Philippe Jauzac

Differential Time-Course Decreases in Nonselective, μ-, δ-, and κ-Opioid Receptors After Focal Cerebral Ischemia in Mice

Background and Purpose—Neuroprotection studies have demonstrated the involvement of opioids in ischemia, and we have previously demonstrated alterations in Bmax of opioidergic receptors after 2 post-MCAO time points in mice. Methods—In the present study, we have investigated in a detailed manner the postischemic time course of variations in [3H]diprenorphine (nonselective), [3H]DAMGO (μ), [3H]DADLE (δ), and [3H]U69593 (κ) relative binding densities after focal cerebral ischemia (0 to 48 hours) in mice. Results—In frontoparietal cortices, our results demonstrate decreases in (1) δ receptor densities at 1 to 3 hours after MCAO, (2) μ and nonselective binding sites at 6 to 12 hours after MCAO, and (3) κ receptor densities between 6 and 24 hours after MCAO. In the rostral part of the infarct border zone, a decrease in δ-receptors was found concomitant with the extension of the infarct core; conversely, the decrease in δ-receptors appeared before (6 to 12 hours) macroscopic histological damage, which occurred ...

The δ-Opioid Receptor Regulates Activity of Ryanodine Receptors in the Human Neuroblastoma Cell Line SK-N-BE

Abstract: Recent studies have demonstrated that opioid agonists affect the cytosolic Ca2+ concentration ([Ca2+]i) either by regulating plasma membrane Ca2+‐channel activity or by mobilizing intracellular Ca2+ stores. The present report documents the [Ca2+]i increase induced by opioid agonists in a human neuroblastoma cell line, SK‐N‐BE, expressing δ‐opioid receptors. In the presence, as well as in the absence, of extracellular Ca2+, opioid agonists enhanced significantly [Ca2+]i, whereas carbachol, known to mobilize specifically inositol 1,4,5‐trisphosphate‐sensitive intracellular Ca2+ stores, acted only in the presence of extracellular Ca2+. The opioid‐induced increase in [Ca2+]i was not affected by treatments modifying the trimeric Gi, Go, and Gs protein transduction mechanisms or the activity of adenylyl cyclase. The Ca2+‐ATPase pump‐inhibiting sesquiterpene lactone, thapsigargin, did not modify the opioid‐induced [Ca2+]i response, whereas it abolished the effects of carbachol. The Ryana speciosa alkaloid, ryanodine, at concentrations known to block endoplasmic reticulum ryanodine receptors, decreased significantly the response to opioids without affecting the effects of carbachol. Thus, our results suggest that, in SK‐N‐BE cells, δ‐opioid receptors mobilize Ca2+ from intracellular ryanodine‐sensitive stores and the mechanism involved is independent of Gi/Go and Gs proteins and protein kinase A activation.

In vitro and in vivo pharmacological profile of UFP‐512, a novel selective δ‐opioid receptor agonist; correlations between desensitization and tolerance

δ‐Opioid receptors (DOP receptors) could represent a novel target in the treatment of depressive disorders. To explore this new field of interest, the development of highly selective DOP receptor agonists is essential. UFP‐512 [H‐Dmt‐Tic‐NH‐CH(CH2‐COOH)‐Bid], was recently shown to behave in vitro as a selective and potent DOP receptor agonist and to promote antidepressant‐ and anxiolytic‐like effects in vivo ( Vergura et al., 2007 ). Here, we have characterized the pharmacological properties of UFP‐512 and established a link between desensitization and tolerance.

