Interaction between the mu-agonist dermorphin and the delta-agonist [D-Ala2, Glu4]deltorphin in supraspinal antinociception and delta-opioid receptor binding

1. In rats, the interaction between the mu-opioid agonist dermorphin and the delta-opioid agonist [D-Ala2, Glu4]deltorphin was studied in binding experiments to delta-opioid receptors and in the antinociceptive test to radiant heat. 2. When injected i.c.v., doses of [D-Ala2, Glu4]deltorphin higher than 20 nmol produced antinociception in the rat tail-flick test to radiant heat. Lower doses were inactive. None of the doses tested elicited the maximum achievable response. This partial antinociception was accomplished with an in vivo occupancy of more than 97% of brain delta-opioid receptors and of 17% of mu-opioid receptors. Naloxone (0.1 mg kg-1, s.c.), and naloxonazine (10 mg kg-1, i.v., 24 h before), but not the selective delta-opioid antagonist naltrindole, antagonized the antinociception. 3. In vitro competitive inhibition studies in rat brain membranes showed that [D-Ala2, Glu4]deltorphin displaced [3H]-naltrindole from two delta-binding sites of high and low affinity. The addition of 100 microM Gpp[NH]p produced a three fold increase in the [D-Ala2, Glu4]deltorphin Ki value for both binding sites. The addition of 10 nM dermorphin increased the Ki value of the delta-agonist for the high affinity site five times. When Gpp[NH]p was added to the incubation medium together with 10 nM dermorphin, the high affinity Ki of the delta-agonist increased 15 times. 4. Co-administration into the rat brain ventricles of subanalgesic doses of dermorphin and [D-Ala2, Glu4]deltorphin resulted in synergistic antinociceptive responses. 5. Pretreatment with naloxone or with the non-equilibrium mu-antagonists naloxonazine and beta-funaltrexamine completely abolished the antinociceptive response of the mu-delta agonist combinations. 6. Pretreatment with the delta-opioid antagonists naltrindole and DALCE reduced the antinociceptive response of the dermorphin-[D-Ala2, Glu4]deltorphin combinations to a value near that observed after the mu-agonist alone. At the dosage used, naltrindole occupied more than 98% of brain delta-opioid receptors without affecting mu-opioid-receptors. 7. These data suggest that in the rat tail-flick test to radiant heat, mu- and delta-opioid agonists co-operate positively in evoking an antinociceptive response. Although interactions between different opioid pathways cannot be excluded, in vitro binding results indicate that this co-operative antinociception is probably mediated by co-activation of the delta-opioid receptors at the cellular level by the mu- and delta-agonist.     

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.     

Antidepressant-like effects of CCK(B) receptor antagonists: involvement of the opioid system

RB 101 (N-[(R,S)-2-benzyl-3-[(S)-2-amino-4-methylthiobutyldithio]-1-oxopr opyl]-L -phenylalaninebenzyl ester), a systemically active inhibitor of enkep halin catabolism, has been shown to elicit antidepressant-like effects in mice, both in the forced-swimming and in the conditioned suppression of the mobility tests. The same type of response has been also observed following administration of the cholecystokinin CCK(B) receptor antagonist L-365,260 ((3R)-(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin -3-yl)-3 -methylphenylurea). In terestingly, the delta-opioid receptor antagonist naltrindole (17-cyclopropylmethyl-6,7-dehydro-4,5alpha-epoxy-3,14-dihydroxy-6, 7,2'-3'-indolomorphinan) blocks the effect of both RB 101 and L-365,260 in the conditioned suppression of the motility test. In this work we have investigated the involvement of the opioid system in the antidepressant response to the CCK(B) receptor antagonist L-365,260 in the forced-swimming test in mice. The effect of L-365,260 was decreased by the delta-opioid receptor antagonist naltrindole. Furthermore, the CCK(B) receptor agonist, BC 264 (Boc-Tyr(OSO3H)-gNle-mGly-Trp-(NMe)Nle-Asp-Phe-NH2), blocked the antidepressant-like effect of RB 101 while CCK-8 (H-Asp-Tyr(OSO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2) enhanced the effect of this drug, probably through stimulation of central CCK(A) receptors, since the CCK(A) receptor antagonist devazepide ((3S)-(-)-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin++ +-3-yl)-1H-indole-2 -carboxamide) abolished the CCK-8-induced potentiation of the RB 101 effect. In addition, RB 101 enhanced the effect of L-365,260. Such an effect was blocked by the delta-opioid receptor antagonist naltrindole. These data further support the involvement of opioid receptors in the antidepressant-type effect induced by CCK(B) receptor blockers and support the hypothesis of a regulatory role of CCK in the activity of the endogenous opioid system. As in other experimental paradigms, CCK(A) and CCK(B) receptor stimulation appears to have opposite effects in modulating opioidergic activity.     

