Reversal of the antinociceptive effects of centrally-administered morphine by the benzodiazepine receptor antagonist Ro 15-1788

The effects of the benzodiazepine receptor antagonist, Ro 15-1788, were examined on analgesia induced by morphine after central (intracerebroventricular, i.c.v., or intrathecal, i.t.) and systemic administration. Analgesia was assessed in squirrel monkeys trained to respond under an electric shock tiltration procedure and in mice using the radiant heat tail-flick test. Central and systemic administration of morphine produced antinociceptive effects that were antagonized by 0.1 mg/kg of naloxone in both species. Ro 15-1788 antagonized the effects of morphine after central (i.c.v. or i.t.) administration but did not alter the effects of morphine given by the systemic route. This novel interaction suggests that Ro 15-1788 may be useful in pharmacologically separating neural substrates subserving opiate analgesia.     

Central mu, delta- and kappa-opioid influences on intestinal water and electrolyte transport in dogs

The effects of intracerebroventricular (i.c.v.) administration of opioid peptides with mu-(DAGO), mu- and delta-(DALAMIDE, DADLE) and kappa-(dynorphin) properties on normal and stimulated (cholera toxin) net fluxes of water, Na+ and K+ through a jejunal Thiry-Vella loop were investigated in conscious dogs. Basal net water absorption was slightly, but significantly (P less than 0.05) increased during i.c.v. infusion of DALAMIDE or DAGO (0.5 ng/kg/min) but not DADLE and dynorphin-(1-13) at the same rate; DALAMIDE and DAGO also markedly reduced (by 72.3 and 79.5% respectively) the secretory effects of cholera toxin (0.4 micrograms/ml). Similar effects were obtained with DALAMIDE and DAGO when injected i.c.v. as a bolus (100 ng/kg) prior to cholera toxin infusion; they were suppressed after i.v. pretreatment with naltrexone (0.3 mg/kg) but also with propranolol (0.2 mg/kg). In contrast, i.v. phentolamine (0.2 mg/kg) and bilateral truncal vagotomy, were unable to block their effects. These results suggest that Met-enkephalin can act centrally to affect intestinal transport of (i) water and (ii) electrolytes in dogs. They act probably at central mu-receptors which are involved in the regulation of intestinal secretion mediated through a central or peripheral beta-adrenergic pathway.     

Enhancement of colonic motor response to feeding by central endogenous opiates in the dog

The role of endogenous opiates in the colonic motor response to feeding has been investigated in four dogs chronically fitted with two strain gages on the proximal and distal colon and a cannula in cerebral lateral ventricle. A daily meal stimulated the colonic motility during 8-10 hrs. The colonic motility index was significantly higher during this period when an enkephalinase inhibitor, tiorphan, was intracerebroventricularly (i.c.v.) administered at a dose of 0.1 mg/kg before the meal. This effect was blocked by a previous i.c.v. administration of naltrexone (0.1 mg/kg) and reproduced by (D-Ala2, Met5) enkephalinamide (DALAMIDE) at a dose of 50 mg/kg. I.c.v. administration of tiorphan or DALAMIDE did not modify the colonic motility in dogs fasted for 48 hrs. The postprandial motility index remained unchanged after intravenous administration of tiorphan or DALAMIDE at the same dosages. These results provide evidence for a central control of the colonic motor response to feeding by endogenous enkephalins in dogs.     

Differential antagonism of mu agonists by U50,488H in the rat

Agonist, and antagonist effects of the proposed kappa opioid agonist, U50,488H (U50) have been studied in an experimental model of seizure activity (flurothyl-induced seizure threshold) (ST) and in the central modulation of spontaneous, volume-induced micturition contractions (bladder motility) (BM) in rats. Intracerebroventricular (i.c.v.) administration of U50 (at the doses tested) did not produce any agonist effect in either ST or in BM. In contrast, i.c.v. administration of [D-Ala2, NMPhe4, Gly-ol]enkephalin (DAGO) or etorphine, agonists with activity at mu opioid receptors, produced an elevation of ST and inhibition of BM. The elevation in ST produced by etorphine (0.004 nmol) was prevented by prior treatment with U50. In contrast, the approximately equieffective elevation in ST resulting from DAGO was not affected by U50 pretreatment. Similarly, pretreatment of rats with U50 antagonized the approximately equieffective BM effects of etorphine, but not those of DAGO. As both DAGO and etorphine are thought to exert their effects via the opiate mu receptor, the results may be consistent with the view that subpopulations of mu receptors exist within the central nervous system; these sites may be differentially associated with the kappa receptor.     

