R.G. Hill

κ-Opioid agonists produce antinociception after i.v. and i.c.v. but not intrathecal administration in the rat

1 Nociceptive thresholds to noxious mechanical (paw pressure) and thermal (tail flick) stimuli were recorded in conscious rats. The effects of three selective κ‐opioid receptor agonists on the responses to these stimuli were determined following intravenous, intracerebroventricular or intrathecal administration. Results were compared with those obtained with morphine. 2 Following intravenous administration PD117302, U50488, U69593 and morphine produced steep parallel dose‐response curves indicating antinociceptive activity when evaluated in the paw pressure test. When U50488 and U69593 were tested at a single dose of 3.3 mg kg−1 no effect was seen in the tail flick test. 3 When given by the intrathecal route only morphine was effective at increasing the nociceptive threshold. PD117302, U50488 and U69593 were without effect in either the paw pressure or tail flick tests when tested at doses up to 100 μg per rat. PD117302 caused flaccid paralysis of the hindlimbs following intrathecal administration at the top dose tested. This effect was not reversible by naloxone. 4 All three κ‐opioid receptor agonists produced naloxone‐reversible antinociception in the paw pressure test, and to a lesser extent in the tail flick test, when injected directly into the third cerebral ventricle with the maximum effect occurring between 5 and 10 min after administration and declining back to control levels by 60 min. Morphine had a much slower onset of action with the peak effect being observed 30 min after dosing. 5 It is concluded that, under our experimental conditions in the rat, the antinociceptive effects of κ‐agonists are likely to be operated via an action at a supraspinal rather than a spinal site.

The cholecystokinin receptor antagonist L364,718 increases food intake in the rat by attenuation of the action of endogenous cholecystokinin

1 To determine the role of endogenous cholecystokinin (CCK) in the regulation of food intake, the effects of the potent CCK receptor antagonist L364,718 were investigated on the intake of a palatable diet in non‐deprived rats. The effect of a single dose of proglumide was also investigated for comparative purposes. In addition, the ability of L364,718 to antagonize the reduction in food intake produced by exogenous cholecystokinin‐octapeptide (CCK8) or bombesin in food‐deprived rats was determined. 2 L364,718 (10–100 μg kg−1, i.p.) increased the intake of palatable diet during the 30 min test period. Proglumide (300 mg kg−1, i.p.) also increased the intake of palatable diet. Conversely, CCK8 (0.5–5 μg kg−1, i.p.) produced a reduction in the intake of the diet. 3 In fasted rats, L364,718 (100 μg kg−1, i.p.) antagonized the reduction in food intake produced by CCK8 (10 μg kg−1, i.p.) but not that produced by bombesin (50 μg kg−1, i.p.). L364,718 did not increase food intake in these animals when measured over a 6 h period. 4 It is concluded that L364,718 is a potent, selective antagonist of the effects of CCK8 on food intake. The observation that L364,718 and proglumide increase the intake of a palatable diet provides some evidence that endogenous CCK is involved in the control of food intake in this model.

Pharmacological profile of PD 117302, a selective κ‐opioid agonist

1 PD 117302, a new nonpeptide opioid compound shown in in vitro studies to be a selective κ‐opioid agonist, has been evaluted in vivo for antinociceptive activity and other effects characteristic of κ‐receptor activation. 2 Dose‐related long lasting antinociception was produced by PD 117302 against a mechanical noxious stimulus in rats following intravenous, subcutaneous or oral administration. 3 PD 117302 was effective in raising the nociceptive threshold to mechanical and chemical but not to thermal noxious stimuli in the mouse. This effect was attenuated in animals pretreated with the opioid antagonist naloxone. 4 In addition to producing antinociception, PD 117302 also caused naloxone‐reversible locomotor impairment and diuresis, effects that are typical of κ‐agonists. 5 PD 117302 did not cause respiratory depression, inhibition of gastrointestinal motility or naloxone‐precipated withdrawal jumping in mice, effects that are associated with actions at the μ‐opioid receptor. 6 The pharmacological profile of PD 117302 in vivo is consistent with in vitro data suggesting that PD 117302 is a selective agonist at the κ‐opioid receptor.

Antinociceptive action of cholecystokinin octapeptide (CCK 8) and related peptides in rats and mice: Effects of naloxone and peptidase inhibitors

Cholecystokinin octapeptide (CCK 8) produced significant antinociception in the tail immersion test in the rat, paw pressure test in the rat and acetylcholine-induced writhing test in the mouse after subcutaneous (s.c.) administration. In the hot plate test, CCK 8 (s.c.) showed antinociceptive activity if the latency to lick was used as the end point but if the latency to jump was recorded the antinociceptive effects were not evident. Cholecystokinin tetrapeptide (CCK 4) was shown to be antinociceptive in the writhing but not in the hot plate test after subcutaneous administration and appeared to be less potent than CCK 8 when tested under the same conditions. Antinociception induced by CCK 8 in the hot plate test (lick) could also be demonstrated after direct intracerebroventricular (i.c.v.) injection and this observation was also made with the CCK-related peptide FMRF amide. Antinociception induced by CCK 8 (which did not appear to be associated with reduced locomotor activity) was evident 5 min after intraventricular injection and was maximal at 10 min, the effect lasting over a 30-45 min period. The antinociceptive effect of CCK 8 was antagonised by naloxone and was potentiated by simultaneous administration of the peptidase inhibitors bestatin, thiorphan and captopril.

