Blockade of Tolerance to Morphine but Not to Opioids by a Nitric Oxide Synthase Inhibitor (Nitric Oxide/Opiate/Dependence/Naloxone Benzoylhydrazone/U50,488H)

Proc. Natl. Acad. Sci. USA Vol. 90, pp. 5162-5166, June 1993 Pharmacology

Blockade of tolerance to morphine but not to opioids by a nitric oxide synthase inhibitor (nitric oxide/opiate/dependence/naloxone benzoylhydrazone/U50,488H)

YURI A. KOLESNIKOV, CHAIM G. PICK, GRAZYNA CISZEWSKA, AND GAVRIL W. PASTERNAK The Cotzias Laboratory of Neuro-Oncology, Memorial Sloan-Kettering Cancer Center, Departments of Neurology and Neuroscience and Pharmacology, Cornell University Medical College, New York, NY 10021 Communicated by Paul A. Marks, February 2, 1993 (received for review December 3, 1992)

ABSTRACT The nitric oxide synthase inhibitor NG-nitro- the NOS inhibitor NO2Arg on the development of tolerance L-arginine (NO2Arg) blocks morphine tolerance in mice. After to morphine and extended these studies to K1 (trans-3,4- implantation of morphine pellets the analgesic response de- dichloro-N-methyl-N-[2-(1-pyrrolindinyl)cyclohexyl]-ben- creases from 100% on the first day to 0% on the third. zeneacetamide; U50,488H) and K3 [naloxone benzoylhydra- Coadministration of NO2Arg along with the pellets markedly zone (NalBzoH)] analgesics. retards the development of tolerance; 60% of mice are analgesic after 3 days, and 50% of mice are analgesic after 5 days. In a daily injection paradigm the analgesic response to mor- MATERIALS AND METHODS phine is reduced from 60% to 0% by 5 days. Concomitant Male CD-1 mice (25-35 g; Charles River Breeding Labora- administration of morphine along with NO2Arg at doses of 2 tories) were used in all studies. Morphine sulfate was ob- mg/kg per day prevents tolerance for 4 weeks. A single tained from Mallinckrodt, and U50,488H was from Upjohn. NO2Arg dose retards morphine tolerance for several days, and NalBzoH was synthesized as previously noted (2), and dosing every 4 days is almost as effective as daily NO2Arg. NO2Arg was purchased from Sigma. NalBzoH was dissolved NO2Arg slowly reverses preexisting tolerance over 5 days in 30% ethanol, which did not produce analgesia in control despite the continued administration of morphine along with mice, and doses are reported as the free base. All other drugs NO2Arg. NO2Arg also reduces dependence and reverses pre- were dissolved in saline. viously established dependence. NO2Arg does not prevent Analgesia was determined by using the radiant heat tail- tolerance to analgesia mediated by the Kc agonist trans-3,4- flick technique (1, 2, 16, 28, 29). After determining baseline dichloro-N-methyl-N-[2-(1-pyrrolindinyl)cyclohexyl]-benzene- latencies, post-treatment tail-flick latencies were determined acetamide (U50,488H) or the K3 agent naloxone benzoylhydra- at 30 min after the opioid, and a maximal latency of10 sec was zone, indicating a selective action of NO in the mechanisms of used to minimize tissue damage. Analgesia was defined as a ,u tolerance and dependence. doubling or greater of baseline values for each mouse. All analgesic values represent at least 10 mice. Extended dura- Chronic use of opioid analgesics invariably leads to toler- tion studies used several groups of at least 10 mice to avoid ance, but the lack of cross tolerance among ,u, K1, and K3 testing animals on >6 days. Dose-response curves were analgesics strongly suggests different mechanisms for the analyzed by using a modification of the BLISS version 20 subtypes (1, 2). Although tolerance may be seen at the level computer program (30), and ED50 values are given with 95% of the receptor and its second-messenger system (3-11), confidence limits. Single-dose comparisons used the Fisher some evidence suggests that morphine tolerance is mediated, exact test. Mean arterial blood pressure was assessed in in part, through activation of antagonistic systems and/or a halothane-anesthetized mice with carotid cannulae con- down-regulation of facilatory ones (12-17). However, chole- nected to a pressure transducer/blood pressure analyzer cystokinin antagonists and antidepressants active against (MicroMed, Louisville, KY). tolerance also potentiate morphine analgesia in naive animals as well, indicating that their actions are not directed specif- ically against tolerance. RESULTS N-methyl-D-aspartate (NMDA) mechanisms have been NO2Arg Actions on Morphine Tolerance. Subcutaneous associated with nociceptive neurons within the spinal cord implantation of a morphine pellet (75 mg of free base) and in the brainstem, and interactions between NMDA produces tolerance (4, 8). All mice are analgesic on the first receptors and opioids have been reported (18-23). Further- day (Fig. 1); the response rapidly drops to no analgesic more, the NMDA antagonists such as MK-801 (11) and the animals by the third day. NO2Arg (4 mg/kg per day) signif- competitive antagonist LY274614 block morphine tolerance icantly reduces the development of tolerance; 50% of the and even aspects of dependence (23). Recent work has mice are analgesic after 5 days. NO2Arg itselfis not analgesic established that many glutamate actions mediated through in this model, and neither acute nor chronic dosing with NMDA receptors result from the subsequent activation of NO2Arg significantly influences the analgesic potency of nitric oxide synthase and the formation of NO (24-26). A morphine in opiate-naive animals (Table 1). brief report indicated that inhibition of nitric oxide synthase We also used a daily injection paradigm that is not depen- (NOS) interfered with the development of dependence (27), dent upon the availability of pellets and, thus, can be ex- and we recently reported the ability of a relatively high dose tended to a wide variety of opioids. Single daily injections of of NG-nitro-L-arginine (NO2Arg) to prevent tolerance to morphine (5 mg/kg) significantly increase the ED50 value of morphine (28). To explore further the role of NO in opioid morphine 2-fold after 5 days, 4-fold by 10 days, and 8-fold by tolerance we have examined the effects of several doses of Abbreviations: NOS, nitric oxide synthase; NO2Arg, NG-nitro-L- The publication costs of this article were defrayed in part by page charge arginine; U50,488H, trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolin- payment. This article must therefore be hereby marked "advertisement" dinyl)cyclohexyl]-benzeneacetamide; NalBzoH, naloxone benzoyl- in accordance with 18 U.S.C. §1734 solely to indicate this fact. hydrazone; NMDA, N-methyl-D-aspartate. 5162 Downloaded by guest on September 28, 2021 Pharmacology: Kolesnikov et al. Proc. Natl. Acad. Sci. USA 90 (1993) 5163

