Compensatory T-Cell Regulation in Unaffected Relatives of SLE Patients, and Opposite IL-2/CD25-Mediated Effects Suggested by Coreferentiality Modeling

In human systemic lupus erythematosus (SLE), diverse autoantibodies accumulate over years before disease manifestation. Unaffected relatives of SLE patients frequently share a sustained production of autoantibodies with indiscriminable specificity, usually without ever acquiring the disease. We studied relations of IgG autoantibody profiles and peripheral blood activated regulatory T-cells (aTregs), represented by CD4(+)CD25(bright) T-cells that were regularly 70-90% Foxp3(+). We found consistent positive correlations of broad-range as well as specific SLE-associated IgG with aTreg frequencies within unaffected relatives, but not patients or unrelated controls. Our interpretation: unaffected relatives with shared genetic factors compensated pathogenic effects by aTregs engaged in parallel with the individual autoantibody production. To study this further, we applied a novel analytic approach named coreferentiality that tests the indirect relatedness of parameters in respect to multivariate phenotype data. Results show that independently of their direct correlation, aTreg frequencies and specific SLE-associated IgG were likely functionally related in unaffected relatives: they significantly parallelled each other in their relations to broad-range immunoblot autoantibody profiles. In unaffected relatives, we also found coreferential effects of genetic variation in the loci encoding IL-2 and CD25. A model of CD25 functional genetic effects constructed by coreferentiality maximization suggests that IL-2-CD25 interaction, likely stimulating aTregs in unaffected relatives, had an opposed effect in SLE patients, presumably triggering primarily T-effector cells in this group. Coreferentiality modeling as we do it here could also be useful in other contexts, particularly to explore combined functional genetic effects.

Potent cyclic antagonists of the complement C5a receptor on human polymorphonulcear leukocytes. Relationships between structures and activity

Human C5a is a plasma protein with potent chemoattractant and pro-inflammatory properties, and its overexpression correlates with severity of inflammatory diseases. C5a binds to its G protein-coupled receptor (C5aR) on polymorphonuclear leukocytes (PMNLs) through a high-affinity helical bundle and a low-affinity C terminus, the latter being solely responsible for receptor activation. Potent and selective C5a antagonists are predicted to be effective anti-inflammatory drugs, but no pharmacophore for small molecule antagonists has yet been developed, and it would significantly aid drug design. We have hypothesized that a turn conformation is important for activity of the C terminus of C5a and herein report small cyclic peptides that are stable turn mimics with potent antagonism at C5aR on human PMNLs. A comparison of solution structures for the C terminus of C5a, small acyclic peptide ligands, and cyclic antagonists supports the importance of a turn for receptor binding. Competition between a cyclic antagonist and either C5a or an acyclic agonist for C5aR on PMNLs supports a common or overlapping binding site on the C5aR. Structure-activity relationships for 60 cyclic analogs were evaluated by competitive radioligand binding with C5a (affinity) and myeloperoxidase release (antagonist potency) from human PMNLs, with 20 compounds having high antagonist potencies (IC50, 20 nM(-1) muM). Computer modeling comparisons reveal that potent antagonists share a common cyclic backbone shape, with affinity-determining side chains of defined volume projecting from the cyclic scaffold. These results define a new pharmacophore for C5a antagonist development and advance our understanding of ligand recognition and receptor activation of this G protein-coupled receptor.

Characterization of the structure of RAMP1 by mutagenesis and molecular modeling

Receptor activity modifying proteins (RAMPs) are a family of single-pass transmembrane proteins that dimerize with G-protein-coupled receptors. They may alter the ligand recognition properties of the receptors (particularly for the calcitonin receptor-like receptor, CLR). Very little structural information is available about RAMPs. Here, an ab initio model has been generated for the extracellular domain of RAMP1. The disulfide bond arrangement (Cys 27-Cys82, Cys40-Cys72, and Cys 57-Cys104) was determined by site-directed mutagenesis. The secondary structure (a-helices from residues 29-51, 60-80, and 87-100) was established from a consensus of predictive routines. Using these constraints, an assemblage of 25,000 structures was constructed and these were ranked using an all-atom statistical potential. The best 1000 conformations were energy minimized. The lowest scoring model was refined by molecular dynamics simulation. To validate our strategy, the same methods were applied to three proteins of known structure; PDB:1HP8, PDB:1V54 chain H (residues 21-85), and PDB:1T0P. When compared to the crystal structures, the models had root mean-square deviations of 3.8 Å, 4.1 Å, and 4.0 Å, respectively. The model of RAMP1 suggested that Phe93, Tyr 100, and Phe101 form a binding interface for CLR, whereas Trp74 and Phe92 may interact with ligands that bind to the CLR/RAMP1 heterodimer. © 2006 by the Biophysical Society.

