Increased Bacterial Load and Expression of Antimicrobial Peptides in Skin of Barrier-Deficient Mice with Reduced Cancer Susceptibility.

Mice lacking three epidermal barrier proteins-envoplakin, periplakin, and involucrin (EPI-/- mice)-have a defective cornified layer, reduced epidermal γδ T cells, and increased dermal CD4(+) T cells. They are also resistant to developing skin tumors. The tumor-protective mechanism involves signaling between Rae-1 expressing keratinocytes and the natural killer group 2D receptor on immune cells, which also plays a role in host defenses against infection. Given the emerging link between bacteria and cancer, we investigated whether EPI-/- mice have an altered skin microbiota. The bacterial phyla were similar in wild-type and EPI-/- skin. However, bacteria were threefold more abundant in EPI-/- skin and penetrated deeper into the epidermis. The major epithelial defense mechanism against bacteria is production of antimicrobial proteins (AMPs). EPI-/- skin exhibited enhanced expression of antimicrobial peptides. However, reducing the bacterial load by antibiotic treatment or breeding mice under specific pathogen-free conditions did not reduce AMP expression or alleviate the abnormalities in T-cell populations. We conclude that the atopic characteristics of EPI-/- skin are a consequence of the defective barrier rather than a response to the increased bacterial load. It is therefore unlikely that the increase in skin microbiota contributes directly to the observed cancer resistance.

Effects of genetic deletion of endogenous opioid system components on the reinstatement of cocaine-seeking behavior in mice.

The repeated cycles of cessation of consumption and relapse remain the major clinical concern in treating drug addiction. The endogenous opioid system is a crucial component of the reward circuit that participates in the adaptive changes leading to relapse in the addictive processes. We have used genetically modified mice to evaluate the involvement of µ-opioid receptor (MOR) and δ-opioid receptor (DOR) and their main endogenous ligands, the enkephalins derived from proenkephalin (PENK) and prodynorphin (PDYN), in the reinstatement of cocaine-seeking behavior. Constitutive knockout mice of MOR, DOR, PENK, and PDYN, and their wild-type littermates were trained to self-administer cocaine or to seek for palatable food, followed by a period of extinction and finally tested on a cue-induced reinstatement of seeking behavior. The four lines of knockout mice acquired operant cocaine self-administration behavior, although DOR and PENK knockout mice showed less motivation for cocaine than wild-type littermates. Moreover, cue-induced relapse was significantly decreased in MOR and DOR knockout mice. In contrast, PDYN knockout mice showed a slower extinction and increased relapse than wild-type littermates. C-Fos expression analysis revealed differential activation in brain areas related with memory and reward in these knockout mice. No differences were found in any of the four genotypes in operant responding to obtain palatable food, indicating that the changes revealed in knockout mice were not due to unspecific deficit in operant performance. Our results indicate that MOR, DOR, and PDYN have a differential role in cue-induced reinstatement of cocaine-seeking behavior.

Epidermal barrier defects link atopic dermatitis with altered skin cancer susceptibility.

Atopic dermatitis can result from loss of structural proteins in the outermost epidermal layers, leading to a defective epidermal barrier. To test whether this influences tumour formation, we chemically induced tumours in EPI-/- mice, which lack three barrier proteins-Envoplakin, Periplakin, and Involucrin. EPI-/- mice were highly resistant to developing benign tumours when treated with 7,12-dimethylbenz(a)anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). The DMBA response was normal, but EPI-/- skin exhibited an exaggerated atopic response to TPA, characterised by abnormal epidermal differentiation, a complex immune infiltrate and elevated serum thymic stromal lymphopoietin (TSLP). The exacerbated TPA response could be normalised by blocking TSLP or the immunoreceptor NKG2D but not CD4+ T cells. We conclude that atopy is protective against skin cancer in our experimental model and that the mechanism involves keratinocytes communicating with cells of the immune system via signalling elements that normally protect against environmental assaults.DOI: http://dx.doi.org/10.7554/eLife.01888.001.

