Concurrent chronic administration of a CCK(B) antagonist can decrease tolerance to the ataxic effects of ethanol.

The effects of chronic administration of a CCK(B) antagonist, CAM1028, were examined on the development of tolerance to ethanol and the appearance of withdrawal signs. Ethanol was given chronically by liquid diet, and none of the dose schedules of CAM1028 affected the amount of ethanol taken in during the chronic treatment. Brain ethanol concentrations were not altered by the administration of CAM1028.The ataxic effects of ethanol were tested on a rotorod, 3 hours after cessation of the ethanol administration, and the last injections of CAM1028 were given 8 hours before withdrawal from the ethanol treatment. When administered at 0.03, 0.1 or 0.3 mg/kg, CAM1028 decreased the extent of tolerance development. Higher doses, 1 and 3 mg/kg, did not alter the tolerance development. Chronic administration of CAM1028 had a small effect in protecting against the effects of ethanol withdrawal that, in contrast to the effects on the tolerance, was seen only at the highest dose, 10 mg/kg, of CAM1028.

Kappa-opioid receptor agonists increase locomotor activity in the monoamine-depleted rat model of parkinsonism.

Excessive glutamate transmission in the basal ganglia is a major factor in the neural mechanisms underlying parkinsonian akinesia. Activation of kappa opioid receptors causes a presynaptic reduction in glutamate release. Kappa opioid receptors are concentrated in those regions of the basal ganglia associated with increased glutamate transmission in parkinsonism. In this study, we use the alpha-methyl-p-tyrosine and reserpine-treated rat model of parkinsonism to investigate whether systemic administration of the kappa opioid agonists enadoline (CI-977) and U69,593 can alleviate the symptoms of parkinsonism either alone or in conjunction with dopamine replacement therapy. We report that, when administered alone, both enadoline and U69,593 can increase locomotion in monoamine-depleted rats. No increase in locomotor activity was seen after kappa opioid agonist administration in non-parkinsonian rats. The responses to kappa opioid agonists were blocked by co-administration of either the nonspecific opioid receptor antagonist naloxone or the selective kappa opioid receptor antagonist nor-binaltorphimine (nor-BNI). An important finding is that when enadoline and L-dopa are administered together, their anti-akinetic properties are synergistic. Thus, the doses of enadoline and L-dopa required to alleviate akinesia when administered together are lower than either administered alone. These data illustrate the importance of kappa opioid receptors in the neural mechanisms controlling voluntary movement and suggest that kappa opioid agonists may have a role as adjuncts to dopamine replacement in the management of Parkinson's disease.

Spermine modulation of specific [3H]-gabapentin binding to the detergent-solubilized porcine cerebral cortex alpha 2 delta calcium channel subunit.

1. Recent studies have identified the [3H]-gabapentin-binding protein, purified from porcine cerebral cortical membranes, as the alpha 2 delta subunit of voltage-sensitive calcium channels (Gee et al., 1996). The present study investigates the influence of the polyamine spermine on specific [3H]-gabapentin binding to detergent-solubilized porcine cerebral cortical membranes. 2. Spermine, spermidine, 1,10 diaminodecane, Mg2+ and Zn2+, all divalent cations, displaced [3H]-gabapentin binding to detergent-solubilized membranes in a concentration-dependent manner with a maximal inhibition of 65-75%. Radioligand binding studies showed that spermine did not directly interact with the [3H]-gabapentin-binding site. Spermine inhibited [3H]-gabapentin binding by interacting with a polyamine-sensitive allosteric site on the membrane protein. The steep concentration-dependence of spermine inhibition of [3H]-gabapentin binding may suggest multi-site co-operativity. 3. Prolonged dialysis of cerebral cortical membranes and Tween 20-solubilized membranes resulted in a > 2.0 fold increase in [3H]-gabapentin binding. The increase in binding was due to the removal of a heat stable, low molecular weight (< 12,000Da) endogenous molecule which influences [3H]-gabapentin binding competitively. 4. Dialysis of detergent-solubilized cerebral cortical membranes also resulted in a decrease in the maximum inhibition of [3H]-gabapentin binding by spermine. Since the rates of the increase in [3H]-gabapentin binding and the loss of the ability of spermine to inhibit [3H]-gabapentin binding on dialysis were different it was inferred that a second endogenous ligand was removed during dialysis. 5. During initial steps of purification of the [3H]-gabapentin-binding protein there was a decrease in the maximum inhibition of [3H]-gabapentin binding by spermine. The loss of the second endogenous molecule during initial purification would reasonably explain the reduction in inhibition of binding by spermine. However, spermine stimulation of [3H]-gabapentin binding to material that eluted from the gel-filtration column later in the purification scheme does not appear to be due to removal of a dialysable endogenous factor or to the dissociation of other calcium channel subunit(s). 6. Adding back dialysate, before or after boiling, to detergent solubilized membranes resulted in a dose-dependent restoration of the inhibition of [3H]-gabapentin binding and of the maximal inhibition [3H]-gabapentin binding by spermine. This result is consistent with the re-addition of two endogenous heat stable ligands. 7. The findings that [3H]-gabapentin binding to the pure alpha 2 delta subunit was stimulated by spermine indicates that the alpha 2 delta subunit of voltage-sensitive calcium channels bears a modulatory spermine site. Such a spermine site has not been identified before. Spermine stimulation of [3H]-gabapentin binding to the purified protein was reversed to inhibition after adding back dialysate. Thus the inhibitory spermine effect in membranes is also probably due to one or more modulatory sites on the alpha 2 delta subunit.

