D-Pen2,D-Pen5]enkephalin Research Articles

Opioid–dopamine interaction in planaria: a behavioral study

The behavioral response of planaria to the exposure to selective opioid agonists was studied. The μ agonist [d-ala 2, N-methyl-Phe 4,Gly 5-ol]enkephalin (DAMGO) and the δ agonist [D-Pen 2, D-Pen 5]enkephalin (DPDPE) failed to alter motor activity at all doses tested. Low doses of the selective κ agonist (±)-trans-U-50-trans-3,4-dichloro- N-methyl- N[2-(1-pyrrodinyl)-cyclohexyl]benzene acetamide methasulphonate (U50, 488) and bremazocine–HCl increased motor activity leading to C-like position (CLP) and screw-like hyperkinesia (SLH). These changes were identical to those seen previously with the exposure to D2 or D1 dopamine receptor agonists, respectively. Higher doses of κ agonists produced the enhancement of CLP and SLH together with robust snake-like movements (SLM). This latter response, that was typical of stimulation of κ opioid receptors, was blocked by co-exposure to naloxone or the selective κ antagonist Nor-binaltorphimine (Nor-BNI). Finally, co-exposure to sulpiride or SH-23390 respectively blocked the CLP or SLH response produced by U50,488 or bremazocine. Our data indicate the presence of κ opioid receptors in planaria and suggest the functional interaction between the opioid and dopamine system in this simple animal model.

Potentiation of anaphylaxis in guinea pig ileal mucosa by a selective δ-opioid receptor agonist

Immediate hypersensitivity reactions in the intestinal mucosa evoke active chloride secretion which enhances the elimination of luminal antigens. The prosecretory actions of histamine and other soluble mediators of anaphylaxis are mediated by submucosal neurons, as are the antisecretory actions of opioid antidiarrheal medications. We tested the hypothesis that the selective δ-opioid receptor agonist [ d-Pen 2, d-Pen 5]enkephalin (DPDPE) alters anaphylaxis-associated ileal anion secretion in vitro. Sheets of ileal mucosa with attached submucosa from guinea pigs sensitized to cow's milk were mounted in Ussing chambers under short-circuit conditions. Mucosal sheets responded to the serosal application of the milk protein, β-lactoglobulin, with a rapid rise in transepithelial short-circuit current (Isc); in contrast, the egg protein, ovalbumin, was without effect. Pretreatment of tissues with the neuronal conduction blocker, saxitoxin, or the H 1 histamine receptor antagonist, diphenhydramine, but not the opioid receptor antagonist, naloxone, significantly reduced mucosal responses to antigen. [ d-Pen 2, d-Pen 5]enkephalin (0.1 μM, serosal addition) decreased baseline Isc, but potentiated mucosal responses to antigen; its effects were abolished in tissues pretreated with naloxone. These results suggest that immediate hypersensitivity reactions in the guinea pig ileal mucosa are mediated by submucosal neural circuits that are phasically modulated by both mast cell products and opioids.

Attenuation of δ opioid receptor-mediated signaling by kainic acid in neural cells: involvement of protein kinase C and intracellular Ca 2+

The potential modulation of opioid receptor signaling by kainic acid (KA) has been investigated in neuroblastoma×glioma NG 108-15 hybrid cells and neuroblastoma SK-N-SH cells. Acute incubation of KA significantly attenuated δ opioid receptor (DOR) signaling induced by the DOR agonist [ d-Pen 2, d-Pen 5]-enkephalin (DPDPE), as measured by activation of G proteins and inhibition of cAMP accumulation. The attenuation by KA was time- and dose-dependent and could be blocked by antagonists of kainate/AMPA receptors, suggesting possible mediation through kainate/AMPA receptors. KA attenuation of DPDPE-stimulated G protein activation was reversed by inhibitors of protein kinase C or by removal of both extracellular Ca 2+ and intracellular Ca 2+. In contrast, NMDA attenuation of DPDPE-stimulated G protein activation was independent of intracellular Ca 2+, indicating that different mechanism(s) may underlie the modulation effect of KA and NMDA. This notion was further supported by the results that KA did not alter nociceptin/orphanin FQ-stimulated G protein activation in NG 108-15 cells but NMDA did. In addition, pretreatment of NG 108-15 cells with antagonists of kainate/AMPA receptors blocked the acute desensitization of DOR signaling. These data provide evidence that KA may be involved in the modulation of opioid receptor signal transduction.