A key role for transmembrane prolines in calcitonin receptor-like receptor agonist binding and signalling:implications for family B G-protein-coupled receptors

Calcitonin receptor like-receptor is a family B G-protein coupled receptor (GPCR). It requires receptor activity modifying protein (RAMP) 1 to give a calcitonin gene-related peptide (CGRP) receptor. Little is known of how members of this receptor family function. Proline residues often form important kinks in alpha-helices. Therefore, all proline residues within the transmembrane helices of the receptor (Pro241, Pro244 in helix 4, Pro275 in helix 5, Pro321 and Pro331 in helix 6) were mutated to alanine. Pro241 Pro275, and Pro321 are highly conserved throughout all family B GPCRs. The binding of CGRP and its ability to stimulate cAMP production were investigated in mutant and wild-type receptors after transient transfection into COS-7 cells with RAMP1. The P321A mutation significantly decreased the pEC(50) for CGRP and reduced its affinity but did not change cell-surface expression. Antagonist binding [CGRP(8-37) and 1-piperidinecarboxamide N-[2-[[5amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3 5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quina zolinyl) (BIBN4096BS)] was little altered by the mutation. Adrenomedullin-mediated signaling was disrupted when P321A was coexpressed with RAMP1, RAMP2, or RAMP3. The P331A mutant produced a moderate reduction in CGRP binding and receptor activation. Mutation of the other residues had no effect on receptor function. Thus, Pro321 and Pro331 are required for agonist binding and receptor activation. Modeling suggested that Pro321 induces a bend in helix 6, bringing its C terminus near that of helix 3, as seen in many family A GPCRs. This is abolished in P321A. P321A-I325P predicted to restore this conformation, showed wild-type activation. Modeling can also rationalize the effects of transmembrane proline mutants previously reported for another family B GPCR, the VPAC(1) receptor.

Diverse functional motifs within the three intracellular loops of the CGRP1 receptor

The CGRP1 receptor exists as a heterodimeric complex between a single-pass transmembrane accessory protein (RAMP1) and a family B G-protein-coupled receptor (GPCR) called the calcitonin receptor-like receptor (CLR). This study investigated the structural motifs found in the intracellular loops (ICLs) of this receptor. Molecular modeling was used to predict active and inactive conformations of each ICL. Conserved residues were altered to alanine by site-directed mutagenesis. cAMP accumulation, cell-surface expression, agonist affinity, and CGRP-stimulated receptor internalization were characterized. Within ICL1, L147 and particularly R151 were important for coupling to Gs. R151 may interact directly with the G-protein, accessing it following conformational changes involving ICL2 and ICL3. At the proximal end of ICL3, I290 and L294, probably lying on the same face of an α helix, formed a G-protein coupling motif. The largest effects on coupling were observed with I290A; additionally, it reduced CGRP affinity and impaired internalization. 1290 may interact with TM6 to stabilize the conformation of ICL3, but it could also interact directly with Gs. R314, at the distal end of ICL3, impaired G-protein coupling and to a lesser extent reduced CGRP affinity; it may stabilize the TM6-ICL3 junction by interacting with the polar headgroups of membrane phospholipids. Y215 and L214 in ICL2 are required for cell-surface expression; they form a microdomain with H216 which has the same function. This study reveals similarities between the activation of CLR and other GPCRs in the role of TM6 and ICL3 but shows that other conserved motifs differ in their function. © 2006 American Chemical Society.

