Antinociceptive effects of RB101, a complete inhibitor of enkephalin-catabolizing enzymes, are enhanced by a cholecystokinin type B receptor antagonist, as revealed by noxiously evoked spinal c-Fos expression in rats

The effects of RB101, a complete inhibitor of enkephalin-catabolizing enzymes, alone or with a selective cholecystokinin (CCK)B receptor antagonist (CI988) or CCK(A) receptor antagonist (devazepide), on carrageenin-induced spinal c-Fos expression were investigated. Spinal c-Fos expression was observed 90 min after intraplantar carrageenin (6 mg/150 microl saline), with Fos-like-immunoreactive neurons preferentially located in the superficial laminae of the spinal dorsal horn. Intravenous RB101 (10, 20 and 40 mg/kg) dose-dependently reduced the number of superficial Fos-like-immunoreactive neurons (r2 = 0.739, P < .0001), with 63 +/- 2% (P < .0001) reduction for the highest dose. These effects were completely blocked by coadministered naloxone. Coadministration of inactive doses of i.v. RB101 (5 mg/kg) and i.p. CI988 (3 mg/kg) significantly and strongly reduced the number of carrageenin-induced, superficial, Fos-like-immunoreactive neurons (55 +/- 5% reduction of control carrageenin c-Fos expression, P < .0001). This effect was blocked by coadministered naloxone. It is important to note that coadministered RB101 and devazepide did not influence spinal c-Fos expression. None of the various drug combinations influenced the carrageenin-induced peripheral edema. These results show that RB101 dose-dependently decreases carrageenin-evoked spinal c-Fos expression. In addition, the effectiveness of RB101 can be revealed by preadministration of the CCK(B) receptor antagonist CI988. Considering the weak opioid side effects obtained with RB101 treatment and the strong increase of its effects by the CCK(B) receptor antagonist, this type of drug combination could have promising therapeutic application in the management of pain in humans.     

Who pays for the state? A reply to Robert Chernomas and Ardeshir Sepehri

To test the possible role of cholecystokinin (CCK) in the decrease of social exploration induced by intraperitoneal (IP) injection of lipopolysaccharide (LPS, 100 microg/kg), mice were pretreated with IP or intracerebroventricular (ICV) injection of the CCKA receptor antagonist L-364,718 (3 mg/kg and 10 microg/kg, respectively) and the CCKB receptor antagonist L-365,260 (1 mg/kg and 10 microg/kg, respectively). L-364,718 and L-365,260 did not alter LPS-induced decrease in social investigation, whatever the route of administration, suggesting that endogenous cholecystokinin does not mediate the effect of proinflammatory cytokines on social exploration in mice.     

Intrathecally administered baclofen for treatment of children with spasticity of cerebral origin

To determine the relative importance of CCK-A, CCK-B, and opioid receptors in mediating the antinociceptive actions of cholecystokinin, we evaluated the actions of selective agonists and antagonists in the mouse hot plate assay. The agonists used were CCK (1-30 nmol i.c.v.), a CCK-A receptor agonist (SNF9019; 0.3-10 nmol i.c.v.), and a CCK-B receptor agonist (SNF9007; 0.3-10 nmol i.c.v.). The antagonists used were the CCK-A receptor antagonist, L364,718 (12.5 nmol i.c.v.), CCK-B receptor antagonist, L365,260 (2.5-25 nmol i.c.v.), and the nonselective opioid receptor antagonist naloxone (1 mg/kg s.c.). CCK and its receptor-selective analogues, SNF9019 and SNF9007, resulted in antinociception that was blocked by naloxone, but was not antagonized by L364,718 or L365,260. In contrast, in positive control experiments, the inhibitory effects of CCK, SNF9019, and SNF9007 on gastrointestinal propulsion in mice were antagonized by identical i.c.v. doses of L364,718 and L365,260. We conclude that centrally administered CCK produces antinociception in the mouse hot plate assay via opioid receptors, but independent of CCK-A or CCK-B receptors. It is necessary to speculate that other CCK receptors, not antagonized by currently available selective antagonists, may exist.     

