-precipitated APD prolongation elicited in chronic opioid-treated DRG neurons [12]. Further support for this view derives from biochemical studies demonstrating that endogenous levels of cyclic

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AMP-dependent GM1 ganglioside are significantly elevated in chronic opioid-treated neurons [73] (see also Refs. in [12,15,16]).

4.4. CTX-B cotreatment blocks kappa as well as mu opioid-induced hyperalgesia in mice The present study demonstrates that the acute hyperalgesic effects of low doses of the specific kappa opioid agonist, U-50, 488H, as well as the mu agonist, morphine, are blocked by CTX-B, thereby unmasking potent opioid analgesia. Hyperalgesia induced in mice by 10 ng / kg U-50, 488H is in agreement with a previous report by Apfel et al. [3] that a low dose of the kappa opioid peptide, dynorphin (0.5 mg / kg, s.c.) resulted in thermal hyperalgesia in mice, measured by a significant decrease in tail-flick latency (tested at 90 min after injection). By contrast, a 100-fold higher dose (50 mg / kg) elicited analgesia [3]. The present study extends Apfel et al.’s report [3] by showing that low-dose kappa opioid-induced hyperalgesia can be selectively blocked by ultra-low doses of the specific kappa opioid antagonist, nor-binaltorphimine and by NTX, as well as by CTX-B. Our results are also consonant with evidence by Gear et al. [30] that the increase in pain observed within 1 h after i.v. injection of a low 5 mg dose of the kappa opioid agonist, nalbuphine in male post-operative dental patients is blocked by cotreatment with 0.5 mg naloxone, unmasking prominent analgesia.

4.5. Efficacy and reversibility of CTX-B in blocking opioid-induced hyperalgesia It is surprising that s.c. injection of doses as low as 10–100 ng of CTX-B can reach sufficient numbers of putative allosteric GM1 binding sites on excitatory opioid receptors on neurons in nociceptive CNS networks so as to block low-dose opioid-induced hyperalgesia in ,30 min after administration. Perhaps some of the CTX-B molecules can enter the spinal cord via dorsal roots and are then transported to opioid receptors on presynaptic terminals of nociceptive DRG neurons in the dorsal horn (see also Ref. [68]). We have suggested a comparable mode of entry into the CNS to account for the similarly surprising and rapid enhancement of excitatory hyperalgesic opioid effects by i.p. injection of low doses of exogenous GM1 in mice [17] (see also Ref. [57]). CTX-B association with GM1-containing membranes appears to result in a localized disturbance of membrane phospholipid packing [41]. Initial disruption of membrane packing by CTX-B has been proposed to be due to the rigid placement of five GM1 molecules within the membrane following association with CTX-B [52]. This could create a geometry in which the area able to be occupied by phospholipids is reduced by 25% or more between the fixed positions of five GM1 molecules bound by the

28

K.-F. Shen, S.M. Crain / Brain Research 919 (2001) 20 – 30

CTX-B pentamer [35,52]. These physicochemical interactions may underlie the efficacy of CTX-B in binding to GM1 sites on excitatory opioid receptors and interfering with GM1-induced switching of opioid receptors from an inhibitory to excitatory mode [15,16]. Interestingly, recombinant CTX-B is being utilized clinically in oral and nasal mucosal vaccines [36,53], and its immunopotentiating capacity as a carrier molecule is considered to be related to its ability to bind to GM1 on the cell membrane of immunocytes [24,38,64]. These clinical studies with oral CTX-B stimulated us to carry out preliminary tests in mice to determine the efficacy of orally administered CTX-B to enhance opioid analgesia. Injection of low-dose morphine (1 mg / kg, s.c.) in mice pretreated with oral CTX-B (via their drinking water) did, in fact, result in prominent long-lasting analgesia, rather than hyperalgesia (Fig. 5A), comparable to the effects observed with s.c. injection of CTX-B plus 0.1–1 mg / kg morphine (Fig. 1). Morphine also showed markedly enhanced analgesic potency in oral CTX-B-treated mice tested with a higher dose of morphine (1 mg / kg, s.c.: Fig. 5B). Our antinociceptive assays in mice cotreated with morphine plus CTX-B demonstrate that CTX-B blockade of opioid hyperalgesia and enhancement of analgesia is effective for .6 h after drug injection (Fig. 3). However, the blocking effect of 10 ng / kg CTX-B on low-dose opioid-induced hyperalgesia was no longer present when the mice were retested 24 h later with opioid alone (Fig. 2B). Nevertheless, the results of chronic daily cotreatment with 0.1 mg / kg CTX-B plus morphine indicate that CTXB blockade of opioid hyperalgesia and tolerance / dependence can be maintained quite effectively even after 5 days (Figs. 3B and 4B).