Anticonvulsant effect of reduced NMDA receptor expression in audiogenic DBA/2 mice.

Pretreatment of DBA/2 mice (n = 14-15 per group) with an 18-mer antisense probe to the NMDA-receptor submit NR1 (2 x 1 micrograms, or 2 x 83 pmol, NR1 antisense probe intracerebroventricularly, -29 and -7 h before testing for seizure response) resulted in almost complete suppression of sound-induced clonic seizures. A saline-treated group gave a 100% seizures response, while the group treated with NR1 antisense probe gave a 7% seizure response to the sound stimulus. The group treated with NR1 nonsense-probe showed no anticonvulsant protection (93% seizure response). The anticonvulsant protection observed following NR1 antisense administration was of relatively short duration, with seizure response gradually returning to control levels 12 to 24 h following the termination of antisense administration. When NR1 receptor levels were assessed by receptor autoradiography ([3H]-MK 801 and -CGP 39653 binding) in the same groups of mice, significant (20%) reductions in NR1 levels were observed in the retrosplenial cortex and the overall cortex. The seizure-induced expression of c-fos and NGFI-A in thalamus, hypothalamus, inferior colliculus and medical geniculate seen in vehicle- and NR1 nonsense-treated mice was completely blocked by NR1 antisense pretreatment.

The antiepileptic agent gabapentin (Neurontin) possesses anxiolytic-like and antinociceptive actions that are reversed by D-serine.

This report describes the activity of the antiepileptic agent gabapentin (Neurontin) in animal models predictive of anxiolysis and analgesia. Gabapentin displayed anxiolytic-like action in the rat conflict test, the mouse light/dark box and the rat elevated X-maze with respective minimum effective doses (MEDs) of 3, 10 and 30 mg/kg. Furthermore, gabapentin also induced behavioural changes suggestive of anxiolysis in the marmoset human threat test with a MED of 30 mg/kg. In the rat formalin test of tonic nociception, gabapentin dose-dependently (30-300 mg/kg) and selectively blocked the late phase with a MED of 100 mg/kg. However, it failed to block carrageenan-induced paw oedema. The intracerebroventricular (ICV) administration of the glycine/NMDA receptor agonist D-Serine, dose-dependently (10-100 micrograms/animal) reversed the antinociceptive action of gabapentin (200 mg/kg, SC). D-Serine (30 micrograms/animal, ICV) also reversed the anxiolytic-like effects (in the light/dark box and the rat elevated X-maze) of gabapentin (30 mg/kg). In contrast, L-Serine (100 micrograms, ICV) failed to block the antinociceptive action of gabapentin. The antinociceptive action of (+)-HA-966 (25 mg/kg, SC), a partial agonist at the glycine/NMDA receptor, was reversed by D-Serine (100 micrograms/animal, ICV). However, D-Serine (100 micrograms/animal, ICV) failed to affect the antinociceptive action of a competitive NMDA receptor antagonist CGS 19755 (3 mg/kg, SC). Gabapentin has negligible affinity for the strychnine insensitive [3H]glycine binding site. This indicates that the interaction between gabapentin and D-Serine may not involve the NMDA receptor complex. Gabapentin may represent a novel type of anxiolytic and analgesic agent.

