Non-Peptide Opioids Differ in Effects on Mu-Opioid (MOP) and Serotonin 1A (5-HT1A) Receptors Heterodimerization and Cellular Effectors (Ca2+, ERK1/2 and p38) Activation

The importance of the dynamic interplay between the opioid and the serotonin neuromodulatory systems in chronic pain is well recognized. In this study, we investigated whether these two signalling pathways can be integrated at the single-cell level via direct interactions between the mu-opioid (MOP) and the serotonin 1A (5-HT1A) receptors. Using fluorescence cross-correlation spectroscopy (FCCS), a quantitative method with single-molecule sensitivity, we characterized in live cells MOP and 5-HT1A interactions and the effects of prolonged (18 h) exposure to selected non-peptide opioids: morphine, codeine, oxycodone and fentanyl, on the extent of these interactions. The results indicate that in the plasma membrane, MOP and 5-HT1A receptors form heterodimers that are characterized with an apparent dissociation constant Kdapp = (440 ± 70) nM). Prolonged exposure to all non-peptide opioids tested facilitated MOP and 5-HT1A heterodimerization and stabilized the heterodimer complexes, albeit to a different extent: Kd, Fentanylapp = (80 ± 70) nM), Kd,Morphineapp = (200 ± 70) nM, Kd, Codeineapp = (100 ± 70) nM and Kd, Oxycodoneapp = (200 ± 70) nM. The non-peptide opioids differed also in the extent to which they affected the mitogen-activated protein kinases (MAPKs) p38 and the extracellular signal-regulated kinase (Erk1/2), with morphine, codeine and fentanyl activating both pathways, whereas oxycodone activated p38 but not ERK1/2. Acute stimulation with different non-peptide opioids differently affected the intracellular Ca2+ levels and signalling dynamics. Hypothetically, targeting MOP–5-HT1A heterodimer formation could become a new strategy to counteract opioid induced hyperalgesia and help to preserve the analgesic effects of opioids in chronic pain

Non-Peptide Opioids Differ in Effects on Mu-Opioid (MOP) and Serotonin 1A (5-HT1A) Receptors Heterodimerization and Cellular Effectors (Ca2+, ERK1/2 and p38) Activation

The importance of the dynamic interplay between the opioid and the serotonin neuromodulatory systems in chronic pain is well recognized. In this study, we investigated whether these two signalling pathways can be integrated at the single-cell level via direct interactions between the mu-opioid (MOP) and the serotonin 1A (5-HT1A) receptors. Using fluorescence cross-correlation spectroscopy (FCCS), a quantitative method with single-molecule sensitivity, we characterized in live cells MOP and 5-HT1A interactions and the effects of prolonged (18 h) exposure to selected non-peptide opioids: morphine, codeine, oxycodone and fentanyl, on the extent of these interactions. The results indicate that in the plasma membrane, MOP and 5-HT1A receptors form heterodimers that are characterized with an apparent dissociation constant Kdapp = (440 ± 70) nM). Prolonged exposure to all non-peptide opioids tested facilitated MOP and 5-HT1A heterodimerization and stabilized the heterodimer complexes, albeit to a different extent: Kd, Fentanylapp = (80 ± 70) nM), Kd,Morphineapp = (200 ± 70) nM, Kd, Codeineapp = (100 ± 70) nM and Kd, Oxycodoneapp = (200 ± 70) nM. The non-peptide opioids differed also in the extent to which they affected the mitogen-activated protein kinases (MAPKs) p38 and the extracellular signal-regulated kinase (Erk1/2), with morphine, codeine and fentanyl activating both pathways, whereas oxycodone activated p38 but not ERK1/2. Acute stimulation with different non-peptide opioids differently affected the intracellular Ca2+ levels and signalling dynamics. Hypothetically, targeting MOP–5-HT1A heterodimer formation could become a new strategy to counteract opioid induced hyperalgesia and help to preserve the analgesic effects of opioids in chronic pain

Mathematical Modeling of the Hypothalamic-Pituitary-Adrenal Axis Dynamics in Rats

