Rat hippocampal neurons are critically involved in physiological improvement of memory processes induced by cholecystokinin-B receptor stimulation.

The involvement in memory processes of the neuropeptide cholecystokinin (CCK) through its interaction with the CCK-B receptors was studied. The two-trial recognition memory task was used. Control animals showed recognition memory after a 2 hr time interval but not after a 6 hr time interval between the two trials. The improving effect of a selective CCK-B agonist, BC 264, intraperitoneally administered (0.3 microgram/kg) in the retrieval phase of the task (6 hr time interval), was also observed after its injection (1 pmol/0.5 microliter) in the dorsal subiculum/CA1 of the hippocampus but not in the caudate/putamen nucleus or in the prefrontal cortex of rats. The CCK-B antagonist L-365,260 injected (10 ng/0.5 microliter) into this region of the hippocampus abolished the improving effect of BC 264 injected intraperitoneally. Furthermore, L-365,260 injected in the hippocampus suppressed the recognition of the novel arm normally found in the controls (2 hr time interval) when it was injected before the acquisition or the retrieval phase of the task. In addition, an increase of the extracellular levels of CCK-like immunoreactivity in the hippocampus of rats during the acquisition and retention phase of the task was observed. Finally, CCK-B receptor-deficient mice have an impairment of performance in the memory task (2 hr time interval). Together, these results support the physiological involvement of the CCKergic system through its interaction with CCK-B receptors in the hippocampus to improve performance of rodents in the spatial recognition memory test.

CCK in anxiety and cognitive processes.

Extensive studies were carried out on the involvement of the CCKergic system in anxiety-, panic- and stress-related behaviour. The stimulation of CCK-A or CCK-B receptors is implicated in the physical and psychological responses of CCK to stress. Furthermore, several selective CCK-B agonists produce anxiogenic-like effects, while CCK-B antagonists induce anxiolytic-like responses in several models of anxiety. However, BC264 a highly selective CCK-B agonist, does not produce anxiogenic-like effects but increases attention and/or memory. These effects are dependent on the dopaminergic systems. Together with biochemical data, this led to the hypothesis of the existence of two CCK-B binding sites, CCK-B1 and CCK-B2, which could correspond to different activation states of a single molecular entity. Investigations into CCK-B1 and CCK-B2 systems might be of critical interest, since only one site, CCK-B1, appears to be responsible for the effects of anxiety. Furthermore, the improvement of attention and/or memory processes by CCK, through CCK-B2 receptors, could offer a new perspective in the treatment of attention and/or memory disorders.

Behavioral profile of CCK2 receptor-deficient mice.

CCK2 receptor-deficient mice were used to investigate in vivo the role of this receptor in behavior. Mutant mice showed a neuromuscular impairment in the traction and rotarod tests but not in the chimney test. Brain cholecystokinin has been shown to participate in stress-related behaviors. However, CCK2 receptor-deficient mice did not show behavioral modifications compared to wild-type mice in the elevated plus maze and in the motility conditioned suppression test, indicating that compensatory mechanisms very likely occur following CCK2 receptor invalidation. On the other hand, a hyperlocomotor activity was observed in actimeter which can be related to an impairment in environmental habituation. Finally, CCK2 receptor-deficient mice showed an impairment of performance in the spontaneous alternation behavior as expected from the opposite effects evoked by CCK2 agonists, supporting the physiological role of CCK2 receptors in attention and/or memory processes. This result is reinforced by the defects observed in these functions after the administration of CCK2 antagonists.

Leptin decreases feeding and exploratory behaviour via interactions with CCK(1) receptors in the rat.