The effect of a benzodiazepine antagonist, RO15-1788, in diazepam dependent rats

A benzodiazepine antagonist, RO15-1788, was administered intragastrically to diazepam-dependent gastric fistula rats and a precipitated abstinence syndrome was observed. The intensity of the RO15-1788 precipitated abstinence syndrome, calculated by the Precipitated Abstinence Scale, increased in intensity in a log-dose manner over a dose range of 1.0 to 15.0 mg/kg of RO15-1788 and plateaued at the 15.0 mg/kg dose. The RO15-1788 precipitated diazepam abstinence syndrome differed both qualitatively and quantitatively from the diazepam withdrawal syndrome.     

Potentiation of opioid analgesia by cocaine: the role of spinal and supraspinal receptors.

These studies examined the effect of cocaine on the analgesia produced by systemically and centrally administered opioid agonists. Cocaine (50 mg/kg, s.c.) increased the analgesic potency of systemic, ICV and IT morphine; and the ICV and IT analgesic effects of the delta selective peptide, [D-Pen2,D-Pen5]enkephalin (DPDPE). Cocaine also increased the analgesic potency of the mu selective ligand [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAGO) administered ICV. However, cocaine did not alter the ED50 for IT DAGO. GC-MS studies indicated that brain cocaine concentration was approximately 3.0 micrograms/g wet weight 45 min following s.c. administration. These results suggest that cocaine-induced increases in opioid analgesic potency are mediated at brain mu and delta receptors and spinal mu receptors. Furthermore, there might be functional differences between spinal and supraspinal sites at which DAGO produces analgesia.     

Ketazocines and morphine: effects on gastrointestinal transit after central and peripheral administration

The mu agonist, morphine, and the prototype kappa agonists, ketocyclazocine and ethylketocyclazocine (EK), were studied for their effects on gastrointestinal transit. Following s.c. administration, both morphine (0.3-3 mg/kg) and ketocyclazocine (0.3-10 mg/kg) antagonized transit of an opaque marker through the small intestines of mice. Morphine (0.1-1 microgram) was also effective after intracerebroventricular (icv) administration in mice whereas ketocyclazocine (0.3-30 micrograms) was not. Similarly, while both morphine (0.3-5 mg/kg) and EK (0.6-10 mg/kg) slowed transit after s.c. injection to rats, only morphine (1-10 micrograms), but not EK (0.3-300 micrograms), was active following icv administration. Icv infusion of the mu benzomorphan, phenazocine (10-100 micrograms), slowed transit in a dose-related manner. These results indicate that there may be an anatomically distinct distribution of receptors for benzomorphan kappa agonists in both the mouse and rat, with these opiate receptors not being located near the lateral cerebral ventricles. The difference in efficacy between morphine and ketazocines in slowing gastrointestinal transit after icv administration to rodents suggests that (a) inactivity in this endpoint is a characteristic of benzomorphan kappa compounds and (b) the model may serve as a useful screen when establishing in vivo profiles of kappa agonists in mice and rats.     

Naloxone blocks 'anxiolytic' effects of neuropeptide Y

Intracerebroventricular injection of neuropeptide Y (NPY) produces potent 'anxiolytic' effects in animal models of anxiety. Administration of opioid receptor antagonists suppresses NPY-induced food intake and thermogenesis. The present study examined whether the opiate antagonist naloxone would also suppress the 'anxiolytic' effects of neuropeptide Y. Following training and stabilization of responding in an operant conflict model of anxiety, rats were injected with either NPY or diazepam. Both NPY (veh., 2, 4, 6 microg, i.c.v.) and chlordiazepoxide (veh., 2, 4, 6 mg/kg, i.p.) produced a dose-dependent increase in punished responding in the conflict test. The 'anxiolytic' effects of NPY were not blocked by the administration of flumazenil (3, 6, 12 mg/kg, i.p.). The administration of naloxone (0.25-2.0 mg/kg, s.c.) antagonized the effects of NPY. Central administration of the selective mu opiate antagonist CTAP (1 microg, i.c.v.) partially blocked NPY-induced conflict responding. These results support the hypothesis that NPY may play an important role in experimental anxiety independent of the benzodiazepine receptor and further implicate the opioid system in the behavioral expression of anxiety.     