Kinetic Study of N-Type Calcium Current Modulation by δ-Opioid Receptor Activation in the Mammalian Cell Line NG108-15

The voltage-dependent inhibition of N-type Ca 2+ channel current by the δ-opioid agonist [ d-pen 2, d-pen 5]-enkephalin (DPDPE) was investigated in the mammalian cell line NG108-15 with 10 μM nifedipine to block L-type channels, with whole-cell voltage clamp methods. In in vitro differentiated NG108-15 cells DPDPE reversibly decreased ω-conotoxin GVIA-sensitive Ba 2+ currents in a concentration-dependent way. Inhibition was maximal with 1 μM DPDPE (66% at 0 mV) and was characterized by a slowing of Ba 2+ current activation at low test potentials. Both inhibition and kinetic slowing were attenuated at more positive potentials and could be relieved up to 90% by strong conditioning depolarizations. The kinetics of removal of inhibition (de-inhibition) and of its retrieval (re-inhibition) were also voltage dependent. Both de-inhibition and re-inhibition were single exponentials and, in the voltage range from −20 to +10 mV, had significantly different time constants at a given membrane potential, the time course of re-inhibition being faster than that of de-inhibition. The kinetics of de-inhibition at −20 mV and of reinhibition at −40 mV were also concentration dependent, both processes becoming slower at lower agonist concentrations. The rate of de-inhibition at +80/+120 mV was similar to that of Ca 2+ channel activation at the same potentials measured during application of DPDPE (∼7 ms), both processes being much slower than channel activation in controls (<1 ms). Moreover, the amplitude but not the time course of tail currents changed as the depolarization to +80/+120 mV was made longer. The state-dependent properties of DPDPE Ca 2+ channel inhibition could be simulated by a model that assumes that inhibition by DPDPE results from voltage- and concentration-dependent binding of an inhibitory molecule to the N-type channel.

Differential desensitization of human δ-opioid receptors by peptide and alkaloid agonists

The efficacy of different opioid agonists to induce acute desensitization of the human δ-opioid receptor-mediated inhibition of cAMP accumulation was investigated in the neuroblastoma cell line SK-N-BE, which endogenously expresses these receptors. While etorphine, a non-selective alkaloid agonist, caused 50% desensitization after a 30-min incubation, the same treatment in the presence of the selective peptide agonists, DPDPE ([ d-Pen 2, d-Pen 5]enkephalin) and deltorphin I (Tyr- d-Ala-Phe-Asp-Val-Val-Gly), almost totally desensitized the δ-opioid receptor-mediated inhibition of adenylyl cyclase. When SK-N-BE cells were prechallenged either with alkaloid or peptide agonist, we observed a cross-desensitization that was less marked when cells were pretreated with peptide agonists and then challenged with etorphine. Taken together, these results demonstrate that human δ-opioid receptors are differentially desensitized by alkaloid and peptide agonists.

Participation of the opioid system in the regulation of prolactin secretion in androgenized rats: effect of ovarian steroids