We assessed the effects of peripheral leptin on anxiety and exploratory behaviour in the elevated plus-maze and in the four-hole box or Y-maze tests, in rats fed 80% of normal daily food intake and rats fed ad libitum. In the Y-maze test, i.p. injection of 0.4 or 1 mg/kg leptin into rationed rats significantly decreased the percentage of spontaneous alternation behaviour and increased the number of visits. In the elevated plus-maze test, rationed rats spent significantly more time in the open arms (aversive part of the maze) than did rats fed ad libitum. This difference in behaviour was abolished by injecting 0.4 mg/kg leptin. In the four-hole box test, i.p. administration of 1 mg/kg leptin significantly reduced the duration and number of hole visits in rationed and ad libitum fed rats. As with leptin inhibition of food intake, these behavioural changes caused by leptin were prevented by a CCK(1) receptor antagonist (L364,718), at a dose that had no effect by itself. Finally, a 20-min stress that increased corticosterone and ACTH levels had no effect on circulating leptin levels and on the leptin content of epididymal fat tissue, stomach and brain. Thus, leptin induces hypoexploration and decreases spontaneous alternation in rats and these effects are partly dependent on nutritional status. These results also suggest that the CCK system may be involved in the induction of these behavioural changes in rats by leptin, via the CCK(1) receptor.

Chronic blockade of D2 but not D1 dopamine receptors facilitates behavioural responses to endogenous enkephalins, protected by kelatorphan, administered in the accumbens in rats.

It has previously been shown that kelatorphan, (R)-3-(N-hydroxycarboxamido-2-benzyl-propanoyl)-L-alanine, a mixed inhibitor of the catabolism of enkephalins, injected into the nucleus accumbens, induced a dose-dependent hyperlocomotion in rats. In this study, the consequence of chronic treatment with sulpiride, a selective D2 dopamine receptor antagonist, SCH 23390, a selective D1 dopamine receptor antagonist, or haloperidol, a nonspecific but preferential D2 receptor antagonist, on the behavioural response induced by acute administration of kelatorphan into the accumbens, has been investigated in rats. The drug SCH 23390 did not modify the behavioural response to kelatorphan, whereas sulpiride and haloperidol induced an increase which was maximal in the third week after the beginning of treatment, a period corresponding to the appearance of the antipsychotic effect of the neuroleptics. This facilitation was reversed by prior administration of the delta-selective antagonist, ICI 174864. These results suggest that the phasic activity of enkephalinergic neurones of the nucleus accumbens and the associated behavioural hyperactivity are facilitated after chronic blockade of the D2 but not the D1 subtypes of dopamine receptor.

The selective CCK-B agonist, BC 264 injected in the antero-lateral part of the nucleus accumbens, reduces the spontaneous alternation behaviour of rats.

The aim of this study was to examine the behavioural effects induced by stimulation of CCK receptors in the nucleus accumbens of the rat, which contains a high density of heterogenously distributed CCK-B receptors. The drug BC264 (Boc-Tyr(SO3H)-gNle-mGly-Trp-(NMe)Nle-Asp-Phe-NH2), a highly potent and selective CCK-B agonist, injected into the postero-median or antero-median n. accumbens did not modify the spontaneous alternation and exploratory behaviour observed in a Y-maze. In contrast, when administered into the antero-lateral part of the n. accumbens, BC264 (0.3-1.0 nmol) reduced alternation to chance level, without changing the number of arm visits. This effect was suppressed by the selective CCK-B antagonist: L365,260, but not by the selective CCK-A antagonist: MK329. The physiological relevance of this effect was supported by the similar responses induced, in the same concentration range, by the CCK8-like agonist, BDNL (Boc diNle28,31CCK7). These results emphasize the functional heterogeneity of the CCK network in the n. accumbens of the rat and the participation of the peptide in the expression of alternation behaviour, through stimulation of CCK-B receptors. They also show that the recently described anxiolytic effects, induced by CCK-B antagonists, do not seem to occur at this level.

Opposing effects of two CCK(B) agonists on the retrieval phase of a two-trial memory task after systemic injection in the rat.