Impaired periamygdaloid-cortex prodynorphin is characteristic of opiate addiction and depression.

Negative affect is critical for conferring vulnerability to opiate addiction as reflected by the high comorbidity of opiate abuse with major depressive disorder (MDD). Rodent models implicate amygdala prodynorphin (Pdyn) as a mediator of negative affect; however, evidence of PDYN involvement in human negative affect is limited. Here, we found reduced PDYN mRNA expression in the postmortem human amygdala nucleus of the periamygdaloid cortex (PAC) in both heroin abusers and MDD subjects. Similar to humans, rats that chronically self-administered heroin had reduced Pdyn mRNA expression in the PAC at a time point associated with a negative affective state. Using the in vivo functional imaging technology DREAMM (DREADD-assisted metabolic mapping, where DREADD indicates designer receptors exclusively activated by designer drugs), we found that selective inhibition of Pdyn-expressing neurons in the rat PAC increased metabolic activity in the extended amygdala, which is a key substrate of the extrahypothalamic brain stress system. In parallel, PAC-specific Pdyn inhibition provoked negative affect-related physiological and behavioral changes. Altogether, our translational study supports a functional role for impaired Pdyn in the PAC in opiate abuse through activation of the stress and negative affect neurocircuitry implicated in addiction vulnerability.

Lack of functional GABAB receptors alters Kiss1 , Gnrh1 and Gad1 mRNA expression in the medial basal hypothalamus at postnatal day 4.

BACKGROUND/AIMS: Adult mice lacking functional GABAB receptors (GABAB1KO) show altered Gnrh1 and Gad1 expressions in the preoptic area-anterior hypothalamus (POA-AH) and females display disruption of cyclicity and fertility. Here we addressed whether sexual differentiation of the brain and the proper wiring of the GnRH and kisspeptin systems were already disturbed in postnatal day 4 (PND4) GABAB1KO mice. METHODS: PND4 wild-type (WT) and GABAB1KO mice of both sexes were sacrificed; tissues were collected to determine mRNA expression (qPCR), amino acids (HPLC), and hormones (RIA and/or IHC). RESULTS: GnRH neuron number (IHC) did not differ among groups in olfactory bulbs or OVLT-POA. Gnrh1 mRNA (qPCR) in POA-AH was similar among groups. Gnrh1 mRNA in medial basal hypothalamus (MBH) was similar in WTs but was increased in GABAB1KO females compared to GABAB1KO males. Hypothalamic GnRH (RIA) was sexually different in WTs (males > females), but this sex difference was lost in GABAB1KOs; the same pattern was observed when analyzing only the MBH, but not in the POA-AH. Arcuate nucleus Kiss1 mRNA (micropunch-qPCR) was higher in WT females than in WT males and GABAB1KO females. Gad1 mRNA in MBH was increased in GABAB1KO females compared to GABAB1KO males. Serum LH and gonadal estradiol content were also increased in GABAB1KOs. CONCLUSION: We demonstrate that GABABRs participate in the sexual differentiation of the ARC/MBH, because sex differences in several reproductive genes, such as Gad1, Kiss1 and Gnrh1, are critically disturbed in GABAB1KO mice at PND4, probably altering the organization and development of neural circuits governing the reproductive axis.

Neurokinin-1 receptor signalling impacts bone marrow repopulation efficiency.

Tachykinins are a large group of neuropeptides with both central and peripheral activity. Despite the increasing number of studies reporting a growth supportive effect of tachykinin peptides in various in vitro stem cell systems, it remains unclear whether these findings are applicable in vivo. To determine how neurokinin-1 receptor (NK-1R) deficient hematopoietic stem cells would behave in a normal in vivo environment, we tested their reconstitution efficiency using competitive bone marrow repopulation assays. We show here that bone marrow taken from NK-1R deficient mice (Tacr1(-/-)) showed lineage specific B and T cell engraftment deficits compared to wild-type competitor bone marrow cells, providing evidence for an involvement of NK-1R signalling in adult hematopoiesis. Tachykinin knockout mice lacking the peptides SP and/or HK-1 (Tac1 (-/-), Tac4 (-/-) and Tac1 (-/-)/Tac4 (-/-) mice) repopulated a lethally irradiated wild-type host with similar efficiency as competing wild-type bone marrow. The difference between peptide and receptor deficient mice indicates a paracrine and/or endocrine mechanism of action rather than autocrine signalling, as tachykinin peptides are supplied by the host environment.