The hypothalamic-pituitary-adrenal (HPA) axis is a dynamic regulatory network of biochemical reactions that integrates and synchronizes the nervous and the endocrine systems functions at the organism level. In order to describe how this vast network of biochemical interactions operates, we have developed a nonlinear eleven-dimensional stoichiometric model that concisely describes key biochemical transformations that comprise the HPA axis in rats. In a stoichiometric model of a biochemical system, the outcomes of complex biochemical pathways are succinctly described by stoichiometric relations. In this representation, substances that initiate, i.e. enter a pathway are regarded to behave as reactants; substances that are generated in a pathway are regarded to behave as products; and the rates at which products of a pathway appear are jointly proportional to the concentrations of the reactants. In order to derive rate constants for specific biochemical reaction pathways, we have resorted to our recently developed nonlinear reaction model that concisely describes biochemical transformations in the HPA axis in humans. In this way, a mathematical framework is developed to describe in the form of a system of ordinary differential equations (ODEs) the integration of biochemical pathways that constitute the HPA axis on chemical kinetics basis. This, in turn, allows us to use numerical simulations to investigate how the underlying biochemical pathways are intertwined to give an integral HPA axis response at the organism level to a variety of external or internal perturbators of the HPA dynamics. Given that the HPA axis is a nonlinear dynamical network, its response is complex and often cannot be intuitively predicted, stoichiometric modeling can be harnessed for gaining additional insights into dynamical functioning of this complex neuroendocrine system.Belgrade, Serbia, June 20-24, 2016 [http://alas.matf.bg.ac.rs/~websites/bioinfo/

Nelinearna laserska skenirajuća mikroskopija hemoglobina i eritrocita

Oslikavanje eritrocita bez obeležavanja pomoću konfokalne ili epifluorescentne mikroskopije je praktično nemoguće jer se fluorescencija od hemoglobina (Hb) ne može detektovati zbog brzih neradijativnih raspada koji su dominantni u odnosu na spontanu emisiju. Sa druge strane, demonstrirana je značajna dvofotonska apsorptivnost Hb u infracrvenoj oblasti1 kao i dvofotonski pobuđena fluorescencija (TPEF) od novoformiranog fotoprodukta2 nakon interakcije ultrakratkih laserskih impulsa sa Hb. TPEF mikroskopija je korišćena u brojnim studijama za oslikavanje Hb i eritrocita bez obeležavanja.3 U ovom radu prikazane su fotofizičke osobine i potencijalne primene novoformiranog fotoprodukta. Kreirane su fluorescentne mikrostrukture pomoću direktnog laserskog upisivanja na tankim Hb slojevima (micropatterning). Fotoprodukt je okarakterisan pomoću različitih (mikro)spektroskopskih tehnika: emisije nakon vofotonske apsorpcije, UV-VIS apsorpcije i spektralnog oslikavanja. Razmatrane su spektroskopske osobine protoporfirina IX (PpIX) pošto je on strukturni deo Hb i potencijalno može biti prekursor za formiranje fotoprodukta. Takođe, Hb je tretiran vodonik peroksidom da bi se uočila optička i spektroskopska sličnost tako dobijenog jedinjenja i fotoprodukta. Potvrđena je dugovremena fotostabilnost fotoprodukta u periodima od nekoliko meseci. Na osnovu toga što je fotoprodukt moguće dobiti na prostorno i vremenski kontrolisan način, izvršeno je fotoobeležavanje i praćenje kretanja pojedinačnih eritrocita u humanoj punoj krvi zdravog donora