Modulation of cocaine-induced motor activity in the rat by opioid receptor agonists

Selective opioid-receptor agonists were tested in combination with cocaine to determine the effect on the motor activity of rats. Cocaine produced dose-dependent increases in locomotor activity (distance traveled). The cocaine-induced increase in locomotor activity was potentiated by the selective delta-opioid receptor agonist [D-Pen2-D-Pen5]enkephalin (DPDPE). This potentiation was blocked by the general opioid receptor antagonist naltrexone, as well as by the selective opioid receptor antagonists beta-FNA (mu-opioid receptor) and naltrindole (delta-opioid receptor). DPDPE also potentiated the increase in locomotor activity produced by the selective dopamine reuptake inhibitor GBR12909, but not that produced by the direct dopamine receptor agonist apomorphine. Cocaine-induced motor activity was potentiated by the activation of central delta-opioid receptors. The synergistic effect seen with delta-opioid receptor activation may involve a mu-opioid receptor component, and is probably mediated via a dopaminergic pathway.     

L-364,718 and L-365,260, two CCK antagonists, have no affinity for central benzodiazepine binding sites in chickens

It has recently been demonstrated that L-365,260, a CCK-B antagonist in mammals, causes an increase in food intake in chickens. In contrast, L-364, 718, a CCK-A antagonist in mammals, shows this effect only at very high dose levels. It has been shown that L-365,260 has very low affinity for chicken CCK receptors. Thus, the mechanism of action of L-365,260 remains unknown. As L-365,260 is a benzodiazepine derivative, one may hypothesize that it would be acting on benzodiazepine binding sites. The aims of this work were to establish the existence of benzodiazepine binding sites in the chicken brain, and to check the possibility that L-365,260 was acting on these receptors, determining the affinity of L-364,718 and L-365,260 for them. We have found specific binding for tritiated flunitrazepam (a benzodiazepine agonist) ([3H]-flunitrazepam) in chicken brain membranes. A single binding site was detected with a Kd of 3.58 +/- 0.97 nM and a Bmax of 451.6 +/- 23.3 fmol/mg protein L-365,260 and L-364,718 exhibited very low affinity for these binding sites (Ki = 1.17 x 10(-6) +/- 0.16 x 10(-6) M and Ki > 10(-5) M, respectively). Thus, these results demonstrate that the increase in food intake caused by L-365,260 in the chicken is not due to a direct action on benzodiazepine receptors. Other possible explanations for its effect are discussed.     

Radiographic verification of implant abutment seating

This study was designed to identify the mechanisms underlying the reduction in food intake in rats. Measurements were made of the release of cholecystokinin (CCK) stimulated by potassium chloride in the hypothalamus after (a) gamma irradiation (60Co), (b) treatment with the CCK-A and CCK-B antagonists L-364,718 and L-365,260 with and without radiation, (c) bilateral abdominal vagotomy, and (d) vagotomy with and without radiation and with and without L-364,718. The concentrations of CCK in hypothalamus perfusate were measured by a radioimmunoassay. Exposure of rats to 1, 3, 5 and 10 Gy (1 Gy/min) increased release of CCK in the hypothalamus in a manner that was dependent on dose. A dose of 5 Gy was chosen for further studies. Intraperitoneal (i.p.) administration of 10, 20 and 50 microg/kg of L-364,718 did not induce significant changes in release of CCK in sham-irradiated animals. However, the drug decreased the release of CCK induced by radiation in a dose-dependent manner. In contrast to L-364,718, 20-50 microg/kg of L-365,260 decreased the release of CCK in the hypothalamus in sham-irradiated animals but did not decrease release of CCK induced by exposure to radiation. Vagotomy produced an insignificant reduction in release of CCK compared to that in sham-irradiated controls. However, vagotomy decreased release of CCK in irradiated rats compared to the irradiated rats without vagotomy. Vagotomy and i.p. administration of 10, 20 and 50 microg/kg of L-364,718 decreased release of CCK in irradiated rats compared to that in irradiated rats without vagotomy. However, i.p. administration of 10, 20 and 50 microg/kg of L-364,718 did not induce significant decreases in release of CCK in the hypothalamus of vagotomized and irradiated animals compared to those in rats that were vagotomized and irradiated but not treated with L-364,718. These results demonstrate that radiation increases the release of CCK in the hypothalamus, and that this effect is inhibited by vagotomy and the administration of a CCK-A receptor antagonist. A CCK-A receptor antagonist may be used to mitigate a radiation-induced deficit in food intake.     