Localization of Usher 1 proteins to the photoreceptor calyceal processes, which are absent from mice.

The mechanisms underlying retinal dystrophy in Usher syndrome type I (USH1) remain unknown because mutant mice lacking any of the USH1 proteins-myosin VIIa, harmonin, cadherin-23, protocadherin-15, sans-do not display retinal degeneration. We found here that, in macaque photoreceptor cells, all USH1 proteins colocalized at membrane interfaces (i) between the inner and outer segments in rods and (ii) between the microvillus-like calyceal processes and the outer segment basolateral region in rods and cones. This pattern, conserved in humans and frogs, was mediated by the formation of an USH1 protein network, which was associated with the calyceal processes from the early embryonic stages of outer segment growth onwards. By contrast, mouse photoreceptors lacked calyceal processes and had no USH1 proteins at the inner-outer segment interface. We suggest that USH1 proteins form an adhesion belt around the basolateral region of the photoreceptor outer segment in humans, and that defects in this structure cause the retinal degeneration in USH1 patients.

Chronic loss of melanin-concentrating hormone affects motivational aspects of feeding in the rat.

Current epidemic obesity levels apply great medical and financial pressure to the strenuous economy of obesity-prone cultures, and neuropeptides involved in body weight regulation are regarded as attractive targets for a possible treatment of obesity in humans. The lateral hypothalamus and the nucleus accumbens shell (AcbSh) form a hypothalamic-limbic neuropeptide feeding circuit mediated by Melanin-Concentrating Hormone (MCH). MCH promotes feeding behavior via MCH receptor-1 (MCH1R) in the AcbSh, although this relationship has not been fully characterized. Given the AcbSh mediates reinforcing properties of food, we hypothesized that MCH modulates motivational aspects of feeding.Here we show that chronic loss of the rat MCH-precursor Pmch decreased food intake predominantly via a reduction in meal size during rat development and reduced high-fat food-reinforced operant responding in adult rats. Moreover, acute AcbSh administration of Neuropeptide-GE and Neuropeptide-EI (NEI), both additional neuropeptides derived from Pmch, or chronic intracerebroventricular infusion of NEI, did not affect feeding behavior in adult pmch(+/+) or pmch(-/-) rats. However, acute administration of MCH to the AcbSh of adult pmch(-/-) rats elevated feeding behavior towards wild type levels. Finally, adult pmch(-/-) rats showed increased ex vivo electrically evoked dopamine release and increased limbic dopamine transporter levels, indicating that chronic loss of Pmch in the rat affects the limbic dopamine system.Our findings support the MCH-MCH1R system as an amplifier of consummatory behavior, confirming this system as a possible target for the treatment of obesity. We propose that MCH-mediated signaling in the AcbSh positively mediates motivational aspects of feeding behavior. Thereby it provides a crucial signal by which hypothalamic neural circuits control energy balance and guide limbic brain areas to enhance motivational or incentive-related aspects of food consumption.

Preprotachykinin-A gene deletion regulates hydrogen sulfide-induced toll-like receptor 4 signaling pathway in cerulein-treated pancreatic acinar cells.