We examined the role of the opioid system on the regulation of prolactin secretion in neonatally androgenized rats and evaluated the participation of ovarian steroids in this regulation. Androgenized rats exhibited an increase of prolactin secretion with higher serum circulating levels in the afternoon (1800) than in the morning (1000). The administration of the opioid antagonist naloxone (2 mg/kg, 30 min before decapitation) reduced serum prolactin levels in both groups. To identify the opioid receptor subtypes involved in this regulation, opioid agonists were administered i.c.v. 15 min before the decapitation (1000). The μ-opioid receptor agonist DAMGO ([ d-Ala 2, NMe-Phe 4, Gly 5-ol]-enkephalin) caused a significant increase in serum prolactin concentration. The selective κ-opioid receptor agonist U-50, 488H ( trans-(±)-3,4-dichloro- N-[2(1-pyrrolidinyl)-cyclohexyl]-benzene acetamide methane sulfonate salt) induced a small but significant increase in serum prolactin levels but no effect was observed after administration of the δ-opioid agonist DPDPE ([ d-Pen 2, d-Pen 5]-enkephalin). The role of oestradiol and the opioid system in the continuous secretion of prolactin was also study. Chronic gonadectomy (3–4 weeks) reduced serum prolactin concentrations measured at 1000 but the administration of naloxone had no effect. Three days of oestrogen treatment (2 μg/rat in oil) restored serum prolactin levels compared with ovariectomized animals and this effect was abolished by naloxone treatment. Interestingly, acute ovariectomy or administration of tamoxifen to intact androgenized rats did not prevent the continuous secretion of prolactin observed in these animals and naloxone treatment reduced serum prolactin levels in both groups of rats. We also examine the participation of adrenal progesterone and the endogenous opioid peptides on the regulation of prolactin levels in androgenized rats. After adrenalectomy, no changes in serum prolactin levels (1000) were observed compared with the control animal and naloxone treatment significantly reduced circulating prolactin levels. Progesterone treatment to intact androgenized rats significantly increased prolactin levels and the administration of naloxone blocked the stimulatory effect of the steroid. These results suggest that the opioid system play a role in the regulation of prolactin secretion in androgenized rats modulated by the persistence of oestrogen action. Moreover, the presence or absence of progesterone did not modify the regulation of prolactin secretion by the opioids. The μ- and κ-opioid receptor subtypes are the ones involved in the modulation of pituitary prolactin secretion.

Modulation of Cocaine-Induced Antinociception by Opioid-Receptor Agonists

Cocaine can produce antinociception in a number of animal models. The present experiments were designed to determine if opioid receptor agonists modulate cocaine-induced antinociception in rats. Cocaine produced a dose-dependent increase in antinociception in the hot-plate, but not paw-pressure, test. The combination of cocaine and morphine or [D-Pen 2, D-Pen 5]enkephalin (DPDPE) produced results no greater than simple additivity in the hot-plate test. However, the combination of cocaine and morphine produced greater antinociception than morphine alone in the paw-pressure test. A low dose of U69,593 potentiated the effects of cocaine in the hot-plate test. In contrast, cocaine attenuated the effect of U69,593 in the paw-pressure test. Both naltrexone and the selective κ-opioid receptor antagonist nor-binaltorphamine (nor-BNI) blocked the potentiation of cocaine-induced antinociception by U69,593. The combination of U69,593 and cocaine can produce superadditive or subadditive effects, depending upon the doses and antinciceptive assay used.

Stereoselective interaction of ketamine with recombinant mu, kappa, and delta opioid receptors expressed in Chinese hamster ovary cells.

The authors examined the interaction of ketamine with recombinant mu, kappa, and delta opioid receptors and recombinant orphan opioid receptors expressed in Chinese hamster ovary cells (CHO-mu, CHO-kappa, CHO-delta, and CHO(ORL1), respectively). CHO-mu, CHO-kappa, and CHO-delta membranes were incubated with the opioid receptor radioligand [3H]diprenorphine at room temperature. Ketamine (racemic, R(-) and S(+)) was included at concentrations covering the clinical range. CHO(ORL1) membranes were incubated with [125I]Tyr(14)nociceptin and racemic ketamine at room temperature. The effects of racemic ketamine and selective opioid receptor agonists (mu: [D-Ala2, MePhe4, Gly(ol)5] enkephalin (DAMGO); kappa: spiradoline or delta: [D-pen2, D-pen5] enkephalin (DPDPE)) on forskolin-stimulated cyclic adenosine monophosphate formation also were examined. Data are mean +/- SEM. Racemic ketamine increased the radioligand equilibrium dissociation constant for [3H]diprenorphine from 85+/-5 to 273+/-11, 91+/-6 to 154+/-16, and 372+/-15 to 855+/-42 pM in CHO-mu, CHO-kappa, and CHO-delta, respectively. The concentration of radioligand bound at saturation was unaffected. In CHO-mu and CHO-kappa cells, racemic ketamine did not slow the rate of naloxone-induced [3H]diprenorphine dissociation. Ketamine and its isomers also displaced [3H]diprenorphine binding to mu, kappa, and delta receptors in a dose-dependent manner, with pKi values for racemic ketamine of 4.38+/-0.02, 4.55+/-0.04, and 3.57+/-0.02, respectively. S(+)-ketamine was two to three times more potent than R(-)-ketamine at mu and kappa receptors. Racemic ketamine displaced [125I]Tyr(14)nociceptin with an estimated affinity constant of 0.5 mM. Racemic ketamine inhibited the formation of cyclic adenosine monophosphate (naloxone insensitive) in a dose-dependent manner (concentration producing 50% inhibition approximately 2 mM) in all cell lines, including untransfected CHO cells. Ketamine (100 microM) reversed DAMGO (mu) and spiradoline (kappa) inhibition of formation of cyclic adenosine monophosphate. Ketamine interacts stereoselectively with recombinant mu and kappa opioid receptors.

