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,1
* Department of Pediatrics and Neonatology, Advocate Lutheran General Children’s Hospital, Park Ridge, Illinois 60068; and Department of Biopharmaceutical Sciences, University of Illinois at Chicago, Chicago, Illinois 60612
1To whom requests for reprints should be addressed at Department of Biopharmaceutical Sciences (M/C 865), The University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612. E-mail: gulati{at}uic.edu
Abstract
The involvement of central endothelin (ET) receptors in neonatal morphine tolerance has been demonstrated. The present study investigates the role of central ET receptors in morphine withdrawal in neonatal rats. The aim was to determine whether activation of G-proteins coupled to opioid and ET receptors by morphine and various ET receptor modulators is affected during morphine withdrawal in neonatal rats. Pregnant female rats were rendered tolerant to morphine by chronic exposure to morphine pellets during 7 days. On Day 8, pellets were removed and rats were allowed to undergo withdrawal for 24 hrs. Rat pups were delivered by cesarean section. G-protein stimulation induced by morphine; ET-1; the ETA receptor antagonist, BMS182874; and the ETB receptor agonist, IRL1620, were determined in the brain of neonatal rats undergoing morphine withdrawal by [35S]GTP
S binding assay. Morphine produced higher (P < 0.05) maximal stimulation of G-protein in the morphine-withdrawal group (83.60%) compared with the placebo group (66.81%). ET-1–induced G-protein stimulation was also altered, and the median effective concentration (EC50) during morphine withdrawal (170.60 nM) was significantly higher than placebo (62.5 nM; P< 0.05). The maximal stimulation induced by the ETA receptor antagonist, BMS182874, in the morphine-withdrawal group (86.07%; EC50 = 31.25 nM) was significantly higher than in the placebo group (EC50 > 1000 nM). The ETB agonist, IRL1620, induced G-protein stimulation was similar in placebo (73.43%, EC50 = 13.26 nM) and morphine-withdrawal groups (75.08%, EC50 = 11.70 nM), respectively. To our knowledge, this is the first report indicating involvement of central ETA receptors in neonatal morphine withdrawal.
Key Words: morphine • BMS182874 (5-[dimethylamino]-N-[3,4-dimethyl-5-isoxazolyd]-1-naphthalenesulfonamide) • IRL1620 (N-Suc-[Glu9, Ala11,15]ET-1[8–21]) • [35S]GTPS-binding withdrawal • endothelin • neonatal rats • central nervous system
Introduction
Because of several reported benefits to neonatal behavior and outcomes from opioid-based analgesia and anesthesia (1–3), opioids are widely used in neonatal pain management. However, chronic use of opioid analgesics results in tolerance and dependence. Iatrogenic opioid dependence was first reported in infants receiving fentanyl during extracorporeal membrane oxygenation (4). Chronic exposure of the fetus during maternal opiate abuse also leads to severe neurologic and behavioral changes. Abrupt cessation of opiates leads to severe withdrawal syndrome, and management of these adverse effects may be a major challenge for the clinician. Recent animal studies have demonstrated that fetal rats and infant rat pups undergo opiate tolerance and physical dependence manifested as withdrawal if the dams are exposed to opiates during pregnancy (5–7).
Morphine and other opioids act by binding to and activating µ-, 
-, and 
-opioid receptors. Conformational changes in opioid receptors initiate signal transduction cascade because of activation of inhibitory G-proteins, Gi
and Go (8–10). During opioid tolerance, opioid receptors are desensitized and there is an upregulation of second messengers, such as adenylate cyclase and cAMP (11). Chronic morphine treatment also results in functional uncoupling of µ-opioid receptors and G-proteins. We propose that endothelin (ET), an endogenous neuropeptide, may be an important factor in mediating opiate tolerance. We have demonstrated the involvement of central ET receptors in morphine analgesia and tolerance. In adult rats, it was found that ETA receptor antagonists significantly potentiated morphine analgesia (12), and restored analgesic response of morphine during tolerance (13). Although peripheral administration of the ETB receptor agonist, IRL1620, was shown to be involved in analgesia through opioid receptors, centrally administered ETB receptor agonists did not produce any effect on morphine analgesia (14). Studies conducted in neonatal rats showed involvement of ET receptors in morphine tolerance (15). We found that ET receptor antagonists did not act on opioid receptors directly, but modulated the action of morphine by acting through G-proteins (15). It was shown that chronic administration of morphine leads to modulation of Gi/Go-coupled receptors. However, corresponding changes in the ET system and involvement of ET receptors in morphine withdrawal in the brain of neonatal rats is not known. Therefore, in the present study, we evaluated the effect of an ETA receptor antagonist and an ETB receptor agonist on G-protein stimulation in neonatal rats undergoing morphine withdrawal, using a [35S]GTPS-binding assay.
Materials and Methods
Neonatal rats harvested from pregnant female Sprague-Dawley rats (Harlan, Indianapolis, IN) rats at term (Day 22 of gestation) were randomly selected from each litter and used for [35S]GTPS binding. Studies were conducted according to guidelines established by Animal Care Committee of University of Illinois at Chicago.
Morphine and placebo pellets were obtained from the National Institute of Drug Abuse, Rockville, MD. Guanosine-5'-diphosphate (GDP) was dissolved in assay buffer (Sigma Aldrich, St. Louis, MO). [35S]GTPS (1000 Ci/mmol; Amersham Pharmacia Biotech, Piscataway, NJ) was diluted in assay buffer. Unlabeled guanosine-5'-o-(3-thio)triphosphate (GTPS) was dissolved in assay buffer (Sigma Aldrich). Morphine sulfate (Mallinckrodt Chemical Co., St. Louis, MO) and IRL1620 (Sigma Aldrich) were dissolved in sterile saline. BMS182874 (Tocris Cookson Inc., Ellisville, MO) was dissolved in 20% dimethylsulfoxide. ET-1 (American Peptides Company Inc., Sunnyvale, CA) was dissolved in 0.1% bovine serum albumin. Dilutions of all drugs were prepared in assay buffer.
