Failure of prolyl‐leucyl‐glycinamide to alter analgesia measured by the Takemori test in morphine‐pretreated rats

Van Ree & de Wied (1976) reported that subcutaneous (s.c.) microgram injections of prolyl-leucyl-glycinamide (PLG), the N-terminal side chain of oxytocin, facilitated the development of morphine tolerance, measured by the Takemori test. It was previously (Takemori et a1 1973; Tulunay & Takemori 1974) noted that as little as one injection of morphine in mice caused an increased sensitivity to naloxone, indicating that the use of naloxone and morphine injections together would provide a very sensitive indicator of tolerance and physical dependence. Van Ree & de Wied found that 1 pg PLG, injected 1 h before a 20 mg kg-' (morning) and again before a 40 mg kg-' (afternoon) morphine HCI treatment injection, attenuated the hotplate analgesia produced the following morning by 80 mg kg-l morphine given with 1 mg kg-' naloxone. Using similar procedures we carried out several experiments which failed to confirm these results. Therefore, the following experiment using several morphine test doses, two PLG treatment doses, and two tests of analgesia, was carried out. The experiment was conducted with 141 experimentally-naive, male Wistar rats (140-160 g), obtained two weeks earlier from Canadian Breeding Laboratories (Constant, Quebec). Maintenance was with free access to food and water under a 0800-2000 h light cycle. One day before the experiment the rats were transferred from group cages to single cages. The experiment, designed after that of van Ree & de Wied (1976), was two days long. On day 1, each rat received intraperitoneally (i.p.) 24 mg kg-1 morphine sulphate (BDH) at lo00 h and 48 mg kg-' at 1700 h, each dose being dissolved in 0.90,6 NaCl (saline) (2 ml kg-'). One h before these injections, each rat was injected S.C. with Q.2 rnl saline containing 0, 1, or 10 pg PLG. The PLG (obtained from Organon, Oss, The Netherlands and Sigma Chemical) was dissolved in a drop of 0,001 M HCI and further diluted with saline. Commencing 0900 h the next day every rat received, at 0.5 h intervals, three hotplate tests, each followed 30 s later by one tailflick test. The hotplate procedure, after van Ree & de Wied (1976), used a stainless-steel hollow plate, maintained at 53 "C to produce 10-12 s response latencies in naive rats. The tailflick procedure was that described by Mucha et al (1978). To avoid tissue damage, both tests had maximum allowable latencies of 60 s. Immediately following the second tailflick test, each rat received one ip . injection on each side of the abdomen. The first consisted of 12, 24, 48, or 96 mg kg-' morphine, and the other 1 mg kg-' naloxone hydrochloride (Endo), each dissolved in saline (2 ml kg-I). * Correspondence. For each rat the two predrug latencies of each test were averaged and the difference between the baseline and the third test was computed. A summary is presented in Table 1. Separate analyses of variance indicated highly significant overall effects of morphine for hotplate (F (3,129) = 34.2, P < 0.001) and tailflick tests (F (3,129) = 54.8, P < 0.001). However, there were no appreciable effects of the PLG: neither overall PLG (F (2,129) = 0.51, F (2,129) = 0.1 1 for hotplate and tailflick, respectively), nor PLG-by-morphine interactions (F (6,129) = 0.24, F (6,129) = 0.35) were significant. These results, therefore, confirmed our initial failures to replicate the finding of van Ree & de Wied (1976) that S.C. administration of PLG before morphine pretreatments reduced the analgesia measured one day later in the Takemori test. Our negative findings cannot be accounted for by procedural variables, since the procedures used were designed to replicate those of van Ree & de Wied (1976). Moreover, we included a higher dose of PLG and a wider range of morphine test doses. Van Ree & de Wied (1976) noted that in pilot experiments PLG changed the log dose/response curve slope, which could result in a crossover of the curves for PLG and control groups. Since all our previous attempts to replicate their findings used only one morphine test dose, additional ones were required to ensure that we had not inadvertently chosen a dose falling at a crossover-point. In addition to this failure to find a PLG-related increase in the degree of morphine tolerance, we have also obtained negative or equivocal results of peptide treatment on morphine tolerance when the morphine was given without naloxone, and when the morphine and PLG (2 ng-20 p g ) were given by intracerebroventricular injection.