The influence of guanyl nucleotide on agonist and antagonist affinity at guinea-pig CCK-B/gastrin receptors: binding studies using [3H]PD140376.

The novel radioligand [3H]PD140376 was used to label receptors that bind cholecystokinin (CCK) and related peptides in membranes prepared from guinea-pig brain and gastric glands. Under control conditions, measurements of the apparent affinity of 11 agonist and 16 antagonist ligands in both tissues revealed a strong positive relationship between the affinity of a compound in either tissue (slope of the regression line = 0.89, r2 = 0.908). Agonists consistently showed higher affinity for sites in gastric glands compared to brain. If agonists were excluded from the analysis, the degree of correspondence between affinities measured in each tissue was almost perfect (slope = 0.93, r2 = 0.986). In the presence of the guanyl nucleotide 5'-guanylimidodiphosphate (GppNHp), agonist affinity in gastric glands, but not brain, was reduced such that there was a direct relationship between binding affinity in each tissue. These data are consistent with the notion that the receptor sites in brain and gastric glands, which recognise CCK and gastrin related compounds, are the same and of the CCK-B/gastrin subtype. The receptors in the two respective tissues, however, do appear to differ in the degree of post-receptor coupling. These findings may explain previously reported differences between gastrin and CCK-B receptors that were based upon binding studies using agonist ligands.

Modulation of the in vivo actions of morphine by the mixed CCKA/B receptor antagonist PD 142898.

The ability of a mixed CCKA/B receptor antagonist PD 142898 (benzenebutanic acid, beta-[[3-(1 H-indol-3-yl)-2-methyl-2-[[[(2-methyl- cyclohexyl)oxy]carbonyl]amino]-1-oxopropyl]amino]-[1 S-[1 alpha [S*(R*)]-2 beta]]) to modulate the antinociceptive, positive reinforcing and gastrointestinal actions of morphine was investigated in the rat. PD 142898 antagonised the development and maintenance of morphine (2.0 mg/kg, s.c.) induced conditioned place preference at 0.1 mg/kg, i.p. However, it potentiated the antinociceptive action of a subthreshold dose of morphine in the radiant tail flick model at doses of 0.001 and 0.01 mg/kg, s.c. Furthermore, PD 142898 (0.0001-1.0 mg/kg, s.c.) also potentiated the antinociceptive action of morphine (1.0 mg/kg, s.c.) against the late phase of formalin response associated with inflammation at the dose of 0.001-1.0 mg/kg. PD 142898 (0.001 mg/kg, s.c.) blocked the development of tolerance to morphine in the formalin test. It failed (0.001-1.0 mg/kg, i.p.) to modulate the inhibitory action of morphine (5.0 mg/kg, s.c.) on gastrointestinal transit as measured using the charcoal meal test. It is argued that the effect of PD 142898 in the conditioned place preference test involves antagonism of CCKA receptors, whilst the potentiation of the antinociceptive action of morphine is mediated via blockade of CCKB receptors. These results suggest that the mixed CCKA/B receptor antagonist may potentiate the analgesic action of morphine, block the development of tolerance without a concomitant increase in constipation and may also reduce the abuse potential of the opiate.

Effect of CCK receptor antagonists on the antinociceptive, reinforcing and gut motility properties of morphine.

