Effect of spinal infusion of l -NAME, a nitric oxide synthase inhibitor, on spinal tolerance and dependence induced by chronic intrathecal morphine in the rat

ELSEVIER

Neuroscience Letters 207 (1996) 33-36

flEgROSCHC[ IETT[gS

Effect of spinal infusion of L-NAME, a nitric oxide synthase inhibitor, on spinal tolerance and dependence induced by chronic intrathecal morphine in the rat S t u a r t D u n b a r * , T o n y L. Y a k s h Department of Anesthesiology 0818, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA

Received 22 December 1995; revised versionreceived 16 February 1996; accepted 16 February 1996

Abstract

Systemic L-NG-nitroarginine methyl ester or L-NAME (LN), a nitric oxide synthase inhibitor, has been reported to attenuate systemic opioid tolerance and withdrawal. Intrathecal co-administration of LN (100 and 500 nmol//~l per h) with spinal morphine produced only a small diminishing attenuation of tolerance and attenuated only one of seven signs of withdrawal. These results show that LN has little effect on morphine tolerance and withdrawal at spinal sites. Keywords: Rat; Nitric oxide synthase inhibition; L-NG-Nitroarginine methyl ester; Spinal; Morphine; Tolerance; Dependence; With-

drawal; Naloxone

Studies have shown that nitric oxide synthase (NOS) inhibitors attenuate tolerance and withdrawal otherwise observed after chronic morphine administration [1,3,5,8, 10-12,20]. Nitric oxide is produced in response to activation of the N-methyl-D-aspartate (NMDA) receptor. NMDA receptor antagonism can attenuate opioid tolerance and dependence in systemic models [2,14,15,21 ] and at spinal sites [4,6]. Accordingly, as for NMDA receptor antagonism, one might expect NOS inhibition to prevent spinal opioid tolerance and dependence also. In this study we use an intrathecal (i.t.) model of continuous coinfusion of morphine with the NOS inhibitor L-Amnitroarginine methyl ester or L-NAME (LN) to assess (I) its spinal effects on spinal morphine tolerance, and (2) its effect on naloxone-precipitated withdrawal. Rats (350-400 g) were implanted under halothane anesthesia with lumbar subarachnoid catheters 8.5 cm in length [22], with the additional modification of a subcutaneous mini-osmotic pump (model 2001 delivering 1/zl/ h; Alza, Palo Alta, CA) and an external loop of catheter [18]. The pump delivers a constant infusion of 1/~l/h for 7 days after an initial activation period in the animal of 4 h. Rats were thereafter housed individually in a 12 h * Corresponding author. Tel.: +1 619 5433597; fax: +1 619 5436070.

light/12 h dark cycle with free access to food and water. Testing on the hot plate (HP) was performed daily from day 0, the day of implantation, to day 7, during the light cycle at 1200 h midday. Rats were assigned to one or other group randomly. All rats were sacrificed with pentobarbital overdose at the end of each study. Drugs used for continuous spinal infusion, dissolved in sterile saline, were: morphine sulfate (MS; Merck, Sharp and Dohme, West Point, PA); and LN (Research Biochemicals International, Natick, MA). Morphine infusion concentration was 20 nmoi//~l per h in all animals. Groups included: saline, LN 500 nmol/h, morphine 20 nmol/h, LN 100 nmol/h + morphine, and LN 500 nmol/h + morphine. The higher dose of LN-500 nmol/h was found to produce extreme weight loss and diarrhea in some animals. In rats infused with saline, LN 500 nmol/h, morphine 20 nmol/h, and LN 500 nmol/h + morphine, the catheter was cut on day 7, flushed with 10/zi of saline, and 24 h later, i.e. on day 8, after baseline latency was obtained, a probe dose of 60 nmol morphine was administered, and latencies measured 15, 30, 60, and 120min after injection. Separate rats from each group were also given naloxone (0.3 mg/kg i.p.) on day 7, and observed for ! h in an observation chamber for signs of withdrawal, i.e. (1) vocalization in response to three separate air puffs,

0304-3940/96/$12.00 © 1996 ElsevierScience Ireland Ltd. All rights reserved PII: S0304-3940(96) 12481 -O

34

S. Dunbar, T.L. Yaksh / Neuroscience Letters 207 (1996) 33-36

100 90

~11

[3

MOR 2 0 n m o l / hr ( n = 1 3 ) LN 5 0 0 n m o l / h r ( n = 8 )

•

~

70

~N

NS(n=8)

•

1K ~

80

'" e~

o ¢

LN 1 0 0 n m o l / h r

+ MOR(n=4)

,[ LN500 nmol/hr+ MI~( n=16)

6O 50 40

*

20 10 0 -10

. 0

1

.

. 2

Infusion

. 3

.