The effects of two selective CCK(B) agonists, BC 264 and BC 197, on memory processes were investigated in rats using a recently developed two-trial recognition memory task. Control animals showed recognition memory after a 2 but not a 6 h time interval between the two trials, thus allowing a memory impairing (2 h) or improving (6 h) effect of pharmacological treatments to be measured. Drugs were injected i.p. before the second trial (retrieval phase). This experimental procedure was first studied with scopolamine and DL-amphetamine, for which a significant deficit after a 2 h interval or improvement after a 6 h interval of performance was observed, respectively. The CCK(B) agonist, BC 264, was ineffective after a 2 h time interval, whereas the dose of 0.3 microg/kg significantly enhanced performance after a 6 h inter-trial interval. In contrast, BC 197 (30 microg/kg) produced a significant disruption of performance after a 2 h inter-trial interval but was without effect after a 6 h time interval. The effects of the two CCK(B) agonists were abolished by pretreatment with a selective CCK(B) antagonist, L365,260 but not by a selective CCK(A) antagonist, L364,718. The present results suggest that CCK(B) receptors display functional heterogeneity and that CCK(B) agonists like BC 264 could offer a new perspective for the treatment of attentional and/or memory deficits.

Peripheral stimulation of CCK-B receptors by BC264 induces a hyperexploration, dependent on the delta opioid system in the nucleus accumbens of rat.

This study analyses the influence of the CCKergic system on the enkephalinergic system in the exploratory behavior of rats, using both behavioral and biochemical approaches. The results show that the increase of the spontaneous alternation behavior induced by the selective CCKB agonist, BC264 (3 microg/kg) was not suppressed by the opioid antagonists, naloxone (100 microg/kg), or naltrindole (300 microg/kg). In contrast, BC264 injected at the same dose induced a hyperlocomotor activity measured in the open-field test, which was antagonized by the selective delta opioid antagonist, naltrindole. BC264 (3 microg/kg) significantly increased the extracellular levels of Met-LI in the anterior part of the nucleus accumbens. Furthermore, local injection of naltrindole (0.25 microg/0.5 microl) in the anterior nucleus accumbens completely suppressed the hyperlocomotion induced by BC264. The behavioral effects induced by BC264 cannot be explained by its interaction with gastrinic receptors mediating gastric acid release, since BC264 produced a long-lasting increase of gastric acid output from conscious gastric fistula rats only at doses 100 times higher than those inducing behavioral modifications. The hyperlocomotion obtained after stimulation by BC264 of probably peripheral CCKB receptors, indicates that this receptor type could participate in the transmission of information between the peripheral system and some regions of the CNS involved in motivations and emotions.

Investigation of the structural parameters involved in the mu and delta opioid receptor discrimination of linear enkephalin-related peptides.

The previous rules proposed for selective recognition of mu and delta opioid receptors by modified enkephalins were investigated through an extensive structure-activity study. Thus, modifications of the sequence of TRIMU 4 (Tyr-D-Ala-Gly-NHCH(CH3)CH2CH(CH3)2, a peptide that exhibits mu selectivity close to that of DAGO (Try-D-Ala-Gly-N(Me)Phe-Gly.ol), were performed for two positions, 2 and 4, critical for mu recognition. The drastic loss of potency following introduction of L-Ala or Aib in position 2 emphasizes the importance of the stereochemistry and the steric size of the X2 amino acid for optimal mu binding. The enhancement of the intrinsic flexibility of the C-terminal alkyl chain of TRIMU 4 through removal of a methyl group leads to TRIMU 5 (Tyr-D-Ala-Gly-NHCH2CH2CH(CH3)2), a peptide with a mu selectivity similar to that of DAGO. In contrast, introduction of an O-tert-butyl Ser2 residue increases affinity for delta receptors. In the hexapeptide series derived from DSLET (Tyr-D-Ser-Gly-Phe-Leu-Thr), a D-Thr2 moiety was shown to be very efficient in improving delta recognition and delta selectivity appeared also to be modulated by the nature of the sixth residue. The potencies of the 24 peptides studied to inhibit the electrically evoked contractions of the GPI or MVD are relatively well correlated with their affinities for brain mu or delta receptors labeled with [3H]DAGO or [3H]DSLET, respectively. Moreover, the analgesic potency (hot plate test) of the peptides is related to their affinity for rat brain mu receptors. The wide range of receptor affinities exhibited by the compounds reported here could be useful to study the physiological role of mu and delta receptors.