Conformational analysis of beta-methyl-para-nitrophenylalanine stereoisomers of cyclo[D-Pen2, D-Pen5]enkephalin by NMR spectroscopy and conformational energy calculations.

Solution conformations of beta-methyl-para-nitrophenylalanine4 analogues of the potent delta-opioid peptide cyclo[D-Pen2, D-Pen5]enkephalin (DPDPE) were studied by combined use of nmr and conformational energy calculations. Nuclear Overhauser effect connectivities and 3JHNC alpha H coupling constants measured for the (2S, 3S)-, (2S, 3R)-, and (2R, 3R)-stereoisomers of [beta-Me-p-NO2Phe4]DPDPE in DMSO were compared with low energy conformers obtained by energy minimization in the Empirical Conformational Energy Program for Peptides (ECEPP/2) force field. The conformers that satisfied all available nmr data were selected as probable solution conformations of these peptides. Side-chain rotamer populations, established using homonuclear (3JH alpha H beta) and heteronuclear (3JH alpha C gamma) coupling constants and 13C chemical shifts, show that the beta-methyl substituent eliminates one of the three staggered rotamers of the torsion angle chi 1 for each stereoisomer of the beta-Me-p-NO2Phe4. Similar solution conformations were suggested for the L-Phe4-containing (2S, 3S)- and (2S, 3R)-stereoisomers. Despite some local differences, solution conformations of L- and D-Phe4-containing analogues have a common shape of the peptide backbone and allow similar orientations of the main delta-opioid pharmacophores. This type of structure differs from several models of the solution conformations of DPDPE, and from the model of biologically active conformations of DPDPE suggested earlier. The latter model is allowed for the potent (2S, 3S)- and (2S, 3R)-stereoisomers of [beta-Me-p-NO2Phe4]DPDPE, but it is forbidden for the less active (2R, 3R)- and (2R, 3S)-stereoisomers. It was concluded that the biologically active stereoisomers of [beta-Me-p-NO2Phe4]DPDPE in the delta-receptor-bound state may assume a conformation different from their favorable conformations in DMSO.

Enkephalin analogs containing 4,4-difluoro-2-aminobutyric acid: synthesis and fluorine effect on the biological activity.

Analogs of Met-enkephalin and [D-Pen2, D-Pen5]enkephalin (DPDPE) containing the partially fluorinated amino acid 4,4-difluoro-2-aminobutyric acid (DFAB) in the 2- or 3-position of the peptide sequence were synthesized and their opioid activities and receptor selectivities were determined in vitro. The linear fluorinated [D-DFAB2, Met5-NH2]enkephalin showed mu and delta agonist potencies comparable to those of natural [Leu5]enkephalin. The partially fluorinated DPDPE analogs behaved differently as compared with their non-fluorinated correlates. While L-amino acid substitution in position 3 of DPDPE usually resulted in higher delta agonist potency than D-amino acid substitution. [D-DFAB3]DPDPE turned out to be a more potent delta agonist than [L-DFAB3]DPDPE. Furthermore, [D-DFAB3]DPDPE showed over 100-fold higher delta agonist potency than [D-Abu3]DPDPE (Abu = 2-aminobutyric acid), indicating that the fluorine substituents interact favorably with a delta opioid receptor subsite.