. 4

duration

. 5

6

7

(days)

Fig. 1. Time course of the antinociceptive effect expressed as % MPE observed with chronic infusion for 7 days of i.t. morphine (MOR), LN 100 nmol/h + morphine, LN 500 nmol/h + morphine, LN 500 nmol/h, or saline (NS). Each line represents the mean +_SE of five or more rats tested daily by HP. *Significance versus morphine group at P < 0.05. or in response to light touch with PE-10 tubing, (2) spontaneous vocalization, (3) abnormal posture indicated by hind paw lift, (4) ejaculation, (5) head shaking, ('wet dog head shakes'), (6) abnormal respiration indicated by intermittent breath holding, and (7) escape attempts, i.e. one or more attempts to escape from the chamber. HP data are expressed as percentage maximum percent effect (% MPE), calculated as follows: % MPE = (infusion or postdrug l a t e n c y - baseline) × 100/(cut-off l a t e n c y - baseline), where post-drug or infusion latency is the response measured at the particular time after initiation of infusion or after i.t. dose of probe drug, baseline is the pre-infusion or pre-probe latency, and the cut off time is 60 s. Where stated, % A U C = area under the dose response curve above baseline after probe dose administration as a percentage of the maximal possible effect × duration of the curve in minutes. Statistics were obtained using computer software programs (Abacus Concepts, StatView, Abacus Concepts, Inc., Berkeley, CA, 1992). Where applicable, absolute HP latencies or calculated % MPE, % A U C and withdrawal studies were analyzed using one- or two-way analysis of variance ( A N O V A ) to detect differences between groups. When differences were found, findings were subjected to a Scheffe F-test (significant at 95%). Unless stated otherwise, single points of comparison were made using a standard paired or unpaired t-test. Differences yielding critical values corresponding to P < 0.05 were considered statistically significant. Results in Fig. 1 show that neither saline, LN, implantation, nor daily HP testing, had any significant effect on HP latencies. All L N + m o r p h i n e co-infused groups achieved maximal increases in latencies after 24 h not significantly different from those of the morphine alone infused group (LN 100 nmol/h + morphine, n = 4, 49 _+

2 s; LN 500 nmol/h + morphine, n = 16, 52 -+ 3 s; versus morphine alone, n -- 13, 53 -+ 3 s; P > 0.l). There was no significant difference between latencies on any one day between the LN 100 nmol/h + morphine infused group and the morphine-alone group throughout the rest of the infusion period (P > 0.I), indicating that LN at this dose did not attenuate the usual loss of morphine effect. There was a modest but significant increase in latencies in the LN 500 nmol/h + morphine group as compared to the morphine-infused group on days 4, 5, and 7 (LN 5 0 0 n m o l / h + m o r p h i n e , 26_+3s, 22_+2s, and 19_+2s; versus morphine, 18-+Is, 16-+ Is, and 15___1s; P < 0.05). Thus, at the higher dose infused, LN had only a small diminishing effect on the development of tolerance. Results of the response to 60 nmol of i.t. morphine on day 8 are shown in Fig. 2a,b. Response was maximal in both saline (n = 4) and LN 500 nmol/h infused rats (n = 4). LN 500 nmol/h infused rats, however, showed a significantly longer duration of action as measured by % AUC than saline-infused rats (90 +_ 2% versus 71 ___2%, Fig. 2b). LN ;3. I

100

80

I

60 %MPE 40

t

20

2

b. 100

80

60

%A U C 40

20 J NS

LN500 Infusion

L N 5 0 0 +MOR

MOR

Group

Fig. 2. Dose response expressed as (a) % MPE, and (b) % AUC, of i.t. morphine (MOR) 60 nmol on day 8, i.e. 24 h after discontinuation of a chronic 7-day infusion of i.t. morphine. LN 500 nmol/h + morphine, LN 500 nmol/h, or saline (NS). Each column represents the mean _+SE of four or more rats. *Significance versus morphine group at P < 0.05.