Source/receptor reconciliation of ambient organic fine particulate matter in Palm Beach County

Airborne particulate matter (PM) is of environmental concern not only in urban but also rural areas that are easily inhalable and have been considered responsible, together with gaseous pollutants, for possible health effects. The objectives of this research study is to generate an extensive data set for ambient PM collected at Belle Glade and Delray Beach that ultimately was used together with published source profiles to predict the contributions of major sources to the overall airborne particle burden in Belle Glade and Delray Beach. ^ The size segregated particle sampling was conducted for one entire year. The samples collected during the months of January and May were further subjected to chemical analysis for organic compounds by Gas Chromatography-Mass Spectrometry. Additional, PM10 sampling was conducted simultaneously with size segregated particle sampling during January and May to analyze for trace elements using Instrumental Neutron Activation Analysis technique. Elements and organic marker compounds were used in Chemical Mass Balance modeling to determine the major source contribution to the ambient fine particle matter burden. ^ Size segregated particle distribution results show bimodal in both sampling sites. Sugarcane pre-harvest burning in the rural site elevated PM10 concentration by about 30% during the sugarcane harvest season compared to sugarcane growing season. Sea salt particles and Saharan dust particles accounted for the external sources. ^ The results of trace element analysis show that Al, Ca, Cs, Eu, Lu, Nd, Sc, Sm, Th, and Yb are more abundant at the rural sampling site. The trace elements Ba, Br, Ce, Cl, Cr, Fe, Gd, Hf, Na, Sb, Ta, V, and W show high abundance at the urban site due to anthropogenic activities except for Na and Cl, which are from sea salt spray. On the other hand, size segregated trace organic compounds measurements show that organic compounds mainly from combustion process were accumulated in PM0.95. ^ In conclusion, major particle sources were determined by the CMB8.2 software as follows: road dust, sugarcane leaf burning, diesel-powered and gasoline powered vehicle exhaust, leaf surface abrasion particles, and a very small fraction of meat cooking. ^

Enhancing non-refractory aerosol apportionment from an urban industrial site through receptor modelling of complete high time-resolution aerosol mass spectra

Receptor modelling was performed on quadrupole unit mass resolution aerosol mass spectrometer (Q-AMS) sub-micron particulate matter (PM) chemical speciation measurements from Windsor, Ontario, an industrial city situated across the Detroit River from Detroit, Michigan. Aerosol and trace gas measurements were collected on board Environment Canada’s CRUISER mobile laboratory. Positive matrix factorization (PMF) was performed on the AMS full particle-phase mass spectrum (PMFFull MS) encompassing both organic and inorganic components. This approach was compared to the more common method of analysing only the organic mass spectra (PMFOrg MS). PMF of the full mass spectrum revealed that variability in the non-refractory sub-micron aerosol concentration and composition was best explained by six factors: an amine-containing factor (Amine); an ammonium sulphate and oxygenated organic aerosol containing factor (Sulphate-OA); an ammonium nitrate and oxygenated organic aerosol containing factor (Nitrate-OA); an ammonium chloride containing factor (Chloride); a hydrocarbon like organic aerosol (HOA) factor; and a moderately oxygenated organic aerosol factor (OOA). PMF of the organic mass spectrum revealed three factors of similar composition to some of those revealed through PMFFull MS: Amine, HOA and OOA. Including both the inorganic and organic mass proved to be a beneficial approach to analysing the unit mass resolution AMS data for several reasons. First, it provided a method for potentially calculating more accurate sub-micron PM mass concentrations, particularly when unusual factors are present, in this case, an Amine factor. As this method does not rely on a priori knowledge of chemical species, it circumvents the need for any adjustments to the traditional AMS species fragmentation patterns to account for atypical species, and can thus lead to more complete factor profiles. It is expected that this method would be even more useful for HR-ToF-AMS data, due to the ability to better understand the chemical nature of atypical factors from high resolution mass spectra. Second, utilizing PMF to extract factors containing inorganic species allowed for the determination of extent of neutralization, which could have implications for aerosol parameterization. Third, subtler differences in organic aerosol components were resolved through the incorporation of inorganic mass into the PMF matrix. The additional temporal features provided by the inorganic aerosol components allowed for the resolution of more types of oxygenated organic aerosol than could be reliably re-solved from PMF of organics alone. Comparison of findings from the PMFFull MS and PMFOrg MS methods showed that for the Windsor airshed, the PMFFull MS method enabled additional conclusions to be drawn in terms of aerosol sources and chemical processes. While performing PMFOrg MS can provide important distinctions between types of organic aerosol, it is shown that including inorganic species in the PMF analysis can permit further apportionment of organics for unit mass resolution AMS mass spectra.