Effects of caerulein and CCK antagonists on tolerance induced to morphine antinociception in mice

Different groups of mice received one daily dose (50 mg/kg) of morphine subcutaneously (SC) for 3, 4 or 5 days to develop tolerance to the opioid. The antinociceptive response of morphine (9 mg/kg) was tested in the hot-plate test 24 h after the last dose of the drug. Tolerance to morphine was obtained in all groups. The group of mice that received morphine for 4 days was employed for the rest of the experiments. Pretreatment of animals with a single dose of caerulein (0.025, 0.05, and 0.1 mg/kg, SC) 30 min prior to receiving morphine (50 mg/kg; during the development of tolerance to the opioid) on day 1, 2, 3, 4 or 5 of morphine administration potentiate antinociception induced by morphine (test dose of 9 mg/kg). The dose of 0.05 mg/kg of caerulein, used 30 min before morphine administration on day 3, was also used to evaluate the effects of antagonists on caerulein-induced decrease in tolerance. The selective cholecystokinin (CCK) receptor antagonists, MK-329 [1-methyl-3-(2 indoloyl)amino-5-phenyl-3H-1,4-benzodiazepin-2-one; 0.25 and 0.5 mg/kg] or L-365,260 [3R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H- 1,4-benzodiazepin-3-yl)-N-(3-methyl-phenyl)urea: 0.25 and 0.5 mg/kg] decreased potentiation of morphine response induced by caerulein. MK-329 or L-365,260, when were injected 35 min before morphine injection during the development of tolerance and on day 3, decreased the tolerance to morphine. A single administration of MK-329 or L-365,260 (in the absence of caerulein) 35 min and 48 h before the test dose of morphine (9 mg/kg) potentiated the antinociception of morphine in nontolerant animals. In conclusion, CCK mechanism(s) may interact with morphine tolerance.     

Cholecystokinin (CCK)-induced stimulation of luteinizing hormone (LH) secretion in adult male rhesus monkeys: examination of the role of CCK in nutritional regulation of LH secretion

Studies were performed with the overall goal of testing the hypothesis that cholecystokinin (CCK), a peptide hormone released from the gastrointestinal tract in response to meal consumption, provides a metabolic signal which modulates LH secretion in response to changes in the body's nutritional intake. In an initial study to document the effects of CCK on LH secretion in adult male rhesus monkeys, sulfated CCK-8 (7 and 15 micrograms/kg) was administered to six monkeys, and blood samples were collected from indwelling venous catheters. The 15-micrograms/kg dose of CCK elicited a rapid release of LH, with peak LH levels of 31.29 +/- 7.19 ng/ml occurring within 5-15 min. To determine the CCK receptor type mediating the effect of CCK on LH secretion, specific CCK type-A (L-364,718) and type-B (L-365,260) receptor antagonists (1 mg/kg) were administered to five monkeys 15 min before CCK administration. The CCK-A antagonist completely blocked LH secretion in response to CCK, whereas the CCK-B antagonist had no effect. To assess whether endogenous CCK, released in response to food intake, stimulates LH secretion, six monkeys were fasted for 1 day and then provided with a normal meal of monkey chow (i.e. a refeed meal) the following day, with either no antagonist, CCK-A antagonist, or CCK-B antagonist administered 30 min before the meal. As previously demonstrated, meal consumption after a brief period of fasting caused a rapid stimulation of pulsatile LH secretion. The refeed meal led to a comparable stimulation of LH secretion regardless of whether monkeys received no antagonist (3.7 +/- 0.44 LH pulses/9 h), CCK-A antagonist (3.33 +/- 0.56 LH pulses/9 h), or CCK-B antagonist (4.0 +/- 0.78 LH pulses/9 h). These results indicate that CCK can stimulate LH secretion in adult male rhesus monkeys, acting via type-A CCK receptors. However, endogenous CCK released in response to meal intake does not appear to be responsible for the meal-induced stimulation of LH secretion that occurs when monkeys are fed a normal meal after a brief period of fasting.     