Rat hippocampal slices in vitro display spontaneous epileptiform activity following long-term organotypic culture

Organotypic cultures of rat hippocampal slices were maintained for periods of up to 12 weeks in vitro. Cultures adopted a two-dimensional architecture whilst retaining the subfields characteristic of intact hippocampal slices. Coventional intracellular onset of spontaneous long-lasting epileptiform activity. Epileptiform activity characteristic of both interictal and ictal events (paroxysmal depolarising shifts, tonic/clonic phases and afterdischarges) was observed in the absence of pharmacological manipulation or of orthodromic stimulation. Epileptiform activity was abolished in the presence of high Mg2+ concentration or tetrodotoxin, agents known to block synaptic transmission. In addition, the frequency of epileptiform events was independent of membrane potential and the amplitude of the paroxysmal depolarising shift (PDS) displayed a near linear relationship with membrane potential. The PDS could be reversed at potentials approaching synaptic equilibrium potential. The N-methyl-D-aspartate (NMDA)-receptor antagonist DL-2-amino-5-phosphonovalerate (DL-APV) dose-dependently reduced both the amplitude and duration of the spontaneous paroxysmal shift, having no effect on the initiation of the event or the resting membrane parameters of the neurone. DL-APV also attenuated a late component of the synaptically evoked excitatory postsynaptic potentials (epsp) not observed in non-epileptiform neurones. Application of GABAA receptor antagonists bicuculline or picrotoxin converted interictal events to ictus. In the presence of these agents, ictal events were up to 90 s in duration. These results suggest that long-term culturing of hippocampal explants leads to an alteration in the balance of excitatory and inhibitory synaptic activity. This allows the expression of an excitatory amino acid depolarisation acting through NMDA receptors which contributes to the generation and maintenance of spontaneous epileptiform activity which is synaptic in origin.

PD117302: a selective agonist for the κ‐opioid receptor

1 A new nonpeptide κ‐opioid compound, a cyclohexyl benzeneacetamide derivative (PD117302), has been synthesized and its affinity for the different types of opioid receptor determined. The ability of PD117302 to modify the activity of the electrically‐stimulated guinea‐pig ileum and rabbit vas deferens has also been evaluated. 2 In binding studies using guinea‐pig brain homogenates, unlabelled PD117302 had a high affinity (Ki = 3.7 nm) at [3H]‐etorphine labelled κ sites and a low affinity at [3H]‐[d‐Ala2, MePhe4, glyol5]‐enkephalin ([3H]‐DAGOL) labelled μ sites (Ki = 408 nm) and [3H]‐SKF 10047 labelled s sites (Ki = 1.8 μm). In bioassay studies, PD117302 was a potent agonist, producing a maximum inhibition of the electrically‐evoked contractions of the guinea‐pig ileum (IC50 = 1.1 nm) and rabbit vas deferens (IC50 = 45 nm) which was naloxone‐reversible. 3 In guinea‐pig brain, [3H]‐PD117302 bound to a high‐affinity opioid binding site with a KD of 2.7 nm and a Bmax of 3.4 pmol g−1 wet weight. The Bmax was found to be less than 50% of the Bmax values for [3H]‐etorphine and [3H]‐bremazocine suggesting that [3H]‐PD117302 may be a specific ligand for a subtype of κ receptor. [3H]‐PD117302 also bound with micromolar affinity to a non‐opioid binding site. 4 Kinetic studies found that [3H]‐PD117302‐specific binding to the high affinity site was saturable, reaching equilibrium within 20 min at 4°C, and reversible, with a half‐life of dissociation of 3.9 min. 5 Several unlabelled compounds with high affinities for the [3H]‐etorphine labelled κ binding site, had comparable affinities when competing for the [3H]‐PD117302‐specific high affinity binding site. In contrast, DAGOL, [d‐Ala2, d‐Leu5] enkephalin (DADLE) and [d‐Pen2, d‐Pen5] enkephalin (DPDPE) had no significant effect on [3H]‐PD117302 binding, suggesting minimal interaction with μ and δ binding sites. 6 In autoradiography studies [3H]‐PD117302 binding sites were found throughout the brain with the greatest density in the striatum, cerebral cortex (layers V‐VI), substantia nigra, and the molecular layer of the cerebellum. Lowest levels were found in the granular layer of the cerebellum, thalamus and cerebral cortex (layers I‐IV).