1. The ability of a selective CCKA receptor antagonist PD 140548 and a selective CCKB receptor antagonist CI-988 (formerly PD 134308) to modulate the various in vivo properties of morphine was investigated in the rat. 2. PD 140548 dose-dependently (0.001-1.0 mg kg-1, i.p.) antagonised the development of conditioned place preference to morphine (2.0 mg kg-1, s.c.). In contrast, CI-988 (0.01-1.0 mg kg-1, i.p.) did not affect this morphine-induced behaviour. Neither of the CCK receptor antagonists blocked or generalised to the morphine (3.0 mg kg-1, i.p.) discriminative stimulus. 3. CI-988 (0.001-10.0 mg kg-1, s.c.) at doses of 0.05 and 0.1 mg kg-1 (s.c.), potentiated the antinociceptive action of a threshold dose of morphine (5.0 mg kg-1, i.p.) in a radiant heat model of acute nociception, the rat tail flick test. Furthermore, at 0.01 mg kg-1 it potentiated the antinociceptive action of morphine (3.0 mg kg-1) during the acute phase of the rat paw formalin test. And at doses of 0.01 and 0.1 mg kg-1 it also potentiated the antinociceptive action of morphine (1.0 mg kg-1) during the tonic phase of the formalin test. However, in both models, higher doses of CI-988 were ineffective. In contrast, PD 140548 (0.001-10 mg kg-1, s.c.) was only active at a dose of 1.0 mg kg-1 (s.c.) and only in the tonic phase of the formalin test. Neither CI-988 nor PD 140548 possessed any intrinsic antinociceptive action in either of the tests. Chronic treatment with CI-988 (0.01 mg kg-1, s.c.) prevented the development of tolerance to morphine antinociception (4 mg kg-1, s.c.) following a 6 day period of twice daily injections of morphine escalating from 1 to 16 mg kg-1 (i.p.). 4. Morphine dose-dependently (1-10 mg kg-1, s.c.) reduced the distance travelled by a charcoal meal in the rat intestine. Neither PD 140548 (0.01-1.0 mg kg-1, i.p.) nor CI-988 (0.01-1.0 mg kg-1, i.p.) potentiated or suppressed this inhibitory action of morphine. 5. In conclusion, the results of the present study indicate that CCKA and CCKB receptors modulate different properties of morphine. Thus, whilst a selective CCKA receptor antagonist blocked the rewarding properties of morphine, a selective CCKB receptor antagonist potentiated the antinociceptive action. However, neither compound displayed a potential for modulating the influence of morphine on gastro-intestinal motility. It is suggested that these findings may have important implications for development of CCK receptor antagonists as analgesic adjuncts to the therapeutic use of morphine.

Comparison of the uptake of [3H]-gabapentin with the uptake of L-[3H]-leucine into rat brain synaptosomes.

1. Gabapentin is a novel anticonvulsant with an unknown mechanism of action. Homogenate binding studies described elsewhere have suggested that [3H]-gabapentin binds to a site in brain similar to the large neutral amino acid (LNAA) uptake site, termed system-L. 2. This study describes an investigation into the uptake of [3H]-gabapentin into a crude synaptosomal preparation from cerebral cortex of rat brain. Characterization studies showed that [3H]-gabapentin is taken up into synaptosomes by a system that is similar to that responsible for the uptake of L-[3H]-leucine. This system is sodium-independent, temperature-sensitive and requires ATP for function. 3. Kinetic studies of [3H]-gabapentin uptake produced a Michaelis constant (KM = 160 microM) similar to that observed for L-[3H]-leucine (KM = 110.3 microM). Vmax values were 837.1 pmol mg-1 protein min-1 and 2.192 nmol mg-1 protein min-1 respectively. 4. Gabapentin and L-leucine mutually inhibit their uptake. Lineweaver-Burke plots of these data demonstrate that inhibition occurs by a competitive mechanism. Further to this the Dixon transformation of the data illustrates that these two substrates share a common uptake site by the similarity between their calculated Ki and KM values (gabapentin inhibition of L-[3H]-leucine uptake: Ki = 160 microM; L-leucine inhibition of [3H]-gabapentin uptake: Ki = 262 microM). 5. Studies into the effect of gabapentin, the system-L-specific ligand 2-(-)-endoamino-bicycloheptane-2-carboxylic acid (BCH), and the system-A-specific ligand alpha-(methyl-amino)-isobutyric acid (MeAIB), on the initial rate of uptake of [3H]-glycine, L-[3H]-glutamate, L-[3H]-glutamine, and L-[3H]-leucine were performed. At 100 microM, gabapentin significantly inhibited initial rate of uptake of [3H]-glycine (29%), L-[3H]-glutamate (22%) and L-[3H]-leucine (40%). 6. Gabapentin is taken up into synaptosomes by a system similar to system-L, responsible for the uptake of large neutral amino acids. Gabapentin will also inhibit the uptake of certain excitatory amino acids in this synaptosomal preparation. The implications of these findings for the mechanism of action for gabapentin are unclear. The data presented here may suggest an intracellular site for mechanism of action for this compound. Similarly changes in levels of amino acid pools may be involved in the mechanism of gabapentin's anticonvulsant action.

Comparison of the autoradiographic binding distribution of [3H]-gabapentin with excitatory amino acid receptor and amino acid uptake site distributions in rat brain.