Metabotropic glutamate receptor agonists stimulate polyphosphoinositide hydrolysis in primary cultures of rat hepatocytes

In phytohemagglutinin (PHA) activated human peripheral blood mononuclear cells. [3H]thymidine uptake and interferon gamma production were increased by the delta-opioid receptor agonist, deltorphin-I (10(-14)-10(-10) M) and by the delta-opioid antagonist naltrindole (10(-13)-10(-9) M). Combination of 10-9 M naltrindole with deltrophin-I (10(-12)-10(-8)M) significantly inhibited the proliferative response but did not affect interferon production.     

The pathways of cell death: oncosis, apoptosis, and necrosis

Differential pulse voltammetry was used to investigate the extracellular dopamine (DA) and DOPAC signal in the anterior part of nucleus accumbens (N.acc.) after microinjection of cholecystokinin (CCK) derivatives into the ventral tegmental area (VTA). Both the mixed CCK(A)/CCK(B) receptor agonist CCK-8s and the selective CCK(B) receptor agonist CCK-4 caused a dose-dependent increase in the DA signal after doses of 10 ng and 100 ng while CCK-8s had no effect on the DOPAC signal. The CCK(A) receptor antagonist L 364,718 (25 microg/kg i.p.) as well as the CCK(B) receptor antagonist L 365,260 (25 microg/kg i.p.) were administered prior to microinjection of 100 ng CCK-8s and L 365,260, but not L 364,718, completely inhibiting the DA increase produced by CCK-8s. Analysis of the tissue levels of DA and its main metabolites in the anterior part of N.acc. revealed no changes after CCK-8s microapplication into VTA. The presented data indicate a CCK(B) receptor-mediated increase in extracellular DA in the anterior N.acc. after microapplication of CCK derivatives into the VTA.     

Selective in vivo binding of [3H]naltriben to delta-opioid receptors in mouse brain

Naltriben (NTB) is a selective antagonist for the putative delta2-opioid receptor. We have determined the regional kinetics and pharmacological profile of [3H]naltriben in vivo in mouse brain. After i.v. administration to CD1 mice, [3H]naltriben uptake and retention were high in striatum, cortical regions and olfactory tubercles, and low in superior colliculi and cerebellum. Robust rank order correlation was found between [3H]naltriben uptake in discrete brain regions and prior delta-opioid receptor binding determinations in vitro and in vivo. [3H]Naltriben binding in vivo was saturable, and was blocked by the delta-opioid receptor antagonist naltrindole, but not by the mu-opioid receptor antagonist cyprodime or the K-opioid receptor agonist (trans)-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]ben zeneacetamide mesylate (U50,488H). (E)-7-Benzylidenenaltrexone (BNTX), a selective antagonist for the putative delta1-opioid receptor, was 9.6- to 12.9-fold less potent than naltriben as an inhibitor of [3H]naltriben binding. Thus, the sites labeled by [3H]naltriben in vivo may correspond to the delta2-opioid receptor subtype. Such assignment is not definitive, particularly considering the 4-fold higher brain uptake of naltriben as compared to (E)-7-benzylidenenaltrexone. Moreover, the regional distribution of [3H]naltriben in brains from CXB-7/BY (CXBK) mice, a strain that shows supraspinal delta1- but not delta2-opioid receptor agonist effects, was quite similar to that found for CD1 mice.     

