Increased Release of Immunoreactive Cholecystokinin Octapeptide By
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European Journal of Pharmacology, 234 (1993) 147-154 147 © 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00 EJP 53006 Increased release of immunoreactive cholecystokinin octapeptide by morphine and potentiation of/z-opioid analgesia by CCK B receptor antagonist L-365,260 in rat spinal cord Yan Zhou, Yu-Hua Sun, Zhi-Wen Zhang and Ji-Shen Han Neuroscience Research Center, Beijing Medical University, Beijing 100083, People's Republic of China Received 2 November 1992, revised MS received 13 January 1993, accepted 19 January 1993 This is the first report showing, in an in vivo study, that systemic morphine produced a marked (89%, P < 0.01) increase of the cholecystokinin octapeptide (CCK-8) immunoreactivity in the perfusate of the rat spinal cord, an effect completely reversed by naloxone. Since CCK-8 has been shown to possess potent anti-opioid activity at a spinal level, a blockade of the spinal cholecystokinin effect would be expected to potentiate opiate analgesia. With tail flick latency as a nociceptive index, it was found that intrathecal (i.t.) injection of a novel CCK B antagonist L-365,260 produced a marked potentiation of the analgesic effect induced by the/~-opioid agonists morphine (4 mg/kg s.c.) or ohmefentanyl (32 ng i.t.). Similar effects were obtained with the CCK a antagonist devazepide at a dose 40-50 times higher than that of L-365,260. Both devazepide and L-365,260 showed a bell-shaped dose-response curve. The results confirm the notion that an increased release of CCK-8 may constitute a self-limiting process for opioid effects at the spinal level, and that it is the CCK B receptor which mediates the anti-opioid effect of CCK-8 in the rat spipal cord. CCK receptor antagonists; CCK-8 immunoreactivity; CCK B receptors; Opiate analgesia; Naloxone 1. Introduction ments designed to test these hypotheses were not al- ways convincing and successful due to a lack of ade- Faris et al. (1983) were the first to show that chole- quate research tools. Thus, proglumide, a weak and cystokinin octapeptide (CCK-8) acts as a specific opiate non-specific CCK receptor antagonist, was shown to antagonist in the rat spinal cord, as evidenced by the potentiate morphine analgesia in one report (Watkins experimental finding that i.t. injection of 3.6 ng of et al., 1985) and to have no effect in another report CCK-8 antagonized opioid analgesia produced by foot- (Lehman et al., 1989). Central administration of chole- shock and morphine. This was confirmed by Han et al. cystokinin antiserum was reported to potentiate anal- (1985), who calculated that the dose of CCK-8 for 50% gesia induced by i.c.v, administered /3-endorphin (Itoh reversal of the effect of morphine (5 mg/kg) analgesia et al., 1985) but not by systemically injected morphine in the rat was 1.7 ng and 3.1 ng by i.c.v, and i.t. routes (Ding et al., 1986), probably due to difficulties with the of administration, respectively. Since CCK-8 has an rapid diffusion of the antibody molecule from the abundant and wide-spread distribution in both brain cerebral ventricle into the brain tissue. and spinal cord, one would naturally anticipate that The development of the potent, specific, non-peptide morphine or other endogenously released opioids CCK receptor antagonists, devazepide and L-365,031 would trigger the release of CCK-8 to counteract the for CCK A receptors (Chang and Lotti, 1986; Evens et opioid effect, and that removal of the effect of chole- al., 1986), and L-365,260 (Lotti and Chang, 1989) and cystokinin would result in an augmentation of opioid PD134308 (Wiesenfeld-Hallin et al., 1990) for CCK B analgesia and/or reversal of opiate tolerance. Experi- receptors, greatly facilitated research in this field. Dourish et al. (1990) have shown recently that mor- phine analgesia is significantly potentiated by L-365,260 Correspondence to: J.S. Han, Neuroscience Research Center, Bei- and by the CCK n antagonist L-365,031 at a 20 times jing Medical University, Beijing 100083, P.R. China. higher dose, suggesting that the CCK B receptor medi- 148 ates the anti-opioid effect. No information, however, is pril (1 ~M) and bestatin (1/zM) was perfused at a rate available concerning the site of action of these two of 1.0 ml/30 min with a push-pull pump. specific antagonists for their anti-opioid activity. Another basic question is whether morphine accel- 2.3. Nociceptive test erates the release of CCK-8 from the CNS. Besides the in vitro studies performed on periaqueductaI grey The rat was restrained in a plastic holder