OBJECTIVE: This study aimed to determine the effect of hydrogen sulfide (H2S) on Toll-like receptor 4 (TLR4)-mediated innate immune signaling in acute pancreatitis (AP) via substance P. METHODS: Male Swiss mice were treated with hourly intraperitoneal injections of cerulein (50 µg/kg) for 10 hours. dl-propargylglycine ([PAG] 100 mg/kg, intraperitoneally), an inhibitor of H2S formation, was administered 1 hour after the induction of AP. Pancreatic acinar cells from male preprotachykinin-A gene-knockout mice (PPTA) and their wild-type counterparts were incubated with or without cerulein (10 M for 60 minutes). To better understand the effect of H2S in inflammation, acinar cells were stimulated with cerulein after addition of H2S donor, sodium hydrosulfide. In addition, cerulein-treated pancreatic acinar cells were pretreated with PAG (30 µM) for 1 hour. RESULTS: The H2S inhibitor PAG eliminated TLR4, interleukin 1 receptor-associated kinase 4, tumor necrosis factor receptor-associated factor 6, and nuclear factor-κB (NF-κB) levels in in vitro and in vivo models of cerulein-induced AP. PPTA gene deletion reduced TLR4, myeloid differentiation factor 88, interleukin 1 receptor-associated kinase 4, tumor necrosis factor receptor-associated factor 6, and NF-κB in cerulein-treated pancreatic acinar cells, whereas administration of sodium hydrosulfide resulted in a further rise in TLR4 and NF-κB levels in cerulein-treated pancreatic acinar cells. CONCLUSION: The present findings show for the first time that in AP, H2S may up-regulate the TLR4 pathway and NF-κB via substance P.

Targeted deletion of the tachykinin 4 gene (TAC4-/-) influences the early stages of B lymphocyte development.

Hemokinin-1 (HK-1), encoded by the TAC4 gene, is a tachykinin peptide that is predominantly expressed in non-neuronal cells, such as immune cells. We have disrupted the mouse TAC4 gene to obtain a better understanding of the actions of HK-1 during hematopoiesis. We demonstrate here that TAC4(-/-) mice exhibit an increase of CD19(+)CD117(+)HSA(+)BP.1(-) "fraction B" pro-B cells in the bone marrow, whereas pre-B, immature, and mature B cells are within the normal range. We show that in vitro cultures derived from TAC4(-/-) bone marrow, sorted "fraction B" pro-B cells or purified long-term reconstituting stem cells, contain significantly higher numbers of pro-B cells compared with controls, suggesting an inhibitory role for HK-1 on developing B cells. Supporting this idea, we show that addition of HK-1 to cultures established from long-term reconstituting stem cells and the newly described intermediate-term reconstituting stem cells leads to a significant decrease of de novo generated pro-B cells. Based on our studies, we postulate that HK-1 plays an inhibitory role in hematopoiesis, and we hypothesize that it may be part of the bone marrow microenvironment that supports and regulates the proliferation and differentiation of hematopoietic cells.

Anxiety- and depressive-like responses and c-fos activity in preproenkephalin knockout mice: oversensitivity hypothesis of enkephalin deficit-induced posttraumatic stress disorder.

The present study used the preproenkephalin knockout (ppENK) mice to test whether the endogenous enkephalins deficit could facilitate the anxiety- and depressive-like symptoms of posttraumatic stress disorder (PTSD). On Day 1, sixteen wildtype (WT) and sixteen ppENK male mice were given a 3 mA or no footshock treatment for 10 seconds in the footshock apparatus, respectively. On Days 2, 7, and 13, all mice were given situational reminders for 1 min per trial, and the freezing response was assessed. On Day 14, all mice were tested in the open field test, elevated plus maze, light/dark avoidance test, and forced swim test. Two hours after the last test, brain tissues were stained to examine c-fos expression in specific brain areas. The present results showed that the conditioned freezing response was significant for different genotypes (ppENK vs WT). The conditioned freezing effect of the ppENK mice was stronger than those of the WT mice. On Day 14, the ppENK mice showed more anxiety- and depressive-like responses than WT mice. The magnitude of Fos immunolabeling was also significantly greater in the primary motor cortex, bed nucleus of the stria terminalis-lateral division, bed nucleus of the stria terminalis-supracapsular division, paraventricular hypothalamic nucleus-lateral magnocellular part, central nucleus of the amygdala, and basolateral nucleus of the amygdala in ppENK mice compared with WT mice. In summary, animals with an endogenous deficit in enkephalins might be more sensitive to PTSD-like aversive stimuli and elicit stronger anxiety and depressive PTSD symptoms, suggesting an oversensitivity hypothesis of enkephalin deficit-induced PTSD.