Rational drug design and synthesis of a highly selective nonpeptide delta-opioid agonist, (4aS*,12aR*)-4a-(3-hydroxyphenyl)-2-methyl- 1,2,3,4,4a,5,12,12a-octahydropyrido[3,4-b]acridine (TAN-67)

We designed highly selective non-peptide agonists for the delta-opioid receptor. On the basis of the "message-address" concept in this field and the accessory site hypothesis, a novel class of heterocycle-fused octahydroisoquinoline derivatives were synthesized. One of these compounds [(4aS*,12aR*)-4a-(3-hydroxyphenyl)-2-methyl-1,2,3,4,4a,5,12, 12a -octahydropyrido[3,4-b]acridine, TAN-67 (2)] showed high selectivity for the delta-opioid receptor (Ki = 1.12 nM) in guinea-pig cerebrum with a 2070-fold lower affinity for the mu-opioid receptor and a 1600-fold lower affinity for the kappa-opioid receptor. TAN-67 was a potent delta-opioid receptor agonist with an IC50 value of 6.61 nM in the mouse vas deferens assay that was reversed by naltrindole (NTI) (Ke = 0.21). Moreover, TAN-67 was shown to have antinociceptive activity following subcutaneous administration in the mouse acetic acid abdominal constriction assay that was antagonized by NTI (delta 1- and delta 2-antagonist) and 7-benzylidinenaltrexone (delta 1-antagonist), but not by naltriben (delta 2-antagonist). This systemically applicable non-peptide agonist will be useful for elucidating the pharmacological properties of the delta-opioid receptor.     

Modulation of fatty acid-binding protein content of adult rat heart in response to chronic changes in plasma lipid levels

Cholecystokinin-octapeptide (CCK8) administered intraperitoneally (i.p.) in rats induces a rapid elevation in serum oxytocin (OT). The receptor subtype mediating this action of CCK was investigated with selective CCK-A and CCK-B receptor agonists and antagonists. CCK8 and A-71623, a potent CCK-A selective agonist, were similar in efficacy and potency for stimulating OT secretion. Both compounds at 10 nmol/kg elicited approximately one-half the response of 100 nmol/kg, which elevated serum OT to approx. 20 to 30-fold above basal level. The potent CCK-B selective agonist, A-63387, at doses up to 100 nmol/kg did not increase serum OT. MK-329, a CCK-A receptor selective antagonist, at a dose of 20 nmol/kg fully inhibited the action of 20 nmol/kg CCK8, while 100 nmol/kg of (R)L-365,260, a CCK-B selective antagonist, had no effect on the CCK8 response. These results, together with previous lesion studies, suggest that vagal CCK-A receptors in the periphery mediate the activation of the oxytocinergic pathway in vivo.     

Camostate- and caerulein-induced delay of gastric emptying in the rat: effect of CCK receptor antagonists

