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The Effect of Vasoactive IntestinalPolypeptide and Pituitary AdenylateCyclase Activating Polypeptide onTolerance to Morphine and Alcohol in Micea

GYULA SZABÓ,b MÓNIKA MÁCSAI, ÉVA SCHEK, AND

GYULA TELEGDY

Department of Pathophysiology, Albert Szent-Györgyi MedicalUniversity, H-6701 Szeged, Hungary

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activatingpolypeptide (PACAP) have been detected in the peripheral and central nervous sys-

tem. The distribution of these peptides throughout the central nervous system suggests theinvolvement of VIP and PACAP in a number of processes including limbic, autonomic,and neuroendocrine functions.

The relationship between pain sensitivity and peptides belonging to secretin-VIP-glucagon family has been examined before upon intrathecal administration. In spinalized,decerebrated rats VIP facilitated the flexor reflex to thermal stimuli and was implicated intransmission of nociceptive information after peripheral nerve damage.1 In normal rats adecrease in the reaction latency in tail-flick test was demonstrated.2 In ovariectomized ratsVIP significantly increased tail-flick latency.3 The effect of VIP proved to be short-lastingin the latest two studies.

The aim of the present study was to investigate the effect of these peptides on analge-sia in intact mice upon peripheral and central administration and to extend these studies byinvestigating the acute interaction of these peptides with morphine, alcohol, and the devel-opment of tolerance to morphine and ethanol.

MATERIALS AND METHODS

Animals

Male, albino mice (25±5 g) of an inbred CFLP strain (SZOTE, Animal Husbandry,Szeged, Hungary) were used. The animals were kept under a standard light-dark cycle withfood and water available ad libitum. At least a week of habituation was allowed beforebeginning the experiments.

aThis work supported by Országos Tudományos Kutatási Alap (T 022230 and T 023169) andEgészségügyi Tudományos Tanács (T 02-670/96 and T 661/1996).

bCorresponding author: G. Szabó, M.D., Ph.D., Associate Professor of Pathophysiology, AlbertSzent-Györgyi Medical University, Semmelweis u. 1, Pf. 531, H-6701 Szeged, Hungary; Tel.: 36-62-455-788; Fax: 36-62-455-695; E-mail: szabog@patph.szote.u-szeged.hu

Surgery

For intracerebroventricular cannulation, mice were anesthetized with sodium pentobar-bital (Nembutal, intraperitoneally) and a polyethylene cannula was inserted into the rightlateral cerebral ventricle and cemented to the skull with cyanoacrylate-containing instantglue. The experiments were started 5 days after intracerebroventricular cannulation.Animals were lightly anesthetized with ether and morphine pellets were implanted subcu-taneously into the sacral area through a small section in the neck area. All procedures werecarried out according to the rules of the Ethical Committee for the Protection of theAnimals (Albert Szent-Györgyi Medical University, Szeged, Hungary)

Treatments

For subcutaneous treatment, VIP or PACAP was dissolved in isotonic saline andinjected in a volume 0.2 ml. For intracerebroventrical treatment, the peptides were dis-solved in artificial cerebrospinal fluid and injected in a volume of 2 ml. In the experimentsfor measuring the antinociceptive effect of morphine, morphine HCl (2.25–5 mg/kg, sub-cutaneous) was used. In chronic tolerance studies, pellets containing 35 mg morphine wereimplanted.

Procedures

For testing the analgesic effect, tail-flick tests were performed after single or repeatedVIP or PACAP pretreatment. The tail of the animals was placed under a centrally focusedradiating heat lamp and the latency of tail avoidance was measured and the degree of anal-gesic effect was calculated in percentage.

Chronic tolerance to morphine was examined on morphine pellet–implanted animals72 h after the implantation without removing the tablets. Five mg/kg morphine was givensubcutaneously after the determination of the threshold of pain. The analgesic effect of thetest dose of morphine was measured at various time intervals. Vasoactive intestinalpolypeptide (VIP) or PACAP was administered 30 min before the implantation of the mor-phine pellets and the treatment was repeated 24, 48, and 72 h later.

For measuring the hypnotic response to ethanol, animals were pretreated with VIP orPACAP (intracerebroventricularly) and 30 min later 4 g/kg ethanol was injected (intraperi-toneally). The duration of the loss of the righting reflex was measured.

For determining the development of rapid tolerance to the hypothermic effect ofethanol, mice were pretreated intraperitoneally with 2 g/kg on day 1 in the morning (8a.m.) and the hypothermic response was detected at various times. Ethanol treatment wasrepeated at 4 p.m. and the development of tolerance to the hypothermic effect of ethanolwas tested the next morning.

Statistical Analysis

Statistical analysis of the data was made by ANOVA. For significant ANOVA values,groups were compared by Tukey’s test for multiple comparisons with unequal cell size. Aprobability level of 0.05 was accepted as indicating significant differences.