1 OCi

Pellets alone

M 75 Pellets + NOArg

0 o-0 50 Ak eCO a) - -- Morphine 25-\ - ---- + NO.Arg 1 mg/kg/d -0- + NO2Arg 2 mg/kg/d

0 1 2 3 4 5 Days Days FIG. 1. Effect of NO2Arg on tolerance to morphine pellets. One group of mice received morphine pellets (75 mg of free base) on day B 100 1 and was tested for analgesia on days 1 (n = 10), 2 (n = 10), and 3 (n = 9). Another group (n = 10) received NO2Arg (4 mg/kg i.p.) on day 0 after which the mice continued to receive daily NO2Arg (4 A A mg/kg i.p.) injections. They received their morphine pellets (75 mg E" 75- of free base) on day 1 and were tested for analgesia on days 1-5. E co 28 days (Table 1). The analgesic response to fixed, daily 0 X Morphine (10 mg/kg) morphine injections decreases from 60% to 0% over 5 days X0 50 A (Fig. 2A). Coadministration of NO2Arg along with morphine .i \+ NOArg (2 mg/kg) prevents this tolerance in a dose-dependent manner. A daily dose of 2 mg/kg per day maintains the analgesic response to 25- a fixed morphine dose for >4 weeks and prevents any significant change in the ED50 value of morphine in the co The lower NO2Arg group after 5 or 10 days (Table 1). NO2Arg 0 dose (1 mg/kg per day) is only partially effective (Fig. 2A). 1 4 7 10 NO2Arg also works against a higher morphine dose (10 Days mg/kg; Fig. 2B). To avoid using supramaximal doses, all dose. FIG. 2. Effect of NO2Arg on tolerance to daily morphine injec- remaining studies used the lower (5 mg/kg) tions. (A) Animals received morphine (5 mg/kg s.c.) alone (o) or with The actions of NO2Arg can be very long lasting (Fig. 3), NO2Arg (i.p.) at doses of 2 mg/kg per day (n = 20; 1 mg/kg at times perhaps due to its ability to irreversibly inhibit NOS (31). A -15 min and 1 hr each day; *) or 1 mg/kg per day (n = 10; 1 mg/kg at -15 min; A). Analgesia was assessed 30 min after opioid admin- Table 1. Effect of acute and chronic morphine and NO2Arg on istration (time 0). Baseline latencies were determined each day for morphine analgesia each mouse before any drug injections and did not vary significantly Morphine, 95% confidence over the days of testing. All points represent at least 10 mice. At 5 Treatment ED50 in mg/kg limits days, the morphine-alone group differed significantly from the 1 mg/kg per day dose (P < 0.05) and from the 2 mg/kg per day dose Morphine alone on day 7 (P < 0.02). (B) Groups of mice (n = 20) received morphine Day 1 4.4 3.6, 5.3 (10 mg/kg s.c.) alone (o) or with daily NO2Arg (v) (2 mg/kg per day Day 5 8.8 6.2, 11.3 i.p.) given 15 min before morphine. Analgesia was assessed as Day 10 17.2 11.8, 23.8 described above. At day 10, the NO2Arg differed significantly (P < Day 28 37.8 26.6, 51.1 0.001) from the morphine-alone group. NO2Arg alone single dose ofNO2Arg retards the appearance oftolerance for Day 1 several days, and NO2Arg given every 4 days maintains the 1 mg/kg 4.6 3.1, 6.7 as well as 2 mg/kg 3.9 2.8, 5.4 analgesic potency ofmorphine approximately daily Day 10 NO2Arg (Fig. 3A). This duration of NO2Arg action is