Effectiveness and selectivity of a heroin conjugate vaccine to attenuate heroin, 6-acetylmorphine, and morphine antinociception in rats: Comparison with naltrexone.

BACKGROUND: One emerging strategy to address the opioid crisis includes opioid-targeted immunopharmacotherapies. This study compared effectiveness of a heroin-tetanus toxoid (TT) conjugate vaccine to antagonize heroin, 6-acetylmorphine (6-AM), morphine, and fentanyl antinociception in rats. METHODS: Adult male and female Sprague Dawley rats received three doses of active or control vaccine at weeks 0, 2, and 4. Vaccine pharmacological selectivity was assessed by comparing opioid dose-effect curves in 50 °C warm-water tail-withdrawal procedure before and after active or control heroin-TT vaccine. Route of heroin administration [subcutaneous (SC) vs. intravenous [IV)] was also examined as a determinant of vaccine effectiveness. Continuous naltrexone treatment (0.0032-0.032 mg/kg/h) effects on heroin, 6-AM, and morphine antinociceptive potency were also determined as a benchmark for minimal vaccine effectiveness. RESULTS: The heroin-TT vaccine decreased potency of SC heroin (5-fold), IV heroin (3-fold), and IV 6-AM (3-fold) for several weeks without affecting IV morphine or SC and IV fentanyl potency. The control vaccine did not alter potency of any opioid. Naltrexone dose-dependently decreased antinociceptive potency of SC heroin, and treatment with 0.01 mg/kg/h naltrexone produced similar, approximate 8-fold decreases in potencies of SC and IV heroin, IV 6-AM, and IV morphine. The combination of naltrexone and active vaccine was more effective than naltrexone alone to antagonize SC heroin but not IV heroin. CONCLUSIONS: The heroin-TT vaccine formulation examined is less effective, but more selective, than chronic naltrexone to attenuate heroin antinociception in rats. Furthermore, these results provide an empirical framework for future preclinical opioid vaccine research to benchmark effectiveness against naltrexone.

A Monoclonal Antibody against 6-Acetylmorphine Protects Female Mice Offspring from Adverse Behavioral Effects Induced by Prenatal Heroin Exposure.

Escalating opioid use among fertile women has increased the number of children being exposed to opioids during fetal life. Furthermore, accumulating evidence links prenatal opioid exposure, including opioid maintenance treatment, to long-term negative effects on cognition and behavior, and presses the need to explore novel treatment strategies for pregnant opioid users. The present study examined the potential of a monoclonal antibody (mAb) targeting heroin's first metabolite, 6-acetylmorphine (6-AM), in providing fetal protection against harmful effects of prenatal heroin exposure in mice. First, we examined anti-6-AM mAb's ability to block materno-fetal transfer of active metabolites after maternal heroin administration. Next, we studied whether maternal mAb pretreatment could prevent adverse effects in neonatal and adolescent offspring exposed to intrauterine heroin (3 × 1.05 mg/kg). Anti-6-AM mAb pretreatment of pregnant dams profoundly reduced the distribution of active heroin metabolites to the fetal brain. Furthermore, maternal mAb administration prevented hyperactivity and drug sensitization in adolescent female offspring prenatally exposed to heroin. Our findings demonstrate that passive immunization with a 6-AM-specific antibody during pregnancy provides fetal neuroprotection against heroin metabolites, and thereby prevents persistent adverse behavioral effects in the offspring. An immunotherapeutic approach to protect the fetus against long-term effects of prenatal drug exposure has not been reported previously, and should be further explored as prophylactic treatment of pregnant heroin users susceptible to relapse.

Comparison of (+)- and (-)-Naloxone on the Acute Psychomotor-Stimulating Effects of Heroin, 6-Acetylmorphine, and Morphine in Mice.