Enkephalin analogs containing 4,4-difluoro-2-aminobutyric acid: Synthesis and fluorine effect on the biological activity

Analogs of Met-enkephalin and [d-Pen2, d-Pen5]enkephalin (DPDPE) containing the partially fluorinated amino acid 4,4-difluoro-2-aminobutyric acid (DFAB) in the 2- or 3-position of the peptide sequence were synthesized and their opioid activities and receptor selectivities were determined in vitro. The linear fluorinated [d-DFAB2, Met5-NH2]enkephalin showed μ and δ agonist potencies comparable to those of natural [Leu5]enkephalin. The partially fluorinated DPDPE analogs behaved differently as compared with their non-fluorinated correlates. While l-amino acid substitution in position 3 of DPDPE usually resulted in higher δ agonist potency than d-amino acid substitution, [d-DFAB3]DPDPE turned out to be a more potent δ agonist than [l-DFAB3]DPDPE. Furthermore, [d-DFAB3]DPDPE showed over 100-fold higher δ agonist potency than [d-Abu3]DPDPE (Abu=2-aminobutyric acid), indicating that the fluorine substituents interact favorably with a δ opioid receptor subsite. © 1998 European Peptide Society and John Wiley & Sons, Ltd.

Effects of opioids on glutamate induced calcium intracellular increase in individually perfused rat hippocampal neurons in vitro

1. 1. Superfusion of cultured hippocampal cells with glutamate (Glu) 0.5 mM for 5 min induces an increase of [Ca 2+ i] that is quickly followed by a recovery to control level. Addition of dynorphin or D-pen 2-D-pen 5-enkephalin (DPDPE) induces a persistence of the elevated [Ca 2+ i], while [D-Ala 2,N-Me-Phe 4, Gly 5-ol]-enkephalin (DAMGO) does not influence it. 2. 2. Superfusion with Glu for 10 min induces a persistent increase of [Ca 2+ i], that is partially reverted by DAMGO, but not affected by dynorphin or DPDPE. 3. 3. The author suggests a differential influence of selective opioids on the Glu-induced [Ca 2+ i] increase.

Vulnerability to stressor-induced disturbances in self-stimulation from the dorsal and ventral A10 area: Differential effects of intraventricular D-Ala 2-Met 5-enkephalinamide, D-Ala 2, N-Me-Phe 4, Gly-Ol 5-enkephalin, and D-Pen 2, D-Pen 5- enkephalin administration

D-Ala 2-Met 5-enkephalinamide (DALA) (1.0 μg/μl) was administered intraventricularly to mice responding for electrical stimulation from the dorsal or ventral aspects of the VTA immediately prior to footshock (Experiment 1). Predictably, footshock reduced self-stimulation from the dorsal but not the ventral VTA immediately, 24, and 168 h following the stressor. Intraventricular DALA administration effected a partial attenuation of stressor-induced self-stimulation reductions from the dorsal VTA immediately and 24 h poststressor. Deficits appeared among DALA-Shocked mice responding for brain stimulation from the ventral VTA during comparable test intervals. The long-term depressant influence of footshock on self-stimulation from the dorsal VTA was abolished among DALA-treated mice and DALA-associated reductions in self-stimulation from the ventral A10 region among stressed mice were not evident 1 week later. Administration of D-Ala 2, N-Me-Phe 4, Gly-Ol 5-enkephalin (DAGO) (0.01 μg/μl) or D-Pen 2, D-Pen 5-enkephalin (DPDPE) (1.0 μg/μl) intraventricularly prior to footshock effected an immediate and a delayed antagonism, respectively, of the stressor on self-stimulation from the dorsal VTA, which persisted for 1 week. Prophylactic administration of 0.001 μg/μl DAGO or 0.01 μg/μl DPDPE prior to the stressor failed to influence self-stimulation from the ventral VTA (Experiment 2). Administration of 0.01 μg/μl DAGO or 1.0 μg/μl DPDPE among mice responding for brain stimulation from the dorsal VTA following footshock produced a weak therapeutic effect immediately poststressor, but effected protracted amelioration of footshock-induced reductions of self-stimulation from the dorsal VTA (Experiment 3). Taken together, μ, δ, and μ-δ activation influenced self-stimulation differentially from the dorsal and ventral VTA according to the temporal order of opioid peptide challenge relative to stressor imposition. These data are discussed with respect to stressors, motivational alterations, and the putative modulating influence of endogenous enkephalin activity in subareas of the VTA.