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.     

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.     

Naltrexone, naltrindole, and CTOP block cocaine-induced sensitization to seizures and death

I.c.v. injection for 9 days of either naltexone (NTX) (5, 10, 20, 40 micrograms/rat) or a selective mu peptide (CTOP) (0.125, 0.25, 0.5, 1, 3, 6 micrograms/rat) or delta (naltrindole) (NLT) (5, 10, 20 micrograms/rat) subtype opioid receptor antagonist affected sensitization to cocaine (COC) (50 mg/kg, i.p.) administered 10 min after. NTX (5 and 40 micrograms/rat), NLT (10 and 20 micrograms/rat), and the peptide CTOP (0.25-0.5 microgram/rat) attenuated seizure parameters (percent of animals showing seizures, mean score and latency) in a day-related manner. The DD50 (days to reach 50% of death) value for COC was 2.69, whereas it was 9.67 and 7.27 for NTX 5 and 40 micrograms/rat, 8.59 for NLT (10 micrograms/rat), and 6.11, 5.95, and 4.30 for CTOP (0.25, 0.5, and 1 microgram/rat respectively). These findings suggest a concurrent involvement of mu- and delta-opioid receptor subtype in COC-induced sensitization to toxic effects.     

Phosphorylation of adenylyl cyclase VI upon chronic delta-opioid receptor stimulation

An immunoprecipitation method was used to measure [32P]phosphate incorporation into the adenylyl cyclase VI protein in Chinese Hamster Ovary (CHO) cells stably expressing the human delta-opioid receptor. Chronic SNC 80 ((+)-4-[(alpha R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N ,N-diethyl-benzamide) 1 microM, 24 h) treatment increased the incorporation of [32P] into a 200 kDa protein band 2.5-fold after gel electrophoresis. The increase in phosphorylation of adenylyl cyclase VI was antagonized by naltrindole (1 microM) and the immunoprecipitation was prevented by the saturation of the antibody with the blocking peptide.     

Modulation of protein kinase C and cAMP-dependent protein kinase by delta-opioid

Modulation of protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) activities by delta-opioid receptor specific agonist [D-Pen2, D-Pen5]-enkephalin (DPDPE) was investigated in neuroblastoma x glioma hybrid NG 108-15 cells. DPDPE activated PKC in a dose-dependent manner, with the maximal response at 5 min. The DPDPE-stimulated PKC activation could be blocked by naltrindole. The activation of PKC by DPDPE was dependent on Ca2+ and was inhibited by chelerythrine chloride (10 microM), but not by H89 (1 microM). Pretreatment of NG 108-15 cells with pertussis toxin (100 ng/ml for 24 h) completely abolished DPDPE-stimulated PKC activation. In contrast to the result from the acute treatment with DPDPE, which had no significant effect on PKA activity, chronic treatment of DPDPE (1 microM for 24 h) increased PKA activity, but reduced the basal activity of PKC. These results demonstrated that DPDPE differentially modulated PKC and PKA activities via a receptor-mediated, PTX sensitive pathway.     