1. Gabapentin is a novel anticonvulsant with an unknown mechanism of action. Recent homogenate binding studies with [3H]-gabapentin have suggested a structure-activity relationship similar to that shown for the amino acid transport system responsible for the uptake of large neutral amino acids (LNAA). 2. The autoradiographic binding distribution of [3H]-gabapentin in rat brain was compared with the distributions for excitatory amino acid receptor subtypes and the uptake sites for excitatory and large neutral amino acids in consecutive rat brain sections. 3. Densitometric measurement of the autoradiographic images followed by normalisation with respect to the hippocampus CA1 stratum radiatum, was carried out before comparison of each binding distribution with that of [3H]-gabapentin by linear regression analysis. The correlation coefficients observed showed no absolute correlation was observed between the binding distributions of [3H]-gabapentin and those of the excitatory amino acid receptor subtypes. The acidic and large neutral amino acid uptake site distributions demonstrated a much closer correlation to the [3H]-gabapentin binding site distribution. The correlation coefficients for D-[3H]-aspartate, L-[3H]-leucine and L-[3H]-isoleucine binding site distributions were 0.76, 0.90 and 0.88 respectively. 4. Concentration-dependent inhibition by unlabelled gabapentin of autoradiographic binding of L-[3H]-leucine and L-[3H]-isoleucine was observed, with non-specific binding levels being reached at concentrations between 10 and 100 microM. 5. Excitotoxic quinolinic acid lesion studies in rat brain caudate putamen and autoradiography were carried out for the amino acid uptake sites mentioned above. The resulting glial infiltration of the lesioned areas was visualized by autoradiography using the peripheral benzodiazepine receptor specific ligand [3H]-PK11195. A significant decrease in binding density in the lesioned area compared with sham-operated animals was observed for D-[3H]-aspartate, L-[3H]-leucine, L-[3H]-isoleucine and [3H]-gabapentin, whilst [3H]-PK11195 showed a significant increase in binding density indicative of glial infiltration into the lesioned area. These results suggest that the gabapentin binding site and the acidic and LNAA uptake site may be present on cell bodies of a neuronal population of cells. 6. From these studies it appears that [3H]-gabapentin, L-[3H]-leucine and L-[3H]-isoleucine bind to the same site in rat brain. The inhibition of [3H]-gabapentin binding by the LNAA uptake system-specific ligand, BCH, suggests that [3H]-gabapentin may label this uptake site, termed system-L. Conversely these ligands could be labelling a novel site that coincidentally has a similar structure-activity relationship to this uptake site. These results suggest a novel mechanistically relevant site of action for gabapentin and may enable further anti-epileptic agents of this type to be developed.

Use of a dipeptide chemical library in the development of non-peptide tachykinin NK3 receptor selective antagonists.

The use of a dipeptide library as the source of a micromolar chemical lead compound for the human tachykinin NK3 receptor is described. The screening of a dipeptide library through a cloned human NK3 receptor binding assay resulted in the identification of Boc(S)Phe(S)PheNH2 (1), which has subsequently been developed, following a 'peptoid' design strategy, into a series of high-affinity NK3 receptor selective antagonists. The structure-activity relationship of the C-terminal portion of this dipeptide lead was first explored and led to the identification of the urea derivative Boc(S)Phe(R)alphaMePheNH(CH2)7NHCONH2 (41, PD157672). This modified dipeptide has a Ke of 7 nM in blocking senktide-induced increases in intracellular calcium levels in human NK3 receptors stably expressed in CHO cells. Subsequent optimization of the N-terminal BocPhe group and the alphaMePhe residue side chain of 41 led to the identification of [S-(R*,S*)]-[2-(2,3-difluorophenyl)-1-methyl-1-[(7-ureidoheptyl)ca r bamoyl]ethyl]carbamic acid 2-methyl-1-phenylpropyl ester (60, PD161182), a non-peptide NK3 receptor selective antagonist. Compound 60 blocks the senktide-evoked increases in intracellular calcium levels in cloned human NK3 receptors stably expressed in CHO cells with Ke of 0.9 nM.

The novel anticonvulsant drug, gabapentin (Neurontin), binds to the alpha2delta subunit of a calcium channel.