Effects of cholecystokinin and related peptides on neuronal activity in the ventromedial nucleus of the rat hypothalamus

1 An investigation into the effects of cholecystokinin octapeptide (CCK‐8S) and its pentapeptide analogue, pentagastrin, on neurones located in ventromedial nuclei of rat hypothalamic slices maintained in vitro has been undertaken. 2 CCK‐8S (0.01‐1.0 μm) applied in the perfusion medium produced a concentration‐dependent increase in firing rate. This effect could be mimicked by pentagastrin and was selectively blocked by L‐364,718, a potent peripheral CCK receptor antagonist that has been shown to possess micro‐molar affinity for central CCK receptors. 3 Intracellular recordings from ventromedial nucleus neurones revealed two distinct populations with comparable resting membrane parameters but differing neuronal activity. One group fired tetrodotoxin (TTX)‐sensitive action potentials spontaneously at resting membrane potential whilst the second group fired action potentials only on injection of depolarizing current and were otherwise silent. 4 Application of CCK‐8S or pentagastrin to spontaneously active neurones produced a small depolarization concomitant with an increase in action potential firing rate but the peptides had no effect on membrane properties of ‘silent’ neurones. 5 These data suggest the existence of at least two populations of neurones in the ventromedial hypothalamus, only one of which is excited by CCK‐8S and pentagastrin.

Actions of the GABAB agonist, (−)‐baclofen, on neurones in deep dorsal horn of the rat spinal cord in vitro

1 The electrophysiological actions of the GABAB agonist, (−)‐baclofen, on deep dorsal horn neurones were studied using an in vitro preparation of the spinal cord of 9–16 day old rat. 2 On all neurones tested, (−)‐baclofen (100nM‐30μm) had a hyperpolarizing action which was associated with a reduction in apparent membrane input resistance. The increase in membrane conductance was dose‐dependent and had a Hill coefficient of 1.0. 3 The (−)‐baclofen‐activated hyperpolarization persisted in the presence of bicuculline (50 μm) and Mg2+(20 mm). 4 The reversal potential of the hyperpolarizing event was estimated at 102 mV and was made less negative by increasing the external concentration of potassium ions. 5 Over the same concentration range, (−)‐baclofen also depressed the polysynaptic composite excitatory postsynaptic potentials (e.p.s.ps) evoked in these neurones by electrical stimulation of the dorsal root entry zone. 6 The potassium channel blockers caesium, applied intracellularly, and barium, applied extracellularly, depressed the postsynaptic response to baclofen but not its effect on e.p.s.ps. 7 We propose that (−)‐baclofen has more than one mechanism of action in spinal dorsal horn: a postsynaptic action mediated via an increase in potassium conductance and a presynaptic action that is not associated with potassium channels and may be mediated via calcium channels. Since previous studies have demonstrated little effect of (−)‐baclofen on transmitter release in spinal cord, it is possible that the postsynaptic hyperpolarizing action of (−)‐baclofen may account for its clinical potency as an anti‐spastic agent.

Ketanserin antagonises the anorectic effect of DL-fenfluramine in the rat

To determine the role played by 5-HT2 receptors in the anorectic action of DL-fenfluramine, the ability of the selective 5-HT2 receptor antagonist ketanserin to block the reduction in food intake produced by this drug was investigated in non-deprived rats. Ketanserin (1 and 2.5 mg/kg i.p.) produced a dose-dependent antagonism of the anorectic effect of DL-fenfluramine (3 mg/kg i.p.). Prazosin (1 mg/kg i.p.) did not antagonise this effect. It is concluded that the anorectic actions of DL-fenfluramine are mediated via 5-HT2 receptors.

Correlation of ontogeny with function of [3H]U69593 labelled κ opioid binding sites in the rat spinal cord

In this study, we have used a variety of in vitro and in vivo techniques to demonstrate the presence, and examine the function, of [3H]U69593 binding sites in the spinal cord of the 9-16-day-old rat in comparison to the adult. Equilibrium binding of [3H]U69593 to homogenates of adult rat spinal cord revealed a single population of non-interacting sites with a maximum binding capacity of 10.4 +/- 1.4 fmol/mg protein and an apparent equilibrium dissociation constant of 2.31 +/- 0.47 nM while in 9-16-day-old cord these parameters were 57.0 +/- 9.4 fmol/mg protein and 2.28 +/- 0.22 nM, respectively. The total binding capacity per cord was 95.8 +/- 8.3 and 121.8 +/- 7.7 fmol/cord for adult and immature rat, respectively. Competition studies using receptor-selective opioid ligands showed that these sites were kappa opioid in nature. Autoradiographical techniques demonstrated a uniform distribution of these sites over transverse sections of 9-16-day-old rat cord. In vitro electrophysiology was performed on spinal cord slice preparations from the 9-16-day-old rat. U69593 (100 nM-1 microM) had no effect on passive membrane properties but produced a naloxone-reversible depression of both spontaneous and electrically evoked activity in dorsal horn neurones. Direct intrathecal injection of U69593 (0.3-10.0 micrograms/animal) into 9-16-day-old rats produced a dose-dependent, naloxone-reversible, antinociception when measured using the paw-pressure test. In conclusion, we have shown that, in contrast to the adult, the spinal cord of the 9-16-day-old rat has a significantly higher concentration of [3H]U69593 binding sites which have functional in vitro and in vivo correlates.