Toll-like receptor 4 (TLR4) signaling is implied in opioid reinforcement, reward, and withdrawal. Here, we explored whether TLR4 signaling is involved in the acute psychomotor-stimulating effects of heroin, 6-acetylmorphine (6-AM), and morphine as well as whether there are differences between the three opioids regarding TLR4 signaling. To address this, we examined how pretreatment with (+)-naloxone, a TLR4 active but opioid receptor (OR) inactive antagonist, affected the acute increase in locomotor activity induced by heroin, 6-AM, or morphine in mice. We also assessed the effect of pretreatment with (-)-naloxone, a TLR4 and OR active antagonist, as well as the pharmacokinetic profiles of (+) and (-)-naloxone in the blood and brain. We found that (-)-naloxone reduced acute opioid-induced locomotor activity in a dose-dependent manner. By contrast, (+)-naloxone, administered in doses assumed to antagonize TLR4 but not ORs, did not affect acute locomotor activity induced by heroin, 6-AM, or morphine. Both naloxone isomers exhibited similar concentration versus time profiles in the blood and brain, but the brain concentrations of (-)-naloxone reached higher levels than those of (+)-naloxone. However, the discrepancies in their pharmacokinetic properties did not explain the marked difference between the two isomers' ability to affect opioid-induced locomotor activity. Our results underpin the importance of OR activation and do not indicate an apparent role of TLR4 signaling in acute opioid-induced psychomotor stimulation in mice. Furthermore, there were no marked differences between heroin, 6-AM, and morphine regarding involvement of OR or TLR4 signaling.

Differential in vitro inhibition of M3G and M6G formation from morphine by (R)- and (S)-methadone and structurally related opioids.

AIMS: To determine the in vitro kinetics of morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) formation and the inhibition potential by methadone enantiomers and structurally related opioids. METHODS: M3G and M6G formation kinetics from morphine were determined using microsomes from five human livers. Inhibition of glucuronide formation was investigated with eight inhibitors (100 microm) and the mechanism of inhibition determined for (R)- and (S)-methadone (70-500 microm) using three microsomal samples. RESULTS: Glucuronide formation displayed single enzyme kinetics. The M3G Vmax (mean+/-SD) was 4.8-fold greater than M6G Vmax (555+/-110 vs. 115+/-19 nmol mg-1 protein h-1; P=0.006, mean of difference 439; 95% confidence interval 313, 565 nmol mg-1 protein h-1). Km values for M3G and M6G formation were not significantly different (1.12+/-0.37 vs. 1.11+/-0.31 mm; P=0.89, 0.02; -0.29, 0.32 mm). M3G and M6G formation was inhibited (P<0.01) with a significant increase in the M3G/M6G ratio (P<0.01) for all compounds tested. Detailed analysis with (R)- and (S)-methadone revealed noncompetitive inhibition with (R)-methadone Ki of 320+/-42 microm and 192+/-12 microm for M3G and M6G, respectively, and (S)-methadone Ki of 226+/-30 microm and 152+/-20 microm for M3G and M6G, respectively. Ki values for M3G inhibition were significantly greater than for M6G for (R)-methadone (P=0.017, 128; 55, 202 microm) and (S)-methadone (P=0.026, 75; 22, 128 microm). CONCLUSIONS: Both methadone enantiomers noncompetitively inhibited the formation of morphine's primary metabolites, with greater inhibition of M6G formation compared with M3G. These findings indicate a mechanism for reduced morphine clearance in methadone-maintained patients and reduced relative formation of the opioid active M6G compared with M3G.

Opiate alkaloids and nitric oxide production in the nematode Ascaris suum.

The tissue distribution, course of secretion, and sex differences of morphine were delineated in Ascaris suum. Nitric oxide (NO) release in various tissues in response to morphine and its metabolite morphine-6-glucuronide (M6G) were also examined. Ascaris suum of both sexes along with their incubation fluid were analyzed for morphine concentrations by high-performance liquid chromatography (HPLC) over a 5-day period. Various tissues were also dissected for HPLC and NO analysis. Morphine was found to be most prevalent in the muscle tissue, and there is significantly more morphine in females than males, probably because of the large amounts present in the female uterus. Morphine (10(-9) M) and M6G (10(-9) M) stimulated the release of NO from muscles. Naloxone (10(-7) M) and N-nitro-L-arginine methyl ester (10(-6) M) blocked (P < 0.005) morphine-stimulated NO release from A. suum muscle tissue. D-Phe-Cys-Tyr-D-Trp-Om-Thr-Pen-Thr-NH2 (CTOP) (10(-7) M) did not block morphine's NO release. However, naloxone could not block M6G-stimulated NO release by muscles, whereas CTOP (10(-7) M) blocked its release. These findings were in seeming contradiction to our earlier inability to isolate a mu opiate receptor messenger RNA by reverse transcriptase-polymerase chain reaction using a human mu primer. This suggests that a novel mu opiate receptor was possibly present and selective toward M6G.

Insights into mu opioid pharmacology the role of mu opioid receptor subtypes.