Reversal of the anti-conflict action of valproate by various GABA and benzodiazepine antagonists

The effects of RO 15-1788, RO 5-3663, picrotoxin and bicuculline on the anti-conflict properties of valproate were studied in rats using a modified Vogel 's conflict test procedure. A low dose of the benzodiazepine (BDZ) antagonist, RO 15-1788 (5 mg/kg), blocked the anti-punishment properties of valproate (400 mg/kg), whereas no antagonism was observed after a high dose (25 mg/kg) of the BDZ antagonist. High doses of RO 5-3663 or picrotoxin also reversed the anti-conflict action of valproate. Bicuculline did not change the effects of valproate in this test situation. The suppressive effect of valproate on locomotor activity was reversed by a low dose (5 mg/kg) of RO 15-1788, but not by the other antagonists. RO 5-3663 was the only antagonist which effectively reversed the muscle relaxant effects of valproate observed in a Rotarod performance test. These findings indicate that various pharmacological actions of valproate may be due to a complex interplay with several sites at the GABA-BDZ-receptor complex.     

Effect of withdrawal of diazepam or morphine treatment on gastric motility (charcoal meal test) in mice: possible role of different central and peripheral receptors

Increased gastrointestinal motility in mice as one of the withdrawal symptoms of commonly abused drugs like diazepam or morphine and its possible mechanism of action was studied. Male Laka mice (20-25 g) were made addict to either diazepam (20 mg/kg, ip for 7 days) or morphine (10 mg/kg, sc for 9 days). Withdrawal symptoms were noted 24 hr after the last injection of diazepam or morphine. The animals were injected with Ro 15-1788 (flumazenil) (1 mg/kg, ip) or naloxone (2 mg/kg, ip) in the respective group to precipitate the withdrawal symptoms. Gastrointestinal motility was assessed by charcoal-meal test. Animals developed tolerance to acute sedative effect of diazepam, and similarly to the acute nociceptive action of morphine. On abrupt cessation of these drugs after chronic treatment the animals showed hyperlocomotion and hyperreactivity in diazepam withdrawal group and hyperalgesia on hot plate in morphine withdrawal groups, respectively. Increase in gastrointestinal motility was observed in all the drug withdrawal groups. Treatment with respective antagonists, Ro 15-1788 (flumazenil) and naloxone precipitated the withdrawal symptoms. The results suggest the involvement of both central and peripheral receptors of benzodiazepines and opioid (mu) receptors in the withdrawal symptoms of the benzodiazepines and morphine, respectively.     

Endothelin-1-induced nociception

Intracerebroventricular (i.c.v.) or intrathecal (i.t.) administration of morphine to mice antagonized the abdominal constriction induced by an i.p. injection of endothelin-1 (ET-1; 0.1 mg/kg). The ED50 values (95% confidence intervals) were 39.3 (16.5-80.2) ng and 1.5 (0.8-4.9) ng, respectively. The antagonism of ET-1-induced abdominal constriction by morphine was blocked by naloxone (1.0 mg/kg, s.c.) or by 24 h pretreatment with beta-funaltrexamine (beta-FNA; 8.84 micrograms, i.c.v.). These results demonstrate for the first time that the stimulus resulting from an i.p. injection of ET-1 is transmitted via ascending (pain) pathways that are subject to attenuation by opioid (mu) receptor activation. Hence, ET-1-induced abdominal constriction is a new pain model which, given the other pharmacology of ET-1, might represent a unique model with potential specific utility for anginal or other visceral pain.     

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).     

Opposite effects of two ligands for peripheral type benzodiazepine binding sites, PK 11195 and RO5-4864, in a conflict situation in the rat

The effects of two drugs acting at the peripheral type benzodiazepine binding sites, PK 11195 and RO5-4864, were examined in shock-induced suppression of drinking in rats. These two compounds have opposite effects : RO5-4864 (3.1-1205 mg/kg i.p.) enhanced whereas PK 11195 (25-50 mg/kg i.p.) decreased the punished responding, and PK 11195 (6.25 mg/kg, a dose which did not alter the punished responding) blocked the proconflict action of RO5-4864 (6.25 and 12.5 mg/kg). The effects of RO5-4864 and PK 11195 were not antagonized by RO15-1788, a selective antagonist of the central benzodiazepine site. In addition, PK 11195 (6.25 mg/kg) did not reverse the proconflict effect of two beta-carbolines : beta-CEE and FG 7142. AS picrotoxin did not change the punished responding, these data imply that the effects of RO5-4864 and PK 11195 on the one hand and those of chlordiazepoxide and beta-carbolines on the other hand are differentially mediated and suggest that the peripheral type benzodiazepine binding sites are involved in this conflict model.     