Development of conformationally constrained linear peptides exhibiting a high affinity and pronounced selectivity for delta opioid receptors.

A series of linear conformationally constrained opioid peptides was designed in an attempt to develop highly selective and potent agonists for the delta opioid receptors. These enkephalin analogues corresponding to the general formula Tyr-D-X(OY)-Gly-Phe-Leu-Thr(OZ) were obtained by incorporating bulky residues (X = Ser or Thr; Y = tert-butyl or benzyl; Z = tert-butyl) into the sequence of the previously reported delta specific agonists DSLET (Tyr-D-Ser-Gly-Phe-Leu-Thr) and DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr). In binding studies based on displacement of mu and delta opioid receptor selective radiolabeled ligands from rat brain membranes, the two constrained hexapeptides, Tyr-D-Ser(O-t-Bu)-Gly-Phe-Leu-Thr (1, DSTBULET) (KI(mu) = 374 nM, Kr(delta) = 6.14 nM, KI(delta)/KI(mu) = 0.016) and in particular Tyr-D-Ser(O-t-Bu)-Gly-Phe-Leu-Thr(O-t-Bu) (7, BUBU) (KI(mu) = 475 nM, KI(delta) = 4.68 nM, KI(delta)/KI(mu) = 0.010) were shown to be among the most potent and selective delta probes reported to date. A roughly similar pattern of selectivity was obtained with the guinea pig ileum and mouse vas deferens bioassays. In addition, the analgesic potency (hot-plate test) of these peptides intracerebroventricularly administered in mice was shown to be significantly related to their mu-receptor affinity.

Social interaction increases the extracellular levels of [Met]enkephalin in the nucleus accumbens of control but not of chronic mild stressed rats.

Chronic application of various mild stress has been shown to decrease the responsiveness to reward in rats. This effect, which was suggested to mimic anhedonia, one of the main symptoms observed in depressive patients, can be measured by various tests. Thus chronic mild stress was shown to reduce the consumption of a palatable sucrose solution, and to decrease the acquisition of preferences for a distinct environment paired with a variety of reinforcing substances. These negative responses could be prevented by chronic treatment with tricyclic or atypical antidepressants. The behavioural changes, induced by exposure to chronic mild stress, were shown to be associated with a number of changes in dopaminergic neurotransmission in the mesolimbic system, especially in the nucleus accumbens. The nucleus accumbens contains a large number of enkephalinergic cell bodies giving rise to local collaterals and axons projecting to the globus pallidus-ventral pallidum region (for review see Ref. 9). Furthermore, there is evidence that this structure is instrumental in mediating the reward effects of exogenous and endogenous opioids (for reviews see Refs 5,7,17). This study was carried out to analyse the possible contribution of the enkephalinergic system in the anhedonic-like state induced by chronic mild stress. Microdialysis was used to study the extracellular levels of [Met]enkephalin-like material in the rostral part of the nucleus accumbens of freely moving rats exposed or not to chronically mild stress. In both groups, the basal levels of [Met]enkephalin-like material were found to be similar. Exposure of the two groups to a congener, increased the extracellular levels of [Met]enkephalin in the controls but not in chronic mild stressed rats. This suggests that the reactivity of the endogenous opioid system could be reduced in stress induced model of anhedonia.

Involvement of brain endogenous cholecystokinin in stress-induced impairment of spatial recognition memory.