Use of mutant mouse lines to investigate origin of gonadotropin-releasing hormone-1 neurons: lineage independent of the adenohypophysis.

Mutant mouse lines have been used to study the development of specific neuronal populations and brain structures as well as behaviors. In this report, single- and double-mutant mice were used to examine the lineage of GnRH-1 cells. GnRH is essential for vertebrate reproduction, with either GnRH-1 or GnRH-3 controlling release of gonadotropins from the anterior pituitary, depending on the species. It is clear that the neuroendocrine GnRH cells migrate from extracentral nervous system locations into the forebrain. However, the embryonic origin of GnRH-1 and GnRH-3 cells is controversial and has been suggested to be nasal placode, adenohypophyseal (anterior pituitary) placode, or neural crest, again dependent on the species examined. We found that mutant mice with either missing or disrupted anterior pituitaries (Gli2(-/-), Gli1(-/-)Gli2(-/-), and Lhx3(-/-)) exhibit a normal GnRH-1 neuronal population and that these cells are still found associated with the developing vomeronasal organ. These results indicate that in mice, GnRH-1 cells develop independent of the adenohypophyseal placode and are associated early with the formation of the nasal placode.

The wnt target jagged-1 mediates the activation of notch signaling by progastrin in human colorectal cancer cells.

The Wnt and Notch signaling pathways are both abnormally activated in colorectal cancer (CRC). We recently showed that progastrin depletion inhibited Wnt signaling and increased goblet cell differentiation of CRC cells. Here, we show that progastrin down-regulation restores the expression by CRC cells of the early secretory lineage marker Math-1/Hath-1 due to an inhibition of Notch signaling. This effect is mediated by a decreased transcription of the Notch ligand Jagged-1, downstream of beta-catenin/Tcf-4. Accordingly, recombinant progastrin sequentially activated the transcription of Wnt and Notch target genes in progastrin-depleted cells. In addition, restoration of Jagged-1 levels in these cells is sufficient to activate Tcf-4 activity, demonstrating the occurrence of a feedback regulation from Notch toward Wnt signaling. These results suggest that progastrin could be instrumental in maintaining the concomitant activation of Wnt and Notch pathways in CRC cells, further highlighting the interest of progastrin targeting for the clinical management of CRC.

Prodynorphin gene deficiency potentiates nalbuphine-induced behavioral sensitization and withdrawal syndrome in mice.

Dynorphin is the presumed endogenous ligand for the kappa-opioid receptor. The dynorphin gene may play a role in psychotropic agent-mediated behavioral changes via dopaminergic modulation. Therefore, in this study, possible involvement of the dynorphin gene in nalbuphine-mediated behavioral responses was examined using prodynorphin (Pdyn) gene knock-out (-/-) mice. Pdyn gene deficiency potentiates nalbuphine-induced behavioral sensitization of locomotor activity and accumbal c-Fos expression. Administration of nalbuphine induced a significant increase in the dialysate dopamine level in the nucleus accumbens. This increase was more pronounced in the Pdyn (-/-) mice than in the wild-type (WT) mice. In addition, Pdyn (-/-) mice were more vulnerable to the naloxone-precipitated withdrawal syndrome (i.e., teeth chattering, wet dog shakes, forepaw tremors, jumping, weight loss, and global withdrawal score) after repeated treatment with nalbuphine than the WT mice. Consistently, nor-binaltorphimine, a kappa-opioid receptor antagonist, significantly potentiated nalbuphine-induced behavioral effects in WT mice, whereas U-50488H, a kappa-opioid receptor agonist, significantly attenuated these changes in Pdyn (-/-) mice in a dose-dependent manner. Our data suggest that the kappa-opioid receptor/dynorphin system is specifically modulated in response to behavioral sensitization and withdrawal signs induced by nalbuphine.