Gabapentin (1-(aminomethyl)cyclohexane acetic acid; Neurontin) is a novel anticonvulsant drug, with a mechanism of action apparently dissimilar to that of other antiepileptic agents. We report here the isolation and characterization of a [3H]gabapentin-binding protein from pig cerebral cortex membranes. The detergent-solubilized binding protein was purified 1022-fold, in a six-step column-chromatographic procedure, with a yield of 3.9%. The purified protein had an apparent subunit Mr of 130,000, and was heavily glycosylated. The partial N-terminal amino acid sequence of the Mr 130,000 polypeptide, EPFPSAVTIK, was identical to that reported for the alpha2delta subunit of the L-type Ca2+ channel from rabbit skeletal muscle (Hamilton, S. L., Hawkes, M. J., Brush, K., Cook, R., Chang, R. J., and Smilowitz, H. M. (1989) Biochemistry 28, 7820-7828). High levels of [3H]gabapentin binding sites were found in membranes prepared from rat brain, heart and skeletal muscle. Binding of [3H]gabapentin to COS-7 cells transfected with alpha2delta cDNA was elevated >10-fold over controls, consistent with the expression of alpha2 delta protein, as measured by Western blotting. Finally, purified L-type Ca2+ channel complexes were fractionated, under dissociating conditions, on an ion-exchange column; [3H]gabapentin binding activity closely followed the elution of the alpha2 delta subunit. [3H]Gabapentin is the first pharmacological agent described that interacts with an alpha2delta subunit of a voltage-dependent Ca2+ channel.

The relationship between glutamate release and cerebral blood flow after focal cerebral ischaemia in the cat: effect of pretreatment with enadoline (a kappa receptor agonist).

The effect of the kappa-opioid agonist enadoline (CI-977) upon the relationship between cerebral blood flow and glutamate release was simultaneously assessed (using microdialysis and hydrogen clearance techniques respectively) at the same anatomical locus in the cerebral cortex (suprasylvian gyrus) after permanent middle cerebral artery (MCA) occlusion in halothane-anaesthetised cats. During controlled graded ischaemia, pretreatment with enadoline (0.3 mg/kg i.v. followed by continuous infusion at 0.15 mg/kg/h), initiated 30 min prior to MCA occlusion, significantly attenuated the marked increases in extracellular glutamate, aspartate and GABA observed in the focal ischaemic penumbra. The present data are consistent with the hypothesis that the neuroprotective efficacy of enadoline in focal cerebral ischaemia is due to inhibition of glutamate release in the ischaemic penumbra.

Bombesin receptors in the brain.

We have shown that in the central nervous system BN receptors are closely associated with 5-HT systems. On a subpopulation of dorsal raphe neurons, NMB receptors are able to depolarize cells by reducing gK+. In one of the target regions of the dorsal raphe 5-HT neurons, the SCN, we have also shown that neurons are excited by BN-related peptides. In the SCN, the GRP receptors excite neurons by two different mechanisms: closure of gK+ and opening of an unidentified cation conductance. Expression of human BN receptors from the brain in CHO cells or Xenopus oocytes shows a very similar pharmacological profile to that seen in the rat brain slice preparations. In the CHO cell line, following BN receptor activation, a major second-messenger path involves hydrolysis of PIP2 by phospholipases to yield IP3, which releases Ca2+ from intracellular stores. In the oocyte expression system, a similar second messenger pathway is clearly apparent, and Ca2+-sensitive gCl- represents the last phase in a cascade of events. The final phase of the mechanism of action in the artificial systems does not involve gK+, suggesting a different second messenger cascade to that in neurons. However, the involvement of phospholipases and their phospholipid products have not been excluded in neurons.

Peptoid CCK receptor antagonists: pharmacological evaluation of CCKA, CCKB and mixed CCKA/B receptor antagonists.