The central fragment of cholecystokinin, CCK8, plays a critical role in stress-related changes in behavior and memory. Therefore, we investigated whether the endogenous cholecystokininergic system is involved in the impairment of attention and/or memory induced by stressful conditions. Plasma corticosterone concentrations increased three-fold and plasma adrenocorticotropin (ACTH); concentrations increased five-fold when rats were maintained in the open arm of an elevated plus maze for 5 min. The same stress conditions impaired spatial recognition in the two-trial memory task. In addition, this stress led to a significant decrease in the extracellular levels of cholecystokinin-like immunoreactivity in the dorsal subiculum/CA1 of the hippocampus and partially suppressed the increase obtained during the acquisition phase of memory. This suggests that the cholecystokininergic system in the hippocampus is involved in stress-induced impairment of spatial recognition memory.

Opposite effects of CCK(B) agonists in grooming behaviour in rats: further evidence for two CCK(B) subsites.

1. The hypothesis of the existence of two CCK(B) receptor subsites, CCK(B1) and CCK(B2) corresponding probably to different coupling states of CCK(B) receptors, was studied by measuring grooming behaviour in rats. 2. The B1 receptor agonist, BC197 (300 microg kg(-1), i.p.) produced a 45-50% decrease in grooming activity, which was prevented by both the CCK(B) receptor antagonists CI-988 (20 microg kg(-1) i.p.) and L-365,260 (200 microg kg(-1), i.p.). 3. In contrast, 3, 10 and 30 microg kg(-1), i.p., of the potent B2 receptor agonist, BC264, enhanced grooming (150-190%). This effect was prevented by previous injection of 75 microg kg(-1) of L-365,260 while higher doses (200 microg kg(-1), i.p.) produced only a partial antagonism. Moreover, CI-988 (20 microg kg(-1), i.p.), showed an opposite effect in potentiating the responses induced by BC264. However, 200 microg kg(-1) of CI-988 tended to suppress the increase of grooming induced by BC264. 4. The effects of BC264 were prevented by the D1 receptor (SCH 23390) and D2 receptor (sulpiride) antagonists, while those of BC197 were only antagonized by sulpiride, emphasizing the existence of a link between peptidergic (CCK) and dopaminergic systems. 5. This study brings additional evidence for the existence of the two CCK(B) receptor subsites and suggests that particular attention should be focused on the selectivity of CCK(B) receptor agonists, notably to explain the fact that some compounds such as Boc-CCK4 induce anxiogenic-like effects while others, including BC264, are devoid of these effects.

Comparison of the behavioural effects induced by administration in rat nucleus accumbens or nucleus caudatus of selective mu and delta opioid peptides or kelatorphan an inhibitor of enkephalin-degrading-enzymes.

The effects of selective agonists for delta opioid receptors: [D-Thr2, Leu5]-enkephalyl-Thr6 (DTLET) and mu receptors: [D-Ala2, MePhe4, Gly-ol5]-enkephalin (DAGO) and of (R)-3-(N-hydroxyl-carboxamido-2-benzylpropanoyl)-L-alanine (kelatorphan), a complete inhibitor of enkephalin degrading enzymes, on the motor activity of rats was examined after their local administration into the nucleus accumbens (NA) or nucleus caudatus (NC). In both structures DTLET dose dependently enhanced locomotor activity as measured in the open-field test. This strong effect was reversed by the selective delta antagonist: ICI 174,864. Contrastingly, DAGO induced hypoactivity followed by hyperactivity 150 min later. This biphasic effect was blocked by systemic injection of naloxone, but not by ICI 174,864. The physiological relevance of these effects was ascertained by the naloxone-reversible stimulatory responses induced by kelatorphan, supporting a role for endogenous enkephalins in the control of behavior through delta receptor stimulation.

Blockade of dopamine receptors reverses the behavioral effects of endogenous enkephalins in the Nucleus caudatus but not in the Nucleus accumbens: differential involvement of delta and mu opioid receptors.