Prodynorphin gene disruption increases the sensitivity to nicotine self-administration in mice.

The endogenous opioid system has been reported to participate in nicotine behavioural responses. The aim of the study was to determine the contribution of the endogenous peptides derived from prodynorphin in acute and chronic nicotine responses, mainly those related to its addictive properties. Locomotion and nociception were evaluated after acute nicotine administration in prodynorphin knockout mice. In addition, nicotine rewarding properties were investigated in the place-conditioning and the intravenous self-administration paradigms. The somatic signs of nicotine withdrawal were also analysed after the injection of the nicotinic antagonist mecamylamine in nicotine-dependent mice. The hypolocomotor and antinociceptive effects induced by acute nicotine administration were not modified in knockout (KO) animals. Nicotine also produced similar conditioned place preference in both genotypes. However, a shift to the left in the percentage of acquisition of intravenous nicotine-self administration was observed in prodynorphin KO mice. Indeed, a significant increase in the number of KO mice acquiring this operant behaviour was revealed when low doses of nicotine were used. Nicotine physical dependence was similar in wild-type and KO animals. These findings reveal a specific role of endogenous peptides derived from prodynorphin in nicotine self-administration, probably through the modulation of its aversive effects.

The gonadotropin-releasing hormone type I receptor is expressed in the mouse cerebellum.

Gonadotropin-releasing hormone (GnRH) is a decapeptide hypothalamic hormone that was named according to its first discovered function--at the head of the neuroendocrine reproductive axis. Numerous other organ systems express GnRH and/or its receptor, although a specific physiological role for GnRH outside of the reproductive axis has yet to be established. Several studies in lower vertebrates have reported GnRH and/or its receptor in the cerebellum. Here, we describe the presence of immunoreactive GnRH receptors in the Purkinje cells of the mammalian cerebellum for the first time. This study provides compelling anatomical evidence for a common link between the cerebellum and the hypothalamo-pituitary axis. Dysfunction of this link occurs in the rare genetic ataxia disorders--Gordon Holmes syndrome and Boucher-Neuhauser syndrome.

Cbln1 regulates rapid formation and maintenance of excitatory synapses in mature cerebellar Purkinje cells in vitro and in vivo.

Although many synapse-organizing molecules have been identified in vitro, their functions in mature neurons in vivo have been mostly unexplored. Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is the most recently identified protein involved in synapse formation in the mammalian CNS. In the cerebellum, Cbln1 is predominantly produced and secreted from granule cells; cbln1-null mice show ataxia and a severe reduction in the number of synapses between Purkinje cells and parallel fibers (PFs), the axon bundle of granule cells. Here, we show that application of recombinant Cbln1 specifically and reversibly induced PF synapse formation in dissociated cbln1-null Purkinje cells in culture. Cbln1 also rapidly induced electrophysiologically functional and ultrastructurally normal PF synapses in acutely prepared cbln1-null cerebellar slices. Furthermore, a single injection of recombinant Cbln1 rescued severe ataxia in adult cbln1-null mice in vivo by completely, but transiently, restoring PF synapses. Therefore, Cbln1 is a unique synapse organizer that is required not only for the normal development of PF-Purkinje cell synapses but also for their maintenance in the mature cerebellum both in vitro and in vivo. Furthermore, our results indicate that Cbln1 can also rapidly organize new synapses in adult cerebellum, implying its therapeutic potential for cerebellar ataxic disorders.

Surfactant protein B deficiency caused by a novel mutation involving multiple exons of the SP-B gene.