more 2 mg/kg 5.4 3.9, 7.4 dramatic with chronic dosing. After administering NO2Arg Chronic morphine and morphine together daily for a week, we stopped the with NO2Arg NO2Arg and continued the daily dosing with morphine alone. Day S 4.6 3.1, 6.7 Little tolerance is seen after morphine is given alone for 10 Day 10 5.8 4.0, 8.7 days. This study also reveals that a single daily dose of Animals tested on day 1 received only a single morphine dose alone NO2Arg (2 mg/kg) is as effective as two divided doses (Fig. or with the stated NO2Arg dose (i.p.) given 15 min before morphine, 3B). and the EDso values were determined. In the morphine-alone groups, NO2Arg also reverses previously established tolerance in mice received daily injections of morphine (5 mg/kg) for 4, 9, or 27 a dose-dependent manner (Fig. 4). After receiving morphine days, and the ED50 was determined on days 5, 10, and 28, respec- alone for 5 days, mice then were given a combination of tively. The chronic morphine with NO2Arg mice received daily morphine with NO2Arg (4 mg/kg per day) starting on the injections of morphine (5 mg/kg) and NO2Arg (2 mg/kg) for 4 and 9 with days, and the ED5o values were determined on days 5 and 10. The sixth day. Although a single day of NO2Arg along chronic NO2Arg group received 2 mg/kg per day in two divided morphine has no effect, analgesia slowly returns with con- doses, as described in Fig. 2A for 9 days. On the 10th day the group tinued administration of both agents. This slow rate of received a single injection of NO2Arg followed 15 min later by the reversal argues strongly against a simple potentiation of challenge morphine dose. morphine potency. Despite the continued administration of Downloaded by guest on September 28, 2021 5164 Pharmacology: Kolesnikov et al. Proc. Natl. Acad Sci. USA 90 (1993) -* Morphine alone -O Morphine A - -c- + NO2Arg (day 1 only) -A- + NO2Arg (days 1, 5 and 9) **4** + NO*Arg 4 mg/kg/d -'A-- + NO2Arg 2 mg/kg/d cn 60 -1:-- Control Ecu

0 COco 1011 E ct ,c 40 a) -0

C 00-0

00) I62C 20 1 5 9 Days B 60- 0 0 3 6 9 12 U) Days E c 40- o- - -o FIG. 4. Effect of NO2Arg on reversal of morphine tolerance. A co0 group of mice (n = 30) received only morphine (5 mg/kg s.c.; o) for 0 5 days. On the sixth day they were divided into three groups. The first ol group (control; o) received no additional drugs until day 10, when the U) mice received the challenge dose of morphine (5 mg/kg s.c.). The °, 20- -*- Morphine + NO2Arg other two groups received NO2Arg doses of2 mg (A) and 4 mg (*)/kg 'a - o Morphine alone per day in two divided doses (-15 min and 1 hr) along with the same morphine dose. Analgesia was determined 30 min after morphine. On day 10, the response of the 4 mg/kg per day dose was significantly greater for the 4 mg/kg per day dose (P < 0.001) and the 2 mg/kg per (J '.. day dose (P < 0.02) compared with animals tested on day S before 1 5 9 13 17 the NO2Arg. The control group did not achieve statistical signifi- Days cance (P > 0.05) compared to day 5.