We have previously (Daugé et al. 1988) demonstrated that injection of the mu agonist [D-Ala2, MePhe4, Gly-ol5]-enkephalin (DAGO) or the delta agonist [D-Thr2, Leu5]-enkephalyl-Thr6 (DTLET) into the rat Nucleus accumbens (N.Acc.), or Nucleus caudatus (N.Caud.) induced a hypoactivity followed by hyperactivity 150 min later in the case of the mu agonist and a hyperactivity in the case of the delta agonist. Moreover, naloxone reversible delta-type responses were obtained by local infusion of kelatorphan, ([(R)-3(N-hydroxylcarboxamido-2-benzylpropanoyl)-L-alanine]), a complete inhibitor of enkephalin catabolism, suggesting a tonic control of the behavioral activity of rat by the endogenous opioid peptides. In this work, the putative involvement of the dopaminergic system in these behavioral responses was investigated by using the DA antagonist thioproperazine. In the N.Acc., the behavioral effects of kelatorphan or of mu or delta agonists were not altered by thioproperazine-induced blockade of dopamine receptors. In contrast, the hyperactivity produced by DTLET or by kelatorphan in the N.Caud. was reversed by thioproperazine while the time-dependent biphasic effect resulting from DAGO injection remained unaffected by the DA antagonist. This blocking effect of thioproperazine is in agreement with the previously described delta-selective enhancement of the release of newly synthesized DA in the striatum but not in the N.Acc.

Involvement of D2 dopamine receptors in the opposing effects of two CCK-B agonists in a spatial recognition memory task: role of the anterior nucleus accumbens.

RATIONALE: A previous study in the rat has shown that systemic injection of two CCK-B agonists, BC264 and BC197, induced opposing effects on the retrieval phase of a spatial recognition memory task. OBJECTIVE: The present study was designed to investigate the mechanisms underlying these effects at the level of the dopaminergic system. METHODS: Rats were injected IPly with BC264 (0.3 microg/kg) or BC197 (30 microg/kg) and with D1 or D2 agonists and antagonists. The cognitive performances of rat were analysed on the retrieval phase of a spatial recognition memory task. The extracellular levels of dopamine were quantified in the anterior nucleus accumbens after injection of BC197 (3, 30 and 300 microg/kg IP), using the microdialysis technique on freely moving rats. Local injection of the D2 antagonist, sulpiride (2.5 ng/microl) was performed in the anterior nucleus accumbens and the cognitive performances analysed following systemic injection of BC264 (0.3 microg/kg). RESULTS: The improvement and the impairment of performance induced respectively by BC264 and BC197 were suppressed by peripheral administration of sulpiride, showing that these opposing effects were both mediated by the stimulation of D2-like receptors. However, different dopaminergic pathways seem to be involved in the effects of the two CCK-B agonists. Indeed, systemic administration of BC197 did not induce the increase of extracellular dopamine levels observed with BC264. Furthermore, local injection of sulpiride, in the anterior nucleus accumbens, completely suppressed the cognitive enhancing effect of BC264. CONCLUSION: These findings suggest that the D2-mediated deficit in the performance induced by BC197 involves brain structures other than the anterior nucleus accumbens. They also demonstrate a critical role of dopaminergic transmission within the anterior nucleus accumbens in the improving effect induced by BC264 in a spatial memory task.

Opioid delta agonists and endogenous enkephalins induce different emotional reactivity than mu agonists after injection in the rat ventral tegmental area.