S. Dunbar, T.L. Yal6~h/ Neuroscience Letters 207 (1996) 33-36

500 nmol/h + morphine (n = 4 ) infused rats showed a greater response as measured by both % MPE and % AUC, than morphine alone infused rats (% MPE: 33 + 2 s versus 18 _.+4 s, P < 0.05, Scheffe; % AUC: 28 _+3% versus 12 + 2%, P < 0.05) (Fig. 2a,b). However, this was significantly less than saline-infused rats (P < 0.05), indicating only a partial return of sensitivity to morphine in this group. In addition, although their behavioral response on the HP appeared appropriate, it is possible that the observed latencies in this group were elevated by a constitutional disturbance in the animal as this higher dose of LN produced diarrhea and extreme weight loss in some animals. Results of the withdrawal studies are shown in Fig. 3. None of the rats infused with saline (n = 4) or with LN 500 nmol/h alone (n = 4) showed any of the seven signs of withdrawal assessed. With the exception of one withdrawal sign, i.e. ejaculation, which was significantly less in L N + m o r p h i n e co-infused groups (P<0.05, Scheffe), there was no significant difference between rats infused with morphine, LN 100 nmol/h + morphine, or LN 500 nmol/h + morphine (P > 0.1), indicating little effect of LN at either dose in attenuating the withdrawal signs assessed. Acute i.t. doses of 500 nmol of LN in both morphine-tolerant and saline-infused rats produced an acute allodynia, i.e. biting and scratching directed at the hind quarters, preventing the assessment of its effect at this dose in acutely reversing tolerance. Systemic administration of N~-nitro-L-arginine (LNNA) [5,10-12] and LN [12] have been reported to attenuate the development of tolerance to systemic morphine administration but not to the kappa agonist U50,488 H [5,11]. The results of the present study, however, fail to support a substantial effect of the NOS inhibitor LN in attenuating tolerance to intrathecal morphine, or

100

BBMOR I~ILN 100+ MOR

liT

i

['-]LN500 D oo+.o.

BINS

6O

% .o 4O

20

0

~ g

Wit hdrawal Sign Pig. 3. "Withdrawal signs after chronic infusion for 7 days o f i.t. morphine (MOR), LN 100 nmol/h + morphine, L N 500 nmol/h + morphine, LN 500 nmol/h, or saline (NS). Each column represents the % of each group o f four or more rats + SE, showing one of the following six signs

of withdrawal: (a) vocalizationin response to air motion or light touch; (b) spontaneous vocalization; (c) abnormal posture; (d) ejaculation; (e) "wet dog head shakes'; (f) escape attempts. *Significance versus morphine group at P < 0.05.

35

in preventing the evolution of a morphine-dependent state at spinal sites. We observed only a small diminishing effect on daily HP measurements at the highest usable dose. Although we saw no additional effect on the second day of infusion in LN + morphine co-infused groups over that of the morphine group, single bolus dose studies have reported that i.t. LN at comparable doses enhances morphine analgesia [16]. Thus, to further assess tolerance, a probe dose of intrathecal morphine was administered 24 h after discontinuation of the 7-day infusion, i.e., in the absence of the NOS inhibitor. To date, all studies have conducted tolerance assessment in the presence of whatever NOS inhibitor is being studied. The current study showed only a partial return of sensitivity to morphine in LN + morphine co-infused rats (Fig. 2a,b). There was also a small but significant prolongation of effect of the probe dose of morphine in rats infused with LN alone (although full dose response curves were not performed) (Fig. 2b), perhaps reflecting an increased sensitivity to, and accounting for, the partial restoration of sensitivity seen over the 7-day infusion on the HP in rats co-infused with LN and morphine (Fig. la,b). This restoration of sensitivity, albeit partial, is similar to that found using the same model of intrathecal morphine tolerance but with MK801 as a co-infusant [4]. Withdrawal is associated with release of glutamate in the spinal cord [7], and the manifestations of that release, which include thermal hyperalgesia, can be blocked by spinal administration of the NMDA receptor antagonist MK801 [4,7]. Many of the effects of activation of the NMDA receptor are mediated through nitric oxide. NMDA-induced thermal hyperalgesia [ 18], as well as that associated with neuropathic pain [9,13], can be blocked by LN. Thus, it is conceivable that NOS inhibition could be active in blocking thermal hyperalgesia associated with the withdrawal state. However, in the present study, LN significantly reduced only one of seven signs of withdrawal assessed. Other studies also report only some signs are suppressed whereas others are not. LN [1,8,12], L-NNA [8,11,12] and L-NMMA (N~monomethyl-L-arginine) [19] administered systemically, either just before naloxone precipitated withdrawal, or over the course of several days prior to withdrawal, have all been reported to attenuate some, but not all signs of withdrawal. Each study measures different withdrawal signs and uses different paradigms, so comparisons are difficult. We conclude that during continued spinal opiate exposure, the changes in spinal function leading to a reduction in opiate responsiveness are not substantially inhibited by continuous blockade of NOS by the highest usable dose of spinal LN. In addition, we conclude that at spinal sites LN only partially attenuates signs of spinal opiate withdrawal. This work was supported by DA02110 (TLY) and by Anesthesiology Training grant NIH T32NS07329 (SD).