FIG. 3. Effect ofdifferent NO2Arg dosing schedules on morphine when we examined the effects ofthis dose ofNO2Arg in mice, analgesia. (A) All animals received daily injections of morphine (5 we find very modest changes in mean arterial blood pressure mg/kg s.c.). One group received only morphine (s). The othergroups (Fig. 7). received NO2Arg (2 mg/kg i.p. 15 min before morphine) given only NO2Arg Actions on Morphine Dependence. In addition to on day 1 (o) or on days 1, 5, and 9 (A). All points represent at least tolerance, chronic administration of NO2Arg attenuates de- 10 mice. At 5 days, the morphine-alone group differed significantly pendence, as measured by naloxone-precipitated jumping from the day-i NO2Arg group (P < 0.05) and the days-i, -5, and -9 NO2Arg group (P < 0.02). (B) One group received morphine (5 mg/kg (Fig. 8). Dependence is assessed both as the percentage of s.c.) every day along with daily injections of NO2Arg days 1-7 (0), animals in a group thatjump and by the total number ofjumps after which the mice received only their daily morphine injections by all animals in a group. In both measures, NO2Arg lowers (o). All points represent at least 10 mice. the incidence of jumping in a dose-dependent manner. NO2Arg also reverses established dependence (Fig. 9). Mice morphine, this return of analgesic sensitivity is at least as challenged with naloxone (1 mg/kg s.c.) after daily admin- rapid as that seen with abstinence. A third group (control) receiving morphine injections for 5 days followed by no 60 --- U50,488H treatments from day 6 until 10 and then rechallenged with a -A- + NO.Arg 8 mg/kg/d single morphine dose (5 mg/kg s.c.) displays less analgesia than either NO2Arg group. E NO2Arg Actions on Tolerance to K Opioids. The ability of co NO2Arg to prevent tolerance is restricted to the u analgesic 40 morphine. Daily injections for 5 days significantly shifts the C4- ED50 for the Kl agonist U50,488H from 4.7 mg/kg (3.1, 6.7) to 13.3 mg/kg (9.2, 19.1; P < 0.05) while the ED50 for 0- NalBzoH significantly shifts from 22.1 mg/kg (11, 80) to 161 mg/kg (103, 240; P < 0.05). NO2Arg does not significantly diminish tolerance to either K agent (Figs. 5 and 6). Like 0'2 morphine, neither acute nor chronic dosing with NO2Arg influences the analgesic sensitivity of mice to U50,488H or NalBzoH (data not shown). 1 ~~~~~~3 5 NO2Arg Actions on Blood Pressure in Mice. NO lowers blood pressure through its actions on the vascular system Days (24-26). Conversely, inhibition of NOS can increase blood FIG. 5. Effect of NO2Arg on Kl tolerance. Mice received the Kl pressure. In rats, we find that a single NO2Arg dose dramat- agonist U50,488H (5 mg/kg s.c.) alone (0) (n = 10) or with NO2Arg ically elevates blood pressure -30 mmHg (1 mmHg = 133 Pa) at 8 mg/kg per day (n = 10; A) daily. Analgesia was measured at 30 (Y.A.K. and G.W.P., unpublished observations). However, min (see text). Downloaded by guest on September 28, 2021 Pharmacology: Kolesnikov et al. Proc. Natl. Acad. Sci. USA 90 (1993) 5165