Pharmacological δ1- and δ2-opioid receptor subtypes in the human neuroblastoma cell line SK-N-BE: no evidence for distinct molecular entities

The two pharmacological δ-opioid receptor subtypes, δ1 and δ2, have been defined on the basis of pharmacological tools but remain to be characterized at the molecular level, since only a single cDNA has been cloned. The present study aimed to investigate the pharmacological properties of δ1- and δ2-opioid subtypes expressed in the human neuroblastoma cell line SK-N-BE and to characterize their putative corresponding mRNAs. Binding experiments using “selective” δ1- and δ2-opioid agonists and antagonists revealed the presence of two binding sites, demonstrating the presence of these δ1-opioid subtypes as they were previously described. The activation of these pharmacological subtypes by the selective agonists induced the incorporation of [α-32P]azidoanilide-GTP into Gαi2/Gα0 subunits with the same efficiency and potency and inhibited adenosine 3′,5′-cyclic monophosphate (cAMP) accumulation with similar efficiency, while their sustained activation for 15 min induced a cross-desensitization. The “selective” δ1 and δ2 antagonists, 7-benzylidenenaltrexone and naltrindole benzofuran, respectively, were found to be as potent in blocking the inhibition of cAMP accumulation induced by both [D-Pen2,5]enkephalin and Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2. The possibility that δ-opioid subtypes could arise from alternative splicing was ruled out by reverse transcription–polymerase chain reaction (RT–PCR) experiments and the sequencing of PCR products, which revealed the presence of a single transcript encoding for the δ-opioid receptor. Different possibilities which could account for the δ-opioid receptor heterogeneity observed in the SN-N-BE cell line are discussed.

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

Abstract 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.

Pharmacological characterization of AR-M1000390 at human delta opioid receptors

We investigated the pharmacological properties of a newly synthesised delta agonist AR-M1000390, derived from SNC-80 ((+)-4-[(alpha R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethyl-benzamide), in the neuroblastoma cell line SK-N-BE expressing only human delta-opioid receptors. Binding and functional experiments showed a weak affinity (K(i) = 106 +/- 34 nM) correlated with a weak potency (EC(50) = 111 +/- 31 nM) to inhibit the forskolin-stimulated cAMP accumulation. Sustained activation of opioid receptors in the presence of the maximal inhibitory concentration of AR-M1000390 produced a rapid and strong desensitization. In order to examine the contribution of internalization and down-regulation in the desensitization processes, binding and functional experiments were conducted in the presence or in the absence of hypertonic sucrose solution to block clathrin-dependent opioid receptor endocytosis. We observed both the inability of AR-M1000390 to down-regulate opioid receptors and the absence of any effect of sucrose on desensitization. The lack of delta-opioid receptor internalization by AR-M1000390 was further corroborated by confocal microscopy using antibodies directed either against the endogenous delta-opioid receptors or the FLAG-tagged delta-opioid receptors stably expressed in the SK-N-BE cells. These data suggest that uncoupling rather than internalization is responsible for delta-opioid receptors desensitization by AR-M1000390.

Reduction of cell proliferation and potentiation of Fas-induced apoptosis by the selective kappa-opioid receptor agonist U50 488 in the multiple myeloma LP-1 cells.

As opioid receptors modulate proliferation and apoptosis of immune cells, we hypothesized that they could reduce malignant haematopoietic cells. After screening, we selected the human multiple myeloma LP-1 cells which express mu- (MOP-) and kappa-opioid receptors (KOP-R). U50 488 produces a modest but significant decrease in viability associated with an arrest in the G0/G1 phase, but not antagonized by NorBNI and not associated with modulation of p21(Cip1), p27(Kip1) or p53 expression. In contrast, no effect was observed with dynorphin, U69 593 and morphine. In conclusion, the anti-proliferative effects of U50 488 are not mediated by KOP-R in the LP-1 cells.

Different kinases desensitize the human δ-opioid receptor (hDOP-R) in the neuroblastoma cell line SK-N-BE upon peptidic and alkaloid agonists