The effect of camostate, a potent releaser of endogenous cholecystokinin (CCK), and of caerulein, an amphibian peptide mimicking the biological actions of CCK, as well as of selective CCK receptor antagonists on gastric emptying of liquids was studied in the rat. Oral administration of camostate (200 mg/kg with the liquid test meal preceded by the same dose 10 min before the meal) significantly delayed gastric emptying of saline, an effect which was completely blocked by previous administration of the CCKA receptor antagonist, devazepide, at a dose (1 mg/kg i.v.) unable to modify the emptying rate when administered alone. Caerulein (0.03-30 nmol/kg i.v.) also delayed the emptying rate in a dose-dependent manner, with an ID50 of 3.94 nmol/kg. The effect of the peptide was also inhibited by devazepide. The CCKB receptor antagonist, L365,260 (3R-(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1, 4-benzodiazepine-3-yl)-N'-(3-methylphenyl)-urea; 3 mg/kg i.v.), was completely unable to modify the CCK (both endogenous and exogenous)-induced delay in gastric emptying. Repeated (7 days) camostate administration did not modify the gastric motor response to endogenous CCK, thus, suggesting that adaptation did not take place. These results demonstrate that endogenous and exogenous CCK delays gastric emptying of liquids through stimulation of CCKA receptors and suggest that adaptation of the gastric motor response to CCK does not occur.     

Protein kinase C-mediated inhibition of transmembrane signalling through CCK(A) and CCK(B) receptors

1. The rat CCK(A) and CCK(B) receptors were stably expressed in Chinese hamster ovary (CHO-09) cells in order to compare modes of signal transduction and effects of protein kinase C (PKC) thereupon. 2. Spectrofluorophotometry of Fura-2-loaded cells revealed that both receptors retained their pharmacological characteristics following expression in CHO cells. Sulphated cholecystokinin-(26-33)-peptide amide (CCK-8-S) increased the cytosolic Ca2+ concentration ([Ca2+]i) in CCK(A) cells, measured as an increase in Fura-2 fluorescence emission ratio, 1000 fold more potently than its non-sulphated form (CCK-8-NS) (EC50 values of 0.19 nM and 0.18 microM, respectively). By contrast, CCK-8-S and CCK-8-NS were equally potent in CCK(B) cells (EC50 values of 0.86 nM and 1.18 nM, respectively). The CCK(A) receptor agonist JMV-180 increased [Ca2+]i only in CCK(A) cells. Likewise, pentagastrin increased [Ca2+]i only in CCK(B) cells. Finally, CCK-8-S-induced Ca2+ signalling through the CCK(A) receptor was most potently inhibited by the CCK(A) receptor antagonist L364,718, whereas the CCK(B) receptor antagonist L365,260 was more potent in CCK(B) cells. 3. Receptor-mediated activation of adenylyl cyclase was measured in the presence of the inhibitor of cyclic nucleotide phosphodiesterase activity, 3-isobutyl-1-methylxanthine. CCK-8-S and, to a lesser extent, CCK-8-NS, but not JMV-180 or pentagastrin, stimulated the accumulation of cyclicAMP in CCK(A) cells. By contrast, none of these agonists increased cyclicAMP in CCK(B) cells. 4. Short-term (3 min) pretreatment with the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) evoked a rightward shift of the dose-response curve for the Ca2+ mobilizing effect of CCK-8-S in both cell lines. In addition, short-term TPA pretreatment markedly reduced CCK-8-S-induced cyclicAMP accumulation in CCK(A) cells. In both cases, the inhibitory effect of TPA was abolished by the PKC inhibitors, GF-109203X and staurosporine, whereas no inhibition was observed with the inactive phorbol ester, 4-alpha-phorbol 12-myristate 13-acetate. 5. During prolonged TPA treatment, the cells gradually recovered from phorbol ester inhibition and in the case of CCK-8-S-induced Ca2+ mobilization complete recovery was achieved after 24 h of TPA treatment. Western blot analysis revealed that this recovery was paralleled by down-regulation of PKC-alpha, suggesting the involvement of this PKC isotype in the inhibitory action of TPA. 6. This study demonstrates that following expression in CHO cells (i) both CCK(A) and CCK(B) receptors are coupled to Ca2+ mobilization, (ii) only CCK(A) receptors are coupled to cyclicAMP formation and (iii) with both receptors signalling is inhibited by PKC.     