Morphine 60 - -- NalBzoH + NO,Arg I mg/kg/d - -*- - + NO.Arg 2 mg/kg/d 75 - + NO,Arg 2 mg/kg/d 15 -A- + NO.Arg 8 mg/kg/d + NO,Arg 8 mg/kg/d coE c X a) 40- cn o Co 0 0 E E 50 *10 a co Cu ':') 0 6a) Cu 20- c 0"O 0) CB a) ._ E 25 5 I2 Ce 1 3 5 E Days m2 FIG. 6. Effect of NO2Arg on K3 tolerance. Animals received the K3 agonist NalBzoH (50 mg/kg s.c.; n = 20) alone (o) or with NO2Arg at 2 mg/kg per day (n = 10; *) or 8 mg/kg per day (n = 10; A) daily. Analgesia was measured at 30 min. FIG. 8. Prevention of precipitated withdrawal by NO2Arg. Groups of mice received morphine (5 mg/kg s.c.) daily for 10 days istration of morphine (5 mg/kg s.c.) for 5 or 10 days display either alone (n = 20) or with NO2Arg at 1, 2, or 8 mg/kg per day (n similar jumping levels. However, naloxone-precipitated = 10 each) according to the dosing schedule outlined in Fig. 1 and in mice for 5 followed by the were then challenged with naloxone (1 mg/kg s.c.). Animals were jumping given morphine days scored for jumping (left ordinate) and by the average number of combination of morphine with NO2Arg (4 mg/kg per day) for jumps per mouse (right ordinate) for a 1-hr period after naloxone an additional 5 days is far less, despite continued morphine challenge. Average number ofjumps per mouse was determined by administration. summing total number ofjumps per group and dividing by number of animals. Percentage ofmicejumping in the 8 mg/kg per day NO2Arg DISCUSSION group differs significantly from the morphine group (P < 0.005). The mechanisms of opioid tolerance remain incompletely NO2Arg alone given acutely or chronically does not affect understood. Opioid actions are modulated by a wide number baseline latencies and does not influence single morphine of antagonistic and facilitory neurotransmitter networks (12- doses. Together, these data support a relatively selective 23), as emphasized by the recent report that the NMDA- action upon a mechanism involved with tolerance rather than antagonist MK-801 (11) and the competitive antagonist a simple potentiation of morphine. This conclusion is further LY274614 (20) block the development ofmorphine tolerance. supported by the studies examining reversal oftolerance. The The close association ofmany NMDA actions with NO led us reversal of established tolerance by NO2Arg is quite slow, to examine the potential role ofNO in opioid tolerance. After taking several days before restoring analgesic sensitivity. demonstrating that NO2Arg attenuates tolerance in a mor- Were NO2Arg simply potentiating morphine, we would have phine pellet model, we extended these studies to a daily expected the immediate reversal of tolerance, such as seen injection paradigm to permit the examination of a broader = Morphine (5 days) range of compounds, particularly K agents. At a daily dose of _ Morhpine (10 days) 2 mg/kg per day NO2Arg prevents the development of 75 - = Morphine + NO2Arg tolerance in the daily injection model for at least 4 weeks, CD extending our prior observation with a higher (8 mg/kg per 0 day) dose (28). Dose-response studies confirm these results. co E E Cu 50h 0. 6 NO2Arg alone 0 C 40- 0) NO2Arg + morphine a) 25 - Morphine alone E 0. E 30- E co. :2 E M 20- -3