Although mu opioids share many pharmacological characteristics, they also reveal many differences. Many approaches over the years have suggested the existence of multiple mu opioid receptors. The unique selectivities of naloxonazine, for example, provided a way of distinguishing mu1from mu2actions. Studies of morphine-6beta-gluruconide suggested that its actions involved yet another mu opioid receptor subtype. The cloning of a mu opioid receptor, MOR-1, provided a way of exploring this possibility at the molecular level. Recent studies have now identified a number of splice variants of this gene that appear to be important in the production of mu opioid analgesia.

14-methoxymetopon, a potent opioid, induces no respiratory depression, less sedation, and less bradycardia than sufentanil in the dog.

UNLABELLED: Opioids of the mu-receptor type depress respiration and induce addiction. At 10-min intervals 14-methoxymetopon (HS-198), which is 20,000 times more potent than morphine in the acethylcholine-writhing test, was given in graded IV doses (3, 6, and 12 microg/kg) to awake, trained canines (n = 7). The following variables were derived: PaO(2), PaCO(2), heart rate (lead II of the electrocardiogram), mean arterial blood pressure, relative changes in the delta domain and the beta domain of the electroencephalogram, the somatosensory evoked potential, and the skin-twitch reflex to electrical stimuli. Thereafter, 20 microg/kg naltrexone was given for reversal. After a washout period, the same animals were exposed to similar doses of sufentanil (SUF) followed by naltrexone. Both opioids induced a dose-related bradycardia and hypotension. The maximal bradycardic effect was 19% after HS-198 and 42% after SUF (P < 0.005). The maximal hypotension was 6% after HS-198 and 20% after SUF (P < 0.01). In the electroencephalogram, power in the delta band increased by 288% after HS-198 and by 439% after SUF (P < 0.01); simultaneously, power in the beta band decreased by 71% and by 95.7%, respectively (P < 0.01). PaO(2) decreased by 41% after SUF and by 4% after HS-198, and PaCO(2) increased by 56.8% and 6.6% in SUF and HS-198, respectively (P < 0.001). Both opioids induced a dose-related depression in the somatosensory evoked potential and increased tolerance to skin-twitch. The maximal effect was 92.7% after SUF and 81.3% after HS-198 was not significant. Naltrexone reversed all changes back to control. Compared with SUF, HS-198 does not induce hypoxia and hypercarbia, induces less hypotension and bradycardia, and induces less sedative effects. IMPLICATIONS: Compared with sufentanil, 14-methoxymetopone does not induce hypoxia and hypercarbia, induces less hypotension and bradycardia, and induces less sedative effects (electroencephalogram). Antinociception is similar to sufentanil (skin-twitch method, amplitude depression in the evoked potential). All effects are reversed by naltrexone. Interaction of kappa-receptor is suggested.

Antagonism of heroin and morphine self-administration in rats by the morphine-6beta-glucuronide antagonist 3-O-methylnaltrexone.

In mice, 3-O-methylnaltrexone blocks the analgesic actions of morphine-6beta-glucuronide and heroin at doses which are inactive against morphine. We found a similar selectivity in rats. 3-O-Methylnaltrexone antagonized the analgesic actions of 6-acetylmorphine in Sprague-Dawley rats and heroin in Wistar rats at doses that were inactive against morphine. Inclusion of a fixed dose of 3-O-methylnaltrexone significantly shifted the analgesic dose-response curves for 6-acetylmorphine and heroin without altering the morphine dose-response curves. In a self-administration model, 3-O-methylnaltrexone treatment significantly increased both heroin and morphine intake during the first hour, suggestive of an antagonist effect. This effect at doses of 3-O-methylnaltrexone which were inactive against morphine analgesia implied a role for the morphine-6beta-glucuronide opioid receptor in the reinforcing properties of heroin and morphine.

3-Methoxynaltrexone, a selective heroin/morphine-6beta-glucuronide antagonist.

Recent work has suggested that heroin and morphine-6beta-glucuronide (M6G) both act through a novel mu opioid receptor subtype distinct from those mediating morphine's actions. This very high affinity 3H-M6G site is selectively competed by 3-methoxynaltrexone. In vivo, 3-methoxynaltrexone (2.5 ng, i.c.v.) selectively antagonizes the analgesic actions of heroin and M6G without interfering with mu (morphine and [D-Ala2,MePhe4,Gly(ol)5]enkephalin), delta ([D-Pen2,D-Pen5]enkephalin), kappa1 (U50,488H) or kappa3 (naloxone benzoylhydrazone) analgesia. In dose-response studies, 3-methoxynaltrexone (2.5 ng, i.c.v.) significantly shifted the ED50 values for heroin and its active metabolite, 6-acetylmorphine, without affecting the morphine curve. These results indicate that 3-methoxynaltrexone selectively blocks a novel 3H-M6G binding site which is responsible for the analgesic actions of heroin and M6G. This ability to selectively antagonize heroin actions opens new possibilities in the development of therapeutics for the treatment of opioid abuse.