Pregnant female rats were rendered tolerant to morphine by a pellet implantation procedure (16). Rats were divided into two groups: group 1 received placebo pellets (n = 4); Group 2 received morphine pellets (n = 4). Each rat was subcutaneously implanted with six pellets during a 7-day period. One morphine pellet was implanted on Day 14 of gestation, two pellets on Day 16 of gestation, and three pellets on Day 18 of gestation. Control rats received placebo pellets containing the same excipients without morphine. On Day 21 of gestation, both placebo and morphine pellets were removed, the incision was closed with sterile wound clips, and rats were allowed to undergo withdrawal from morphine for 24 hrs. Pregnant females on Day 22 of gestation (at term) were anesthetized using 1% isoflurane anesthesia, and rat pups were delivered by cesarean section. Pups were sacrificed by cervical section immediately, the cerebellums were removed, and the brains were stored at –70°C until analysis. Neonatal rats from both groups were randomly selected and used for [35S]GTPS binding in neuronal membranes.
[35S]GTPS binding was performed according to the procedure described earlier (17), using approximately 100 µg protein in each sample. The total volume in each tube was 0.5 ml, containing 0.35 ml of homogenate, various concentrations of drugs (morphine, ET-1, BMS182874, and IRL1620), 30 µM GDP, 100 nM [35S]GTPS, and assay buffer. The concentration range for morphine, ET-1, BMS182874, and IRL1620 was 0.98–1000 nM. Nonspecific binding was measured using 10 µM unlabeled GTPS. Specific binding was expressed as femtomoles per milligram of protein (mean ± SEM).
Data was analyzed by one-way analysis of variance (ANOVA) followed by a Bonferroni test. A level of P < 0.05 was considered significant.
Results
Morphine and ET-1 produced concentration-dependent increases in [35S]GTPS binding. Basal GTP binding was in the range of 0.14 ± 0.01 to 0.89 ± 0.40 fmol/mg protein. Basal binding was statistically similar (P > 0.05) in placebo and morphine-withdrawal groups. Maximal GTP binding in the morphine-withdrawal group (83.60 ± 5.77%) was significantly higher (P < 0.05) as compared with the placebo group (66.81 ± 1.51%). An median effective concentration (EC50) value for morphine-stimulated GTP binding in morphine-withdrawal group was 28.78 ± 0.17 nM (Table 1
), which was significantly lower than the EC50 value in the placebo group (170.60 ± 0.10 nM). ET-1–induced GTP binding was attenuated in the morphine-withdrawal group; however, at concentrations greater than the EC50 value, the G-protein activation was increased. Maximal stimulation in the placebo and morphine-withdrawal group was 74.88 ± 1.50% and 87.16 ± 4.83%, respectively. The EC50 value in the morphine-withdrawal group was 84.79 ± 0.04 nM. This was significantly higher than the EC50 value in the placebo group (20.66 ± 0.12 nM; Table 1).
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S binding in the placebo group was greater than 1000 nM, whereas the EC50 value in the morphine-withdrawal group (33.20 ± 3.29 nM) was significantly lower (P < 0.05) compared with the placebo group. The ETB receptor agonist, IRL1620, produced a maximal stimulation of 73.43 ± 8.89% in the placebo group, which was similar (P > 0.05) to that of the morphine-withdrawal group (75.08 ± 5.41%). The EC50 values for IRL1620-stimulated [35S]GTPS binding in the placebo (16.34 ± 0.12 nM) and morphine-withdrawal (15.80 ± 0.11 nM) groups were similar (Table 1
). Discussion
Mechanisms involved in withdrawal after chronic opioid administration are extremely complex, involving changes in opiate signal transduction and interactions between opiate and nonopiate systems (18). In the present study, we investigated the effect of morphine withdrawal G-protein activation of opioid and ET receptors in the brain of neonatal rats.
Studies show that infant rat pups and fetal rats experience opiate tolerance if dams are exposed to opiates during pregnancy (19). We found that morphine-induced stimulation of GTP binding was higher, whereas ET-1–induced stimulation of GTP binding was lower in neonatal rats undergoing morphine withdrawal compared with the placebo group. These findings clearly implicate a role of central ET receptors in morphine withdrawal in neonatal rats. It was further found that the ETA receptor antagonist, BMS182874, did not affect GTP binding in normal rats, but significantly increased GTP binding in neonatal rats undergoing morphine withdrawal. Our results indicate that these changes taking place in G-protein–coupling mechanisms are restored by ETA receptor antagonists.
Although opioid receptor and ET receptor coupling to G-proteins is affected in a similar manner in morphine tolerance, the present findings suggest that opioid receptors and ET receptors are affected differently during withdrawal. The development of opioid dependence and withdrawal may involve complex interactions between several neurotransmitter systems having opposing actions on the G-protein system (20). Neonates have been found to have significantly different characteristics of receptors and concentration of neurotransmitters compared with adults, and extensive postnatal developmental changes take place in the central nervous system. Based on the findings of the present study, we speculate that ETA receptors may play a role in morphine withdrawal in neonatal rats. It is conduced that ETA receptor antagonists restore coupling of G-proteins to opioid receptors, which may play an important part in opiate tolerance and withdrawal.
Footnotes
The present study was funded by Chicago Labs Inc., Chicago, IL.
Received for publication September 9, 2005. Accepted for publication December 12, 2005.
References
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