Monica Baiula

Agonist-Regulated Internalization and Desensitization of the Human Nociceptin Receptor Expressed in CHO Cells

Abstract. In this study, we examined agonist-induced internalization, recycling and signalling (measure of cAMP levels) of the cloned human nociceptin receptor (hNOP) expressed in CHO-K1 cells. Internalization was proven by a receptor-binding assay on viable cells. The agonist nociceptin/orphanin FQ (NC) promoted rapid internalization of the hNOP receptor (≈78% of cell surface receptors were lost after 2 min exposure to 1 μM NC) in a clathrin- and ATP-dependent manner. Internalization was more rapid and marked in CHO-K1 cells than, as we previously reported, in SK-N-BE cells. This difference may be related to higher levels of β-arrestin isoforms detected in CHO-K1 than in SK-N-BE cells. hNOP receptor internalization was partially reversible and recycling occurred in the presence of the agonist; receptor recycling was dependent on okadaic acid-sensitive phosphatases and was blocked by monensin. Confocal microscopy analysis confirmed the internalization and the recycling back to the p lasma membrane of an epitope-tagged hNOP receptor expressed in CHO-K1 cells. These receptors underwent rapid desensitization upon agonist challenge: NC efficacy in inhibiting forskolin-stimulated cAMP production was significantly reduced 10 min after exposure and correlated with the rate of receptor internalization. Moreover, we observed that blockade of hNOP receptor recycling by monensin would cause a more prolonged and relevant desensitization of this receptor. Thus, the dynamic cycle between hNOP receptor activation, internalization and recycling determines the activity of this receptor on the cell surface.

Peripheral antinociceptive effects of the cyclic endomorphin-1 analog c[YpwFG] in a mouse visceral pain model

We previously described a novel cyclic endomorphin-1 analog c[Tyr-D-Pro-D-Trp-Phe-Gly] (c[YpwFG]), acting as a mu-opioid receptor (MOR) agonist. This study reports that c[YpwFG] is more lipophilic and resistant to enzymatic hydrolysis than endomorphin-1 and produces preemptive antinociception in a mouse visceral pain model when injected intraperitoneally (i.p.) or subcutaneously (s.c.) before 0.6% acetic acid, employed to evoke abdominal writhing (i.p. ED(50)=1.24 mg/kg; s.c. ED(50)=2.13 mg/kg). This effect is reversed by the selective MOR antagonist β-funaltrexamine and by a high dose of the mu(1)-opioid receptor-selective antagonist naloxonazine. Conversely, the kappa-opioid receptor antagonist nor-binaltorphimine and the delta-opioid receptor antagonist naltrindole are ineffective. c[YpwFG] produces antinociception when injected i.p. after acetic acid (ED(50)=4.80 mg/kg), and only at a dose of 20mg/kg did it elicit a moderate antinociceptive response in the mouse, evaluated by the tail flick assay. Administration of a lower dose of c[YpwFG] (10mg/kg i.p.) apparently produces a considerable part of antinociception on acetic acid-induced writhes through peripheral opioid receptors as this action is fully prevented by i.p. naloxone methiodide, which does not readily cross the blood-brain barrier; whereas this opioid antagonist injected intracerebroventricularly (i.c.v.) is not effective. Antinociception produced by a higher dose of c[YpwFG] (20mg/kg i.p.) is partially reversed by naloxone methiodide i.c.v. administered. Thus, only at the dose of 20mg/kg c[YpwFG] can produce antinociception through both peripheral and central opioid receptors. In conclusion, c[YpwFG] displays sufficient metabolic stability to be effective after peripheral administration and demonstrates the therapeutic potential of endomorphin derivatives as novel analgesic agents to control visceral pain.

Antiangiogenic Effect of Dual/Selective α5β1/αvβ3 Integrin Antagonists Designed on Partially Modified Retro-Inverso Cyclotetrapeptide Mimetics

Recent evidence highlighted the role of alpha(5)beta(1) integrin in angiogenesis and in regulating alpha(v)beta(3) integrin function. As a consequence, selective alpha(5)beta(1) integrin inhibitors or dual alpha(5)beta(1)/alpha(v)beta(3) integrin inhibitors are considered promising candidates for the development of cancer therapeutic agents. In this paper, we describe the synthesis and pharmacological characterization of a minilibrary of cyclotetrapeptide mimetics containing a PMRI Arg-Gly-Asp sequence. In particular, c[(R)-betaPhepsi(NHCO)Asppsi(NHCO)Gly-Arg] (3) displayed a good activity in inhibiting the alpha(v)beta(3) integrin-mediated cell adhesion of fibronectin or vitronectin, as well as the adhesion of fibronectin to the alpha(5)beta(1) integrin. Interestingly, the diastereomeric compound c[(S)-betaPhepsi(NHCO)Asppsi(NHCO)Gly-Arg] (2) maintained a good efficacy in inhibiting alpha(5)beta(1) integrin while gaining a certain selectivity over alpha(v)beta(3) integrin. These two integrin antagonists significantly inhibited bFGF-induced human endothelial cell tube formation at submicromolar concentrations. Conformational analysis and Molecular Docking calculations suggest that the different alpha(5)beta(1) versus alpha(v)beta(3) selectivity of 2 and 3 can be rationalized on the basis of the alternative display of the aromatic side chain adjacent to Asp.