Differential blockade of morphine and morphine-6 beta-glucuronide analgesia by antisense oligodeoxynucleotides directed against MOR-1 and G-protein alpha subunits in rats.

An antisense oligodeoxynucleotide directed against the 5'-untranslated region of MOR-1 blocks the analgesic actions of the mu 1 analgesics morphine and [D-Ala2,D-Leu5]enkephalin (DADL) when they are microinjected into the periaqueductal gray. In contrast, morphine-6 beta-glucuronide (M6G) analgesia is unaffected by this treatment. Antisense oligodeoxynucleotides directed against distinct Gi alpha subunits also distinguish between morphine and M6G analgesia. A probe targeting Gi alpha 2 blocks morphine analgesia, as previously reported, but is inactive against M6G analgesia. Conversely, an antisense oligodeoxynucleotide against Gi alpha 1 inhibits M6G analgesia without affecting morphine analgesia. The antisense oligodeoxynucleotide directed against G(o)alpha is ineffective against both compounds. These results confirm the prior association of Gi alpha 2 with morphine analgesia and strongly suggests that M6G acts through a different opioid receptor, as revealed by its insensitivity towards the MOR-1 antisense probe and differential sensitivity towards G-protein alpha subunit antisense oligodeoxynucleotides.

Modulation of metabolic effects of morphine-6-glucuronide by morphine-3-glucuronide.

Modification of pharmacological effects of morphine by its glucuronides has been recently reported. Morphine-6-glucuronide (M6G) is a more potent opioid agonist than morphine, whereas morphine-3-glucuronide (M3G) has no opioid effects and has been suggested to be an antagonist of morphine's antinociceptive and respiratory depressive effects. This study addressed the metabolic effects of direct central nervous system administration of M3G and its interaction with the hyperglycemic effects of M6G. Hormonal and whole body glucose metabolic effects of M3G, M6G, and M3G + M6G ICV administration were studied in conscious unrestrained chronically catheterized rats. Whole body glucose kinetics were assessed with a primed constant intravenous infusion of 3[3H]glucose in rats injected intracerebroventricularly (ICV) with H2O (5 microliters), M3G (1 microgram), M6G (1 microgram), or M3G (1 microgram) + M6G (1 microgram). A significant rise in plasma glucose level was observed after ICV injection of M6G (28%), and M3G + M6G (41%), but not after M3G as compared to time-matched H2O control. Early increases in the rate of glucose appearance (Ra) and whole body glucose utilization (Rd) were observed (58% and 48%, respectively) 30 min after M3G + M6G administration, whereas the increases after M6G injection were progressive and reached values 47% higher than basal 180 min after injection. M3G administration enhanced the M6G induced increase in plasma glucose level (+21%), Ra (+29%), Rd (+26%), and plasma lactate level (+21%). Though no significant hormonal change was observed in H2O, M3G, and M6G injected animals, the combination of M3G + M6G resulted in a significant increase in circulating catecholamine levels with no alterations in plasma corticosterone, insulin, and glucagon.(ABSTRACT TRUNCATED AT 250 WORDS)

The excitatory effects of morphine-3-glucuronide are attenuated by LY274614, a competitive NMDA receptor antagonist, and by midazolam, an agonist at the benzodiazepine site on the GABAA receptor complex.

Administration of morphine-3-glucuronide (M3G) by the intracerebroventricular (i.c.v.) route in doses of 2-8 micrograms produced a marked dose-dependent behavioural excitation in adult Sprague-Dawley rats. When LY274614 (1-50 ng i.c.v.) or midazolam maleate (25-50 micrograms i.c.v.) was administered 20 min prior to a maximal excitatory dose of M3G (7 micrograms), all excitatory behaviours were reduced. In contrast, when LY274614 (200 ng i.c.v.) was given after M3G (7 micrograms), it did not reduce all of the excitatory behaviours. Since we have also shown in in vitro binding studies that M3G has very low affinity for the known binding sites on the N-methyl-D-aspartate (NMDA) and the gamma-amino-butyric acid (GABAA) receptor complexes (1), the results of this study suggest that the anti-convulsant compounds, LY274614 and midazolam, are functional antagonists of the excitatory effects of M3G. LY274614 (1-50 ng i.c.v.) and midazolam (25-50 micrograms i.c.v.) did not produce significant behavioural excitation and phenyclidine (PCP)-type effects were not observed. This contrasts with the NMDA receptor antagonists CGS19755 and MK801, where PCP-type effects have been reported to interfere with behavioural assessment. Morphine hydrochloride in a maximal analgesic dose (50 micrograms i.c.v.), did not reduce the excitation score of a maximal excitatory dose of M3G (7 micrograms), lending support to the view that M3G's excitatory effects are elicited through a non-opioid mechanism.