Several novel cholecystokinin (CCK) receptor ligands with differing degrees of receptor selectivity were characterised in both in vitro and in vivo models. In radioligand binding assays, the dipeptoid PD 135666 ((benzenebutanoic acid, beta-[[3-(1H-indol-3-yl)-2-methyl-1-oxo-2- [[(tricyclo[3.3.1.1(3,7)]dec-2-yloxy)carbonyl]amino]propyl] amino],-[R-(+*,S*)]) selectively inhibited [125I]Bolton Hunter CCK-8 binding to CCKB receptors in mouse cerebral cortex (CCKB IC50 = 0.1 nM) but was weaker as an inhibitor of CCKA receptor binding in the rat pancreas (IC50 = 26 nM). In contrast, its enantiomer PD 140548 ((benzenebutanoic acid, beta-[[3-(1H-indol-3-yl)-2-methyl-1-oxo-2-[[(tricyclo[3.3.1.1(3,7] dec-2-yloxy)carbonyl]amino]propyl] amino],-[S-(R*,S*)) displayed the reverse selectivity (CCKA IC50 = 2.8 nM, CCKB IC50 = 260 nM). PD 142898 ([benzenebutanic acid, beta-[[3-(1H-indol-3-yl)-2-methyl-2-[[[(2- methylcyclohexyl)oxy]carbonyl]amino]-1-oxopropyl]amino]-,[1S-[1 alpha[S*(R*)],2 beta ]]) possessed nanomolar affinity for both receptor subtypes (CCKB IC50 = 4.2 nM, CCKA IC50 = 3.8 nM) whereas its corresponding enantiomer PD 142896 ([benzenebutanic acid, beta-[[3-(1H-indol-3-yl)-2-methyl-2-[[[(2- methylcyclohexyl)oxy]carbonyl]amino]-1-oxopropyl]amino]-, [1R-[1 alpha[S*(R*)],2 beta]]) displayed 147-fold selectivity for the CCKA receptor (CCKA IC50 = 7.9 nM, CCKB IC50 = 1160 nM). The pyrazolidinone PD 141479 (trans-5-(2-chlorophenyl)-3-oxo-4-phenyl-N-[4- (trifluoromethyl)phenyl]-1-pyrazolidinecarboxamide) was found to interact selectively with the CCKB receptor (CCKB IC50 = 36 nM, CCKA IC50 = 1100 nM). PD 140548, PD 142896, PD 135666 and PD 142898 antagonised the CCKA receptor-mediated contraction of guinea pig gall bladder with respective pA2 values of 7.2, 7.4, 6.6 and 8.5. In the rat elevated X-maze, PD 135666 and PD 141479, together with the mixed CCKA/B receptor antagonist PD 142898 produced anxiolytic effects with respective minimum effective doses (MEDs) of 0.01, 0.001 and 0.01 mg/kg s.c. Furthermore, the selective CCKB receptor antagonist CI-988 (0.01-1 mg/kg) and PD 142898 (0.001-0.1 mg/kg), dose dependently induced behavioural changes suggestive of anxiolysis in the marmoset human threat test with respective MED values of < 0.01 and < 0.001 mg/kg s.c. In contrast, compounds with the CCKA selective profile were either inactive in the two behavioural models or showed activity only at doses of 1 mg/kg and above. These data suggest that the anxiolytic effects of CCK receptor antagonists parallel their affinity for the CCKB rather than the CCKA receptor.

Functional implications of kappa opioid receptor-mediated modulation of glutamate transmission in the output regions of the basal ganglia in rodent and primate models of Parkinson's disease.

Parkinson's disease is characterized by an increased excitatory amino acid transmission in the internal segment of the globus pallidus and the substantia nigra pars reticulata. The effects of the kappa receptor agonist enadoline (CI-977) on glutamate transmission were investigated in vitro. Enadoline reduced the K(+)-evoked release of glutamate from slices of substantia nigra in a concentration-dependent manner (maximum effect: 78% inhibition at 200 microM). This effect was blocked by the selective kappa receptor antagonist nor-binaltorphimine. The endogenous ligand for kappa receptors is thought to be dynorphin. Dynorphin released from terminals of striato-pallidal and striato-nigral pathways might thus act as an endogenous modulatory agent on glutamatergic transmission in the basal ganglia. In vivo experiments were carried out in rodent and primate models of Parkinson's disease to assess the potential of manipulating kappa receptors as a potential treatment for Parkinson's disease. Enadoline reduced reserpine-induced akinesia when injected in the entopeduncular nucleus of the rat. Similarly, injections of CI-977 in the internal segment of globus pallidus (GPi) of the MPTP-treated marmoset alleviated parkinsonian symptoms and allowed the animal to recover its locomotor activity. This suggest that reducing the overactive glutamatergic transmission in the output regions of the basal ganglia by activating kappa receptors might potentially form the basis of a novel anti-parkinsonian therapy.

The cholecystokinin-induced increase in intracellular calcium in AR42J cells is mediated by CCKB receptors linked to internal calcium stores.