BACKGROUND: Inability to produce surfactant protein (SP)-B causes fatal neonatal respiratory disease. Homozygosity for a frameshift mutation (121ins2) in the gene encoding SP-B (SFTPB) is the predominant but not the exclusive cause of disease. OBJECTIVES: To report a novel mutation in the SFTB gene. METHODS: We analyzed tracheal aspirates, lung tissue obtained by in vivo lung biopsy and DNA from a newborn infant with lethal respiratory failure. RESULTS: DNA analysis revealed a large homozygous genomic deletion encompassing exon 7 and 8 of SFTPB gene, a mutation we described as c.673-1248del2959. The parents were both heterozygous carriers. Analysis of the SP profile in tracheal aspirates and lung tissue by immunohistochemistry, routine and electron microscopy supported the diagnosis of SP-B deficiency and suggested that this large mutation might lead to abnormal routing and processing of proSP-B and proSP-C. CONCLUSIONS: This report shows that SP-B deficiency can also be caused by a multi exon deletion in the SFTPB gene and this finding emphasizes the importance of using modern DNA analysis techniques capable of detecting multi exon deletions.

Use of preproenkephalin knockout mice and selective inhibitors of enkephalinases to investigate the role of enkephalins in various behaviours.

RATIONALE: The most simple and efficient method to study the physiological role of enkephalins is to increase the lifetime of these endogenous opioid peptides by inhibiting their inactivating enzymes. Enkephalins are degraded by the concomitant action of two metallopeptidases: neutral endopeptidase (NEP, EC3.4.21.11) and aminopeptidase N (APN, EC3.4.11.2), both enzymes releasing inactive metabolites. OBJECTIVES: Potent dual inhibitors have been developed, such as RB101. However, NEP and APN have a broad specificity and can cleave various peptides in vitro. Therefore, it was essential to investigate the specific involvement of enkephalins in the various pharmacological responses induced by dual inhibitors. MATERIALS AND METHODS: We compared the pharmacological responses induced by RB101 in wild-type and preproenkephalin-deficient mice (Penk1-/-) using several behavioural assays. RESULTS: In all the tests used (hot plate test, force swim test, castor-oil-induced diarrhoea), RB101 induced strong effects in wild-type animals, whereas slight effects were observed in Penk1-/- animals. These residual effects are blocked by pre-administration of the opioid antagonist naloxone, supporting the involvement of the opioid receptors in the responses observed. CONCLUSIONS: The pharmacological effects induced by dual inhibitors acting on both NEP and APN are mainly due to the protection of the endogenous enkephalins at supraspinal and peripheral levels. It could be speculated that the residual effects observed in Penk1-/- mice after RB101 administration could be due to the direct action of other opioid peptides or through an indirect effect involving the protection of other peptide substrates of NEP or APN, as substance P or angiotensin.

Essential role of mu opioid receptor in the regulation of delta opioid receptor-mediated antihyperalgesia.

Analgesic effects of delta opioid receptor (DOR) -selective agonists are enhanced during persistent inflammation and arthritis. Although the underlying mechanisms are still unknown, membrane density of DOR was shown to be increased 72 h after induction of inflammation, an effect abolished in mu opioid receptor (MOR) -knockout (KO) mice [Morinville A, Cahill CM, Kieffer B, Collier B, Beaudet A (2004b) Mu-opioid receptor knockout prevents changes in delta-opioid receptor trafficking induced by chronic inflammatory pain. Pain 109:266-273]. In this study, we demonstrated a crucial role of MOR in DOR-mediated antihyperalgesia. Intrathecal administration of the DOR selective agonist deltorphin II failed to induce antihyperalgesic effects in MOR-KO mice, whereas it dose-dependently reversed thermal hyperalgesia in wild-type mice. The antihyperalgesic effects of deltorphin II were blocked by naltrindole but not d-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP) suggesting that this agonist was mainly acting through DOR. SNC80-induced antihyperalgesic effects in MOR-KO mice were also attenuated as compared with littermate controls. In contrast, kappa opioid receptor knockout did not affect deltorphin II-induced antihyperalgesia. As evaluated using mice lacking endogenous opioid peptides, the regulation of DOR's effects was also independent of beta-endorphin, enkephalins, or dynorphin opioids known to be released during persistent inflammation. We therefore conclude that DOR-mediated antihyperalgesia is dependent on MOR expression but that activation of MOR by endogenous opioids is probably not required.