Morphine-6-beta-D-glucuronide respiratory pharmacodynamics in the neonatal guinea pig.

Morphine-6-beta-D-glucuronide (M6G) is a metabolite of morphine with opioid activity in adults. No data are available, however, on the developmental pharmacology of M6G including investigation of the respiratory effects of M6G in the neonate. A randomized, placebo-controlled study comparing the time-action, dose-response and potency of the respiratory effects of M6G to morphine was done using a nonanesthetized neonatal guinea pig model and a noninvasive computerized plethysmograph technique. Respiration was measured while the neonate breathed room air followed by 5% CO2 in air. M6G (0.5-5.0 mg/kg) and morphine (1.5-15 mg/kg) administered subcutaneously decreased ventilation in 3-, 7- and 14-day-old neonatal guinea pigs given a 5% CO2 challenge. During CO2 inhalation, time-to-peak action for M6G occurred 21 min later than for morphine. At maximal ventilatory depression on day 3, a dose of 1.5 mg/kg morphine or M6G decreased minute ventilation while breathing 5% CO2 by 30% compared to placebo. Ventilation also decreased as a function of age in both placebo and drug-treated animals. The percent respiratory depression relative to placebo remained constant for a given dose of morphine as the neonate aged, but not for M6G, which increased in potency. M6G was equipotent to morphine on day 3 after birth, but was 8-fold more potent by day 7. This increase in potency persisted through day 14. The increased potency of M6G that accompanies aging may be caused by either a change in M6G disposition or a change in opioid receptors during development of the neonatal guinea pig.

Intrathecal morphine-3-glucuronide does not antagonize spinal antinociception by morphine or morphine-6-glucuronide in rats.

Morphine or morphine-6-glucuronide either alone or in combination with morphine-3-glucuronide was administered intrathecally to rats. Antinociceptive effects were evaluated with the tail flick and the hot plate tests. Motor function was tested using the rotarod test. Estimated ED50 from the dose-response curves for morphine and morphine-6-glucuronide showed about a 30 times more potent antinociceptive effect of morphine-6-glucuronide compared with morphine. Morphine-3-glucuronide had no antinociceptive effect. Simultaneous administration of morphine-3-glucuronide 5.0 micrograms did not show any significant effect on antinociception induced by morphine 1.0 microgram or morphine-6-glucuronide 0.05 microgram.

Effects of morphine metabolites on micturition in normal, unanaesthetized rats.

1. By means of continuous cystometry in normal, unanaesthetized rats, the effects on micturition of intrathecally (i.t.) administered morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G), the two main metabolites of morphine, were studied and compared with those of i.t. morphine. 2. Both M6G (0.01, 0.1, and 0.5 microgram) and M3G (5 micrograms) were found to have significant effects on micturition. Like morphine (0.1, 0.5, and 10 micrograms), M6G was able to inhibit the micturition reflex, and produce urinary retention and dribbling incontinence in a dose-dependent manner. The potency of M6G for inhibiting micturition was approximately 10 times higher than that of morphine, and the duration of its effect was longer. All effects of M6G could be reversed by naloxone. 3. M3G (5 micrograms) facilitated the micturition reflex, resulting in decreases in bladder capacity and micturition volume, and an increase in spontaneous contractile activity. Pretreatment with naloxone (10 micrograms), which by itself had no effect on micturition, enhanced the facilitatory effects of M3G. In addition, M3G tended to counteract the inhibitory effects of both morphine and M6G on micturition. M3G (5 micrograms) also produced an excitatory behavioural syndrome. 4. It is concluded that in rats, i.t. M3G has excitatory effects on micturition and behaviour, probably not mediated via opioid receptors. I.t M6G has a potent inhibitory effect on micturition mediated by stimulation of opioid receptors. It may have effects on somatosensory afferent input in lower doses than those required for effects on micturition.