Нелинеарна ласерска скенирајућа микроскопија хемоглобина и еритроцита

Oslikavanje eritrocita bez obeležavanja pomoću konfokalne ili epifluorescentne mikroskopije je praktično nemoguće jer se fluorescencija od hemoglobina (Hb) ne može detektovati zbog brzih neradijativnih raspada koji su dominantni u odnosu na spontanu emisiju. Sa druge strane, demonstrirana je značajna dvofotonska apsorptivnost Hb u infracrvenoj oblasti1 kao i dvofotonski pobuđena fluorescencija (TPEF) od novoformiranog fotoprodukta2 nakon interakcije ultrakratkih laserskih impulsa sa Hb. TPEF mikroskopija je korišćena u brojnim studijama za oslikavanje Hb i eritrocita bez obeležavanja.3 U ovom radu prikazane su fotofizičke osobine i potencijalne primene novoformiranog fotoprodukta. Kreirane su fluorescentne mikrostrukture pomoću direktnog laserskog upisivanja na tankim Hb slojevima (micropatterning). Fotoprodukt je okarakterisan pomoću različitih (mikro)spektroskopskih tehnika: emisije nakon dvofotonske apsorpcije, UV-VIS apsorpcije i spektralnog oslikavanja. Razmatrane su spektroskopske osobine protoporfirina IX (PpIX) pošto je on strukturni deo Hb i potencijalno može biti prekursor za formiranje fotoprodukta. Takođe, Hb je tretiran vodonik peroksidom da bi se uočila optička i spektroskopska sličnost tako dobijenog jedinjenja i fotoprodukta. Potvrđena je dugovremena fotostabilnost fotoprodukta u periodima od nekoliko meseci. Na osnovu toga što je fotoprodukt moguće dobiti na prostorno i vremenski kontrolisan način, izvršeno je fotoobeležavanje i praćenje kretanja pojedinačnih eritrocita u humanoj punoj krvi zdravog donora.Осликавање еритроцита без обележавања помоћу конфокалне или епифлуоресцентне микроскопије је практично немогуће јер се флуоресценција од хемоглобина (Хб) не може детектовати због брзих нерадијативних распада који су доминантни у односу на спонтану емисију. Са друге стране, демонстрирана је значајна двофотонска апсорптивност Хб у инфрацрвеној области1 као и двофотонски побуђена флуоресценција (ТPEF) од новоформираног фотопродукта2 након интеракције ултракратких ласерских импулса са Хб. ТPEF микроскопија је коришћена у бројним студијама за осликавање Хб и еритроцита без обележавања.3 У овом раду приказане су фотофизичке особине и потенцијалне примене новоформираног фотопродукта. Креиране су флуоресцентне микроструктуре помоћу директног ласерског уписивања на танким Хб слојевима (micropatterning). Фотопродукт је окарактерисан помоћу различитих (микро)спектроскопских техника: емисије након двофотонске апсорпције, UV-VIS апсорпције и спектралног осликавања. Разматране су спектроскопске особине протопорфирина IX (PpIX) пошто је он структурни део Хб и потенцијално може бити прекурсор за формирање фотопродукта. Такође, Хб је третиран водоник пероксидом да би се уочила оптичка и спектроскопска сличност тако добијеног једињења и фотопродукта. Потврђена је дуговремена фотостабилност фотопродукта у периодима од неколико месеци. На основу тога што је фотопродукт могуће добити на просторно и временски контролисан начин, извршено је фотообележавање и праћење кретања појединачних еритроцита у хуманој пуној крви здравог донора.Knjiga sažetaka: Treći Kongres biologa Srbije, Zlatibor, Srbija 21 - 25. 9. 2022

Interactions of ultrashort laser pulses with hemoglobin: Photophysical aspects and potential applications

Hemoglobin (Hb), a life-sustaining and highly abundant erythrocyte protein, is not readily fluorescent. A few studies have already reported Two-Photon Excited Fluorescence (TPEF) of Hb, however, the mechanisms through which Hb becomes fluorescent upon interaction with ultrashort laser pulses are not completely understood. Here, we characterized photophysically this interaction on Hb thin film and erythrocytes using fluorescence spectroscopy upon single-photon/two-photon absorption, and UV-VIS single-photon absorption spectroscopy. A gradual increase of the fluorescence intensity, ending up with saturation, is observed upon prolonged exposure of Hb thin layer and erythrocytes to ultrashort laser pulses at 730 nm. When compared to protoporphyrin IX (PpIX) and oxidized Hb by H2O2, TPEF spectra from a thin Hb film and erythrocytes showed good mutual agreement, broad peaking at 550 nm, supporting hemoglobin undergoes degradation and that same fluorescent specie(s) originating from the heme moiety are generated. The uniform square shaped patterns of the fluorescent photoproduct exhibited the same level of the fluorescence intensity even after 12 weeks from the formation, indicating high photoproduct stability. We finally demonstrated the full potential of the formed Hb photoproduct with TPEF scanning microscopy towards spatiotemporally controlled micropatterning in HTF and single human erythrocyte labelling and tracking in the whole blood

Alpha-Gal on the Protein Surface Hampers Transcytosis through the Caco-2 Monolayer

Transepithelial transport of proteins is an important step in the immune response to food allergens. Mammalian meat allergy is characterized by an IgE response against the carbohydrate moiety galactosyl-α-1,3-galactose (α-Gal) present on mammalian glycoproteins and glycolipids, which causes severe allergic reactions several hours after red meat consumption. The delayed reaction may be related to the processing of α-Gal carrying proteins in the gastrointestinal tract. The aim of this study was to investigate how protein glycosylation by α-Gal affects the susceptibility to gastric digestion and transport through the Caco-2 cell monolayer. We found that α-Gal glycosylation altered protein susceptibility to gastric digestion, where large protein fragments bearing the α-Gal epitope remained for up to 2 h of digestion. Furthermore, α-Gal glycosylation of the protein hampered transcytosis of the protein through the Caco-2 monolayer. α-Gal epitope on the intact protein could be detected in the endosomal fraction obtained by differential centrifugation of Caco-2 cell lysates. Furthermore, the level of galectin-3 in Caco-2 cells was not affected by the presence of α-Gal glycosylated BSA (bovine serum albumin) (BSA-α-Gal). Taken together, our data add new knowledge and shed light on the digestion and transport of α-Gal glycosylated proteins