The possible role of opioid receptor heterogeneity in the biphasic changes in locomotion (activation and inhibition) induced by non-selective opiates such as morphine, has been investigated by measuring the behaviour of rats exposed to different environments after injection into the ventral tegmental area, of selective mu (DAGO) or delta (DTLET, DSTBULET, BUBU) opioid agonists and of kelatorphan, a complete inhibitor of enkephalin metabolism. delta agonists or kelatorphan-induced hyperactivity in a familiar (actimeter), unfamiliar (four-hole box) and a fear inducing (open-field) environment. These effects were suppressed by naloxone and delta selective antagonists (ICI 174, 864 2 mg/kg SC, naltrindole 7 nmol in the ventral tegmental area). Moreover, the delta agonists and endogenous enkephalins protected by kelatorphan did not affect the emotional state of rats measured in an elevated plus maze. Infused into the ventral tegmental area, DAGO also enhanced locomotion in the actimeter but in contrast to delta agonists and kelatorphan, the mu agonist decreased activity in the open-field and the four-hole box. The hypoactivity observed in these tests could be related to an enhanced emotionality produced by mu receptor stimulation, as shown by the significant decrease in the number of visits and time spent in open arms of the elevated plus maze. Naloxone (0.3 mg/kg SC) but not delta selective antagonists, blocked the various responses induced by DAGO.

Cognitive enhancing effects in young and old rats of pBC264, a selective CCK(B) receptor agonist.

RATIONALE: The implication of CCK(B) receptors in cognitive processes is far from fully understood. OBJECTIVE: The present study investigated the effect of propionyl-BC264, a selective agonist of CCK(B) receptors, in young and old rats. METHODS: Cognitive functions were studied in a two-trial recognition memory task developed in our laboratory. RESULTS: It was shown that propionyl-BC264 enhanced information processing in young as well as in old rats when injected (10 microg/kg; IP) immediately after the acquisition phase and before the retrieval trial but not before the acquisition trial. This cognitive enhancing effect was blocked by prior administration of L 365,260, a selective CCK(B) receptor antagonist. CONCLUSIONS: In view of the fact that BC264 is devoid of anxiogenic effects, it could be of value in the treatment of cognitive impairments associated with both normal and pathological ageing.

Synthesis, biochemical and pharmacological properties of BUBUC, a highly selective and systemically active agonist for in vivo studies of delta-opioid receptors.

Based on the results of conformational studies of linear and cyclic delta-opioid peptides such as BUBU [Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr(OtBu)] and DPLPE c[Tyr-D-Pen-Gly-Phe-Pen], a new enkephalin-related peptide, Tyr-D-Cys(StBu)-Gly-Phe-Leu-Thr(OtBu) (BUBUC) was synthesized and tested for its opioid activity and selectivity at both the peripheral and central levels. Amongst all the synthetic compounds described so far, BUBUC appears to be the most highly delta-selective probe [KI (mu) = to 2980 nM, KI (delta): 2.9 nM, KI (mu)/KI (delta) approximately 1000]. This selectivity was confirmed by the results of pharmacological studies, including measurements of supraspinal analgesia and behavioral changes in mice. In the later test, BUBUC was shown to increase the rearing activity after IV administration at very low concentrations (0.1 mg/kg) and this effect was reversed by the delta-selective antagonist naltrindole. No antinociceptive response was observed at a 10-fold higher concentration. Thanks to its enzymatic stability and its hydrophobicity. BUBUC is the first systemically active, highly selective delta agonist and should therefore be useful to characterize the physiological role of delta-opioid receptors.

Synthesis and biological activity of Boc [Nle28, Nle31]CCK27-33, a highly potent CCK8 analogue.

A new CCK8 related peptide, Boc[Nle28,Nle31]CCK27-33 (Boc[diNle]CCK7) was synthesized and tested for cholecystokinic activity, at both the peripheral and the central level. This analogue, protected against both chemical oxidation and enzymatic degradation by aminopeptidases, was shown to be equipotent to CCK8 in releasing amylase from rat pancreas fragments. In addition, the EC50 values of Boc[diNle]CCK7 in the guinea pig gallbladder and ileum contraction assays (3.2 nM and 3.0 nM respectively) were similar to those of CCK8 (6.0 nM and 2.0 nM). Moreover both Boc[diNle]CCK7 and CCK8 elicited similar effects on the open field test over the same concentrations range. These results demonstrate the ability of Boc[diNle]CCK7 to be a suitable tool for investigating the physiological role of native CCK8.