The effect of centrally administered CCK-receptor antagonists on food intake in rats

Cholecystokinin (CCK) receptors are classified as two subtypes, designated CCK(A) and CCK(B), and both subtypes are found in brain and peripheral tissues of rats. CCK-8 has been shown to act peripherally to reduce meal size, and this satiating action can be blocked by CCK(A)-receptor antagonists. Recent evidence suggests that, in addition to the peripheral action of CCK, central CCK mechanisms may also be involved in satiety. Central administration of proglumide, a mixed CCK-receptor antagonist (CCK(A) > CCK(B)) has been shown to increase food intake and block the satiating effect of peripherally administered CCK-8 (15). In an attempt to replicate and extend these results, rats were given injections of proglumide or selective CCK-receptor antagonists into the lateral ventricle prior to a peripheral injection of CCK-8 or saline. Only proglumide stimulated an increase in 30-min test meal intake and attenuated the satiating effect of CCK-8. Two selective CCK(A)-receptor antagonists, lorglumide and devazepide, did not increase intake significantly when given alone, and they did not attenuate the effect of peripherally administered CCK-8. The selective CCK(B)-receptor antagonist, L365,260, reduced intake at all doses tested except the lowest. The lowest dose did not increase intake when given alone and did not attenuate the inhibitory effect of CCK on test-meal intake. Finally, a combination of devazepide and L365,260 did not increase intake or block the effect of peripherally administered CCK-8. These results suggest that CCK released by neurons in the brain and acting on central CCK(A)- and CCK(B)-receptors is not necessary for the control of meal size or for the satiating effect of peripherally administered CCK-8 in rats under our experimental conditions.     

Kappa1- and kappa2-opioid receptors mediating presynaptic inhibition of dopamine and acetylcholine release in rat neostriatum

1. The effects of selective opioid receptor agonists and antagonists on N-methyl-D-aspartate (NMDA, 10 microM)-induced release of [3H]-dopamine and [14C]-acetylcholine (ACh) from superfused neostriatal slices were studied to investigate the possible occurrence of functional kappa-opioid receptor subtypes in rat brain. 2. The kappa receptor agonists (-)-ethylketocyclazocine ((-)-EKC), U69593 and the endogenous opioid peptide dynorphin A1-13 caused a naloxone-reversible inhibition of NMDA-induced [3H]-dopamine release, with pD2 values of about 9, 8.5 and 8.2, respectively, whereas both the mu agonist Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO) and the delta agonist D-Pen2-D-Pen5-enkephalin (DPDPE) were ineffective in this respect. The inhibitory effect of submaximally effective concentrations of dynorphin A1-13, U69593 and (-)-EKC on NMDA-induced [3H]-dopamine release were not changed by the delta1/delta2-opioid receptor antagonist naltrindole (up to a concentration of 1 microM, but reversed by the kappa receptor antagonist nor-binaltorphimine (nor-BNI), with an IC50) as low as 0.02 nM, indicating the involvement of U69593-sensitive kappa1-opioid receptors. 3. NMDA-induced [14C]-ACh release was reduced in a naloxone-reversible manner by DPDPE (pD2 about 7.2), dynorphin A1-13 (pD2 6.7) and EKC (pD2 6.2), but not by U69593 and DAMGO. The inhibitory effect of a submaximally effective concentration of DPDPE, unlike those of dynorphin A1-13 and (-)-EKC, on NMDA-induced [14C]-ACh release was antagonized by naltrindole with an IC50 of 1 nM, indicating the involvement of delta-opioid receptors in the inhibitory effect of DPDPE. On the other hand, the inhibitory effects of dynorphin A1-13 and (-)-EKC on [14C]-ACh release were readily antagonized by nor-BNI with an IC50 of about 3 nM. A 100 fold higher concentration of nor-BNI also antagonized the inhibitory effect of DPDPE, indicating the involvement of U69593-insensitive kappa2-opioid receptors in the inhibitory effects of dynorphin A1-13 and (-)-EKC. 4. Although naloxone benzoylhydrazone (NalBzoH), displaying high affinity towards the putative kappa3-opioid receptor, antagonized the inhibitory effects of dynorphin A1-13 and (-)-EKC on [3H]-dopamine and [14C]-ACh release as well as that of U69593 on [3H]-dopamine release, it displayed a low apparent affinity (IC50 about 100 nM) in each case. 5. In conclusion, whereas activation of kappa1-opioid receptors causes presynaptic inhibition of NMDA-induced dopamine release, kappa2 receptor activation results in inhibition of ACh release in rat neostriatum. As such, this study is the first to provide unequivocal in vitro evidence for the existence of functionally distinct kappa-opioid receptor subtypes in the brain.     