with its (Rattray and Delleroche, 1987), substantia nigra (Be- hind legs protruding. The nociceptive threshold was noliel et al., 1990) and spinal cord (Benoliel et al., measured by the latency of the tail flick response, 1991), the most commonly cited in vivo study is the elicited by radiant heat applied to the lower 1/3 of the preliminary observation of Tang et al. (1984) that the tail. The mean tail flick latency of three measurements addition of morphine (1 p~M) to the fluid perfusing rat taken at 5-rain intervals at the start of the experiment spinal subarachnoid space increases the cholecys- was taken as the basal threshold. The tail flick latency tokinin content of the perfusate. Since they did not taken at 10-min intervals after drug administration is present a statistical analysis in their preliminary report, expressed as the percentage change from basal tail it seemed worthwhile to re-evaluate this important flick latency, with a cutoff limit of + 150% to avoid issue in order to ascertain the extent and the time unnecessary skin damage. course of this reaction. On the basis of the above mentioned information 2.4. Radioimmunoassay of CCK-8 the present study was performed to clarify the follow- ing issues: (a) whether systemic injection of a conven- Cholecystokinin was extracted as reported by Mar- tional dose of morphine (5 mg/kg s.c.) would indeed ley and Rehfeld (1984). Aliquots of spinal perfusate accelerate the release of CCK-8 immunoreactivity from (1.0 ml) were collected in polyethylene tubes in an the rat spinal cord, and (b)whether the novel CCK A ice-water bath and then heated for 10 min in a water receptor antagonist devazepide and the CCK B recep- bath at 95°C. After cooling, the aliquots were cen- tor antagonist L-365,260 would potentiate opioid anal- trifuged at 10000 ×g for 15 min. Supernatants were gesia after i.t. injection and, if so, what is the dose ratio kept at -20°C and dried in a vacuum dryer. The between the two antagonists. residue was taken up in 0.2 ml H20 for the radioim- munoassay of CCK-8. The cholecystokinin antiserum, which was kindly provided by Dr. J.S. Hong (NIEHS, 2. Materials and methods USA), showed no detectable cross reactivity with [MetS]enkephalin, /3-endorphin, gastrin, CCK-7, CCK- 2.1. 1.t. injection of drugs 4, or unsulfated CCK-8. The radioimmunoassay proce- dure for cholecystokinin in the present study was simi- Male Wistar rats weighing 200-250 g were anes- lar to that suggested by Amersham Inc. On the basis of thetized with chlorohydrate (0.4 g/kg i.p.). The i.t. 10 consecutive assays, the sensitivity of the assay was cannula was implanted as described by Yaksh and 0.4 fmol/ml, with 4.6% intra-assay and 9.7% inter-as- Rudy (1976). A PE-10 polyethylene catheter was im- say variations. planted through the cisterna magna down to the sub- arachnoid space for 7.5 cm to reach the upper border 2.5. Drugs of the lumbar enlargement of the spinal cord. The outer part of the catheter was plugged and fixed to the 125I-Bolting Hunter-CCK-8 was purchased from skin after the wound was closed. Experiments with i.t. Amersham Inc. (UK). CCK-8 was a gift from Squibb injection of drugs started about 20-26 h after the and Sons, Inc. Morphine HC1 was purchased from surgical operation. Drugs were injected via the catheter Qinhai Drug Company (China). Naloxone HC1 was at a volume of 10 /zl, followed by 10 /zl of normal from Sigma (USA). Ohmefentanyl HCI (N-[1-(/3-hy- saline to flush out the cannula. Injection was com- droxy-fl-phenethy)-3-methyl-4-piperidyl]-N-phenylpro- pleted within 30 s. pionamide) was obtained from the Shanghai Institute of Materia Medica. Devazepide (3S(-)-N-(2,3-dihy- 2.2. Perfusion of the subarachnoid space of the spinal dro-l-methyl-2-oxo-5-phenyl-l-H- 1,4-benzodiazepin-3- cord yl)-H-indole-2-carboxamide) and L-365,260 (3R(+)-N- (2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodia- Rats were anesthetized with chlorohydrate, and a zepin-3-yl)N'-(3-methyl-phenyl)urea) were donated by PE-10 catheter was inserted 7.5 cm down the subarach- Dr. R.F. Freidinger of the Merck, Sharp and Dohme noid space through the cisterna magna. Another Research Laboratories (USA). Devazepide and L- polyethylene tubing (PE-50) was inserted 1 cm into the 365,260 were first dissolved in dimethylsulfoxide cisterna. Artificial cerebrospinal fluid containing capto- (DMSO) and then diluted with propylene glycol to a 149 final ratio of 20% DMSO" 80% propylene glycol (v/v). Radioimmunoassay revealed that in the control group Morphine HC1, naloxone HC1 and ohmefentanyl were (fig. 1, left panel) the content of CCK-8 immunoreac- dissolved in normal saline.