0 I5 10- FIG. 9. Reversal of precipitated withdrawal in morphine- dependent mice by NO2Arg. Three groups of mice (n = 10) received ( morphine (5 mg/kg s.c.) daily. Withdrawal was precipitated in one 0 15 30 45 60 group after only 5 days (n = 10) and in another group after 10 days Time, min (n = 20; see above). The last group (n = 10) continued to receive morphine for 10 days but also received NO2Arg (2 mg/kg i.p. at times FIG. 7. Effect of NO2Arg on mean arterial blood pressure. -15 min and 60 min) for days 6-10, at which time withdrawal was Groups of mice were anesthetized, and mean arterial blood pressure precipitated. Animals were scored forjumping (left ordinate) and by (MABP) was measured. One group (n = 7; *) received NO2Arg alone average number ofjumps per mouse (right ordinate) for a 1-hr period (2 mg/kg i.p.), a second (n = 5; o) received morphine alone (5 mg/kg after naloxone challenge. Average number ofjumps per mouse was s.c.), and the third (n = 5; A) received both NO2Arg and morphine determined by summing total number of jumps per group and at the same doses. Mean arterial blood pressure was continuously dividing by number of animals. Percentage of mice jumping in the monitored for 60 min. The SEM among animals was <7% for all NO2Arg group differs significantly from the 10-day morphine group points and typically under 3%. No statistical differences were noted. (P < 0.005). Downloaded by guest on September 28, 2021 5166 Pharmacology: Kolesnikov et al. Proc. Natl. Acad. Sci. USA 90 (1993) with proglumide and the antidepressants (12-16). This slow E. F. & Pasternak, G. W. (1990) J. Pharmacol. Exp. Ther. 255, reversal also indicates that tolerance involves steps beyond 769-774. the activation of NOS. If NO were the direct mediator of 3. Blanchard, S. G. & Chang, K. J. (1988) in The Opiate Recep- tolerance, inhibition of its synthetic enzyme would immedi- tors, ed. Pasternak, G. W. (Humana, Clifton, NJ), pp. 425-439. ately restore sensitivity. Most likely, NO elicits changes in 4. Law, P. Y., Hom, D. S. & Loh, H. H. (1983) Mol. Pharmacol. additional systems that then counteract analgesic mecha- 25, 413-424. 5. Lahti, R. A. & Collins, R. J. (1978) Eur. J. Pharmacol. 51, nisms, and the slow return of sensitivity reflects the down- 185-186. regulation of these systems. 6. Danks, J. A., Tortella, F. C., Long, J. B., Bykov, V., Jacob- The effective NO2Arg doses are relatively low, far less than son, A. E., Rice, K. W., Holaday, J. W. & Rothman, R. B. those expected to block NOS activity by 50%o (31) and are (1988) Neuropharmacology 27, 965-974. associated with very modest changes in mean arterial blood 7. Sharma, S. K., Klee, W. A. & Nirenberg, M. (1975) Proc. pressure. However, these blood pressure effects may be Natl. Acad. Sci. USA 72, 3092-3096. species selective because we observe far greater elevations in 8. Smith, A. P., Law, P. Y. & Loh, H. H. (1988) in The Opiate rats (Y.A.K. and G.W.P., unpublished observations). The Receptors, ed. Pasternak, G. W. (Humana, Clifton, NJ), pp. prolonged effects of NO2Arg are consistent with its irrevers- 441-485. ible inhibition of constitutive NOS in brain (26, 31). Chronic 9. Kennedy, C. & Henderson, G. (1991) Mol. Pharmacol. 40, administration of low doses of irreversible inhibitors should 1000-1005. produce an increasing level ofenzymatic blockade over time. 10. Puttfarcken, P., Werling, L. L. & Cox, B. M. (1988) Mol. NO2Arg given daily for 7 days prevents Pharmacol. 33, 520-527. tolerance for 10 days 11. Trujillo, K. A. & Akil, H. (1991) Science 251, 85-87. after its cessation, the longest persistent action seen. 12. Watkins, L. R., Kinscheck, I. B. & Mayer, D. J. (1984) Sci- These actions on tolerance are restricted to the ,u analgesic ence 224, 395-399. morphine. Tolerance to the K, analgesic U50,488H and the K3 13. Faris, P. L., Komisaruk, B. R., Watkins, L. R. & Mayer, analgesic NalBzoH is unaffected by NO2Arg, implying very D. L. (1983) Science 219, 310-312. different mechanisms for their tolerance. These distinctions 14. Dourish, C. T., Hawley, D. & Iversen, S. D. (1988) Eur. J. are consistent with the existence of discrete analgesic mech- Pharmacol. 147, 469. anisms for the subtypes and the absence of cross tolerance 15. Botney, M. & Fields, H. L. (1983) Ann. Neurol. 