Family history as a risk factor for ulcerative colitis-associated colon cancer in cotton-top tamarin

1. Debate exists as to the nature of antidepressant-induced antinociception. It is unclear whether antidepressants are inherently antinociceptive, are able to potentiate opioid antinociception or both. We have used the acetic acid induced abdominal constriction assay in mice to investigate antidepressant-induced antinociception. 2. All the antidepressants tested (s.c.) produced dose-dependent protection against acetic acid-induced abdominal constriction. Similarly, morphine and aspirin were also effective antinociceptive agents in this nociceptive assay. 3. Opioid antagonists, naloxone (0.5 mg kg(-1), s.c.) and naltrindole (1 mg kg(-1), s.c.), shifted the dose-response relationships to the right for each of the antidepressant agents (dothiepin, amitriptyline, sibutramine, (+)-oxaprotiline and paroxetine). In this context the naloxone dose-ratios were 1.95, 3.90, 2.32, 4.50 and 2.65, with naltrindole dose-ratios of 4.36, 17.00, 4.28, 11.48 and 2.65 were obtained, respectively. Naloxone also shifted the morphine dose-response relationship to the right, by a factor of 2.62, whilst naltrindole had no effect upon morphine antinociception. Aspirin antinociception remained unaffected by both opioid antagonists. 4. The enkephalin catabolism inhibitor acetorphan, by itself, produced no activity in this test at a dose of 10 mg kg(-1) (s.c.). However, at higher doses, acetorphan produced a linear dose-response relationship against acetic acid-induced abdominal constriction. 5. When acetorphan was administered before either the antidepressants or morphine, there was a clear potentiation of the antidepressant- or morphine-induced antinociception. However, acetorphan had no effect on aspirin antinociception. 6. Since neither of the opioid antagonists were able to attenuate, nor was acetorphan able to potentiate, aspirin antinociception, we concluded that the mechanism of antidepressant-induced antinociception is different from that of the non-steroidal anti-inflammatory drugs. 7. These data are consistent with the view that antidepressants may induce endogenous opioid peptide release, as shown by the acetorphan study. In this context, the ability of naltrindole to displace the antidepressant dose-response relationship to the right without affecting morphine antinociception, implicates the delta-opioid receptor and endogenous opioid peptides in antidepressant-induced antinociception.     

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.     

Antinociceptive effect of L-arginine in diabetic mice

The antinociceptive effect of L-arginine in streptozotocin-induced diabetic mice was examined. Although s.c. administration of L-arginine produced a dose-dependent inhibition of the tail-flick response in both non-diabetic and diabetic mice, the antinociceptive response was greater in diabetic mice than in non-diabetic mice. The antinociceptive effects of L-arginine in both diabetic and non-diabetic mice were significantly antagonized by s.c. administration of naltrindole, a selective delta-opioid receptor antagonist. However, neither beta-funaltrexamine, a selective mu-opioid receptor antagonist, nor nor-binaltorphimin ++, a selective kappa-opioid receptor antagonist, significantly affected the antinociceptive effect of L-arginine in diabetic and non-diabetic mice. These results suggest that L-arginine produces a marked antinociceptive effect in diabetic mice through the activation of delta-opioid receptors.     

Modulation of integrin alpha-v-beta-1 expression on human tumor cells by leukemia inhibitory factor (LIF) and oncostatin M (OSM)

Corticostatins/defensins are a family of cationic peptides recently isolated from phagocytotic cells of the myeloid lineage. Natural killer (NK) cells are spontaneously cytotoxic large granular lymphocytes that are involved in immunosurveillance against cancer and infections. Their activity is modulated by hormones of the hypothalamic-pituitary-adrenal axis. We wished to determine whether two human corticostatins/defensins, HP-1 and HP-4, are able to change in vitro the spontaneous NK activity of human peripheral blood mononuclear cells (PBMC) and the responses either to the stimulatory cytokines immune interferon (IFN-gamma) or interleukin 2 (IL-2) and to the inhibitory hormone cortisol. NK cell activity was measured in a 4-h direct cytotoxicity assay with K562 cells as a target. HP-1 and HP-4 (10 (-8) -10 (-9) M) significantly inhibited the spontaneous and cytokine-inducible NK activity, and increased the cortisol-dependent inhibition. Radioimmunoassay of HPLC purified fractions obtained from sonicated NK cells showed HP-1 in the two cell preparations examined. We also evaluated the effects of HP-1 and HP-4 (10 (-8) M -10(-9) M) upo IFN-gamma and interleukin 6 (IL-6) production by PBMC stimulated with phytohemagglutinin (PHA) or concanavalin A (ConA). IFN-gamma was titrated with the biological assay using WISH cells as indicators and vescicular stomatitis virus (VSV) as the challenge virus. IL-6 was measured using an enzyme amplified sensitivity immunoassay. Both HP-1 and HP-4 significantly reduced cytokine production. Our data indicate that corticostatins/defensins are novel modulators of lymphocyte functions in vitro. Their immunodepressing properties could add complexity and plasticity to hypothalamic-pituitary-adrenal-cytokine circuits in vivo.     