Estimation of the affinity of naloxone at supraspinal and spinal opioid receptors in vivo: studies with receptor selective agonists

The apparent affinity of naloxone at cerebral and spinal sites was estimated using selective mu [D-Ala2, Gly-o15]-enkephalin (DAGO) and delta [D-Pen2, D-Pen5]enkephalin] (DPDPE) opioid agonists in the mouse warm water tail-withdrawal test in vivo; the mu agonist morphine was employed as a reference compound. The approach was to determine the naloxone pA2 using a time-dependent method with both agonist and antagonist given intracerebroventricularly (i.c.v.) or intrathecally (i.th.); naloxone was always given 5 min before the agonist. Complete time-response curves were determined for each agonist at each site in the absence, and in the presence, of a single, fixed i.c.v. or i.th. dose of naloxone. From these i.c.v. or i.th. pairs of time-response curves, pairs of dose-response lines were constructed at various times; these lines showed decreasing displacement with time, indicative of the disappearance of naloxone. The graph of log (dose ratio-1) vs. time was linear with negative slope, in agreement with the time-dependent form of the equation for competitive antagonism. From this plot, the apparent pA2 and naloxone half-life was calculated at each site and against each agonist. The affinity of naloxone was not significantly different when compared between agonists after i.c.v. administration. A small difference was seen between the affinity of i.th. naloxone against DPDPE and DAGO; the i.th. naloxone pA2 against morphine, however, was not different than that for DPDPE and DAGO. The naloxone half-life varied between 6.6 and 16.9 min, values close to those previously reported for this compound. These results suggest that the agonists studied may produce their i.c.v. analgesic effects at the same receptor type or that alternatively, the naloxone pA2 may be fortuitously similar for mu and delta receptors in vivo. Additionally, while the affinity of naloxone appears different for the receptors activated by i.th. DAGO and DPDPE, further work may be necessary before firm conclusions regarding the nature of the spinal analgesic receptor(s) can be drawn.     

Comparison of the discriminative stimulus effects of midazolam after intracranial and peripheral administration in the rat.

Rats were trained in a two-lever drug discrimination paradigm to discriminate midazolam (0.32 mg/kg, i.p. or 1.0 mg/kg, i.p.) from the no-drug condition. After completion of i.p. and s.c. midazolam generalization gradients (0.032-1.0 mg/kg), rats were surgically implanted with unilateral cannulae into the lateral ventricles. Intracerebroventricular (i.c.v.) doses of 1.1-44.2 micrograms midazolam were delivered to unrestrained rats. Midazolam produced dose-dependent increases in drug-appropriate responding by all three routes of administration, but was 2.4- to 4.3-fold more potent when given i.c.v. than when given s.c. or i.p. Midazolam, over the dose range tested, did not produce substantial decreases in response rate by any route of administration. The discriminative-stimulus effect of i.c.v. midazolam was blocked by peripherally administered flumazenil, and such antagonism was surmounted by a 2- to 5-fold increase in the i.c.v. midazolam dose. Taken together, these data suggest that the discriminative-stimulus effects of midazolam are mediated via central benzodiazepine (BZ) receptors.     

Alteration of brain stem auditory evoked potentials after intracerebroventricular administration of met-enkephalin in rabbits.

Brainstem auditory evoked potentials (BAEPs) were elicited by binaural click stimulation and recorded from the rabbits with chronically implanted electrodes and a cannula for intracerebroventricular injection (i.c.v.). 400 BAEPs were averaged off line. The registration was carried out before and after i.c.v. injection of met-enkephalin (2.5 or 25 nmol), naloxone (20 micrograms), or i.v. injection of morphine (1.0, 2.0, 5.0 mg/kg b.w.). Enkephalin caused shortening of interpeak latency time, naloxone caused its lengthening, while the effect of morphine was not unidirectional. Enkephalin caused increase in the surface area below the negative peaks located in the range of 4.5-7.5 ms from the first positive peak, naloxone caused its decrease while the effect of morphine was also in this respect not unidirectional. It is concluded that opiate receptors are involved in the modulation of the auditory brainstem responses.     

Effects of morphine on hexamethonium-sensitive and -resistant excitatory responses of stomach to stimulation of vagal trunk in cats

Electrical stimulation of the vagal trunk with 10 Hz in frequency, 3 ms in duration and 15 volt in intensity for 10 s in cats produced an excitatory response of the stomach and the response was composed of two phases, an initial rapid excitation during stimulation period and the late multi-peak response after stimulation period. The initial response was inhibited by the administrations of hexamethonium (10 mg/kg, i.v.) and atropine (100 micrograms/kg, i.v.). The late response was not inhibited by hexamethonium but was inhibited by atropine (100 micrograms/kg, i.v.). The hexamethonium-sensitive initial excitation was not affected by the administration of morphine and gamma-aminobutyric acid (GABA). On the other hand, the hexamethonium-resistant late response was attenuated by the treatment with morphine (1 to 10 mg/kg, i.v.) and GABA (100 to 500 micrograms/kg, i.v.). Such inhibitory actions of morphine and GABA on the late response were antagonized by picrotoxin. From these results, it was concluded that morphine might inhibit specifically the hexamethonium-resistant late excitatory response of the stomach without affecting the hexamethonium-sensitive initial excitatory response and the inhibitory effect of morphine on the late response of stomach might be due to action of GABA released from the intramural neurons of gastric walls in cats.     