Changes in intracellular levels of free [Ca2+]i were monitored in cell suspensions and either single cells or cell clusters of the rat pancreatic tumour cell line AR42J grown on cover slips. Increases in free [Ca2+]i were seen when the bathing medium contained cholecystokinin octapeptide sulphated (CCK) or CCKB receptor agonists. Responses to CCK agonists were repeatable and reversed on washout. The responses to cholecystokinin and pentagastrin could be blocked by selective CCKB receptor antagonists but not a CCKA receptor antagonist. Depleting internal Ca2+ stores with thapsigargin blocked the response to pentagastrin suggesting that the response was mediated by Ca2+ release from internal stores. The rapid run down of the pentagastrin response in the absence of extracellular Ca2+ shows that replenishment of internal stores by extracellular Ca2+ is important in maintaining the CCK response.

Characterization of tachykinin mediated increases in [Ca2+]i in Chinese hamster ovary cells expressing human tachykinin NK3 receptors.

The nature of the senktide response of the human NK3 receptor expressed in Chinese hamster ovary cells was characterised using the Ca2+ sensitive dye Fura-2 and imaging methods. Application of the NK3 receptor agonist senktide caused an increase in [Ca2+]i in the cells. The profile for NK3 receptor agonists was that senktide was more potent than [beta-Ala8]neurokinin A-(4-10) which was more potent than [Sar9,Met(O2)11]substance P. SR 48968 was a poor antagonist of the senktide response in intact cells confirming the weak affinity of this agent for the NK3 receptor (IC50 of approximately 1 microM) shown in binding assays. The NK3 receptor mediated increase in intracellular Ca2+ was independent of [Ca2+]o, blocked by the microsomal Ca2+ ATPase inhibitor thapsigargin and the phospholipase C inhibitor U73122 but not by ryanodine. Thus the source of the Ca2+ was probably a ryanodine insensitive, inositol triphosphate sensitive intracellular store.

Different types of bombesin receptors on neurons in the dorsal raphe nucleus and the rostral hypothalamus in rat brain slices in vitro.

The actions of the peptides bombesin (BN), gastrin releasing peptide (GRP), neuromedin C (NMC), litorin and neuromedin B (NMB) were studied on neurons in slices of rat brain maintained in vitro to determine the BN receptor type present in different brain areas. Intracellular and extracellular recordings were made from hypothalamic neurons on the border of the periventricular nucleus (PVN) and suprachiasmatic nucleus (SCN) and from mesencephalic 5-HT sensitive neurons in the dorsal raphe nucleus. In the region of the brain containing the SCN and PVN, BN and the BN-related peptides excited 31 out of 77 neurons on which they were tested. There was little difference in the potency of the BN-related peptides as excitants of neurons, the EC50 being about 10 nM. The response to the peptides usually lasted between 5 and 15 min with little sign of desensitization. Using NMC, GRP and NMB as agonists, the equilibrium constant for the GRP receptor antagonist [D-Phe6]-BN-(6-13)-ethylamide was approximately 10 nM. The response to the peptides fully recovered on washout of the antagonist. The CCKB/gastrin receptor antagonist CI-988 (1 microM) had no effect on either GRP- or NMC-mediated excitation. In the dorsal nucleus 40 of 75 neurons were sensitive to the BN-related peptides. BN, [Tyr4]-BN, NMB and litorin, were 10-20 times more potent than GRP and NMC. The responses to the BN-related peptides were not blocked by the selective GRP receptor antagonists [D-Phe6]-BN-(6-13)-methylester, [DF5Phe6][D-Ala11]-BN-(6-13)-methylester and [D-Phe6]-BN-(6-13)- ethylamide.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential modulation of kappa and mu opioid antinociception by the glycine/NMDA receptor agonist D-serine.

D-Serine, a selective agonist for the strychnine-insensitive glycine allosteric site associated with the NMDA receptor-ion channel complex, was found to modulate differentially the antinociception produced by kappa and mu-opioid receptor agonists in the rat formalin test. D-Serine (100 micrograms, i.c.v.) attenuated the antinociception produced by the selective kappa-opioid agonist, enadoline (0.003-0.1 mg kg-1, s.c.) against the tonic, but not acute, phase of the formalin response. Conversely, D-serine potentiated the antinociception produced by morphine (0.3-10 mg kg-1, s.c.) against both the acute and tonic phases. These results demonstrate an important interaction between the opioid and NMDA/glycine systems in the control of nociceptive information possibly at different levels of the neuraxis.