4.6. Cotreatment of mice with whole CTX blocks excitatory opioid receptor functions by CTX-A-mediated interference with Gs-coupling, whereas CTX-B interferes with GM1 -regulation of these receptors Intracerebroventricular injection of whole CTX in mice has been reported to markedly enhance the analgesic potency of morphine, presumably by ‘impairing the function of Gs [stimulatory] transducer proteins’ [55]. Furthermore, intrathecal injection of CTX in mice inhibited the ‘antianalgesic action’ of dynorphin which appeared to be ‘mediated by activation of [excitatory] Gs-coupled opioid receptors’ [4]. These interpretations are supported by evidence that: (1) i.c.v. injection of mice with antibodies directed against Gs a also enhanced morphine antinociception [56]; and (2) downregulation of the Gs a protein by intrathecal injection of antisense oligonucleotides in mice resulted in blockade of acute low-dose morphine-induced hyperalgesia [23] and tolerance during chronic morphine treatment [22]. The studies with whole CTX in mice are consistent with selective blockade of excitatory opioid

receptor functions by CTX-A-mediated interference with Gs-coupling of these receptors, as previously demonstrated with low doses of whole CTX or CTX-A on DRG neurons in culture [59] (Section 1). By contrast, the present study shows that cotreatment with the non-toxic B subunit of CTX can result in remarkably similar blockade of excitatory opioid receptor functions by selective interference with GM1-regulation of these Gs-coupled receptors.

4.7. Concluding remarks Although the results of the present study show that CTX-B selectively blocks excitatory Gs-coupled, but not inhibitory Gi / Go-coupled, mu and kappa opioid receptor functions, CTX-B will probably also interfere with other subtypes of opioid receptors as well as with non-opioid receptors that are regulated by GM1, e.g. Gs-coupled delta opioid receptors [75,76], Gs-coupled b-adrenergic receptors [50], Gs-coupled 5-HT receptors [2,5] and Gs-coupled prostaglandin E 1 receptors [74]. CTX-B might also interfere with other physiological roles of GM1 in the nervous system, e.g. neurotrophic activities [25,26,43]. On the other hand, we are not aware of evidence that low doses of CTX-B do, in fact, alter non-opioid Gs-coupled receptor functions or have significant aversive side-effects on nonopioid systems in vivo. The results of the present study suggest, therefore, that cotreatment with CTX-B may be useful as an alternative or supplement to ultra-low-dose opioid receptor antagonists for selectively blocking excitatory opioid receptor functions so as to enhance the potency of opioid analgesics and attenuate their tolerance / dependence liability. We have demonstrated that cotreatment of mice with CTX-B blocks excitatory opioid receptor-mediated hyperalgesia over a wide range of doses without attenuating inhibitory opioid receptor-mediated analgesia. By contrast, the dosage of NTX or other opioid receptor antagonists needs to be formulated within a more critical range so as to block high-affinity excitatory opioid receptors mediating hyperalgesia while minimizing attenuation of lower-affinity inhibitory opioid receptors mediating analgesia [16,18,62]. Nevertheless, the remarkable similarities, as well as the significant differences in mode of action, between the effects of cotreatment of mice with bimodally-acting opioid agonists plus CTX-B vs. ultra-low-dose NTX may provide valuable insights into improved methods for enhancing analgesic potency and attenuating tolerance / dependence liability in the clinical treatment of pain.

Acknowledgements This study was supported by a research grant from Pain Therapeutics Inc., CA.

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