13, 160-164. between K drugs and morphine (1-3, 8, 29). It is unlikely that 16. Pick, C. G., Paul, D., Eison, M. S. & Pasternak, G. W. (1992) NO2Arg acts through interfering with learning processes Eur. J. Pharmacol. 211, 375-381. associated with tolerance (32-35) because this would pre- 17. Spiegel, K., Kourides, I. A. & Pasternak, G. W. (1983) Ann. sumably block tolerance to the K drugs as well. Neurol. 13, 462-465. The effects of NO2Arg on morphine tolerance and 18. Chapman, V. & Dickenson, A. H. (1991) Brain Res. 573, 321. depen- 19. Moore, P. K., Oluyomi, A. O., Babbedge, R. C., Wallace, J. & dence likely reflect the inhibition of NOS normally induced Hart, S. L. (1991) Br. J. Pharmacol. 102, 198-202. by activation of NMDA receptors, although a sequential 20. Tiseo, P. J. & Inturrisi, C. E. (1993) J. Pharmacol. Exp. Ther., pathway has not yet been proven. Immunohistochemically, in press. NOS is observed within the amygdala, central gray, spinal 21. Chen, L. & Huang, L. H. (1991) Neuron 7, 319-326. tract of the trigeminal nerve and the substantia gelatinosa of 22. Dougherty, P. M. & Willis, W. D. (1991) BrainRes. 542,15-22. the spinal cord (36-38), regions containing opioid receptors 23. Higgins, G. A., Nguyen, P. & Sellers, E. M. (1992) Life Sci. 50, and important in the production ofanalgesia. However, these PL167-PL172. areas of colocalization are quite limited, and the overall 24. Bredt, D. S. & Snyder, S. H. (1992) Neuron 8, 3-11. distribution of NOS does not correspond to that of ,u recep- 25. Garthwaite, J., Charles, S. L. & Chess-Williams, R. (1988) tors or any other known opioid receptor subtype. Thus, it is Nature (London) 336, 385-388. unlikely that NO plays a widespread role in opioid actions. 26. Xie, Q., Cho, H. J., Calaycay, J., Mumford, R. A., Swiderek, K. M., Lee, T. D., Ding, A., Troso, T. & Nathans, C. (1992) Our observation that NO2Arg also acts against dependence Science 256, 225-228. confirms a prior report (27) and provides further evidence 27. Kimes, A. S., Vaupel, D. B., Bruckner, M. & London, E. D. linking tolerance to dependence. However, tolerance in vivo (1991) Soc. Neurosci. Abstr. 17, 214.12. appears distinct from biochemical mechanisms seen in tissue 28. Kolesnekov, Y. A., Pick, C. G. & Pasternak, G. W. (1992) culture studies (7-11). Although tolerance to morphine de- Eur. J. Pharmacol. 221, 399-400. velops in the SK-N-BE (2) cell line (K. M. Standifer, J. 29. Gistrak, M. A., Paul, D., Hahn, E. F. & Pasternak, G. W. Cheng, and G.W.P., unpublished observations), NO2Arg (1989) J. Pharmacol. Exp. Ther. 251, 469-476. does not affect it. Thus, tolerance is a complex mixture of 30. Umans, J. G. & Inturrisi, C. E. (1981) J. Pharmacol. Exp. events. Although NMDA receptors and NOS are important Ther. 218, 409-415. in our other mechanisms at 31. Dwyer, M. A., Bredt, D. S. & Snyder, S. H. (1991) Biochem. model, the level of the receptor Biophys. Res. Commun. 176, 1136. and second-messenger systems may play significant roles in 32. Wikler, A. (1980) Opioid Dependence: Mechanisms and Treat- different situations. ment (Plenum, New York). 33. Siegel, S. (1976) Science 193, 322-325. We appreciate the support provided by Drs. J. B. Posner and R. 34. Baker, T. B. & Tiffany, S. T. (1985) Psychol. Rev. 92, 78-108. Hawks. This work was supported, in part, by a grant from the 35. Goudie, A. J. & Demeliweek, C. (1986) in Behavioral Analysis National Institute on Drug Abuse (DA07242) to G.W.P. Y.A.K. was ofDrug Dependence, eds. Goldberg, S. R. & Stolerman, I. P. supported, in part, by funds granted from the Norman and Rosita (Academic, Orlando, FL), pp. 225-286. Winston Foundation and the Michael and Ethel Cohen Fellowship 36. Bredt, D. S., Huang, P. M. & Snyder, S. H. (1990) Nature Fund. G.W.P. is a recipient of a Research Scientist Development (London) 347, 768-770. Award from the National Institute on Drug Abuse (DA00138). 37. Snyder, S. H. & Bredt, S. (1991) Trends Pharmacol. Sci. 12, 125-128. 1. Pasterak, G. (1993) Clin. Neuropharmacol. 16, 1-18. 38. Bredt, D. S., Glatt, C. E., Hwang, P. M., Fotuhi, M., Dawson, 2. Paul, D., Levison, J. A., Howard, D. H., Pick, C. G., Hahn, T. M. & Snyder, S. H. (1991) Neuron 7, 615-624. Downloaded by guest on September 28, 2021