The C-Truncated δ-Opioid Receptor Underwent Agonist-Dependent Activation and Desensitization

The agonist-dependent activation and desensitization of a recombinant analog of δ-opioid receptor lacking the carboxyl-terminal (C-terminal) 31 residue peptide segment were discussed. The cDNA of the C-truncated δ-opioid receptor was created by polymerase chain reaction (PCR), stably expressed in Chinese hamster ovary cells and characterized by binding assay and function assay. Agonist [D-Pen2,D-Pen5]-enkephalin (DPDPE) stimulated the specific [35S]GTPγS binding to membrane fragments of cells expressing the C-truncated and the wild-type δ-opioid receptors in different levels dose-dependently.EC50values of DPDPE on truncated and wild-types in stimulation were very similar. Agonist-dependent desensitization, which could be blocked by protein kinase inhibitor staurosporine (Stau), was observed after pretreating truncated receptors with 1 μM DPDPE for 10 min, the same as wild-types. The results reveal that the C-terminal of the δ-opioid receptor is not involved in controlling the G-protein activation and phosphorylation-related functional desensitization.

Functional Blockade of Opioid Analgesia by Orphanin FQ/Nociceptin

Orphanin FQ/nociceptin (OFQ/N) is a recently identified neuropeptide with high affinity for the orphan opioid receptor. OFQ/N blocked morphine analgesia in mice in a dose-dependent manner, as well as the analgesic actions of [ d-Pen 2, d-Pen 5]enkephalin (DPDPE), morphine-6β-glucuronide, trans-3,4-dichloro- N-[2-(1-pyrrolindinyl)-cyclohexyl]-benzeneacetamide, methane sulfonate hydrate (U50,488H) and naloxone benzoylhydrazone. These actions are anti-analgesic, because OFQ/N also blocked clonidine analgesia and OFQ/N was inactive against the inhibition of gastrointestinal transit by morphine. Although OFQ/N was quite potent in these paradigms, two truncated forms, OFQ/N(1-11) and OFQ/N(1-7), were inactive. An antisense oligodeoxynucleotide targeting the first coding exon of KOR-3, the mouse homolog of the orphan opioid receptor, effectively prevented the anti-opioid actions of OFQ/N, confirming the importance of the orphan opioid receptor in this action.

N-Methyl-D-aspartate attenuates opioid receptor-mediated G protein activation and this process involves protein kinase C.

The effects of N-methyl-D-aspartate (NMDA) on opioid receptor-mediated G protein activation were explored in neuroblastoma X glioma hybrid (NG108-15) cells. Treatment of the cells with NMDA resulted in a remarkable attenuation of [35S]guanosine-5'-O-(3-thio)triphosphate binding stimulated by [D-Pen2,D-Pen5]-enkephalin (DPDPE), a delta-opioid receptor agonist. The effects of NMDA were dose and time dependent with an IC50 value of 5 nM and could be blocked by NMDA receptor antagonists. After NMDA treatment, the DPDPE dose-response curve shifted to the right (EC50 value increased approximately 7-fold, from 6 to 40 nM), and the maximal response induced by DPDPE was reduced by approximately 60%. The effects of NMDA were reversible, and the DPDPE response could recover within 60 min. The functional responses of delta-, mu-, and kappa-opioid receptors in primarily cultured neurons also were attenuated significantly by NMDA treatment. The inhibitory effects of NMDA on opioid receptor-mediated G protein activation could be blocked by coadministration of the protein kinase C (PKC) inhibitors or by elimination of the extracellular Ca2+. Correspondingly, NMDA treatment of NG108 cells significantly elevated cellular PKC activity and stimulated Gialpha2 phosphorylation. Transient transfection into NG108-15 cells of the wild-type Gialpha2 and a mutated Gialpha2 (Ser144Ala) resulted in a 2-fold increase in DPDPE-stimulated G protein activation. The DPDPE responses were greatly inhibited by NMDA treatment in the wild-type Gialpha2-transfected cells but much less affected in the mutant Gialpha2-transfected cells. In summary, NMDA attenuates opioid receptor/G protein coupling, and this process requires activation of PKC.