Mathematical Modeling of Perifusion Cell Culture Experiments

In perifusion cell cultures, the culture medium flows continuously through a chamber containing immobilized cells and the effluent is collected at the end. In our main applications, gonadotropin releasing hormone (GnRH) or oxytocin is introduced into the chamber as the input. They stimulate the cells to secrete luteinizing hormone (LH), which is collected in the effluent. To relate the effluent LH concentration to the cellular processes producing it, we develop and analyze a mathematical model consisting of coupled partial differential equations describing the intracellular signaling and the movement of substances in the cell chamber. We analyze three different data sets and give cellular mechanisms that explain the data. Our model indicates that two negative feedback loops, one fast and one slow, are needed to explain the data and we give their biological bases. We demonstrate that different LH outcomes in oxytocin and GnRH stimulations might originate from different receptor dynamics. We analyze the model to understand the influence of parameters, like the rate of the medium flow or the fraction collection time, on the experimental outcomes. We investigate how the rate of binding and dissociation of the input hormone to and from its receptor influence its movement down the chamber. Finally, we formulate and analyze simpler models that allow us to predict the distortion of a square pulse due to hormone-receptor interactions and to estimate parameters using perifusion data. We show that in the limit of high binding and dissociation the square pulse moves as a diffusing Gaussian and in this limit the biological parameters can be estimated.

Receptor activity-modifying protein-directed G protein signaling specificity for the calcitonin gene-related peptide family of receptors

The calcitonin gene-related peptide (CGRP) family of G protein- coupled receptors (GPCRs) is formed through the association of the calcitonin receptor-like receptor (CLR) and one of three receptor activity-modifying proteins (RAMPs). Binding of one of the three peptide ligands, CGRP, adrenomedullin (AM), and intermedin/adrenomedullin 2 (AM2), is well known to result in aGαs-mediated increase in cAMP. Here we used modified yeast strains that couple receptor activation to cell growth, via chimeric yeast/Gα subunits, and HEK-293 cells to characterize the effect of different RAMP and ligand combinations on this pathway. We not only demonstrate functional couplings to both Gαs and Gαq but also identify a Gαi component to CLR signaling in both yeast and HEK-293 cells, which is absent in HEK-293S cells. We show that the CGRP family of receptors displays both ligand- and RAMPdependent signaling bias among the Gαs, Gαi, and Gαq/11 pathways. The results are discussed in the context of RAMP interactions probed through molecular modeling and molecular dynamics simulations of the RAMP-GPCR-G protein complexes. This study further highlights the importance of RAMPs to CLR pharmacology and to bias in general, as well as identifying the importance of choosing an appropriate model system for the study of GPCR pharmacology.

Impaired Compensatory Beta-cell Function And Growth In Response To High-fat Diet In Ldl Receptor Knockout Mice.

In this study, we investigated the effect of low density lipoprotein receptor (LDLr) deficiency on gap junctional connexin 36 (Cx36) islet content and on the functional and growth response of pancreatic beta-cells in C57BL/6 mice fed a high-fat (HF) diet. After 60 days on regular or HF diet, the metabolic state and morphometric islet parameters of wild-type (WT) and LDLr-/- mice were assessed. HF diet-fed WT animals became obese and hypercholesterolaemic as well as hyperglycaemic, hyperinsulinaemic, glucose intolerant and insulin resistant, characterizing them as prediabetic. Also they showed a significant decrease in beta-cell secretory response to glucose. Overall, LDLr-/- mice displayed greater susceptibility to HF diet as judged by their marked cholesterolaemia, intolerance to glucose and pronounced decrease in glucose-stimulated insulin secretion. HF diet induced similarly in WT and LDLr-/- mice, a significant decrease in Cx36 beta-cell content as revealed by immunoblotting. Prediabetic WT mice displayed marked increase in beta-cell mass mainly due to beta-cell hypertrophy/replication. Nevertheless, HF diet-fed LDLr-/- mice showed no significant changes in beta-cell mass, but lower islet-duct association (neogenesis) and higher beta-cell apoptosis index were seen as compared to controls. The higher metabolic susceptibility to HF diet of LDLr-/- mice may be explained by a deficiency in insulin secretory response to glucose associated with lack of compensatory beta-cell expansion.

Peripheral P2x7 Receptor-induced Mechanical Hyperalgesia Is Mediated By Bradykinin.