SZABÓ et al.: MORPHINE AND ETHANOL TOLERANCE 567

RESULTS AND DISCUSSION

Animals treated with VIP (2 pg–2 µg subcutaneously, 200 pg–200 ng intracerebroven-tricularly) displayed an increase in tail-flick latency, supporting the observation ofKomisaruk and colleagues.3 However, the effect was not dose dependent and no differencewas observed in the effective doses between peripheral and central administration. Peptideadministration resulted in a longer lasting effect, compared to earlier studies2,3 and animalstreated with intracerebroventricular VIP displayed longer lasting analgesia (TABLE 1).

VIP pretreatment (subcutaneous or intracerebroventricular) significantly diminishedthe analgesic effect of a single morphine injection. The analgesic effect of VIP (subcuta-neous or intracerebroventricular) can be prevented by naloxone pretreatment or decreasedif naloxone was administered after the maximal analgesic effect of VIP (45 min) wasreached. Naloxone pretreatment significantly reduced the morphine-induced analgesia inpeptide-naive animals. VIP pretreatment reduced the analgesic response of morphine,however VIP treatment could not reduce the analgesic response below the level of the con-trol animals.

Animals injected with PACAP itself did not display analgesia or decrease of the anal-gesic response to an acute morphine challenge. Subchronic treatment with PACAP for 3days diminished the acute analgesic effect of morphine.

VIP (intracerebroventricular) blocked the development of chronic tolerance to mor-phine, but did not affect naloxone-precipitated withdrawal symptoms (loss of body weight,decrease in body temperature, and naloxone-induced withdrawal jumps). PACAP has noeffect on the development of tolerance to morphine and on naloxone-induced withdrawalsigns (TABLE 2).

Intracerebroventricular administration of PACAP and VIP does not influence the dura-tion of loss of the righting reflex.

568 ANNALS NEW YORK ACADEMY OF SCIENCES

TABLE 1. The effect of VIP on analgesic response in tail-flick test

Dose Subcutaneous Administration Intracerebroventricular Administration

0 -2.09 ± 1.10a 0.34 ± 0.890.02 pg -1.08 ± 5.970.2 pg 5.98 ± 3.602 pg 14.24 ± 3.23*20 pg 9.44 ± 1.87*200 pg 19.68 ± 2.90* 13.89 ± 1.82*2 ng 18.99 ± 2.26* 14.98 ± 1.62*20 ng 17.00 ± 2.11* 11.36 ± 0.76*200 ng 15.21 ± 1.99* 16.49 ± 1.49*2 µg 16.56 ± 2.91*

aMean ± S.E.M. and *p < 0.05 compared to control group.

TABLE 2. The effect of VIP on development of chronic tolerance to morphine

Time Morphine Tolerant + Saline Morphine Tolerant + VIP

30 min -1.64 ± 1.46a 12.58 ± 3.04*60 min 20.80 ± 3.72 32.58 ± 3.54*90 min 15.50 ± 2.51 24.89 ± 3.09*120 min 1.01 ± 2.39 8.17 ± 1.31*

aMean ± S.E.M. and *p < 0.05 compared to morphine tolerant + saline group.

Note: The effect of VIP on chronic tolerance to morphine was investigated on animals with mor-phine pellet implantation. VIP treatment caused a significant increase in tail-flick latency as com-pared to tolerant control group at all time points.

Acute treatment with PACAP (intracerebroventricular) enhanced, VIP (subcutaneous)decreased the hypothermic response to ethanol on day 1, but did not influence the devel-opment of tolerance to the hypothermic effect of ethanol.

Subchronic treatment with PACAP (intracerebroventricular) decreased the hypothermicresponse of an acute ethanol challenge and a similar effect was observed on the developmentof tolerance. VIP (subcutaneous) decreased the hypothermic response, but the developmentof tolerance was not affected. Subchronic subcutaneous treatment with PACAP does notinfluence the development of tolerance to the hypothermic effect of ethanol.

The results suggest that peptides of the secretin-VIP-glucagon family can modulate thedevelopment of tolerance to morphine and ethanol.

REFERENCES

1. WIESENFELD-HALLIN, Z. 1987. Intrathecal vasoactive intestinal polypeptide modulates spinalreflex excitability primarily to cutaneous thermal stimuli in rats. Neurosci. Lett. 80: 293–297.

2. CRIDLAND, R. A. & L. J. HENRY. 1988. Effects of intrathecal administration of neuropeptides ona spinal nociceptive reflex in the rat: VIP, galanin, CGRP, TRH, somatostatin and angiotensinII. Neuropeptides 11: 23–32.

3. KOMISARUK, B. R., C. BANAS, S. B. HELLER, B. WHIPPLE, G. F. BARBATO & F. JORDAN. 1988.Analgesia produced by vasoactive intestinal polypeptide administered directly to the spinalcord in rats. Ann. N.Y. Acad. Sci. 527: 650–654.

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