Effect of atrial natriuretic peptide and cyclic GMP phosphodiesterase inhibition on collagen synthesis by adult cardiac fibroblasts

1. Cardiac fibroblasts play an important role in the pathophysiology of cardiac remodelling induced by hypertension and myocardial infarction by undergoing proliferation and depositing extracellular matrix proteins such as collagen. We have examined the effects of atrial natriuretic peptide (ANP) on proliferation and collagen synthesis by adult rat and human cardiac fibroblasts in culture. 2. In cells from both species radioligand studies using 125I-ANP suggested that the majority of binding sites (> 85%) were non-guanylyl cyclase-linked (NPR-C subtype). Nonetheless ANP (10(-9) to 10(-6) M), in the presence of zaprinast, an inhibitor of phosphodiesterase 5 (PDE5), increased fibroblast cyclic GMP levels 3-5 fold in a concentration-dependent manner (P < 0.05). 3. ANP (10(-11) to 10(-6) M), a NPR-C ligand, C-ANF4-23 (10(-11) to 10(-6) M) and zaprinast alone had no significant effect on either basal or serum-stimulated DNA synthesis or fibroblast number. In combination with zaprinast (10(-5) M), however, ANP (10(-9) to 10(-6) M) but not C-ANF4-23 (10(-7) M) inhibited markedly both basal and stimulated fibroblast mitogenesis, an effect reproduced by 8-bromo-cyclic GMP (10(-5) to 10(-3) M). 4. Collagen synthesis, determined by measuring hydroxyproline levels, was stimulated with transforming growth factor-beta1 (40 pM), angiotensin II (10(-7) M) or 2% foetal bovine serum. The increase in collagen production, normalised by cell number, was reduced dramatically (to at or near basal production) by ANP (10(-9) to 10(-7) M) but not C-ANF4-23 (10(-7) M) in the presence of zaprinast. Again 8-bromo-cyclic GMP (10(-5) to 10(-3) M) reproduced the effect. 5. ANP is capable of inhibiting collagen synthesis in adult rat and human cardiac fibroblasts via cyclic GMP, a property unmasked and enhanced by inhibition of PDE5.     

Pancreatic polypeptide stimulates rat adrenal glucocorticoid secretion by activating the adenylate cyclase-dependent signaling pathway

Pancreatic polypeptide (PP) concentration-dependently raised basal corticosterone and cyclic-AMP production of dispersed rat zona fasciculata/reticularis adrenocortical cells, maximal effective concentration being 10(-7) M. 10(-7) M PP also significantly enhanced submaximally (10[-12]/10[-11] M), but not maximally (10[-9]/10[-8] M) ACTH-stimulated corticosterone and cyclic-AMP release. Corticosterone responses to PP were abolished by the specific protein kinase A (PKA) antagonist H-89 (10[-5] M). The selective ACTH-receptor antagonist corticotropin-inhibiting peptide (10[-6] M) annulled corticosterone response to 10(-9) M ACTH, but not to 10(-7) M PP. Collectively, our present findings indicate that PP stimulates glucocorticoid secretion of rat adrenal glands, acting through specific receptors coupled, like those of ACTH, with the adenylate cyclase/PKA-dependent signaling pathway.     