P2X7 receptors play an important role in inflammatory hyperalgesia, but the mechanisms involved in their hyperalgesic role are not completely understood. In this study, we hypothesized that P2X7 receptor activation induces mechanical hyperalgesia via the inflammatory mediators bradykinin, sympathomimetic amines, prostaglandin E2 (PGE2), and pro-inflammatory cytokines and via neutrophil migration in rats. We found that 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate triethylammonium salt (BzATP), the most potent P2X7 receptor agonist available, induced a dose-dependent mechanical hyperalgesia that was blocked by the P2X7 receptor-selective antagonist A-438079 but unaffected by the P2X1,3,2/3 receptor antagonist TNP-ATP. These findings confirm that, although BzATP also acts at both P2X1 and P2X3 receptors, BzATP-induced hyperalgesia was mediated only by P2X7 receptor activation. Co-administration of selective antagonists of bradykinin B1 (Des-Arg(8)-Leu(9)-BK (DALBK)) or B2 receptors (bradyzide), β1 (atenolol) or β2 adrenoceptors (ICI 118,551), or local pre-treatment with the cyclooxygenase inhibitor indomethacin or the nonspecific selectin inhibitor fucoidan each significantly reduced BzATP-induced mechanical hyperalgesia in the rat hind paw. BzATP also induced the release of the pro-inflammatory cytokines tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6 and cytokine-induced neutrophil chemoattractant-1 (CINC-1), an effect that was significantly reduced by A-438079. Co-administration of DALBK or bradyzide with BzATP significantly reduced BzATP-induced IL-1β and CINC-1 release. These results indicate that peripheral P2X7 receptor activation induces mechanical hyperalgesia via inflammatory mediators, especially bradykinin, which may contribute to pro-inflammatory cytokine release. These pro-inflammatory cytokines in turn may mediate the contributions of PGE2, sympathomimetic amines and neutrophil migration to the mechanical hyperalgesia induced by local P2X7 receptor activation.

Icatibant, An Inhibitor Of Bradykinin Receptor 2, For Hereditary Angioedema Attacks: Prospective Experimental Single-cohort Study.

Hereditary angioedema (HAE) with C1 inhibitor deficiency manifests as recurrent episodes of edema involving the skin, upper respiratory tract and gastrointestinal tract. It can be lethal due to asphyxia. The aim here was to evaluate the response to therapy for these attacks using icatibant, an inhibitor of the bradykinin receptor, which was recently introduced into Brazil. Prospective experimental single-cohort study on the efficacy and safety of icatibant for HAE patients. Patients with a confirmed HAE diagnosis were enrolled according to symptoms and regardless of the time since onset of the attack. Icatibant was administered in accordance with the protocol that has been approved in Brazil. Symptom severity was assessed continuously and adverse events were monitored. 24 attacks in 20 HAE patients were treated (female/male 19:1; 19-55 years; median 29 years of age). The symptoms were: subcutaneous edema (22/24); abdominal pain (15/24) and upper airway obstruction (10/24). The time taken until onset of relief was: 5-10 minutes (5/24; 20.8%); 10-20 (5/24; 20.8%); 20-30 (8/24; 33.4%); 30-60 (5/24; 20.8%); and 2 hours (1/24; 4.3%). The time taken for complete resolution of symptoms ranged from 4.3 to 33.4 hours. Adverse effects were only reported at injection sites. Mild to moderate erythema and/or feelings of burning were reported by 15/24 patients, itching by 3 and no adverse effects in 6. HAE type I patients who received icatibant responded promptly; most achieved improved symptom severity within 30 minutes. Local adverse events occurred in 75% of the patients.

The Analgesic Effect Of Dipyrone In Peripheral Tissue Involves Two Different Mechanisms: Neuronal K(atp) Channel Opening And Cb(1) Receptor Activation.

Dipyrone (metamizole) is an analgesic pro-drug used to control moderate pain. It is metabolized in two major bioactive metabolites: 4-methylaminoantipyrine (4-MAA) and 4-aminoantipyrine (4-AA). The aim of this study was to investigate the participation of peripheral CB1 and CB2 cannabinoid receptors activation in the anti-hyperalgesic effect of dipyrone, 4-MAA or 4-AA. PGE2 (100ng/50µL/paw) was locally administered in the hindpaw of male Wistar rats, and the mechanical nociceptive threshold was quantified by electronic von Frey test, before and 3h after its injection. Dipyrone, 4-MAA or 4-AA was administered 30min before the von Frey test. The selective CB1 receptor antagonist AM251, CB2 receptor antagonist AM630, cGMP inhibitor ODQ or KATP channel blocker glibenclamide were administered 30min before dipyrone, 4-MAA or 4-AA. The antisense-ODN against CB1 receptor expression was intrathecally administered once a day during four consecutive days. PGE2-induced mechanical hyperalgesia was inhibited by dipyrone, 4-MAA, and 4-AA in a dose-response manner. AM251 or ODN anti-sense against neuronal CB1 receptor, but not AM630, reversed the anti-hyperalgesic effect mediated by 4-AA, but not by dipyrone or 4-MAA. On the other hand, the anti-hyperalgesic effect of dipyrone or 4-MAA was reversed by glibenclamide or ODQ. These results suggest that the activation of neuronal CB1, but not CB2 receptor, in peripheral tissue is involved in the anti-hyperalgesic effect of 4-aminoantipyrine. In addition, 4-methylaminoantipyrine mediates the anti-hyperalgesic effect by cGMP activation and KATP opening.