Delta-opioid receptors on astroglial cells in primary culture: mobilization of intracellular free calcium via a pertussis sensitive G protein

Astrocytes in primary culture from rat cerebral cortex were probed concerning the expression of δ-opioid receptors and their coupling to changes in intracellular free calcium concentrations ([Ca 2+] i). Fluo-3 or fura-2 based microspectrofluorometry was used for [Ca 2+] i measurements on single astrocytes in a mixed astroglial-neuronal culture. Application of the selective δ-opioid receptor agonist, [ d-Pen 2, d-Pen 5]-enkephalin (DPDPE), at concentrations ranging from 10 nM to 100 μM, induced concentration-dependent increases in [Ca 2+] i (EC 50=114 nM). The responses could be divided into two phases, with an initial spike in [Ca 2+] i followed by either oscillations or a sustained elevation of [Ca 2+] i. These effects were blocked by the selective δ-opioid receptor antagonist ICI 174 864 (10 μM). The expression of δ-opioid receptors on astroglial cells was further verified immunohistochemically, using specific antibodies, and by Western blot analyses. Pre-treatment of the cells with pertussis toxin (100 ng/ml, 24 h) blocked the effects of δ-opioid receptor activation, consistent with a G i- or G o-mediated response. The sustained elevation of [Ca 2+] i was not observed in low extracellular Ca 2+ and was partly blocked by nifedipine (1 μM), indicating the involvement of L-type Ca 2+ channels. Stimulating neurons with DPDPE resulted in a decrease in [Ca 2+] i, which may be consistent with the closure of the plasma membrane Ca 2+ channels on these cells. The current results suggest a role for astrocytes in the response of the brain to δ-opioid peptides and that these opioid effects in part involve altered astrocytic intracellular Ca 2+ homeostasis.

Effect of Chronic Administration of [D-Pen 2, D-Pen 5]Enkephalin on the Activity of Nitric Oxide Synthase in Brain Regions and Spinal Cord of Mice

Bhargava, H. N. and Y. J. Cao. Effect of chronic administration of [D-Pen 2, D-Pen 5]enkephalin on the activity of nitric oxide synthase in brain regions and spinal cord of mice. Peptides 19(1) 113–117, 1998.—The effect of multiple intracerebroventricular (ICV) injections of [D-Pen 2, D-Pen 5]enkephalin (DPDPE), a δ-opioid receptor agonist, on the activity of nitric oxide synthase (NOS) was determined in the brain regions and spinal cord of the mouse. Male Swiss Webster mice were injected twice daily with DPDPE (20 μg/mouse, ICV) or its vehicle for 4 days. This procedure has previously been shown to induce tolerance to the antinociceptive actions of DPDPE in mice. On day 5, the animals treated with DPDPE were either sacrificed 20 min after an ICV injection of DPDPE (tolerant) or without any injection (abstinent i.e., 16 h after the last injection of DPDPE). NOS activity in brain regions (cortex, striatum, hippocampus, midbrain, pons/medulla, hypothalamus and cerebellum) and spinal cord was determined by the rate of conversion of arginine into citrulline. Tolerance to DPDPE was associated increases in NOS activity in midbrain (49%) and pons/medulla (32%) and decreases in cerebellum (28%) and spinal cord (44%). However, NOS activity was unchanged in the cortex, corpus striatum, hippocampus and hypothalamus. On the other hand, during abstinence from DPDPE, NOS activity increased in midbrain (84%) and hypothalamus (35%) but decreased in cerebral cortex (27%), cerebellum (27%) and spinal cord (20%). NOS activity was unchanged in the corpus striatum, hippocampus and pons/medulla. Previous studies from this laboratory had demonstrated that chronic administration of μ- and κ-opioid receptor agonists results in increases NOS activity in certain brain regions and that tolerance to μ- and κ-, but not to δ-opioid receptor agonists, is attenuated by NOS inhibitors. The present studies, for the first time, demonstrate decreases in NOS activity in certain brain regions and spinal cord of mice treated chronically with δ-opioid receptor agonist. Furthermore, these findings may explain the inability of NOS inhibitors to attenuate tolerance to DPDPE in mice.