Regulation of spontaneous activity of the delta-opioid receptor: studies of inverse agonism in intact cells

Adenylyl cyclase activity was measured following labelling of the cellular ATP pool with [3H]adenine in intact Rat-1 fibroblasts that had been stably transfected to express the murine delta-opioid receptor (clone D2). Basal [3H]cyclic AMP accumulation was low and was increased substantially by the addition of the diterpene forskolin. The synthetic enkephalin D-Ala2,D-Leu5 enkephalin (DADLE) produced strong inhibition of forskolin-amplified [3H]cyclic AMP production, whereas the delta-opioid ligand ICI174864 augmented forskolin-amplified adenylyl cyclase activity. Naloxone was unable to mimic the effects of ICI174864, and coincubation of the cells with these two ligands attenuated the effect of ICI174864. The EC50 (9.4 +/- 0.6 x 10(-8) M) for ICI174864 augmentation of forskolin-stimulated adenylyl cyclase was equal to its estimated Ki. Pertussis toxin pretreatment of clone D2 cells prevented both this effect of ICI174864 and the inhibition produced by DADLE. Use of a Cytosensor microphysiometer demonstrated that treatment of clone D2 cells with DADLE increased and that with ICI174864 decreased the basal rate of cellular proton extrusion. By using these two distinct experimental strategies, ICI174864 was shown to function in a manner anticipated for an inverse agonist, demonstrating that such effects can be observed in intact cells and are not restricted to assays performed on membrane preparations.     

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.     

Endothelin-induced prostacyclin production in rat aortic rings is mediated by protein kinase C

Endothelin (ET) is a vasoconstrictor peptide released from endothelial cells that is known to cause prostaglandin release. The mechanism remains unclear. To determine whether the protein kinase C (PKC) signaling pathway is stimulated by endothelin, we pretreated rat aortic rings with either PKC activator or inhibitors and measured the release of prostacyclin (PGI2) by radioimmunoassay. ET (10(-9) M) produced a 10-fold increase in PGI2 release. Pretreatment with 10(-9) M of three different PKC inhibitors, 1-(5-isoquinolinesulfonyl)piperazine(CL), staurosporine, and 1-(5-isoquinolinesulfonyltmethyl)piperazine (H7), blocked ET-induced PGI2 release. ET-induced PGI2 release was also blocked by pretreatment with inhibitors of either phospholipase A2 7,7-dimethyleicosadienoic acid or trifluoromethyl ketone analogue) (10(-9) M) or cyclooxygenase (indomethacin) (10(-9) M). We conclude that ET activates PKC, which activates phospholipase A2, which liberates arachidonic acid, which increases PGI2 production and release.     

Distribution of fasting serum insulin measured by enzyme immunoassay in an unselected population of 4,032 individuals. Reference values according to age and sex. D.E.S.I.R. Study Group. Données Epidémiologiques sur le Syndrome d''Insulino-Résistance

The effects of tumor necrosis factor alpha (TNF-alpha) on arachidonic acid (AA) metabolism were investigated by prelabeling the human osteoblastic osteosarcoma cell line, G292, with [3H]AA. TNF-alpha differentially stimulates cyclooxygenase and lipoxygenase pathways of AA metabolism in a dose response manner in the cells. The highest concentration of TNF-alpha (10(-8)M) significantly increased the cyclooxygenase pathway, with prostaglandin E2 (PGE2) being a major product. However, at the lowest concentration (10(-10)M) of TNF-alpha, 15-hydroxyeicosatetraenoic acid (HETE) production was significantly increased, with no significant effects on the other identifiable products. When the concentration of TNF-alpha was increased to 10(-9) M leukotriene B4 (LTB4), 15-, 12-, and 5-HETE were significantly increased. The calcium ionophore A23187 (10(-6) M) significantly increased 15-HETE production, without significantly affecting cyclooxygenase metabolites. However, a combination of TNF-alpha (10(-8)M) and A23187 (10(-6)M) caused an inhibitory effect on each agent-induced PGE2 or 15-HETE production.     

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.