Toll-like Receptor 4 Activation Promotes Cardiac Arrhythmias By Decreasing The Transient Outward Potassium Current (ito) Through An Irf3-dependent And Myd88-independent Pathway.

Cardiac arrhythmias are one of the main causes of death worldwide. Several studies have shown that inflammation plays a key role in different cardiac diseases and Toll-like receptors (TLRs) seem to be involved in cardiac complications. In the present study, we investigated whether the activation of TLR4 induces cardiac electrical remodeling and arrhythmias, and the signaling pathway involved in these effects. Membrane potential was recorded in Wistar rat ventricle. Ca(2+) transients, as well as the L-type Ca(2+) current (ICaL) and the transient outward K(+) current (Ito), were recorded in isolated myocytes after 24 h exposure to the TLR4 agonist, lipopolysaccharide (LPS, 1 μg/ml). TLR4 stimulation in vitro promoted a cardiac electrical remodeling that leads to action potential prolongation associated with arrhythmic events, such as delayed afterdepolarization and triggered activity. After 24 h LPS incubation, Ito amplitude, as well as Kv4.3 and KChIP2 mRNA levels were reduced. The Ito decrease by LPS was prevented by inhibition of interferon regulatory factor 3 (IRF3), but not by inhibition of interleukin-1 receptor-associated kinase 4 (IRAK4) or nuclear factor kappa B (NF-κB). Extrasystolic activity was present in 25% of the cells, but apart from that, Ca(2+) transients and ICaL were not affected by LPS; however, Na(+)/Ca(2+) exchanger (NCX) activity was apparently increased. We conclude that TLR4 activation decreased Ito, which increased AP duration via a MyD88-independent, IRF3-dependent pathway. The longer action potential, associated with enhanced Ca(2+) efflux via NCX, could explain the presence of arrhythmias in the LPS group.

Endocytosis Of Tight Junctions Caveolin Nitrosylation Dependent Is Improved By Cocoa Via Opioid Receptor On Rpe Cells In Diabetic Conditions.

Retinal pigment epithelium cells, along with tight junction (TJ) proteins, constitute the outer blood retinal barrier (BRB). Contradictory findings suggest a role for the outer BRB in the pathogenesis of diabetic retinopathy (DR). The aim of this study was to investigate whether the mechanisms involved in these alterations are sensitive to nitrosative stress, and if cocoa or epicatechin (EC) protects from this damage under diabetic (DM) milieu conditions. Cells of a human RPE line (ARPE-19) were exposed to high-glucose (HG) conditions for 24 hours in the presence or absence of cocoa powder containing 0.5% or 60.5% polyphenol (low-polyphenol cocoa [LPC] and high-polyphenol cocoa [HPC], respectively). Exposure to HG decreased claudin-1 and occludin TJ expressions and increased extracellular matrix accumulation (ECM), whereas levels of TNF-α and inducible nitric oxide synthase (iNOS) were upregulated, accompanied by increased nitric oxide levels. This nitrosative stress resulted in S-nitrosylation of caveolin-1 (CAV-1), which in turn increased CAV-1 traffic and its interactions with claudin-1 and occludin. This cascade was inhibited by treatment with HPC or EC through δ-opioid receptor (DOR) binding and stimulation, thereby decreasing TNF-α-induced iNOS upregulation and CAV-1 endocytosis. The TJ functions were restored, leading to prevention of paracellular permeability, restoration of resistance of the ARPE-19 monolayer, and decreased ECM accumulation. The detrimental effects on TJs in ARPE-19 cells exposed to DM milieu occur through a CAV-1 S-nitrosylation-dependent endocytosis mechanism. High-polyphenol cocoa or EC exerts protective effects through DOR stimulation.