36

S. Dunbar, T.L. Yaksh / Neuroscience Letters 207 (1996) 33-36

[1] Adams, M.L., Kalicki, J.M., Meyer, E.R. and Cicero, T.J., Inhibition of the morphine withdrawal syndrome by a nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester, Life Sci., 52 (1993) 245-249. [2] Ben Eliyahu, S., Marek, P., Vaccarino, A.L., Mogil, J.S., Sternberg, W.F. and Liebeskind, J.C., The NMDA receptor antagonist M K-801 prevents long-lasting non-associative morphine tolerance in the rat, Brain Res., 575 (1992) 304-308. [3] Cappendijk, S.L., de Vries, R. and Dzoljic, M.R., Inhibitory effect of nitric oxide (NO) synthase inhibitors on naloxone-precipitated withdrawal syndrome in morphine-dependent mice, Neurosci. Lett., 162 (1993) 97-100. [4] Dunbar, S.A. and Yaksh, T.L., Intrathecal MK801 attenuates intrathecal morphine tolerance and withdrawal in the rat, Anesthesiology, 81 (1994) A838. [5] Elliott, K., Minami N., Kolesnikov, Y.A., Pasternak, G.W. and Inturrisi, C.E., The NMDA receptor antagonists, LY274614 and MK-801 and the nitric oxide synthase inhibitor, NG-nitro-L arginine, attenuate analgesic tolerance to the mu-opioid morphine but not to kappa opioids, Pain, 56 (1994) 69-75. [6] Gutstein, H.B. and Trujillo, K.A., MK801 inhibits the development of morphine tolerance in the rat at spinal sites, Brain Res., 626 (1993) 332-334. [7] Jhamandas, K., Marsala, M. and Yaksh, T.L., Spinal release of Lglutamate during naloxone precipitated opioid withdrawal in the unanesthetized rat, Soc. Neurosci. Abstr., 20 (1994) 753. [8] Kimes, A.S., Vaupel, D.B. and London, E.D., Attenuation of some signs of opioid withdrawal by inhibitors of nitric oxide synthase, Psychopharmacology, 112 (1993) 521-524. [9] Kitto, K.F., Haley, J.E. and Wilcox, G.L., Involvement of nitric oxide in spinally mediated hyperalgesia in the mouse, Neurosci. Lett., 148 (1992) I-5. [10] Kolesnikov, Y.A., Pick, C.G. and Pasternak, G.W., NG-Nitro-L arginine prevents morphine tolerance, Eur. J. Pharmacol., 221 (1992) 399-400. [11] Kolesnikov, Y.A.. Pick, C.G., Ciszewska, G. and Pasternak, G.W., Blockade of tolerance to morphine but not to kappa opioids

[12]

[13]

[14]

[15]

[16]

[17] [18]

[19]

[20]

[21]

[22]

by a nitric oxide synthase inhibitor, Proc. Natl. Acad. Sci. USA, 90 (1993) 5162-5166. Majeed, N.H., Przewlocka, B., Machelska, H. and Przewlocki, R., Inhibition of nitric oxide synthase attenuates the development of morphine tolerance and dependence in mice, Neuropharmacology, 33 (1994) 189-192. Malmberg, A.B. and Yaksh, T.L., Spinal nitric oxide synthesis inhibition blocks NMDA induced thermal hyperalgesia and produces antinociception in the formalin test in rats, Pain, 54 (1993) 291-300. Marek, P., Ben Eliyahu, S., Gold, M. and Liebeskind, J.C., Excitatory amino acid antagonists (kynurenic acid and MK-801) attenuate the development of morphine tolerance in the rat, Brain Res., 547 (1991) 77-81. Marek, P., Ben Eliyahu, S., Vaccarino, A.L. and Liebeskind, J.C., Delayed application of MK-801 attenuates development of morphine tolerance in rats, Brain Res., 558 (1991) 163-165. Przewlocki, R., Machelska, H. and Przewlocka, B., Inhibition of nitric oxide synthase enhances morphine antinociception in the rat spinal cord, Life Sci., 53 (1993) 1-5. Sorkin, L.S., NMDA evokes an LN sensitive spinal release of glutamate and citrulline, NeuroReport, 4 (1993) 479-482. Sosnowski, M. and Yaksh, T.L., Differential cross tolerance between intrathecal morphine and sufentanil in the rat, Anesthesiology, 73 (1990) 1141-1147. Thorat, S.N., Reddy, P.L. and Bhargava, H.N., Evidence for the role of nitric oxide in kappa-opiate tolerance in mice, Brain Res., 621 (1993) 171-174. Thorat, SN., Barjavel, M.J., Matwyshyn, G.A. and Bhargava, H.N., Comparative effects of NG-monomethyl-L-arginine and MK-801 on the abstinence syndrome in morphine-dependent mice, Brain Res., 642 (1994) 153-159. Trujillo, K.A. and Akil, H., Inhibition of morphine tolerance and dependence by the NMDA receptor antagonist MK-801, Science, 251 (1991) 85-87. Yaksh, T.L. and Rudy, T.A., Chronic cathetherization of the subarachnoid space, Physiol. Behav., 7 (1976) 1032-1036.