Contribution of α4β1 integrin to the antiallergic effect of levocabastine

Levocabastine is an antiallergic drug acting as a histamine H1-receptor antagonist. In allergic conjunctivitis (AC), it may also antagonize up-regulation of the intercellular adhesion molecule-1 (ICAM-1) expressed on epithelial conjunctival cells. However, little is known about its effects on eosinophils, important effector cells in AC. The adhesion molecule integrin alpha(4)beta(1) is expressed in eosinophils; it interacts with the vascular cell adhesion molecule-1 (VCAM-1) and fibronectin (FN) in vascular endothelial cells and contributes to eosinophil activation and infiltration in AC. This study provides evidence that in a scintillation proximity assay levocabastine (IC(50) 406 microM), but not the first-generation antihistamine chlorpheniramine, displaced (125)I-FN binding to human integrin alpha(4)beta(1) and, in flow cytometry analysis, levocabastine antagonized the binding of a primary antibody to integrin alpha(4) expressed on the Jurkat cell surface. Levocabastine, but not chlorpheniramine, binds the alpha(4)beta(1) integrin and prevents eosinophil adhesion to VCAM-1, FN or human umbilical vascular endothelial cells (HUVEC) in vitro. Similarly, levocabastine affects alpha(L)beta(2)/ICAM-1-mediated adhesion of Jurkat cells. In a model of AC levocabastine eye drops reduced the clinical aspects of the late-phase reaction and the conjunctival expression of alpha(4)beta(1) integrin by reducing infiltrated eosinophils. We propose that blockade of integrin-mediated cell adhesion might be a target of the antiallergic action of levocabastine and may play a role in preventing eosinophil adhesion and infiltration in AC.

Mapracorat, a selective glucocorticoid receptor agonist, causes apoptosis of eosinophils infiltrating the conjunctiva in late-phase experimental ocular allergy

Background Mapracorat, a novel nonsteroidal selective glucocorticoid receptor agonist, has been proposed for the topical treatment of inflammatory disorders as it binds with high affinity and selectivity to the human glucocorticoid receptor and displays a potent anti-inflammatory activity, but seems to be less effective in transactivation of a number of genes, resulting in a lower potential for side effects. Contrary to classical glucocorticoids, mapracorat displays a reduced ability to increase intraocular pressure and in inducing myocilin, a protein linked to intraocular pressure elevation. Allergic conjunctivitis is the most common form of ocular allergy and can be divided into an early phase, developing immediately after allergen exposure and driven primarily by mast cell degranulation, and a late phase, developing from 6–10 hours after the antigen challenge, and characterized by conjunctival infiltration of eosinophils and other immune cells as well as by the production of cytokines and chemokines. Methods In this study, mapracorat was administered into the conjunctival sac of ovalbumin (OVA)-sensitized guinea pigs 2 hours after the induction of allergic conjunctivitis, with the aim of investigating its activity in reducing clinical signs of the late-phase ocular reaction and to determine its mechanism of anti-allergic effects with respect to apoptosis of conjunctival eosinophils and expression of the chemokines C-C motif ligand 5 (CCL5), C-C motif ligand 11 (CCL11), and interleukin-8 (IL-8) and the proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Results Mapracorat, administered into the conjunctival sac of OVA-sensitized guinea pigs 2 hours after allergen exposure, was effective in reducing clinical signs, eosinophil infiltration, and eosinophil peroxidase activity in the guinea pig conjunctiva; furthermore, it reduced conjunctival mRNA levels and protein expression of both CCL5 and CCL11. Mapracorat was more effective than dexamethasone in increasing, in conjunctival sections of OVA-treated guinea pigs, apoptotic eosinophils. Conclusion Mapracorat displays anti-allergic properties in controlling the late phase of ocular allergic conjunctivitis and is a promising candidate for the topical treatment of allergic eye disorders.

Development of isoxazoline-containing peptidomimetics as dual αvβ3 and α5β1 integrin ligands.

Isoxazoline‐containing peptidomimetics, designed to be effective αvβ3 and α5β1 integrin ligands, were synthesized through an original procedure involving N,O‐bis(trimethylsilyl)hydroxyamine conjugate addition to alkylidene acetoacetates, followed by intramolecular hemiketalization. To mimic the RGD recognition sequence, basic and acidic terminal appendages were introduced, and the final products were tested in cell adhesion inhibition assays. All the synthesized compounds proved to be excellent ligands for both integrin receptors, and a strong influence on intracellular signaling and phosphorylation pathways was demonstrated by evaluation of fibronectin‐induced phosphorylation of ERK. The molecular basis of the observed inhibitory activity was suggested on the results of docking experiments.