In a previous work, we described a differential desensitization of the human delta-opioid receptor (hDOP-R) by etorphine (a non-selective and alkaloid agonist) and delta-selective and peptidic agonists (DPDPE ([D-Pen(2,5)]enkephalin) and deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH(2))) in the neuroblastoma cell line SK-N-BE (Allouche et al., Eur. J. Pharmacol., 371, 235, 1999). In the present study, we explored the putative role of different kinases in this differential regulation. First, selective chemical inhibitors of PKA, PKC and tyrosine kinases were used and we showed a significant reduction of etorphine-induced opioid receptor desensitization by the bisindolylmaleimide I (PKC inhibitor) while genistein (tyrosine kinase inhibitor) was potent to impair desensitization induced by the different agonists. When the PKA was inhibited by H89 pretreatment, no modification of opioid receptor desensitization was observed whatever the agonist used. Second, we further studied the role of G protein-coupled receptor kinases (GRKs) and by using western-blot experiments we observed that only the GRK2 isoform was expressed in the SK-N-BE cells. Next, the neuroblastoma cells were transfected with the wild type GRK2 or its dominant negative mutant GRK2-K220R and the inhibition on cAMP level was determined in naïve and agonist-pretreated cells. We showed that over-expression of GRK2-K220R totally abolished etorphine-induced receptor desensitization while no effect was observed with peptidic agonists and over-expression of GRK2 selectively impaired cAMP inhibition promoted by etorphine suggesting that this kinase was involved in the regulation of hDOP-R activated only by etorphine. Third, correlation between functional experiments and phosphorylation of the hDOP-R after agonist activation was assessed by western-blot using the specific anti-phospho-DOP-R Ser(363) antibody. While all agonists were potent to increase phosphorylation of opioid receptor, we showed no impairment of receptor phosphorylation level after PKC inhibitor pretreatment. Upon agonist activation, no enhancement of receptor phosphorylation was observed when the GRK2 was over-expressed while the GRK2-K220R partially reduced the hDOP-R Ser(363) phosphorylation only after peptidic agonists pretreatment. In conclusion, hDOP-R desensitization upon etorphine exposure relies on the GRK2, PKC and tyrosine kinases while DPDPE and deltorphin I mediate desensitization at least via tyrosine kinases. Although the Ser(363) was described as the primary phosphorylation site of the mouse DOP-R, we observed no correlation between desensitization and phosphorylation of this amino acid.

ßarrestin1-biased agonism at human δ-opioid receptor by peptidic and alkaloid ligands

We have previously reported on the differential regulation of the human δ-opioid receptor (hDOR) by alkaloid (etorphine) and peptidic (DPDPE and deltorphin I) ligands, in terms of both receptor desensitization and post-endocytic sorting. Since ßarrestins are well known to regulate G protein-coupled receptors (GPCRs) signaling and trafficking, we therefore investigated the role of ßarrestin1 (the only isoform expressed in our cellular model) in the context of the hDOR. We established clonal cell lines of SK-N-BE cells over-expressing ßarrestin1, its dominant negative mutant (ßarrestin1(319-418)), and shRNA directed against endogenous ßarrestin1. Interestingly, both binding and confocal microscopy approaches demonstrated that ßarrestin1 is required for hDOR endocytosis only when activated by etorphine. Conversely, functional experiments revealed that ßarrestin1 is exclusively involved in hDOR desensitization promoted by the peptides. Taken together, these results provide substantial evidence for a ßarrestin1-biased agonism at hDOR, where ßarrestin1 is differentially involved during receptor desensitization and endocytosis depending on the ligand.

Maximal densities of mu, delta, and kappa receptors are differentially altered by focal cerebral ischaemia in the mouse.

Abstract Though opioids are known to have neuroprotective properties, little information is available on the functional state of opioidergic receptors following focal cerebral ischaemia. The present study investigated the evolution of the Bmax and Kd for [3H]DAMGO, [3H]DADLE, and [3H]U69,593, respectively, for the μ, δ, and κ opioidergic receptors after permanent focal cerebral ischaemia in mice. While the various Kd were unchanged, μ and δBmax values were precociously decreased in frontoparietal cortices, earlier than κ receptors, reflecting infarct extension with time. The Bmax values for μ and δ receptors were also altered in non-infarcted tissues, such as tissues at risk (e.g., temporal auditory cortex) and exofocal (e.g., contralateral and non-infarcted) cortices. These results suggest that, in non-infarcted areas, the observed changes reflect functional modifications to focal ischaemia.