Morphine-3-glucuronide may functionally antagonize morphine-6-glucuronide induced antinociception and ventilatory depression in the rat.

The effects of the major morphine metabolites, morphine-3-glucuronide and morphine-6-glucuronide, on nociception were assessed by the tail-flick, hot-plate and writhing tests in the rat. Morphine-3-glucuronide (M3G) 1.1 x 10(-9) mol (0.5 micrograms) or saline was injected intracerebroventricularly (i.c.v.) or intrathecally (i.t.) followed by a second injection of 2.0 x 10(-10) mol (0.1 microgram) or 2.0 x 10(-11) mol (0.01 microgram) morphine-6-glucuronide (M6G) 10 min later. Administration of M3G (i.c.v.) significantly attenuated the antinociceptive effects of M6G in the hot-plate test. After i.t. administration, the antinociceptive effect of M6G in all three tests was significantly reduced in the M3G pretreated group compared to the group receiving saline. The ventilatory effects of 4.0 x 10(-9)-1.0 x 10(-8) mol (2-5 micrograms) M6G and 1.7-2.2 x 10(-8) mol (8-10 micrograms) M3G given i.c.v. were studied by a whole-body plethysmographic technique in halothane anaesthetized rats. Separate groups of rats received M3G followed by M6G injection or vice versa. In animals receiving M3G there was a prevention or attenuation of the M6G induced depression of respiratory frequency, tidal volume and minute ventilation compared to control groups receiving M6G in combination with saline. These results show that M3G may functionally antagonize the central antinociceptive effects as well as the ventilatory depression induced by M6G. Interestingly, M3G was more potent in antagonizing the M6G-induced analgesia after i.t. administration than that after i.c.v. administration, which may suggest that the spinal cord is more sensitive to the non-opioid excitatory effects of M3G than supraspinal structures.

Morphine-3-glucuronide--a potent antagonist of morphine analgesia.

In this study, morphine-3-glucuronide (M3G), the major plasma and urinary metabolite of morphine, was shown to be a potent antagonist of morphine analgesia when administered to rats by the intra-cerebroventricular (i.c.v.) route. The antagonism of morphine analgesia was observed irrespective of whether i.c.v. M3G (2.5 or 3.0 micrograms) was administered 15 mins prior to or 15 mins after i.c.v. morphine (20 micrograms). When M3G (10mg) was administered intraperitoneally (i.p.) to rats 30-40 mins prior to morphine (1.5mg i.p.), the analgesic response was significantly reduced compared to administration of morphine (1.5mg i.p.) alone. It was further demonstrated that i.c.v. M3G (2.0 micrograms) antagonized the analgesic effects of subsequently administered i.c.v. morphine-6-glucuronide (0.25 micrograms).

U50,488H differentially antagonizes the bladder effects of mu agonists at spinal sites.

The mu antagonist property of the kappa agonist U50,488H was studied at the spinal level, using motility of the rat urinary bladder as an endpoint in vivo. Intrathecal (i.th.) administration of the mu agonists [D-Ala2,NMePhe4,Gly-ol]enkephalin (DAGO), [N-MePhe3,D-Pro4]enkephalin (PL017), morphine and normorphine, as well as the delta agonist [D-Pen2,D-Pen5]enkephalin (DPDPE), resulted in an equieffective inhibition of volume-initiated contractions of the urinary bladder. In contrast, i.th. administration of U50,488H, a highly selective kappa agonist, had no effect on bladder motility. Pretreatment of rats with i.th. U50,488H prior to agonist administration, blocked the suppression of spontaneous bladder activity induced by equieffective i.th. does of morphine and normorphine, but failed to alter the inhibitory effect of the mu agonists DAGO and PL017, or that of the delta agonist DPDPE. The finding that U50,488H differentially antagonized the identical bladder effects of several mu agonists suggests the presence of mu receptor subtypes (mu isoreceptors) in the rat spinal cord, which may be involved in the regulation of bladder function.

NMDA receptor antagonist D-APV depresses excitatory activity produced by normorphine in rat hippocampal slices.

Both pentylenetetrazole and normorphine increased CA1 pyramidal cell sensitivity to afferent stimulation and caused the appearance of synchronous afterpotentials (secondary spikes). D-2-Amino-5-phosphonovalerate (D-APV) attenuated secondary spikes induced by both drugs, but had no effect on the change in excitability of the primary population spike. These results suggest that both opiates and GABA receptor antagonists are able to unmask NMDA receptors in the in vitro rat hippocampus slice preparation.