The CCKB antagonist, L-365,260, attenuates fear-potentiated startle

The neuropeptide cholecystokinin (CCK), via the CCKB receptor, increases behaviors associated with anxiety in laboratory animals and humans. The present experiment assessed the role of endogenous CCKB function in fear-potentiated startle, a test of "anxiety" in rats. The amplitude of the acoustic startle response is potentiated if preceded by a stimulus that has been previously paired with shock. Pretreatment with the CCKB antagonist L-365,260 (0, 0.1, 1.0, and 10.0 mg/kg, IP) did not affect baseline acoustic startle amplitudes, but dose-dependently decreased fear-potentiated startle. These results indicate that the specific attenuation of fear-potentiated startle induced by L-365,260 was not due to a general decrease in motor responsivity. The present findings are consistent with the effects of CCKB antagonists in other tests measuring anxiety in animals.     

Dopamine depletion augments endogenous opioid-induced locomotion in the nucleus accumbens using both mu 1 and delta opioid receptors

The aim of this study is to analyze further the opioid receptor subtypes involved in the augmentation of behavioral activity after dopamine depletion in the nucleus accumbens of rats. Initially, the opioid receptors involved in the augmentation of locomotion produced by endogenous opioids were evaluated by microinjection of kelatorphan, an inhibitor of proteolytic enzymes that inactivates enkephalin, with or without specific antagonists for mu 1 or delta-opioid receptors, naloxonazine or naltrindole, respectively. Kelatorphan produced a dose-dependent increase in horizontal photocell counts and vertical movements. At all doses examined the behavioral response was augmented in rats sustaining accumbal dopamine lesions. The augmentation in dopamine-depleted rats was partially blocked by naloxonazine or naltrindole. Since the motor stimulant response to intra-accumbens microinjection of the delta-opioid agonist, [D-penicillamine2,5]-enkephalin, was not augmented in a previous study, we tested the behavioral response to a new endogenous delta-opioid agonist, [D-Ala2] deltorphin I. The locomotor response to deltorphin was slightly augmented in dopamine-depleted rats. These data suggest that the augmentation in the motor response elicited by endogenous opioids after dopamine lesions in the nucleus accumbens involves both mu 1, and delta-opioid receptors.     

Naltrindole administration during the preweanling period and manipulation affect adrenocortical reactivity in young rats

The effect of a daily injection of the delta-selective opioid antagonist naltrindole (1 mg/kg), from birth to postnatal day 19, on basal and post-stress corticosterone levels in 25-day old rats of both sexes was investigated. The effects of manipulation were studied by including two control groups, one group received daily injections of saline and a second one was not manipulated. The stress protocol consisted of a 3 min swimming session in water at 20 degrees C. Corticosterone determinations were performed by radioimmunoassay. Control non-manipulated animals showed a significant increase in corticosterone levels in response to stress. Manipulation decreased basal hormone levels in females and prevented the stress-induced rise in corticosterone in males. Functional blockade of the delta-receptor during the preweanling period by the naltrindole treatment inhibited the corticosterone response to stress in females. The results indicate the existence of sex differences in the effects of manipulation on hypothalamus-pituitary-adrenal axis activity and the involvement of the delta-opioid receptor in the modulation of the adrenocortical response to stress during the postnatal period.