Transcriptional activation of human mu-opioid receptor gene by insulin-like growth factor-I in neuronal cells is modulated by the transcription factor REST.

The human mu‐opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional repression. We investigated whether insulin‐like growth factor I (IGF‐I), which affects various aspects of neuronal induction and maturation, regulates OPRM1 transcription in neuronal cells in the context of the potential influence of REST. A series of OPRM1‐luciferase promoter/reporter constructs were transfected into two neuronal cell models, neuroblastoma‐derived SH‐SY5Y cells and PC12 cells. In the former, endogenous levels of human mu‐opioid receptor (hMOPr) mRNA were evaluated by real‐time PCR. IGF‐I up‐regulated OPRM1 transcription in: PC12 cells lacking REST, in SH‐SY5Y cells transfected with constructs deficient in the REST DNA binding element, or when REST was down‐regulated in retinoic acid‐differentiated cells. IGF‐I activates the signal transducer and activator of transcription‐3 signaling pathway and this transcription factor, binding to the signal transducer and activator of transcription‐1/3 DNA element located in the promoter, increases OPRM1 transcription. We propose that a reduction in REST is a critical switch enabling IGF‐I to up‐regulate hMOPr. These findings help clarify how hMOPr expression is regulated in neuronal cells.

Expression of the repressor element‐1 silencing transcription factor (REST) is influenced by insulin‐like growth factor‐I in differentiating human neuroblastoma cells

The repressor element‐1 (RE‐1) silencing transcription factor (REST) interacts with an RE‐1 cis element and represses the transcription of neuron‐specific genes in neuronal progenitors but is down‐regulated in post‐mitotic neurons. We report that REST expression is modified, in a time‐dependent manner, in SH‐SY5Y neuroblastoma cells exposed to insulin‐like growth factor I (IGF‐I), a polypeptide hormone affecting various aspects of neuronal induction and maturation. REST is increased in cells treated with IGF‐I for 2 days and then declines in 5‐day‐treated cells concomitant with a progressive neurite extension. To investigate any role played by REST in neurodifferentiation by IGF‐I, we employed an antisense oligonucleotide (AS‐ODN) complementary to REST mRNA. In AS‐ODN‐treated cells, the effects elicited by IGF‐I on cell proliferation are not influenced whereas a marked decrease of REST significantly increases neurite elongation without any gross perturbation of neurogenesis. Synapsin I and βIII‐tubulin gene promoters contain an RE‐1 motif and their transcription is repressed by REST; both of them are increased in cells exposed to IGF‐I for 5 days and further elevated by AS‐ODN treatment. A parallel increase of growth cone‐associated protein 43, a protein chosen as a neuronal marker not directly regulated by REST, is also observed. Therefore, REST is elevated during early steps of neural induction by IGF‐I and could contribute to down‐regulate genes not yet required by the differentiation program while it declines later for the acquisition of neural phenotypes. These results suggest a model in which differentiating neuroblastoma cells determine their extent of neurite outgrowth on the basis of REST disappearance.

Modulation of αvβ3- and α5β1-integrin-mediated adhesion by dehydro-β-amino acids containing peptidomimetics

A novel class of low molecular weight ligands of αvβ₃ and α₅β₁ integrins, that possess a dehydro-β-amino acid as conformationally constrained core, linked to the pharmacophoric moieties mimicking the RGD recognition sequence, have been synthesized through a very simple protocol. Cell adhesion assays and integrin-mediated signaling activation experiments suggested a good affinity of these compounds toward both integrin receptors. Moreover, further elongation with two glycine units allowed to obtain an excellent dual inhibitor. Structural models for αvβ₃ integrin-ligand binding confirmed that the dehydro-β-amino derivatives are able to act as an electrostatic clamp by establishing several stabilizing interactions with the receptor.

Therapeutic Targeting of Eosinophil Adhesion and Accumulation in Allergic Conjunctivitis

Considerable evidence indicates that eosinophils are important effectors of ocular allergy. Increased worldwide prevalence of allergic eye pathologies has stimulated the identification of novel drug targets, including eosinophils and adhesion molecules. Accumulation of eosinophils in the eye is a key event in the onset and maintenance of allergic inflammation and is mediated by different adhesion molecules. Antihistamines with multiple mechanisms of action can be effective during the early and late phases of allergic conjunctivitis by blocking the interaction between β1 integrins and vascular cell adhesion molecule (VCAM)-1. Small molecule antagonists that target key elements in the process of eosinophil recruitment have been identified and reinforce the validity of α4β1 integrin as a therapeutic target. Glucocorticoids are among the most effective drugs for ocular allergy, but their use is limited by adverse effects. Novel dissociated glucocorticoids can prevent eosinophil accumulation and induce apoptosis of eosinophils, making them promising candidates for ophthalmic drugs. This article reviews recent understanding of the role of adhesion molecules in eosinophil recruitment in the inflamed conjunctiva along with effective treatments for allergic conjunctivitis.