Behavioral effects and benzodiazepine antagonist activity of Ro 15-1788 (flumazepil) in pigeons

The selective benzodiazepine receptor antagonist, Ro 15-1788, produced behavioral effects in pigeons at doses at least 100 times lower than those previously reported to possess intrinsic pharmacological activity in mammals. In contrast to its effects in mammalian species, in pigeons, Ro 15-1788 does not exhibit partial agonist activity. Key-peck responses of pigeons were studied under a multiple fixed-interval 3-min, fixed-interval 3-min schedule in which the first response after 3-min produced food in the presence of red or white keylights. In addition, every 30th response during the red keylight produced a brief electric shock (punishment). Under control conditions, punished responding was suppressed to 30% of unpunished response levels. Ro 15-1788 (0.01 mg/kg, i.m.) increased unpunished response rates by 33% without affecting rates of punished responding. Doses of 0.1 to 1.0 mg/kg Ro 15-1788 produced dose-related decreases in both punished and unpunished responding. As is characteristic of other benzodiazepines, midazolam (0.1 and 0.3 mg/kg, i.m.) markedly increased punished responding but had little effect on rates of unpunished responding. Ro 15-1788 antagonized the increases in punished responding and also reversed the rate-decreasing effects of higher doses of midazolam. However, the effectiveness of Ro 15-1788 as a benzodiazepine antagonist was limited by its intrinsic activity: rate-decreasing doses of Ro 15-1788 were unable to completely reverse behavioral effects of midazolam. Midazolam was an effective antagonist of the behavioral effects of Ro 15-1788 (up to 0.1 mg/kg) but midazolam did not influence the rate-decreasing effects of 1.0 mg/kg Ro 15-1788 across a 100-fold dose range. In the pigeon, the behavioral effects of relatively low doses of Ro 15-1788 (0.01-0.1 mg/kg) appear to be related to benzodiazepine receptor mechanisms, whereas other systems appear to be involved in the effects of higher doses.     

Effects of Opiates on High-Affinity Ca2+,Mg2+-ATPase in Brain Membrane Subfractions

The effect of a single administration of morphine sulfate (15 mg/kg, s.c. or 30 mg/kg, i.p., 30 min) on Ca2+-stimulated Mg2+-dependent ATPase activity was investigated in synaptosomal plasma membranes (SPM) prepared from rat cortex. Morphine produced a significant decrease in Ca2+,Mg2+-ATPase activity in synaptosomal fractions (SPM 1 + 2) known to contain a high density of opiate receptors and calmodulin-dependent Ca2+,Mg2+-ATPase. However, in another subpopulation (SPM 3) that contains fewer opiate receptors and less enzyme activity, no such decrease in the enzyme activity was observed after the opiate administration. The decrease in Ca2+,Mg2+-ATPase activity seen in SPM 1 + 2 was specifically antagonized by the opiate antagonist naloxone hydrochloride (2 mg/kg, s.c.) when given 15 min before morphine administration. Mg2+-ATPase was not altered either by morphine or by a naloxone-morphine combination. These findings give further evidence for the role of intracellular Ca2+ in mediating many of the acute effects of opiates.     

Prolonged in vivo antagonism of central mu- and delta-opioid receptor activity by beta-funal trexamine

beta-Funaltrexamine (beta-FNA) was tested in the spinal cord and supraspinally against inhibition of reflex bladder contractions produced in the anesthetized rat by the opioid-receptor selective agonists [D-Ala2, MePhe4, Gly (ol)5]enkephalin (DAGO, mu-agonist) and [D-Pen2, D-Pen5] enkephalin (DPDPE, delta-agonist). All agents were microinjected either intracerebroventricularly (i.c.v.) or intrathecally (i.t.). beta-FNA (1-8 micrograms) produced long-lasting antagonism of both DAGO and DPDPE. Complete recovery from its effects was only observed some 24-32 h later. Higher doses of beta-FNA (4 and 8 micrograms i.t.) produced short-lived agonistic activity though the selectivity of this was not determined. It was concluded that beta-FNA was a potent, long-lasting antagonist at central opioid receptors in vivo but was unselective for the mu and delta opioid receptor.