Modulation by calcium/calmodulin-dependent protein kinase II of functional response of delta opioid receptor in neuroblastoma x glioma hybrid (NG108-15) cells

The potential modulation of opioid receptor signaling by calcium/calmodulin-dependent protein kinase II (CaMKII) has been investigated in NG108-15 cells. Both CaMKII specific inhibitors used, KN62 and KN93, time- and dose-dependently blocked inhibition of cAMP accumulation by [D-Pen 2, D-Pen 5]enkephalin (DPDPE), with an ic 50 of about 1.2 μM and 0.8 μM, respectively. In the presence of 1 μM KN62 or KN93, the DPDPE dose-response curve shifted to the right ( ic 50 from 0.7 to 20 nM for KN62 and from 0.65 to 10 nM for KN93, respectively), and the maximal response was also significantly reduced. KN92, an inactive analogue of KN93, showed no significant impact, while ionomycin, an activator of CaMKII, greatly potentiated the opioid receptor response, suggesting that the effects of KN62, KN93 and ionomycin were likely mediated through CaMKII. In addition, KN62 did not affect ligand binding, receptor/Gi coupling, or basal and forskolin-stimulated adenylyl cyclase activity, suggesting its possible interference in the Gi/adenylyl cyclase interaction. Furthermore, a CaMKII inhibitor potently blocked the functional responses of other Gi-coupled receptors (m4 muscarinic and alpha2 adrenergic receptors) tested, but not that of Gs-coupled receptors (prostaglandin E1 and adenosine receptors). Our results clearly demonstrate that CaMKII modulates the signaling of opioid receptor and other Gi-coupled receptors.

Etorphine inhibits cell growth and induces apoptosis in SK-N-SH cells: involvement of pertussis toxin-sensitive G proteins

Opiates have been used extensively in the treatment of pain but with the severe side effect of addiction, which is believed to be related to opiates' direct (primary) or indirect (secondary) neurotoxicity. In this study, the effects of opioids on cell growth and apoptosis have been examined in human neuroblastoma cell line SK-N-SH. Etorphine, a wide-spectrum and potent agonist of opioid receptors, was found to significantly inhibit cell growth and to induce apoptosis. The inhibitory and apoptotic activities of etorphine followed a dose- and time-dependent manner. The more specific agonists of opioid receptors such as morphine, [ d-Ala 2, N-Me-Phe 4, Gly 5-ol]-enkephalin (DAGO), [ d-Pen 2, d-Pen 5]-enkephalin (DPDPE), dynorphin A and nociceptin/orphanin FQ did not show similar toxic activities under the same conditions. In addition, the effects of etorphine could not be blocked by the opioid receptor antagonist naloxone, suggesting that the effects of etorphine might not be mediated by a classical opioid receptor. However, pretreatment of SK-N-SH cells with pertussis toxin (PTX) blocked the inhibition of cell growth and apoptosis induced by etorphine, indicating the involvement of PTX-sensitive G proteins in the processes. It was also shown that etorphine-induced apoptosis was prevented by actinomycin D (AD) and interleukin-1 β converting enzyme inhibitor I. Interestingly, etorphine was similarly potent to inhibit growth of pheochromocytoma (PC12) cells but less effective in SH-SY5Y neuroblastoma cells and C6 glioma cells. We propose that inhibition of cell growth and induction of apoptosis may be one mechanism of opioid neurotoxicity.